Shiga Toxigenic Escherichia Coli Isolates Research Articles

Isolation and characterization of bacteriophage Ib_pec2 against shigatoxigenic Escherichia coli.

This study was aimed to isolate and characterize bacteriophage against drug-resistant, shigatoxigenic Escherichia coli (STEC), one of the zoonotic, food-borne organisms associated with ruminants, mainly cattle. STEC were isolated (n = 35) from neonatal calves, dairy workers, and the surrounding environment and their antimicrobial resistance pattern was studied. Out of the 35 isolates tested, 17 isolates were found to be multidrug resistant to important antibiotics like ampicillin, amoxicillin-clavulanate, ciprofloxacin, streptomycin, and tetracycline. Bacteriophage namely Ib_pec2 was isolated against one of the STEC isolates and its morphology, genetic and proteomic characterization was done. Morphological analysis by TEM revealed bacteriophages belonging to myoviridae family. The genetic characterization of g23 gene revealed that the bacteriophage belonged to Tequatrovirus of myoviridae family. Proteomic analysis was able to identify five proteins identical to Tequatrovirus of myoviridae family. One-step growth curve experiment revealed a latency period of 40 min and a burst size of 893 pfu/bacteria. Temperature and pH ranging from 40°C to 50°C, pH6-8, respectively. Phage could able to lyse majority of the STEC isolates. STEC are commensal organisms in the gastrointestinal tract of ruminants but are pathogenic in humans. Bacteriophages can be used as alternatives to antibiotics to control bacterial growth in ruminants and prevent its further spillage in the environment.

Effect of the vaccination against Shiga toxin 2e in a farm with history of oedema disease, caused by atypical Escherichia coli producing Shiga toxin (STEC)

Oedema disease of weaned piglets is caused by shigatoxigenic Escherichia coli (STEC), typically harbouring the stx2e gene and F18 adhesins. The aim of this study was to assess the effect of a commercially available oedema disease vaccine on the zootechnical performance, mortality and individual antibiotic treatment in a herd, in which non-typical STEC strains without F18 adhesin have been identified. The zootechnical performance (average daily gain, total weight gain), mortality and individual antibiotic treatment were compared between vaccinated and non-vaccinated control piglets in a monocentric field efficacy study, which was performed using two groups in a parallel, randomised design. A significantly higher average daily gain and total weight gain were recorded in the vaccinated piglets in comparison to the controls. The lower morbidity, mortality and antibiotic treatment in piglets in the vaccine group were not statistically significant. As a conclusion, the positive effect of the vaccination was confirmed in the herd with prevalent STEC not harbouring F18 adhesin. The vaccine was, therefore, also effective against oedema disease caused by such unusual STEC isolates, under the conditions of this study.

Shiga-toxigenic Escherichia coli from animal food sources in Mauritius: Prevalence, serogroup diversity and virulence profiles

