Contents

Gram-positive

Difficult or Tricky Antibiotic Resistance Phenotypes to Recognize

Dr Koh Tse Hsien Department of Pathology Singapore General Hospital

Staphylococcus aureus with penicillinase production Vancomycin Resistant Enterococci (VRE) Staphylococcus aureus with reduced susceptibility to glycopeptides (hVISA, VISA, VRSA)

Gram-negative
ampC Cephalosporinases ( AmpC and pAmpC) Carbapenemases (IMP, VIM, KPC, and OXA-48)

Mechanisms of Resistance
Target modification

Penicillinase in S. aureus

Antibiotic sequestration

Enzymatic inactivation

Penicillinase production

Vancomycin susceptibility in S. aureus

Vancomycin-intermediate S. aureus (VISA)

First described in Japan in 1997 Intermediate resistant (MIC 4-8 mg/L) Hetero-intermediate resistant (MIC <4 mg/L) Increased production of cell wall precursors and PBP 2 Reduced cross linking in cell wall (less target for vancomycin) Lower growth rates and thicker cell walls (vancomycin sequestration) Prolonged exposure to vancomycin
MRSA VISA

Micky LEONG (Ms) :: Laboratory Technologist, Electron Microscopy Unit YLLSOM

Vancomycin intermediate Staphylococcus aureus

Lab detection of VISA

Not detected by routine disk or automated methods Etest (consistently one twofold dilution higher) Broth microdilution

EUCAST
MIC breakpoint (mg/L) S Vancomycin 2 R> 2 Zone diameter breakpoint (mm) S R< -

1. Glycopeptide MICs are method dependent and should be determined by broth microdilution (reference ISO 20776). S. aureus with vancomycin MIC values of 2 mg /L are on the border of the wild type MIC distribution and there may be an impaired clinical response. The resistant breakpoint has been reduced to 2 mg /L to avoid reporting "GISA" isolates intermediate as serious infections with "GISA" isolates are not treatable with increased doses of vancomycin or teicoplanin.

The clinical significance of vancomycin minimum inhibitory concentration in Staphylococcus aureus infections: A systematic review and meta-analysis High vancomycin MIC (1.5 mg/L by Etest) associated with higher mortality rate This was driven by BSI with vancomycin MIC of 2 mg/L by Etest Higher vancomycin MIC values (1.5 mg/L by Etest) predicted treatment failure Etest recommended for all MRSA BSI
van Hal et al. CID 2012; 54: 755-71

CLSI
MIC breakpoint (mg/L) S Vancomycin 2 I 4-8 R 16 Zone diameter breakpoint (mm) S I R -

18) MIC tests should be performed to determine the susceptibility of all isolates of staphylococci to vancomycin. The disk test does not differentiate vancomycin-susceptible isolates of S. aureus from vancomycin-intermediate isolatesThe vancomycin 30-g disk test detects S. aureus isolates containing the vanA vancomycin resistance gene (VRSA). Such isolates will show no zone of inhibition around the disk (zone = 6 mm)

How does your lab measure the MIC?

Etest 0.5-1 dilution higher than broth microdilution (gold standard) Automated MIC 1-2 dilutions lower than gold standard Most vancomycin outcome studies involved Etest

Vitek versus Etest (92 isolates)

Vitek

Etest Dr Tan TY, Dept of Lab Medicine, Changi General Hospital

(Hetero) hVISA

Lab detection of hVISA

Population analysis Macrodilution Etest (not true MIC) Etest GRD MHA5T screening plate (5 g/ml teicoplanin) hVISA may predict potential failure of daptomycin therapy

FIG. 8. Examples of Etest methodology used to detect hVISA

Vancomycin-resistant S. aureus (VRSA)

In-vitro transfer of VanA in 1992 Michigan (June 2002), Pennsylvania (Sept 2002) MIC 1024 mg/L, 32 mg/L VanA positive Plasmid-mediated Detected by disk diffusion but may not be detected by automated methods

Howden, B. P. et al. 2010. Clin. Microbiol. Rev. 23(1):99-139

FIG. 1. Disk diffusion and Etest analysis of the PA-VRSA isolate on Mueller-Hinton agar

Vancomycin-resistant Enterococci

Tenover, F. C. et al. 2004. Antimicrob. Agents Chemother. 48(1):275-280

Vancomycin-resistant Enterococcus
VanA Vanc MIC mg/L Teic MIC Mg/L Species Genetic determinant Transferable 64->1000 Resistant VanB 4-1024 Resistant VanC 2-32 Intermediate 0.5 Susceptible
E. Gallinarum E. Casseiflavus E. flavescens

