Article

Second Generation Subtyping: A Proposed PulseNet Protocol for Multiple-Locus Variable-Number Tandem Repeat Analysis of Shiga Toxin–Producing Escherichia coli O157 (STEC O157)

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Abstract

Most bacterial genomes contain tandem duplications of short DNA sequences, termed "variable-number tandem repeats" (VNTR). A subtyping method targeting these repeats, multiple-locus VNTR analysis (MLVA), has emerged as a powerful tool for characterization of clonal organisms such as Shiga toxin-producing Escherichia coli O157 (STEC O157). We modified and optimized a recently published MLVA scheme targeting 29 polymorphic VNTR regions of STEC O157 to render it suitable for routine use by public health laboratories that participate in PulseNet, the national and international molecular subtyping network for foodborne disease surveillance. Nine VNTR loci were included in the final protocol. They were amplified in three PCR reactions, after which the PCR products were sized using capillary electrophoresis. Two hundred geographically diverse, sporadic and outbreak- related STEC O157 isolates were characterized by MLVA and the results were compared with data obtained by pulsed-field gel electrophoresis (PFGE) using XbaI macrorestriction of genomic DNA. A total of 139 unique XbaI PFGE patterns and 162 MLVA types were identified. A subset of 100 isolates characterized by both XbaI and BlnI macrorestriction had 62 unique PFGE and MLVA types. Although the clustering of isolates by the two subtyping systems was generally in agreement, some discrepancies were observed. Importantly, MLVA was able to discriminate among some epidemiologically unrelated isolates which were indistinguishable by PFGE. However, among strains from three of the eight outbreaks included in the study, two single locus MLVA variants and one double locus variant were detected among epidemiologically implicated isolates that were indistinguishable by PFGE. Conversely, in three other outbreaks, isolates that were indistinguishable by MLVA displayed multiple PFGE types. An additional more extensive multi-laboratory validation of the MLVA protocol is in progress in order to address critical issues such as establishing epidemiologically relevant interpretation guidelines for the MLVA data.

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... subtyping techniques helps to increase the discriminative power of PFGE. 13,18,27,28 Another genotyping method that has been developed to discriminate STEC from multiple sources is multiple locus variable-number tandem repeat analysis (MLVA) ( Table 1). 13,18,27,28 MLVA amplifies short regions of repeated DNA sequences, known as variable number tandem repeat (VNTR), differing in size, location and number of copies. ...
... 13,18,27,28 Another genotyping method that has been developed to discriminate STEC from multiple sources is multiple locus variable-number tandem repeat analysis (MLVA) ( Table 1). 13,18,27,28 MLVA amplifies short regions of repeated DNA sequences, known as variable number tandem repeat (VNTR), differing in size, location and number of copies. 28 When selecting VNTR loci for MLVA, the stability of the locus is important for results interpretation. ...
... 13,18,27,28 MLVA amplifies short regions of repeated DNA sequences, known as variable number tandem repeat (VNTR), differing in size, location and number of copies. 28 When selecting VNTR loci for MLVA, the stability of the locus is important for results interpretation. The advantage of using MLVA as a typing method is that it is rapid and high throughput and allows the discrimination of certain strains by non-typeable PFGE. ...
Article
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Shiga toxin-producing Escherichia coli (STEC) is an enteric pathogen linked to outbreaks of human gastroenteritis with diverse clinical spectra. In this review, we have examined the currently methodologies and molecular characterization techniques for assessing the phenotypic, genotypic and functional characteristics of STEC O157 and non-O157. In particular, traditional culture and isolation methods, including selective enrichment and differential plating, have enabled the effective recovery of STEC. Following recovery, immunological serotyping of somatic surface antigens (O-antigens) and flagellum (H-antigens) are employed for the classification of the STEC isolates. Molecular genotyping methods, including multiple-locus variable-number tandem repeat analysis, arrays, and whole genome sequencing, can discriminate the isolate virulence profile beyond the serotype level. Virulence profiling is focused on the identification of chromosomal and plasmid genes coding for adhesins, cytotoxins, effectors, and hemolysins to better assess the pathogenic potential of the recovered STEC isolates. Important animal reservoirs are cattle and other small domestic ruminants. STEC can also be recovered from other carriers, such as mammals, birds, fish, amphibians, shellfish and insects. Finally, antimicrobial resistance in STEC is a matter of growing concern, supporting the need to monitor the use of these agents by private, public and agricultural sectors. Certain antimicrobials can induce Shiga toxin production and thus promote the onset of severe disease symptoms in humans. Together, this information will provide a better understanding of risks associated with STEC and will aid in the development of efficient and targeted intervention strategies.
... It allows the direct comparison of the PFGE data generated between the different laboratories that make up the network. Currently, this network of laboratories is complementing the results of PFGE with MLVA and WGS methodologies [156][157][158]. ...
... The development achieved in the genomic sciences has suggested that other techniques could give more precise information to aid outbreak investigations. Since the first decade of 2000′s several methodologies as the MLVA and WGS (Fig. 1) has been evaluated and implemented for bacterial subtyping [22,[41][42][43][44][156][157][158][180][181][182][183][184][185][186][187][188][189]. ...
... The WGS provides greater discriminatory power and precision than PFGE and MLVA and is able to reveal information phylogenetically relevant [22,[41][42][43]156,158,[180][181][182][183][184][185][186][187]189] (Table 3). Although the costs of setup and per test of WGS procedures have dropped dramatically due to the development of benchtop automatic sequencers, they still surpass the instrumentation cost for fingerprinting by PFGE [186,187]. ...
Article
Pulsed Field Gel Electrophoresis (PFGE) has been considered for many years the ‘gold-standard’ for characterizing many pathogenic organisms as well as for subtyping bacterial species causing infection outbreaks. This article reviews the basic principles of PFGE and it includes the main advantages and limitations of the different electrode configurations that have been used in PFGE equipment and their influence on the DNA electrophoretic separation. Remarkably, we summarize here the most relevant theoretical and practical aspects that we have learned for more than 20 years developing and using the miniaturized PFGE systems. We also discussed the theoretical aspects related to DNA migration in PFGE agarose gels. It served as the basis for simulating the DNA electrophoretic patterns in CHEF mini gels and mini-chambers during experimental design and optimization. A critical comparison between standard and miniaturized PFGE systems, as well as the enzymatic and non-enzymatic methods for intact immobilized DNA preparation, is provided throughout the review. The PFGE current applications, advantages, limitations and future challenges of the methodology are also discussed.
... The string of allele types for the target VNTR loci can be called an MLVA type. MLVA is advantageous, as the procedure is simple and rapid, and the data obtained are portable and discrete to enable comparison among multiple laboratories (8). ...
... The amount of DNA prepared by heat extraction was adjusted so that the resulting signals of the Data analysis of MLVA Fragment data were evaluated and converted to repeat copy numbers using GeneMapper software (Thermo Fisher Scientific K.K.) and GeneMarker software (SoftGenetics LLC, PA, USA). The data were imported to BioNumerics software and analyzed as previously described (8). Repeat copy number for those with no PCR product was designated as -2. Simpson's diversity index (D) and 95% confidence intervals (CI) were calculated according to the formula described previously (16). ...
Article
Non-O157 Shiga toxin-producing Escherichia coli (STEC) are a growing concern for public health. The number of sporadic cases and outbreaks due to non-O157 STEC are increasing. Molecular subtyping that discriminates the isolates rapidly with high resolution is essential to identify clusters of cases and/or detect and investigate outbreaks. Multiple-locus variable-number tandem repeat analysis (MLVA) is one of the most useful typing methods for isolates responsible for foodborne diseases. In Japan, serogroups O26, O111, O103, O121, O145, O165, and O91 are frequently isolated or associated with severe cases of non-O157 STEC infections. In this study, we designed an MLVA scheme (MLVA43) by adding 26 loci to an MLVA scheme (MLVA17) previously developed by our group for O157, O26, and O111 using 17 loci; the current scheme focused on serogroups O103, O121, O145, O165, and O91. Discriminatory power of MLVA43 was comparable to that of pulsed-field gel electrophoresis (PFGE) for serogroups O103, O145, O165, and O91, and superior to that of PFGE for O121. MLVA43 identified more profiles than did MLVA17, except for serogroup O111 with 707 isolates. The new MLVA43 scheme would enable rapid identification of clusters and outbreaks, which will help rapid measure against non-O157 STEC infections.
... Despite the historical usefulness and robustness of PFGE, it has been widely demonstrated by the scientific community that the method does not consistently provide optimal discrimination, particularly for highly clonal strains, resulting in outbreak investigations of seemingly unrelated cases and in some instances, discriminates among epidemiological related isolates, obscuring useful linkages and cluster detection [3,8,9]. To enhance resolution, multilocus variable-number tandem-repeat analysis (MLVA) was implemented (in Canada, as a supplemental subtyping tool, and elsewhere as a primary tool), and has been extremely valuable in discriminating among closely related isolates that would otherwise be indistinguishable by PFGE [4,[10][11][12][13][14][15]; however, optimal resolution is often achieved when the results of these typing methods are combined and interpreted together [5], rendering characterization of foodborne bacterial pathogens to be laborious, time-consuming and expensive, which has prohibited widespread adoption of both methods in many public health laboratories [16,17]. Additionally, MLVA protocols have only been developed for selected pathogens (i.e., Salmonella Enteritidis, Salmonella Typhimurium and Escherichia coli O157:H7) within the PulseNet Canada network, further hindering its overall effectiveness [17]. ...
