RESUMO
Shiga toxin-producing Escherichia coli (STEC) are pathogens of significant public health concern. Several studies have confirmed that cattle are the main reservoir of STEC in Argentina and other countries. Although Shiga toxins represent the primary virulence factors of STEC, the adherence and colonization of the gut are also important in the pathogenesis of the bacteria. The aim of this study was to analyze and to compare the presence of putative virulence factors codified in plasmid -katP, espP, subA, stcE- and adhesins involved in colonization of cattle -efa1, iha- in 255 native STEC strains isolated from different categories of cattle from different production systems. The most prevalent gene in all strains was espP, and the less prevalent was stcE. katP was highly detected in strains isolated from young and rearing calves (33.3%), while subA was predominant in those isolated from adults (71.21%). Strains from young calves showed the highest percentage of efa1 (72.46%), while iha showed a high distribution in strains from rearing calves and adults (87.04 and 98.48% respectively). It was observed that espP and iha were widely distributed throughout all strains, whereas katP, stcE, and efa1 were more associated with the presence of eae and subA with the eae-negative strains. A great proportion of eae-negative strains were isolated from adults -dairy and grazing farms- and from rearing calves -dairy and feedlot-, while mostly of the eae-positive strains were isolated from dairy young calves. Data exposed indicate a correlation between the category of the animal and the production systems with the presence or absence of several genes implicated in adherence and virulence of STEC.
Assuntos
Bovinos/microbiologia , Infecções por Escherichia coli/veterinária , Toxinas Shiga/biossíntese , Escherichia coli Shiga Toxigênica/genética , Fatores de Virulência/genética , Virulência/genética , Adesinas Bacterianas/genética , Animais , Argentina , Toxinas Bacterianas/genética , Infecções por Escherichia coli/microbiologia , Proteínas de Escherichia coli/genética , Proteína Reguladora de Resposta a Leucina/genética , Metaloendopeptidases/genética , Plasmídeos/genética , Serina Endopeptidases/genética , Escherichia coli Shiga Toxigênica/crescimento & desenvolvimento , Escherichia coli Shiga Toxigênica/isolamento & purificação , Escherichia coli Shiga Toxigênica/patogenicidade , Subtilisinas/genéticaRESUMO
Verotoxin-producing E. coli (VTEC) O157:H7 is the dominant serotype isolated from patients with HUS and, Argentina has the highest rate of HUS in the world. Molecular typing had allowed to identify subpopulations related to the origin and virulence of O157:H7 strains. Our aim was to perform a genetic characterization of 43 O157:H7 strains isolated in Argentine mostly from cattle and humans in order to establish the potential public health risk. For it, we used a combination of molecular subtyping methods in order to identify clade 8_rhsA (C3468G), LSPA-6 and virulence profiles and, a cytotoxicity assay on Vero cell. All isolates carried the clade 8 SNP variant and 98% of them belonged to lineage I/II (2% lineage II). Isolates were grouped into eleven nle profiles, 46% were positive for all nle genes, while the remaining isolates, except two, showed incomplete OI-71, particularly lacked nleF. All isolates showed the plasmid profile ehxA-espP-katP-stcE and harbored ehaA, elfA, iha and lpfA variants lpfA1-3 and lpfA2-2 and, ECSP_0242. The frequencies of the remaining ECSP genes were 95% ECSP_2687, 88% ECSP_3286, 86% ECSP_3620, 53% ECSP_2870/2872 and 44% ECSP_1733. All O157:H7 strains, except the isolate identified as lineage II, were cytotoxic on Vero cells. Among Argentinean strains, most genetic markers occur at equal relative frequencies among clinical and bovine isolates, showing diversity mostly in nle genes profiles. The belonging of the isolates to hypervirulent clade 8 and lineage I/II, the high prevalence of nle and putative virulence factors genes, would allow assigning most O157:H7 strains of this region a high risk to public health.
Assuntos
Doenças dos Bovinos/microbiologia , Escherichia coli O157/fisiologia , Proteínas de Escherichia coli/genética , Síndrome Hemolítico-Urêmica/microbiologia , Toxinas Shiga/biossíntese , Animais , Argentina/epidemiologia , Aderência Bacteriana/genética , Bovinos , Doenças dos Bovinos/epidemiologia , Análise por Conglomerados , Escherichia coli O157/classificação , Genótipo , Síndrome Hemolítico-Urêmica/epidemiologia , Humanos , Fenótipo , Plasmídeos/genética , Virulência/genética , Fatores de Virulência/genéticaRESUMO
The present study was designed to determine the relationships among biofilm formation, cellular stress and release of Shiga toxin (Stx) by three different clinical Shiga toxin-producing Escherichia coli (STEC) strains. The biofilm formation was determined using crystal violet stain in tryptic soy broth or thioglycollate medium with the addition of sugars (glucose or mannose) or hydrogen peroxide. The reactive oxygen species (ROSs) were detected by the reduction of nitro blue tetrazolium and reactive nitrogen intermediates (RNI) determined by the Griess assay. In addition, the activities of two antioxidant enzymes, superoxide dismutase (SOD) and catalase (CAT), were studied. For the cytotoxicity studies, Vero cells were cultured with Stx released of STEC biofilms. The addition of sugars in both culture mediums resulted in an increase in biofilm biomass, with a decrease in ROS and RNI production, low levels of SOD and CAT activity, and minimal cytotoxic effects. However, under stressful conditions, an important increase in the antioxidant enzyme activity and high level of Stx production were observed. The disturbance in the prooxidant-antioxidant balance and its effect on the production and release of Stx evaluated under different conditions of biofilm formation may contribute to a better understanding of the relevance of biofilms in the pathogenesis of STEC infection.