Van A VRE

16-512 0.5 Resistant Susceptible

E. Faecium E. faecalis E. Faecium E. faecalis

Acquired Yes

Acquired Yes

Intrinsic No

VSE

Van B VRE

VRE

VRE Lab Detection

Disk diffusion-incubate full 24 h, view with transmitted light for hazy zones MIC methods Screening plates (vancomycin 6 mg/L) Multiplex PCR to detect vanA, vanB

Identify all VRE to species level

vanC motile E. gallinarum

Mechanisms of Resistance
Loss of membrane permeability

EUCAST
MIC breakpoint (mg/L) S Cefotaxime 1 1 1 1 R> 2 2 4 4 Zone diameter breakpoint (mm) S 20 23 22 24 R< 17 20 19 21

Enzymatic inactivation

Ceftriaxone Ceftazidime Cefepime

CLSI
MIC breakpoint (mg/L)
inner membrane periplasmic space outer membrane

Zone diameter breakpoint (mm) S 26 23 21 18 I 23-25 20-22 18-20 15-17 R 22 19 17 14

S Cefotaxime
Efflux

I 2 2 8 16

R 4 4 16 32

1 1 4 8

Ceftriaxone Ceftazidime Cefepime

EUCAST
1. The cephalosporin breakpoints for Enterobacteriaceae will detect all clinically important resistance mechanisms (including ESBL and plasmid mediated AmpC). Some isolates that produce beta-lactamases are susceptible or intermediate to 3rd or 4th generation cephalosporins with these breakpoints and should be reported as tested, i.e. the presence or absence of an ESBL does not in itself influence the categorisation of susceptibility. In many areas, ESBL detection and characterisation is recommended or mandatory for infection control purposes.

Rationale for change

PD modeling suggests cephalosporin breakpoints reduced to 1-4 mg/L, possible to obtain serum concentrations of cephalosporins above MIC for the required 4050% of dosage interval Animal experiments show MIC better predictor of outcome than mechanism Reports of clinical failure correlated with higher MICs.

CLSI
(7) Following evaluation of PK-PD properties, limited clinical data, and MIC distributions, revised interpretive criteria for cephalosporins (cefazolin, cefotaxime, ceftazidime, ceftizoxime, and ceftriaxone) and aztreonam were first published in January 2010 (M100-S20) and are listed in this table. Cefazolin interpretive criteria were revised again in June 2010 and are listed below. Cefepime and cefuroxime (parenteral) were also evaluated; however, no change in interpretive criteria was required for the dosages indicated below. When using the current interpretive criteria, routine ESBL testing is no longer necessary before reporting results (ie, it is no longer necessary to edit results for cephalosporins, aztreonam, or penicillins from susceptible to resistant). However, ESBL testing may still be useful for epidemiological or infection control purposes. For laboratories that have not implemented the current interpretive criteria, ESBL testing should be performed as described in Table 2A Supplemental Table 1.

Class C beta-lactamases
Gene normally found in most Gram negatives except Klebsiella and Salmonella Cephalosporinases Not inhibited by clavulanate, tazobactam (except M. morgannii) Resistant to cefoxitin Cefepime not generally affected Chromosomal Generally expressed at low level but are inducible

AmpC in Gram-negative bacilli

ESCHAPPM
Enterobacter cloacae/aerogenes Serratia marcescens Citrobacter freundii Hafnia alvei Aeromonas hydrophila/caviae Providencia stuartii/rettgeri

Induction vs Stable Derepression

Induction
transient switching on of -lactamase synthesis in response to an inducer (cefoxitin, imipenem)

Stable Derepression
permanent hyperproduction of the lactamase independent of an inducer

Morganella morganii

Chromosomal AmpC in Enterobacter spp.

K. Pneumoniae Would you treat this patient with ceftriaxone?

Augmentin Ceftriaxone Ceftriaxone

Aztreonam Cefoxitin Ceftoxitin

Ceftriaxone

De-repressed mutant
De-repressed mutants

Original strain

Cefoxitin

pAmpC DHA-1

DHA in Klebsiella pneumoniae

>32 mg/L-

1mg/L susceptible-

-1.5 mg/L

De-repressed mutant

Original strain

CLSI
(8) Enterobacter, Citrobacter, and Serratia may develop resistance during prolonged therapy with third-generation cephalosporins as a result of derepression of AmpC -lactamase. Therefore, isolates that are initially susceptible may become resistant within three to four days after initiation of therapy. Testing of repeat isolates may be warranted.

