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from diagnosis, the seeds of better health
chromID MRSA
chromID MRSA* has been designed for the screening of Methicillin-Resistant Staphylococcus aureus (MRSA). It helps healthcare units actively reinforce MRSA surveillance culture and control healthcare-associated infections through implementing patient isolation and reinforced hygiene procedures. (JCM 10/04). chromID MRSA (patent) enables the direct identification of MRSA strains based on alpha-glucosidase-producing colonies and the presence of an antibiotic, cefoxitin. chromID MRSA enables the immediate identification of Methicillin-Resistant S. aureus through the growth of green colonies after 18-24 hour incubation**; a green colour is characteristic and specific of MRSA colonies. It also provides excellent performance for the screening of MRSA in terms of nutrient capacity, detection sensitivity and colouration specificity (JCM 02/06). The presence of cefoxitin enables the detection of MRSA strains, including hetero-resistant strains. chromID MRSA can be used for direct screening and/or after an enrichment step (JCM 04/06), chromID MRSA enables the selective inhibition of most bacteria not belonging to the genus Staphylococcus, as well as yeasts. It is also extremely easy-to-read (JCM 04/07) and ready-to-use. chromID MRSA enables culture and isolation and identification on the same medium.
*chromID MRSA is the new name for MRSA ID. **See technical sheet for further information.
Contents
ARTICLES
Comparison of four chromogenic media for culture based screening of
Methicillin-Resistant Staphylococcus aureus
JOURNAL OF MEDICAL MICROBIOLOGY, April 2007 A. Cherkaoui et al.
02
Combined Use of Pastorex Staph-Plus and Either of Two New Chromogenic

Agars, MRSA ID and CHROMagar MRSA, for Detection of Methicillin-Resistant Staphylococcus aureus
JOURNAL OF CLINICAL MICROBIOLOGY, January 2007 V. Compernolle et al.
03
Evaluation of three chromogenic media (MRSA-ID, MRSA-Select and

CHROMagar MRSA) and ORSAB for surveillance cultures of Methicillin-Resistant Staphylococcus aureus
CLINICAL MICROBIOLOGY AND INFECTION, April 2006 I. Nahimana et al.
04
Performance of MRSA ID, a New Chromogenic Medium for Detection of

JOURNAL OF CLINICAL MICROBIOLOGY, February 2006 B. M. W. Diederen et al.
05
Development and Evaluation of a Chromogenic Agar Medium for

JOURNAL OF CLINICAL MICROBIOLOGY, October 2004 J. D. Perry et al.
06
POSTERS
ECCMID / Munich (Germany) / April, 2007 In vitro evaluation of MRSA screening methods
07
S. Bcher et al.
ECCMID / Munich (Germany) / April, 2007 Comparison of 3 Chromogenic media for rapid detection of Methicillin-Resistant Staphylococcus aureus (MRSA) from screening swabs in hospitalised patients
08
C. Nonhoff et al.
ECCMID / Munich (Germany) / April, 2007 An evaluation of MRSA ID: A new Chromogenic Medium for the Isolation and Identification of Methicillin-Resistant Staphylococcus aureus
09
A. Davies et al.
ECCMID / Munich (Germany) / April, 2007 Comparison of Chromogenic Agar in the detection of MRSA
11
A. Curry et al.
ICAAC / San Francisco (USA) / September, 2006 A Multicentre Evaluation of Two Chromogenic Media for the Isolation of MethicillinResistant Staphylococcus aureus from Clinical Samples
12
N. Bendridi et al.
ICAAC / San Francisco (USA) / September, 2006 Evaluation of 3 Chromogenic media for the detection of Methicillin-Resistant Staphylococcus aureus (MRSA) Strains from Belgian hospitalized patients
14
C. Nonhoff et al.
M. J. Bruins et al.
ECCMID / Nice (France) / April, 2006 Evaluation of chromogenic bioMrieux MRSA ID medium for direct detection of Methicillin-Resistant Staphylococcus aureus from surveillance cultures and from clinical samples.
17
C. Snchez-Carrillo et al.
P. Ciragil et al.
ECCMID / Nice (France) / April, 2006 Comparative evaluation of the chromogenic MRSA ID media with standard procedure for screening of Methicillin-Resistant Staphylococcus aureus carriers and detection of high level resistance to Mupirocin by disk diffusion
21
I. Mansoor et al.
N. Fanjat et al.
ECCMID / Copenhagen (Denmark) / April, 2005 Multiresistant bacteria screening: Clinical evaluation of MRSA ID, a new chromogenic medium for the screeningof Methicillin-Resistant Staphylococcus aureus
25
M. E. Reverdy et al.
ECCMID / Copenhagen (Denmark) / April, 2005 Comparison of MRSA ID medium and enrichment broth culture for detection of Methicillin-Resistant Staphylococcus aureus carriers by muco-cutaneous surveillance cultures
27
C. Nonhoff et al.
chromID VRE
RICAI / Paris (France) / December, 2005 Comptability of MRSA ID, a new Chromogenic Medium dedicated to MRSA carriage, with identification, susceptibility testing and other rapid tests
23
chromID ESBL
ECCMID / Nice (France) / April, 2006 Detection of nasal carriage of Staphylococcus aureus and Methicillin-Resistant Staphylococcus aureus in general population with a new Chromogenic medium: MRSA ID AGAR
19
chromID MRSA
ECCMID / Nice (France) / April, 2006 Optimisation of screening for Methicillin-Resistant Staphylococcus aureus by using the Chromogenic medium MRSA ID
16
April, 2007
JOURNAL OF MEDICAL MICROBIOLOGy (2007), 56, 500503 DOI 10.1099/jmm.0.46981-0
Comparison of four chromogenic media for culturebased screening of Methicillin-Resistant Staphylococcus aureus
Abdessalam Cherkaoui,1 Gesuele Renzi,1 Patrice Franc ois2 and Jacques Schrenzel1,2
1
Clinical Microbiology Laboratory, University of Geneva Hospitals (HUG), Service of Infectious Diseases, CH-1211 Geneva 14, Switzerland 2 Genomic Research Laboratory, University of Geneva Hospitals (HUG), Service of Infectious Diseases, CH-1211 Geneva 14, Switzerland
Methicillin-Resistant Staphylococcus aureus (MRSA) are responsible for nosocomial and community-acquired infections. Detection of MRSA is of utmost importance for the adaptation of infection control and therapeutic strategies. To date, selective agar plates constitute the standard routine method for reliable detection of this worldwide infectious problem. The performance of four different chromogenic media was evaluated in this study for MRSA detection and identification on > 240 consecutive swab samples. The results indicate that primary plating on MRSASelect or MRSA ID is more sensitive than screening with oxacillin-based culture media. In addition, the utilization of cefoxitin- or cephamycin-containing plates reduces significantly the number of required confirmatory tests. Several selective agar plates allowing the identification of S. aureus are commercially available. However, their respective performances under real conditions of utilization are heterogeneous, underlining the absence of a gold standard medium for MRSA screening.
KEY POINTS

MRSA Select or MRSA ID (new name chromID MRSA) are convenient & time-saving for the presumptive identification of MRSA in routine use. Interpretation of colony colours is easy.
02
January, 2007
Vol. 45, No. 1
JOURNAL OF CLINICAL MICROBIOLOGY, Jan. 2007, p. 154158 0095-1137/07/$08.00 0 doi:10.1128/JCM.01115-06

Copyright 2007, American Society for Microbiology. All Rights Reserved.
Combined Use of Pastorex Staph-Plus and Either of Two New Chromogenic Agars, MRSA ID and CHROMagar MRSA, for Detection of Methicillin-Resistant Staphylococcus aureus
Veerle Compernolle, Gerda Verschraegen, and Geert Claeys*
Department of Microbiology, Ghent University Hospital, Ghent, Belgium
Received 31 May 2006/Returned for modification 25 August 2006/Accepted 24 October 2006
We describe the search toward a fast and reliable strategy to detect and confirm the presence of Methicillin-Resistant Staphylococcus aureus (MRSA) in screening samples. First, we evaluated the sensitivities and specificities of oxacillin resistance screening agar (ORSA) with enrichment (tryptic soy broth [TSB] and ORS [TSB-ORSA]) and without enrichment (ORSA), MRSA ID (MRSA ID) plates, and CHROMagar MRS (C MRSA) plates, all of which were inoculated with equal volumes of a suspension made by emulsifying screening swabs. Whereas the sensitivities after 48 h were similar for all media tested (77% for MRSA ID and ORSA; 73% for C MRSA and ORSA after enrichment [TSB-ORSA]), the specificities of MRSA ID (98% after 24 h and 94% after 48 h) and C MRSA (98% after 24 h and 90% after 48 h) were superior to the specificities of ORSAs (92% after 24 h and 83% after 48 h) and TSB-ORSA (86% after 24 h and 81% after 48 h). Subsequently, the performance of the Pastorex Staph-Plus agglutination test with presumptive MRSA isolates taken directly from chromogenic agars (direct Pastorex agglutination) was compared to that of the Pastorex Staph-Plus agglutination test with isolates from blood agar subcultures (conventional Pastorex agglutination). When the direct Pastorex agglutination test on MRSA ID plates was combined with Gram staining, the direct Pastorex agglutination test with samples from MRSA ID plates was as reliable as the conventional Pastorex agglutination test with samples from blood agar subcultures from MRSA ID plates. In contrast, the direct Pastorex agglutination test with samples from C MRSA plates gave false-negative results. Finally, we calculated the processing times of the four different strategies, namely, (i) enrichment in TSB supplemented with NaCl, subsequent culture on ORSA, and the conventional Pastorex agglutination test; (ii) direct inoculation of ORSA combined with conventional Pastorex agglutination test; (iii) direct inoculation of MRSA ID plate combined with Gram staining and the direct Pastorex agglutination test; and (iv) direct inoculation of C MRSA plates combined with Gram staining and the direct Pastorex agglutination test. We concluded that the use of MRSA ID in combination with Gram staining and the direct Pastorex agglutination test is faster and more specific than the other strategies tested.
KEY POINTS

The use of both MRSA ID (new name chromID MRSA) or CHROMagar MRSA reduced the workload and speed up the processing time. The use of MRSA ID (new name chromID MRSA) in combination with the direct Pastorex agglutination is faster and more specific than the other strategies tested.
03
chromID VRE
chromID ESBL
chromID MRSA
April, 2006
CLINICAL MICROBIOLOGY AND INFECTION ORIGINAL ARTICLE 10.1111/j.1469-0691.2006.01534.x
Evaluation of three chromogenic media (MRSA-ID, MRSA-Select and CHROMagar MRSA) and ORSAB for surveillance cultures of Methicillin-Resistant Staphylococcus aureus
I. Nahimana, P. Francioli and D. S. Blanc
Hospital Preventive Medicine, University Hospital of Lausanne, Lausanne, Switzerland
ABSTRACT
Screening specimens were homogenised in saline 0.9% w v before either direct inoculation or following enrichment in broth on three chromogenic media (MRSA-ID, CHROMagar MRSA and MRSA Select) and ORSAB medium for the detection of Methicillin-Resistant Staphylococcus aureus (MRSA). In total, 102 of 466 specimens yielded MRSA on at least one medium. After incubation for 16 - 18 h, the sensitivity was 51%, 59%, 47% and 65% on MRSA-ID, CHROMagar MRSA, ORSAB and MRSA Select, respectively, compared with 82%, 75%, 67% and 80%, respectively, after 42 h, and 93%, 95%, 79% and not tested, respectively, following broth enrichment. There were significantly more MRSA colonies on MRSA-Select after 16 - 18 h than on ORSAB or MRSA ID (p 0.001 and 0.0022, respectively), whereas there were more MRSA colonies after 42 h on MRSA-ID and MRSA-Select than on ORSAB (p 0.0004 and 0.012, respectively). The specificity of the media for identifying MRSA based on the colour of colonies after incubation for 1618 h was 100%, 99%, 99% and 100%, respectively, compared with 98%, 97%, 98% and 98%, respectively, after 42 h, and 100%, 99%, 100% and not tested, respectively, following broth enrichment. The speed of detection (mean time to report a positive result) was 1.65, 1.72, 2.31 and 1.35 days, respectively. For each of the three media tested following enrichment, the use of an enrichment broth increased the detection rate of MRSA by 1624%. Keywords: Chromogenic media, detection, enrichment, Methicillin-Resistant Staphylococcus aureus, performance, screening specimens Original Submission: 4 October 2005; Revised Submission: 27 March 2006; Accepted: 17 April 2006 Clin Microbiol Infect
KEY POINTS

The enrichment broth increased the detection of MRSA by 16 to 24% for all media. The fact that no additional tests were needed with MRSA ID (new name chromID MRSA) and MRSA Select makes their use advantageous.
04
February, 2006
Vol. 44, No. 2
JOURNAL OF CLINICAL MICROBIOLOGY, Feb. 2006, p. 586588 0095-1137/06/$08.00 0 doi:10.1128/JCM.44.2.586588.2006

Performance of MRSA ID, a New Chromogenic Medium for Detection of Methicillin-Resistant Staphylococcus aureus
chromID MRSA
05
Bram M. W. Diederen,1,2* Marie-Louise van Leest,1 Inge van Duijn,1 Piet Willemse,1 Peter H. J. van Keulen,1 and Jan A. J. W. Kluytmans1
Laboratory for Microbiology and Infection Control, Amphia Hospital, Breda, The Netherlands,1 and Laboratory of Medical Microbiology, St. Elisabeth Hospital, Tilburg, The Netherlands2
Received 1 July 2005/Returned for modification 23 August 2005/Accepted 6 November 2005
KEY POINTS

MRSA ID (new name chromID MRSA) is sensitive and specific (differentiation MSSA and MRSA) in vitro. MRSA ID (new name chromID MRSA) enables the detection of a large number of different MRSA strains. With MRSA ID (new name chromID MRSA), optimal results can be obtained within a day.
chromID VRE
chromID ESBL
MRSA ID was evaluated for its ability to identify Methicillin-Resistant Staphylococcus aureus. A well-defined collection of staphylococci was used (n=998). The sensitivity after 24 h was 96.4%, increasing to 98.8% after 48 h. The specificity was 98.2% after 24 h and decreased to 89.7% after 48 h.
October, 2004
Vol. 42, No. 10
JOURNAL OF CLINICAL MICROBIOLOGY, Oct. 2004, p. 45194523 0095-1137/04/$08.00 0 DOI: 10.1128/JCM.42.10.45194523.2004

Development and Evaluation of a Chromogenic Agar Medium for Methicillin-Resistant Staphylococcus aureus
John D. Perry,* Amie Davies, Lynne A. Butterworth, Andrew L. J. Hopley, Audrey Nicholson, and F. Kate Gould
Department of Microbiology, Freeman Hospital, Newcastle upon Tyne, United Kingdom
Received 9 April 2004/Accepted 21 June 2004
We describe here the development and evaluation of MRSA ID, a new chromogenic agar medium for the specific isolation and identification of Methicillin-Resistant Staphylococcus aureus (MRSA). We used S. aureus ID (bioMrieux, La Balme Les Grottes, France) and supplemented it with various antimicrobials, including cefoxitin, ciprofloxacin, oxacillin, and methicillin. Cefoxitin proved to be superior to the other antimicrobials for the selection of MRSA from other strains of S. aureus. MRSA ID (consisting of S. aureus ID supplemented with 4 mg of cefoxitin/liter) was evaluated by the use of 747 swabs from various clinical sites. All specimens were also cultured on CHROMagar MRSA and oxacillin resistance screening agar base (ORSAB) and in selective mannitol broth (SMB). A total of 85 MRSA strains were isolated by a combination of all methods. After 22 to 24 h of incubation, 80% of the MRSA strains were isolated as green colonies on MRSA ID, compared with 59 and 62% of the strains that were isolated as colored colonies on CHROMagar MRSA and ORSAB, respectively. After 48 h of incubation, 89, 72, and 78% of the MRSA strains were isolated on MRSA ID, CHROMagar MRSA, and ORSAB, respectively. Sixty-five percent of the strains were isolated by growth in SMB. The specificities of MRSA ID, CHROMagar MRSA, ORSAB, and SMB were 99.5, 99.3, 97.9, and 92.8%, respectively, after 22 to 24 h of incubation. We conclude that MRSA ID is a sensitive and specific medium for the isolation and identification of MRSA.
KEY POINTS

S. aureus ID (new name chromID S. aureus) has shown a high sensitivity & specificity for the isolation of S. aureus. Cefoxitin proved to be superior to the other antiobiotics for the selection of MRSA. MRSA ID (new name chromID MRSA), consisting of chromID S. aureus supplemented with cefoxitin is a sensitive and specific medium for the isolation and identification of MRSA.
06
ECCMID / April, 2007

Munich (Germany) poster P1606
In vitro evaluation of MRSA screening methods

S. Bcher1, R. Smyth2, G. Kahlmeter2, R. Skov1
1 2
Staphylococcal Laboratory, National Center for Antimicrobials and Infection Control, Statens Serum Institut (SSI), Denmark Department of Clinical Microbiology, Vxj, Sweden
INTRODUCTION & OBJECTIVES:

Detection of MRSA in screening samples has proven difficult and laborious. Use of enrichment broth is known to increase sensitivity, but requires an extra day of incubation. Several brands of chromogenic agars have recently become available and are said to give good results from direct inoculation and 24h incubation. There is a lack of published studies of the use of enrichment broths in combination with these new selective agars. This study uses a large collection of diverse MRSA strains and a variety of CoNS tested with four different selective agars and two different enrichment broths.
DISCUSSION
Preliminary investigations showed that some MRSA strains would not grow in the MSB. We therefore developed a semi-selective broth with 3.5 mg/L cefoxitin and only 20 mg/L aztreonam as 75 mg/L of aztreonam in combination with cephalosporin was too inhibitory for some MRSA strains. This in vitro investigation on a highly diverse collection supported these findings: 24 MRSA isolates (25%) representing 9 of 13 clonal complexes did not grow in the MSB. All 97 MRSA isolates were able to grow in the TSB. This suggests that the antibiotic content is too high in the MSB. Chromogenic agars: All 3 chromogenic agars tested to support growth of the tested MRSA isolates. A recent study, on clinical samples, by Nahimana et al. (Clin Microbiol Infect. 2006 Dec;12 (12):1168-74) showed that broth enrichment in combination with chromogenic agars led to an increased sensitivity of 14-26%. Although all chromogenic agars supported growth of all the MRSA isolates in this collection, the findings above suggest that competition between bacteria in clinical samples may delay or inhibit MRSA growth, suggesting that an enrichment step is necessary to optimize sensitivity on clinical samples.
MATERIAL AND METHODS

Strains tested: 97 MRSA strains, representing 13 clonal complexes, including many heterogenic isolates. 52 Methicillin-Sensitive Staphylococcus aureus (MSSA). 49 Methicillin-Resistant coagulase-negative Staphylococci (MRCNS). Enrichment broths: TSB with 2.5% salt, 20 mg/L aztreonam and 3.5 mg/L cefoxitin (TSB). Phenol red mannitol broth with 75 mg/L aztreonam and 5mg/L ceftizoxime (MSB). (Wertheim et al. JCM, 2001,vol.39). Selective agars: MRSA SSI (Statens Serum Institut) MRSA ID (bioMrieux) MRSA Select (Bio-Rad) Mannitol salt agar with 4 mg/L cefoxitin (MSA) Reference agar: 5% blood agar Method: Direct inoculation: 20 CFU of each strain was inoculated directly onto each of the agar media. After 18 and 48h incubation at 35-36C, colonies on the solid media were counted. Enrichment: 20 CFU of each strain was inoculated into TSB and MSB. After 18-24h incubation 20l was subcultured onto each of the selective agars and a blood agar. After another 18h at 35-36C, growth and colony colour was registered.
RESULTS OF PLATES:
Agar plates Direct inoculation Sens. MRSA SSI MRSA ID MRSA Select MSA Blood agar 98% 98% 97% 78%/91%* 100% Spec. 99% 98% 99% 71% Inoculation following 18 24 h of enrichment in broth TSB sens. 99% 99% 99% 99% 100% TSB spec. 96% 94% 92% 64% 59% MSB sens. 74% 72% 74% 70% 75% MSB spec. 98% 94% 95% 77% 58%
*Following 24 and 48 hours of incubation, respectively.
RESULTS OF ENRICHMENT BROTHS:

MSB: Sensitivity 75% and visible growth (turbidity and/or colour change) was observed in only 48%. Specificity was 58%. TSB: Sensitivity 100% and visible growth was recorded in 99% of the TSB broths. Specificity was 59%.
MRSA SSI MRSA ID MRSA Select MSA
MSB enrichment displayed a low sensitivity (75%). Growth in MSB was only visible in 48% of the broths and only few gave colour change after 24h. TSB enrichment displayed had high sensitivity (100%). Only one MRSA strain did not grow to 107 108 after overnight incubation. The low specificity for both broths was primarily caused by growth of MRCNS. All 3 chromogenic MRSA agars supported growth of the tested MRSA, even when using low inocula, and specificity was high with all. The MSA plate required 48 hours incubation (or a preceding period of enrichment) to provide acceptable results. Although our study implies that the chromogenic agars do not perform better when preceded by enrichment, there is a need for a comparative clinical study.
KEY POINTS
The 3 chromogenic media supported growth of the MRSA tested, even when using low inocula. This study was performed using collection strains, therefore a comparative clinical study is required.
07
chromID VRE
CONCLUSION
chromID ESBL
chromID MRSA

Comparison of three chromogenic media for rapid detection of Methicillin-Resistant Staphylococcus aureus (MRSA) from screening swabs in hospitalised patients
Claire Nonhoff, O. Denis, A. Brenner, P. Buidin, C. Thiroux and Marc J. Struelens
Reference Laboratory for Staphylococci ULB-Erasme Hospital, Brussels, Belgium
OBJECTIVES
To compare the performance of MRSA-ID (bioMrieux), MRSA-Screen (Oxoid) and MRSA-Select (Bio-Rad) media for detection of MRSA in muco-cutaneous swab samples from patients admitted to a 860-bed teaching hospital.
Table 2: Distribution of MRSA isolates by patients (n = 45)

N of patients 21 10 6 4 1 1 1 1

MRSA-ID +/18h +/36h 0 0 +/18h +/36h 0 +/36h
Primary plates MRSA-Screen MRSA-Select +/18h +/36h 0 +/36h +/18h +/36h 0 +/36h +/18h +/36h 0 0 +/18h +/36h +/36h 0
Enrichment + + + 0 0 0 0 +