Article
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Background Rapid and accurate identification of Verotoxigenic Escherichia coli (VTEC) O157:H7 is dependent on well-established, standardized and highly discriminatory typing methods. Currently, conventional subtyping tests for foodborne bacterial pathogen surveillance are rapidly being replaced with whole-genome sequencing (WGS) in public health laboratories. The capacity of WGS to revolutionize global foodborne disease surveillance has positioned this tool to become the new gold standard; however, to ensure evidence standards for public health decision making can still be achieved, the performance of WGS must be thoroughly validated against current gold standard methods prior to implementation. Here we aim to verify the performance of WGS in comparison to pulsed-field gel electrophoresis (PFGE) and multiple-locus variable-number tandem repeat analysis (MLVA) for eight retrospective outbreaks of VTEC O157:H7 from the Canadian perspective. Since real-time implementation and routine use of WGS in public health laboratories is highly reliant on standardized data analysis tools, we also provide a comparative analysis of two popular methodologies for WGS analyses; an in-house developed single nucleotide variant phylogenomics (SNVPhyl) pipeline and the BioNumerics whole genome multilocus sequence typing (wgMLST) tool. To provide a useful and consistent starting point for examining laboratory-based surveillance data for VTEC O157:H7 in Canada, we also aim to describe the number of genetic differences observed among outbreak-associated isolates. Results WGS provided enhanced resolution over traditional subtyping methods, and accurately distinguished outbreak-related isolates from non-outbreak related isolates with high epidemiological concordance. WGS also illuminated potential linkages between sporadic cases of illness and contaminated food, and isolates spanning multiple years. The topologies generated by SNVPhyl and wgMLST were highly congruent with strong statistical support. Few genetic differences were observed among outbreak-related isolates (≤5 SNVs/ < 10 wgMLST alleles) unless the outbreak was suspected to be multi-strain. Conclusions This study validates the superiority of WGS and indicates the BioNumerics wgMLST schema is suitable for surveillance and cluster detection of VTEC O157:H7. These findings will provide a useful and consistent starting point for examining WGS data for prospective laboratory-based surveillance of VTEC O157:H7, but however, the data will continue to be interpreted according to context and in combination with epidemiological and food safety evidence to inform public-health decision making in Canada. Electronic supplementary material The online version of this article (10.1186/s12864-018-5243-3) contains supplementary material, which is available to authorized users.
... MLVA has been developed for typing of various microbial species including Escherichia coli O157, Salmonella enterica, Pseudomonas aeruginosa, and A. baumannii (21)(22)(23). In the current study, MLVA-8 Orsay scheme was performed in order to trace the epidemiology of the carbapenem-resistant A. baumannii isolates and for bacterial differentiation at the strain level. ...
... In STRAf 3A and 3C three and two mutations were found, respectively, suggesting that these repeats are potentially less stable. It is known that the variability of some tandem repeats is much higher than others (Bustamante et al., 2013) and that the number of repeat units within a STR can change very rapidly during an outbreak (Hyytiä-Trees et al., 2006;Noller et al., 2006). Importantly, the three mutations in STRAf marker 3A were found in one isolate, demonstrating that the former definition of microvariation for the STRAf assay would not have been valid for this marker if similar variations would have been found in an outbreak. ...
Article
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More than a decade ago a short tandem repeat-based typing method was developed for the fungus Aspergillus fumigatus. This STRAf assay is based on the analysis of nine short tandem repeat markers. Interpretation of this STRAf assay is complicated when there are only one or two differences in tandem repeat markers between isolates, as the stability of these markers is unknown. To determine the stability of these nine markers, a STRAf assay was performed on 73-100 successive generations of five clonally expanded A. fumigatus isolates. In a total of 473 generations we found five times an increase of one tandem repeat unit. Three changes were found in the trinucleotide repeat marker STRAf 3A, while the other two were found in the trinucleotide repeat marker STRAf 3C. The di- or tetranucleotide repeats were not altered. The altered STRAf markers 3A and 3C demonstrated the highest number of repeat units (≥50) as compared to the other markers (≤26). Altogether, we demonstrated that 7 of 9 STRAf markers remain stable for 473 generations and that the frequency of alterations in tandem repeats is positively correlated with the number of repeats. The potential low level instability of STRAf markers 3A and 3C should be taken into account when interpreting STRAf data during an outbreak.
... Multi-locus variable number tandem repeat analysis (MLVA) typing was carried out on the positive E. coli O157:H7 isolates according to the system developed by Hyytia-Trees et al. (2006), using eight loci (excluding VNTR10) and with the following modifications: the VNTR 34, 9, 19 and 36 forward primers were labelled with FAM, VNTR 25 and 37 with HEX and VNTR 3 and 17 with NED. Qiagen Multiplex PCR Master Mix (Qiagen, Vienna, Austria) was used for the two multiplex PCR reactions with the forward and reverse primer concentrations VNTR 3 1Á0 lmol l À1 , VNTR 34 0Á1 lmol l À1 , VNTR 9 0Á1 lmol l À1 , VNTR 25 0Á05 lmol l À1 , VNTR 17 0Á15 lmol l À1 , VNTR 19 0Á015 lmol l À1 , VNTR 36 0Á012 lmol l À1 and VNTR 37 0Á015 lmol l À1 . ...
Article
Aim To investigate the survival of E. coli O157:H7 and Salmonella Typhimurium in cowpats on pasture in a temperate Nordic climate. Methods and Results The study consists of two parts, the first part using artificially created cowpats inoculated with E. coli O157:H7 and Salmonella Typhimurium and the second part using cowpats from empty pastures on which cattle herds positive for E. coli O157:H7 had grazed six month previously. Artificial cowpats were created, placed in an outdoor field station in June, August and October, and sampled over one year. E. coli O157:H7 and Salmonella Typhimurium were analysed by standard culture methods. The results showed viable E. coli O157:H7 and Salmonella Typhimurium in the sampled cowpats throughout the 365‐day sample period for the June trial, 250 days for the August trial and 40‐70 days for the October trial. In addition, 200 natural cowpats were sampled from eight pastures that had previously held E. coli O157:H7 positive cattle herds. Five positive E. coli O157:H7 isolates were obtained, all with the same multi‐locus variable number tandem repeat analysis (MLVA) pattern as had been found on the pasture the previous grazing season. Conclusions E.coli O157:H7 and Salmonella Typhimurium can survive in cowpats up to a year and persist throughout a winter season. Therefore there is a possibility that cowpats can act as a reservoir and be a source of re‐infection of Salmonella or E. coli O157:H7 in cattle between grazing seasons. Significance and Impact of the Study The obtained results can provide valuable information for managing the risk posed by zoonotic pathogens originating from farm environments. This article is protected by copyright. All rights reserved.
... We found a wide genetic diversity among O157:H7 isolates through MLVA, agreeing with previous studies [20,23,24]. A total of 36 MLVA profiles was observed, 33 of which were unique. ...
Article
Verotoxin-producing Escherichia coli O157:H7 is the dominant serotype isolated from patients with hemolytic-uremic syndrome (HUS) and, Argentina has the highest rate of HUS in the world. However, not all O157:H7 isolates have the same ability to infect and cause disease in humans. It has been postulated that O157:H7 strains integrate subpopulations related to the origin and virulence. In order to study the population structure and genetic diversity of VTEC O157:H7 from Argentina, a combination of molecular subtyping methods such as multiple loci VNTR analysis (MLVA), single nucleotide polymorphisms (SNP) and phylogroups assignment were used. According to MLVA, high genetic diversity was found among strains isolated from cattle, humans and food. On the other hand, 92% of the isolates presented the allele tir 255 T>A T and 95% were assigned to phylogroup E. We did not find a significant association between the isolates origin and the allele T presence (P>0,05) postulated as significantly overrepresented in human isolates. Our results show that human and cattle VTEC O157:H7 isolates from Argentina are a homogeneous group and, although it presents high genetic diversity in relation to their MLVA and virulence profiles, it is not possible to distinguish divergent populations. The presence in all the strains of a high number of T3SS effectors genes and the no association of genetic subtypes with strain source, is an alert about the potential risk in public health that VTEC O157:H7 cattle strains possess and, at less, a partial explication about the high incidence of HUS in Argentina.
... Each unique allelic string was designated a unique MLVA type (MT). A dendrogram was constructed by UPGMA clustering based on categorical coe cient analysis [27,29]. ...