Shiga-toxigenic Escherichia coli (STEC) are important human pathogens associated with diarrhea and in some cases haemorrhagic colitis. Contaminated food derived from cattle and wildlife species are often associated with disease outbreaks. In this study, we report the prevalence, serogroup diversity and virulence profiles of STEC strains derived from cattle, rusa deer and pig. Of the 422 samples analyzed, STEC were detected in 40% (80/200) of cattle, 27.0% (33/122) of deer and 13.0% (13/100) of pigs. STEC isolates belonged to 38 O-serogroups whereby 5.2% (24/462) of the isolates belonged to clinically important EHEC-7 serogroups: O26 (n = 2), O103 (n = 1), O145 (n = 3) and O157 (n = 18). Fourteen serogroups (O26, O51, O84, O91, O100, O104, O110, O117, O145, O146, O156, O157, O177 and ONT) displayed multiple virulence profiles. We also identified two serovars (O117 and O119) in deer which are not well-documented in epidemiological surveys. 73.7% (28/38) of recovered O-serogroups are known to be associated with serious human illnesses including haemolytic uremic syndrome (HUS) and bloody diarrhea. STEC isolates harboring single genotypes stx1, stx2, eae and hlyA accounted for 3.0% (14/462), 9.1% (42/462), 47.6% (220/462) and 1.7% (8/462) of all STEC isolates screened, respectively. Virulence combinations stx1 and stx2 were harboured by 1.3% of isolates while strains with genetic profiles eae/hlyA were the second most prevalent amongst STEC isolates. The full known virulent genotypes (stx2/eae, stx1/stx2/eae, stx1/stx2/hlyA and stx2/eae/hlyA) were present in 22 of the 462 STEC strains. A total of 10 different virulence patterns were recovered amongst animal species. Phylogeny of the gnd gene showed that amongst STEC strains, serovar O100 outlined the main cluster. Fourteen (n = 14) different sequence types (STs) were identified from a panel of twenty (n = 20) STEC isolates. One of the isolate (PG007B) possessed a unique ST (adk 10, fumC 693, gyrB 4, icd 1, mdh 8, purA 8, recA 2) that could not be assigned using MLST databases. None of the ST's recovered in deer were observed in domestic species. Our findings shows that food associated animals found on the tropical island of Mauritius carry a diversity of STEC strains with many serovars known to be associated with human disease. This report indicates that increased awareness, surveillance and hygienic attention at critical stages of the human food chain are warranted.

SMRT sequencing reveals differential patterns of methylation in two O111:H- STEC isolates from a hemolytic uremic syndrome outbreak in Australia

In 1995 a severe haemolytic-uremic syndrome (HUS) outbreak in Adelaide occurred. A recent genomic analysis of Shiga toxigenic Escherichia coli (STEC) O111:H- strains 95JB1 and 95NR1 from this outbreak found that the more virulent isolate, 95NR1, harboured two additional copies of the Shiga toxin 2 (Stx2) genes encoded within prophage regions. The structure of the Stx2-converting prophages could not be fully resolved using short-read sequence data alone and it was not clear if there were other genomic differences between 95JB1 and 95NR1. In this study we have used Pacific Biosciences (PacBio) single molecule real-time (SMRT) sequencing to characterise the genome and methylome of 95JB1 and 95NR1. We completely resolved the structure of all prophages including two, tandemly inserted, Stx2-converting prophages in 95NR1 that were absent from 95JB1. Furthermore we defined all insertion sequences and found an additional IS1203 element in the chromosome of 95JB1. Our analysis of the methylome of 95NR1 and 95JB1 identified hemi-methylation of a novel motif (5′-CTGCm6AG-3′) in more than 4000 sites in the 95NR1 genome. These sites were entirely unmethylated in the 95JB1 genome, and included at least 177 potential promoter regions that could contribute to regulatory differences between the strains. IS1203 mediated deactivation of a novel type IIG methyltransferase in 95JB1 is the likely cause of the observed differential patterns of methylation between 95NR1 and 95JB1. This study demonstrates the capability of PacBio SMRT sequencing to resolve complex prophage regions and reveal the genetic and epigenetic heterogeneity within a clonal population of bacteria.

Characterization of Bacteriophages Targeting Non-O157 Shiga Toxigenic Escherichia coli

