Dynamic of mutational events in variable number tandem repeats of Escherichia coli O157:H7.

VNTRs regions have been successfully used for bacterial subtyping; however, the hypervariability in VNTR loci is problematic when trying to predict the relationships among isolates. Since few studies have examined the mutation rate of these markers, our aim was to estimate mutation rates of VNTRs specific for verotoxigenic E. coli O157:H7. The knowledge of VNTR mutational rates and the factors affecting them would make MLVA more effective for epidemiological or microbial forensic investigations. For this purpose, we analyzed nine loci performing parallel, serial passage experiments (PSPEs) on 9 O157:H7 strains. The combined 9 PSPE population rates for the 8 mutating loci ranged from 4.4 × 10(-05) to 1.8 × 10(-03) mutations/generation, and the combined 8-loci mutation rate was of 2.5 × 10(-03) mutations/generation. Mutations involved complete repeat units, with only one point mutation detected. A similar proportion between single and multiple repeat changes was detected. Of the 56 repeat mutations, 59% were insertions and 41% were deletions, and 72% of the mutation events corresponded to O157-10 locus. For alleles with up to 13 UR, a constant and low mutation rate was observed; meanwhile longer alleles were associated with higher and variable mutation rates. Our results are useful to interpret data from microevolution and population epidemiology studies and particularly point out that the inclusion or not of O157-10 locus or, alternatively, a differential weighting data according to the mutation rates of loci must be evaluated in relation with the objectives of the proposed study.

Distribution of additional virulence factors related to adhesion and toxicity in Shiga toxin-producing Escherichia coli isolated from raw products in Argentina.

UNLABELLED: A total of 73 Shiga toxin-producing Escherichia coli (STEC) isolates, belonging to 25 serotypes and isolated from raw products in Argentina, were examined for the occurrence of genes responsible for bacterial adhesions to intestine, ehaA (EHEC autotransporter), lpfAO113 (long polar fimbriae), sab (STEC autotransporter [AT] contributing to biofilm formation), ecpA (E. coli common pilus), hcpA (haemorrhagic coli pilus), elfA (E. coli laminin-binding fimbriae), sfpA (sorbitol-fermenting EHEC O157 fimbriae plasmid-encoded) and of the toxigenic gene cdt-V (cytolethal distending toxin). Our study showed different adhesin profiles that are not linked to one specific serotype and that all analysed isolates possess, besides stx genes, some adherence genes. Several of the isolates contained also multiple toxin genes. The results of the present work alert the presence of genes coding for additional adhesins and cdt-V toxin in LEE-negative STEC strains that occur in foods, and this traits could increase their pathogenic potential. SIGNIFICANCE AND IMPACT OF THE STUDY: Meat products are one of the main vehicles of Shiga toxin-producing E. coli, and the presence of genes coding for additional adhesins and toxins could increase their pathogenic potential. There is a need for a more detailed characterization of the strains in regard to these extra virulence factors.

Detection of Shiga toxin-producing Escherichia coli by sandwich enzyme-linked immunosorbent assay using chicken egg yolk IgY antibodies.

Enterohemorrhagic Escherichia coli (EHEC), a subset of Shiga toxin producing E. coli (STEC) is associated with a spectrum of diseases that includes diarrhea, hemorrhagic colitis and a life-threatening hemolytic-uremic syndrome (HUS). Regardless of serotype, Shiga toxins (Stx1 and/or Stx2) are uniformly expressed by all EHEC, and so exploitable targets for laboratory diagnosis of these pathogens. In this study, a sandwich ELISA for determination of Shiga toxin (Stx) was developed using anti-Stx2B subunit antibodies and its performance was compared with that of the Vero cell assay and a commercial immunoassay kit. Chicken IgY was used as capture antibody and a HRP-conjugated rabbit IgG as the detection antibody. The anti-Stx2B IgY was harvested from eggs laid by hens immunized with a recombinant protein fragment. Several parameters were tested in order to optimize the sandwich ELISA assay, including concentration of antibodies, type and concentration of blocking agent, and incubation temperatures. Supernatants from 42 STEC strains of different serotypes and stx variants, including stx(2EDL933), stx(2vha), stx(2vhb), stx(2g), stx(1EDL933), and stx(1d) were tested. All Stx variants were detected by the sandwich ELISA, with a detection limit of 115 ng/ml Stx2. Twenty three strains negative for stx genes, including different bacteria species, showed no activity in Vero cell assay and produced negative results in ELISA, except for two strains. Our results show that anti-Stx2B IgY sandwich ELISA could be used in routine diagnosis as a rapid, specific and economic method for detection of Shiga toxin-producing E. coli.