Assuntos
Biofilmes/crescimento & desenvolvimento , Infecções por Escherichia coli/etiologia , Escherichia coli Shiga Toxigênica/fisiologia , Escherichia coli Shiga Toxigênica/patogenicidade , Animais , Catalase/metabolismo , Chlorocebus aethiops , Meios de Cultura , Infecções por Escherichia coli/metabolismo , Infecções por Escherichia coli/microbiologia , Escherichia coli O157/patogenicidade , Escherichia coli O157/fisiologia , Humanos , Espécies Reativas de Nitrogênio/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Toxinas Shiga/biossíntese , Toxinas Shiga/toxicidade , Superóxido Dismutase/metabolismo , Células VeroRESUMO
Toxin synthesis by Shiga toxin-producing Escherichia coli (STEC) appears to be coregulated through the induction of the integrated bacteriophages that encode the toxin genes. These phages might be the principal means for the dissemination and release of Shiga toxins. We evaluated the effect of three common food preservatives, potassium sorbate, sodium benzoate, and sodium propionate, on the propagation of the phages and Shiga toxins. We tested each preservative at four concentrations, 1, 1.25, 2.5, and 5 mg/ml, both on free phages and on lysogenic phages in bacteria. We also evaluated the expression of a lambdoid phage, which was exposed to increasing concentrations of preservatives, by measuring ß-galactosidase activity from SPC105, a transductant strain. Furthermore, we tested the effect of the preservatives on cytotoxigenic activity of Shiga toxin on Vero cells. We detected an increase of the inhibitory effect of the phage lytic activity, both in lysogenic and free phages, as the preservative concentration increased. However, the inhibition was higher on the lysogenic phages release than on free phages. Sodium benzoate and potassium sorbate were about equal at inhibiting phages; they were more effective than sodium propionate. A significant decrease of lacZ expression, encoded in a lambda phage, was observed. We also found a reduction in Shiga toxin titer caused by exposure of E. coli O157:H7 to 5 mg/ml sodium benzoate or potassium sorbate. These results imply that these three preservatives, used to inhibit microbial spoilage of foods, also act to inhibit lytic activity and dispersion of a phage carrying the gene encoding powerful Shiga cytotoxins. Also notable was the inactivation of Shiga toxin activity, although this effect was detected using concentrations of preservatives greater than those allowed by the Argentine Food Code.
Assuntos
Bacteriófagos/efeitos dos fármacos , Conservantes de Alimentos/farmacologia , Lisogenia , Toxinas Shiga/biossíntese , Escherichia coli Shiga Toxigênica/efeitos dos fármacos , Escherichia coli Shiga Toxigênica/virologia , Animais , Bacteriófago lambda/efeitos dos fármacos , Chlorocebus aethiops , Relação Dose-Resposta a Droga , Propionatos/farmacologia , Benzoato de Sódio/farmacologia , Ácido Sórbico/farmacologia , Células VeroRESUMO
BACKGROUND AND AIMS: Shiga-like toxin (Stx)-producing Escherichia coli (STEC) infection is an ongoing health issue that can lead to serious complications, including hemolytic uremic syndrome (HUS) and death. This study assessed demographic and epidemiologic information of STEC infection among Argentinean children. METHODS: A prospective surveillance of 2435 screened children (age, 0.5-15 years) presenting with watery diarrhea and/or bloody diarrhea was undertaken to evaluate the clinical course of STEC infection. RESULTS: Prevalence of STEC infection was 4.1% among subjects presenting with watery diarrhea for ≤ 5 days' duration, bloody diarrhea for ≤ 36 hours' duration, or both. Incidence of STEC infection was significantly higher in the subjects with bloody diarrhea. Ninety-three STEC+ children underwent further evaluation, of whom 8 (8.6%) developed HUS. White blood cells, particularly neutrophils, were abnormally elevated at screening in 5 of 8 HUS subjects. Quantifiable serum Stx-2 values were noted within 24 to 48 hours after the onset of bloody diarrhea in 3 HUS subjects using a validated chemiluminescence assay, with levels quickly dissipating by HUS onset. CONCLUSIONS: Results suggest that young STEC-positive children with bloody diarrhea and exhibiting neutrophilic leukocytosis in the early course of their diarrhea are at risk for HUS progression. The observation of measurable concentrations of Stx-2 levels in the early post-bloody-diarrhea period and rapid dissipation at the time of HUS onset requires further evaluation.