Patients and specimen collection From March to June 2006, 1002 screening swabs from nares (n = 726), throat (n = 116), perineum (n = 111) and skin (n = 49) were obtained fom 639 hospitalized patients. This sample size was calculated to allow detection of 10% variation in sensitivity between media. Microbiological methods Swabs were homogeneized by vortexing into 1 ml of sterile saline. One hundred microliter were inoculated into Brain-Heart-Infusion broth supplemented with NaCl 7% (SB), MRSA ID, MRSA-Screen and MRSASelect. SB broths were sub-cultured after 18 h incubation onto the three chromogenic media. Plates were incubated at 35C and examined after and for primary plates and after 18h for enrichmnt broth. Colonies suspected by morphological and colour examination were identified by coagulase test. Resistance to oxacillin was determined by the cefoxitin disk method according to the CLSI recommendations. Identification of MRSA was confirmed by multiplex PCR for the 16S rRNA, nuc and mecA genes. Sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) were analysed based on colony colour and morphology and PCR results.
Ten (14.7%) isolates from 6 (13.3%) patients only detected after enrichment Ten (14.7%) isolates from 7 (15.5%) patients only detected on primary agar plates
Fig. 1: Distribution of MRSA isolates by specimen type (n = 68)

Other 16%
Perineum 19%
Nare 41%
Throat 24%
Fig. 2: Distribution of MRSA/MSSA isolates and normal flora

400 N of specimen 300 200 100 0
36h Enrichment 18h
MRSA-ID
MRSA-Screen MRSA-Select
RESULTS
Table 1: Performance of the three chromogenic media
Medium MRSA-ID Incub. time 18h 36h enrichment 18h 36h enrichment 18h 36h enrichment True + MRSA 31 51 58 30 55 58 31 49 58 False 0 Sens.a Spec.b PPVc NPVd MSSA % % % % 0 1 2 6 26 24 0 0 0 45.6 75.0 85.3 44.1 80.9e 85.3 45.6 72.1e 85.3 100 99.9 99.8 99.4 97.2 97.4 100 100 100 100 98.0f 96.7f 83.3 67.9f 70.7f 100 100f 100f 96.2 98.2 98.9 96.1 98.9 98.9 96.2 98.0 98.9
RS
A-
ID M RS A-
Sc
re
en M RS A-
Se
lec
t M RS
A-
ID M RS A-
Sc
re
en M RS A-
Se
lec
t M RS
A-
ID M RS A-
Sc
re
en M RS A-
Se
lec
MRSA
MSSA
Normal flora
CONCLUSIONS
1. The three chromogenic media similarly demonstrated insufficient sensitivity but high specificity after 18h. Therefore, re-incubation of direct plates for 18h and subculture of enrichment broth appeared desirable for optimal recovery of MRSA. 2. The sensitivity of MRSA-Screen was marginally better than that of MRSA-Select after 36h (p=0.045). 3. The specificity of MRSA-ID and MRSA-Select was excellent after 36h or enrichment step and significantly better than MRSA-Screen (p<0.001) due to the growth of many MSSA isolates on the latter medium. 4. Only 22 (48.9%) patients were detected after 18h incubation.
MRSA-Screen
MRSA-Select
a d
Sens.: sensitivity; b Spec.: specificity; c PPV: positive predictive value; NPV: negative predictive value; e p=0.045; f p<0.001
KEY POINTS

At 18 hrs incubation, the three media show a similar level of performance. Re-incubation for 18 hrs is preferable. At 36 hrs incubation, the specificity of MRSA ID (new name chromID MRSA) & MRSA Select was excellent.
08

Munich (Germany) poster R2151
An Evaluation of MRSA ID: A New Chromogenic Medium for the Isolation and Identification of Methicillin-Resistant Staphylococcus aureus
A. Davies, J.D. Perry, L.A. Butterworth, A. Nicholson & F.K. Gould
Microbiology Department, Freeman Hospital, Newcastle upon Tyne, NE7 7DN, UK
BACKGROUND
Methicillin-Resistant Staphylococcus aureus (MRSA) has emerged as a nosocomial pathogen of major world-wide importance (8) and an increasingly frequent cause of community-acquired infection (10). Despite much debate, laboratory-based screening for MRSA colonisation of patients and health care workers remains a cornerstone of infection control measures to limit the spread of this organism (13). A wide range of methods has evolved for the detection of MRSA in the clinical laboratory. Mannitol salt agar supplemented with oxacillin is widely used (7) but has shown limited sensitivity (5) and specificity (4) in some studies. A modified version of mannitol salt agar is Oxacillin Resistance Screening Agar base (ORSAB) but independent studies have revealed similar limitations regarding sensitivity and specificity (1-3). Two studies have reported the adaptation of CHROMagar Staph aureus for the specific isolation of MRSA by inclusion of methicillin or oxacillin but there are no reports of the efficacy of these media with clinical samples. S. aureus ID, is a recently developed chromogenic agar and has been shown to have high sensitivity and specificity for the isolation of S. aureus (11). S. aureus form green colonies on this medium due to production of alpha glucosidase and the medium is highly selective against non-staphylococci, including enterococci. The aim of this study was to evaluate the performance of S. aureus ID supplemented with 4 mg/l cefoxitin for the specific isolation of MRSA from clinical specimens. This new medium was designated MRSA ID. Aims: To evaluate the performance of MRSA ID against CHROMagar MRSA and ORSAB for the isolation of MRSA from 192 nasal swabs referred to our laboratory for MRSA screening.
Fig. 1. Typical appearance of MRSA on MRSA ID.
Table 1: Typical appearance of MRSA on MRSA ID.

Medium Total strains MRSA ID CHROMagar MRSA ORSAB No.a of MRSA strain 28 25 (26) 20 (25) 21 (26) % Sensitivitya 20-22h 48h 89 71 75 93 89 93 % Specificitya 20-22h 48h 100 100 98.8 84.3 97.7 96.5
a Numbers in parentheses indicate total strains in 48 h of incubation.
RESULTS
A total of 28 strains (14.6 %) was isolated on at least one of the three media (see table 1). After 20-22 h incubation 25 of these strains (89 %) were isolated as coloured colonies on MRSA ID compared with 20 (71 %) on CHROMagar MRSA and 21 (75 %) on ORSAB medium. After 48 h incubation, 26 strains (93 %) were isolated as coloured colonies on MRSA ID and ORSAB medium compared with 25 (89 %) on CHROMagar MRSA. No strains of Methicillin-sensitive S. aureus (MSSA) were isolated on any of the media. The chromogenic reactions of all three media were highly specific for MRSA after 20-22 h incubation. The specificities of the media were 100 %, 100 % and 98.8 % after 20-22 h for MRSA ID, CHROMagar MRSA and ORSAB respectively. After 48 hours, some strains of coagulase negative staphylococci generated pale green colonies on MRSA ID however these strains were clearly distinguishable from S. aureus colonies. The typical appearance of MRSA colonies on MRSA ID is shown in Fig. 1.
METHOD
S. aureus ID was provided by bioMrieux, La Balme Les Grottes, France and supplemented with 4 mg/l cefoxitin to produce MRSA ID. ORSAB (CM1008) was obtained from Oxoid Ltd., Basingstoke, UK and supplemented with an antibiotic supplement (SRO195) in exact accordance with manufacturers instructions. CHROMagar Staph. aureus was obtained as a dehydrated medium from M-Tech Diagnostics, Warrington, UK and supplemented with a designated batch of antibiotic supplement provided by the supplier to produce CHROMagar MRSA. Culture plates of each type were prepared containing 20 ml of agar. A total of 192 nasal swabs from distinct hospital patients were each emulsified in 750 microlitres of saline (0.85 %). A 50 l sample of the resulting suspension was inoculated onto MRSA ID, CHROMagar MRSA and ORSAB medium. The inoculum was spread for single colonies and all media were incubated at 37C. The culture plates were examined for the presence of MRSA after 20-22 h incubation and were examined again after a total of 48 h incubation. All suspect colonies of MRSA on all media were confirmed using standard methods including the tube coagulase test and detection of the mecA resistance determinant.
DISCUSSION
MRSA ID showed a substantially better performance than any of the other media tested and its sensitivity after 20-22 h incubation was comparable to either CHROMagar MRSA or ORSAB medium incubated for 48 hours. The two strains that were not recovered on MRSA ID produced no more than two colonies on either of the other media. It is therefore possible that these strains were not present in the inoculum used for the MRSA ID medium.
09
chromID VRE
chromID ESBL
chromID MRSA
An Evaluation of MRSA ID: A New Chromogenic Medium for the Isolation and Identification of Methicillin-Resistant Staphylococcus aureus
There has been renewed interest in the use of cefoxitin for differentiation of MRSA from MSSA. Mougeot et al. (9) demonstrated that cefoxitin was more effective than oxacillin plus salt for the detection of MRSA strains using disc susceptibility testing. Felten et al. (6) compared a standardised oxacillin 5 g disc susceptibility test with a cefoxitin disc susceptibility test against 83 MRSA strains including 26 with low-level resistance. The authors reported absolute discrimination of all of these strains from 69 MSSA strains using the cefoxitin disc test at 37C with a low inoculum. Using the same semi-confluent inoculum, the oxacillin disc test showed a sensitivity of 41 % for detection of MRSA. The authors also reported that a concentration of 4 mg/l cefoxitin allowed growth of all MRSA strains and inhibition of all MSSA strains. These results suggest that MRSA strains grow much more readily in the presence of cefoxitin compared with oxacillin, possibly due to enhanced induction of the penicillin binding protein, PBP2, by cefoxitin (12). MRSA ID, which exploits these apparent advantages of cefoxitin, has been shown to be a highly effective medium for the isolation of MRSA from nasal swabs and compares well with other available media.
REFERENCES: 1. Apfalter et al. 2002. Performance of a new chromogenic oxacillin resistance screen medium (Oxoid) in the detection and presumptive identification of Methicillin-Resistant Staphylococcus aureus. Diagn. Microbiol. Infect. Dis. 44:209-211. 2. Becker et al. Oxacillin Resistance Screening Agar Base for detection of Methicillin-Resistant Staphylococcus aureus. J. Clin. Microbiol. 40:4400-4401. 3. Blanc et al. 2003. Evaluation of a novel medium for screening specimens from hospitalized patients to detect Methicillin-Resistant Staphylococcus aureus. J. Clin. Microbiol.41:3499-3502. 4. Davies & Zadik. 1997. Comparison of methods for the isolation of Methicillin-Resistant Staphylococcus aureus. J. Clin. Pathol. 50:257-258. 5. Davies et al. 2000. Methicillin-Resistant Staphylococcus aureus: evaluation of five selective media. Br. J. Biomed. Sci. 57:269-272. 6. Felten et al. 2002. Evaluation of three techniques for detection of low-level Methicillin-Resistant Staphylococcus aureus (MRSA): a disk diffusion method with cefoxitin and moxalactam, the Vitek 2 system, and the MRSA-Screen latex agglutination test. J. Clin. Microbiol. 40:2766-2771. 7. Gorss, E. B. 1992. Prospective, focused surveillance for oxacillin-resistant Staphylococcus aureus, p. 11.15.1-11.15.2. In H. D. Isenberg (ed.), Clinical microbiology procedures handbook. American Society for Microbiology, Washington, D.C. 8. Harbarth et al. 2001. Control of multiply resistant cocci: do international comparisons help? Lancet Infect. Dis. 4:251-261. 9. Mougeot et al. 2001. Staphylococcus aureus: nouvelle dtection de la rsistance intrinsque par la mthode de diffusion. Pathol. Biol.49:199-204. 21 (26)7593 98.8 12. Rohrer et al. 2001. Improved methods for detection of Methicillin-Resistant Staphylococcus aureus. Eur. J. Clin. Microbiol. Infect. Dis. 20:267-270. 13. Rubinovitch et al. 2001. Screening for Methicillin-Resistant Staphylococcus aureus in the endemic hospital: what have we learned? J. Hosp. Infect. 47:9-18.
CONCLUSIONS
MRSA ID is a highly effective medium for the detection of MRSA from nasal swabs and has an excellent sensitivity and specificity after 20-22 h incubation.
KEY POINTS
The 3 chromogenic media supported growth of the MRSA tested, even when using low inocula. This study was performed using collection strains, therefore a comparative clinical study is required.
10

Comparison of Chromogenic Agar in the Detection of MRSA

A.Curry, M. Walsh, M. Matheson, A. OReilly, S. Knowles
Microbiology, National Maternity Hospital, Holles Street, Dublin2, Ireland
Ireland has one of the highest rates of MRSA in Europe along with the United Kingdom and Southern European countries. The annual report from EARSS for 2005 showed that in Ireland 41.8% of Staphylococcus aureus isolates were MRSA, which follows trends from previous years. In general, the majority of Irish hospitals have an endemic problem with hospital acquired infection due to MRSA.
CRITERIA FOR AGAR SELECTION

Sensitivity, specificity, ease of reading under lighting conditions in our laboratory. MRSA ID grew as large green colonies on a clear agar - easy to read - MRSA Select grew as small pink colonies easy to read in large numbers but easily missed when a scanty growth - Presence of Enterococci (green/pink) on both agars distorted white colonies of CoNS: MRSA ID-easy to distinguish MRSA Select-Enterococci appeared like MRSA - 6 isolates of cefoxitin resistant GNB grew on both agars - One isolate of Methicillin-Sensitive Staphylococcus aureus on both agars Fig. 2: Photos of organisms isolated on both chromogenic agars
PURPOSE
To compare two different chromogenic agars for the isolation of MRSA in order to find the most appropriate agar for use in our laboratory.
METHOD
300 patients screens: - Nasal - Umbilical MRSA screen - Groin Each screen was pooled and broken into Brain Heart Infusion enrichment broth, incubated aerobically at 37C for 24 hours. Subcultured onto MRSA ID and MRSA Select chromogenic agar, incubated aerobically at 37C for 24 hours. Checked for the presence of green (MRSA ID) or pink (MRSA Select) colonies. All negative screens incubated for further 24 hours and checked for green or pink colonies. All green or pink colonies had confirmatory tests for MRSA - PBP2 latex agglutination - Cefoxitin disc resistance
MRSA ID
MRSA Select MRSA GNB MSSA
CONCLUSIONS
Both agars were equally sensitive and specific. It was decided that under our laboratory condition, MRSA ID chromogenic agar was the better agar for the isolation of MRSA. However, due to the presence of one isolate of MSSA, confirmatory tests are required.
RESULTS
Fig. 1: Organisms isolated on MRSA ID and MRSA Select chromogenic agar
MRSA ID vs MRSA Select 12 10
No. Isolated
8 6 4 2 0 MRSA GNB Organism Isolated MRSA ID MRSA Select N=300 Screens MSSA
KEY POINTS

Both agars were equally sensitive and specific. MRSA ID (new name chromID MRSA) enabled the growth of large green colonies on a clear agar. MRSA ID (new name chromID MRSA) was easier to read.
11
chromID VRE
chromID ESBL
chromID MRSA
ICAAC / September, 2006

San Francisco (USA) poster D0820
A Multicentre Evaluation of Two Chromogenic Media for the Isolation of Methicillin-Resistant Staphylococcus aureus from Clinical Samples
N. Bendridi (1), Am. Freydiere (1) B. Willinger (2), M. Manafi (2), JD. Perry (3) and FK. Gould (3)
(1)
Laboratoire de Bactriologie, Hpital Debrousse, Hospices Civils de Lyon, Lyon, France. (2) Division of Clinical Microbiology, Institute of Hygiene and Medical Microbiology, University of Vienna, Vienna . (3) Department of Microbiology, Freeman Hospital, Newcastle upon Tyne, United Kingdom
ABSTRACT
Background: Two commercially available chromogenic agars for the detection of Methicillin-Resistant Staphylococcus aureus (MRSA) were evaluated with clinical samples in a multicentre study in Newcastle (UK), Lyon (France: Fr) and Vienna (Austria: Au). Methods: A total of 910 clinical samples (UK:346, Fr:226, Au:338) were cultured onto MRSA ID and CHROMagar MRSA and also onto conventional non-chromogenic media routinely used in each laboratory. Chromogenic agars were examined after both 24 and 48 h incubation at 37C. Results were checked by the detection of penicillinbinding protein 2a and/or the mecA gene. A wide range of sample types were chosen for testing including wound swabs, sputa, urines, blood cultures and general screening swabs. Results: A total of 152 isolates of MRSA were recovered in the three centres (UK:30, Fr:28, Au:94). The sensitivity of detection of MRSA ID was 87%, 86% and 41% after 24 hours and 100%, 89% and 97% after 48 hours in the UK, France and Austria respectively. Corresponding results for CHROMagar MRSA were 83%, 86% and 44% after 24 hours and 87%, 86% and 84% after 48 hours. Conventional non-chromogenic agars resulted in the isolation of 57%, 93% and 93% respectively in the three laboratories. Conclusions: The combined results show little difference between the chromogenic agars after 24 h incubation. MRSA ID showed a higher sensitivity than CHROMagar MRSA after 48 h incubation in all three laboratories. MRSA strains isolated in Vienna were frequently isolated on both media without showing the expected chromogenic reaction after 24 hours and the isolation rate was therefore comparatively low. Chromogenic agars offer a useful adjunct to conventional media for the isolation of MRSA from clinical samples. The sensitivity and specificity of the two chromogenic media for the various sample types in each laboratory will be presented.
isolation and identification of MRSA strains must be rapid. Methods to detect MRSA in clinical samples should have high sensitivity and specificity and achieve isolation of MRSA in one single step (2). The purpose of this study was to evaluate the performance of 2 selective media, the MRSA ID (bioMrieux, France) and the CHROMagar MRSA (CHROMagar Microbiology, Paris) in comparison with conventional non-chromogenic media routinely used in each laboratory, using a wide range of samples collected in three European laboratories.

Chromogenic media
MRSA ID medium (bioMrieux) chromogenic substrate + cefoxitin: 4 mg/l
CHROMagar MRSA medium (CHROMagar Microbiology) chromogenic substrate + cephamycin
INTRODUCTION
Methicillin-Resistant Staphylococcus aureus (MRSA) is a nosocomial pathogen of major importance (1), and the incidence of infections caused by MRSA increases. Because of its high transmissibility, its multiple antibiotic resistance, and its consequences on patients management,
Identification: Typical colonies (green) on MRSA ID; and (pink) on CHROMagar MRSA were checked by conventional tests (Gram staining, catalase test, positive agglutination with Pastorex Staph Plus (Bio-Rad) or Staph Slidex Plus (bioMrieux) and/or API ID 32 Staph (bioMrieux). Susceptibility testings: A cefoxitin disk (30 g) diffusion method on Mueller Hinton agar or a ATB Staph panel (bioMrieux) was systematically performed. In case of discordant result between the MRSA media and conventional susceptibility testings a latex agglutination with the MRSA Slidex reagent (bioMrieux) and/or a mecA gene detection by PCR was performed. Samples and inoculation: From March 2005 to April 2006, a total of 910 clinical samples from the United Kingdom (n = 346), France (n = 226), and Austria (n = 338) including wounds, sputa, urines, blood were streaked (10l) onto MRSA ID, CHROMagar MRSA and conventional media routinely used in each laboratory. The plates were incubated aerobically at 37C and examined after both 24 and 48 h.
Table 1: Number of MRSA strains isolated on the different media, sensitivities and specificities after 24 and 48 h of incubation
Number of MRSA strains isolated after 24h (48h) of incubation Total specimens 910 U.K. France Austria 12 346 226 338 Total MRSA 152 30 28 94 18 26 87 25 (26) 24 (24) 41 (79) 28 (30) 24 (25) 39 (91) Conventional CHROMagar MRSA MRSA ID Sensitives and specificities in % after 24h (48h) of incubation CHROMagar MRSA Se 83 (87) 86 (86) 41 (97) Sp 99 (93) 97 (97) 95 (84) Se MRSA ID Sp
87 (100) 99 (82) 88 (89) 97 (96)
41 (97) 100 (97)
RESULTS AND DISCUSSION

The results of detection, sensitivity and specificity in the three laboratories are reported in table 1. Conventional non-chromogenic agars result in the isolation of 57%, 93% and 93% of MRSA strains, respectively in the three laboratories. The results obtained with CHROMagar MRSA in UK and France (sensitivities 83 and 86%) after 24h of incubation are slightly inferior to the sensitivities of about 95% obtained by Kircher et al. (2004), Flayhart et al. (2005) and Loulergue et al. (2006) with various clinical samples (3, 4. 5). Perry et al., (2004) evaluating MRSA ID and CHROMagar MRSA reported sensitivities of 89 and 72% respectively with various clinical samples (6). Reverdy et al. (2005)(7) and Nonhoff et al. (2005)(8) evaluating the MRSA ID medium with nasal swabs reported sensitivities of 93 % and 64 % respectively after incubation for 24h. Thus, significant difference in sensitivities may exist from one study to another. In this collaborative study where the 3 laboratories used the same experimental procedure, significant differences in the sensitivities for both chromogenic media were observed in Austria after incubation for 24 h (about 41% in Austria vs about 85% both in UK and France). Are these differences related to the nature of the samples or to the potential clonality of the strains ?
CONCLUSIONS
CHROMagar MRSA and MRSA ID agar show similar good results of sensitivity and specificity for the detection of Methicillin-Resistant Staphylococcus aureus after 24 h of incubation in two of the 3 laboratories. As in other studies, for the third laboratory the sensitivity is optimal only after 48 h of incubation with a decreased specificity.
SPUTUM Total samples U.K. France Austria 123 180 15 CHROMagar MRSA 12 (12) 18 (18) 3 (4) MRSA ID Total samples 39 7 10
BLOOD CULTURE CHROMagar MRSA 1 (1) 1 (1) 3 (3) MRSA ID Total samples 137 39 313
Other SAMPLES* CHROMagar MRSA 9 (10) 5 (5) 40 (74) MRSA ID
12 (13) 19 (20) 3 (4)
1 (1) 1 (1) 3 (3)
10 (13) 5 (5)
* Other samples included wound swabs, pus, genital samples, nasal and oropharyngal samples, bone samples, urines, .
The limited number of other samples in both the UK and the French laboratories, do not allow to determine any potential influence of the nature of the samples on the sensitivity of the media. The potential influence of the clonality of the strains remains to be evaluated.
1. Archer GL (1998) Clin. Infect. Dis 26, 1179-1181. 2. Simor AE, Goodfellow J, Louie L, et al. (2001) J. Clin. Microbiol 37, 2789-2792. 3. Kircher S, Dick N, Ritter V, et al.(2004). abstr. C120, p. 104. In Abstracts of the 104rd Meeting, American Society for Microbiology, New Orleans, Louisiana. 4. Flayhart D, Hindler JF, Bruckner DA, et al. (2005) J. Clin. Microbiol 43, 5536-5540. 5. Loulergue J, de Gialluly C, Morange V, et al. (2006) Eur. J. Clin. Microbiol. Infect. Dis. 25: 40-409. 6. Perry JD, Davies A, Butterworth LA, et al. (2004) J. Clin. Microbiol, 42, 4519-4523. 7. Reverdy ME, Roger-Dalbert C, Bobichon D, et al. (2004). Clin. Microbiol. Infect. 10 (Suppl. 3): 350 8. Nonhoff C, Struelens MJ, Brenner A, et al. (2005) Clin. Microbiol. Infect. 11 (Suppl 2): 443.
KEY POINTS