Short communication: characterization of Shiga toxin-producing Escherichia coli isolated from newborn, milk-fed, and growing calves in Argentina.

Shiga toxin-producing Escherichia coli (STEC) cause foodborne pathogenic disease that is shed in the feces of cattle. The aim of this study was to evaluate how early young calves are colonized by STEC strains, potentially pathogenic for humans, and the prevalence in different calf categories. From 808 rectal swabs analyzed by PCR, 38% were stx positive. The prevalence in newborn (<24 h from birth), milk-fed (<2-mo-old), and growing calves (2-8 mo old) were 25, 43, and 58%, respectively. Forty different STEC serotypes were found among isolates from newborn, milk-fed, and growing calves that shed STEC strains potentially pathogenic for humans. The STEC strains could be acquired early from mothers, enabling the infection of other animal categories and confirming the risk to public health.

Seasonal variation of HUS occurrence and VTEC infection in children with acute diarrhoea from Argentina.

In order to study the seasonality of haemolytic uraemic syndrome (HUS) and verotoxigenic Escherichia coli (VTEC) infection in children, 437 patients under 6 years of age with acute diarrhoea were studied, 8% of whom progressed to HUS. VTEC was found in 10% of all of the stool samples analysed and seasonal occurrence of HUS (p < 0.01) was confirmed. VTEC infection was more prevalent in warm months, although the differences were not statistically significant. Moreover, a significant difference in the detection of O157:H7 serotype and in the vt profile between cold and warm months (autumn and winter; spring and summer, respectively) was established. The O157:H7 serotype was isolated more frequently during warm months. Moreover, a predominance of vt (2) was noted, which was partially replaced by the combination of vt (1) with vt (2) in the cold season. The results of this study indicate the seasonal variation of the disease and the presence of serotype O157:H7 and the vt types. They also reinforce the need to develop prevention programmes considering the seasonal pattern of the disease, which would generate an impact on public health. Control strategies of the pathogen in cattle in the most risky season of the year would also be of benefit.

Enteropathogenic Escherichia coli contamination at different stages of the chicken slaughtering process.

Enteropathogenic Escherichia coli is a foodborne pathogen that produces potentially fatal infant diarrhea, noticeably in developing countries. The aim of this study was to detect EPEC contamination by PCR at different stages of the chicken slaughtering process. We collected swabs from chicken cloacae and washed carcasses (external and visceral cavity) during the slaughtering process in 3 sampling occasions. Unwashed eviscerated carcasses were also sampled (at the visceral cavity) in the second and third sampling occasions. Enteropathogenic Escherichia coli was detected in 6 to 28% of cloacal samples, 39 and 56% of unwashed eviscerated carcasses, and 4 to 58% of washed carcasses. None of the samples were positive for bfpA, suggesting contamination with atypical EPEC. The detection of EPEC at different stages of the chicken slaughtering process showed that the proportion of contaminated samples remained or even increased during processing. In addition, the high proportion of contaminated carcasses during chicken processing represents a risk for the consumers and a challenge to improve procedures for those working in the sanitary control service.

Multiplex PCR assay for the detection of five putative virulence genes encoded in verotoxigenic Escherichia coli plasmids.