Assuntos
Diarreia/epidemiologia , Infecções por Escherichia coli/epidemiologia , Síndrome Hemolítico-Urêmica/epidemiologia , Toxina Shiga II/biossíntese , Toxinas Shiga/biossíntese , Escherichia coli Shiga Toxigênica/isolamento & purificação , Adolescente , Argentina/epidemiologia , Criança , Diarreia/diagnóstico , Diarreia/microbiologia , Infecções por Escherichia coli/diagnóstico , Infecções por Escherichia coli/microbiologia , Feminino , Síndrome Hemolítico-Urêmica/diagnóstico , Síndrome Hemolítico-Urêmica/microbiologia , Humanos , Lactente , Masculino , Reação em Cadeia da Polimerase , Vigilância da População/métodos , Prevalência , Fatores de Risco , Toxina Shiga II/genética , Toxinas Shiga/genética , Escherichia coli Shiga Toxigênica/patogenicidadeRESUMO
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.
Assuntos
Doenças dos Bovinos/microbiologia , Infecções por Escherichia coli/veterinária , Variação Genética , Carne/microbiologia , Toxinas Shiga/biossíntese , Toxinas Shiga/genética , Escherichia coli Shiga Toxigênica/isolamento & purificação , Animais , Bovinos , Infecções por Escherichia coli/microbiologia , Proteínas de Escherichia coli/biossíntese , Proteínas de Escherichia coli/classificação , Proteínas de Escherichia coli/genética , Expressão Gênica , Genótipo , Fenótipo , Toxinas Shiga/classificação , Escherichia coli Shiga Toxigênica/genéticaRESUMO
In Argentina, Escherichia coli O157:H7/NM (STEC O157) is the prevalent serotype associated with hemolytic uremic syndrome (HUS), which is endemic in the country with more than 400 cases per year. In order to estimate the prevalence and characteristics of STEC O157 in beef cattle at slaughter, a survey of 1,622 fecal and carcass samples was conducted in nine beef exporting abattoirs from November 2006 to April 2008. A total of 54 samples were found positive for STEC O157, with an average prevalence of 4.1% in fecal content and 2.6% in carcasses. Calves and heifers presented higher percentages of prevalence in feces, 10.5 and 8.5%, respectively. All STEC O157 isolates harbored stx(2) (Shiga toxin 2), eae (intimin), ehxA (enterohemolysin), and fliC(H7) (H7 flagellin) genes, while stx(1) (Shiga toxin 1) was present in 16.7% of the strains. The prevalent (56%) stx genotype identified was stx(2) combined with variant stx(2c (vh-a)), the combination of which is also prevalent (>90%) in STEC O157 post-enteric HUS cases in Argentina. The clonal relatedness of STEC O157 strains was established by phage typing and pulsed-field gel electrophoresis (PFGE). The 54 STEC isolates were categorized into 12 different phage types and in 29 XbaI-PFGE patterns distributed in 27 different lots. STEC O157 strains isolated from 5 of 21 carcasses were identical by PFGE (100% similarity) to strains of the fecal content of the same or a contiguous bovine in the lot. Five phage type-PFGE-stx profiles of 10 strains isolated in this study matched with the profiles of the strains recovered from 18 of 122 HUS cases that occurred in the same period.
Assuntos
Matadouros , Bovinos/microbiologia , Escherichia coli O157 , Fezes/microbiologia , Toxinas Shiga/biossíntese , Animais , Argentina/epidemiologia , Tipagem de Bacteriófagos , Análise por Conglomerados , Contagem de Colônia Microbiana , Qualidade de Produtos para o Consumidor , DNA Bacteriano/análise , Escherichia coli O157/classificação , Escherichia coli O157/genética , Escherichia coli O157/isolamento & purificação , Escherichia coli O157/metabolismo , Genótipo , Síndrome Hemolítico-Urêmica/epidemiologia , Síndrome Hemolítico-Urêmica/microbiologia , Humanos , Epidemiologia Molecular , Fenótipo , Prevalência , Toxinas Shiga/genéticaRESUMO
The prevalence, serotypes and virulence profiles of Shiga toxin-producing Escherichia coli (STEC) were investigated in 205 healthy beef and dairy cattle, and 106 goats reared in the southeastern region of Minas Gerais State, Brazil. The prevalence of STEC was 57.5% (61/106) in goats, 39.2%, (40/102) in beef cattle and 17.5% (18/103) in dairy cattle. Among the 514 STEC isolates, 40 different serotypes were found and some of them were identified in a specific host. STEC isolates harboring stx1 corresponded to 15.6% (28/180), 26.7% (16/60) and 24.1% (66/274) in beef cattle, dairy cattle and goats, respectively. stx2 was found in 30% (54/180), 53.3% (32/60) and 34.7% (95/274) of beef and dairy cattle, and goats. stx1 plus stx2 sequences were harbored by 54.4% (98/180), 20% (12/60) and 41.2% (113/274) of beef cattle, dairy cattle and goats, respectively. The eae sequence was found in 15% (9/60) and 0.6% (1/180) of STEC isolates from dairy and beef cattle, respectively, and the toxB gene was found only in one O157:H7 strain isolated from beef cattle. Strains with the genetic profiles stx2 ehxA iha saa and stx1 stx2 ehxA iha saa were the most prevalent among STEC isolates from cattle. Profiles stx1 stx2 ehxA iha, stx2, and stx1 iha accounted for 75.5% (207 /274) of the STEC isolates from goats. While STEC strains carrying either stx2 alone or associated with stx1 were found more frequently in cattle, those harboring sequences stx1c and stx2d alone or associated with stx1c predominated in goats. Our data show a diversity of STEC strains in food-producing animals, most of them carrying genes linked to severe forms of human diseases.
