Characterization of the flanking region of the Shiga toxin operon in Stx2a bacteriophages reveals a diversity of the NanS-p sialate O-acetylesterase gene.

Shiga toxin-producing E. coli (STEC) are diarrheagenic strains that can cause bloody diarrhea and hemolytic-uremic syndrome. Their main virulence factor, the Shiga toxin (Stx), is encoded by phages integrated into the bacterial chromosome. Stx phages are widely diverse and carry many genes with limited or unknown function. As the toxin subtype Stx2a is associated with highly pathogenic strains, this study was mainly focused on the characterization of the stx flanking region of Stx2a phages. Of particular interest was a sialate O-acetylesterase (NanS-p), which has been described previously to be encoded downstream stx in some phage genomes and may confer a growth advantage for STEC. Complete DNA sequences of Stx2a phages and prophages were retrieved from the GenBank database, and the genomic regions from anti-terminator Q to holin S genes were bioinformatically analyzed. Predicted NanSp sequences from phages encoding other Stx subtypes were also studied. Additionally, expression of nanS-p was quantified by qPCR in strains selected from our laboratory collection. The analysis of Stx2a phage genomes showed that all carried the Q, stx2a, nanS-p and S genes, but with allele diversity and other sequence differences. In particular, sequence differences were detected in each of the three domains of NanS-p esterases encoded by Stx2a phages and other Stx phages; however, nanS-p was not identified in the Stx2e, Stx2f and Stx2g phages analyzed. The expression of nanS-p increased in most stx2a-positive strains under phage inducing conditions, as was previously shown for stx2a. As the present work showed diversity at the Q-S region among Stx phages, and particularly in the encoded NanS-p enzyme, future studies will be necessary to evaluate if NanS-p variants differ in their activity and to assess the impact of the absence of nanS-p in certain Stx phages.

Comparable stx2a expression and phage production levels between Shiga toxin-producing Escherichia coli strains from human and bovine origin.

Shiga toxin-producing Escherichia coli (STEC) can cause diarrhoea and severe diseases in humans, such as haemolytic uraemic syndrome. STEC virulence is considered to correlate with the amount of Shiga toxins (Stx) produced, especially Stx2, whose subtype Stx2a is most frequently associated with high virulence. Stx are encoded in prophages, which play an important role in STEC pathogenesis. The aim of this study was to evaluate stx2a expression levels and Stx2a phage production using qPCR and the double-agar-layer method in 29 STEC strains, corresponding to serotypes O26:H11 (6), O91:H21 (1), O145:H- (11) and O157:H7 (11), isolated from cattle and humans. Results were then tested for possible associations with serotype, origin or some genetic features. We observed heterogeneous levels of stx2a expression and Stx2a phage production. However, statistical comparisons identified a higher stx2a expression in response to mitomycin C in strains isolated from cattle than in those from humans. At the same time, compared to stx2a /stx2c strains, stx2a strains showed a higher increase in phage production under induced conditions. Notably, most of the strains studied, regardless of serotype and origin, carried inducible Stx2a phages and evidenced expression of stx2a that increased along with phage production levels under induced conditions.

Serotipos, perfiles de virulencia y subtipos de stx en Escherichia coli productor de toxina Shiga aislados de productos de pollo / Serotypes, virulence profiles and stx subtypes of Shigatoxigenic Escherichia coli isolated from chicken derived products

Shigatoxigenic Escherichia coli (STEC) is a foodborne pathogen that causes hemolytic uremic syndrome (HUS) and the consumption of chicken products has been related to some HUS cases. We performed a non-selective isolation and characterization of STEC strains from retail chicken products. STEC isolates were characterized according to the presence of stx1, stx2, eae, saa and ehxA; stx subtypes and serotypes. Most of them carried stx2, showing subtypes associated with severe human disease. Although reported in other avian species, the stx2f subtype was not detected. The isolates corresponded to different serotypes and some of them, such as O22:H8, O113:H21, O130:H11, O171:H2 and O178:H19, have also been identified among STEC isolated from patients suffering from diarrhea, hemorrhagic colitis, HUS, as well as from cattle. Considering the virulence profiles and serotypes identified, our results indicate that raw chicken products, especially hamburgers sold at butcheries, can be vehicles for high-risk STEC strains.

