As part of a community-based study to determine colonisation prevalence of multi-drug resistant bacteria, stool specimens were collected from members of 88 households living in Siem Reap, Cambodia. All culture work was done at the clinical microbiology laboratory Angkor Hospital for Children (AHC), Siem Reap. This laboratory participates in national and international (World Health Organization Invasive Bacterial–Vaccine Preventable Diseases) external quality assurance schemes .
For this evaluation, 20 sequential study stool specimens from five households were processed immediately on receipt in the laboratory. Faecal slurries were prepared by emulsifying a small amount stool, picked up using a sterile swab, in 1.5 ml of sterile tryptone soya broth (Oxoid, Basingstoke, UK)—10% glycerol storage medium. These slurries were cultured immediately and then following 4–5 days (Frozen #1) and 172–3 days (~ 6 months, Frozen #2) storage at − 80 °C.
For culture, 10 µL of fresh or defrosted faecal slurry was streaked onto both CHROMagar ESBL, for ESBL detection, and KPC, for CPM detection, plates (CHROMagar, Paris, France) and incubated overnight at 37 °C under aerobic conditions. The CHROMagar plates were prepared in-house following the manufacturer's instructions, including positive and negative quality control with appropriate American Type Culture Collection and in-house bacterial strains.
From the fresh specimen culture plates, presence or absence of pink (suspected E. coli) and blue (suspected Klebsiella sp., Enterobacter sp., Citrobacter sp.–KEC group) colonies was recorded. From the frozen specimen culture plates, colony presence or absence was recorded and then one pink and one blue colony (of the dominant morphotype, if colonial variation was noted) was picked from each plate for formal identification by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-ToF; VITEK MS, bioMerieux, Marcy L’Etoile, France) and antimicrobial susceptibility testing (AST) by disk diffusion. The antibiotics tested were amoxicillin-clavulanate, ampicillin, cefpodoxime, ceftazidime, ceftriaxone, chloramphenicol, ciprofloxacin, gentamicin, meropenem, nitrofurantoin, and sulphamethoxazole-trimethoprim. The 2019 version of the Clinical Laboratory Standards Institute guidelines were used to interpret AST results .
Differences in detection of pink or blue colonies over time was assessed using the Chi-squared for trend test, with p values of < 0.05 being considered statistically significant. For comparison of AST profiles, "intermediate" results were re-assigned as "susceptible" to give a binary readout ("susceptible" or "resistant") for each isolate-drug combination.