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Background: The natural hosts of Shigella are conventionally humans and other primates; however, the host range of Shigella has been shown to expand to many animals. Although Shigella is becoming a huge threat to animals, there is limited information on the genetic background of local strains. The purpose of this study was to assess the presence of virulence factors and the molecular characteristics of S. flexneri isolated from calves with diarrhea. Methods: From 2014 to 2016, 54 S. flexneri isolates were collected from diarrhea, and their biochemical characteristics were determined according to API20E and virulence factors via PCR. The molecular characteristics of the isolates were studied by MLST, MLVA and PFGE. Results: Fifty-four S. flexneri isolates possessed four typical biochemical characteristics of Shigella. The prevalences of ipaH, virA, ipaBCD, ial, sen, set1A, and set1B were 100%, 100%, 77.78%, 79.63%, 48.15% and 48.15%, respectively. None of the studied strains possessed the stx gene. Regarding the differences in virulence factor distributions, the 54 S. flexneri isolates fell into seven gene profile types. Among these VTs, VT4 and VT6 were the most common, accounting for 74.07% of all VTs. MLVA based on 8 VNTR loci discriminated the isolates into 39 different MTs, PFGE based on NotI digestion divided the 54 isolates into 31 PTs, and MLST based on 15 housekeeping genes differentiated the isolates into 7 STs, with 1 ST (ST227) being novel. Conclusion: Our findings provide baseline information on the distribution of virulence genes in and the molecular characteristics of S. flexneri collected from diarrheal calves, which is a potential threat to public safety. These data will be important for addressing clinical and epidemiological issues regarding Shigellosis.
... Each unique allelic string was designated a unique MLVA type (MT). A dendrogram was constructed by UPGMA clustering based on categorical coe cient analysis [33,45]. ...
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Full-text available
Background The natural hosts of Shigella are conventionally humans and other primates; however, the host range of Shigella has been shown to expand to many animals. Although Shigella is becoming a huge threat to animals, there is limited information on the genetic background of local strains. The purpose of this study was to assess the presence of virulence factors and the molecular characteristics of S. flexneri isolated from calves with diarrhea. Results Fifty-four S. flexneri isolates possessed four typical biochemical characteristics of Shigella. The prevalences of ipaH, virA, ipaBCD, ial, sen, set1A, set1B and stx were 100%, 100%, 77.78%, 79.63%, 48.15%, 48.15% and 0, respectively. MLVA based on 8 VNTR loci discriminated the isolates into 39 different MTs, PFGE based on NotI digestion divided the 54 isolates into 31 PTs, and MLST based on 15 housekeeping genes differentiated the isolates into 7 STs. Conclusion Our findings of this study have enriched our knowledge of the molecular characteristics of S. flexneri collected from diarrheal calves, which will be important for addressing clinical and epidemiological issues regarding Shigellosis.
... In both studies, isolates from sporadic cases and outbreaks were included and MLVA provided with discriminatory power comparable to PFGE; moreover, in some cases differentiated strains within PFGE clusters. Keys et al. (2005) developed an eight-TR protocol that was subsequently optimized by Hyytia-Trees et al. (2006) and adopted by PulseNet as an alternative or complement to PFGE. ...
Chapter
Accurate identification of the infection source and the transmission route are necessary for the effective implementation of preventive measures against microbial food-borne pathogens. Advances in the field of molecular biology has allowed the development of sophisticated techniques able to detect differences at genomic level and through which studies of epidemiological nature may be conducted. Techniques, such as pulsed-field gel electrophoresis (PFGE), multilocus variable number of tandem repeats analysis (MLVA), and multilocus sequence typing (MLST) have been thoroughly studied and extensively applied. These techniques are characterized by specific strengths and weaknesses that need to be taken into consideration before any conclusion is drawn. In this chapter all information related to typing approaches of Listeria monocytogenes, Salmonella serovars, Escherichia coli O157:H7, and Campylobacter spp. are integrated and critically discussed.
... The PFGE technique has shown limited discriminatory power in subtyping some highly clonal serotypes (e.g., S. enterica serotype Enteritidis and S. enterica serotype Hadar; Swaminathan et al., 2001;Lukinmaa et al., 2004;Hyytiä-Trees et al., 2006). Additionally, PFGE often lacks discriminatory power to partition strains into epidemiologically meaningful clusters. ...
Article
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This study investigated the prevalence, serovar distribution, antimicrobial resistance, and pulsed field gel electrophoresis (PFGE) typing of Salmonella enterica isolated from Lake Zapotlán, Jalisco, Mexico. Additionally, the association of the presence of Salmonella with physicochemical and environmental parameters was analyzed using Pearson correlation analysis and principal component analysis (PCA). Salmonella spp. were identified in 19 of 63 (30.15%) samples. The prevalence of Salmonella was positively correlated with air temperature, electrical conductivity, pH, and dissolved oxygen and negatively correlated with relative humidity, water temperature, turbidity, and precipitation. The predominant serotype identified was Agona (68.48%), followed by Weltevreden (5.26%), Typhimurium (5.26%), and serogroup B (21.05%). Overall, the highest detected antimicrobial resistance was toward colistin (73.68%), followed by sulfamethoxazole (63.15%), tetracycline (57.89%), nalidixic acid (52.63%), and trimethoprim (52.63%). All Salmonella strains were genetically diverse, with a total of 11 XbaI and four BlnI profiles on PFGE. The use of these two enzymes allowed differentiate strains of Salmonella of the same serotype. The results obtained in this study contribute to a better understanding of the Salmonella spp. ecology in an endorheic subtropical lake and provide information for decision makers to propose and implement effective strategies to control point and non-point sources of pathogen contamination.
... MLVA typing of eight VNTR loci was performed on a total of 168 confirmed E. coli O157 isolates previously by Jones et al. (32), using the method described in Hyvtiä-Trees et al. (35). The fragment size of PCR products representing eight separate loci was determined using an automated ABI3730 DNA analyzer (Applied Biosystems), and the number of repeats was calculated using known data for the reference strain EDL933. ...
Article
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Shiga toxin-producing Escherichia coli (STEC) O157 is an important foodborne pathogen that can be transmitted to humans both directly and indirectly from the feces of beef cattle, its primary reservoir. Numerous studies have investigated the shedding dynamics of E. coli O157 by beef cattle; however, the spatiotemporal trends of shedding are still not well understood. Molecular tools can increase the resolution through the use of strain typing to explore transmission dynamics within and between herds and identify strain-specific characteristics that may influence pathogenicity and spread. Previously, the shedding dynamics and molecular diversity, through the use of multilocus variable number of tandem repeat analysis (MLVA) of STEC O157, were separately investigated in an Australian beef herd over a 9-month study period. Variation in shedding was observed over time, and 33 MLVA types were identified. The study presented here combines the two datasets previously published with an aim to clarify the relationship between epidemiological variables and strain types. Three major genetic clusters (GCs) were identified that were significantly associated with the location of the cattle in different paddocks. No significant association between GCs and individual cow was observed. Results from this molecular epidemiological study provide evidence for herd-level clonal replacement over time that may have been triggered by movement to a new paddock. In conclusion, this study has provided further insight into STEC O157 shedding dynamics and pathogen transmission. Knowledge gaps remain regarding the relationship of strain types and the shedding dynamics of STEC O157 by beef cattle that could be further clarified through the use of whole-genome sequencing.
... Each unique allelic string was designated a unique MLVA type (MT). A dendrogram was constructed by UPGMA clustering based on categorical coefficient analysis [27,29]. ...
Preprint
Full-text available
Background: The natural hosts of Shigella are conventionally humans and other primates; however, the host range of Shigella has been shown to expand to many animals. Although Shigella is becoming a huge threat to animals, there is limited information on the genetic background of local strains. The purpose of this study was to assess the presence of virulence factors and the molecular characteristics of S. flexneri isolated from calves with diarrhea. Methods: From 2014 to 2016, 54 S. flexneri isolates were collected from diarrhea, and their biochemical characteristics were determined according to API20E and virulence factors via PCR. The molecular characteristics of the isolates were studied by MLST, MLVA and PFGE. Results: Fifty-four S. flexneri isolates possessed four typical biochemical characteristics of Shigella. The prevalences of ipaH, virA, ipaBCD, ial, sen, set1A, and set1B were 100%, 100%, 77.78%, 79.63%, 48.15% and 48.15%, respectively. None of the studied strains possessed the stx gene. Regarding the differences in virulence factor distributions, the 54 S. flexneri isolates fell into seven gene profile types. Among these VTs, VT4 and VT6 were the most common, accounting for 74.07% of all VTs. MLVA based on 8 VNTR loci discriminated the isolates into 39 different MTs, PFGE based on NotI digestion divided the 54 isolates into 31 PTs, and MLST based on 15 housekeeping genes differentiated the isolates into 7 STs, with 1 ST (ST227) being novel. Conclusion: Our findings provide baseline information on the distribution of virulence genes in and the molecular characteristics of S. flexneri collected from diarrheal calves, which is a potential threat to public safety. These data will be important for addressing clinical and epidemiological issues regarding Shigellosis.