Escherichia coli productor de toxina de Shiga (STEC) es un patógeno transmitido por alimentos que causa el síndrome urémico hemolítico (SUH). Algunos casos de SUH están relacionados con el consumo de productos de pollo. Se realizó el aislamiento no selectivo y la caracterización de cepas STEC provenientes de productos de pollo atendiendo a la presencia de stx1, stx2, eae, saa y ehxA, subtipos de stx y serotipos. La mayoría de los aislamientos portaba stx2 y subtipos de stx asociados con enfermedades graves en humanos. Aunque se ha detectado en otras especies aviares, el subtipo stx2f no se encontró. Se detectaron diferentes serotipos, entre ellos O22:H8, O113:H21, O130:H11, O171:H2 y O178:H19, también identificados como STEC aislados de pacientes con diarrea, colitis hemorrágica y SUH, y de ganado bovino. Teniendo en cuenta los perfiles de virulencia y los serotipos identificados, nuestros resultados indican que los productos de pollo crudos, especialmente las hamburguesas que se venden en las carnicerías, pueden ser vehículos de cepas STEC de alto riesgo.

Serotypes, virulence profiles and stx subtypes of Shigatoxigenic Escherichia coli isolated from chicken derived products.

Shigatoxigenic Escherichia coli (STEC) is a foodborne pathogen that causes hemolytic uremic syndrome (HUS) and the consumption of chicken products has been related to some HUS cases. We performed a non-selective isolation and characterization of STEC strains from retail chicken products. STEC isolates were characterized according to the presence of stx1, stx2, eae, saa and ehxA; stx subtypes and serotypes. Most of them carried stx2, showing subtypes associated with severe human disease. Although reported in other avian species, the stx2f subtype was not detected. The isolates corresponded to different serotypes and some of them, such as O22:H8, O113:H21, O130:H11, O171:H2 and O178:H19, have also been identified among STEC isolated from patients suffering from diarrhea, hemorrhagic colitis, HUS, as well as from cattle. Considering the virulence profiles and serotypes identified, our results indicate that raw chicken products, especially hamburgers sold at butcheries, can be vehicles for high-risk STEC strains.

Effect of the Food Additives Sodium Citrate and Disodium Phosphate on Shiga Toxin-Producing Escherichia coli and Production of stx-Phages and Shiga toxin.

Induction and propagation of bacteriophages along the food production chain can represent a significant risk when bacteriophages carry genes for potent toxins. The aim of this study was to evaluate the effect of different compounds used in the food industry on the growth of Shiga toxin-producing Escherichia coli (STEC) and the production of stx-phage particles and Shiga toxin. We tested the in vitro effect of lactic acid, acetic acid, citric acid, disodium phosphate, and sodium citrate on STEC growth. A bacteriostatic effect was observed in most of treated cultures. The exceptions were those treated with sodium citrate and disodium phosphate in which similar growth curves to the untreated control were observed, but with reduced OD600 values. Evaluation of phage production by plaque-based assays showed that cultures treated with sodium citrate and disodium phosphate released phages in similar o lower levels than untreated cultures. However, semi-quantification of Stx revealed higher levels of extracellular Stx in STEC cultures treated with 2.5% sodium citrate than in untreated cultures. Our results reinforce the importance to evaluate if additives and other treatments used to decrease bacterial contamination in food induce stx-phage and Stx production.

Genetic characterization of Shiga toxin-producing Escherichia coli O26:H11 strains isolated from animal, food, and clinical samples.

The Shiga-toxin producing Escherichia coli (STEC) may cause serious illness in human. Here we analyze O26:H11 strains known to be among the most reported STEC strains causing human infections. Genetic characterization of strains isolated from animal, food, and clinical specimens in Argentina showed that most carried either stx 1a or stx 2a subtypes. Interestingly, stx 2a-positive O26:H11 rarely isolated from cattle in other countries showed to be an important proportion of O26:H11 strains circulating in cattle and food in our region. Seventeen percent of the isolates harbored more than one gene associated with antimicrobial resistance. In addition to stx, all strains contained the virulence genes eae-ß, tir, efa, iha, espB, cif, espA, espF, espJ, nleA, nleB, nleC, and iss; and all except one contained ehxA, espP, and cba genes. On the other hand, toxB and espI genes were exclusively observed in stx 2-positive isolates, whereas katP was only found in stx 1a-positive isolates. Our results show that O26:H11 STEC strains circulating in Argentina, including those isolated from humans, cattle, and meat products, present a high pathogenic potential, and evidence that cattle can be a reservoir of O26:H11 strains harboring stx 2a.