Non-O157 Shiga toxigenic Escherichia coli (STEC) are an important group of foodborne pathogens, implicated in several outbreaks and recalls in the past 2 decades. It is therefore crucial to devise effective control strategies against these pathogens. Bacteriophages present an attractive alternative to conventional pathogen control methods in the food industry. Bacteriophages, targeting non-O157 STEC (O26, O45, O103, O111, O121, O145), were isolated from beef cattle operations in Oklahoma. Their host range and lytic ability were determined against several ( n = 21) non-O157 STEC isolates, by using the spot-on-lawn assay. Isolated phages were purified, and their morphology was determined under a transmission electron microscope. Infection kinetics of selected phages ( n = 19), particularly adsorption rate, rise period, latent period, and burst size, were determined. Phages were also evaluated for stability at a wide pH range (1 to 11) and temperature range (-80 to 90°C). In total, 45 phages were isolated and classified into Myoviridae, Siphoviridae, or Tectiviridae. The phages had a latent period between 8 and 37 min, a rise period between 19 and 40 min, and a large burst size (12 to 794 virions per infected cell), indicating high lytic activity. Tested phages were stable at pH 5 to 9 for 24 h, whereas a decrease in phage titer was observed at pHs 1, 2, and 11. Phages were stable at 40 and 60°C, except for O103-specific phages. At 70°C, all the phages lost viability after 20 min, except three phages targeting O26 and O121 and one phage targeting O45 and O111 STEC, which remained viable for 60 min. All the phages lost activity after 10 min at 90°C, except one each of O26 and O121 STEC-infecting phages that remained viable for 60 min. Phages remained stable for 90 days under refrigerated (4°C) and frozen (-20 and -80°C) storage. Characterization of phages, targeting diverse non-O157 STEC serotypes, could help in the development of effective biocontrol strategies for this group of pathogens in the food industry.

Prevalence and antimicrobial resistance pattern of Shiga toxigenic Escherichia coli in diarrheic buffalo calves.

Aim:Aim of the study was to investigate the prevalence, virulence gene profiles, and antimicrobial resistance pattern of Shiga toxigenic Escherichia coli (STEC) in diarrheic buffalo calves from Andhra Pradesh and Telangana States.Materials and Methods:A total of 375 fecal samples from diarrheic buffalo calves of 1-7, 8-30, 31-60, and 61-90 days age were collected from which STEC were isolated, and virulence genes were detected using multiplex polymerase chain reaction. The antimicrobial resistance of isolates was tested by disk diffusion method.Results:The prevalence of E. coli associated diarrhea in buffalo calves was 85.04%, of which 35.01% was STEC origin. In STEC, the combination of eaeA and, hlyA virulence genes was highest (42.45%) followed by stx1 (16.04%), stx1, stx2 and hlyA (13.21%), stx2 (12.64%), stx1, eae and hlyA (9.43%) and stx1 and hlyA (6.6%) genes were detected. Highest antimicrobial resistance was observed for tetracycline (63.21%) and ampicillin (48.11%), while chloramphenicol, gentamycin (96.33%) and imipenem (99.06%) antibiotics are susceptible. Multidrug resistance was detected in 69.81% of the STEC isolates from diarrheic buffalo calves.Conclusion:Higher prevalence of eaeA and hlyA genes carrying isolates of STEC may be a serious zoonotic threat and increased prevalence of multidrug resistance in E. coli may necessitate stringent selection of appropriate antimicrobial agent in treating buffalo calf diarrhea cases.

Biofilm formation, invasiveness and colicinogeny in locus of enterocyte and effacement negative O113:H21 Shigatoxigenic Escherichia coli.

Although Shiga toxins (Stx) are well-established virulence traits of O113:H21 Shigatoxigenic Escherichia coli (STEC) strains, a shortage in the knowledge of other virulence properties that may contribute to pathogenesis may exist in this serotype. This study investigated biofilm, invasiveness and colicinogeny capabilities in O113:H21 STEC isolated in Brazil, mostly from animal reservoirs. A search for genes that were reported to participate in the process of biofilm formation was also performed. The 34 O113:H21 STEC isolates analysed were assayed for biofilm production in polystyrene microplates. Genes for biofilm were investigated by PCR. Invasion of cell lineages was assessed in gentamicin protection assays and colicinogeny was investigated by phenotypic tests. Fifty per cent of the strains were biofilm formers, and 35% exhibited an invasive behaviour. The pattern of distribution of biofilm-related genes did not correlate with biofilm phenotypes observed, and a high percentage of the investigated strains were able to secrete colicins. Ability to form biofilm, invasiveness and colicinogeny is demonstrated for the first time in a collection of O113:H21 STEC. The ability to express three additional phenotypes besides Stx production may be a factor influencing the pathogenicity and persistence potential of O113:H21 STEC.

