Antimicrobial activity of Lactiplantibacillus plantarum against shiga toxin-producing Escherichia coli.

AIMS: The aim of the present work was to characterize the Lactiplantibacillus sp. LP5 strain, isolated from pork production, and identify bacteriocin-like inhibitory substances produced by this strain. METHODS AND RESULTS: In this study, LP5 was identified by species-specific PCR and 16S rRNA sequencing. Additionally, bacterial growth kinetics, antimicrobial activity, the detection of genes related to plantaricin production, and the genetic expression of plantaricins were determined. Lactiplantibacillus sp. LP5 was identified as Lactiplantibacillus plantarum. The well-diffusion test using cell-free supernatants (CFS), neutralized CFS, CFS treated with catalase, and CFS treated with proteinase K showed that inhibitory effects on a Shiga toxin-producing Escherichia coli (STEC) strain were produced by bacteriocins. The PCR technique allowed the detection of genes encoding E/F plantaricins, as well as J/K and whole genome sequencing, and bacteriocin mining analysis allowed us to confirm the presence of these plantaricins. CONCLUSIONS: We can conclude that the inhibitory effect of L. plantarum LP5 isolated from pigs against the STEC EDL933 strain could be associated with the bacteriocins production and represents a potential use as a probiotic strain.

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.

Seropositivity to Shiga toxin 2 among Argentinian urban and rural residents. Association with sociodemographic and exposure factors.

BACKGROUND: Shiga toxin-producing Escherichia coli (STEC) are enteric pathogens that cause hemolytic-uremic syndrome (HUS). Ruminants, especially cattle, are their main reservoir. This study describes the seroepidemiology of STEC in rural and urban populations in Argentina, a country with a high HUS incidence. METHODS: A cross-sectional study was performed in patients without gastrointestinal symptoms. IgG antibodies against Stx2 were detected by western blotting. RESULTS: Anti-Stx2 antibodies were detected in 14.56% of serum samples, more frequently in rural (19.38%) than urban residents (12%). Seropositivity was associated with lower socioeconomic status (SES). Among the other variables considered, thawing homemade hamburgers before cooking them, and the lack of knowledge about HUS were also associated with seropositivity. A multivariate logistic regression analysis performed with the variables that were statistically significant showed that only the SES index remained significant. As SES was measured based on several variables, we further analyzed each one of them and found that the lack of a high education level was statistically associated with seropositivity. CONCLUSIONS: The present findings have implications for STEC prevention efforts, highlighting the importance of considering SES and risks factors linked to different SES levels when targeting consumer-level public health interventions.

Calidad microbiològica de la carne picada y detección de patógenos en muestras ambientales de carnicerías de la ciudad de Tandil, provincia de Buenos Aires,Argentina / Microbiological quality of fresh ground beef and detection of pathogens in environmental samples taken from butcher shops in the city of Tandil, Buenos Aires Province, Argentina

Resumen Este estudio evaluó las condiciones higiénico-sanitarias de carnicerías de la ciudadde Tandil (provincia de Buenos Aires) mediante una estimación del riesgo basada en encuestasdirigidas a revisar las buenas prácticas de manufactura y de higiene de los establecimientos. Seutilizó una escala de 1 a 100 para clasificar a los establecimientos en las categorías de riesgoalto (0-40), riesgo moderado (41-70) y riesgo bajo (71-100). A su vez, se evaluó la presencia deSalmonella spp., Staphylococcus aureus, Escherichia coli productor de toxina Shiga (STEC) encarne bovina picada y en muestras ambientales como mesada, cuchilla, picadora y manos delcarnicero. Las muestras se tomaron una sola vez e inmediatamente se refrigeraron y transpor-taron al laboratorio para su análisis. En el período de estudio todas las carnicerías (100) fueronclasificadas como de «riesgo bajo¼ y con buenas condiciones higiénico-sanitarias. No obstante,el 75% de las muestras de carne picada no cumplió con al menos uno de los criterios microbiológicos establecidos en el Artículo 255 del Código Alimentario Argentino. Se sugiere estableceruna estrategia tendiente a identificar los desvíos e implementar un plan de mejoras continuasen las carnicerías de la ciudad de Tandil.

Abstract The aim of this work was to evaluate the hygienic-sanitary conditions of butcher shops in Tandil, Buenos Aires Province, by estimating the risk based on good manufacturing and hygiene practices, through surveys of the establishments. The analysis was performed using a scale of 1-100, and classifying them as high risk (0-40), moderate risk (41-70) or low risk (71-100). The presence of Salmonella spp., Staphylococcus aureus and Shiga toxin-producing Escherichia coli (STEC) from both, ground beef and environmental samples such as countertop, cleaver, mincer and butcher's hands, taken at butcher shops was also evaluated. Sampling was performed only once and immediately refrigerated and transported to the laboratory for analysis. All butcher shops evaluated (100) were classified as "low risk'' with good hygienic-sanitary conditions. However, 75% of the ground beef samples analyzed did not meet at least one of the microbiological criteria established in the Código Alimentario Argentino [Argentine Food Code], article 255. We propose to establish a strategy to identify deviations and implement a plan for continuous improvement in butcher shops of Tandil city.