Shiga toxins and stx phages: highly diverse entities.

Shiga toxins are the main virulence factors of a group of Escherichia coli strains [Shiga toxin-producing E. coli (STEC)] that cause severe human diseases, such as haemorrhagic colitis and haemolytic-uraemic syndrome. The Shiga toxin family comprises several toxin subtypes, which have been differentially related to clinical manifestations. In addition, the phages that carry the Shiga toxin genes (stx phages) are also diverse. These phages play an important role not only in the dissemination of Shiga toxin genes and the emergence of new STEC strains, but also in the regulation of Shiga toxin production. Consequently, differences in stx phages may affect the dissemination of stx genes as well as the virulence of STEC strains. In addition to presenting an overview of Shiga toxins and stx phages, in this review we highlight current knowledge about the diversity of stx phages, with emphasis on its impact on STEC virulence. We consider that this diversity should be taken into account when developing STEC infection treatments and diagnostic approaches, and when conducting STEC control in reservoirs.

[Characterization of enteropathogenic Escherichia coli (EPEC) strains isolated during the chicken slaughtering process]. / Caracterización de cepas de Escherichia coli enteropatogénico (EPEC) aisladas durante el proceso de faena de pollos.

In Argentina, EPEC is one of the most prevalent agents isolated from children with diarrhea. Because contamination with this pathotype could occur during slaughter, the aim of this study was to isolate and characterize EPEC strains obtained from live animals (cloacae), eviscerated carcasses, washed carcasses and water from chillers. Twenty nine isolates of atypical EPEC were characterized. These isolates presented a wide variety of serotypes, some of which (O2:H40, O8:H19 and O108:H9) had been reported in other animal species. Serotype O45:H8, previously isolated from children with diarrhea was also found. Isolates of serotypes O2:H40, O108:H9 and O123:H32 were detected at different stages of the slaughtering process, suggesting that the process is not adequately performed. This latter fact highlights the importance of reinforcing control and hygienic measures at different stages of the chicken slaughtering process in order to reduce microbial contamination.

Caracterización de cepas de Escherichia coli enteropatogénico (EPEC) aisladas durante el proceso de faena de pollos / Characterization of enteropathogenic Escherichiia coli (EPEC) strains isolated during the chicken slaughtering process

En Argentina, Escherichia coli enteropatogénico (EPEC) es uno de los agentes más prevalentes aislados de niños con diarrea. Debido a que la contaminación con este patotipo en productos de pollo podría ocurrir durante el proceso de faena, nos planteamos como objetivo aislar y caracterizar EPEC de muestras de animales vivos (cloacas), carcasas evisceradas sin lavar, carcasas lavadas y agua del tanque de enfriamiento. Se caracterizaron 29 aislamientos de EPEC que presentaron una amplia variedad de serotipos, algunos de los cuales (O2:H40, O8:H19 y O108:H9) han sido informados en otras especies animales. También se encontró el serotipo O45:H8, aislado con anterioridad de niños con diarrea. Se detectaron aislamientos de los serotipos O2:H40, O108:H9 y O123:H32 en distintas etapas del proceso de faena, lo que sugiere que el procesamiento no se realiza en forma adecuada. Se torna necesario reforzar las medidas de control e higiene en las distintas etapas del proceso para disminuir la contaminación microbiana

In Argentina, EPEC is one of the most prevalent agents isolated from children with diarrhea. Because contamination with this pathotype could occur during slaughter, the aim of this study was to isolate and characterize EPEC strains obtained from live animals (cloacae), eviscerated carcasses, washed carcasses and water from chillers. Twenty nine isolates of atypical EPEC were characterized. These isolates presented a wide variety of serotypes, some of which (O2:H40, O8:H19 and O108:H9) had been reported in other animal species. Serotype O45:H8, previously isolated from children with diarrhea was also found. Isolates of serotypes O2:H40, O108:H9 and O123:H32 were detected at different stages of the slaughtering process, suggesting that the process is not adequately performed. This latter fact highlights the importance of reinforcing control and hygienic measures at different stages of the chicken slaughtering process in order to reduce microbial contamination

Caracterización de cepas de Escherichia coli enteropatogénico (EPEC) aisladas durante el proceso de faena de pollos. / [Characterization of enteropathogenic Escherichia coli (EPEC) strains isolated during the chicken slaughtering process].