... Each unique allelic string was designated a unique MLVA type (MT). A dendrogram was constructed by UPGMA clustering based on categorical coefficient analysis [27,29]. ...
Preprint
Full-text available
Background: The natural hosts of Shigella are conventionally humans and other primates; however, the host range of Shigella has been shown to expand to many animals. Although Shigella is becoming a huge threat to animals, there is limited information on the genetic background of local strains. The purpose of this study was to assess the presence of virulence factors and the molecular characteristics of S. flexneri isolated from calves with diarrhea. Methods: From 2014 to 2016, 54 S. flexneri isolates were collected from diarrhea, and their biochemical characteristics were determined according to API20E and virulence factors via PCR. The molecular characteristics of the isolates were studied by MLST, MLVA and PFGE. Results: Fifty-four S. flexneri isolates possessed four typical biochemical characteristics of Shigella. The prevalences of ipaH, virA, ipaBCD, ial, sen, set1A, and set1B were 100%, 100%, 77.78%, 79.63%, 48.15% and 48.15%, respectively. None of the studied strains possessed the stx gene. Regarding the differences in virulence factor distributions, the 54 S. flexneri isolates fell into seven gene profile types. Among these VTs, VT4 and VT6 were the most common, accounting for 74.07% of all VTs. MLVA based on 8 VNTR loci discriminated the isolates into 39 different MTs, PFGE based on NotI digestion divided the 54 isolates into 31 PTs, and MLST based on 15 housekeeping genes differentiated the isolates into 7 STs, with 1 ST (ST227) being novel. Conclusion: Our findings provide baseline information on the distribution of virulence genes in and the molecular characteristics of S. flexneri collected from diarrheal calves, which is a potential threat to public safety. These data will be important for addressing clinical and epidemiological issues regarding Shigellosis.
... Gorgé et al. have proposed a simplified version of the method adapted for Shigella isolates based on standard electrophoresis using VNTRs with TRs with long lengths, but each VNTR is amplified in a separate PCR (8). Several other combinations of VNTR have been proposed, and all of these require either fluorescent dyes or specific electrophoresis (capillary electrophoresis systems, polyacrylamide gels with silver staining) (10)(11)(12)(13)(14)(15)(16). ...
Article
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Fast typing methods that can easily and accurately distinguish clonal groups and unrelated isolates are of particular interest for microbiologists confronted with outbreaks or performing epidemiological studies. Highly discriminatory universal methods, like PFGE, optical mapping, or WGS, are expensive and/or time-consuming. MLST is useful for phylogeny but is less discriminatory and requires sequencing facilities. PCR methods, which are fast and easy to perform, also have drawbacks. Random PCRs and REP-PCR are universal but lack reproducibility. Other PCR methods may lack the 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 that we have developed combines the advantages of standard PCR (simple, fast, and inexpensive) with the high discriminatory power of MLVA and permits the typing of all E. coli isolates (either intestinal or extraintestinal pathogenic isolates as well as commensal isolates).
... It concerns a PCR-based method that amplifies multicopy DNA tandem repeats across the genome (Lindstedt et al., 2003). MLVA is considered less laborious, easier to standardize, and in general more discriminatory than PFGE (Hyytia-Trees et al., 2006). However, to date, optimization and standardization of a protocol that is suitable for the wide range of STEC serogroups is not established yet (Karama and Gyles, 2010). ...
... Genotyping with eight MLVA loci was performed and amplification of the VNTR target loci was modified to be a single reaction with a final volume of 10 μl that included 1 μl of 10X PCR Mg 2+ free buffer (Invitrogen, Carlsbad, CA, USA), 2 μM MgCl 2 , 1 U of Platinum Taq polymerase (Invitrogen), 0.2 mM of PCR Nucleotide Mix (Roche Applied Sciences), 1 μl of DNA template, and primers at concentrations of 0.6, 0.2, 0.12, 0.36, 0.6, 0.02, 0.012, and 0.03 μm to amplify VNTR3, 9,25,34,17,19,36, and 37, respectively. Primers and reagents were supplied by Takara Holdings, Japan [39]. ...
Article
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Background: The Shiga toxin-producing Escherichia coli (STEC) represented a great risk to public health. In this study, 60 STEC strains recovered from broiler and duck fecal samples, cow's milk, cattle beef, human urine, and ear discharge were screened for 12 virulence genes, phenotypic and genotypic antimicrobial resistance, and multiple-locus variable-number tandem-repeat analysis (MLVA). Results: The majority of strains harbored Shiga toxin 1 (stx 1) and stx 1d , stx 2 and stx 2e , and ehxA genes, while a minority harbored stx 2c subtype and eaeA. We identified 10 stx gene combinations; most of strains 31/60 (51.7%) exhibited four copies of stx genes, namely the stx 1 , stx 1d , stx 2 , and stx 2e , and the strains exhibited a high range of multiple antimicrobial resistance indices. The resistance genes blaCTX-M-1 and blaTEM were detected. For the oxytetracycline resistance genes, most of strains contained tetA, tetB, tetE, and tetG while the tetC was present at low frequency. MLVA genotyping resolved 26 unique genotypes; genotype 21 was highly prevalent. The six highly discriminatory loci DI = 0.9138 are suitable for the preliminary genotyping of STEC from animals and humans.
... PFGE analysis was performed using a protocol modified from the Centers for Disease Control and Prevention PulseNet (Hyytia-Trees et al., 2006). Briefly, astA + E. coli was cultured on eosin methylene blue (EMB) agar overnight at 37°C. ...
Article
The aim of this study was to compare the sequence of the astA gene found in 8 Korean and 11 Japanese Escherichia coli isolates. Conventional PCR was used to amplify the astA gene from the chromosomal and plasmid DNA preparation samples of each isolate using commercial DNA extraction kits. Cloning of the PCR products, sequence analysis, and pulse field gel electrophoresis (PFGE) were sequentially performed. An identical copy of astA in each isolate were found for 8 Korean and 8 Japanese E. coli strains isolated from bovine, porcine, and healthy human carriers. Among these, 1 Korean and 4 Japanese isolates carried a stop mutation at residue 16. Three Japanese outbreak strains (V199, V638, and 96-127-23) carried multiple clones of astA gene with multiple amino acids changes at residues 11, 16, 20, 23, 30, 33, and 34. Compared with the non-diarrheal isolates, clonal diversity and sequence variations of the astA gene in outbreak isolates may be associated with virulence potential of EAST1.
... This type of O157:H7 has previously been shown to be a common cause of both domestic and imported cases of human O157:H7 illness in Sweden (Soderlund et al. 2014). Molecular typing by MLVA (Hyytia-Trees et al. 2006) showed a close relationship between all pattern A isolates (profiles NA-7-14-5-6-6-6-7 (n = 3), NA-7-14-5-6-7-5-7 (n = 1) and NA-7-14-6-6-7-5-7 (n = 2)), suggesting this is an example of a recent clonal expansion and spread between farms and The O157:H7 strain containing vtx2c::IS629 was grown in six replicates on two different types of solid media. After 7 days of incubation ddPCR analysis was performed in triplicate for each sample (undiluted) to detect vtx2c genes from which the IS element had been excised, and in one replicate of each sample (diluted 1:1000) to determine the number of O157 genome copies. ...
Article
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There are several anecdotal reports of insertion sequence (IS)-element inactivation of verotoxin genes among enterohaemorrhagic Escherichia coli of the serotype O157:H7, a pathogen causing severe gastrointestinal disease in infected humans. These insertions can be expected to drastically reduce the virulence of the bacteria. IS-element inactivation has been shown to be reversible in model systems, suggesting the possibility of spontaneous restoration of virulence. In the present study, traditional and high-throughput sequencing was used to characterize three patterns of IS629 inactivation of verotoxin 2 genes in EHEC O157:H7, caused by insertion or insertion followed by partial deletion. At least one of the patterns of inactivation appears to have persisted several years among cattle O157:H7, indicating it has no major effect on fitness in the animal reservoir. Digital PCR was used to directly quantify the reversal rates of the insertional inactivation of a selected isolate under laboratory conditions. Inserts were found to be absent from in the order of 1/10⁵ of individual genomes, with significantly higher loss frequencies observed in cultures under nutrient poor conditions. We conclude that strains with this type of inactivation found in food or animal samples should be considered a threat to human health, and may pose a challenge for PCR-based detection methods.
... Each unique allelic string was designated a unique MLVA type. A dendrogram was constructed by UPGMA clustering based on categorical coefficient analysis [35,49]. ...