MRSA ID (new name chromID MRSA) & CHROMagar MRSA show similar good results in 2 of the 3 laboratories. For the third laboratory, the sensitivity is optimal only after 48 h of incubation with a decreased specificity.
13
chromID VRE
chromID ESBL
30 (71)
chromID MRSA
Table 2: Number of MRSA strains isolated on the 2 chromogenic media, after 24 h and (48 h) of incubation, according to the sample type in the 3 laboratories

Evaluation of Three Chromogenic Media for the Detection of Methicillin-Resistant Staphylococcus aureus (MRSA) Strains from Belgian Hospitalized Patients
C. Nonhoff, O. Denis and M. J. Struelens
Laboratoire de Rfrence MRSA - Staphylocoques, Universit Libre de Bruxelles-Erasme Hospital, Brussels, Belgium
UPADTED ABSTRACT
Background : Rapid detection of MRSA carriers among hospitalized patients is essential for control of nosocomial transmission. The performance of new chromogenic agar media, MRSA ID (MRSA ID, bioMrieux), MRSA Chromogenic Agar (MRSA Screen, Oxoid) and MRSA Select (MRSA Select, Bio-Rad) for the detection of Methicillin-Resistant Staphylococcus aureus (MRSA) strains was evaluated on a genotypically diverse collection of strains from Belgian hospitals. Methods : Four sets of S. aureus strains identified by triplex PCR for 16S rRNA, mecA and nuc genes, were included : 87 MRSA isolates representing a Belgian epidemic (n = 60) and sporadic clones (n = 27), low-level MRSA isolates with oxacillin MIC ranging from 0.5 to 16 mg/l (n=29) and/or falsely susceptible by oxacillin screen agar or cefoxitin disk diffusion, community-acquired MRSA (CA MRSA) harboring the Panton-Valentin leukocidin (PVL) gene (n=39) and MethicillinSusceptible S. aureus (MSSA) (n=50). Using a 10 l calibrate loop, a 10-fold dilution of a 0.5 McFarland inoculum of each strain was streaked onto each plate. Plates were examined after 18 and 36 h incubation for typical colony morphology and color production. Results : After 18 h incubation, the sensitivity was 96.1%, 94.8% and 95.5% for MRSA ID, MRSA Screen and MRSA Select, respectively, and reached 97.4% and 96.8% for MRSA Screen and MRSA Select after 36 h. Specificity after 18 h was 100% for MRSA ID and MRSA Select and 96% for MRSA Screen. After 36 h, the specificity of MRSA Screen decreased to 76%. Few MRSA isolates showed atypical colour (white) on the three media: 2%, 3.2% and 2.6% for MRSA-ID, MRSA Screen and MRSA Select, respectively. Conclusions : All three chromogenic media demonstrated good sensitivity and specificity for the detection of MRSA strains isolated in Belgian hospitals. The specificity of MRSA Screen decreased markedly after prolonged incubation. There is a need to compare the performance of these chromogenic media for the screening of MRSA carriers on surveillance specimens.
of this study was to evaluate the performance of three chromogenic agar media, MRSA ID (bioMrieux), MRSA Chromogenic Agar (Oxoid) and MRSA Select (Bio-Rad) on a collection of MSSA and MRSA well characterized.
METHODS
Collection of bacterial isolates Four sets of S. aureus strains identified by triplex PCR for 16S rRNA, mecA and nuc genes, were included: - Set 1: 87 MRSA isolates representing Belgian epidemic (n = 60) and sporadic (n = 27) clones - Set 2: 29 low-level MRSA isolates with oxacillin MIC ranging from 0.5 to 16 g/ml and/or falsely susceptible by oxacillin screen agar or cefoxitin disk diffusion - Set 3: 39 community-acquired MRSA (CA-MRSA) harboring the Panton-Valentin leukocidin (PVL) gene - Set 4: 50 Methicillin-Susceptible S. aureus (MSSA) Procedure Using a 10 l calibrate loop, a 10-fold dilution of a 0.5 McFarland of each strain was streaked onto each plate. Plates were examined after 18 and 36 h incubation for typical colony morphology, according to the manufacturers recommendations. Characteristic MRSA colonies (fig 1) MRSA ID (bioMrieux, Marcy lEtoile, France) : large green colonies MRSA Screen (Oxoid, Basingstoke, UK) : blue-jean colonies MRSA Select (Bio-Rad, Marnes-la-Coquette, France) : small pink colonies Fig. 1: Characteristic MRSA colonies on chromogenic media
MRSA ID
INTRODUCTION
Methicillin-Resistant Staphylococcus aureus (MRSA) is a major nosocomial pathogen responsible for a wide spectrum of infections including pneumonia, bacteremia and wound infections. In Belgian acute-care hospitals, 33% of S. aureus strains isolated from blood culture in patients hospitalized were resistant to Methicillin in 2004 (http://www.earss.rivm.nl/). MRSA infections have also been reported in the community in several parts of the world. Early identification of MRSA carriers is essential for implementation of decontamination and isolation measures to prevent dissemination in healthcare setting. Recently, selective media which contain chromogenic enzyme substrates, have been developed for the isolation and rapid identification of MRSA. The purpose MRSA Select
MRSA Screen
14
RESULTS
Table 1: Characteristics and results of the 4 sets of S. aureus strains
MRSA strains Oxa MIC N g/ml Medium 8-256 MRSA ID N of strains showing Incubation Growth Characteristic Atypical Time N (%) colonies4 colonies5 86 (98.9) 86 (98.9) 86 (98.9) 87 (100) 86 (98.9) 87 (100) 39 (100) 39 (100) 39 (100) 39 (100) 39 (100) 39 (100) 27 (93.1) 27 (93.1) 27 (93.1) 28 (96.6) 27 (93.1) 27 (93.1) 0 (0) 1 (2) 2 (4) 12 (24) 0 (0) 1 (2) 86 86 85 87 86 87 39 39 39 39 39 39 24 24 23 25 23 24 0 1 2 12 0 1 0 0 1 0 0 0 0 0 0 0 0 0 3 3 4 3 4 3 0 0 0 0 0 0
CONCLUSIONS
1. After 18 h incubation, all three chromogenic media demonstrated excellent sensitivity and specificity for the detection of MRSA strains isolated in Belgian hospitals. 2. The specificity of MRSA Screen decreased markedly after prolonged incubation. This was not found with the other media. 3. Studies to compare these chromogenic media on surveillance specimens are required to confirm this performance.
HA-MRSA1 87
16 h 36 h MRSA Screen 16 h 36 h MRSA Select 16 h 36 h MRSA ID 16 h 36 h MRSA Screen 16 h 36 h MRSA Select 16 h 36 h 16 h 36 h MRSA Screen 16 h 36 h MRSA Select 16 h 36 h
CA-MRSA2 39
4-256
LLR-MRSA3 29 0.5 - 16 MRSA ID
MSSA
50 0.25 - 1 MRSA ID
16 h 36 h MRSA Screen 16 h 36 h MRSA Select 16 h 36 h
Table 2: Performance of the three chromogenic media

Medium MRSA ID MRSA Screen MRSA Select Incubation time 16 h 36 h 16 h 36 h 16 h 36h Sensitivity (%) 96.1 96.1 94.8 97.4 95.5 96.8 Specificity (%) 100 98 96 76
After 18 h incubation, all three chromogenic media demonstrated excellent and comparable sensitivity. The specificity after 18 h was 100% for MRSA ID and MRSA Select and 96% for MRSA Screen. After 36 h, the specificity of MRSA Screen decreased dramatically to 76% due to the growth of a significant proportion of MSSA isolates (24%). Few MRSA isolates, essentially strains showing low oxacillin MIC ( 16 g/ml), showed atypical colour (white colonies) on the three media : 2%, 3.2% and 2.6% for MRSA ID, MRSA Screen and MRSA Select, respectively.
KEY POINTS

At 18 hrs incubation, the three media show excellent performance. At 36 hrs incubation, the specificity of MRSA ID (new name chromID MRSA) & MRSA Select was excellent. This study was performed using collection strains, therefore a comparative clinical study is required.
15
chromID VRE
100 98
chromID ESBL
1 - HA-MRSA : epidemic (n = 60) and sporadic (n = 27) types of hospital-associated MRSA 2 - CA-MRSA : community-acquired MRSA 3 - LLR : low-level resistant MRSA to oxacillin (Oxa) 4 - Characteristic colonies : green on MRSA ID, blue jean on MRSA Screen, pink on MRSA Select 5 - Atypical colonies : white colonies on chromogenic media
chromID MRSA

Nice (France) poster P1430
Optimisation of screening for Methicillin-Resistant Staphylococcus aureus by using the chromogenic medium MRSA ID
M.J. Bruins, M.J. Egbers, G.J.H.M. Ruijs, M.J.H.M. Wolfhagen
Isala klinieken, Zwolle, The Netherlands
ABSTRACT
The incidence of Methicillin-Resistant Staphylococcus aureus in hospitals in the Netherlands is still low, because patients and staff at risk are routinely screened for MRSA. We tried to optimise our MRSA screening protocol by substituting the currently used CHROMagar Staph. aureus with the new MRSA ID medium. MRSA ID was found te be a 100% sensitive, easy to interpret medium. Using MRSA ID shortened the turnaround time with 24 hours and reduced costs.
Confirmation - Methicillin-Resistance on Columbia sheep blood agar with 6 mg/l oxacillin. - Methicillin-Resistance with methicillin strip on Mueller Hinton agar. - VITEK 2 GP and AST-N020 cards. - PCR for mecA and sa442 genes.
RESULTS
Total number of cultures: 449. Total number of patients: 190. Table 1: MRSA ID after 24 h.
MRSA ID after 24 h. MRSA + 29 14 0 406 29 420
INTRODUCTION AND OBJECTIVES

In Dutch hospitals, a strict search and destroy policy to control the transmission of Methicillin-Resistant Staphylococcus aureus (MRSA) has so far kept the incidence under 1%. Hospitalised patients and health care workers at risk are routinely screened for carriership of MRSA. To shorten patient isolation periods and control costs, screening methods need to be rapid, highly sensitive, specific and inexpensive. We prospectively analysed whether using the new chromogenic medium MRSA ID (bioMrieux, Marcy lEtoile, France) instead of the currently used CHROMagar Staph. aureus (CHROMagar, Paris, France) improved our MRSA screening protocol with respect to turnaround time and workload.
Green + Total
Sensitivity 100 % - Specificity 97 %
Total 43 406 449
Table 2: MRSA ID after 48 h.

Green + Total
Total 78 371 449

Culture Swabs submitted for MRSA screening were: - Inoculated into mannitol broth with 2.5% NaCl. - Incubated 18-24 hrs. at 35C. - Subcultured onto: 1. MRSA ID Incubation at 35C. Reading after 24 and 48 hrs. MRSA: green colonies.
Table 3: CHROMagar staph. aureus after 48 h.

Green + Total
Total 78 371 449
Remarks: 1) MRSA ID: lower specificity after 48 hrs. (Table 2). 2) CHROMagar Staph. aureus: three positives missed because of overgrowth of Methicillin-susceptible Staphylococcus aureus and other flora (Table 3).
2. CHROMagar Staph. aureus
CONCLUSIONS
1) MRSA ID: After 24 hrs.: sensitivity 100%, specificity 97%. Easy to interpret. 2) Use of MRSA ID instead of CHROMagar Staph. aureus: Turnaround time MRSA screening protocol 24 hrs shorter. Less confirmative testing, thus lower costs.
Incubation at 35C. Reading after 48 hrs. Staphylococcus aureus: mauve colonies
KEY POINTS

MRSA ID (new name chromID MRSA) enables a MRSA screening protocol in 24 hrs. Easy to interpret. Less confirmative testing, thus lower costs.
16

Evaluation of chromogenic bioMrieux MRSA ID medium for direct detection of Methicillin-Resistant Staphylococcus aureus from surveillance cultures and from clinical samples
Carlos Snchez-Carrillo*, Emilia Cercenado, Mercedes Marn, Marisa Rivera, Julia Jensen, Mara Guembe, Emilio Bouza
Servicio de Microbiologa. Hospital General Universitario Gregorio Maran. Madrid. Spain
ABSTRACT
Objectives: Rapid detection of Methicillin-Resistant Staphylococcus aureus (MRSA) from surveillance cultures and from clinical samples is crucial in order to reduce the transmission and to control the spread of MRSA. We evaluate a new selective and differential chromogenic medium, bioMrieux MRSA ID (CB-MRSA) medium (bioMrieux, Marcy lEtoile. France) which enables recovery and concomitant identification of MRSA strains directly from nasal swab specimens and from other clinical samples. Methods: A total of 530 clinical samples were inoculated to CB-MRSA and mannitol-salt agar (MSA, Tec-Laim. Madrid. Spain): 452 surveillance nasal swab specimens obtained from the anterior nares and 78 other samples (57 wounds, 21 bronchial aspirates). Green colonies on CB-MRSA and mannitol-positive on MSA at 24 h and 48 h were identified by the MicroScan system (Dade Behring) using Pos Combo 2SA panels and by detection of the mecA and femA genes by PCR. Results: A total of 202 S. aureus isolates were recovered; of these 67 (33%) were oxacillin susceptible (MSSA) and 134 (66%) were oxacillin resistant (MRSA). On CB-MRSA, 132/134, or 98.5% of MRSA were recovered, whereas recovery on MSA was 82.8% (111/134) (p<0,0001). The overall sensitivity, specificity, positive predictive value and negative predictive value of CB-MRSA were: 98.5%, 98.2%, 94.9%, and 99.4%, respectively; whereas, the corresponding values for MSA were: 82.8%, 99.7%, 99.1%, and 94.5%. Seven false positive readings on CB-MRSA were detected (one nasal swab and 6 other samples) and corresponded to Enterobacter cloacae (3 isolates), MSSA (1), Methicillin-Resistant coagulase-negative staphylococci (1), Candida spp. (1), and Bacillus spp.(1). Fifty-eight MRSA isolates (43%) were detected on CB-MRSA at 24 h; 42% at 48 h, and in the remaining 15% of MRSA, readings were only performed at 48 h. On MSA followed by conventional methods for the final identification of MRSA, identification was delayed to 72 h in 14% of cases, and to more than 72 h in 86%. Conclusions: In this study, CB-MRSA was superior to conventional methods for recovery of MRSA from nasal swabs and from other clinical samples, and shortened the time to detection of MRSA to 24 or 48 h. The enhanced recovery of MRSA using this medium and the identification of most MRSA isolates at 24 h without additional susceptibility testing, will benefit the patients and decrease costs associated to infections caused by MRSA.
INTRODUCTION
Methicillin-Resistant Staphylococcus aureus (MRSA) remains an important nosocomial pathogen. The presence of MRSA in an institution is clearly associated with longer hospital stay, more days of antibiotic administration, and higher costs. Early detection of MRSA carriers is crucial for infection control aiming to appropriately apply isolation precautions. Detection of MRSA by standard microbiological methods is timeconsuming since it first requires the isolation of S. aureus within mixed flora samples before assessing their level of resistance to Methicillin.
OBJECTIVES
To evaluate a new selective and differential chromogenic medium, MRSA ID (bioMrieux, Marcy lEtoile, France) which enables recovery and concomitant identification of MRSA strains directly from nasal swab specimens and from other clinical samples.
were used in this study. - 452 surveillance nasal swab specimens - 57 wounds - 21 bronchial aspirates Specimens were obtained and then transported using Amies transport medium swabs (Invasive sterile Eurotubo Collection swab) or using a sterile device. Samples were inoculated onto MRSA ID and onto mannitol-salt agar (MSA, Tec-Laim. Madrid, Spain). Wounds and bronchial aspirate were also inoculated onto appropriate media in order to detect other microorganisms. MRSA ID and MSA were incubated in air at 35C. Readings were performed at 24 and 48 h of incubation.
Samples MRSA ID Green colonies POSITIVE for MRSA Confirmation: MicroScan mec A + fem A Colour other than green: POSITIVE for MRSA Mannitol-salt agar (MSA) Colonies other than yellow: NEGATIVE Yellow colonies MRSA screening test Columbia agar Confirmation: MicroScan mec A + fem A
17
chromID VRE
chromID ESBL
METHODS I 530 clinical samples referred to our laboratory for MRSA screening
chromID MRSA
Media utilizing chromogenic enzyme substrates have been developed to try to detect MRSA with high sensitivity and specificity combined with a short results reporting time.
Evaluation of chromogenic bioMrieux MRSA ID medium for direct detection of Methicillin-Resistant Staphylococcus aureus from surveillance cultures and from clinical samples
METHODS II
Yellow colonies on MSA (indicating S. aureus) were subcultured onto Columbia blood agar and onto Mueller-Hinton oxacillin-agar (S-MRSA) following the CLSI guidelines. Further identification and susceptibility were performed by the MicroScan system (Dade Behring. Sacramento. CA). Green colonies on MRSA ID (indicating MRSA), were considered to be positive and were subcultured onto Columbia blood agar for further identification and susceptibility testing by the MicroScan system. The absence of growth or colonies with colours other than green were considered negative. Recovery and identification of MRSA from MSA and from MRSA ID using PCR for the detection of the mecA and femA genes was considered the reference method. Colonies resembling MRSA in both media (MRSA ID and MSA+S-MRSA) and confirmed by PCR as MRSA were considered to be true positives. Colonies resembling MRSA in both media (MRSA ID and MSA+S-MRSA) but not confirmed by PCR as MRSA were considered to be false positives. If a sample was negative for MRSA on test media, the result was assumed to be a true negative. If a test failed to grow MRSA but MRSA was confirmed by any other test, the result was considered to be a false negative. Fifty-eight MRSA isolates (43%) were detected on MRSA ID at 24 h. At 48 h, 42% of MRSA isolates were detected, and in the remaining 15%, readings were only performed at 48 h. Recovery on MSA followed by identification by conventional methods, delayed the identification of MRSA to 72 h in 14% of cases, and to more than 72 h in 86%. Only seven false positive readings on MRSA ID were detected (one nasal swab and 6 other samples): Enterobacter cloacae (3) Methicillin-Susceptible S. aureus (1) Methicillin-Resistant coagulase-neg. staphylococci (1) Candida spp. (1) Bacillus spp. (1)
E. cloacae
Methicillin-Resistant coagulase negative staphylococci
RESULTS I
Table 1: Recovery of MSSA and MRSA isolates on MSA and MRSA ID
Media MSA MRSA ID Total
p< 0.0001
No. (%) of MSA 67 (100%) 1 (1.4%) 67 (100%)
No. (%) of MRSA 111 (82.8%) 132 (98.5%) 134 (100%)

Bacillus spp. Methicillin-Resistant S. aureus
CONCLUSIONS
In this study, MRSA ID was superior to conventional methods for the recovery of MRSA from nasal swabs and from other clinical samples.
Table 2: Overall results for MSA and MRSA ID

MSA Sensitivity Specificity Positive predictive value Negative predictive value 82.8% 99.7% 99.1% 94.5% MRSA ID 98.5% 98.2% 94.9% 99.4%
The use of MRSA ID shortened the time to detection of MRSA to 24 or 48 h. The enhanced recovery of MRSA using this medium and the identification of most MRSA isolates at 24 h without additional susceptibility testing, will benefit the patients and decrease costs associated to infections caused by MRSA.
RESULTS II
Table 3: Nasal culture results for MSA and MRSA ID
MSA Sensitivity Specificity Positive predictive value Negative predictive value 86.2% 99.7% 98.9% 95.8% MRSA ID 98.3% 99.7% 99.2% 99.4%
KEY POINTS

MRSA ID (new name chromID MRSA) shortened MRSA time detection to 24 or 48 h. The identification of most MRSA isolates at 24 h without additional tests will benefit patients and decrease costs associated to infections caused by MRSA.
18

Detection of nasal carriage of Staphylococcus aureus and Methicillin-Resistant Staphylococcus aureus in general population with a new Chromogenic medium: MRSA ID AGAR
Pinar Ciragil, Mustafa Gul, Murat Aral, Sumeyra Alkis
Department of Clinical Microbiology, Kahramanmaras Sutcu Imam University, Faculty of Medicine, Kahramanmaras, Turkey
INTRODUCTION
Staphylococcus aureus (S. aureus) is one of the most frequently identified pathogens in clinical laboratories and is responsible for a variety of mild to life-threatening infections. Methicillin-Resistant Staphylococcus aureus (MRSA), known as a nosocomial pathogen, has been isolated in community-acquired infections since 1980s (CA-MRSA). Carriage of S. aureus in the nose appears to play a key role in the epidemiology and pathogenesis of infection. The prevalence and incidence of S. aureus nasal carriage vary according to the population studied. Since the carrier status is asymptomatic and transmission can occur from any individual colonized with MRSA, measurement of the rate of MRSA carriage through a population-based study may be helpful to estimate the potential for the spread of MRSA in the community. Rapid identification is essential to limit the spread of MRSA among patients and also in the community. Conventional methods to detect resistance to oxacillin such as disc diffusion or agar dilution require 24 hours of incubation, however they may fail to detect oxacillin resistance when phenotypic expression of an isolate is heterogenious. PCR-based methods have recently developed and now detect the mecA gene which and requires special equipment and cannot be performed in most routine microbiology laboratories. Thus, new screening media for the improved detection of MRSA have recently been developed. In this study we assessed a new chromogenic medium MRSA ID agar (bioMrieux, France) for the prevalence of, and risk factors for, CA- MRSA and S. aureus nasal carriage in the general population.
RESULTS
Out of 609 samples, a total of 133 (21.8%) samples were positive for nasal S. aureus colonization. After 18-24 hours of incubation 19 (3.1%) of the samples were identified as MRSA with green colonies on MRSA ID agar. However, CBA identified 18 (2.9%) MRSA isolates with a combination of tests, including tube coagulase tests and ATB STAPH 5 in 48 hrs. There was an excellent agreement between both CBA and MRSA ID media according to the kappa test (Table 1). Characteristics of subjects for nasal colonization with MRSA are shown in table 2. No significant risk factors were observed among MRSA carriers (p>0.05), except a history of antimicrobial use. Thirteen (6.3%) MRSA carriers had reported antimicrobial use within the 3 months, while 6 (1.5%) had no antimicrobial use reported (p=0.001) (Table 3). Table 1: Agreement between MRSA ID agar and CBA for MRSA detection
N of subjects MRSA ID Agar Negative Positive 587 4 3 590 15 19 Total 591 18 609
Negative Blood agar Positive Total

