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Appl Environ Microbiol. 2019 Jan 4. pii: AEM.02812-18. doi: 10.1128/AEM.02812-18. [Epub ahead of print]

Rapid and simple universal Escherichia coli genotyping method based on Multiple-Locus Variable-Number of Tandem Repeats Analysis using single-tube multiplex PCR and standard gel electrophoresis.

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Service de Microbiologie, Centre National de Référence associé Escherichia coli, Hôpital Robert-Debré, AP-HP, Paris, France.
IAME, UMR 1137, INSERM, Université Paris Diderot, Sorbonne Paris Cité, Paris, France.
Institut Pasteur, Unité des Bactéries Pathogènes Entériques, Centre National de Référence des Escherichia coli, Shigella et Salmonella, Paris, France.
Service de Microbiologie, Centre National de Référence associé Escherichia coli, Hôpital Robert-Debré, AP-HP, Paris, France.


We developed a multiplex PCR method based on Multiple-Locus Variable-number of tandem repeats (VNTR)-Analysis (MLVA) designed for rapid typing of E. coli and Shigella isolates The method amplifying seven VNTRs doesn't require sequencing capillary nor fluorescent dyes. Amplification products are simply loaded on a standard agarose gel for electrophoresis and banding patterns are analyzed visually. We evaluated the method on 220 strains belonging to different collections: ECOR collection (n=72), O1:K1 isolates causing neonatal meningitis (n=38), extended-spectrum beta-lactamase-producing fecal isolates belonging to the worldwide ST131 clone (n=38), Shiga-toxin producing E. coli (STEC) of serogroups O157:H7 (n=21) and O26 (n=16, 8 belonging to an outbreak), 27 Shigella isolates (22 S. sonnei including 5 epidemic strains) and 8 reference strains. The performances were compared to MLST, the DiversiLab automated REP-PCR, PFGE and whole genome sequencing. On the ECOR collection, we found 66 different profiles. Among the clonal group O1:K1, 14 different profiles were identified. For the 37 STECs, we had 23 profiles, 1 corresponding to the 8 epidemic strains. We found 19 profiles amongst the 27 Shigella isolates, 1 corresponding to the epidemic strain. The method was able to recognize strains of the ST131 clone, to distinguish O16 and O25b serogroups and identified 15 different MLVA types among them. This method allows a simple, fast and inexpensive typing of E. coli / Shigella that can be carried out in any laboratory equipped for molecular biology with a discriminating power superior to MLST and DiversiLab REP-PCR, but slightly lower than PFGE.IMPORTANCEFast typing methods that could easily and accurately distinguish clonal groups and unrelated isolates are of particular interest for microbiologists confronted to outbreaks or performing epidemiological studies. Highly discriminant universal methods like PFGE, optical mapping or WGS are expensive and/or time consuming. MLST is useful for phylogeny but less discriminant and requires sequencing facilities. PCR methods which are fast and easy to perform have also their drawbacks. Random PCRs and REP-PCR are universal but lack reproducibility. Other PCR methods may lack discriminatory power to differentiate isolates during outbreaks. MLVA combines the advantages of PCR methods with a high discriminatory power but in its standard form requires sequencing capillary electrophoresis. The method we have developed combines the advantages of standard PCR (simple, fast and inexpensive) with the high discriminatory power of MLVA and permits to type all E. coli (pathogenic either intestinal and extra-intestinal as well as commensal).


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