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Background The natural hosts of Shigella are typically humans and other primates, but it has been shown that the host range of Shigella has expanded to many animals. Although Shigella is becoming a major threat to animals, there is limited information on the genetic background of local strains. The purpose of this study was to assess the presence of virulence factors and the molecular characteristics of S. flexneri isolated from calves with diarrhea. Results Fifty-four S. flexneri isolates from Gansun, Shanxi, Qinghai, Xinjiang and Tibet obtained during 2014 to 2016 possessed four typical biochemical characteristics of Shigella. The prevalences of ipaH, virA, ipaBCD, ial, sen, set1A, set1B and stx were 100 %, 100 %, 77.78 %, 79.63 %, 48.15 %, 48.15 and 0 %, respectively. Multilocus variable number tandem repeat analysis (MLVA) based on 8 variable number of tandem repeat (VNTR) loci discriminated the isolates into 39 different MLVA types (MTs), pulsed field gel electrophoresis (PFGE) based on NotI digestion divided the 54 isolates into 31 PFGE types (PTs), and multilocus sequence typing (MLST) based on 15 housekeeping genes differentiated the isolates into 7 MLST sequence types (STs). Conclusions The findings from this study enrich our knowledge of the molecular characteristics of S. flexneri collected from calves with diarrhea, which will be important for addressing clinical and epidemiological issues regarding shigellosis.
... Genotyping with eight MLVA loci was performed and amplification of the VNTR target loci was modified to be a single reaction with a final volume of 10 μl that included 1 μl of 10X PCR Mg 2+ free buffer (Invitrogen, Carlsbad, CA, USA), 2 μM MgCl 2 , 1 U of Platinum Taq polymerase (Invitrogen), 0.2 mM of PCR Nucleotide Mix (Roche Applied Sciences), 1 μl of DNA template, and primers at concentrations of 0.6, 0.2, 0.12, 0.36, 0.6, 0.02, 0.012, and 0.03 μm to amplify VNTR3, 9,25,34,17,19,36, and 37, respectively. Primers and reagents were supplied by Takara Holdings, Japan [39]. ...
Article
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Background The Shiga toxin-producing Escherichia coli (STEC) represented a great risk to public health. In this study, 60 STEC strains recovered from broiler and duck fecal samples, cow’s milk, cattle beef, human urine, and ear discharge were screened for 12 virulence genes, phenotypic and genotypic antimicrobial resistance, and multiple-locus variable-number tandem-repeat analysis (MLVA). Results The majority of strains harbored Shiga toxin 1 ( stx 1 ) and stx 1d , stx 2 and stx 2e , and ehx A genes, while a minority harbored stx 2c subtype and eae A. We identified 10 stx gene combinations; most of strains 31/60 (51.7%) exhibited four copies of stx genes, namely the stx 1 , stx 1d , stx 2 , and stx 2e , and the strains exhibited a high range of multiple antimicrobial resistance indices. The resistance genes bla CTX-M-1 and bla TEM were detected. For the oxytetracycline resistance genes, most of strains contained tet A, tet B, tet E, and tet G while the tet C was present at low frequency. MLVA genotyping resolved 26 unique genotypes; genotype 21 was highly prevalent. The six highly discriminatory loci DI = 0.9138 are suitable for the preliminary genotyping of STEC from animals and humans. Conclusions The STEC isolated from animals are virulent, resistant to antimicrobials, and genetically diverse, thus demands greater attention for the potential risk to human.
... Each unique allelic string was designated a unique MLVA type (MT). A dendrogram was constructed by UPGMA clustering based on categorical coe cient analysis [27,29]. ...
Preprint
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Background: The natural hosts of Shigella are conventionally humans and other primates; however, the host range of Shigella has been shown to expand to many animals. Although Shigella is becoming a huge threat to animals, there is limited information on the genetic background of local strains. The purpose of this study was to assess the presence of virulence factors and the molecular characteristics of S. flexneri isolated from calves with diarrhea. Methods: From 2014 to 2016, 54 S. flexneri isolates were collected from diarrhea, and their biochemical characteristics were determined according to API20E and virulence factors via PCR. The molecular characteristics of the isolates were studied by MLST, MLVA and PFGE. Results: Fifty-four S. flexneri isolates possessed four typical biochemical characteristics of Shigella. The prevalences of ipaH, virA, ipaBCD, ial, sen, set1A, and set1B were 100%, 100%, 77.78%, 79.63%, 48.15% and 48.15%, respectively. None of the studied strains possessed the stx gene. Regarding the differences in virulence factor distributions, the 54 S. flexneri isolates fell into seven gene profile types. Among these VTs, VT4 and VT6 were the most common, accounting for 74.07% of all VTs. MLVA based on 8 VNTR loci discriminated the isolates into 39 different MTs, PFGE based on NotI digestion divided the 54 isolates into 31 PTs, and MLST based on 15 housekeeping genes differentiated the isolates into 7 STs, with 1 ST (ST227) being novel. Conclusion: Our findings provide baseline information on the distribution of virulence genes in and the molecular characteristics of S. flexneri collected from diarrheal calves, which is a potential threat to public safety. These data will be important for addressing clinical and epidemiological issues regarding Shigellosis.
... Each unique allelic string was designated a unique MLVA type (MT). A dendrogram was constructed by UPGMA clustering based on categorical coe cient analysis [33,45]. ...
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Background The natural hosts of Shigella are conventionally humans and other primates; however, the host range of Shigella has been shown to expand to many animals. Although Shigella is becoming a huge threat to animals, there is limited information on the genetic background of local strains. The purpose of this study was to assess the presence of virulence factors and the molecular characteristics of S. flexneri isolated from calves with diarrhea. Results Fifty-four S. flexneri isolates possessed four typical biochemical characteristics of Shigella. The prevalences of ipaH, virA, ipaBCD, ial, sen, set1A, set1B and stx were 100%, 100%, 77.78%, 79.63%, 48.15%, 48.15% and 0, respectively. MLVA based on 8 VNTR loci discriminated the isolates into 39 different MTs, PFGE based on NotI digestion divided the 54 isolates into 31 PTs, and MLST based on 15 housekeeping genes differentiated the isolates into 7 STs. Conclusion Our findings of this study have enriched our knowledge of the molecular characteristics of S. flexneri collected from diarrheal calves, which will be important for addressing clinical and epidemiological issues regarding Shigellosis.
... Each unique allelic string was designated a unique MLVA type (MT). A dendrogram was constructed by UPGMA clustering based on categorical coefficient analysis [27,29]. ...
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Full-text available
Background: The natural hosts of Shigella are conventionally humans and other primates; however, the host range of Shigella has been shown to expand to many animals. Although Shigella is becoming a huge threat to animals, there is limited information on the genetic background of local strains. The purpose of this study was to assess the presence of virulence factors and the molecular characteristics of S. flexneri isolated from calves with diarrhea. Methods: From 2014 to 2016, 54 S. flexneri isolates were collected from diarrhea, and their biochemical characteristics were determined according to API20E and virulence factors via PCR. The molecular characteristics of the isolates were studied by MLST, MLVA and PFGE. Results: Fifty-four S. flexneri isolates possessed four typical biochemical characteristics of Shigella. The prevalences of ipaH, virA, ipaBCD, ial, sen, set1A, and set1B were 100%, 100%, 77.78%, 79.63%, 48.15% and 48.15%, respectively. None of the studied strains possessed the stx gene. Regarding the differences in virulence factor distributions, the 54 S. flexneri isolates fell into seven gene profile types. Among these VTs, VT4 and VT6 were the most common, accounting for 74.07% of all VTs. MLVA based on 8 VNTR loci discriminated the isolates into 39 different MTs, PFGE based on NotI digestion divided the 54 isolates into 31 PTs, and MLST based on 15 housekeeping genes differentiated the isolates into 7 STs, with 1 ST (ST227) being novel. Conclusion: Our findings provide baseline information on the distribution of virulence genes in and the molecular characteristics of S. flexneri collected from diarrheal calves, which is a potential threat to public safety. These data will be important for addressing clinical and epidemiological issues regarding Shigellosis.
... ). A dendrogram was constructed by UPGMA clustering based on categorical coe cient analysis[35,50].DNA ngerprinting was performed by PFGE with the restriction enzyme NotI (TaKaRa; Japan) according to the international standards set by the CDC. PFGE images were photographed with a Universal Hood II (Bio-Rad; USA) and analyzed with BioNumerics using the Dice similarity coe cient, unweighted pair-group method with the arithmetic mean (UPGMA) and 1.0% band position tolerance. ...
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Background The natural hosts of Shigella are conventionally humans and other primates; however, the host range of Shigella has been shown to expand to many animals. Although Shigella is becoming a huge threat to animals, there is limited information on the genetic background of local strains. The purpose of this study was to assess the presence of virulence factors and the molecular characteristics of S. flexneri isolated from calves with diarrhea. Results Fifty-four S. flexneri isolates possessed four typical biochemical characteristics of Shigella. The prevalences of ipaH, virA, ipaBCD, ial, sen, set1A, set1B and stx were 100%, 100%, 77.78%, 79.63%, 48.15%, 48.15% and 0, respectively. MLVA based on 8 VNTR loci discriminated the isolates into 39 different MTs, PFGE based on NotI digestion divided the 54 isolates into 31 PTs, and MLST based on 15 housekeeping genes differentiated the isolates into 7 STs. Conclusion Our findings of this study have enriched our knowledge of the molecular characteristics of S. flexneri collected from diarrheal calves, which will be important for addressing clinical and epidemiological issues regarding Shigellosis.
