Sample Receiving and Recording

On receipt of shrimp, all packages were inspected to ensure that samples were not
compromised (no leaks, melting etc). Samples were then immediately transferred to our -80℃
to preserve the integrity of the samples until they are processed. At the time of sample
processing, laboratory identifiers are assigned (ex. CBC01…) and metadata (retail origin, site of
retail outlet, product description/brand, country of origin, farmed vs. wild caught if listed and
raw vs. cooked) are recorded. Each product was also photographed.

Bacterial Culture and Isolation

All products were screened for:
• Staphylococcus aureus (particularly interested in MRSA)
• Escherichia coli
• Salmonella species
• Vibrio cholerae, Vibrio vulnificus and Vibrio parahaemolyticus
• ESBL producing Enterobacteriaceae – this is a bacterial family which comprised of a
large number of clinically important bacterial genera including: Escherichia spp.,
Klebsiella spp., Enterobacter spp., among others.

Samples were processed using a series of selective bacteriological media (agars and broths) to
maximize the recovery of the organisms of interest. These selective media are inhibitory to
many “background” organisms and also contain ingredients which highlights the growth of
organisms of interest (ex. on CHROMagar ESBL, E. coli forms pink colonies while other
Enterobacteriaceae are blue).

Briefly, 3 shrimp were added to a sterile sample bag and washed in 250ml of buffered peptone
water (BPW). The shrimp + BPW mixture was vigorously shook/massaged to liberate bacteria
living/on the sample. The BPW was then added to a series of broths/agars for selective culture
(see flowchart). Colonies from selective agars resembling the organisms of interest were sub-
cultured to blood agar, this allows “classical” colony morphology to be observed and is the
media from which bacteria are identified. From blood agar, bacteria were identified using
biochemical tests (ex. for E. coli the indole and citrate tests) and positively identified organisms
were frozen at -80℃ for future characterization. In parallel, 3 shrimp were also added to 250ml
of alkaline saline peptone water (ASPW) and similarly washed. The entire 250ml volume was
pre-incubated at 37℃ for 6 hours. Following this pre-incubation step, 1ml was added to 9ml of
sterile ASPW and incubated overnight at 42℃. ASPW broth was then plated on CHROM ID
Vibrio and suspect colonies were sub-cultured to blood agar, isolates which were oxidase test
positive were then identified by MALDI-TOF and frozen at -80℃ for future characterization.

The bacterial isolation/identification process used for this investigation is a 6 day procedure.
Antimicrobial Susceptibility Testing
Antimicrobial susceptibility testing will be done using a broth microdilution method. A
commercial test system will be utilized (Beckman Coulter MicroScan panels). We will be using
different panels for Gram-positive (Staphylococcus aureus) and Gram-negative bacteria (E. coli,
Salmonella spp., Vibrio spp. and other Enterobacteriaceae). These panels will be selected to
allow us to identify key resistance phenotypes such as methicillin resistant S. aureus (MRSA)
and broad spectrum ß-lactamase (such as ESBLs and carbapenemases) producing E. coli.

Antimicrobial susceptibility testing is a highly standardized laboratory technique and we will be

following the guidelines of the Clinical and Laboratory Standards Institute (CLSI) for conducting
and interpreting our test results. For quality control, we will include a number of reference
bacterial strains in our experiments (Staphylococcus aureus ATCC 29213, Enterococcus faecalis
ATCC 29212, Escherichia coli 25923 and Pseudomonas aeruginosa ATCC 27853) as appropriate.
 GLOSSARY OF TERMS
ASPW - ALKALINE SALINE PEPTONE A high pH, high salt media used for selecting Vibrio species
WATER
ATCC American Type Culture Collection – an organization which
maintains cultures of reference strains used as laboratory
controls
BLOOD AGAR A non-selective agar that supports the growth of many
bacteria
BIOCHEMICAL TEST A test which identifies particular metabolic properties of a
bacteria which is used for bacterial identification
BRILLIANT GREEN AGAR A selective agar used for the isolation of Salmonella
BPW - BUFFERED PEPTONE WATER A pH controlled media used to support the growth of enteric
bacteria
CHROMAGAR ESBL A selective and differential media used to identify a wide
variety of Enterobacteriaceae which are resistant to 3rd
generation cephalosporins (particularly ESBL producers).
Resistant organisms such Klebsiella spp., would be identified
on this media.
CHROM ID VIBRIO A selective and differential media used to identify Vibrio
species
EOSIN METHYLENE BLUE AGAR A selective and differential media which facilitates the
(EMB) isolation of Escherichia coli
MACCONKEY BROTH A selective broth which facilitates the isolation of Escherichia
coli
MALDI-TOF A mass spectrometry based technique for the identification of
bacterial species. Matrix Assisted Laser Desorption/Ionization
Time of Flight Mass Spectrometry.
MANNITOL SALT AGAR (MSA) A selective and differential media which facilitates the
isolation of Staphylocccus species
RAPPAPORT BROTH A selective broth which facilitates the isolation of Salmonella
STAPHYLOCOCCUS BROTH An in house prepared selective broth containing tryptone,
7.5% NaCl, mannitol and yeast extract which facilitates the
isolation of Staphylococcus species
TETRATHIONATE BROTH A selective broth which facilitates the isolation of Salmonella
XLD AGAR A selective agar used for the isolation of Salmonella