In Argentina, EPEC is one of the most prevalent agents isolated from children with diarrhea. Because contamination with this pathotype could occur during slaughter, the aim of this study was to isolate and characterize EPEC strains obtained from live animals (cloacae), eviscerated carcasses, washed carcasses and water from chillers. Twenty nine isolates of atypical EPEC were characterized. These isolates presented a wide variety of serotypes, some of which (O2:H40, O8:H19 and O108:H9) had been reported in other animal species. Serotype O45:H8, previously isolated from children with diarrhea was also found. Isolates of serotypes O2:H40, O108:H9 and O123:H32 were detected at different stages of the slaughtering process, suggesting that the process is not adequately performed. This latter fact highlights the importance of reinforcing control and hygienic measures at different stages of the chicken slaughtering process in order to reduce microbial contamination.

Characterization of Shiga toxin-producing Escherichia coli O130:H11 and O178:H19 isolated from dairy cows.

Shiga toxin-producing E. coli (STEC) are isolated from human patients with bloody diarrhea, hemorrhagic colitis (HC), and hemolytic uremic syndrome (HUS). In the last years, the infections with non-O157 serotypes are increasing their frequency of association with human disease. STEC produce Shiga toxin (Stx) and other virulence factors that could contribute to human pathogenesis. Cattle are the main reservoir and the transmission to humans is through the consumption of undercooked meat, non-pasteurized dairy products, and vegetables or water contaminated with feces. We have previously determined that O130:H11 and O178:H19 serotypes were the most prevalent in dairy cows from Argentina. In the present study, 37 and 25 STEC isolates from dairy cows belonging to O130:H11 and O178:H19 serotypes, respectively, were characterized regarding to their cytotoxicity on Vero cells, stx subtypes, presence of sab and typing by multiple-locus variable-number tandem repeat analysis (MLVA). All strains demonstrated a cytotoxic effect, and in O130:H11 isolates, stx2EDL933 was the predominant subtype. In O178:H19 isolates the main stx2 subtype was stx2vha. The sab gene was detected in 65 and 24% of the isolates belonging to O130:H11 and O178:H19, respectively. Only one MLVA profile was identified among the O130:H11 isolates meanwhile 10 MLVA profiles were detected among the O178:H19 isolates which were grouped in two main clusters. In conclusion, our data show that O130:H11 and O178:H19 STEC isolates encode virulence factors associated with severe human disease and both serotypes should be considered for routinely testing. Our subtyping experiments showed that isolates could be distinguished based on the stx2 subtype and the presence/absence of sab gene, and for isolates belonging to O178:H19, also when the MLVA type was considered. However, MLVA subtyping of O130:H11 isolates will require the development of more specific markers.

Differences in Shiga toxin and phage production among stx(2g)-positive STEC strains.

Shiga toxin-producing Escherichia coli (STEC) are characterized by the production of Shiga toxins (Stx) encoded by temperate bacteriophages. Stx production is linked to the induction of the phage lytic cycle. Several stx variants have been described and differentially associated with the risk of developing severe illness. The variant named stx(2g) was first identified in a STEC strain isolated from the faeces of healthy cattle. Analysis of stx(2g)-positive strains isolated from humans, animals, and environmental sources have shown that they have a close relationship. In this study, stx(2g)-positive STEC isolated from cattle were analyzed for phage and Stx production, with the aim to relate the results to differences observed in cytotoxicity. The presence of inducible phages was assessed by analyzing the bacterial growth/lysis curves and also by plaque assay. Bacterial growth curves in the absence of induction were similar for all isolates, however, notably differed among induced cultures. The two strains that clearly evidenced bacteriolysis under this condition also showed higher phage titers in plaque assays. However, only the phage plaques produced by one of these strains (FB 62) hybridized with a stx(2)-probe. Furthermore, the production of Stx was evaluated by enzyme immunoassay (EIA) and Western immunoblotting in overnight supernatants. By EIA, we detected Stx only in supernatants of FB 62, with a higher signal for induced than uninduced cultures. By immunoblotting, Stx2 could be detected after induction in all stx(2g)-positive isolates, but with lower amounts of Stx2B subunit in those supernatants where phages could not be detected. Taking into account all the results, several differences could be found among stx(2g)-positive strains. The strain with the highest cytotoxic titer showed higher levels of stx(2)-phages and toxin production by EIA, and the opposite was observed for strains that previously showed low cytotoxic titers, confirming that in stx(2g)-positive strains Stx production is phage-regulated.