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Pleurotus ostreatus is one of the most important edible mushrooms. Many cultivars have been bred to meet consumer needs. The identification of cultivars based on the morphological characteristics is restricted because fruiting bodies are frequently capricious due to environmental conditions; accordingly, sequence-based methods are required. A total of 546 simple sequence repeat primers derived from the P. ostreatus genome were screened, and one primer, JHH_SSR-184, was found to show polymorphisms on the major cultivars in Korea. The sequences of the polymorphic loci showed variable-number tandem repeat loci-like features enabling cultivar specificity. Thus, these loci might be applicable to discriminate P. ostreatus cultivars.
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Shiga toxin producing Escherichia coli O157:H7 (STEC O157) is naturally found in the gastrointestinal tract of cattle and can cause severe disease in humans. There is limited understanding of the population dynamics and microevolution of STEC O157 at herd level. In this study, isolates from a closed beef herd of 23 cows were used to examine the population turnover in the herd. Of the nine STEC O157 clades previously described, clade 7 was found in 162 of the 169 isolates typed. Multiple locus variable number tandem repeat analysis (MLVA) analysis differentiated 169 isolates into 33 unique MLVA types. Five predominant MLVA types were evident with most of the remaining types containing only a single isolate. MLVA data suggest that over time clonal replacement occurred within the herd. Genome sequencing of 18 selected isolates found that the isolates were divided into four lineages, representing four different ‘clones' in the herd. Genome data confirmed clonal replacement over time and provided evidence of cross transmission of strains between cows. The findings enhanced our understanding of the population dynamics of STEC O157 in its natural host that will help developing effective control measures to prevent the spread of the pathogen to the human population. This article is protected by copyright. All rights reserved.
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This study examined the potential pathogenicity of Shiga toxin-producing Escherichia coli (STEC) in feces of sika deer by PCR binary typing (P-BIT), using 24 selected STEC genes. A total of 31 STEC strains derived from sika deer in 6 prefectures of Japan were O-serotyped and found to be O93 (n=12), O146 (n=5), O176 (n=3), O130 (n=3), O5 (n=2), O7 (n=1), O96 (n=1), O116 (n=1), O141 (n=1), O157 (n=1) and O-untypable (n=1). Of the 31 STEC strains, 13 carried both stx1 and stx2, 5 carried only stx1, and 13 carried one or two variants of stx2. However, no Stx2 production was observed in 3 strains that carried only stx2: the other 28 strains produced the appropriate Stx. P-BIT analysis showed that the 5 O5 strains from two wild deer formed a cluster with human STEC strains, suggesting that the profiles of the presence of the 24 P-BIT genes in the deer strains were significantly similar to those in human strains. All of the other non-O157 STEC strains in this study were classified with strains from food, domestic animals and humans in another cluster. Good sanitary conditions should be used for deer meat processing to avoid STEC contamination, because STEC is prevalent in deer and deer may be a potential source of STEC causing human infections.
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During 2007-2010, 13 545 confirmed human VTEC infections and 777 haemolytic uraemic syndrome (HUS) cases were reported in the EU; isolates from 85 % of cases were not fully serotyped and therefore could not be classified using the Karmali seropathotype concept. Seropathotype group D covered 5 % of isolates from fully serotyped cases; 14 cases (0.7 %) belonged to seropathotype group E, defined by Karmali et al. (2003) as non-human only. Isolates from around 27 % of cases could not be assigned. There were no HUS cases reported for the serotypes in groups D and E but 17 HUS cases could not be assigned. The health outcome was reported for only a fraction of confirmed cases. About 64 % of patients presented with only diarrhoea; VTEC infection resulted in HUS in around 10 % of cases. The new ISO/TS 13136:2012 standard improves the detection of VTEC in food. An alternative concept based on the detection of verocytotoxins alone or genes encoding such verocytotoxins does not provide a sound scientific basis on which to assess risk to the consumer because there is no single or combination of marker(s) that fully define a ‘pathogenic’ VTEC. Strains positive for verocytotoxin 2 gene(vtx2)- and eae (intimin production)- or [aaiC (secreted protein of EAEC) plus aggR (plasmid-encoded regulator)] genes are associated with higher risk of more severe illness than other virulence gene combinations. The 2011 O104:H4 outbreak demonstrated the difficulty of predicting the emergence of ‘new’ pathogenic VTEC types by screening only for the eae gene or by focusing on a restricted panel of serogroups. A molecular approach utilising genes encoding virulence characteristics additional to the presence of vtx genes has been proposed.
Chapter
Molecular diagnostic techniques are based on the detection of a fragment of genetic material (nucleic acids - i.e. DNA or RNA) which is unique to the target organism and, as such, they are highly specific. Many methods include an amplification step for the target DNA/RNA. Microarrays were originally used for the study of gene expression, but oligonucleotide DNA microarrays have been developed and applied to the detection of a number of food-borne pathogens including E. coli O157:H7, Listeria monocytogenes, and Campylobacter. In the food industry, molecular characterisation and typing of isolates can give scientific evidence about where a pathogenic or spoilage micro-organisms is entering the chain, where cross-contamination may be occurring, and can demonstrate if particular strains are endemic in a factory environment, allowing focused and effective risk-based management decisions. This chapter describes a range of genotypic techniques for the detection of bacteria in food.
Chapter
The discipline of molecular epidemiology applied to infectious disease uses the tools of molecular microbiology to characterize the distribution and determinants of infectious disease in human and nonhuman animal populations. Advancements made in this discipline have paralleled the advancements made in molecular microbiology to subtype microbes. This chapter describes genotyping techniques that are frequently used in epidemiologic investigations, including outbreak investigations, disease surveillance, healthcare-associated infection control, and other public health activities. The application of “next-generation sequencing” technology to epidemiologic investigations will discuss the unique advantages this new technology offers but also its challenges. Examples of the use of these techniques to investigate bacterial infectious diseases are highlighted. These examples will emphasize those studies that contributed to the implementation of new public health measures that otherwise could not have been made.
Chapter
A series of outbreaks of infection with Shiga toxin (or verotoxin [VT])‐producing Escherichia coli or enterohemorrhagic E. coli (EHEC) O157:H7 occurred in Japan in 1996, the largest outbreak occurring in primary schools in Sakai City, Osaka Prefecture, where more than 7,500 cases were reported ( 1 ). Although the reason for the sudden increase in the number of reports of EHEC isolates in 1996 is not known, the number of reports has grown to more than 3,000 cases per year since 1996 from an average of 105 cases reported each year during the previous 5‐year period (1991–1995) ( 2 ). Despite control measures instituted since 1996, including designating EHEC infection as a notifiable disease, and the disease being monitored effectively through nationwide surveillance, the number of reports remains high, around 3,800 cases per year ( Fig. 1 ). Serogroup O157 predominates over other EHEC serogroups, but isolation frequency of non‐O157 EHEC has gone up slightly over the past few years. Non‐O157 EHEC has caused outbreaks where consumption of a raw beef dish was the source of the infection and some fatal cases were occurred. Laboratory surveillance consisting of prefectural and municipal public health institutes (PHIs) and the National Institute of Infectious Diseases has contributed to finding not only multiprefectural outbreaks but also recognizing sporadic cases that could have been missed as an outbreak without the aid of molecular subtyping of EHEC isolates. This short overview presents recent information on the surveillance of EHEC infections in Japan.
Article
Multilocus variable-number tandem-repeat analysis (MLVA) is a widely accepted molecular typing tool for enterohemorrhagic Escherichia coli (EHEC). However, ensuring the accuracy of MLVA data among multiple laboratories remains difficult. We developed a method of constructing adjusted look-up tables, which are necessary for MLVA profiling, at each laboratory using a regression analysis based on electrophoresis data from 24 in-house reference strains. On performing MLVA against 51 EHEC O157 isolates, the repeat numbers of 46 isolates were determined accurately using the look-up table with a 99% prediction interval, an outcome superior to that when using a 95% prediction interval. For the remaining five isolates, although the electrophoresis size fell outside the look-up table, we were able to predict the repeat number accurately by extrapolation or the nearest values of the look-up table. Our approach provides more accurate results than a nonadjusted conventional look-up table for calibrating MLVA profiles.