Kappa: 0.805, p=0.000

Study population A total of 609 apparently healthy subjects who accompanied the patients from the community were evaluated for the prevalence of nasal S. aureus colonization and to identify risk factors associated with S. aureus and CA-MRSA colonization. Data on age, sex, underlying chronic diseases (i.e. diabetes, renal disease, gastrointestinal disease) hospitalization and history of surgical operations, and antimicrobial use within the last 3 months were recorded. Nasal cultures Nasal cultures were performed and inoculated on Columbia with 5% sheep blood agar (CBA) and a new chromogenic medium: MRSA ID agar. Culture plates were incubated at 37C in aerobic conditions and interpreted after 18-24 hours. Suspected colonies (green on MRSA ID, and pigmented on CBA) were confirmed: S. aureus using a coagulase test and Methicillin-Resistance using mini API, ATB STAPH 5 (bioMrieux, France). White or very pale green colonies were identified as coagulasenegative staphylococci (CNS). MRSA ID agar (Figure 1) is a selective chromogenic medium. It contains a chromogenic substrate of alphaglucosidase and cefoxitin which favour the identification of MRSA (green colonies). A selective mixture inhibits most bacteria not belonging to the genus Staphylococcus, as well as yeasts. Figure 1: MRSA isolates on MRSA ID agar
19
Table 2: Characteristics of subjects for nasal colonization with Methicillin-Resistant Staphylococcus aureus (MRSA)
With MRSA Variable Age (years) 14-40 > 41 Sex Female Male
p<0.05 is significant
Without MRSA Colonization (n=590)
Colonization (n=19)
11(2.8) 8 (3.7)
383 (97.2) 207 (96.3)
529
8 (2.7) 11 (3.5)
286 (97.3) 304 (96.5)
584
chromID VRE
N of subjects
chromID ESBL
chromID MRSA
Detection of nasal carriage of Staphylococcus aureus and Methicillin-Resistant Staphylococcus aureus in general population with a new Chromogenic medium: MRSA ID AGAR
Table 3: Risk factors for nasal colonization with Methicillin-Resistant Staphylococcus aureus (MRSA) N of subjects With MRSA
Risk factors Colonization (n=19) Without MRSA Colonization (n=590) P
CONCLUSIONS
CA-MRSA colonization may vary according to risk factors and geographical region. Antimicrobial use may increase the colonization of MRSA in apparently healthy subjects (Table3). The increasing penetration of CA-MRSA in the community requires methods for rapid and accurate diagnosis. The new MRSA ID medium enables a reliable and rapid detection of MRSA without any need of additional identification tests. After 24-48 hrs of incubation CNS isolates gave white or very pale green colonies, so these were easily distinguished from MRSA strains which form large green colonies. Identification of MRSA strains with MRSA ID is faster and easier than CBA. Consequently MRSA ID may be used instead of CBA for the identification of MRSA strains.
Hospitalization within 1 year Positive Negative 11(2.8) 8 (3.7) 383 (97.2) 207 (96.3) .529
Surgical operation within 1 year Positive Negative 8 (2.7) 11 (3.5) 286 (97.3) 304 (96.5) .584
History of antimicrobial use Positive Negative Underlying diseases Positive Negative

p<0.05 is significant
13 (6.3) 6 (1.5)
195 (93.8) 395 (98.5)
.001
2 (1.8) 17 (3.4)
111 (98.2) 479 (96.6)
.360
KEY POINTS

MRSA ID (new name chromID MRSA) enables reliable and rapid detection of MRSA without any additional tests. After 24-48 hrs with MRSA ID (new name chromID MRSA), CNS isolates gave white or very pale green colonies, so these were easily distinguished from MRSA colonies which form large green colonies.
20

Comparative evaluation of the chromogenic MRSA ID media with standard procedure for screening of Methicillin-Resistant Staphylococcus aureus carriers and detection of high level resistance to Mupirocin by disk diffusion
Iqbal Mansoor, Jean Brouillard, Stphanie Cannoot, Claudine Ducastel
Rseau Hospitalier de Mdecine Sociale. Belgium
ABSTRACT
Objectives: The procedure for screening patients with Methicillin-Resistant Staphylococcus aureus (MRSA) is very laborious. It includes the use of agar plates and selective enrichment broth (SEB). Results are available to clinicians only after 48-96h, thus delaying isolation meaures needed for preventing nosocomial transmission of MRSA. In this study we evaluated the yield of direct plating specimens on MRSA ID without SEB. Moreover high level resistance to mupirocin was determined by disk diffusion (200g) and MICs. Methods: A total of 523 specimens were cultured : 487 nasal and 36 wounds swabs. To overcome inoculation bias, a random table was used. Direct plating on MRSA ID with a disk of mupirocin (200g) was compared with our procedure which includes a Columbia ANC blood agar (COL) and a Na Cl 7.5% SEB. Plates were read after 24/48h and broths subcultured on COL after 24h. All colonies of Staphylococcus on the COL and green colonies on MRSA ID plates were identified with the Slidex Staph Plus (SSP) latex. Oxacillin susceptibility was done with an oxacillin (6g/ml) agar screen method (OAS). Mupirocin inhibition zone diameters (IZD) were recorded for MRSA strains isolated from clinical samples and from 14 well caracterized mup A strains. Mupirocin MICs were performed with Etest strips for MRSA strains with IZD <20mm . Results: A total of 39 (7%) MRSA were isolated by a combination of all methods. The overall sensitivities for primary plating were 77 (87%) for COL and 90 (100%) for MRSA ID after 24 (48h) of incubation time respectively. The SEB in combination with COL gave a yield of 92%. The specificities for the MRSA ID plates after 24 (48h) incubation were 99.8 (98.3%). No Methicillin-susceptible Staphylococcus aureus was isolated on the MRSA ID while 65 (12%) with our routine procedures. The number of SSP and OSA tests done was 281 (54%), 149 (28%) for routine procedure and 48 (9%), 39 (7%) for direct plating on MRSA ID respectively (p<0.05). The mupirocin IZD range from 6 to 50 mm and 6 to 14 mm for clinical and mup A strains. Mupirocin MICs for strains (19) tested were >1024g/ml. Conclusion: The use of MRSA ID with 24h of incubation time is as sensitive as standard procedure for MRSA screening. Green colonies appearing after 48h should be further identified and susceptibility testing performed. High dose mupirocin disks could be a good alternative to E-test for detecting high level resistance to mupirocin.
INTRODUCTION AND OBJECTIVES

Screening for MRSA carriers in high risk wards is essentiel for preventing nosocomial acquisition of MRSA. Selection of procedure should be based not only on the yield of the test but also on the TAT. With the standard procedure, results are available to clinicians after 48-96h, thus delaying the recommended measures to be implemented in case of MRSA carriers. The use of PCR could be an alternative but is laborious and costly. This study compared the yieldof direct plating on MRSA ID with standard procedure which includes an enrichment broth. The predictive value of the inhibition zone diameters for detecting high level resistance to Mupirocin by disk diffusion with Mupirocin (200g) disks on the MRSA ID was evaluated. The number of confirmatory tests needed in each arm of the study was evaluated.

Specimens : 487 nasal, 36 wound swabs. Standard procedure: direct plating on Columbia ANC (COL) and enrichment broth with Na Cl 7.5%. Broths were subcultured on COL after 16-24 h Inncubation at 35C in ambient air. Reading of plates after 24 / 48h for primary plating and after 24h for subcultured plates. Investigational arm: direct plating on MRSA ID plates with a Mupirocin disk (200g). Reading of plates after 20 - 24h and 48h. Identification of green colonies on the MRSA ID and probable staphylococcal colonies on the COL with Slidex Staph Plus latex. Oxacillin susceptibility was done with an oxacillin screen agar (6g/ml) Mupirocin inhibition zone diameters were recorded for the clinical MRSA strains and for 14 mup A strains. To avoid inoculation bias, the order of plating was determined by a random table.
RESULTS
39 (7%) MRSA were isolated : 29 nasal ; 10 wounds swabs. The yield of the MRSA ID was 90 (100%) after 24/48h of incubation. The specificities of the MRSA ID were 99.8 (98.3%) after 24/48h. No Methicillin sensible S. aureus was isolated on the MRSA ID while 65 (12%) with our routine procedures. 3 false negative oxacillin susceptibility were observed with the Oxa-screen agar. Latex agglutinations were performed with the Slidex Staph Plus in 281 (54%) and 149 (28% ) cases for routine and MRSA ID plates respectively. Oxacillin susceptibility testings were performed in 48 (9%) and 39 (7%) cases for the routine and MRSA ID plates. Mupirocin Inhibition zone diameters (IZD) recorded for 14 well caracterized mupA and for the clinical MRSA strains isolated ranged from 6-14 and 6-50mm respectively. Mupirocin MICs for 19 strains with IZD < 14 were all > 1024g/ml.
21
chromID VRE
chromID ESBL
chromID MRSA
Comparative evaluation of the chromogenic MRSA ID media with standard procedure for screening of Methicillin-Resistant Staphylococcus aureus carriers and detection of high level resistance to Mupirocin by disk diffusion
Yield 24 h 48 h MRSA ID 35 (90%) 39 (100) COLANC 30 (77) 34 (87) Broth 36 (92)
35 30 25 20 15 10 5 0 <6 MIC > 1024 mup A clin. samples < 14 > 30 mm
CONCLUSIONS Plating on MRSA ID and reading after 24h of incubation is as sensitive

as the standard procedure with enrichment broths. Confirmation of species identity and susceptibility testing should be performed on green colonies grown on the MRSA ID plates after 48h of incubation. High dose Mupirocin disks could be a good alternative to E tests for detecting high level resistance.
KEY POINTS

After 24 hrs, MRSA ID (new name chromID MRSA) is as sensitive as the standard procedure. High dose Mupirocin disks could be a good alternative for detecting high level resistance.
22
RICAI / December, 2005

Paris (France) poster 463/73P
Comptability of MRSA ID, a new Chromogenic Medium dedicated to MRSA carriage, with identification, susceptibility testing and other rapid tests
N.Fanjat N.Garcia D.Robichon
bioMrieux, La Balme les Grottes, France
MRSA ID is a new chromogenic medium dedicated to the screening of MRSA carriage. The green colouration is characteristic of MRSA colonies. The purpose of this study is to evaluate the compatibility between MRSA ID and the bioMrieux identification and susceptibility products and other rapid tests. Results are compared with those obtained from conventional media.
Table I: Influence of MRSA ID as a pre-culture medium on the identification of MRSA strains (results in %)
MRSA ID COS 1 2 4 1 88 12 2 4 MRSA ID COS 1 2 3 4 MRSA ID COS 1 2 3 4 3 4 1 82 2 2 3 4

Figure 1: Reagent testing for 50 MRSA strains, after pre-culture on MRSA ID and Columbia Agar + 5% sheep blood (COS) or Trypcase Soy Agar + 5% sheep blood (TSS)
COS MRSA ID
16
No undentified strains
MRSA ID COS 1 2 3 4 MRSA ID
1 82 2
1 82 2
16
16
24h at 37C
24h at 37C
1 82 2
TSS
MRSA ID
Identification scale 1 Correct identification to one choice 2 Low discrimination 3 Undentified 4 Misidentified
Correct identification
24h at 37C
24h at 37C
The rapid tests show a correlation of 100% between the results obtained on MRSA ID and COS (Table II).
Identification Testing - VITEK GPI Susceptibility Testing - VITEK GPS 504
MRSA ID S. aureus ATCC 43300 Incubation 18-24 hours
2. Susceptibility testing: Regarding the results obtained on Rapid ATB STAPH and ATB STAPH strips (Tables III and IV), a category agreement rate superior or statistically equivalent to 90% is observed for all drugs. Nevertheless, there is a trend to induce a higher resistance for glycopeptides: teicoplanin and/or vancomycin. Table II: Influence of MRSA ID as a pre-culture medium on the identification of MRSA strains (results in %)
Reagents COS Results MRSA ID 50 strains 50 strains + 50 strains + 1 strain 49 strains + 50 strains + 50 strains 50 strains + 50 strains + 1 strain 49 strains + 50 strains +
RESULTS
1. Identification: Compared to the reference medium, the use of MRSA ID as a pre-culture medium has no influence on the MRSA identification (Table I). The results show an agreement rate of 100% for RAPIDEC Staph and VITEK GPI and 98% for API Staph, VITEK 2 ID-GPC and GP. The correct identification rates on MRSA ID (identification to one choice or in low discrimination) are 88% for RAPIDEC Staph, 100% for API Staph and VITEK GPI, 98% for VITEK 2 ID-GPC and 96% for VITEK 2 GP.
Oxidase Reagent ID color Catalase Rabbit plasma Slidex Staph Plus Slidex MRSA Detection
23
chromID VRE
chromID ESBL
Identification Testing - RAPIDEC Staph - API Staph - VITEK 2 ID-GPC - VITEK 2 GP ID 32 STAPH not tested
Identification Testing - Rapid ATB STAPH - ATB STAPH - VITEK 2 AST-P523
Identification Testing - Oxidase Reagent - ID color Catalase - Rabbit plasma (detection of staphylocoagulase) - Slidex Staph Plus - Slidex MRSA Detection
COS 1 2 3 4
16
chromID MRSA
Comptability of MRSA ID, a new Chromogenic Medium dedicated to MRSA carriage, with identification, susceptibility testing and other rapid tests
Table III: Category agreements between MRSA ID and the reference medium (%)
Kanamycin Erythromycin Nitrofurantoin Rifampicin Ofloxacin Pristinamycin Lincomycin Fosfomycin Chloramphenicol Oxacillin Teicoplanin Other drugs Minocyclin Lincomycin Fostomycin Vancomycin Cotrimoxadol Kanamycin Gentamicin Tobramicin Pefloxacin Teicoplanin Other drugs
Figure 2: Influence of MRSA ID as a pre-culture medium on glycopedtide MIC determinations

Distribution of the dilution deviation on VITEK GPS 504 100 90 80 98 94 100 70 60 50 40 30 20 10 0 -3 -2 -1 0 1 Dilution range 2 3
98
96
96 92 90 88 100
Table IV: Distribution of the category deviation for glycopeptides

Vancomycin SX XR SR RX XS RS Teicoplanin 2 3 1 Vancomycin Teicoplanin 2 1
Vancomycin
Teicoplanin
Distribution of the dilution deviation on VITEK 2 AST-P523 100 90 80 70 60 50 40 30 20 10 0
X The growth index value is too close to the threshold value
With the VITEK and VITEK 2 systems (Table V and Figure 2), the MIC agreements within 1 dilution are at least 96% except for Teicoplanin on AST-P523: MICs are overestimated for this drug. Moreover, MICs are one dilution overestimated for vancomycin on GPS 504 and AST-P523. However, in all cases, categorizations are not affected (using CA-SFM or NCCLS MIC breakpoints). Table V: MIC agreements ( 1 dilution) between MRSA ID and the reference medium (%)
VITEK GPS 504 Oxacillin Erythromycin Lincomycin Vancomycin Chloramphenicol Trim./ Sulfam. Fusidic acid Other drugs VITEK 2 AST-P523 Benzypenicillin Clindamycin Erythromycin 98 Pristinamycin Teicoplanin 98 Other drugs 100
-3
-2
-1
0 1 Dilution range
98
CONCLUSION
According to the study results, MRSA ID is fully compatible with the other bioMrieux identification products and different rapid tests. Concerning susceptibility, the results obtained with glycopeptides have to be interpreted carefully since a tendency towards too many resistant results has been observed. This point has been included in the package insert of the medium. Regarding the other antibiotics tested, the susceptibility testing results can be used to determine the resistance profile of the MRSA strains.
96 100
KEY POINTS
MRSA screening & diagnosis : a complete offer with culture, identification, detection of bacterial resistance, ID / AST automation.
24

Copenhagen (Denmark) poster P1383
Multiresistant bacteria screening: Clinical evaluation of MRSA ID, a new chromogenic medium for the screening of Methicillin-Resistant Staphylococcus aureus
M. E. Reverdy1, S. Orenga2, J. M. Roche2, S. Delorme2, J. Etienne1
National Reference Laboratory for Staphylococci, Hospital E. Herriot, Lyon, France, 2bioMrieux, La Balme & Craponne, France
ABSTRACT
Nasal carriage of Methicillin-Resistant S. aureus (MRSA) is an important cause of nosocomial infection. Screening of patients during hospital admission is recommended to reduce the incidence of MRSA. The aim of the study was to evaluate the new MRSA ID media (bioMrieux) for rapid isolation and identification of MRSA. MRSA ID was compared to ORSAB medium (Oxoid). Columbia CNA sheep blood agar (COL) was used as the reference method. A total of 278 nasal swabs were inoculated directly on the media which were incubated 18-24 hrs and 48 hrs at 35C. Green colonies on MRSA ID, blue on ORSAB and suspected S. aureus colonies on COL were regarded as presumptive MRSA isolates. Confirmation was performed using a coagulase test and PCR for mecA gene. A total of 45 swabs were positive for MRSA. After 18-24 hrs incubation, 42, 38 and 42 MRSA strains were isolated on MRSA ID, ORSAB and COL respectively. MRSA isolates produced colonies of intense colour on chromogenic media. The sensitivity of detection of MRSA ID (93.3%) was higher than ORSAB (84.4%). The number of false positives (FP) on ORSAB was higher (25 coagulase-negative Staphylococci (CNS) and 2 Methicillin-Sensitive S. aureus) compared to MRSA ID (3 CNS). If only taking into account colony colour, the predictive positive value (PPV) was 93.3% for MRSA ID, 58.5% for ORSAB. If the coagulase test result was included, the PPV value of ORSAB became 95%. After 48 hrs, MRSA ID enabled the recovery of 1 MRSA strain more (43 strains) whereas 5 strains more were isolated on ORSAB (43 strains). However, the PPV values of both media decreased because MRSA ID produced many pale green colonies of CNS, and ORSAB in the same way. Depending on the interpretative reading of the laboratory, FP on MRSA ID were easily differentiated from MRSA isolates. MRSA ID enables rapid and definitive identification of MRSA isolates. Sensitivity of MRSA ID after 18-24 hrs was higher than that of ORSAB. According to the local epidemiology, a reading of MRSA ID after 48 hrs could be performed. In this case, green colonies should be confirmed S. aureus. The use of MRSA ID does not require any supplementary testing after 18-24 hrs, whereas additional tests are necessary for ORSAB media.

A total of 278 nasal swabs were inoculated directly on MRSA ID, ORSAB, and columbia CNA sheep blood agar used as reference method. Culture plates were incubated at 35C in aerobic conditions and interpreted after both 18-24 hrs and 48 hours. Suspected colonies (green on MRSA ID, blue on ORSAB and pigmented on COL) were confirmed S. aureus by coagulase test and MRSA by detection of mecA gene Figure 1: MRSA culture on MRSA ID
After 18-24 hours and 48 hours respectively, a total of 43 and 45 of MRSA were isolated on any of the three media (Figure 2). 18-24 hours results: 42 (93.3%) of the 45 strains were isolated as intense green colonies on MRSA ID and 38 (84.4%) on ORSAB medium (Figure 3). If only taking into account colony colour, 3 false positives (FP) were found on MRSA ID so the positive predictive value (PPV) was 93.3%, and 27 FP were found on ORSAB so the PPV was 58.5%. If the coagulase test result was included, the PPV value of ORSAB became 95% (Figure 4). Three false positives (FP) found on MRSA ID were due to CNS which produced pale green colonies; these CNS were easily distinguishable from MRSA strains which generated intense green colonies. On ORSAB, the two remaining FP after coagulase test corresponded to two Methicillin-Sensitive S. aureus (MSSA). The size of MRSA colonies on MRSA ID medium was larger than the one observed on ORSAB. This confers to MRSA ID the advantage of an high legibility and allowed the storage of MRSA strains without any subculture. After 18-24 hours of incubation, MRSA ID shows the best level of fertility, sensitivity and specificity, without any supplementary testing (whereas coagulase test is necessary for ORSAB media). 48 hours results: The sensitivity of both chromogenic media reached 95.6% (one MRSA strain more on MRSA ID and five on ORSAB (Figures 2 and 3). However MRSA ID produced many pale green colonies of CNS, and ORSAB in the same way. So, at 48h, the coagulase test must be performed for the two media.
INTRODUCTION
Nosocomial infections are a major concern for most hospitals, in particular MRSA infections. Screening of MRSA carriage on hospital admission and contact isolation of colonized patient are considered as the most efficient measure to control MRSA diffusion. MRSA ID (bioMrieux, France) is a new, readyto-use, chromogenic medium designed for rapid identification of MRSA. Direct identification of MRSA strains is based on the expression of alphaglucosidase revealed by green-coloured colonies and the presence of an antibiotic, cefoxitin. The aim of the study was to evaluate the biological performances of MRSA ID with clinical samples.
25
chromID VRE
chromID ESBL
RESULTS
chromID MRSA
Multiresistant bacteria screening: Clinical evaluation of MRSA ID, a new chromogenic medium for the screening of Methicillin-Resistant Staphylococcus aureus
Figure 2
50 40 30 20 10 0 42 38 42 43 43 43 43 45
CONCLUSIONS
Within 18-24 hours, MRSA ID enables a reliable and rapid detection of MRSA without additional identification test. According to our local epidemiology, the incubation for 48 h was not relevant during this study. In the event of a 48 hrs reading, green colonies should be confirmed S. aureus. This study indicates that performances of MRSA ID in terms of fertility, sensitivity and specificity were superior to the ORSAB medium.
18 - 24 h 48 h Fertility (number of SARM isolated) MRSA ID ORSAB COL ANC Any
Figure 3
93.3 100 80 60 40 20 0 18 - 24 h Sensitivity (%) MRSA ID ORSAB 48 h 84.4 95.6 95.6
Figure 4
93.3 100 80 60 40 20 0 colour only coagulase test for ORSAB only 18 - 24 h Specificity (%) MRSA ID ORSAB coagulase test for both media 48 h 58.5 93.3 95 93.5 95.6
KEY POINTS

MRSA ID (new name chromID MRSA) enables rapid and definitive identification of MRSA isolates. Sensitivity of MRSA ID (new name chromID MRSA) after 18-24 hrs was higher than the routine tests used. MRSA ID (new name chromID MRSA) does not require any supplementary testing after 18-24 hrs.
26

Copenhagen (Denmark) poster P1379
Comparison of MRSA ID medium and enrichment broth culture for detection of Methicillin-Resistant Staphylococcus aureus carriers by muco-cutaneous surveillance cultures
C. Nonhoff, A. Brenner, N. Legros, C. Thiroux, M. J. Struelens and O. Denis
Reference Laboratory for Staphylococci ULB-Erasme Hospital, Brussels, Belgium
BACKGROUND
Methicillin-Resistant Staphylococcus aureus (MRSA) infections are an important clinical and epidemiological problem in hospitals. The major mode of MRSA transmission is contact from MRSA colonised or infected patient to another one via transiently colonised hands of healthcare workers. Therefore, the early identification of MRSA carriers is essential for implementation of infection control measures to prevent dissemination.
Table 1: Sensitivity, specificity, positive and negative predictive values of MRSA ID and SBA before and after enrichment
False + Medium Incubation True + Sensitivity Specificity time (MRSA) MSSAa CNSb (%) (%) Without enrichment MRSA ID1 24h 35 0 1 63.6 100 48h 54 2 53 98.2 99.5 54.6 88.1 SBA1 24h 30 52 NAc 48h 38 62 NA 69.1 85.8 After enrichment MRSA ID2 24h 51 4 2 92.7 99.1 48h 53 6 20 96.4 98.6 SBA2 24h 49 60 NA 89.1 86.2 48h 49 62 NA 89.1 85.8
a c
PPV (%) 100 96.4 36.6 38.0 92.7 89.8 45.0 44.1
NPV (%) 100 100 100 100 100 100 100 100
OBJECTIVES
To evaluate the performance of the new chromogenic agar, MRSA ID medium (bioMrieux), for detection of MRSA from surveillance cultures of muco-cutaneous swab specimens in patients admitted to a 860-bed teaching hospital.

