Date Published: September 25, 2008
Publisher: BioMed Central
Author(s): Shuyu Wu, Eirini Chouliara, Lars Bogø Jensen, Anders Dalsgaard.
Screening and enumeration of antimicrobial resistant Escherichia coli directly from samples is needed to identify emerging resistant clones and obtain quantitative data for risk assessment. Aim of this study was to evaluate the performance of 3M™ Petrifilm™ Select E. coli Count Plate (SEC plate) supplemented with antimicrobials to discriminate antimicrobial-resistant and non-resistant E. coli.
A range of E. coli isolates were tested by agar dilution method comparing the Minimal Inhibitory Concentration (MIC) for eight antimicrobials obtained by Mueller-Hinton II agar, MacConkey agar and SEC plates. Kappa statistics was used to assess the levels of agreement when classifying strains as resistant, intermediate or susceptible.
SEC plate showed that 74% of all strains agreed within ± 1 log2 dilution when comparing MICs with Mueller-Hinton II media. High agreement levels were found for gentamicin, ampicillin, chloramphenicol and cefotaxime, resulting in a kappa value of 0.9 and 100% agreement within ± 1 log2 dilution. Significant variances were observed for oxytetracycline and sulphamethoxazole. Further tests showed that the observed discrepancy in classification of susceptibility to oxytetracycline by the two media could be overcome when a plate-dependent breakpoint of 64 mg/L was used for SEC plates. For sulphamethoxazole, SEC plates provided unacceptably high MICs.
SEC plates showed good agreement with Mueller-Hinton II agar in MIC studies and can be used to screen and discriminate resistant E. coli for ampicillin, cephalothin, streptomycin, chloramphenicol, cefotaxime and gentamicin using CLSI standardized breakpoints, but not for sulphamethoxazole. SEC plates can also be used to discriminate oxytetracycline-resistant E. coli if a plate-dependent breakpoint value of 64 mg/L is used.
Escherichia coli is the most common intestinal bacterium of the Enterobacteriacae family and its presence outside the intestine is often used as an indicator of faecal pollution and in surveillance programs of antimicrobial resistance. Screening and enumeration of antimicrobial resistant E. coli directly from samples, e.g., food or animal specimens, is needed to identify emerging resistance clones and to obtain quantitative data for epidemiological investigations . MacConkey agar has been widely used as selective medium for isolation and enumeration of E. coli due to its low cost and high selectivity [2-4]. However, further identification of isolates is necessary since many species of Enterobacteriaceae can grow on MacConkey agar and colony characteristics are not sufficient to correctly identify E. coli . More recently Petrifilm™ system (3M Microbiology Products, St. Paul., MN, USA) was developed for direct enumeration of E. coli . The 3M™ Petrifilm™ Select E. coli Count Plate (SEC plate) is a sample-ready culture medium system consisting of plastic film with grids that are coated with selective agents, nutrients and gelling agent. Gel contains a β-glucuronidase indicator for confirmed detection of E. coli . Only E. coli colonies are conspicuous on SEC plates, eliminating need for colony identification and allowing for a direct enumeration of E. coli. SEC plates have undergone some validation for enumeration of E. coli in food and water samples [8,9]. However, no previous studies have tested the feasibility of using SEC plates to discriminate antimicrobial-resistant E. coli. The main objective of this study was therefore to evaluate the SEC plate supplemented with antimicrobials to discriminate antimicrobial-resistant and non-resistant E. coli in a comparison study between SEC plate and Mueller-Hinton II agar, the standard medium commonly used for antimicrobial susceptibility testing in E. coli, and for which international recognized breakpoints and cut-off values for antimicrobial resistance have been defined .
In a pilot study with a range of strains belonging to Enterobactericeae and other bacterial species (including E. coli, Salmonella, Shigella, Klebsiella, Citrobacter and Enterobacter), we confirmed high specificity of SEC plates for isolation of E. coli, as only E. coli gave conspicuous growth on SEC plates (results not shown). SEC plate was designed for direct and simple quantitative detection of E. coli[8,9]. Typical E. coli on SEC plates appear as blue-green colonies as it has been found that about 97% of E. coli produce β-glucuronidase which reacts with an indicator dye in the plate media to produce dark green to blue-green colonies. Colonies other than E. coli are not conspicuous because they are colorless or have a light grey-beige color. The usefulness in using β-glucuronidase activity for identification of E. coli has been confirmed by Schraft and coworkers who reported almost identical colony counts based on β-glucuronidase activity and on classical biochemical reactions.
This study provides the first information that the SEC plate medium is suitable to discriminate antimicrobial-resistant and non-resistant E. coli using CLSI standardized breakpoints. It appears feasible to use this medium to screen and enumerate resistant E. coli due to its high specificity and the good agreement obtained in MIC studies when compared with Mueller-Hinton II agar for most antimicrobials (ampicillin, cephalothin, streptomycin, chloramphenicol, cefotaxime and gentamycin). SEC plates may also be used to enumerate oxytetracycline-resistant E. coli if a plate-dependent breakpoint of 64 mg/L is used. However, SEC plate was not found suitable to select for sulphamethoxazole-resistant E. coli.
The authors declare that they have no competing interests.
WS performed the experiment and was responsible for the data analysis and writing the manuscript. EC participated in the study design and experimental work. AD and LJE were involved in the study design and revising the manuscript critically. All authors read and approved the final manuscript.