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.

Prevalence of Shiga-toxigenic <em>Escherichia coli</em> in Mauritian dairy cattle

Shiga-toxigenic Escherichia coli (STEC) are important human pathogens (1). They are characterized by their ability to pro­duce Shiga toxins (stx1 and stx2). Seven STEC have been shown to withstand food processing procedures that are expected to ensure food safety. Clinical symptoms associated with STEC infection can vary from abdominal cramps and acute bloody diarrhea to more severe aftereffects including hemorrhagic coli­tis, hemolytic uremic syndrome and thrombocytopenic purpura, which can lead to kidney failure and death.Dairy cattle, which excrete STEC in their feces, are a major source of STEC infection (2). Humans become infected with STEC through direct contact with infected animals or by inges­tion of contaminated water, raw and unpasteurized milk, meat products, and/or plant-derived products (4–6). The objectives of this study were to estimate both cow-level and farm-level point prevalence estimates of STEC fecal shedding in Mauritian dairy cattle and to characterize putative STEC isolates based on their virulence factors.A cross-sectional study was conducted to investigate the preva­lence of STEC in the dairy cattle population of Mauritius. Fecal samples were collected from 150 individual dairy cattle from 38 dairy farms located throughout the nine district regions of the island. Collected samples were enriched in modified Tryptic Soy broth followed by isolation on CHROMagarTM STEC (3). Suspected isolates were streaked onto EMB agar, further puri­fied on nutrient agar and subsequently cryopreserved in glyc­erol until further investigation. Putative isolates were charac­terized using molecular techniques (7, 8) for the presence of chromosomal sequences encoding Shiga toxin genes (stx1 and stx2), the intimin protein (eaeA) and the plasmid-encoded hemo­lysin (hlyA).Out of the 38 farm samples, 29 farms (76%) were found to be positive for presumptive STEC isolates. From the 150 fecal samples collected, 111 (74%) were found to harbor presump­tive STEC isolates (Table I). Polymerase-chain-reaction- (PCR-) based characterization has confirmed the presence of STEC in a number of fecal samples. Results obtained so far indicate that STEC are common members of the gut microbiome of dairy cattle in Mauritius.Presumptive STEC isolates are currently being screened with PCR targeting stx1, stx2, eaeA and hlyA genes. This epidemiological study on STEC is the first of its kind in Mauritius and in the Indian Ocean region. It aims at providing new information concerning the presence of STEC in Mauritian dairy cattle. It involves the use of the latest chromogenic agar (CHROMagarTM STEC) available on the market. This culture medium has been designed for the detection of a wide range of STEC from different sources. The study highlights the importance of implementing proper sanitary measures at the dairy farm level to prevent cross contamination of milk and the surrounding environment.

GeneSippr: a rapid whole-genome approach for the identification and characterization of foodborne pathogens such as priority Shiga toxigenic Escherichia coli.

The timely identification and characterization of foodborne bacteria for risk assessment purposes is a key operation in outbreak investigations. Current methods require several days and/or provide low-resolution characterization. Here we describe a whole-genome-sequencing (WGS) approach (GeneSippr) enabling same-day identification of colony isolates recovered from investigative food samples. The identification of colonies of priority Shiga-toxigenic Escherichia coli (STEC) (i.e., serogroups O26, O45, O103, O111, O121, O145 and O157) served as a proof of concept. Genomic DNA was isolated from single colonies and sequencing was conducted on the Illumina MiSeq instrument with raw data sampling from the instrument following 4.5 hrs of sequencing. Modeling experiments indicated that datasets comprised of 21-nt reads representing approximately 4-fold coverage of the genome were sufficient to avoid significant gaps in sequence data. A novel bioinformatic pipeline was used to identify the presence of specific marker genes based on mapping of the short reads to reference sequence libraries, along with the detection of dispersed conserved genomic markers as a quality control metric to assure the validity of the analysis. STEC virulence markers were correctly identified in all isolates tested, and single colonies were identified within 9 hrs. This method has the potential to produce high-resolution characterization of STEC isolates, and whole-genome sequence data generated following the GeneSippr analysis could be used for isolate identification in place of lengthy biochemical characterization and typing methodologies. Significant advantages of this procedure include ease of adaptation to the detection of any gene marker of interest, as well as to the identification of other foodborne pathogens for which genomic markers have been defined.