Patients and specimen collection From September to November 2004, 491 screening swabs from nares (n = 363), throat (n = 47), perineum (n = 46) and skin (n = 35) were obtained fom 331 hospitalized patients. Microbiological methods Swabs were homogeneized by vortexing into 1 ml of sterile saline solution. One hundred microliter were inoculated into Brain-Heart-Infusion broth supplemented with NaCl 7.5% (SBHI), 5% sheep blood Columbia agar (SBA1) and MRSA ID (MRSA ID1) agar. SBHI broth was sub-cultured after 24 h incubation onto a secondary SBA (SBA2) and a secondary MRSA ID (MRSA ID2). Plates were incubated at 35C and examined after 24 and 48 h. Colonies suspected by morphological and colour examination were tested with Pastorex Staph-Plus (Bio-Rad). Colonies showing positive agglutination were further identified by coagulase production in human plasma. Resistance to oxacillin was determined by the cefoxitin disk method according to the NCCLS recommendations. Identification of MRSA was confirmed by multiplex PCR for the 16S rRNA, nuc and mecA genes. Sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) were analysed based on colony colour and morphology and positive agglutination test. MRSA ID (Figure 1) is a selective medium containing cefoxitin that inhibits the majority of bacteria other than Methicillin-Resistant Staphylococcus and yeasts. S. aureus forms green colonies on this chromogenic medium due to the production of alpha-glucosidase.
MSSA = Methicillin-susceptible Staphylococcus aureus, bCNS = coagulase-negative staphylococci, NA = not applicable
Figure 1: MRSA isolate on MRSA ID medium
Medium MRSA ID1
Incubation Time 24h
48h
MRSA ID2
24h
48h
CONCLUSIONS
1. MRSA ID is a sensitive and specific medium for screening of MRSA muco-cutaneous carriage in hospitalized patients. Sensitivity is enhanced after 48 h incubation (63.6% versus 98.2%) with a very slight decrease in specificity (100% versus 99.5%). 2. The colour of the colonies is assessed best when the plates are inspected upside down. 3. After 48 h incubation, very pale green colonies were in the great majority (> 95%) CNS isolates; these colonies were easily distinguishable from MRSA strains. 4. Combination of MRSA ID with enrichment broth did not increase its sensitivity and therefore enrichment is not necessary.
RESULTS
Figure 2: Distribution of MRSA isolates by specimen type (n = 55)
Other 10% Perineum 27% Nare 47%
Throat 16%
KEY POINTS

MRSA ID (new name chromID MRSA) is a sensitive and specific medium. 48 hrs incubation increased the yield by 36%, and the additional phase of enrichment broth is not required. Therefore, gain in time-to-result of 24 hrs, and an important simplification of methodology for MRSA screening.
27
chromID VRE
Colour MRSA Green 9 Pale green 18 Very pale green 8 Green 50 Pale green 3 Very pale green 1 Green 43 Pale green 8 Very pale green 0 Green 51 Pale green 2 Very pale green 0
Species MSSA 0 0 0 2 0 0 3 1 0 5 1 0
CNS 1 0 0 2 4 47 1 0 1 3 0 20
chromID ESBL
Table 2: Distribution of staphylococcal isolates on MRSA ID medium by colour and incubation time
chromID MRSA
chromID ESBL
chromID ESBL agar is a completely new and innovative chromogenic medium designed specifically for the Screening of the Extended Spectrum Beta Lactamase producing Enterobacteria (ESBL). chromID ESBL combines the selective growth of ESBL and the direct identification of E.coli, bacterial group of KESC (Klebsiella, Enterobacter, Serratia and Citrobacter) and bacterial group of Proteeae (Proteus, Providencia, Morganella). Selective growth of ESBL producer: Enriched with a rich nutrient base including a variety of peptones and a mixture of antibiotics. This mixture includes Cefpodoxime (article JCM 02/07), an antibiotic recognised as being the marker of choice for this resistance mechanism. Direct identification: Two chromogenic substrates aided by a natural substrate enable the direct identification of the most frequently found ESBL Enterobacteria. Escherichia coli: immediate pink to burgundy colouration Klebsiella, Enterobacter, Serratia, Citrobacter (KESC): immediate green to browny-green colouration Proteeae (Proteus, Providencia, Morganella): immediate dark to light brown colouration. (Poster RICAI 2006 n 0362) chromID ESBL agar is extremely easy-to-read and allows the detection of mixed culture. (Poster ECCMID 2007 n 505) chromID ESBL agar is easy-to-use (Poster RICAI 2006 n 0362) and reduces the unnecessary confirmations. (Poster ICAAC 2006 n 0457, Poster RICAI 2006 n 0362, article JCM 02/07) chromID ESBL agar is fully compatible with identification and AST Vitek reagents as well as Oxydase reagent and confirmation diffusion disks methods. (Poster RICAI 2006 n 0361) chromID ESBL has a superior sensitivity than conventional methods. (Poster ICAAC 2006 n 0457, Poster RICAI 2006 n 0362, Poster ECCMID 2007 n 505) and a shorter-time to results (Poster ECCMID 2007 n 505, article JCM 02/07). Genotypic characterization of resistance mechanisms of the ESBL strains shows that chromID ESBL is able to detect all types of ESBL as TEM, SHV & CTX-M. (article JCM 02/07)
Contents
ARTICLES
Evaluation of a New Selective Chromogenic Agar Medium for Detection
of Extended-Spectrum -Lactamase-Producing Enterobacteriaceae
JOURNAL OF CLINICAL MICROBIOLOGY, February 2007 Y. Glupczynski et al.
31
POSTERS
ECCMID / Munich (Germany) / April, 2007 Screening of multidrug-resistant bacteria: clinical evaluation of a novel chromogenic medium chromIDTM ESBL for the screening of ESBL-producing Enterobacteriaceae
32
M. Husson et al.
RICAI / Paris (France) / December, 2006 Compatibility of chromID ESBL, a new chromogenic medium, with identification, susceptibility testing reagents and other tests
34
N. Fanjat et al.
RICAI / Paris (France) / December, 2006 Evaluation of chromID ESBL medium for the detection of extended-spectrum beta-lactamase (ESBL) producing enterobacteria
36
A. Cady et al.
ICAAC / San Francisco (USA) / September, 2006 Novel chromogenic medium from bioMrieux for detection of ESBL-producing Enterobacteriaceae
38
Y. Glupczynski et al.
February, 2007
JOURNAL OF CLINICAL MICROBIOLOGY, Feb. 2007, p. 501505 Vol. 45, No. 2
0095-1137/07/$08.00 0 doi:10.1128/JCM.02221-06 Copyright 2007, American Society for Microbiology. All Rights Reserved.
Evaluation of a New Selective Chromogenic Agar Medium for Detection of Extended-Spectrum -Lactamase-Producing Enterobacteriaceae
Youri Glupczynski,* Catherine Berhin, Caroline Bauraing, and Pierre Bogaerts
Laboratoire de Bactriologie, Cliniques Universitaires UCL de Mont-Godinne, B-5530 Yvoir, Belgium
Received 30 October 2006/Accepted 8 December 2006
A novel chromogenic agar medium (ESBL-Bx; bioMrieux, Marcy lEtoile, France) was compared to Mac-Conkey agar supplemented with 2 mg ceftazidime/liter (MCKC) for the selective isolation and presumptive identification of extended-spectrum -lactamase (ESBL)-producing Enterobacteriaceae directly from clinical samples. Of a total of 644 clinical specimens (including 551 fecal samples), 496 yielded no growth and 148 yielded growth on one or both media. Overall, 44 ESBL-producing Enterobacteriaceae strains (Escherichia coli [n = 17], Enterobacter aerogenes [n = 17], Klebsiella spp. [n 5], and Citrobacter freundii [n = 5]) were isolated from 37 specimens by a combination of both methods after 18 to 24 h of incubation. The sensitivities were 97.7 and 84.1% for ESBL-Bx and MCKC, respectively, with 43 ESBL-positive strains isolated as colored colonies from 36 specimens on ESBL-Bx versus 37 ESBL-positive organisms isolated from 32 specimens on MCKC. The specificities by specimens were 89 and 91% for ESBL-Bx and MCKC, respectively. On either one of the two media, natural AmpC-hyperproducing Enterobacter spp. (n = 25) and Citrobacter spp. (n = 14) were the most common false positives as well as non-ESBL-producing Klebsiella oxytoca (n = 18) on ESBL-Bx and Morganella morganii (n =10) on MCKC. We conclude that ESBL-Bx is a sensitive and specific medium for the isolation of ESBL-producing Enterobacteriaceae from clinical samples. The main advantages of ESBL-Bx over MCKC reside in its chromogenic character and its sensitivity and selectivity, which enabled the recovery and presumptive identification of most ESBL-producing Enterobacteriaceae within 24 h and reduced by 27% the need for unnecessary identification and confirmation of ESBL testing when disregarding all colorless colonies growing on this medium.
KEY POINTS
Cefpodoxime is the marker of choice for the ESBL resistance mechanism. chromID ESBL reduces the need for unnecessary identification. chromID ESBL enables the identification of mixed cultures. TEM-derived, SHV and CTX-M enzymes are detected.

31
chromID VRE
chromID ESBL
chromID MRSA

Screening of multidrug-resistant bacteria: clinical evaluation of a novel chromogenic medium chromIDTM ESBL for the screening of ESBL-producing Enterobacteriaceae
M. Husson1, S. Ghirardi2, M. Grard1, A. Dediste1
1
Laboratoire de bactriologie, CHU Saint-Pierre, 1000 Bruxelles, Belgium 2bioMrieux, Craponne, France
ABSTRACT
Objectives: Prevention of Health Care associated infections relies on the development and follow up of wise infection controls programs. Screening of extended-spectrum beta-lactamase producing Enterobacteriaceae (ESBLE) is one of the methods to eradicate the Multi Drug Resistant (MDR) bacteria. The aim of the study was to evaluate the new chromogenic medium chromID ESBL (bioMerieux, Craponne, France) for the rapid isolation and identification of ESBLE. Methods: chromID ESBL was compared to a home made method combining a Mac Conkeyagar plate with a disk of ceftazidime(McC). A total of 173 at risk hospitalized patients were tested; 73 rectal swabs, 52 respiratory secretions, 48 urines were directly inoculated on the 2 media which were incubated during 18-24 hrs and 48 hrs at 35C-37C. Typical colonies growing on chromID ESBL and on McC were identified. Confirmation of the presence of ESBLs was performed using disk diffusion methods (according to CA-SFM and/or CLSI standards). Results: A total of 19 specimens were positive for ESBLE, corresponding to 20 ESBLE strains: Escherichia coli (n=13), Klebsiella spp. (n=3), Enterobacter aerogenes (n=2), Enterobacter cloacae (n=2). After 24 hrs incubation, the 19 specimens were positive on chromID ESBL, whereas only 13 were positive on McC. Negative predictive value (NPV) for chromID ESBL and McCwas respectively 100% [97.32% ; 100%] and 96% [91.37% ; 98.17%], corresponding to 6 false negative on McC. After 24 hrs, 9 specimens on chromID ESBL and 17 on McC showed a characteristic growth but were not confirmed ESBL positive (false positives). The specificity was superior for chromID ESBL than for McC (respectively 94% and 89%), with no statistical significant difference. Selectivity towards non ESBLE was higher for chromID ESBL. Conclusion: The new chromID ESBL, combining two chromogenic substrates, one natural substrate and a selective agent of ESBL character, is well positioned as a screening test in order to exclude ESBLE carriage within 24 hrs and to participate to the development and follow up of infection control programs.

173 clinical specimens : 73 rectal swabs, 52 broncho-tracheal aspirates and 48 urines originating from at risk hospitalized patients coming from geriatrics, ICU, internal medicine and neonatology wards. Direct inoculation in parallel onto: - chromID ESBL (bioMrieux, Marcy lEtoile, France) - a McCagar plate (BD, Erembodegem, Belgium) with a paper disk containing 30g ceftazidime (Oxoid, Basingstoke, UK) in the first quadrant of the plate. Incubation in air at 37C. First reading after 18 to 24h incubation and a subsequent reading after 48h incubation. Recording of the presence of suspect colonies, density of growth, morphology and type and intensity of coloration of the colonies were recorded. Suspect colonies were any colored colony on chromID ESBL and any colony growing in the inhibition zone around the ceftazidime disk (arbitrary diameter of 22mm). Any suspected colony was identified and tested for susceptibility by the Vitek2 with GN and AST-N046 cards (bioMrieux, Marcy lEtoile, France). Confirmation of ESBL production was performed with combined double disks or synergy test according to CLSI and/or CA-SFM guidelines. Aspect of the colonies
McC + ceftazidime with colonies in inhibition zone 2 spp. Klebsiella
E. coli + Klebsiella
OBJECTIVES
To assess the fertility of the chromID ESBL agar plate; its ability to detect ESBLE, the specificity of coloration of the colonies and the selectivity of the plate towards yeasts and bacteria other than ESBLE (data not detailed because not statistically significant).
32
RESULTS AFTER 24H INCUBATION

chromID ESBL ESBLE Other bacteria No growth Total # specimens 19 28 126 173 McC+ceftaz 13 17 143 173
9 FP from which 8 are oxidase +
After 24 h incubation chromID ESBL McC+ceftaz
True Pos 19 13
Faise Neg 0 6
Sensitivity % 100 68.42
IC 95% [82.61%;100%] [45.57%;84.47%]
For expression of a phenotype True Pos chromID ESBL McC+ceftaz 145 143 Faise Neg 9 17 Specificity % 94.16 89.38 IC 95% [89.14%;96.93%] [83.51%;93.32%]
Pos.Pred.Val chromID ESBL McC+ceftaz 67.86% 43.33%
Neg.Pred.Va I 100% 95.97% NS NS
CONCLUSIONS
The new chromID ESBL is well positioned as a screening test in order to exclude ESBLE carriage within 24 hr. The majority of FP brown coloured colonies can immediately be ruled out by an oxidase test. After a positive screening the presence of ESBL has to be confirmed as described by CLSI or CA-SFM but the very good NPV allows implementing of hygiene measures before these results are available.
REFERENCES 1. Glupczynski et al. 2007. Evaluation of a New Selective chromogenic agar Medium for Detection of Extended-spectrum B-lactamase-producing Enterobacteriaceae. JCM.45:501-505. 2. Paterson DL, BonomoRA. 2005 Extended-spectrum -lactamases: a clinical update.CMR.18(4): 657-86.
KEY POINTS

Detection of mixed cultures. Shorter time to result.

33
chromID VRE
chromID ESBL
chromID MRSA

Paris (France) poster 361
Compatibility of chromID ESBL, a new chromogenic medium, with identification, susceptibility testing reagents and other tests
Fanjat N., Garcia N., Pincus D., Robichon D., Zambardi G.
bioMrieux, La Balme Les Grottes, France
INTRODUCTION
chromID ESBL is a new chromogenic medium dedicated to the screening of extended-spectrum -lactamase-producing Enterobacteria (ESBLs). chromID ESBL enables the direct identification of Escherichia coli, Proteeae and the Klebsiella-Enterobacter-Serratia-Citrobacter group (1). The purpose of this study is to evaluate the compatibility of chromID ESBL with bioMrieux identification and susceptibility products, the Oxidase reagent and the ESBL confirmation disk diffusion methods. Results are compared with those obtained from reference media.
ID 32 E chromID ESBL 1 2 TSA 1 72 2 2 4 12 3 4 4 VITEK GNI+ chromID ESBL 1 2 TSS 1 56 2 4 38 3 4
3 4 2
ID 32 GN chromID ESBL 1 2 COS 1 80 2 2 14 3 4 2 VITEK 2 GN chromID ESBL 1 2 COS 1 90 2 2 8 3 4

=> Correct Identification
4 2

Figure 1: Reagent testing after pre-culture on chromID ESBL and reference medium
chromID ESBL Reference medium
18-24h at 37C 50 Enterobacteria strains
18-24h at 37C 50 Enterobacteria strains + 10 Pseudomonas
Identification scale 1 Correct identification to one choice 2 Low discrimination 3 Unidentified 4 Misidentified
Identification Testing - API 20 E - rapid ID 32 E - ID 32 E - ID 32 GN - VITEK GNI+ - VITEK 2 GN
Susceptibility Testing (CA-SFM breakpoints) - rapid ATB E - ATB G- VITEK GNS 506 - VITEK 2 AST-NO17 - VITEK 2 AST-NO41
Rapid Test - Oxidase Reagent
ESBL confirmation disk diffusion methods - CA-SFM recommendations - CLSI recommendations
1.2 Compatibility with the Oxidase Reagent The 10 Pseudomonas strains show a positive result as expected. On the other 50 Enterobacteria strains, the results obtained on chromID ESBL are correlated with those expected for 49 strains. One strain is oxidase false positive, both on chromID ESBL and the reference medium.
Reference medium : Trypcase Soy Agar + 5% sheep blood (TSS) for VITEK GNI+ and GNS 506 Trypcase Soy Agar (TSA) for ID 32 E Columbia Agar + 5% sheep blood (COS) for the other tests
2. SUSCEPTIBILITY TESTING:
2.1 Compatibility with susceptibility reagents Regarding the results obtained on rapid ATB E and ATB G- strips (Table II), a category agreement rate superior or statistically equivalent to 90% is observed for all drugs. Moreover, there is no trend to induce a higher susceptibility or resistance and the phenotypes given by the expert system are well correlated. With the VITEK and VITEK2 systems (Table III), the MIC agreements 1 dilution are at least 90% except for Cefotaxime on GNS 506. However, the MIC deviation distribution shows no significant tendency for this molecule (Figure 2). Discrepancies concerning categorisation are observed for Cefotaxime and Nitrofurantoin on GNS 506 and Amoxicillin/Clavulanic acid on AST-N017 (Table II). The agreement rates of these drugs are statistically inferior to 90%. These discrepancies are principally due to MICs close to the breakpoints. Nevertheless, for these three drugs (Table IV) and the others, there is no trend to induce a higher susceptibility or resistance and the expertise is not affected. Results obtained for the ESBL test on GNS 506 and AST-N041 with chromID ESBL are 100% correlated with those obtained from the reference medium. Table II: Category agreements between chromID ESBL and the reference medium (%)
rapid ATB E Amoxicillin / Clavulanic ac. Ticarcillin / Clavulanic ac. Nitrofurantoin Other drugs
6 strains not included (no growth in the control cupule)
RESULTS 1 IDENTIFICATION:
1.1 Compatibility with identification reagents Compared to the reference medium, the use of chromID ESBL as a pre-culture medium has no influence on the identifications (Table I). The results show an agreement rate of 100% for API 20 E, 98% for VITEK 2 GN, 94% for ID 32 GN and VITEK GNI+, 90% for rapid ID 32 E and 86% for ID 32 E. The correct identification rates on chromID ESBL (identifications to one choice or in low discrimination) are statistically equivalent to those obtained on the reference medium. They are 100% for API 20 E and VITEK 2 GN, 98% for VITEK GNI+ and ID 32 GN, 94% for ID 32 E and 92% for rapid ID 32 E. Table I: Influence of chromID ESBL as a pre-culture medium on the identifications (results in %)
API 20 E chromID ESBL 1 2 COS 1 76 2 24 3 4 3 4 rapid ID 32 E chromID ESBL 1 2 COS 1 78 2 4 10 3 4 3 6 2 4
ATB G88.6 86.4 90 Ceftazidime Piperacillin / Tazobactam Cefotaxime Other drugs 88 84 82 90
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VITEK 2 AST-N017 + AST-N041 Nitrofurantoin 88 Cefotaxime 84 Amoxicillin / Clavulanic ac. 78 Other drugs 90
VITEK GNS 506
Amoxicillin / Clavulanic ac. Tobramycin Pefloxacin Ceftazidime Nitrofurantoin Cefotaxime Other drugs
1 strain not included (not in the data base)
88.7 83.7 77.6 67.3 90
2.2 Compatibility with the ESBL confirmation disk diffusion methods With the ESBL confirmation disk diffusion methods, using CA-SFM and CLSI recommendations, the ESBL phenotype is confirmed for the 10 strains tested after isolation on chromID ESBL.
Table III: MIC agreements (1 dilution) between

