Multiple-Locus Variable-Number Tandem-Repeats Analysis of Escherichia coli O157 using PCR multiplexing and multi-colored capillary electrophoresis

dShiga toxin-producing Escherichia coli (STEC) is a heterogeneous group of bacteria causing disease ranging from asymptomatic carriage and mild infection to hemolytic uremic syndrome (HUS). Here, we describe patients with STEC infection and characterize the STEC strains detected in our laboratory by use of PCR for stx 1 , stx 2 , and eae from 1996 through 2011. Patient information was collected from referral forms and from the Norwegian Surveillance System for Communicable Diseases. STEC isolates were characterized with respect to serogroup or serotype, selected potential virulence genes, and multilocus variable-number tandemrepeat analysis (MLVA) genotype. STEC strains were isolated from 138 (1.09%) of 12,651 patients tested. STEC strains of serogroups O26, O103, O121, O145, and O157 were the most frequent. These serogroups, except non-sorbitol-fermenting O157, were also the most frequent among the 11 patients (all <5 years old) who developed HUS. Twenty-four STEC strains were classified as being HUS associated based on an epidemiological link to a HUS case, including an MLVA genotype identical to that of the STEC strain. The age of the patient (<5 years) and the genes eae and stx 2a were significantly associated with HUS-associated STEC (P < 0.05 for each parameter), while stx 1 was associated with non-HUS-associated STEC (P < 0.05). All of the potential virulence genes analyzed, except ehxA, were significantly more frequent among HUS-associated than non-HUS-associated strains (P < 0.05 for each gene). However, these genes were also present in some non-HUS-associated STEC strains and could therefore not reliably differentiate between HUS-associated and non-HUS-associated STEC strains.

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“…Two MLVA protocols, one for the O157 serogroup and one for all E. coli isolates, were used for MLVA typing (28)(29)(30) at the NRL, NIPH.…”

Section: Methodsmentioning

confidence: 99%

PCR-Based Detection and Molecular Characterization of Shiga Toxin-Producing Escherichia coli Strains in a Routine Microbiology Laboratory over 16 years

et al. 2014

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“…Multilocus variable-number tandem-repeat analysis (MLVA) is an effective, high-throughput typing method that measures hypervariable tandem repeats (TRs) at various locations in the genome (8,13,16,22). However, any change in the rate of hypervariability in these TRs is problematic when trying to predict the phylogenetic relationships among isolates.…”

mentioning

confidence: 99%

Effects of Environmental Stress on Stability of Tandem Repeats in Escherichia coli O157:H7

Cooley¹,

Carychao²,

Nguyen³

et al. 2010

Appl Environ Microbiol

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Multilocus variable-number tandem-repeat analysis (MLVA) is used for source tracking Escherichia coli O157:H7 in agricultural environments. Tandem repeats were stable after limited replication but changed after exposure to irradiation, elevated temperatures, and starvation conditions. The pO157 plasmid was frequently lost under these stress conditions. Environmental stresses may increase phylogenetic diversity as measured by MLVA.Escherichia coli O157:H7 is a pathogen causing serious disease that survives in a variety of hosts and environments, many of which are part of our food production system (24). Multilocus variable-number tandem-repeat analysis (MLVA) is an effective, high-throughput typing method that measures hypervariable tandem repeats (TRs) at various locations in the genome (8,13,16,22). However, any change in the rate of hypervariability in these TRs is problematic when trying to predict the phylogenetic relationships among isolates. Alterations in mutation rates due to stress in several studied systems have been previously reported (2,6,9,10,15,20,25,28) and recently reviewed (18). Mutation rate changes likely occur through the induction of a general-stress-response sigma factor (rpoS), leading to genome-wide instability through the activity of a strand-slippage mechanism (SSM) during replication (30) and/or to reduced fidelity of repair after DNA damage (SOS response and mismatch repair) (7, 21). Mutation rates have been shown to differ between MLVA loci (13,16,22) and between strains (31). Therefore, we hypothesized that environmental stress changes the mutation rate at MLVA TRs.Recovery of bacteria stressed in natural environments. Twenty-eight isolates of E. coli O157:H7 strain RM1484 (apple juice outbreak strain) were recovered from Arabidopsis thaliana seed about 60 days after 28 independently contaminated plants were planted as previously described (4). Hence, the bacteria survived in the phyllosphere during growth and maturation of the plant and seed. Ninety-six RM1484 isolates were also recovered from creek water (CW) after incubation for 7 days at 15°C. Each strain was analyzed by MLVA for 11 loci as previously published (Table 1) (3). No differences were found at any of the 11 loci in the recovered isolates compared to RM1484 (Table 2). These results are consistent with previous results that showed Erwinia amylovora repeat structures to be stable after log-term storage in sterile water at 25°C and passage in plants (26). Additionally, strain RM1484 was recovered from soil after growth of contaminated lettuce for 4 weeks at either 15°C or 23°C; the soil was then allowed to remain fallow and dry for an additional 4 weeks. One isolate from the 23°C soil and two isolates from the 15°C soil contained TR insertions (Table 2). Low nutrient levels in the phyllosphere or CW correlated with decreased DNA replication compared to the rhizosphere results (1). Detection of TR changes exclusively in rhizosphere isolates and not in phyllosphere and CW isolates substantiates the conclusion that replicat...