Characterization of Shiga Toxigenic Escherichia coli O157 and Non-O157 Isolates from Ruminant Feces in Malaysia.

Shiga toxigenic Escherichia coli (STEC) O157 and several other serogroups of non-O157 STEC are causative agents of severe disease in humans world-wide. The present study was conducted to characterize STEC O157 and non-O157 serogroups O26, O103, O111, O121, O45, and O145 in ruminants in Malaysia. A total of 136 ruminant feces samples were collected from 6 different farms in Peninsular Malaysia. Immunomagnetic beads were used to isolate E. coli O157 and non-O157 serogroups, while PCR was used for the detection and subtyping of STEC isolates. STEC O157:H7 was isolated from 6 (4%) feces samples and all isolates obtained carried stx 2c, eaeA-γ1, and ehxA. Non-O157 STEC was isolated from 2 (1.5%) feces samples with one isolate carrying stx 1a, stx 2a, stx 2c, and ehxA and the other carrying stx 1a alone. The presence of STEC O157 and non-O157 in a small percentage of ruminants in this study together with their virulence characteristics suggests that they may have limited impact on public health.

Synanthropic rodents as possible reservoirs of shigatoxigenic Escherichia coli strains

Shigatoxigenic Escherichia coli (STEC) strains are worldwide zoonotic pathogen responsible for different cases of human disease including hemolytic uremic syndrome (HUS). Transmission of STEC to humans occurs through the consumption of food and water contaminated by faeces of carriers and by person-to-person contact. The objective of this study was two-fold: (1) to investigate whether synanthropic rodents are possible reservoirs of STEC in the urban area and (2) whether a particular genus out of synanthropic rodent is the principal carrier of STEC. One hundred and forty-five rodents were captured in Buenos Aires City. Screening for stx1/stx2 and rfbO157 was done by PCR from the confluence zone. STEC isolates were further characterized with biochemical tests by standard methods. Additional virulence factors (eae, ehxA, and saa) were also determined by PCR. Forty-one of the rodents were necropsied and sample of kidney and small and large intestine were taken for histopathological diagnosis. The samples sections were stained with hematoxylin-eosin, and observed by light microscopy to evaluate the systemic involvement of these species in natural infections. STEC was isolated from seven out of 27 suspect animals at screening. The following genotypes were found in the STEC strains: stx1/stx2/ehxA (1), stx2 (4), stx2/ehxA (1), stx2/ehxA/eae (1). Neither gross nor microscopic lesions compatible with those produced by Shiga toxin were observed in the studied organs of necropsied rodents. The bivariate analysis including the 145 rodent's data showed that the isolation of STEC is associated positively to Rattus genus. This synanthropic species may play a role in the transmissibility of the agent thus being a risk to the susceptible population. Their control should be included specifically in actions to dismiss the contamination of food and water by STEC in the urban area, as additional strategies for epidemiological control.

Isolation of Shiga toxigenic Escherichia coli from butcheries in Taquaritinga city, State of São Paulo, Brazil

Shiga toxigenic Escherichia coli (STEC) has been implicated as the cause of several human diseases. Samples (ground beef, grinding-machines and human hands) from 23 butcheries were assayed for E. coli using standard microbiological methods, and 287 isolates were submitted to polymerase chain reaction for the detection of stx 1, stx 2 and eae genes. Four STEC isolates were recovered, two from ground beef and two from grinding-machines; all harbored the stx 2 gene and were negative for the eae gene. All E. coli isolates including the four STEC were screened for antibiotic resistance. High levels of resistance against several antimicrobial agents were detected; those most commonly observed were to tetracycline (76.6%), amoxicillin (64.1%) and cephalothin (58.8%). Such high levels of antimicrobial resistance highlight the need for a more rational use of these agents in cattle.