Assuntos
Doenças dos Bovinos/microbiologia , Infecções por Escherichia coli/veterinária , Doenças das Cabras/microbiologia , Escherichia coli Shiga Toxigênica/classificação , Escherichia coli Shiga Toxigênica/isolamento & purificação , Fatores de Virulência/genética , Animais , Brasil/epidemiologia , Bovinos , Doenças dos Bovinos/epidemiologia , Reservatórios de Doenças/veterinária , Infecções por Escherichia coli/epidemiologia , Infecções por Escherichia coli/microbiologia , Feminino , Doenças das Cabras/epidemiologia , Cabras , Humanos , Masculino , Filogenia , Prevalência , Sorotipagem , Toxinas Shiga/biossíntese , Toxinas Shiga/genética , Escherichia coli Shiga Toxigênica/patogenicidade , Especificidade da EspécieRESUMO
The aim of this study was to characterize Shiga toxigenic Escherichia coli (STEC) by PCR using strains isolated from ham, beef, and cattle in Colombia. A total of 189 E. coli strains were tested for the presence of the uidA, stx1, and stx2 genes, and identification was confirmed by the automated PCR BAX system for E. coli O157:H7. Genes encoding Shiga-like toxins (stx) were found in eight (6.06%) of 132 strains previously isolated from minced beef; four (50%) of these strains yielded amplification products for both toxin genes (stx1 and stx2), and four (50%) yielded products only for the stx2 toxin. None of the strains analyzed were positive by PCR for the presence of the single base-pair mutation in the uidA gene from E. coli O157:H7; these results were confirmed by the BAX system analysis. A multiplex PCR assay was standardized for the three genes. Results from this study confirmed previous data about the low prevalence of E. coli O157:H7 and Shiga-like toxins in Colombia and is the first known report of the prevalence of non-O157 enterohemorrhagic E. coli in this country.
Assuntos
Contaminação de Alimentos/análise , Produtos da Carne/microbiologia , Reação em Cadeia da Polimerase/métodos , Toxinas Shiga/genética , Escherichia coli Shiga Toxigênica/isolamento & purificação , Colômbia , Escherichia coli O157/genética , Escherichia coli O157/isolamento & purificação , Escherichia coli O157/metabolismo , Microbiologia de Alimentos , Mutação , Prevalência , Toxina Shiga I/biossíntese , Toxina Shiga I/genética , Toxina Shiga II/biossíntese , Toxina Shiga II/genética , Toxinas Shiga/biossíntese , Escherichia coli Shiga Toxigênica/genética , Escherichia coli Shiga Toxigênica/metabolismoRESUMO
AIMS: To determine the suitability of eight different commercial broth media for Shiga toxin (Stx) production. METHODS AND RESULTS: Shiga toxin-producing Escherichia coli (STEC) strains producing Stx1 or Stx2 were grown at 37 degrees C (250 rev min(-1)) for 24 h in brain heart infusion broth, E. coli broth, Evans medium, Luria-Bertani broth, Penassay broth, buffered-peptone water, syncase broth and trypticase soy broth. Toxin production was measured by enzyme-linked immunosorbent assay (ELISA) in polymyxin-treated cell pellets and/or supernatants of cultures, ELISA optical densities reached 1 when isolates were grown for 2-4 h in E. coli broth in the presence of antibiotic. Besides, a collection of STEC-expressing Stx strains was evaluated and the Stx production was assayed in the supernatants and in polymyxin-treated pellets of bacterial growth after 4 h of cultivation in E. coli broth in the presence of antibiotic. CONCLUSIONS: The most suitable medium for Stx production was E. coli broth when the bacterial isolates were grown for 4 h in the presence of ciprofloxacin and the Stx production is detected in the supernatant. SIGNIFICANCE AND IMPACT OF THE STUDY: This study presents the first comprehensive comparison of different broth media with regard to Stx production to establish optimal culture conditions for STEC detection in routine diagnostic laboratories.
Assuntos
Meios de Cultura , Escherichia coli/metabolismo , Toxinas Shiga/biossíntese , Animais , Bovinos , Chlorocebus aethiops , Ensaio de Imunoadsorção Enzimática , Escherichia coli/patogenicidade , Células VeroRESUMO
The presence of Shiga toxin-producing Escherichia coli (STEC) in water buffaloes is reported for the first time in South America. The prevalence of STEC ranged from 0 to 64% depending on the farm. STEC isolates exhibiting the genetic profiles stx(1)stx(2)ehxA iha saa and stx(2)ehxA iha saa predominated. Of the 20 distinct serotypes identified, more than 50% corresponded to serotypes associated with human diseases.