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Introduction. The Philippines, comprising three island groups, namely, Luzon, Visayas and Mindanao, experienced an increase in cholera outbreaks in 2016. Previous studies have shown that Vibrio cholerae isolates obtained from the Philippines are novel hybrid El Tor strains that have evolved in the country and are clearly distinct from those found in Mozambique and Cameroon. Gap statement. The characterization of the strains isolated from outbreaks has been limited to phenotypic characteristics, such as biochemical and serological characteristics, in most previous studies. Aim. We performed multilocus variable-number tandem repeat (VNTR) analysis (MLVA) for V. cholerae isolates obtained from 2015 to 2016 to further characterize and understand the emergence and dissemination of the strains in the Philippines. Methodology. A total of 139 V . cholerae O1 Ogawa biotype El Tor isolates were obtained from the Philippines during diarrhoeal outbreaks in 18 provinces between 2015 and 2016. VNTR data were analysed to classify the MLVA profiles where the large-chromosome types (LCTs) were applied for grouping. Results. We identified 50 MLVA types among 139 isolates originating from 18 provinces, and 14 LCTs. The distribution of the LCTs was variable, and a few were located in specific areas or even in specific provinces. Based on eBURST analysis, 99 isolates with 7 LCTs and 32 MLVA types belonged to 1 group, suggesting that they were related to each other. LCT A was predominant ( n =67) and was isolated from Luzon and Visayas. LCT A had 14 MLVA types; however, it mostly emerged during a single quarter of a year. Eight clusters were identified, each of which involved specific MLVA type(s). The largest cluster involved 23 isolates showing 3 MLVA types, 21 of which were MLVA type A-14 isolated from Negros Occidental during quarter 4 of 2016. Comparative analysis showed that almost all isolates from the Philippines were distinct from those in other countries. Conclusions. The genotypic relationship of the V. cholerae isolates obtained during outbreaks in the Philippines was studied, and their emergence and dissemination were elucidated. MLVA revealed the short-term dynamics of V. cholerae genotypes in the Philippines.
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Single-nucleotide polymorphisms (SNPs) are one of the most common forms of genetic variation and as such are powerful tools for the identification of bacterial strains, their genetic diversity, phylogenetic analysis, and outbreak surveillance. In this study, we used 15 sets of SNP-containing primers to amplify and sequence the target Escherichia coli. Based on the combination of the 15-sequence primer sets, each SNP site encompassing forward and reverse primer sequences (620–919 bp) were aligned and an SNP-based marker was designed. Each SNP marker exists in at least two SNP sites at the 3′ end of each primer; one natural and the other artificially created by transition or transversion mutation. Thus, 12 sets of SNP primers (225–488 bp) were developed for validation by amplifying the target E. coli. Finally, a temperature gradient triplex PCR kit was designed to detect target E. coli strains. The selected primers were amplified in three genes (ileS, thrB, and polB), with fragment sizes of 401, 337, and 232 bp for E. coli O157:H7, E. coli, and E. coli O145:H28, respectively. This allele-specific SNP-based triplex primer assay provides serotype-specific detection of E. coli strains in one reaction tube. The developed marker would be used to diagnose, investigate, and control food-borne E. coli outbreaks.
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Enterohemorrhagic Escherichia coli O157 (EHEC) causes severe complications such as hemolytic uremic syndrome. Contaminated ready-to-eat (RTE) food is one of the vehicles of multijurisdictional outbreaks of foodborne disease worldwide. Multijurisdictional (covering cities, towns, and villages) outbreaks of EHEC are usually linked to an increase in cases, and here we describe such an outbreak involving 29 cases in October 2017 in the Niigata Prefecture. After prefecture-wide active case finding, we conducted a case-control study of 29 cases with eligible data who tested positive for EHEC. To determine the association of the outbreak with risk factors, we compared these cases with 38 controls selected from family and acquaintances who were both symptom free and tested negative for EHEC. The largest number of cases was in the 20-29-year age group (7/29; 24%) and most were women (20/29; 69%). All 29 cases had an identical or similar multilocus variable number tandem-repeat analysis (MLVA) profile. Of these, 76% (22/29) had consumed some type of grilled skewered meat. Also, 69% (20/29) had consumed grilled skewered meat produced by company X. EHEC infection was strongly associated with the consumption of grilled skewered meat produced by any food processing company (odds ratio [OR] = 11.8, confidence interval [95% CI]: 3.7-37.4) and by company X (OR = 9.8, 95% CI: 3.2-30.7). At company X, the skewered meat was grilled to 95°C and then removed from the grilling area to meat trays. The meat trays were not sufficiently washed and disinfected. Testing indicated that the facility was negative for EHEC but four asymptomatic employees tested positive for EHEC. Company X was temporarily closed and voluntarily recalled the foods. We recommend that all employees sufficiently wash and disinfect meat trays to prevent contamination of RTE food, avoid cross-contamination of grilled skewered meat through the environment by regularly cleaning the facility, and appropriately practice self-health care.
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Cholera is an infectious disease of major concern in Vietnam and other Asian countries. In 2009, there was a large outbreak of cholera in northern Vietnam. To investigate relationships among isolates of the causative pathogen Vibrio cholerae in this region since 2007, we carried out a multilocus variable-number tandem repeat analysis (MLVA) of 170 isolates collected between 2007 and 2009. A total of 24 MLVA types were identified using seven loci. Five clones (1–5) were identified using five loci of the large V. cholerae chromosome; clones 1 and 2 were major, and the others were minor. Clone 1 isolates were responsible for the 2009 outbreak. A shift in the predominant clone occurred between 2007 and 2009, with clone 1 likely derived from clone 2. Moreover, the former was less diverse than the latter, suggesting a single source of cholera dissemination. Epidemiological data indicated a wavelet prior to the large outbreak, suggesting that drinking water source or food chain became contaminated during dissemination. Our results reveal the utility of MLVA for analysis of V. cholerae isolates within a relatively short period and broaden our understanding of its transmission and response to cholera.
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Escherichia coli O157:H7 is a major cause of food-borne illness in the United States. Outbreak detection involves traditional epidemiological methods and routine molecular subtyping by pulsed-field gel electrophoresis (PFGE). PFGE is labor-intensive, and the results are difficult to analyze and not easily transferable between laboratories. Multilocus variable-number tandem repeat (VNTR) analysis (MLVA) is a fast, portable method that analyzes multiple VNTR loci, which are areas of the bacterial genome that evolve quickly. Eighty isolates, including 21 isolates from five epidemiologically well-characterized outbreaks from Pennsylvania and Minnesota, were analyzed by PFGE and MLVA. Strains in PFGE clusters were defined as strains that differed by less than or equal to one band by using XbaI and the confirmatory enzyme SpeI. MLVA was performed by comparing the number of tandem repeats at seven loci. From 6 to 30 alleles were found at the seven loci, resulting in 64 MLVA types among the 80 isolates. MLVA correctly identified the isolates from all five outbreaks if only a single-locus variant was allowed. MLVA differentiated strains with unique PFGE types. Additionally, MLVA discriminated strains within PFGE-defined clusters that were not known to be part of an outbreak. In addition to being a simple and validated method for E. coli O157:H7 outbreak detection, MLVA appears to have a sensitivity equal to that of PFGE and a specificity superior to that of PFGE.
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Traditional and molecular typing schemes for the characterization of pathogenic microorganisms are poorly portable because they index variation that is difficult to compare among laboratories. To overcome these problems, we propose multilocus sequence typing (MLST), which exploits the unambiguous nature and electronic portability of nucleotide sequence data for the characterization of microorganisms. To evaluate MLST, we determined the sequences of approximately 470-bp fragments from 11 housekeeping genes in a reference set of 107 isolates of Neisseria meningitidis from invasive disease and healthy carriers. For each locus, alleles were assigned arbitrary numbers and dendrograms were constructed from the pairwise differences in multilocus allelic profiles by cluster analysis. The strain associations obtained were consistent with clonal groupings previously determined by multilocus enzyme electrophoresis. A subset of six gene fragments was chosen that retained the resolution and congruence achieved by using all 11 loci. Most isolates from hyper-virulent lineages of serogroups A, B, and C meningococci were identical for all loci or differed from the majority type at only a single locus. MLST using six loci therefore reliably identified the major meningococcal lineages associated with invasive disease. MLST can be applied to almost all bacterial species and other haploid organisms, including those that are difficult to cultivate. The overwhelming advantage of MLST over other molecular typing methods is that sequence data are truly portable between laboratories, permitting one expanding global database per species to be placed on a World-Wide Web site, thus enabling exchange of molecular typing data for global epidemiology via the Internet.
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Pulsed-field gel electrophoresis (PFGE) has been used extensively in epidemiological investigations of bacteria, especially during food-borne outbreaks or nosocomial infections. The relationship between similarities in PFGE patterns and true genetic relatedness is poorly understood. In this study, computer-simulated populations of Escherichia coli isolates were created by mutating the sequence of E. coli K-12 strain MG1655. The simulated populations of isolates were then digested, again through simulation, with different restriction enzymes and were analyzed for their relatedness by different techniques. Errors associated with band determination and band matching were incorporated into the analyses, as both of these error types have been shown to affect PFGE interpretations. These errors increased the apparent similarities of the isolates. The use of multiple enzymes improved the fidelity between the results of PFGE analyses and the true sequence similarities. These findings, when they are combined with results from laboratory studies, emphasize the need for the inclusion of multiple enzymes and additional epidemiological data in order to make more accurate interpretations.