VITEK GNS 506 Cefotaxime Other drugs
1 strain not included (not in the data base)
chromID ESBL E. coli ESBL +
CA-SFM E. coli ESBL +
CLSI
71.4 90
VITEK 2 AST-N017 + AST-N041 All drugs 90
CONCLUSIONS
According to the study results, the new chromID ESBL is fully compatible with bioMrieux products for identification and susceptibility testing. The Oxidase Reagent and the ESBL confirmation disk diffusion methods performed after isolation on chromID ESBL show an optimal compatibility.
Table IV: Distribution of the category deviation

GNS 506 Cefotaxime Nitrofurantoin SI IR SR RI IS RS 4 4 1 3 3 1 2 3 2 4 AST-N017 Amoxicillin / Clavulanic ac. 2 4 5 -
REFERENCE 1 Novel chromogenic medium from bioMrieux for detection of ESBL-producing Enterobacteriaceae. Poster #D-0457 ICAAC September 27-30, 2006 San Francisco USA.
80 70 60 50 40 30 20 10 0
-4
-3
-2
-1 0 1 Dilution range
KEY POINTS

chromID ESBL is fully compatible with all VITEK technology reagents: ID and AST. chromID ESBL is fully compatible with the Oxidase Reagent and the ESBL confirmation disk diffusion methods.
35
chromID VRE
chromID ESBL
Figure 2: Influence of chromID ESBL as a pre-culture medium on Cefotaxime MIC determination on VITEK GNS 506
chromID MRSA

Evaluation of chromID ESBL medium for the detection of extended-spectrum beta-lactamase (ESBL) producing enterobacteria
A . Cady1, A. Carrer2, H. Rglier-Poupet1, N. Fortineau2, J.M. Adam1, P. Nordmann2, C. Poyart1.
1
Service de Bactriologie, CHU Cochin ; 2Service de Bactriologie, CHU Bictre, France
REVISED ABSTRACT:
The purpose of this study was to evaluate the performances of chromID ESBL medium (bioMrieux) for the presumptive identification of ESBL-producing enterobacteria by comparing it to ESBL medium (biplate Drigalski+cefotaxime and Mac Conkey+ ceftazidime; AES). The manufacturers recommendations stipulate that all the bacteria that grow on AES ESBL medium and all those that are stained on chromID ESBL medium are presumed to produce ESBL. 765 samples (rectal swabs, bronchial secretions and urine) from patients at a risk of carrying ESBL in two university hospitals in the Paris region were inoculated on these media. After 24 hrs and 48 hrs of incubation at 37C, bacterial growth and chromID ESBL coloration were detected by means of synergy tests and additional tests if required (cloxacillin agar). Out of 765 samples, 32 were positive with at least one ESBL-producing GNB. The ESBL-producing enterobacteria detection sensitivities at 24 hrs and 48 hrs are 85.3% and 91.2% , respectively, for chromID ESBL medium, as opposed to 85.3% for 85.3% for the AES ESBL medium. The 4 false negatives on chromID ESBL at 24 hrs correspond to colorless colonies: 2 Escherichia coli, 1 Citrobacter freundii and 1 Proteus mirabilis, an oxidase test makes it possible to continue to screen for ESBL. The positive predictive values at 24 hrs and 48 hrs are 38.7% and 28.4 %, respectively, for chromID ESBL medium and 15.85% and 11.65% for the AES ESBL medium. Cephalosporinasehyperproducing enterobacteria, chromosomal BL-hyperproducing Klebsiella oxytoca and Pseudomonas aeruginosa produced colored colonies on chromID ESBL. At 24 hrs and 48 hrs, the specificity of chromID ESBL is significantly greater than that for the AES ESBL medium. It is increased if an oxidase test is used to eliminate some P. aeruginosa which appear with a brown or green color on chromID ESBL. In conclusion, chromID ESBL medium is the first chromogenic medium enabling the simple and rapid detection of patients carrying ESBL.
Figure 1: Appearance of colonies on chromID ESBL agar a. E. coli; b. K. pneumoniae
MATERIALS AND METHOD

From May to July 2006, 765 samples (urine, respiratory secretions, rectal swabs) from 547 patients were inoculated on bioMrieux chromID ESBL and AES ESBL. These patients, at a risk of carrying ESBL E, were hospitalized in the intensive care, geriatric and surgery departments of 2 university hospitals in the Paris region (Hpital Bictre and Groupe Hospitalier Cochin-Saint-Vincent de Paul). 50ml of the urine and respiratory samples were inoculated in a quadrant on each of the media. The rectal swabs were homogenized in 1 ml of sterile physiological saline solution. Ten microliters of the suspension obtained was then inoculated on the agars under test. After 24 hrs and 48 hrs of incubation at 37C, the growth intensity and color of the bacterial colonies was determined. On chromID ESBL medium, the colonies presumed to produce ESBL had the following colors: pink or burgundy for E. coli (Figure 1a), blue or green for Klebsiella, Enterobacter, Serratia and Citrobacter spp. (Figure 1b), brown or chestnut brown for Proteus, Providencia and Morganella spp. (Figure 1c). All the bacteria growing on either medium were identified independently using the VITEK2 system or an API 20E strip and, for all the enterobacteria, the presence of ESBL was tested by means of synergy tests and additional tests if required (25 mg/l cloxacillin agar). For the AES medium, the microorganisms isolated on Mac Conkey ceftazidime and/or Drigalski cefotaxime were identified. The manufacturers recommendations stipulate that, all the bacteria growing on AES ESBL medium and all those colored on chromID ESBL medium are presumed to produce ESBL. Figure 2: Breakdown of the 33 ESBL-producing bacteria
9.1 % 6.1 % 3.0 % 6.1 % 3.0 % 48.5 %
INTRODUCTION
Multi-resistant bacteria, particularly extended-spectrum beta-lacatamaseproducing enterobacteria (ESBL E) are frequently the source of potentially highly epidemic hospital-acquired infections. In order to limit their dissemination, early screening of patients carrying these ESBL E is required, to be able to set up suitable hygiene measures rapidly. bioMrieuxs chromID ESBL agar, the first chromogenic medium enabling presumptive identification of ESBL E according to the color of the colonies, was developed specifically for this purpose. The purpose of this study was to evaluate the performances of chromID ESBL medium (detection sensitivity and specificity with respect to yeasts and bacteria other than ESBL E), by comparing them to those of AES ESBL medium (biplate Mac Conkey + ceftazidime and Drigalski + cefotaxime).
24.2 %
Escherichia coli Klebsiella pneumoniae Enterobacter cloacae Enterobacter aerogenes Citrobacter freundii Citrobacter koseri Proteus mirabilis
RESULTS
Of the 765 samples analyzed, 32 were positive for ESBL E including two different ESBL E bacteria. The breakdown of the ESBL E bacterial species is given in figure 2. They mainly consist of E. coli (n=16, 48.5%), Klebsiella pneumoniae (n=8; 24.2%) and enterobacteria from the Enterobacter (n=3; 9.1%), Citrobacter (n=5; 15.2%) and Proteus (n=1; 3%) genera.
36
Table 1: TP, FP, FN, Sensitivity, PPV , NPV

Incubation time 24 H 48 H Medium chromID ESBL ESBL AES chromID ESBL ESBL AES Number of microorganisms True False False positives positives negatives 29 28 31 28 46 154 78 220 4 5 2 5 Sensitivity 87.88% 84.85% 93.94% 84.85% Positive predictive value 38.7% 15.4% 28.4% 11.3% Number of samples True False negatives negatives 692 605 663 552 3 5 1 5 Negative predictive value 99.57% 99.18% 99.85% 99.1%
DETECTION SENSITIVITY
At 24 hrs, chromID ESBL medium shows a better detection sensitivity (accounting for the ESBL bacteria, irrespective of the suspect color on chromID ESBL) than the AES medium (87.9% as opposed to 84.8%). The 4 false negatives on chromID ESBL are two E. coli and one P. mirabilis (colorless) and one iridescent C. freundii (Figure 3), an oxidase test makes it possible to continue to screen for ESBL. On the other hand, the 5 false negatives on the AES medium correspond to bacteria that did not grow on the medium. An improvement in the sensitivity is observed at 48 hrs for chromID ESBL medium only: 93.9% (one E. coli and the C. freundii displayed the expected color) (Table 1). Figure 3: False negatives on chromID ESBL a. colorless E. coli; b. iridescent C. freundii
Figure 4: Breakdown of strains resulting in unnecessary confirmations

6 8 1
On chromID ESBL medium: n=46
18
13
Pseudomonas spp. Enterobacter spp. Escherichia coli Morganella morganii Klebsiella spp Hafnia alvei Acinetobacter spp. Stenotrophomonas maltophilia Enterococcus faecalis Serratia marcescens Others On AES ESBL medium: n=154
8 8 10
3 2
5 73
POSITIVE PREDICTIVE VALUE

The detection specificities, evaluated using the calculation of the positive predictive values (PPV) for the chromID and AES ESBL media are 38.7% and 15.4% at 24 hrs, respectively (p<0.05). Forty-six strains on chromID ESBL correspond to non-ESBL-producing microorganisms and grow on the medium with a suspect color and result in a number of unnecessary ESBL confirmations. The breakdown is shown in figure 4. They essentially consist of Pseudomonas aeruginosa (brown or green-colored colonies) and enterobacteria hyperproducing their natural cephalosporinase (Enterobacter spp, E. coli, H. alvei) or penicillinase (K. oxytoca). On AES, there are three times more unnecessary confirmations (n=154) essentially represented by P. aeruginosa.
13 24
SELECTIVITY
For each medium at 24 hrs and 48 hrs, the growth intensity of all the microorganisms other than ESBL E, representing the ancillary flora, was evaluated. The study was performed on the 733 samples not positive for ESBL E. At 24 hrs, 88.67% of the samples inoculated on chromID ESBL are sterile as opposed to 82.5% on AES medium. When an ancillary flora is observed, i.e. on 139 samples, its growth intensity is less on chromID ESBL than on the AES medium in 47.5% of cases.
CONCLUSIONS
chromID ESBL displays a satisfactory sensitivity of 87.9% at 24 hrs and its specificity is significantly superior to that of the AES ESBL medium. The existence of non-colored ESBL E strains and the presence of colored P. aeruginosa strains could suggest the implementation of an oxidase test in order to increase the efficiency and the performance of the medium. In conclusion, chromID ESBL medium is easy to use and is the first chromogenic medium chromID, a simple, rapid and reliable detection of patients carrying ESBL E. allows the early implementation of hygiene and isolation measures.
NEGATIVE PREDICTIVE VALUE

The negative predictive value (NPV) of chromID ESBL medium is equally good as that of the AES medium. In fact, the absence of colored colonies on this medium corresponds, in 99.57% of cases, to the absence of ESBL in the sample. The NPV is improved after 48 hrs of incubation (99.85%).
KEY POINTS

Reduction of unnecessary confirmations. Ease-of-use due to chromogenic detection of strains. Better recovery of ESBL strains.
37
chromID VRE
chromID ESBL
chromID MRSA

Novel chromogenic medium from bioMrieux for detection of ESBL-producing Enterobacteriaceae

Y. Glupczynski1, C. Berhin1, C. Bauraing1, P. Bogaerts1
1
Laboratoire de bactriologie, Cliniques Universitaires U.C.L. Mont-Godinne, 5530 Yvoir, Belgium
ABSTRACT
Background: Rapid detection of extended-spectrum -lactamase producing Enterobacteriaceae (ESBLE) from surveillance cultures is crucial in order to control spread of ESBLs. We assessed a new chromogenic medium prototype (ESBL-Bx; bioMrieux, La Balme-les-Grottes, France) that enables isolation and presumptive identification of ESBLE directly from fecal or other samples. Methods: During 10 months, 460 patients hospitalized for >48 h were evaluated for ESBLE carriage. A total of 644 samples (561 fecal, 63 lower respiratory tract, 20 others) were plated on ESBL-Bx and on MacConkey agar supplemented with ceftazidime (2mg/L) (MCKC). All colonies growing on ESBL-Bx and on MCKC, after 18-24 h, were identified and tested for susceptibility by the VITEK2 (bioMrieux). ESBL confirmation was performed by combination double disks (ceftazidime and cefotaxime with or without clavulanic acid; Oxoid). Characterization of resistance mechanisms was determined by IEF and PCR of TEM, SHV, CTX-M, OXA, and AmpC genes with amplicon sequencing. Results: A total of 135 Enterobacteriaceae isolates were recovered from 112 specimens (17.4%) on all media; of these, 37 (33.0%) samples had ESBLE (44 isolates) and 77 grew non-ESBLE (91 isolates). On ESBL-Bx, 36 (97.3%) clinical samples containing at least one ESBLE were recovered, whereas recovery on MCKC was 32 (86.5%).; By organisms, 43/44 (97.7%) ESBLE were found on ESBL-Bx versus 37/44 (84.1%) on MCKC. ESBLE detected were: E. coli (n=17); E. aerogenes (n=17), Klebsiella spp. (n=5), and C. freundii (n=5). On both media, natural AmpC hyperproducing Enterobacter and Citrobacter spp. (n=39) were false positive as well as non-ESBL producing K. oxytoca (n=18) on ESBL-Bx and M. morganii (n=10) on MCKC. Conclusion: ESBL-Bx showed superior sensitivity to MCKC. The chromogenic and selective characters of ESBL-Bx enhanced recovery and identification of most ESBLE within 24 h and reduced unnecessary confirmations.
identify ESBL-producing Gram-negative bacilli from contaminated samples more easily and reliably, selective media should achieve identification and detection of ESBL-producing organisms in one single step. The purpose of this study was to evaluate the sensitivity and specificity of a novel prototype of selective chromogenic agar medium (ESBL-Bx) that enables isolation and presumptive identification of ESBL-producing Enterobacteriaceae (ESBLE) directly from fecal samples and other clinical specimens.

Clinical samples from patients hospitalized in general medicine, surgery, ICU and hematology wards for >48 h were obtained for ESBL screening. Each specimen was homogenized in 1 ml of sterile saline and 50 l aliquots were plated on a selective ESBL chromogenic medium (ESBL-Bx; bioMrieux, La Balme-les-Grottes, France) and onto Mac Conkey agar (Oxoid, Basingstoke, UK) supplemented with 2 mg/L ceftazidime (MCKC). Media were incubated aerobically at 37C. After 18-24 h incubation, plates were scored as either positive or negative for growth and coloration of colonies on ESBL-Bx was recorded. Density of growth was scored semi-quantitatively (<10 colonies; +1; +2; +3; +4 according to the number of quadrants of the agar plates on which growth was observed). All colonies growing on ESBL-Bx and on MCKC after 18-24 h were subcultured onto Columbia blood agar and were identified and tested for susceptibility by VITEK2 (GN cards and AST-N020 cards; bioMrieux). Confirmation of ESBL was performed by combined double disks (ceftazidime and cefotaxime alone and with clavulanic acid) according to CLSI guidelines. Genotypic characterization of resistance mechanisms was determined by IEF and PCR of TEM, SHV, CTX-M and AMPC, genes with amplicon sequencing. The gold standard for ESBL was defined by combined positive results of phenotypic and genotypic tests.
RESULTS
A total of 644 clinical specimens (561 stools, 63 sputum and bronchial aspirates, 20 miscellaneous) from 460 patients were plated in parallel on ESBL-Bx and MCKC. Four hundred ninety-six of these yielded no growth while bacterial growth on one or both media was recorded for 148 specimens. Overall, 135 Enterobacteriaceae isolates were recovered from 112 specimens (17.4%) on all media (Table 1); of these, 37 (33.0%) samples grew 44 ESBLE isolates (Table 2) and 75 grew 91 non-ESBLE isolates (Table 3). On ESBL-Bx, 36 clinical samples (sensitivity: 97.3%) containing at least one ESBLE were recovered, whereas recovery on MCKC was 32/37 (sensitivity 86.5%) (p=0.22); by organisms, 43 ESBLE (97.7%; [IC95% 87.9% - 99.6%]) were found on ESBL-Bx Vs. 37 (84.1%; [IC95% 70.3% - 92.2%]) ) on MCKC (ns). Fifty eight specimens grew 68 non-ESBL producing Enterobacteriaceae isolates on ESBL-Bx (specificity by specimens: 90.4% [549/607]) and 50 samples grew 57 non-ESBL producing Enterobacteriaceae on MCKC (specificity by specimens:91.8% [557/607]).
INTRODUCTION
Extended-spectrum -lactamase (ESBL)-producing Enterobacteriaceae have emerged worldwide as nosocomial pathogens of major importance (1). The recent emergence and spread of novel types of community-acquired ESBLs poses an additional challenge for clinical microbiological laboratories to improve their detection (2,3). Several phenotypic tests have been recommended for screening and confirmation of ESBLs but these are usually performed on isolated organisms following culture and antibiotic susceptibility testing (4, 5). Laboratory-based detection of patients infected or colonized with ESBLproducing organisms by surveillance cultures has proven useful to control nosocomial outbreaks (1, 6, 7). Methods to detect ESBL in clinical samples should have high sensitivity and specificity combined with a short time to reporting of results. To
38
On both media, natural AmpC hyperproducing Enterobacter spp. and Citrobacter spp. (n=39) were false-positive as well as K-OXY K. oxytoca hyperproducers (n=18) on ESBL-Bx and M. morganii (n=10) on MCKC (Table 3). Colourless colonies mainly corresponding to non-fermenters were identified in 35 specimens (41 microorganisms in 33 specimens on ESBL-Bx and 30 microorganisms in 26 specimens on MCKC) (Table 3). Table 1: Number and types/groups of bacerial isolates recovered from 644 clinical samples on two different selective media.
Groups/species Escherichia coli KES group
a
detected on the two selective media

Chromogenic ESBL medium (ESBL-Bx) 11 0 18 10 14 9 1 2 2 1 43 MAcConkey + Any ceftazidime medium (MCKC) 12 1 2 5 12 12 0 10 0 1 37 17 1 20 10 15 13 1 10 2 2 44
Groups/species Escherichia coli KES groupa K. pneumoniae K. oxytoca E. aerogenes E. cloacae Citrobacter spp. C. freundii C. koseri PMP groupb M. morganii P. vulgaris Other H. alvei Total N of Non-ESBL Enterobacteriaceae
a b
Chromogenic ESBL medium (ESBL-Bx) 28 64 15 4 111 40 1
MAcConkey + Any ceftazidime medium (MCKC) 24 42 17 10 96 30 0 34 68 19 14 135 44 1
Citrobacter spp. PMP groupb Total N Enterobacteriaceae Non-fermentersc Othersd

a
Stool sample (E. coli, K. pneumoniae)
Rectal swab (E. coli, K. pneumoniae)
CONCLUSION
The ESBL-Bx prototype agar showed superior sensitivity to the MacConkeyceftazidime (MCKC) which represents the conventional method. The chromogenic and selective characters of ESBL-Bx enhanced recovery and identification of most ESBL-producing Enterobacteriaceae within 18 to 24 h and reduced unnecessary confirmations. Further studies with a wider range of clinical samples are required to confirm the value and clinical usefulness of the ESBL-Bx selective chromogenic medium taking into account the possible occurrence of wide variations in the epidemiology of ESBL between countries.
Fig 1a & 1b. Growth of ESBL-producing E. coli (pink/bordeaux-like colonies) and K. pneumoniae (green colonies) isolates from stool (Fig 1a) and from rectal swab (Fig1b) after overnight culture on chromogenic ESBL-Bx medium. Table 2: Total number of ESBL-positive Enterobacteriaceae strains detected on the two selective media
Groups/species Escherichia coli KES groupa K. pneumoniae K. oxytoca E. aerogenes Citrobacter spp. C. freundii Total N ESBL
a
Chromogenic ESBL medium (ESBL-Bx) 17 3 2 16 5 43
MAcConkey + Any ceftazidime medium (MCKC) 13 3 2 14 5 37 17 4 2 17 5 44
REFERENCES 1. Paterson DL, Bonomo RA. Extended-spectrum -lactamases: a clinical update. Clin. Microbiol. Rev. 2005; 18: 657-686. 2. Ben-Ami R, Schwaber MJ, Navon-Venezia S, et al. Influx of extended-spectrum -lactamaseproducing Enterobacteriaceae into the hospital. Clin. Infect. Dis. 2006; 42: 325-34. 3. Livermore DM, Hawkey PM. CTX-M: the changing face of ESBLs in the UK. J. Antimicrob. Chmother. 2005; 56: 451-4. 4. Pfaller MA. Segreti J. Overview of the epidemiological profile and laboratory detection of extended-spectrum -lactamases. Clin. Infect. Dis. 2006; 42 (Suppl 4): S153-S163. 5. Performance standards for antimicrobial susceptibility testing; Fifteenth informational supplement. Clinical and Laboratory Standards Institute (CLSI); 2005: Vol 25 N1 (M100S15), Wayne, PA, USA. 6. Lucet JC,, Decre D, Fichelle A et al. Control of a prolonged outbreak of extended-spectrum beta-lactamase-producing Enterobacteriaceae in a universiy hospital. Clin. Infect. Dis. 1999; 29: 1411-8.
Klebsiella - Enterobacter - Serratia group
Table 3: Total number of non ESBL-producing Enterobacteriaceae
KEY POINTS

Increased sensitivity in comparison with conventional methods. Reduction in unnecessary confirmations.