The aim was to perform a pentavalent PCR assay for the detection of putative virulence genes encoded in VTEC plasmids, katP, espP, subA, stcE, and ehxA. The five-specific primer pairs used in the assay do not interfere with each other and generate amplification products of 914, 774, 556, 399, and 262 bp. It was selected at random 39 strains belonged to 20 serotypes in order to evaluate the multiplex in a wide variety of strains. The results of this study indicate that it is possible to perform simultaneous amplification and search for recognized plasmid-encoded virulence markers from different E. coli serotypes and apply this technique to the genetic characterization of E. coli strains isolated from reservoirs, foods or patients. This complementary technique is a useful tool to detect interstrain differences for epidemiological studies and to provide information that could be related to the risk of human infection.

Reduction of Adherence of E. coli O157:H7 to HEp-2 Cells and to Bovine Large Intestinal Mucosal Explants by Colicinogenic E. coli.

Enterohemorrhagic E. coli strains (EHEC) had emerged as foodborne pathogens and cause in human diarrhea and hemolytic-uremic syndrome. Because of the widespread distribution of EHEC serotypes and O157 and non-O157 in cattle population, its control will require interventions at the farm level such as the administration of probiotics that produce inhibitory metabolites. E. coli O157:H7 shows tissue tropisms for the gastrointestinal tract (GIT) of cattle. The aim of this study was to test the ability of a colicinogenic E. coli (isolated from bovine) to reduce the adherence of E. coli O157:H7 to HEp-2 cells and to GIT of cattle. We inoculated HEp-2 cells and bovine colon explants with both kinds of strains. Colicinogenic E. coli was able to reduce the adherence of E. coli O157:H7 to HEp-2 cells and to bovine tissues.

Characterization of Shiga toxin-producing Escherichia coli isolated from dairy cows in Argentina.

AIMS: To feno-genotypically characterize the Shiga toxin-producing Escherichia coli (STEC) population in Argentinean dairy cows. METHODS AND RESULTS: From 540 STEC positive samples, 170 isolates were analyzed by multiplex PCR and serotyping. Of these, 11% carried stx1, 52% stx2 and 37% stx1/stx2. The ehxA, saa and eae were detected in 77%, 66% and 3%, respectively. Thirty-five per cent of strains harboured the profile stx1, stx2, saa, ehxA and 29% stx2, saa, ehxA. One hundred and fifty-six strains were associated with 29 different O serogroups, and 19 H antigens were distributed among 157 strains. STEC O113:H21, O130:H11 and O178:H19 were the most frequently found serotypes. The STEC O157:H7 were detected in low rate and corresponded to the stx2(+) , eae(+) , ehxA(+) virulence pattern. CONCLUSIONS: We detected a diversity of STEC strains in dairy cattle from Argentina, most of them carrying genes linked to human disease. SIGNIFICANCE AND IMPACT OF THE STUDY: The non-O157 STEC serotypes described in this study are associated worldwide with disease in humans and represent a risk for the public health. For this, any microbiological control in dairy farms should be targeted not only to the search of O157:H7 serotype.

Occurrence of Shiga toxin-producing E. coli (STEC) on carcasses and retail beef cuts in the marketing chain of beef in Argentina.

Argentina has the highest incidence of HUS in the world. HUS is produced by STEC O157 and non-O157. Cattle's faeces and hides are sources of STEC contamination of carcasses during slaughter. We investigated the presence of STEC in carcasses and cuts of meat in the marketing chain in an agricultural city located in Buenos Aires Province (Argentina). In this study, the detection of the stx gene was used as an indicator of carriage of meat with STEC. In carcasses, we detected 12.34% and 18.64% of STEC at the slaughter and sanitary control cabin (place where carcasses arrive from slaughters located outside the city), respectively. These percentages increased at butcheries (24.52%). The 25% of retail beef cuts were STEC-positive with significant differences among the different cuts of meat (chuck: 12.12%, rump roast: 12.12% and minced beef: 40.74%). The stx2 gene was the predominant gene detected in all samples at different levels of the commercialization meat chain.