Assuntos
Proteínas de Escherichia coli , Escherichia coli/imunologia , Escherichia coli/patogenicidade , Toxinas Shiga/biossíntese , Virulência/genética , Animais , Brasil , Búfalos , Bovinos/microbiologia , Reservatórios de Doenças , Escherichia coli/genética , Escherichia coli/isolamento & purificação , Sorotipagem , Toxinas Shiga/genéticaRESUMO
The distribution of virulence markers related to cytolethal distending toxin-V (CDT-V), subtilase cytotoxin (SubAB), the enterohemorrhagic Escherichia coli factor for adherence (Efa1), the adhesin similar to IrgA (Iha), the long polar fimbriae (LpfO113), the autoagglutinating adhesin (Saa), and the protein required for full expression of adherence of O157:H7 Sakai strain (ToxB) was investigated in 121 Shiga toxin-producing E. coli (STEC) strains isolated in Brazil. STEC strains were isolated from human infections (n=49), cattle (n=68) and ground meat samples (n=4). Overall, the lpfA(O113), iha, efa1, saa, and toxB sequences were observed in 89.2%, 87.6%, 47.1%, 43%, and 13.2% of the strains, respectively. The genes efa1 (96.6%) and toxB (27%) were only identified among eae-positive strains, while saa (83.8%), cdt-V (12.9%), and subAB (48.4%) just occurred in eae-negative STEC strains. STEC strains harboring cdt-V and subAB were for the first time described in the South American subcontinent. In addition, the simultaneous presence of cdt-V and subAB has not been previously reported, nor the presence of subAB in STEC O77, O79, O105, O174, and O178 serogroups. A diversity of virulence profiles was observed among the STEC strains studied. The most prevalent profile observed among eae-positive STEC strains mainly isolated from humans was eae efa1 iha lpfA(O113), whereas iha lpfA(O113) saa ehxA subAB prevailed among eae-negative STEC strains, mostly isolated from cattle and foods.
Assuntos
Adesinas de Escherichia coli/biossíntese , Infecções por Escherichia coli/veterinária , Escherichia coli/química , Toxinas Shiga/biossíntese , Fatores de Virulência , Adesinas de Escherichia coli/genética , Adesinas de Escherichia coli/metabolismo , Animais , Brasil , Escherichia coli/classificação , Escherichia coli/isolamento & purificação , Escherichia coli/patogenicidade , Infecções por Escherichia coli/epidemiologia , Infecções por Escherichia coli/microbiologia , Proteínas de Escherichia coli/metabolismo , Virulência/genéticaRESUMO
AIMS: To investigate the prevalence and characteristics of Shiga toxin-producing Escherichia coli (STEC) in cattle from Paraná State, southern Brazil. METHODS AND RESULTS: One hundred and seven faeces cattle samples were cultured on Sorbitol-MacConkey agar. Escherichia coli colonies were tested for production of Shiga toxin using Vero-cell assay. A high prevalence (57%) of STEC was found. Sixty-four STEC were serotyped and examined for the presence of stx(1), stx(2), eae, ehxA and saa genes and stx(2) variants. The isolates belonged to 31 different serotypes, of which three (O152:H8, O175:H21 and O176:H18) had not previously been associated with STEC. A high prevalence of stx(2)-type genes was found (62 strains, 97%). Variant forms found were stx(2), stx(2c), stx(2vhb), stx(2vO111v/OX393) and a form nonclassifiable by PCR-RFLP. The commonest genotypes were stx(2)ehxA saa and stx(1)stx(2)ehxA saa. CONCLUSIONS: A high frequency of STEC was observed. Several strains belong to serotypes previously associated with human disease and carry stx(2) and other virulence factors, thus potentially representing a risk to human health. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study of STEC in Paraná State, and its findings emphasize the need for proper cattle handling to prevent human contamination.
Assuntos
Bovinos/microbiologia , Escherichia coli/classificação , Escherichia coli/isolamento & purificação , Toxinas Shiga/biossíntese , Animais , Brasil , Chlorocebus aethiops , Escherichia coli/patogenicidade , Fezes/microbiologia , Genótipo , Carne , Fenótipo , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , Sorotipagem , Toxinas Shiga/genética , Células Vero , Virulência/genéticaRESUMO
AIMS: To determine the prevalence of Shiga toxin-producing Escherichia coli (STEC) and serotypes and virulence markers of the STEC isolates from beef and dairy cattle in Rio Grande do Sul, Brazil. METHODS AND RESULTS: Faecal samples from beef cattle were collected at slaughterhouses. The isolates were submitted to colony hybridization assay with specific DNA probes for stx1, stx2 and eae genes, and serotyped for the identification of O and H antigens. Thirty-nine per cent of beef cattle surveyed harboured at least one STEC strain. Among the distinct serotypes identified, 10 were shared by both beef and dairy cattle. Most of the strains isolated harboured stx2. Genotypic and phenotypic profiles allowed the identification of 34 and 31 STEC strains, isolated from beef and dairy cattle, respectively. Serotypes O10:H14, O15:H21, O96:H21, O119:H4, O124:H11, O128:H21, O137:H-, O141:H19, O159:H42, O160:H2 and O177:H11, identified in this study, have not been previously reported as STEC isolated from cattle. CONCLUSIONS: Cattle are an important reservoir of STEC strains associated with human diseases in South America. SIGNIFICANCE AND IMPACT OF THE STUDY: Determining the prevalence, genotypic profile and serotypes of STEC strains isolated from cattle enables the prediction of possible risk for public health.