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THE genomes of all eukaryotes contain tracts of DNA in which a single base or a small number of bases is repeated. Expansions of such tracts have been associated with several human disorders including the fragile X syndrome1. In addition, simple repeats are unstable in certain forms of colorectal cancer, suggesting a defect in DNA replication or repair2-4. We show here that mutations in any three yeast genes involved in DNA mismatch repair (PMS1, MLH1 and MSH2) lead to 100- to 700-fold increases in tract instability, whereas mutations that eliminate the proof-reading function of DNA polymerases have little effect. The meiotic stability of the tracts is similar to the mitotic stability. These results suggest that tract instability is associated with DNA poly-merases slipping during replication, and that some types of colo-rectal cancer may reflect mutations in genes involved in DNA mismatch repair.
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A large collection of good genetic markers is needed to map the genes that cause human genetic diseases. Although nearly 400 polymorphic DNA markers for human chromosomes have been described, the majority have only two alleles and are thus uninformative for analysis of genetic linkage in many families. A few known marker systems, however, detect loci that respond to restriction enzyme cleavage by producing a fragment that can have many different lengths. This polymorphism is due to variation in the number of tandem repeats of a short DNA sequence. Because most individuals will be heterozygous at such loci, these markers will provide linkage information in almost all families. Ten oligomeric sequences derived from the tandem repeat regions of the myoglobin gene, the zeta-globin pseudogene, the insulin gene, and the X-gene region of hepatitis B virus, were used to develop a series of single-copy probes. These probes revealed new, highly polymorphic genetic loci whose allele sizes reflected variation in the number of tandem repeats.
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The genomes of all eukaryotes contain tracts of DNA in which a single base or a small number of bases is repeated. Expansions of such tracts have been associated with several human disorders including the fragile X syndrome. In addition, simple repeats are unstable in certain forms of colorectal cancer, suggesting a defect in DNA replication or repair. We show here that mutations in any three yeast genes involved in DNA mismatch repair (PMS1, MLH1 and MSH2) lead to 100- to 700-fold increases in tract instability, whereas mutations that eliminate the proof-reading function of DNA polymerases have little effect. The meiotic stability of the tracts is similar to the mitotic stability. These results suggest that tract instability is associated with DNA polymerases slipping during replication, and that some types of colorectal cancer may reflect mutations in genes involved in DNA mismatch repair.
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The mechanisms underlying the evolution and emergence of new bacterial pathogens are not well understood. To elucidate the evolution of pathogenic Escherichia coli strains, here we sequenced seven housekeeping genes to build a phylogenetic tree and trace the history of the acquisition of virulence genes. Compatibility analysis indicates that more than 70% of the informative sites agree with a single phylogeny, suggesting that recombination has not completely obscured the remnants of ancestral chromosomes. On the basis of the rate of synonymous substitution for E. coli and Salmonella enterica (4.7 x 10(-9) per site per year), the radiation of clones began about 9 million years ago and the highly virulent pathogen responsible for epidemics of food poisoning, E. coli O157:H7, separated from a common ancestor of E. coli K-12 as long as 4.5 million years ago. Phylogenetic analysis reveals that old lineages of E. coli have acquired the same virulence factors in parallel, including a pathogenicity island involved in intestinal adhesion, a plasmid-borne haemolysin, and phage-encoded Shiga toxins. Such parallel evolution indicates that natural selection has favoured an ordered acquisition of genes and the progressive build-up of molecular mechanisms that increase virulence.
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PulseNet, the national molecular subtyping network for foodborne disease surveillance, was established by the Centers for Disease Control and Prevention and several state health department laboratories to facilitate subtyping bacterial foodborne pathogens for epidemiologic purposes. PulseNet, which began in 1996 with 10 laboratories typing a single pathogen (Escherichia coli O157:H7), now includes 46 state and 2 local public health laboratories and the food safety laboratories of the U.S. Food and Drug Administration and the U.S. Department of Agriculture. Four foodborne pathogens (E. coli O157:H7; nontyphoidal Salmonella serotypes, Listeria monocytogenes and Shigella) are being subtyped, and other bacterial, viral, and parasitic organisms will be added soon.
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A multi-virulence-locus sequence typing (MVLST) scheme was developed for subtyping Listeria monocytogenes, and the results obtained using this scheme were compared to those of pulsed-field gel electrophoresis (PFGE) and the published results of other typing methods, including ribotyping (RT) and multilocus sequence typing (MLST). A set of 28 strains (eight different serotypes and three known genetic lineages) of L. monocytogenes was selected from a strain collection (n > 1,000 strains) to represent the genetic diversity of this species. Internal fragments (ca. 418 to 469 bp) of three virulence genes (prfA, inlB, and inlC) and three virulence-associated genes (dal, lisR, and clpP) were sequenced and analyzed. Multiple DNA sequence alignment identified 10 (prfA), 19 (inlB), 13 (dal), 10 (lisR), 17 (inlC), and 16 (clpP) allelic types and a total of 28 unique sequence types. Comparison of MVLST with automated EcoRI-RT and PFGE with ApaI enzymatic digestion showed that MVLST was able to differentiate strains that were indistinguishable by RT (13 ribotypes; discrimination index = 0.921) or PFGE (22 profiles; discrimination index = 0.970). Comparison of MVLST with housekeeping-gene-based MLST analysis showed that MVLST provided higher discriminatory power for serotype 1/2a and 4b strains than MLST. Cluster analysis based on the intragenic sequences of the selected virulence genes indicated a strain phylogeny closely related to serotypes and genetic lineages. In conclusion, MVLST may improve the discriminatory power of MLST and provide a convenient tool for studying the local epidemiology of L. monocytogenes.
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Escherichia coli O157:H7, a Shiga toxin-producing E. coli, has been the causative agent of many cases of severe, often life-threatening foodborne illness. Because of the importance of E. coli O157:H7 to public health, many molecular typing methods have been developed to determine its transmission routes and source of infection during epidemiological investigations. Pulsed-field gel electrophoresis (PFGE) is currently used by public health organizations to track infections of E. coli O157:H7 and other foodborne pathogens. In this study, we compared the ability of PFGE, multilocus sequence typing (MLST), and repetitive-element PCR (Rep-PCR) to distinguish among 92 E. coli O157:H7 isolates from cattle, food, and infected humans. Several virulence genes, including the intimin gene (eaeA), the hemolysin gene (hlyA), and the H7 fimbrial gene (fliC), and a housekeeping gene for beta-glucuronidase (uidA) were included in MLST. Rep-PCR reactions were performed using a commercially available typing kit (Bacterial Barcodes Inc., Houston, Tex.) with the provided Uprime-RI primer set. Results of the study indicated that PFGE provided the most discrimination among the techniques, identifying 72 distinct PFGE profiles for the isolates; Rep-PCR elucidated 14 different profiles, whereas MLST generated five profiles. Additionally, there did not appear to be any correlation among the typing methods examined in this study. Therefore, to date, PFGE remains the technique of choice for molecular subtyping of E. coli O157:H7.
Article
The Multiple-Locus Variable-Number Tandem-Repeats Analysis (MLVA) method is currently being used as the primary typing tool for Shiga-toxin-producing Escherichia coli (STEC) O157 isolates in our laboratory. The initial assay was performed using a single fluorescent dye and the different patterns were assigned using a gel image. Here, we present a significantly improved assay using multiple dye colors and enhanced PCR multiplexing to increase speed, and ease the interpretation of the results. The different MLVA patterns are now based on allele sizes entered as character values, thus removing the uncertainties introduced when analyzing band patterns from the gel image. We additionally propose an easy numbering scheme for the identification of separate isolates that will facilitate exchange of typing data. Seventy-two human and animal strains of Shiga-toxin-producing E. coli O157 were used for the development of the improved MLVA assay. The method is based on capillary separation of multiplexed PCR products of VNTR loci in the E. coli O157 genome labeled with multiple fluorescent dyes. The different alleles at each locus were then assigned to allele numbers, which were used for strain comparison.
Article
Evaluation of the Escherichia coli genome for variable number tandem repeat (VNTR) loci in order to provide a subtyping tool with greater discrimination and more efficient capacity. Twenty-nine putative VNTR loci were identified from the E. coli genomic sequence. Their variability was validated by characterizing the number of repeats at each locus in a set of 56 E. coli O157:H7/HN and O55:H7 isolates. An optimized multiplex assay system was developed to facility high capacity analysis. Locus diversity values ranged from 0.23 to 0.95 while the number of alleles ranged from two to 29. This multiple-locus VNTR analysis (MLVA) data was used to describe genetic relationships among these isolates and was compared with PFGE (pulse field gel electrophoresis) data from a subset of the same strains. Genetic similarity values were highly correlated between the two approaches, through MLVA was capable of discrimination amongst closely related isolates when PFGE similar values were equal to 1.0. Highly variable VNTR loci exist in the E. coli O157:H7 genome and are excellent estimators of genetic relationships, in particular for closely related isolates. Escherichia coli O157:H7 MLVA offers a complimentary analysis to the more traditional PFGE approach. Application of MLVA to an outbreak cluster could generate superior molecular epidemiology and result in a more effective public health response.
Multilo-cus sequence typing: a portable approach to the
  • M C Maiden
  • J A Bygraves
  • E Feil
Maiden, M.C., J.A. Bygraves, E. Feil, et al. 1998. Multilo-cus sequence typing: a portable approach to the