Seasonal variation of Shiga toxin-encoding genes (stx) and detection of E. coli O157 in dairy cattle from Argentina.

AIMS: To study the seasonal variation of Shiga toxin-encoding genes (stx) and to investigate the presence of Shiga toxin-producing Escherichia coli (STEC) O157 in cattle belonging to five dairy farms from Argentina. METHODS AND RESULTS: Rectal swab samples were collected from 360 dairy cows in each season and 115 and 137 calves in autumn and in spring, respectively. The stx were investigated by multiplex PCR and it was used as the indicator for STEC. Samples positives for stx were tested by PCR for eae-gamma1 of E. coli O157 and then subjected to IMS (immunomagnetic separation). In positive animals significant differences in the prevalence of stx between warm and cold seasons were detected. In warm seasons, stx1 + stx2 increased and stx1 decreased, independently of the animal category. The prevalence of STEC O157 in cows and calves were 0.2% and 0.8%, respectively. CONCLUSIONS: This work provides new data about the occurrence of stx and STEC O157 in dairy herds from Argentina and suggests a relationship between the type of stx and season of year. SIGNIFICANCE AND IMPACT OF STUDY: The detection of STEC O157 and the seasonality of stx and its types provide an opportunity to improve control strategies designed to prevent contamination of food products and transmission animal-person.

Escherichia coli with anti-O157:H7 activity isolated from bovine colon.

AIMS: To isolate bacteria from bovine gastrointestinal tract and investigate their inhibitory effect on Escherichia coli O157:H7 in vitro. METHODS AND RESULTS: A total of 2400 bacterial colonies were isolated from cattle colonic mucous membrane. Thirteen strains demonstrated the ability to inhibit the growth of E. coli O157:H7. From these, seven were screened for the presence of virulence factors as: stx(1), stx(2), ehxA, eae, st1a and lt1 by polymerase chain reaction. The selected bacteriocin-producing bacteria showed susceptibility to most of the antibiotics used. CONCLUSIONS: The strains of E. coli isolated, which exhibit inhibitory activity on E. coli O157:H7 growth by the production of inhibitory substances, may be useful in the control of this pathogen in reservoirs. An important characteristic of these strains was the absence of any of the virulence factors assayed and the susceptibility to most of the antibiotics used for Gram-negative bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: These microorganisms might be used as probiotic bacteria to reduce the carriage of E. coli O157:H7 in cattle, thus limiting the contamination of carcasses at slaughter and subsequently the contamination of foods and the transfer of this pathogen to man.

Hypobiosis induction alters the protein profile of Ostertagia ostertagi (Nematoda: Trichostrongylidae).

The appearance of variations in the protein profile of Ostertagia ostertagi (Stiles, 1892) infective larvae (L3), which were induced by hypobiosis triggering factors, was evaluated by means of SDS-PAGE and densitometric analysis. Area integration analyses of their protein profiles was carried out between 66 and 77 kDa. Important quantitative variations were identified in the protein levels of the induced larvae, where a 5.25 fold increase compared to the control was observed. Two 75.4 and 70 kDa protein bands were found which exceeded the control profile by 4.5 and 44 fold, respectively. This fact suggests that the changes brought about in the process of hypobiosis induction are restricted. This work demonstrates changes at a molecular level corresponding with biological changes induced by conditions causing O. ostertagi hypobiosis.

Toxigenic Escherichia coli isolated from pigs in Argentina.