39
chromID VRE
chromID ESBL
chromID MRSA
Klebsiella - Enterobacter - Serratia group (also includes Hafnia alvei) b Proteus - Morganella - Providencia group c Non-fermenters: P. aeruginosa, Stenotrophomonas maltophilia, Acinetobacter spp. d Others: Enterococcus faecium (VRE): 1
Klebsiella - Enterobacter - Serratia group Proteus - Morganella - Providencia group
chromID VRE
Characteristic colouration of colonies with: - Bluish-green colour = E. faecalis - Violet colour = E. faecium
chromID VRE agar is a completely new and innovative media designed for the screening of Vancomycin-Resistant enterococci. It enables the direct and definitive identification of E. faecium and E. faecalis showing acquired Vancomycin-Resistance (JCM Publication, May 2007). chromID VRE (patents pending) contains two chromogenic substrates - Poster ICAAC 2006 (alpha-Glucosidase & beta-Galactosidase) and vancomycin (8mg/l) which enable: Specific and selective isolation & detection of acquired Vancomycin-Resistant enterococci. Direct identification (see protocol) of E. faecium and E. faecalis after 24 hour incubation
The selective mixture inhibits: - enterococci with natural resistance (ie. E. casseliflavus & E. gallinarum) (JCM Publication, May 2007) - most Gram-negative and Gram-positive bacteria, as well as yeasts. chromID VRE agar is fully compatible with all VITEK technology reagents: identification and Antiobiotic Susceptibility Testing (Poster ASM 2007 C 137). Greater Simplicity Ready-to-use medium. Specific chromogenic media for the screening of VRE Immediate differentiation of E. faecium and E. faecalis Culture + isolation + identification on the same medium Greater Reliability - A Complete Solution Excellent performance for the screening of VRE in terms of nutrient capacity and detection sensitivity Optimised selectivity for true specificity
Contents
ARTICLES
Evaluation of a Novel Chromogenic Agar Medium for Isolation and Differentiation
of Vancomycin-Resistant Enterococcus faecium and Enterococcus faecalis Isolates
JOURNAL OF CLINICAL MICROBIOLOGY, May 2007 N. A. Ledeboer et al.
43
POSTERS / ORAL PRESENTATIONS

ASM / Toronto (Canada) / May, 2007 Compatibility of chromID VRE, a new Chromogenic Medium, with Identification and Susceptibility Testing Reagents
44
N. Fanjat et al.
ECCMID / Munich (Germany) / April, 2007 A new chromogenic agar medium, chromID VRE, to screen for VancomycinResistant Enterococcus faecium and Enterococcus faecalis
44
N. A. Ledeboer et al.
ECCMID / Munich (Germany) / April, 2007 A novel chromogenic medium for Vancomycin-Resistant Enterococcus faecium and Enterococcus faecalis detection
47
Dr G. Cuzon et al.
RICAI / Paris (France) / December, 2006 chromID VRE, the first chromogenic medium for the detection of enterococci with acquired vancomycin resistance
49
J. Delmas et al.
ICAAC / San Francisco (USA) / September, 2006 Evaluation of a novel chromogenic agar medium from bioMrieux to screen for Vancomycin-Resistant Enterococcus faecium and faecalis
51
N. A. Ledeboer et al.
May, 2007
JOURNAL OF CLINICAL MICROBIOLOGY, May 2007, p. 15561560 Vol. 45, No. 5 0095-1137/07/$08.00 0 doi:10.1128/JCM.02116-06
Evaluation of a Novel Chromogenic Agar Medium for Isolation and Differentiation of Vancomycin-Resistant Enterococcus faecium and Enterococcus faecalis Isolates
Nathan A. Ledeboer,1Kingshuk Das,1Michael Eveland,2Cline Roger-Dalbert,3Sandrine Mailler, 3 Sonia Chatellier,3and William Michael Dunne1*
Department of Pathology and Immunology, Washington University School of Medicine,1 and Barnes-Jewish Hospital,2 St. Louis, Missouri, and Microbiology Research and Development, bioMerieux, La-Balme-les-Grottes, France3
Received 15 October 2006/Returned for modification 20 December 2006/Accepted 15 February 2007
The development of reliable and rapid methods for the identification of patients colonized with Vancomycin-Resistant enterococci (VRE) is central to the containment of this agent within a hospital environment. To this end, we evaluated a prototype chromogenic agar medium (VRE-BMX; bioMrieux, Marcy lEtoile, France) used to recover VRE from clinical specimens. This medium can also identify isolated colonies as either VancomycinResistant Enterococcus faecium or Enterococcus faecalis, based on distinct colony colors. We compared the performance of VRE-BMX with bile esculin azide agar supplemented with vancomycin (BEAV). For this study, 147 stool samples were plated on each test medium and examined after 24 and 48 h of incubation. At 24 h, the sensitivity and specificity of each medium were as follows: BEAV, 90.9% and 89.9%, respectively; VRE-BMX, 96.4% and 96.6%, respectively. The positive predictive values (PPV) of VRE-BMX and BEAV at 24 h were 89.8% and 80.7%, respectively. VRE-BMX provided the identification of 10 isolates of Vancomycin-Resistant E. faecalis and 4 isolates of Vancomycin-Resistant E. faecium that were not recovered by BEAV. Further, VRE-BMX was capable of identifying patients colonized with both E. faecium and E. faecalis, a feature useful for infection control purposes that is not a function of BEAV. In terms of the recovery of Vancomycin-Resistant E. faecium and E. faecalis, the sensitivity and PPV were as follows: BEAV, 75.7% and 74.6%, respectively; VRE-BMX, 95.5% and 91.3%, respectively. In this initial evaluation, we found that VRE-BMX provided improved recovery of VRE from stool specimens, with the added advantage of being able to differentiate between Vancomycin-Resistant E. faecalis and E. faecium. Extending the incubation period beyond 24 h did not significantly improve the recovery of VRE and resulted in decreased specificity.
KEY POINTS
chromID VRE is able to identify and differentiate VRE.faecium from VRE.faecalis, while inhibiting growth of Vancomycin-Susceptible Enterococcus spp. chromID VRE reliably identifies enterococci to species level and confirms glycopeptide resistance. Therefore, it is not necessary to pursue with additional tests for identification and susceptibility testing.
43
chromID VRE
chromID ESBL
chromID MRSA
ASM / May, 2007

Toronto (Canada) poster C137
Compatibility of chromIDTM VRE, a New Chromogenic Medium, with Identification and Susceptibility Testing Reagents
N. Fanjat1, N. Garcia1, D.H. Pincus2, G. Zambardi1, D. Robichon1
bioMrieux, La Balme Les Grottes, France, 2 bioMrieux, Hazelwood, MO, USA
ABSTRACT
Background: chromIDTM VRE is a new chromogenic medium dedicated for the screening of Vancomycin-Resistant Enterococcus (VRE) carriage. It enables the isolation of acquired VRE and the direct identification of Enterococcus faecalis and E. faecium. Compatibility studies of chromID VRE with bioMrieux identification and susceptibility products (API, ID32, ATB strips, VITEK and VITEK 2 cards) were performed. Results were compared with those obtained on the reference media. Methods: One hundred strains (VanA or VanB) were subcultured on both chromID VRE and the reference medium (Columbia sheep blood agar or Trypcase-soy agar sheep blood). Identification and susceptibility testing were performed according to the manufacturers recommendations. Results: When compared with the reference media, identification testing was not affected by isolation on chromID VRE. Correct identification rates were 100% for VITEK GPI and VITEK 2 GP, and 78% for API 20 STREP (with many misidentifications obtained). For rapid ID 32 STREP, correct identification was 76% (with a statistical difference noted). Concerning the ATB STREP strip, category agreements were superior to 90% for all the drugs validated for enterococci. Moreover, there was no trend to induce a higher susceptibility or resistance. With VITEK (GPS 514 susceptibility card), category agreements and MICs were superior or statistically equivalent to 90% for all drugs. The trend to higher susceptibility obtained from chromID VRE with ampicillin and benzylpenicillin was acceptable because false resistances were observed from the reference medium. With VITEK 2 (AST-P532 susceptibility card), no statistical difference between categorizations was observed, except for erythromycin for which chromID VRE favorably increased the resistance expression level. Except for erythromycin, there was no trend to induce higher resistance with the other drugs. Conclusion: According to the results obtained during this study, chromID VRE is compatible with bioMrieux identification and susceptibility products, except for rapid ID32 STREP. Moreover, API 20 Strep is not recommended.

Figure 1: Test scheme from chromID VRE and reference culture
chromID VRE Reference medium
18-24h at 37C
18-24h at 37C
100 Enterococcus strains Vanco R (Van A or Van B)
50 Enterococcus strains
Identification Testing - API 20 E - rapid ID 32 STREP - VITEK GPI - VITEK 2 GP
Susceptibility Testing ( CA-SFM breakpoints) - ATB STREP - VITEK GPS 514 - VITEK 2 AST-P532
mediaReference medium: Trypcase Soy Agar + 5% sheep blood (TSS) for VITEK GPI and GPS 514 Columbia Agar + 5% sheep blood (COS) for the other tests
RESULTS
Identification (table 1): chromID VRE had no negative influence on the identifications obtained with VITEK and VITEK 2 cards. The results showed an agreement rate of 88% for VITEK GPI and 94% for VITEK 2 GP. The correct identification rates on chromID VRE, 100% for both products, were equal or better than those obtained from the reference medium. chromID VRE decreased the performance of API and ID 32 identification strips, due to either unidentified or misidentified strains. With API 20 Strep, 10 E. faecium were misidentified as Leuconostoc spp., and 4 E. faecalis as E. avium after incubation for 4 h. With ID 32 STREP, 5 E. faecium were unidentified and 7 were in low discrimination with E. casseliflavus and E. gallinarum. The agreement rate and the correct identification rate (identifications to one choice or in low discrimination) were 78% for API 20 Strep and 76% for rapid ID 32 STREP. For the latter, there was a statistical difference with results obtained from the reference medium. Table 1: Influence of chromID VRE on identification results (%)
API 20 STREP chromID VRE 1 2 COS 1 70 3 2 1 3 4 4 VITEK GPI chromID VRE 1 2 COS 1 84 4 2 6 4 3 2 4
Identification key: 1 One choice 2 Low discrimination 3 Unidentified 4 Misidentified
4 10 2 2 6
rapid ID 32 STREP chromID VRE 1 2 3 COS 1 65 7 10 2 1 3 3 8 4 VITEK 2 GP chromID VRE 1 2 COS 1 94 2 2 4 3 4
INTRODUCTION
chromID VRE is a new chromogenic medium dedicated to the screening of VRE carriage. The detection of this resistance is important for the prevention, the epidemiology and the emergence of Vancomycin-Resistant enterococci and staphylococci. chromID VRE enables the isolation of acquired Vancomycin-Resistant enterococci and the direct identification of Enterococcus faecalis and E. faecium. The purpose of this study was to evaluate the compatibility of chromID VRE with bioMrieux identification and antimicrobial susceptibility products. Results are compared with those obtained from reference media.
=> Correct Identification
44
Susceptibility (Tables 2-4): Regarding the results obtained on the ATB STREP strip, a category agreement rate superior or statistically equivalent to 90% was observed for all drugs validated for enterococci. Moreover, there was no trend to induce a higher susceptibility or resistance. With VITEK GPS 514, category and essential (1 dilution) agreements were superior or statistically equivalent to 90% for all drugs. With fosfomycin, discrepancies were principally due to MICs close to the breakpoints. The trend to higher susceptibility obtained from chromID VRE with ampicillin and benzylpenicillin was acceptable because false resistances were observed on the reference medium based on consensus results. With VITEK 2 AST-P532, no statistical difference between categorizations was observed except for erythromycin for which chromID VRE favorably increased the inducible resistance expression (Figure 2). Concerning categorizations, no significant discrepancies were observed except for erythromycin for which the agreement rate was statistically inferior to 90%. Table 2: Essential agreements (%) of chromID VRE vs. reference medium
VITEK GPS 514 Ampicillin Other drugs 88 90 VITEK 2 AST-P532 Ceftazidime Other drugs 82 90
Figure 2: Influence of chromID VRE on erythromycin MICs (w/VITEK 2 AST-P532)

40 35 Number of strains 30 25 20 15 10 5 0 -3
-2
-1
0 1 Dilution range
CONCLUSIONS
According to the study results, the new chromID VRE is fully compatible with bioMrieux VITEK and VITEK 2 products for identification and with all bioMrieux products for susceptibility testing. On the other hand, chromID VRE is not recommended with API and ID identification reagents.
Table 3: Category agreements (%) of chromID VRE vs. reference medium

VITEK GPS 514 Benzylpenicillin Chloramphenicol Fosfomycin Teicoplanin Other drugs 86 88 82 88 90 VITEK 2 AST-P532 Erythromycin Nitrofurantoin Trimethoprim / Sulfam. Other drugs 76 88 88 90
Table 4: Distribution of the category deviation for discrepant tests

GPS 514 Benzylpenicillin SI IR SR RI IS RS 5 1 1 Fosfomycin 2 7 Teicoplanin 1 1 2 2 AST-P532 Erythromycin 2 7 3
KEY POINTS

chromID VRE is fully compatible with all VITEK technology reagents: ID and AST. chromID VRE is not recommended to be used with API and ID strips.
45
chromID VRE
chromID ESBL
chromID MRSA

Munich (Germany) oral presentation 0111
A new chromogenic agar medium, chromID VRE, to screen for Vancomycin-Resistant Enterococcus faecium and Enterococcus faecalis
Nathan A. Ledeboer, Robert J. Tibbetts, Wm. Michael Dunne
Department of Pathology and Immunology, Washington University School of Medicine, St Louis, Missouri, USA
BACKGROUND
Description of Genus - Gram-positive cocci - Vancomycin-Resistance VanA: High-level vancomycin and teicoplanin resistance VanB: Moderate to high-level resistance to vancomycin, no resistance to teicoplanin Transmission - Direct contact with infected patients - Health Care Workers Colonization - Organism initially colonizes the intestinal tract and can be carried asymptomatically Diagnosis - Specimens Stool, Rectal Swabs - Media Bile Esculin Azide vancomycin Agar - Biochemicals Automated Systems Antimicrobial susceptibility patterns Chromogenic Agar - Principle Chromogen is added to media that will react with specific enzyme complex in E. faecium and E. faecalis to color colonies - E. faecalis is blue / green - E. faecium is purple 8 g/mL vancomycin added to medium to select for vancomycin resistance - Released by bioMrieux worldwide, soon to be released in Canada and the United States
RESULTS
100 95 90 85 80 75 70 65 60 55 50 24
chromID VRE
Sensitivity of Media for VRE
100 90 80 70 60 50 40 30 20 10 0
Specificity of Media for VRE
% Sensitive
48
BEAV
% Sensitive
24
chromID VRE
48
BEAV
Length of incubation (in hours)
Growth of Contaminants
60
Number of isolates
50 40 30 20 10 0
FALSE POSITIVE ISOLATES
COLORLESS ISOLATES
24
48
Length of incubation (in hours) chromID VRE BEAV
24
48

Enrolled 120 stool specimens submitted for VRE screen Specimens standardized in saline and plated to BEAV and chromID VRE Plates read at 24 and 48 h ID and antimicrobial susceptibilities were performed typically colored colonies Confirmed results with PCR E. faecium E. faecalis
CONCLUSIONS
chromID VRE offers equivalent sensitivity to BEAV in isolating and identifying VRE in feces. - Incubation for 48 h does not significantly improve sensitivity of either chromID VRE or BEAV. Specificity of chromID VRE is significantly greater than BEAV in detecting E. faecalis and E. faecium. Growth of contaminants that may be interpreted as false positives is greater on BEAV versus chromID VRE. Molecular techniques enhance detection of many agents and decrease laboratory turn-around time. Molecular methods often add a level of cost and complexity that are not in line with the relevance of the targeted organism. Critically evaluate importance of diagnosing organism versus costs.
ACKNOWLEDGEMENTS Washington University: Wm. Michael Dunne - Robert J. Tibbetts All Studies were approved by Washington University Human Studies Committee
KEY POINTS

chromID VRE, the new state-of-the-art medium for the screening of Vancomycin-Resistant Enterococci. Chromogenic substrates added to the medium colour colonies: E. faecalis is blue/green and E. faecium is purple.
46

Munich (Germany) oral presentation 0104
A novel chromogenic medium for Vancomycin-Resistant Enterococcus faecium and Enterococcus faecalis detection
Dr G. Cuzon, Dr T. Naas, Dr N. Fortineau, Pr P. Nordmann
Hpital Bictre, South Paris Medical School, France
03/2006 12/2005 07/2006
Vancomycin-Resistant Enterococci (VRE): History 1956: Discovery of vancomycin (USA) 1964: First clinical use (USA) 1969: First in vitro Vancomycin-Resistant E. faecium strains. [Toala, Am J Med Sci 1969] 1988: First clinical isolates, Europe [Leclercq, NEJM 1988; Uttley, Lancet 1988 ] Then worldwide spread USA +++ South-East Asia (Korea +++) 2003: first case of vancomycin resistance transferred from VRE to MRSA [Chang, NEJM 2003] Epidemiology in Europe Proportion of Glycopeptide resistant Enterococci isolates in participating countries in 2005 (EARSS)
5 Clinical
01/2007
t
INTRODUCTION
2004
5 Screening 2006 10 March 2005 August 2005 15 February 2005 n = 123 patients 2007
08/2004
E. faecium
Nosocomial outbreak of VRE at Bictre Hospital, France (end 2004 - beginning 2007) vanA genotype but vanD phenotype Heterogeneous expression of glycopeptide resistance [Naas et al., JCM, 2005] TEC E KAN AMX VA RA SXT CM PT PIP STR GM IPM P OX CTX LVX LZD FOS FT C E. faecium Bct-1 TE OPT
No Data <1% 1 -5 % 5 - 10 % 10 - 25 % 25 - 50 % > 50 %
Clinical Impact Enterococci = intestinal flora 2 main species: E. faecium, E. faecalis Infections: mostly in immunocompromised patients Colonization: reservoir for transmission of VRE Consequences: increase in mortality, length of stay and cost of hospitalization [Edmond, CID, 1996; Webb, CID, 2001]. Genetic Support (acquired) vanA (+++) - Transposon- encoded - High level resistance to vancomycin and teicoplanin vanB (++) - Moderate to high level resistance to vancomycin - No resistance to teicoplanin vanD, vanE, vanG
Recommandations of the Hospital Infection Control Practice Advisory Commitee Screening: All hospitalized patients in infected wards At the admission Once a week Previously colonised or infected patients following their readmission
OBJECTIFS
Culture Selective media with vancomycin: Enterococcossel vancomycin Agar Medium Identification of isolates Strains for epidemiology Time consuming Labor intensive Molecular techniques Multiplex PCR Rapid Identification of isolates Detection of resistance genes Cost Living or dead bacteria?
Objectives Evaluation, over a 6-week period in routine settings, of a new chromogenic medium ChromID VRE (VRE ID) (bioMrieux, France) in comparison with Enterococcossel vancomycin Agar Medium (EVA) (Becton Dickinson).
47
chromID VRE
E. faecalis
Selection of a homogeneous expression of resistance with pre-enrichment in broth containing vancomycin
chromID ESBL
No Data <1% 1 -5 % 5 - 10 % 10 - 25 % 25 - 50 % > 50 %
chromID MRSA
A novel chromogenic medium for Vancomycin-Resistant Enterococcus faecium and Enterococcus faecalis detection
METHODS
Rectal swabs 491 rectal swabs (351 patients) Bile 40 % Vancomycin 3g/mL Colistin Amphotericin B
Overnight broth enrichment (24h)
Enterococcossel Agar Vancomycin Medium
37C, 48 h Aerobic atmosphere
chromID VRE
Negative
Colonies with typical aspect
Negative
STOP
STOP
Cocci Gram +, catalase Antimicrobial susceptibility testing (diffusion method), Vancomycin plus Teicoplanin MIC (E-Test), Identification by VITEK 2 and genotyping
RESULTS
168 specimens yielded growth 33 VRE isolates from 11 patients vanA genotype
chromID VRE E. faecium E. faecalis 30 2 EVA 29 2 Total 31 2
Sensitivity and specificity

Medium VRE ID EAV Sensitivity % 96.9 93.9 IC 95% 84.3-99.5 80.0-98.4 Specificity % 99.4 83.4 IC 95% 98.1-99.8 79.7- 86.6
Sensitivity: VRE ID = EVA Specificity: VRE ID > EVA
Positive and negative predictive values

PPV Medium VRE ID EAV TP 32 31 FP 3 78 TN 462 FN 1 % 91.4 IC 95% 77.3 - 97.1 20.9 - 38.1 % 99.8 99.5 NPV IC 95% 98.8 - 99.9 98.1 - 99.9
388 2 28.7 TP: true positive FP: false positive TN: true negative FN: false negative PPV: VRE ID > EAV NPV: VRE ID = EAV
Interfering flora chromID VRE - No characteristic strains Enterococcossel Agar vancomycin - Strains with typical aspect of VRE = false positive +++ naturally Vancomycin-Resistant Gram positives (Lactobacillus, E. gallinarum, E. casseliflavus)
CONCLUSION
chromID VRE: - Detection of VRE - Identification of E. faecium and E. faecalis Only colonies of interest are further studied Time and cost saving chromID VRE was capable of detecting a vanA genotype with a vanD phenotype (low level expression). 24 h broth enrichment was necessary using both subculture media for efficient VRE detection of the epidemic clone.
ACKNOWLEDGEMENTS Universit PARIS-SUD XI - Assistance Hpitaux Publique de Paris Dr. T. Naas Dr. N. Fortineau Pr. P. Nordmann
KEY POINTS
With chromID VRE, only colonies of interest needed to be further studied : time and cost saving. chromID VRE was capable of detecting a vanA genotype with a vanD phenotype (low level expression). 24-hr broth enrichment was necessary for using both subculture media for efficient VRE detection of the epidemic clone.
48

chromID VRE, the first chromogenic medium for the detection of enterococci with acquired Vancomycin-Resistance
J. Delmas, C. Schweitzer, F. Robin, S. Trinquet, R.
Laboratoire de Bactriologie clinique, CHU de Clermont-Ferrand, France
ABSTRACT
The screening of patients carrying vancomycin-resistant enterococci (VRE) is recommended in countries with high prevalence levels in order to prevent hospital-acquired VRE infections. The selective media used to date are based on bile esculin, such as the Becton Dickinson agar EnterococcosselTM (8mg/l vancomycin). chromID VRE agar (bioMrieux, France) is a new selective medium for detecting VRE in fecal and urine samples. This medium is original in that it makes it possible to identify Enterococcus faecium and Enterococcus faecalis strains on agar using chromogenic substrates. The purpose of this study was to compare the abilities of chromID VRE and EnterococcosselTM media to isolate VRE in fecal samples. Therefore, for two months, we tested over 1000 samples from patients hospitalized at the Clermont-Ferrand University Hospital. The study was performed with readings of the selective media at 24 and 48 hours of incubation with or without prior enrichment. The results demonstrate that the sensitivity of chromID VRE chromogenic medium is equivalent to that of the bile esculin medium, while its selectivity was higher. In particular, it makes it possible to eliminate the naturally Vancomycin-Resistant E. gallinarum and E. casseliflavus enterococcal species, and Gram-positive bacteria. In conclusion, chromID VRE medium is a chromogenic medium that is easy to use for the rapid and selective isolation of VRE in fecal samples, making it possible to set up hygiene and isolation measures.
RESULTS
Sensitivity chromID VRE versus BD: No significant difference in sensitivity between the 2 media. 22 Vancomycin-Resistant Enterococcus faecium strains (> 256 mg/l, vanA) were isolated on these media (see table 1). Enrichment versus non-enrichment: Increase in sensitivity of 45.5% for chromID VRE and 59.1% for BD after enrichment (p < 0.05). Incubation for 24 hrs versus 48 hrs: No significant difference in sensitivity between the 2 media. Figure 1: Appearance of species most frequently isolated on chromID VRE medium
INTRODUCTION
The frequency of the isolation of Vancomycin-Resistant enterococci (VRE) in hospital-acquired infections has increased in France since 2004 (1, 2). Therefore, they need to be monitored. The screening strategies for patients carrying VRE are based on the use of medium containing vancomycin (3). However, there is no consensus on the medium or method to use. The purpose of this study was to compare the performances of the new selective VRE ID medium (bioMrieux) to EnterococcosselTM agar (Becton Dickinson).
E. faecium
E. faecalis False positives
MATERIALS AND METHODS