[Microbiological quality of fresh ground beef and detection of pathogens in environmental samples taken from butcher shops in the city of Tandil, Buenos Aires Province, Argentina]. / Calidad microbiológica de la carne picada y detección de patógenos en muestras ambientales de carnicerías de la ciudad de Tandil, provincia de Buenos Aires, Argentina.

The aim of this work was to evaluate the hygienic-sanitary conditions of butcher shops in Tandil, Buenos Aires Province, by estimating the risk based on good manufacturing and hygiene practices, through surveys of the establishments. The analysis was performed using a scale of 1-100, and classifying them as high risk (0-40), moderate risk (41-70) or low risk (71-100). The presence of Salmonella spp., Staphylococcus aureus and Shiga toxin-producing Escherichia coli (STEC) from both, ground beef and environmental samples such as countertop, cleaver, mincer and butcher's hands, taken at butcher shops was also evaluated. Sampling was performed only once and immediately refrigerated and transported to the laboratory for analysis. All butcher shops evaluated (100) were classified as "low risk" with good hygienic-sanitary conditions. However, 75% of the ground beef samples analyzed did not meet at least one of the microbiological criteria established in the Código Alimentario Argentino [Argentine Food Code], article 255. We propose to establish a strategy to identify deviations and implement a plan for continuous improvement in butcher shops of Tandil city.

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.

Identification and detection of iha subtypes in LEE-negative Shiga toxin-producing Escherichia coli (STEC) strains isolated from humans, cattle and food.

LEE-negative Shiga toxin-producing Escherichia coli (STEC) strains are important cause of infection in humans and they should be included in the public health surveillance systems. Some isolates have been associated with haemolytic uremic syndrome (HUS) but the mechanisms of pathogenicity are is a field continuos broadening of knowledge. The IrgA homologue adhesin (Iha), encoded by iha, is an adherence-conferring protein and also a siderophore receptor distributed among LEE-negative STEC strains. This study reports the presence of different subtypes of iha in LEE-negative STEC strains. We used genomic analyses to design PCR assays for detecting each of the different iha subtypes and also, all the subtypes simultaneously. LEE-negative STEC strains were designed and different localizations of this gene in STEC subgroups were examinated. Genomic analysis detected iha in a high percentage of LEE-negative STEC strains. These strains generally carried iha sequences similar to those harbored by the Locus of Adhesion and Autoaggregation (LAA) or by the plasmid pO113. Besides, almost half of the strains carried both subtypes. Similar results were observed by PCR, detecting iha LAA in 87% of the strains (117/135) and iha pO113 in 32% of strains (43/135). Thus, we designed PCR assays that allow rapid detection of iha subtypes harbored by LEE-negative strains. These results highlight the need to investigate the individual and orchestrated role of virulence genes that determine the STEC capacity of causing serious disease, which would allow for identification of target candidates to develop therapies against HUS.

Antimicrobial Resistance in Class 1 Integron-Positive Shiga Toxin-Producing Escherichia coli Isolated from Cattle, Pigs, Food and Farm Environment.

The aim of this study was to investigate the presence of class 1 integrons in a collection of Shiga toxin-producing Escherichia coli (STEC) from different origins and to characterize pheno- and genotypically the antimicrobial resistance associated to them. A collection of 649 isolates were screened for the class 1 integrase gene (intI1) by Polymerase chain reaction The variable region of class 1 integrons was amplified and sequenced. Positive strains were evaluated for the presence of antimicrobial resistance genes with microarray and for antimicrobial susceptibility by the disk diffusion method. Seven out of 649 STEC strains some to serogroups, O26, O103 and O130 isolated from cattle, chicken burger, farm environment and pigs were identified as positive for intl1. Different arrangements of gene cassettes were detected in the variable region of class 1 integron: dfrA16, aadA23 and dfrA1-aadA1. In almost all strains, phenotypic resistance to streptomycin, tetracycline, trimethoprim/sulfamethoxazole, and sulfisoxazole was observed. Microarray analyses showed that most of the isolates carried four or more antimicrobial resistance markers and STEC strains were categorized as Multridrug-resistant. Although antimicrobials are not usually used in the treatment of STEC infections, the presence of Multridrug-resistant in isolates collected from farm and food represents a risk for animal and human health.

Recent Advances in Shiga Toxin-Producing Escherichia coli Research in Latin America.

Pathogenic Escherichia coli are known to be a common cause of diarrheal disease and a frequently occurring bacterial infection in children and adults in Latin America. Despite the effort to combat diarrheal infections, the south of the American continent remains a hot spot for infections and sequelae associated with the acquisition of one category of pathogenic E. coli, the Shiga toxin-producing E. coli (STEC). This review will focus on an overview of the prevalence of different STEC serotypes in human, animals and food products, focusing on recent reports from Latin America outlining the recent research progress achieved in this region to combat disease and endemicity in affected countries and to improve understanding on emerging serotypes and their virulence factors. Furthermore, this review will highlight the progress done in vaccine development and treatment and will also discuss the effort of the Latin American investigators to respond to the thread of STEC infections by establishing a multidisciplinary network of experts that are addressing STEC-associated animal, human and environmental health issues, while trying to reduce human disease. Regardless of the significant scientific contributions to understand and combat STEC infections worldwide, many significant challenges still exist and this review has focus in the Latin American efforts as an example of what can be accomplished when multiple groups have a common goal.

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 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.

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.

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.

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.