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“…The increasing availability of whole-genome sequences is an invaluable source of VNTRs, which has opened the way to multiple-locus VNTR analysis (MLVA) for the typing of bacteria. MLVAs have been proposed so far for Bacillus anthracis (17), Yersinia pestis (27), Francisella tularensis (10), Mycobacterium tuberculosis (16), Legionella pneumophila (28), Pseudomonas aeruginosa (24), Escherichia coli O157:H7 (19), and Salmonella enterica subsp. enterica serovars Typhimurium (18) and Typhi (20).…”

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confidence: 99%

Variable Number of Tandem Repeats in Salmonella enterica subsp. enterica for Typing Purposes

et al. 2004

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The genomic sequences of Salmonella enterica subsp. enterica strains CT18, Ty2 (serovar Typhi), and LT2 (serovar Typhimurium) were analyzed for potential variable number tandem repeats (VNTRs). A multiplelocus VNTR analysis (MLVA) of 99 strains of S. enterica supsp. enterica based on 10 VNTRs distinguished 52 genotypes and placed them into four groups. All strains tested were independent human isolates from France and did not reflect isolates from outbreak episodes. Of these 10 VNTRs, 7 showed variability within serovar Typhi, whereas 1 showed variability within serovar Typhimurium. Four VNTRs showed high Nei's diversity indices (DIs) of 0.81 to 0.87 within serovar Typhi (n ‫؍‬ 27). Additionally, three of these more variable VNTRs showed DIs of 0.18 to 0.58 within serovar Paratyphi A (n ‫؍‬ 10). The VNTR polymorphic site within multidrugresistant (MDR) serovar Typhimurium isolates (n ‫؍‬ 39; resistance to ampicillin, chloramphenicol, spectinomycin, sulfonamides, and tetracycline) showed a DI of 0.81. Cluster analysis not only identified three genetically distinct groups consistent with the present serovar classification of salmonellae (serovars Typhi, Paratyphi A, and Typhimurium) but also discriminated 25 subtypes (93%) within serovar Typhi isolates. The analysis discriminated only eight subtypes within serovar Typhimurium isolates resistant to ampicillin, chloramphenicol, spectinomycin, sulfonamides, and tetracycline, possibly reflecting the emergence in the mid-1990s of the DT104 phage type, which often displays such an MDR spectrum. Coupled with the ongoing improvements in automated procedures offered by capillary electrophoresis, use of these markers is proposed in further investigations of the potential of MLVA in outbreaks of salmonellosis, especially outbreaks of typhoid fever.

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Multiple-Locus Variable-Number Tandem-Repeats Analysis of Escherichia coli O157 using PCR multiplexing and multi-colored capillary electrophoresis

Cited by 74 publications

References 8 publications

PCR-Based Detection and Molecular Characterization of Shiga Toxin-Producing Escherichia coli Strains in a Routine Microbiology Laboratory over 16 years

PCR-Based Detection and Molecular Characterization of Shiga Toxin-Producing Escherichia coli Strains in a Routine Microbiology Laboratory over 16 years

Effects of Environmental Stress on Stability of Tandem Repeats in Escherichia coli O157:H7

Variable Number of Tandem Repeats in Salmonella enterica subsp. enterica for Typing Purposes

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