Assuntos
Bovinos/microbiologia , Escherichia coli/classificação , Escherichia coli/patogenicidade , Toxinas Shiga/biossíntese , Animais , Brasil , Indústria de Laticínios , Reservatórios de Doenças , Escherichia coli/isolamento & purificação , Escherichia coli/metabolismo , Infecções por Escherichia coli , Fezes/microbiologia , Humanos , Carne/microbiologia , Sorotipagem , Toxinas Shiga/genética , Fatores de Virulência/genéticaRESUMO
The aim of the present study was to investigate the occurrence of Escherichia coli O157:H7 and other Shiga toxin-producing E. coli (STEC) in 'Castellano' cheese, a non-cooked and hard or semi-hard Spanish cheese made from ewe's milk. A total of 83 raw milk cheese samples with different ripening times (2.5, 6 and 12 months) were taken at 30 cheese factories. Samples were examined for the presence of STEC using in the first stage the Association of Official Analytical Chemists (AOAC) official method number 997.11, and then, in the second stage, isolates were tested for virulence genes using genotypic (PCR) methods. Three STEC strains were detected in two samples (2.4%) of 'Castellano' cheese, one with 2.5 and the other one with 12 month-ripening period. From those STEC isolates, two were identified as E. coli O14 and the third presented an O-specific polysaccharide not-groupable serologically (ONG). PCR showed that all isolates were characterized by harbouring the Shiga toxin (stx) stx1 gene and by the absence of the genes for stx2, eaeA, and ehxA virulence factors. This study revealed the potential of STEC to survive in long-ripened-hard cheeses.
Assuntos
Queijo/microbiologia , Escherichia coli O157/isolamento & purificação , Escherichia coli/isolamento & purificação , Contaminação de Alimentos/análise , Toxinas Shiga/biossíntese , Animais , Qualidade de Produtos para o Consumidor , Escherichia coli/metabolismo , Escherichia coli O157/metabolismo , Humanos , Leite/microbiologia , Ovinos , Espanha , Fatores de Tempo , VirulênciaRESUMO
A cross-sectional study was conducted to determine the prevalence and characteristics of verocytotoxigenic Escherichia coli (VTEC) on 25 dairy farms each located in Waller field and Carlsen field farming areas in Trinidad. On each selected farm, faecal samples were collected from milking cows, calves and humans; rectal swabs were obtained from pet farm dogs; bulk milk was sampled as well as effluent from the milking parlour. Escherichia coli was isolated from all sources on selective media using standard methods. Isolates of E. coli were subjected to slide agglutination test using E. coli O157 antiserum, vero cell cytotoxicity assay to detect verocytotoxin (VT) and heat labile toxin (LT) production, the polymerase chain reaction (PCR) to detect VT genes, and the dry spot test to screen for E. coli O157 and non-O157 strains. In addition, faecal samples from animal and human sources were tested for VT genes using PCR. Of a total of 933 E. coli isolates tested by the slide test, eight (0.9%) were positive for the O157 strain. The vero cell cytotoxicity assay detected VT-producing strains of E. coli in 16.6%, 14.6%, 3.2% and 7.1% of isolates from cows, calves, farm dogs and humans respectively (P < 0.05; chi(2)). For LT production, the highest frequency was detected amongst isolates of E. coli from calves (10.8%) and the lowest (0.0%) amongst isolates from humans and bulk milk (P < 0.05; chi(2)). Of the 61 VT-producing isolates by vero cell cytotoxicity assay tested by PCR, the VT, LT and eae genes were detected in 62.3%, 4.9% and 1.6% respectively (P < 0.05; chi(2)). Amongst the 45 E. coli isolates that were VT positive (vero cell) or VT-gene positive by PCR, 2.2%, 2.2%, 4.4% and 6.7% belonged to non-O157 strains O91, O111, O103 and O157, respectively, as determined by the Dry spot test. Detection of VTEC strains in milk and dairy animals poses a health risk to consumers of milk originating from these farms. In addition, the demonstration of VTEC strains in humans, VT gene in faecal samples and E. coli isolates as well as non-O157 VTEC strains of E. coli are being documented for the first time in the country.