The presence of porcine toxigenic E. coli (ETEC, VTEC) in 28 piggeries (5% of total) of the central and northeast region of Argentina was studied for a better understanding of the epidemiology of porcine strains. Samples were taken by rectal swabs from healthy piglets and from those with diarrhoea, in addition to their dams. Between 5-10 colonies were isolated from each one of 223 animals sampled from 1992 to 1997. By using specific primers each strain was screened by PCR for VT1, VT2all, VT2e, STIa, and LTI toxin genes. Only strains positive for any of the toxins mentioned above were screened for STb. Their O serogroups were determined by agglutination. All of the above enterotoxins and verocytotoxins were found in E. coli isolated from the animals. The STIa gene was detected in E. coli isolated from 27/127 piglets with diarrhoea, in comparison with LTI (4/127 pigs). No toxin gene was amplified from E. coli isolated from either healthy piglets or their dams. When strains isolated from 48 piglets without diarrhoea but showing delayed growth were analysed by PCR, their toxin profile was determined to be VT1 (1/48 piglets), VT2all (5/48), STIa (1/48), LTI (3/48) and VT2e (3/48). Serogroup O64 prevailed among ETEC; O138 prevailed for ETEC/VTEC strains. This is the first extensive study regarding porcine toxigenic E. coli in Argentina and constitutes an important database for the implementation of prevention measures.

Virulence genotypes and serotypes of verotoxigenic Escherichia coli isolated from cattle and foods in Argentina. Importance in public health.

Virulence factors of Verotoxin-producing Escherichia coli (VTEC) strains isolated from hamburgers and ground beef were studied in Argentina by PCR. Their virulence profiles were correlated with those corresponding to strains isolated from calves and adult cattle. Most virulent profiles (VTs+ eae+ Mp+) were present in E. coli from healthy and diarrheic calves corresponding to O5:H-, O5:H27, O20:H?, O26:H11, O38:H?, O103:H-, O103:H2, O111:H-, O118:H16, O165:H-serotypes. The presence of the eae gene was significantly more frequent among VTEC strains isolated from calves (20/26; 76%) than from adult cattle (1/39; 2.5%) (p < 0.005). VT2+ eae- E. coli was prevalent in foods and adult cattle at slaughterhouse. The prevalence of the eae gene was similar between VTEC strains isolated from meat (0/21) and adult cattle (1/39; 2.5%) which constitutes the main population processed at slaughterhouses in Argentina. Serotyping showed that VTEC strains were distributed among 31 serotypes, some of which (O20:H19, O91:H21, O113:H21, O116:H21, O117:H7, O171:H2, OX3:H21) were shared between bovine and food strains. These O serogroups have been isolated from cases of haemorrhagic colitis (HC) and haemolyticuraemic syndrome (HUS) in humans in several continental European countries. This study confirms the role of cattle as a reservoir of many VTEC serotypes other than O157:H7 and represents a base for future diagnostic, prevention and control strategies of EHEC in this country. In addition, this study affirms the advantages of PCR-based screening of E. coli isolates given the finding of so many verotoxin-producing strains.

A DNA fragment of Leptospira interrogans encodes a protein which shares epitopes with equine cornea.

Horses infected with Leptospira interrogans present several clinical disorders, one of them being recurrent uveitis. An antigenic relationship between this bacterium and equine cornea has been described in previous studies. With the aim to make progress on defining the molecular basis and pathogenesis of equine recurrent uveitis, here we describe the cloning of one DNA fragment from a Leptospira interrogans serovar pomona genomic lambda gt11 library. Although there are references of transcription of leptospiral genes in E. coli from their own leptospiral promoters, in this recombinant construction the leptospiral DNA was located under the control of lacZ promoter since no expression could be detected in the absence of IPTG. This clone, isolated by expression screening with polyclonal serum raised against equine corneal proteins, encodes a 90 kDa protein of L. interrogans which crossreacts with equine cornea as proved Western-blotting. Antibodies directed against this leptospiral protein strongly recognised a 66 kDa equine corneal protein, one of those recognised by an anti-equine cornea serum. Our findings suggest that an immune response to 90 kDa protein participates in pathogenesis of equine uveitis.

Prevalence of bovine verotoxin-producing Escherichia coli in Argentina.