CDS method
E. cloacae, E. aerogenes, C. freundii, inducible pAmpC
High frequency of derepressed mutants (10-5 to 10-6) Report R to all cephalosporins Test cefepime and carbapenems Do not attempt to test other -lactams

S. marcescens

BSAC (EUCAST?)
Enterobacter spp., Citrobacter freundii, Serratia spp. and Morganella morganii. If susceptible in- vitro, the use of monotherapy of cefotaxime should be discouraged, owing to the risk of selection of resistance, or suppress the susceptibility testing result for this agent.

No derepressed mutants with ceftazidime, tazocin and aztreonam Derepressed mutants with other -lactams including cefotaxime.

H. alvei, P. stuartii, P. rettgeri and M. morganii

Very low mutation rate (10-8) Test and report accordingly

Inducible plasmid AmpC (DHA) in K. pneumoniae?

http://web.med.unsw.edu.au/cdstest/

Carbapenemases
Class A
KPC

Carbapenemases in Gramnegative bacilli

Class B
IMP, VIM, NDM

Class C Class D
OXA-48

EUCAST
MIC breakpoint (mg/L) S Ertapenem Imipenem Meropenem 0.5 2 2 R> 1 8 8 Zone diameter breakpoint (mm) S 25 22 22 R< 22 16 16

EUCAST
1. The carbapenem breakpoints for Enterobacteriaceae will detect all clinically important resistance mechanisms (including the majority of carbapenemases). Some isolates that produce carbapenemase are categorised as susceptible with these breakpoints and should be reported as tested, i.e. the presence or absence of a carbapenemase does not in itself influence the categorisation of susceptibility. In many areas, carbapenemase detection and characterisation is recommended or mandatory for infection control purposes.

CLSI
24) Following evaluation of PK-PD properties, limited clinical data, and MIC distributions that include recently described carbapenemase-producing strains, revised interpretive criteria for carbapenems were first published in June 2010 (M100-S20-U) and are listed below. Because of limited treatment options for infections caused by organisms with carbapenem MICs or zone diameters in the intermediate range, clinicians may wish to design carbapenem dosage regimens that use maximum recommended doses and possibly prolonged intravenous infusion regimens, as has been reported in the literature.1-4 Consultation with an infectious diseases practitioner is recommended for isolates for which the carbapenem MICs or zone diameter results from disk diffusion testing are in the intermediate or resistant ranges. Until laboratories can implement the current interpretive criteria, the modified Hodge test (MHT) should be performed as described in the updated Table 2A Supplemental Table 3. After implementation of the current interpretive criteria, the MHT does not need to be performed other than for epidemiological or infection control purposes (refer to Table 2A Supplemental Table 2). The following information is provided as background on carbapenemases in Enterobacteriaceae that are largely responsible for MICs and zone diameters in the new intermediate and resistant ranges, and thus the rationale for setting revised carbapenem breakpoints: The clinical effectiveness of carbapenem treatment of infections produced by isolates for which the carbapenem MIC or disk diffusion test results are within the new intermediate (I) range is uncertain due to lack of controlled clinical studies.

CLSI
MIC breakpoint (mg/L) S Ertapenem Imipenem Meropenem 0.5 1 1 I 1 2 2 R 2 4 4 Zone diameter breakpoint (mm) S 22 23 23 I 19-21 20-22 20-22 R 18 19 19

NDM-1

DM23092 IMP+

DM23092 IMP+

Modified Hodge test

pAmpC

pAmpC

pAmpC

OXA-48

Outcomes of infections caused by carbapenemaseproducing K. pneumoniae according to treatment regimen

Carbapenemase-producing K. pneumoniae: (when) might we still consider treating with carbapenems?

Carbapenem MIC 4 mg/L High-dose prolonged infusion Used in combination with another active compound

A B C D E F G

combination more than 1 active drug including carbapenem combination more than 1 active drug excluding carbepenem monotherapy with aminoglycoside monotherapy with carbapenem monotherapy with tigecycline monotherapy with colistin Tzouvekelis et al. CMR 2012 25: 682inappropriate therapy 707

Daikos and Markogiannakis CMID 2011; 17: 1135-1141

My take
Detection of mechanism may still have role for tertiary care hospitals with resistance problems and complicated patients Combination therapy usually including carbapenem for CP-CRE Should we be doing more MICs? Automated system may not be equivalent to MIC. Etest practical option for most labs