Assuntos
Doenças dos Bovinos/epidemiologia , Infecções por Escherichia coli/veterinária , Escherichia coli , Filogenia , Toxinas Shiga/biossíntese , Testes de Aglutinação , Animais , Bovinos , Doenças dos Bovinos/microbiologia , Doenças dos Bovinos/transmissão , Chlorocebus aethiops , Estudos Transversais , Indústria de Laticínios/estatística & dados numéricos , Cães , Escherichia coli/classificação , Escherichia coli/isolamento & purificação , Escherichia coli/metabolismo , Infecções por Escherichia coli/epidemiologia , Infecções por Escherichia coli/microbiologia , Infecções por Escherichia coli/transmissão , Fezes/microbiologia , Feminino , Humanos , Leite/microbiologia , Reação em Cadeia da Polimerase , Prevalência , Trinidad e Tobago/epidemiologia , Células Vero , ZoonosesRESUMO
Shiga toxin-producing Escherichia coli (STEC), is the most important recently emerged group of foodborne pathogens. Ruminants, especially cattle, have been implicated as a principal reservoir of STEC, undercooked ground beef and raw milk being the major vehicles of foodborne outbreaks. Enteropathogenic E. coli (EPEC) strains are defined as eae-harboring diarrheagenic E. coli that possess the ability to form A/E lesions on intestinal cells and that do not possess Shiga toxin genes. In order to determine the occurrence, serotypes and virulence markers of STEC and EPEC strains, 546 fecal samples from 264 diarrheic calves and 282 healthy calves in beef farms in São Paulo, Brazil, were screened by PCR. STEC and EPEC were isolated in 10% and 2.7% of the 546 animals, respectively. Although IMS test was used, the STEC serotype O157:H7 was not detected. The most frequent serotypes among STEC strains were O7:H10, O22:H16, O111:H(-), O119:H(-) and O174:H21, whereas O26:H11, O123:H11 and O177:H11 were the most prevalent among EPEC strains. In this study, serotypes not previously reported were found among STEC strains: O7:H7, O7:H10, O48:H7, O111:H19, O123:H2, O132:H51, O173:H(-), and O175:H49. The eae gene was detected in 25% of the STEC and 100% of EPEC strains. The intimin type theta/gamma2 was the most frequent among STEC, whereas the intimin beta1 was the most frequent intimin type among EPEC strains. To our knowledge, this is the first report of the occurrence of the new intimin muB in one strain of animal origin. This new intimin was detected in one atypical EPEC strain of serotype O123:H? isolated from diarrheic cattle. The enterohemolysin (ehxA) was detected in 51% of the STEC and 80% of the EPEC strains, whereas STEC autoagglutinating adhesin (saa) virulence gene was detected only in those STEC strains negative for eae gene. All 15 bovine EPEC strains isolated in this study were negative for both eaf and bfp genes. Our data shows that in Brazil cattle are not only a reservoir of STEC and atypical EPEC, but also a potential source of infection in humans, since the important STEC serotypes previously described and associated with severe diseases in humans, such as O111:H(-), O113:H21, O118:H16, and O174:H21 were isolated.
Assuntos
Doenças dos Bovinos/microbiologia , Infecções por Escherichia coli/veterinária , Escherichia coli/classificação , Escherichia coli/patogenicidade , Toxinas Shiga/biossíntese , Adesinas Bacterianas/genética , Animais , Brasil , Bovinos , Doenças dos Bovinos/epidemiologia , Reservatórios de Doenças/veterinária , Escherichia coli/genética , Escherichia coli/isolamento & purificação , Infecções por Escherichia coli/epidemiologia , Infecções por Escherichia coli/microbiologia , Proteínas de Escherichia coli , Fezes/microbiologia , Humanos , Carne/microbiologia , Filogenia , Reação em Cadeia da Polimerase , Sorotipagem , Virulência/genéticaRESUMO
The present study was conducted in Lima Metropolitana to evaluate the prevalence of Shiga toxin-producing Escherichia coli (STEC) O157:H7 in raw beef, raw ground beef, soft cheese and fresh vegetables, sampled at different markets in the city. Between October 2000 and February 2001, 407 food samples were collected from different markets in the 42 districts of Lima Metropolitana. Samples were assayed for E. coli O157 by selective enrichment in modified Tryptic Soy Broth containing novobiocin, followed by immunomagnetic separation (IMS) and plating onto sorbitol MacConkey agar supplemented with cefixime and potassium tellurite. Fifty (12.3%) of 407 food samples resulted positive for E. coli O157 isolation (23 of 102 ground beef; 15 of 102 beef meat; eight of 102 soft cheese and four of 101 fresh vegetables). Thirty-five E. coli O157 isolates were further analysed for the presence of virulence genes. All 35 were positive by PCR for O157 rfbE, fliCh7, eae-gamma1 and ehxA genes. In addition, genes encoding Shiga toxins were detected in 33 of 35 isolates, five isolates (14%) encoded stx(1), stx(2), and 28 (80%) stx2 only. The isolates were of seven different phage types (PT4, PT8, PT14, PT21, PT34, PT54, and PT87) with three phage types accounting for 80% of isolates: PT4 (15 isolates), PT14 (8 isolates), and PT21 (5 isolates). Interestingly, the majority (31 of 35; 89%) of E. coli O157:H7 isolates characterized in this study belonged mainly to the phage types previously found in STEC O157:H7 strains associated with severe human disease in Europe and Canada. Pulsed-field gel electrophoresis (PFGE) of 32 isolates revealed 14 XbaI-PFGE groups (I to XIV) of similarity >85%, with 23 (72%) isolates grouped in five clusters. Some isolates from different districts presented a high clonal relatedness. Thus, PFGE group VIII clustered eleven strains from nine different districts. The broad range of PFGE subtypes found in this study demonstrates the natural occurrence of many genetic variants among STEC O157:H7 spread in Lima.