Faecal swabs obtained from 126 calves and 118 cows in Argentina were investigated for the presence of verotoxin-producing Escherichia coli (VTEC). VTEC strains were recovered from 10 (23%) of 43 calves with diarrhoea, from 24 (29%) of 83 healthy calves, from 40 (44%) of 91 healthy cows waiting at the slaughterhouse, and from 6 (22%) of 27 healthy grazing cattle. PCR showed that 21 (9%) of animals carried VT1+ strains, 49 (20%) VT2+ strains and 10 (4%) VT1+ VT2+ strains. VT1+ strains predominated among calves (16% versus 0.8%; p < 0.001). The presence of eae gene was significantly more frequent among VTEC strains isolated from calves (78%; 46/59) than from cows (2%; 1/65) (p < 0.001). Furthermore, eae gene was more prevalent in VT1+ strains (97%; 32/33) than in VT2+ strains (14%; 10/70) (p < 0.001) and in VT1+ VT2+ strains (24%; 5/21) (p < 0.001). Sorbitol negative high virulent strains serogroups O157 were not detected. This study indicates that cattle are a reservoir of VTEC strains, and that eae gene is associated with VT1+ strains that are predominating among young animals. Fortunately, only adult animals are taken to the slaughterhouse, among which VTEC strains negative for eae gene are predominating.

Differentiation of pathogenic and non-pathogenic leptospires by means of the polymerase chain reaction.

A polymerase chain reaction was carried out to detect pathogenic leptospires isolated from animals and humans in Argentina. A double set of primers (G1/G2, B64-I/B64-II), described before, were used to amplify by PCR a DNA fragment from serogroups belonging to Leptospira interrogans but did not allow to detect saprophytic strains isolated from soil and water (L. biflexa). This fact represents an advantage since it makes possible the differentiation of pathogenic from non-pathogenic leptospires in cultures. The sensitivity of this assay has been determined, allowing to detect just only 10 leptospires in the reaction tube. Those sets of primers generated either a 285 bp or 360 bp fragment, depending on the pathogenic strain.

Detection of an antigenic protein of Leptospira interrogans which shares epitopes with the equine cornea and lens.

A protein epitope which is involved in an antigenic relationship between equine ocular tissues and Leptospira interrogans was detected in homogenates of the bacterium. The antigenic determinant was harboured on a peptide structure which was shown to be sensitive to the action of denaturing and reducing agents by means of Western blotting. The outer surface of the leptospires appeared to be free of this epitope as was proved by dot-blot and electron microscopic studies.

[Detection of enterotoxigenic Escherichia coli adhesion antigens. Study of their expression in different culture media]. / Detección de antígenos de adhesión en Escherichia coli enterotoxigénica. Estudio de la expresión en diferentes medios de cultivo.

Polyclonal antibodies were raised in rabbits against the adherence antigens K88ab, ac, ad K99 and F41 of enterotoxigenic Escherichia coli (ETEC). Their specificity for the antigen was tested by using homologous and heterologous E. coli strains, which had been grown under permissive and nonpermissive conditions for pili expression and also confronted to a purified extract of each fimbrial adhesin. Optimal conditions for agglutinating bacterial suspensions were established for both antisera and antigens. Expression of adhesins was studied in several culture media, which are being commonly used in clinical bacteriology. K99, K88ab and K88ad were satisfactorily expressed in both Minca broth and solid media on T.S.I. Mac Conkey and E.M.B. K88ac was not expressed on G1253 strain when grown on T.S.I. or in Mac Conkey when the inoculum was obtained from E.M.B. However, Mac Conkey allowed K88ac expression when inoculum had been grown on Luria-Bertani. On the other hand, F41 was only satisfactorily expressed in Minca broth, failing in being detected by agglutination in the remaining culture media. These results allow to use alternative media, beyond the recommended Minca broth, to detect adhesins K99, K88ab, and K88ad on ETEC isolates.