Subtyping of STEC by MLVA in Argentina.

Shiga toxin-producing Escherichia coli (STEC) causes serious human illness such as hemolytic uremic syndrome (HUS). Argentina has the world's highest rate of this syndrome, which is the leading cause of acute renal failure among children. E. coli O157:H7 is the most common cause of HUS, but a substantial and growing proportion of this illness is caused by infection due to non-O157 strains. Multiple-locus variable-number tandem repeat analysis (MLVA) has become an established technique to subtype STEC. This review will address the use of routine STEC subtyping by MLVA in order to type this group of isolates and to get insight into the genetic diversity of native STEC. With regard to these objectives we modified and adapted two MLVA protocols, one exclusive for O157 and the other, a generic E. coli assay. A total of 202 STEC isolates, from different sources and corresponding to 20 serotypes, have been MLVA genotyped in our laboratory. In our experience, MLVA constitutes a very sensitive tool and enables us to perform an efficient STEC subtyping. The diversity found in many serotypes may be useful for future epidemiological studies of STEC clonality, applied to O157 as well as to non-O157 isolates.

Molecular characterization of Shiga toxin-producing Escherichia coli isolated from the environment of a dairy farm.

Environmental samples were taken from ground, cattle water troughs, and feeders from a dairy farm with different STEC prevalence between animal categories (weaning calves, rearing calves, and dairy cows). Overall, 23 % of samples were positive for stx genes, stx(2) being the most prevalent type. Isolates were analyzed by PCR monoplex to confirm generic E. coli and by two multiplex PCR to investigate the presence of stx(1), stx(2), eae, saa, ehxA, and other putative virulence genes encoded in STEC plasmids: katP, espP, subA, and stcE. The toxin genes were subtyped and the strains were serotyped. The ground and the environment of the rearing calves were the sites with the highest number of STEC-positive samples; however, cattle water troughs and the environment of cows were the places with the greater chance of finding stx(2EDL933) which is a subtype associated with serious disease in humans. Several non-O157 STEC serotypes were detected. The serotypes O8:H19; O26:H11; O26:H-; O118:H2; O141:H-; and O145:H- have been asociated with human illness. Furthermore, the emergent pathogen STEC O157:H- (stx(1)-ehxA-eae) was detected in the environment of the weaning calves. These results emphasize the risk that represents the environment as source of STEC, a potential pathogen for human and suggest the importance of developing control methods designed to prevent contaminations of food products and transmission from animal to person.

Occurrence of subtilase cytotoxin and relation with other virulence factors in verocytotoxigenic Escherichia coli isolated from food and cattle in Argentina

We investigated the presence of the gene of subtilase cytotoxin (SubAB), described in certain highly virulent verocytotoxigenic E. coli strains, in isolates from Argentina and its relation with other virulence factors. The gene subA was present in eae-negative strains mostly associated with saa, vt2 and ehxA genes.

Genetic characterization of non-O157 verocytotoxigenic Escherichia coli isolated from raw beef products using multiple-locus variable-number tandem repeat analysis.

Verocytotoxigenic Escherichia coli (VTEC) can produce serious human illness linked to the consumption of contaminated food, mainly of bovine origin. There is growing concern about non-O157 VTEC serotypes, which in some countries cause severe infections in a proportion similar to O157:H7 strains. As several epidemiological studies indicated the important role of meat as the major vehicle in the transmission of this pathogen to human consumers, our aim was to investigate the genetic diversity among non-O157:H7 VTEC isolated from raw beef products. We performed a multiple-locus variable-number tandem repeat (VNTR) analysis (MLVA), and to our knowledge, this is the first time that VTEC serotypes O8:H19, O112:H2, O113:NM, O171:NM, ONT:H7, ONT:H19, and ONT:H21 were typed by this method. MLVA typing grouped the total number of strains from this study (51) into 21 distinct genotypes, and 11 of them were unique. Several MLVA profiles were found in different serotypes, O178:H19 being the most variable. The isolates could be principally discriminated by alleles of three of seven loci studied (CVN001, CVN004, and CVN014), and on the other hand, CVN003 rendered null alleles in all the isolates. As some VNTR markers might be serotype specific, it is possible that the implementation of new VNTR loci will increase intraserotype discrimination.