Assuntos
Escherichia coli O157 , Contaminação de Alimentos/análise , Microbiologia de Alimentos , Toxinas Shiga/biossíntese , Animais , Tipagem de Bacteriófagos , Bovinos , Queijo/microbiologia , Contagem de Colônia Microbiana , Qualidade de Produtos para o Consumidor , Eletroforese em Gel de Campo Pulsado , Escherichia coli O157/genética , Escherichia coli O157/metabolismo , Escherichia coli O157/patogenicidade , Humanos , Separação Imunomagnética , Carne/microbiologia , Produtos da Carne/microbiologia , Peru , Toxinas Shiga/análise , Verduras/microbiologia , Virulência/genéticaRESUMO
Shiga toxin-producing Escherichia coli (STEC) strains isolated from healthy cattle (O111:NM, seven strains; O111:H8, three strains) in Brazil were studied and compared to previously characterized human strains in regard to their phenotypic and genotypic characteristics to evaluate their pathogenic potential. Most bovine STEC O111 strains were isolated from dairy calves, and strains with genotypes stx1 alone and stx1/stx2 (variant stx2) occurred in different regions. Irrespective of the stx genotype, all strains were positive for eae theta, alpha variants of tir, espA and espB, and for ler, qseA, iha, astA and efa1 genes. Only one strain was negative for EHEC-hlyA and all strains were negative for iha, saa and espP genes and for EAF and bfpA, genetic markers of EPEC. Except for the presence of stx2, bovine strains showed the same profile of putative virulence genes found among the human strains. Similar biochemical behavior was identified among the strains analysed. Two bovine STEC strains produced the localized adherence (LA) phenotype in 6-h tests with Caco-2 (human enterocyte) cells. Intimate attachment (judged by the FAS test) was found in 9 out of 10 bovine strains as it was observed for the human STEC strains. RAPD-PCR analysis showed two distinct RAPD groups among the STEC O111 strains examined. Despite the relative low frequency of STEC O111 strains recovered from cattle no differences in their pathogenic potential were observed compared to some strains isolated from human diarrhea, suggesting that healthy cattle may be a potential source of infection for humans in Brazil.
Assuntos
Doenças dos Bovinos/microbiologia , Infecções por Escherichia coli/veterinária , Escherichia coli/patogenicidade , Toxinas Shiga/genética , Fatores de Virulência/análise , Animais , Aderência Bacteriana , Brasil , Células CACO-2 , Bovinos , Doenças dos Bovinos/transmissão , Reservatórios de Doenças/veterinária , Escherichia coli/classificação , Escherichia coli/genética , Infecções por Escherichia coli/microbiologia , Infecções por Escherichia coli/transmissão , Marcadores Genéticos , Genótipo , Humanos , Fenótipo , Técnica de Amplificação ao Acaso de DNA Polimórfico/métodos , Técnica de Amplificação ao Acaso de DNA Polimórfico/veterinária , Toxinas Shiga/biossíntese , Virulência/genética , Fatores de Virulência/genéticaRESUMO
The contamination of beef carcasses with Shiga toxin-producing O157:H7 and non-O157 Escherichia coli (STEC) obtained from a slaughter plant in Guadalajara, Mexico was investigated. A total of 258 beef carcasses were sampled during a 12-month period. All samples were assayed for STEC by selective enrichment in modified tryptone soy broth supplemented with cefixime, cefsulodin and vancomycin, followed by plating on Sorbitol MacConkey Agar supplemented with cefixime and tellurite (CT-SMAC). Simultaneously, all samples were assayed by immunomagnetic separation (IMS) and plated on CT-SMAC and CHROMagar. The presence of the stx1, stx2, eaeA and hly933 genes, recognized as major virulence factors of STEC, was tested for O157:H7 and non-O157 E. coli isolates by multiplex polymerase chain reaction (PCR). STEC was detected in two (0.8%) samples. One of these STEC isolates corresponded to the serotype O157:H7 showing stx2, eaeA and hyl933 genes. The other isolate corresponded to non-O157 STEC and only had the stx1 gene. Thirteen carcasses (5%) were positive for nonmotile E. coli O157 and 7 (2.7%) were positive for E. coli O157:H7. The presence of O157:H7 and non-O157 STEC on beef carcasses in this slaughter plant in Guadalajara, Mexico, emphasizes the importance of implementing the Hazard Analysis and Critical Control Point (HACCP) system, as well as the need for implementing, evaluating, and validating antimicrobial interventions to reduce the presence of potential pathogenic microorganisms.