Samples: Over a 2-month period (July and August), 1007 fecal samples (861 swabs and 146 stools) were collected from patients hospitalized at the Clermont-Ferrand university hospital. Inoculation media: Two media, containing 8 mg/l vancomycin, were use to isolate VRE: - chromID VRE agar: a chromogenic medium intended to detect and identify E. faecium and E. faecalis enterococci with acquired Vancomycin-Resistance, - EnterococcoselTM agar (Becton Dickinson): a selective Bile-esculin medium (BD) intended to detect E. faecium and E. faecalis enterococci with acquired Vancomycin-Resistance. Brain-Heart Infusion Broth supplemented with 3 mg/l vancomycin (BHIBV) was used as the enrichment medium. Screening method: the swabs were discharged in 700 l of physiological saline solution; the stool samples were suspended in 10 ml of physiological saline solution. - Direct inoculation of 50 l of suspension on the chromID VRE, BD and BHIBV media.
Gram-negative bacilli
Yeasts
Positive and negative predictive values chromID VRE versus BD: No significant difference between the 2 media for the PPV and NPV (at 24 hrs, 48 hrs, with or without enrichment). See table 1. Enrichment versus non-enrichment with reading of selective media at 24 hrs: - No significant difference for PPV, - However, the NPV for both media is increased significantly by enrichment. Incubation for 24 hrs versus 48 hrs: - The PPV of both media is decreased after 48 hrs of incubation. - No significant difference for the NPV for both media.
49
chromID VRE
chromID ESBL
chromID MRSA
- After 24 hrs of enrichment, inoculation of 50 l of BHIBV on the two chromID VRE and BD agars. All the cultures were incubated at 37C under aerobic conditions. The colonies were examined at 24 hrs and subsequently at 48 hrs. Identification and detection of glycopeptide resistance: The characteristic colonies were blue to green-blue in color for E. faecalis, and purple for E. faecium on chromID VRE; they were dark brown to black on BD. - These colonies were analyzed by means of Gram staining. The identification and antibiotic susceptibility testing were then performed on VITEK 2 (bioMrieux) using CPS ID3 agar (bioMrieux). - The confirmation of the identification of the enterococci and the typing of the van gene responsible for vancomycin resistance were performed with the GenoType Enterococcus test (Biocentric, Bandol, France) Statistical study: This was based on the MacNemar test. A value of p< 0.05 was considered to be significant.
chromID VRE, the first chromogenic medium for the detection of enterococci with acquired Vancomycin-Resistance
Table 1: Results in terms of specificity, sensitivity, positive and negative predictive value
24h with enrich w/o enrich 48h wiyh enrich w/o enrich VRE BD VRE BD VRE BD VRE BD TP 22 22 12 9 22 22 12 13 FP 48 89 17 25 108 137 147 116 TN 937 896 968 960 877 848 838 869 FN 0 0 10 13 0 0 10 9 Sen % 100 100 54.55 40.91 100 100 54.55 59.09 Sp % 95.13 90.96 98.27 97.46 89.04 86.09 85.08 88.22 PPV 31.43 19.82 41.38 26.47 16.92 13.84 7.55 10.08 NPV 100.00 100.00 98.98 98.66 100.00 100.00 98.82 98.97
TP: true positives, FP: false positives, TN: true negatives, FN: false negatives, Sen: sensitivity, Sp: specificity, PPV: positive predictive value, NPV: negative predictive value
Specificity However, the specificity of chromID VRE medium was higher than BD agar after enrichment (p < 0.05). Enrichment versus non-enrichment: Enrichment decreased the specificity of both media significantly at 24 hrs. Incubation for 24 hrs versus 48 hrs: incubation for 48 hrs decreased the specificity of both media significantly. False-positive enterococci (see figures 1, 2): - E. gallinarum and E. casseliflavus enterococci: 0 % on chromID VRE and 5.7 % on BD.
- Vancomycin-Susceptible enterococci: at 24 hrs of incubation, 0 % irrespective of the medium; at 48 hrs: 2. % on chromID VRE and 0.6 % on BD. Other false positives: Gram-negative bacilli and yeasts on chromID VRE and Gram-positive bacteria on BD (see figure 2). Specificity at 24 hrs, after Gram staining: 100 % for chromID VRE; 17.24 % for BD. Gram staining made it possible to eliminate almost all the false positives for chromID VRE medium. In this way, numerous spiking and identification tests are avoided with chromID VRE medium.
Figure 2: Breakdown of false positives isolated on chromID VRE and Bile esculin (BD) agars and role of Gram in VRE isolation
180 160 140 120 100 80 60 40 20 0 chromID VRE BD chromID BD VRE 24hrs 48hrs without enrichissement chromID VRE BD chromID BD VRE 24hrs 48hrs with enrichissement chromID VRE BD chromID BD VRE 24hrs 48hrs without enrichissement chromID VRE BD chromID VRE 24hrs 48hrs with enrichissement BD
Number of false positives (without Gram) yeasts Vancomycin-Susceptible enterococci Uncertain Gram bacilli Gram-negative bacilli "Van C enterococci
Number of false positives remaining after Gram reading Gram-positive bacilli Other Gram-positive cocci
CONCLUSION
chromID VRE versus BD: The sensitivity of chromID VRE agar is the same as that of BD agar. However, false positives such as lactobacilli, Pediococci, E. gallinarum and E. casseliflavus enterococci are not detected on chromID VRE medium. In addition, the chromogenic staining of chromID VRE medium combined with Gram staining identifies E. faecium and E. faecalis species. Due to these characteristics, chromID VRE medium is very specific and easy to use. Enrichment versus non-enrichment: Enrichment improves the sensitivity of both media considerably, but it decreases their specificity.
However, Gram staining makes it possible to obtain the same specificity with chromID VRE medium with or without enrichment. Incubation for 24 hrs versus 48 hrs: incubating the agars for 48 hrs did not increase the sensitivity of both media and decreased their specificity. Therefore, chromID VRE agar would appear to be an effective medium for the rapid and selective isolation and identification of chromID VRE Van A phenotype strains, which are currently the most frequent. The performance of this medium may be improved considerably through the use of an enrichment medium and Gram staining on positive cultures.
KEY POINTS
Sensitivity of both media tested is similar. The chromID VRE medium combined with Gram staining identifies E. faecium and E. faecalis species. False positives (lactobacilli, Pediococci, E. gallinarum and E. casseliflavus) are not detected on chromID VRE. chromID VRE medium is very specific and easy-to-use.
50

San Francisco (USA) poster D-819
Evaluation of a novel chromogenic agar medium from bioMrieux to screen for Vancomycin-Resistant Enterococcus faecium and faecalis
Nathan A. Ledeboer1, Kingshuk Das1, Michael Eveland2, and Wm. Michael Dunne1
1
Department of Pathology and Immunology, Washington University School of Medicine and 2Barnes-Jewish Hospital, Saint Louis, Missouri, USA
ABSTRACT
Background: The development of reliable and rapid methods for the identification of patients colonized with Vancomycin-Resistant enterococci (VRE) is central to the prevention of person-to-person transmission of this agent within the hospital environment. To this end, we evaluated a prototype chromogenic agar medium VRE-BMX designed by bioMrieux (La Balme les Grottes, FR.) vs. our conventional method (Bile esculin azide agar with vancomycin; BEAV) to recover VRE from clinical specimens and identify the isolated colonies as either Enterococcus faecium (VREfm) or E. faecalis (VREfs) based on different colony color. Results: At 24 hours, the sensitivity and specificity were: BEAV: 90.9%, 84.8%; VRE-BMX: 96.4%, 93.5%. The positive predictive values for identification of positive samples by the chromogenic medium and BEAV at 24 hours were: VRE-BMX 89.8%, BEAV 78.1%. In addition, VRE-BMX identified 10 isolates of VREfs and 4 isolates of VREfm that were not recovered using BEAV. Furthermore, the VRE-BMX was capable of identifying patients colonized with both VREfm and VREfs - a feature useful for epidemiology follow-up that is not available with BEAV. So if we focus on both VREfm and VREfs isolated, the sensitivity and PPV values were: BEAV 75.7%, 74.6% and VRE-BMX 95.5% and 91.3%. Conclusions: We conclude that the prototype formulation of chromogenic agar provides improved recovery of VRE from stool specimens and the added advantage of differentiation between VREfs and VREfm with an increase of specificity towards enterococci intrinsically resistant to vancomycin. Extended incubation beyond 24 hours did not significantly improve recovery of VRE and resulted in a decreased specificity.
The clinical impact of VRE has been examined in several studies, with the most notable consequences being increased mortality, increased lengths of hospital stay, as well as increased costs of hospitalization associated with VRE infection. For example, one retrospective case-control study demonstrated a 6% increase in mortality, and an average of 6-day increased length of hospitalization in patients infected or colonized with VRE, in comparison to controls without documented VRE involvement. Recognizing the impact of VRE, the Hospital Infection Control Practices Advisory Committee (HICPAC) of the Centers for Disease Control and Prevention (CDC) in 1995 created recommendations to curb the epidemic of VRE infection in hospital settings. In addition to addressing healthcare workers as vectors for transmission, and recommending judicious use of antibiotics such as vancomycin, HICPAC recommended protocols for the early detection of VRE and precautionary isolation of involved patients. This poster describes the use of a culture-based VRE detection method that allows rapid detection not allowed by conventional culture methods, but conveys similar benefits to traditional culture methods in regard to sensitivity, specificity, and potential for susceptibility and epidemiological studies.

Study enrollment and collection of clinical specimens Study subjects were selected from inpatients at Barnes-Jewish Hospital, a 1,442-bed tertiary care hospital in St. Louis, MO. Patients were considered for inclusion in the study if they had fecal specimens had been submitted for VRE detection or Clostridium difficile ELISA. These criteria were chosen to select a patient population at high risk for VRE colonization. Specimens submitted for this study were limited to fecal specimens collected in sterile containers. Collected specimens were transported and stored at 4C up to 24 h before being plated on the control or experimental medium. This study was approved by the Washington University Human Studies Committee. Media and culture conditions We compared the performance of the experimental chromogenic medium (VRE-BMX; bioMrieux, LaBalme, France) with bile esculin azide agar supplemented with 6 mg/mL vancomycin (BEAV; Remel, Lenexa, KS). VRE-BMX and BEAV plates were directly inoculated by submerging a sterilized inoculating loop in the specimen and subculturing using the quadrant method. Plates were incubated at 35C and examined for growth at 24 and 48 h time points. VRE-BMX plates with purple or green colonies were presumed positive for E. faecium or E. faecalis, respectively. BEAV plates with black colonies were presumed positive for VRE. Following observation of the colony morphologies, individual unique colonies were subcultured onto both CPS 3 ID medium (bioMrieux) and Columbia blood agar plates for confirmatory identification and antimicrobial susceptibility testing using the VITEK 2 automated identification and susceptibility system (bioMrieux). Multiplex PCR for detection of glycopeptide resistance genes and identification of E. faecium and E. faecalis DNA was extracted per previously published methods. Multiplex PCR was adapted from the method previously described by Depardieu et al (J. Clin. Microbiol. 2004, 43, 3390-3397). Amplified products were detected and quantified by electrophoresis on a 2100 BioAnalyzer system (Agilent, Massy, France) according to the manufacturers instructions.
51
INTRODUCTION
Enterococcus species are members of the normal intestinal flora, and are the most common aerobic Gram-positive cocci found in the large bowel. The organisms have gained more notoriety, however, as nosocomial pathogens, now grouped as the third most common bloodborne pathogen in the United States. At least one factor accounting for this pathogenicity is their intrinsic resistance to a variety of antimicrobial agents, including cephalosporins, co-trimoxazole, clindamycin, and beta-lactamase resistant penicillins. However, an even more alarming pattern arose in 1988, as plasmid-mediated resistance to vancomycin was first described. The most common acquired resistance-conferring enzyme is vanA, which is a transposon-mediated, inducible gene conferring high-level resistance to vancomycin and teicoplanin, and can be located on plasmid or chromosomal DNA. Glycopeptide resistance mediated by vanB is less common, and confers resistance to vancomycin, but not Teicoplanin. Less common acquired genes conferring Vancomycin-Resistance include vanD, vanE, vanF, and vanG, which encode moderate to low-level resistance to glycopeptides.
chromID VRE
chromID ESBL
chromID MRSA
Evaluation of a novel chromogenic agar medium from bioMrieux to screen for Vancomycin-Resistant Enterococcus faecium and faecalis
Figure 1 VRE-BMX medium plated with E. faecalis or E. faecium and Bile Esculin Azide agar plated with E. faecium. Figures 1A and 1B demonstrate ability of VRE-BMX medium to speciate E. faecium and E. faecalis based on color of the colony. On VRE-BMX colonies of E. faecalis will appear blue-green and colonies of E. faecium will appear purple. For purposes of this study, fecal specimens were directly plated to VRE-BMX and BEAV and observed for growth at 24 and 48 hours. Figure 1A Figure 1B Figure 1C Figure 3 Specificity of VRE-BMX and BEAV for VRE after 24 and 48 h.
Specificity of Media for VRE 100 90 80 70
% Specific
BEFORE PCR
AFTER PCR
60 50 40 30 20 10 0
Figure 2 Results from sensitivity analysis of VRE-BMX and BEAV at 24 and 48 hours. Figure 2A demonstrates sensitivity of each medium for identification of VRE (E. faecalis or E. faecium). Figure 2B demonstrates sensitivity for E. faecalis and E. faecium, respectively. Figure 2A
Sensitivity of Media for VRE 98 96 94
% Sensitive
24
48 24 Length of incubation (in hours) VRE ID BEAV
48
BEFORE PCR
AFTER PCR
92 90 88 86 84
Figure 4 Growth of contaminants on each medium at 24 and 48 hours. Contaminants are classified as potential false positive isolates and colorless isolates. Isolates with any hue of blue-green or purple were classified as potential false positives on VRE-BMX. Isolates with a black hue on BEAV were classified as potential false positives. Isolates most likely to cause false positive results include: Candida spp., Enterobacter spp., on VRE-BMX and Enterococcus spp. other than E. faecalis or E. faecium, Gram-positive rods, Streptococcus spp. on BEAV.
Growth of Contaminants 80 70
Number of isolates
FALSE POSITIVE ISOLATES
COLORLESS ISOLATES
60 50 40 30 20 10
24
48 24 Length of incubation (in hours) VRE ID BEAV
48
Figure 2B
100 90 80 70
% Sensitive
Sensitivity of Media for E. faecium or E. Faecalis
24
48 VRE ID BEAV
24
48
60 50 40 30 20 10 0 24 48 24 48
Length of incubation (in hours) VRE ID E. faecium VRE ID E. faecalis 52 BEAV E. faecium BEAV E. faecalis
Table 1A
Medium 24h VRE-BMX BEAV 48h VRE-BMX BEAV TP 51 48 53 51 FP 7 13 24 20 TN 86 80 68 72 FN 3 6 2 4 % 87.9 78.7 68.8 71.8 PPV CI95% [ 76.9% ; 94.1% ] [ 66.6% ; 87.2% ] [ 57.6% ; 78.2% ] [ 60.2% ; 81.1% ] NPV % 96.6 93.0 97.1 94.7 CI95% [ 90.4% ; 98.9% ] [ 85.4% ; 96.8% ] [ 90.0% ; 99.2% ] [ 87.0% ; 98.0% ]
Table 1A and 1B Positive and negative predictive values for VRE-BMX and BEAV. Table 1A demonstrates PPV and NPV of each medium before PCR analysis to confirming the presence of vanA or vanB in each isolate identified as Table 1B
Medium 24h VRE-BMX BEAV 48h VRE-BMX BEAV TP 53 50 55 53 FP 6 12 21 18 TN 86 80 68 71 FN 2 5 3 5
Enterococcus spp. Table 1B demonstates PPV and NPV after PCR analysis. Results confirm increased inclubation of BEAV or VRE-BMX does not improve sensitivity or PPV of media.
% 89.8 80.7 72.4 74.7
PPV CI95% [ 79.3% ; 95.3% ] [ 68.9% ; 88.7% ] [ 61.2% ; 81.3% ] [ 63.2% ; 83.4% ]
NPV % 97.7 94.1 95.8 93.4 CI95% [ 91.9% ; 99.4% ] [ 86.8% ; 97.5% ] [ 88.1% ; 98.6% ] [ 85.3% ; 97.2% ]
CONCLUSIONS
chromID ESBL
53
VRE-BMX represents the first chromogenic medium to identify VRE and differentiate E. faecium from E. faecalis. VRE-BMX offers improved sensitivity versus BEAV in isolating and identifying VRE in feces: - BEAV provides identification of VRE, VRE-BMX provides species level identification of E. faecalis and E. faecium for improved tracking of nosocomial epidemics - Incubation for 48 h does not significantly improve sensitivity of either VRE-BMX or BEAV Specificity of BEAV and VRE-BMX are not significantly different.
Growth of contaminants that may be interpreted as false positives is slightly greater on BEAV versus VRE-BMX: - Presence of contaminants potentially causing false positives significantly increases at 48 h on both media types Growth of colorless contaminants is greater on VRE-BMX. VRE-BMX is more likely to predict positive and negative VRE colonization at 24 h versus BEAV: - Incubation of plates for 48 h decreases PPV and NPV for both medium types.
KEY POINTS

chromID VRE is the first chromogenic medium to identify and differentiate VRE.faecium from VRE.faecalis. chromID VRE provides improved recovery of VRE from stool specimens as well as increased specificity towards enterococci intrinsically resistant to vancomycin.
chromID VRE
chromID MRSA
Notes
06-07 / 002GB99122A / This document is not legally binding. bioMrieux S.A. reserves the right to modify specifications without notice / BIOMRIEUX, the blue logo, chromID, from diagnosis the seeds of better health, VITEK, ATB, API, Rapidec and Slidex are used, pending and/or registered trademarks belonging to bioMrieux S.A. or one of its subsidiaries / bioMrieux S.A. RCS Lyon 673 620 399 / Photos : C. Ganet, N. Bouchut, Getty Images / Printed in France / TL McCANN SANT Lyon / RCS Lyon B 398 160 242
www.biomerieux.com
bioMrieux S.A. 69280 Marcy lEtoile France Tel.: 33 (0)4 78 87 20 00 Fax: 33 (0)4 78 87 20 90

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from diagnosis, the seeds of better health

Combined Use of Pastorex Staph-Plus and Either of Two New Chromogenic

Evaluation of three chromogenic media (MRSA-ID, MRSA-Select and

Performance of MRSA ID, a New Chromogenic Medium for Detection of

Development and Evaluation of a Chromogenic Agar Medium for

JOURNAL OF CLINICAL MICROBIOLOGY, Jan. 2007, p. 154158 0095-1137/07/$08.00 0 doi:10.1128/JCM.01115-06

Received 31 May 2006/Returned for modification 25 August 2006/Accepted 24 October 2006

JOURNAL OF CLINICAL MICROBIOLOGY, Feb. 2006, p. 586588 0095-1137/06/$08.00 0 doi:10.1128/JCM.44.2.586588.2006

Received 1 July 2005/Returned for modification 23 August 2005/Accepted 6 November 2005

JOURNAL OF CLINICAL MICROBIOLOGY, Oct. 2004, p. 45194523 0095-1137/04/$08.00 0 DOI: 10.1128/JCM.42.10.45194523.2004

Received 9 April 2004/Accepted 21 June 2004

ECCMID / April, 2007

In vitro evaluation of MRSA screening methods

INTRODUCTION & OBJECTIVES:

MATERIAL AND METHODS

*Following 24 and 48 hours of incubation, respectively.

RESULTS OF ENRICHMENT BROTHS:

ECCMID / April, 2007

Table 2: Distribution of MRSA isolates by patients (n = 45)

MRSA-ID +/18h +/36h 0 0 +/18h +/36h 0 +/36h

MATERIAL AND METHODS

Fig. 1: Distribution of MRSA isolates by specimen type (n = 68)

Fig. 2: Distribution of MRSA/MSSA isolates and normal flora

36h Enrichment 18h

ECCMID / April, 2007

Fig. 1. Typical appearance of MRSA on MRSA ID.

Table 1: Typical appearance of MRSA on MRSA ID.

a Numbers in parentheses indicate total strains in 48 h of incubation.

ECCMID / April, 2007

Comparison of Chromogenic Agar in the Detection of MRSA

CRITERIA FOR AGAR SELECTION

MRSA Select MRSA GNB MSSA

ICAAC / September, 2006

MATERIAL AND METHODS

MRSA ID medium (bioMrieux) chromogenic substrate + cefoxitin: 4 mg/l

CHROMagar MRSA medium (CHROMagar Microbiology) chromogenic substrate + cephamycin

87 (100) 99 (82) 88 (89) 97 (96)

41 (97) 100 (97)

RESULTS AND DISCUSSION

Other SAMPLES* CHROMagar MRSA 9 (10) 5 (5) 40 (74) MRSA ID

12 (13) 19 (20) 3 (4)

1 (1) 1 (1) 3 (3)

ICAAC / September, 2006

LLR-MRSA3 29 0.5 - 16 MRSA ID

16 h 36 h MRSA Screen 16 h 36 h MRSA Select 16 h 36 h

Table 2: Performance of the three chromogenic media

ECCMID / April, 2006

INTRODUCTION AND OBJECTIVES

Total 43 406 449

Table 2: MRSA ID after 48 h.

Total 78 371 449

MATERIAL AND METHODS

Table 3: CHROMagar staph. aureus after 48 h.

Total 78 371 449

2. CHROMagar Staph. aureus

Incubation at 35C. Reading after 48 hrs. Staphylococcus aureus: mauve colonies

ECCMID / April, 2006

Methicillin-Resistant coagulase negative staphylococci

No. (%) of MSA 67 (100%) 1 (1.4%) 67 (100%)

No. (%) of MRSA 111 (82.8%) 132 (98.5%) 134 (100%)

Table 2: Overall results for MSA and MRSA ID