Verotoxins in bovine and meat verotoxin-producing Escherichia coli isolates: type, number of variants, and relationship to cytotoxicity.

In this study, we determined vt subtypes and evaluated verotoxicity in basal as well as induced conditions of verotoxin-producing Escherichia coli (VTEC) strains isolated from cattle and meat products. Most (87%) of the 186 isolates carried a vt(2) gene. Moreover, the vt(2) subtype, which is associated with serious disease, was present in 42% of our VTEC collection. The other vt subtypes detected were vt(1), vt(1d), vt(2vha), vt(2vhb), vt(2O118), vt(2d) (mucus activatable), and vt(2g). A total of 41 (22%) of the isolates possessed more than one vt subtype in its genome, and among them the most frequent combination was vt(1)/vt(2), but we also observed multiple combinations among vt(2) subtypes. Differences in verotoxicity titers were found among a selection of 54 isolates. Among isolates with a single vt(2) variant, those carrying the vt(2) subtype had high titers under both uninduced and induced conditions. However, the highest increase in cytotoxicity under mitomycin C treatment was detected among the strains carrying vt(2vha) or vt(2hb) variants. Notably, the isolates carrying the vt(1) subtype showed a lesser increase than that of most of the vt(2)-positive VTEC strains. Furthermore, the presence of more than one vt gene variant in the same isolate was not reflected in higher titers, and generally the titers were lower than those for strains with only one gene variant. The main observation was that both basal and induced cytotoxic effects seemed to be associated with the type and number of vt variants more than with the serotype or origin of the isolate.

Occurrence of subtilase cytotoxin and relation with other virulence factors in verocytotoxigenic Escherichia coli isolated from food and cattle in Argentina.

We investigated the presence of the gene of subtilase cytotoxin (SubAB), described in certain highly virulent verocytotoxigenic E. coli strains, in isolates from Argentina and its relation with other virulence factors. The gene subA was present in eae-negative strains mostly associated with saa, vt2 and ehxA genes.

Genetic diversity of O157:H7 and non-O157 verocytotoxigenic Escherichia coli from Argentina inferred from multiple-locus variable-number tandem repeat analysis (MLVA).

Although serotype O157:H7 has been implicated in most cases of haemolytic-uraemic syndrome (HUS), there is growing concern about non-O157 serotypes of verocytotoxigenic Escherichia coli (VTEC). Multiple-locus variable-number tandem repeat analysis (MLVA) has been focused on the specific typing of O157:H7 isolates, but recently, a generic MLVA assay for E. coli and Shigella has been developed. We performed a study of the polymorphism in 7 generic VNTR loci both in VTEC O157:H7 and non-O157 isolates from Argentina, in order to asses the ability of the method to type this group of isolates and to get insight into their genetic diversity. Sixty-four isolates from cattle, patients with diarrhoea, and contaminated food belonging to 8 different serotypes were studied. All of them could be typed by this method and revealed 41 different MLVA genotypes. The MLVA dendrogram showed 2 main clusters which corresponded to O157:H7 and non-O157, respectively. Our results confirm the suitability of this MLVA method for analyzing VTEC isolates belonging to several serotypes, both O157:H7 as well as non-O157, highlight the genetic variability of the O157:H7 serotype and the need of additional research in order to find more VNTR loci that could allow a higher discrimination among non-O157 VTEC.

Molecular characterization of Verocytotoxigenic Escherichia coli O157: H7 isolates from Argentina by Multiple-Loci VNTR analysis (MLVA)

The aim of this work was to adapt described MLVA protocols to the molecular typing and characterization of VTEC O157:H7 isolates from Argentina. Nine VNTR loci were amplified by PCR showing diversity values from 0.49 to 0.73. Nine MLVA profiles were observed and the cluster analysis indicated both unrelated and closely related VTEC O157:H7 strains. In spite of the limited number of isolates studied, the panel of VNTR used made it possible to perform a first approach of the high genetic diversity of native strains of O157:H7 by MLVA.