Rapid and high resolution genotyping of all Escherichia coli serotypes using 10 genomic repeat-containing loci

Division of Infectious Diseases control, Norwegian Institute of Public Health, Lovisenberggata 8, P.O. Box 4404 Nydalen, N-0403 Oslo, Norway.
Journal of microbiological methods (Impact Factor: 2.03). 11/2011; 88(1):134-9. DOI: 10.1016/j.mimet.2011.11.003
Source: PubMed


Our laboratory has previously published two multiple-locus variable-number tandem-repeats analysis (MLVA) methods for rapid genotyping of Escherichia coli (E. coli), which are now in routine use for surveillance and outbreak detection. The first assay developed was specific for E. coli O157:H7; however this assay was not suitable for genotyping other E. coli serotypes. A new generic MLVA-assay was then developed with the capability of genotyping all E. coli serotypes. This generic E. coli MLVA (GECM7) was based on polymorphism in seven variable number of tandem repeats (VNTR) loci. GECM7 worked well with the majority of E. coli serotypes; however we wanted to increase the resolution for this method based in part of comparison with PFGE typing of E. coli O26:H11, where PFGE appeared to display higher resolution. The GECM7 method was improved by adding three new repeat-loci to a total of ten (GECM10), and a considerable increase in resolution was observed (from 296 to 507 genotypes on the same set of strains).

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Available from: Kjersti Haugum, Nov 24, 2014
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    • "STEC O157 strains were analyzed by an 11-loci MLVA method described previously (Cooley et al., 2010). Non-O157 STEC strains were analyzed by the 10-loci MLVA method which includes 7 loci initially described for E. coli (Lindstedt et al., 2007; Cooley et al., 2013) and 3 additional loci including a CRISPR locus (Løbersli et al., 2012). Briefly, overnight cultures were boiled and aliquots were used as template in multiplex PCR reactions with fluorescently labeled primers to amplify fragments containing tandem repeats. "
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    ABSTRACT: Produce contaminated with enteric pathogens is a major source of foodborne illness in the United States. Lakes, streams, rivers, and ponds were sampled with Moore swabs bi-monthly for over 2 years at 30 locations in the vicinity of a leafy green growing region on the Central California Coast and screened for Shiga toxin producing Escherichia coli (STEC), Salmonella enterica, and Listeria monocytogenes to evaluate the prevalence and persistence of pathogen subtypes. The prevalence of STEC from 1386 samples was 11%; 110 samples (8%) contained E. coli O157:H7 with the highest prevalence occurring close to cattle operations. Non-O157 STEC isolates represented major clinical O-types and 57% contained both shiga toxin types 1 and 2 and intimin. Multiple Locus Variable Number Tandem Repeat Analysis of STEC isolates indicated prevalent strains during the period of study. Notably, Salmonella was present at high levels throughout the sampling region with 65% prevalence in 1405 samples resulting in 996 isolates with slightly lower prevalence in late autumn. There were 2, 8, and 14 sites that were Salmonella-positive over 90, 80, and 70% of the time, respectively. The serotypes identified most often were 6,8:d:-, Typhimurium, and Give. Interestingly, analysis by Pulsed Field Gel Electrophoresis indicated persistence and transport of pulsotypes in the region over several years. In this original study of L. monocytogenes in the region prevalence was 43% of 1405 samples resulting in 635 individual isolates. Over 85% of the isolates belonged to serotype 4b with serotypes 1/2a, 1/2b, 3a, 4d with 4e representing the rest, and there were 12 and 2 sites that were positive over 50 and 80% of the time, respectively. Although surface water is not directly used for irrigation in this region, transport to the produce can occur by other means. This environmental survey assesses initial contamination levels toward an understanding of transport leading to produce recalls or outbreaks.
    Frontiers in Cellular and Infection Microbiology 05/2014; 4:30. DOI:10.3389/fcimb.2014.00030 · 3.72 Impact Factor
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    • "The diversity index (D N ) was calculated for each locus using the formula D N = 1 − ∑(fr a )2, where fr a is the allelic frequency (Noller et al., 2003). MLVA profiles were defined either taking into account the alleles at each of the 7 loci proposed by Lindstedt et al. (2007)— MLVA G7 —or including also the alleles for the 3 loci proposed by Løbersli et al. (2012)—MLVA G10 . Dendrograms were constructed using UPGMA cluster analysis implemented by software START Vs. 1.0.5 (Joley et al., 2001). "
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    ABSTRACT: Multiple locus variable number tandem repeats (VNTRs) analysis (MLVA) has become a reliable tool, able to establish genetic relationships for epidemiological surveillance and molecular subtyping of pathogens such as verotoxigenic Escherichia coli (VTEC). This emerging pathogen whose primary reservoir is the cattle causes severe disease in humans, such as hemorrhagic colitis and hemolytic uremic syndrome. With the aim of comparing a recently proposed MLVA assay with that used routinely in our laboratory, we analyzed a set of VTEC isolates (n = 72) obtained from meat using both assays. All samples could be typed by the new MLVA assay, and an increase in the number of distinct profiles (31-43) was observed. However, intraserotype resolution was not significantly enhanced; thus, the incorporation of more VNTR loci is still needed to achieve a greater discrimination among non-O157:H7 serotypes.
    Diagnostic microbiology and infectious disease 10/2013; 78(4). DOI:10.1016/j.diagmicrobio.2013.10.017 · 2.46 Impact Factor
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    • "improved by incorporating more loci. Recently, Løbersli et al. (2012) improved that method by adding three new repeat-loci to a total of 10. They applied it and observed a considerable increase in resolution, of 71%, using the three new loci. "
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    ABSTRACT: Shiga toxin-producing Escherichia coli (STEC) causes serious human illness such as hemolytic uremic syndrome (HUS). Argentina has the world's highest rate of this syndrome, which is the leading cause of acute renal failure among children. E. coli O157:H7 is the most common cause of HUS, but a substantial and growing proportion of this illness is caused by infection due to non-O157 strains. Multiple-locus variable-number tandem repeat analysis (MLVA) has become an established technique to subtype STEC. This review will address the use of routine STEC subtyping by MLVA in order to type this group of isolates and to get insight into the genetic diversity of native STEC. With regard to these objectives we modified and adapted two MLVA protocols, one exclusive for O157 and the other, a generic E. coli assay. A total of 202 STEC isolates, from different sources and corresponding to 20 serotypes, have been MLVA genotyped in our laboratory. In our experience, MLVA constitutes a very sensitive tool and enables us to perform an efficient STEC subtyping. The diversity found in many serotypes may be useful for future epidemiological studies of STEC clonality, applied to O157 as well as to non-O157 isolates.
    Frontiers in Cellular and Infection Microbiology 08/2012; 2:111. DOI:10.3389/fcimb.2012.00111 · 3.72 Impact Factor
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