Attachment of Shiga toxigenic Escherichia coli to stainless steel

Shiga toxigenic Escherichia coli (STEC) are important foodborne pathogens causing gastrointestinal disease worldwide. Bacterial attachment to food surfaces, such as stainless steel may lead to cross contamination of foods and subsequent foodborne disease. A variety of STEC isolates, including E. coli O157:H7/H− strains, were grown in planktonic (broth) and sessile (agar) culture, following which initial attachment to stainless steel was determined using epifluorescence microscopy. Experiments were performed to determine whether the number of bacteria attached to stainless steel differed between STEC strains and between the two modes of growth. No relationship was found between STEC strains and the number of bacteria attached to stainless steel. Five STEC strains, including one non-toxigenic O157 isolate, attached in significantly greater ( p < 0.05) numbers to stainless steel following growth in planktonic culture compared to sessile culture. In contrast, two clinical strains of O157:H7 attached in significantly greater ( p < 0.05) numbers following growth in sessile culture compared to planktonic culture. Thirteen out of twenty E. coli strains showed no significant difference ( p > 0.05) in attachment when grown in planktonic or sessile culture. The change of interfacial free energy between the bacterial strains and stainless steel was calculated and the influence of free energy in attachment was determined. Although a significant variation ( p < 0.05) in free energy values was found between STEC strains, no correlation was found between free energy values and bacterial counts on stainless steel. In addition, no correlation was also found between bacterial hydrophobicity and surface charge values or production of surface structures (type I fimbriae or flagella) (previously determined) with the number of bacteria attached to stainless steel. The results of this study suggest that different growth conditions (planktonic and sessile) can influence the attachment of STEC to stainless steel. Factors other than bacterial physicochemical properties and these surface structures may also influence STEC attachment to stainless steel.

Attachment of Shiga Toxigenic Escherichia coli to Beef Muscle and Adipose Tissue

Shiga toxigenic Escherichia coli (STEC) serotypes are important foodborne pathogens that cause gastrointestinal disease worldwide. An understanding of how STEC strains attach to surfaces may provide insight into the potential persistence of and contamination with STEC in food environments. The initial attachment of a selection of STEC serotypes to beef muscle and adipose tissue was evaluated for isolates grown in planktonic and sessile culture. Initial experiments were performed to determine whether attachment differed among STEC strains and between the two modes of growth. Viable counts were obtained for loosely and strongly attached cells, and the strength of attachment (Sr) was calculated. All bacterial isolates grown in sessile culture attached in higher numbers to muscle and adipose tissue than did bacteria in planktonic cultures. For all attachment assays performed, mean concentrations for loosely attached cells were consistently higher than concentrations for strongly attached cells. The mean concentrations for strongly attached bacteria for planktonic and sessile cultures were significantly higher (P < 0.05) on adipose than on muscle tissue. However, some strains of STEC, particularly those from sessile culture, did not differ in their attachment to muscle or adipose tissue. Sr values were not significantly different (P > 0.05) among STEC isolates for all assays. No correlation was found between bacterial hydrophobicity and surface charge values (previously determined) and production of surface structures, viable counts, and Sr values. STEC grown in planktonic and sessile culture seems to behave differently with respect to attachment to muscle and adipose tissue. Cells in sessile culture may have a greater potential to strongly attach to meat surfaces.

Multiplex PCR for Direct Detection of Shiga Toxigenic Escherichia coli Strains Producing the Novel Subtilase Cytotoxin

We have recently described a novel AB5 subtilase cytotoxin produced by certain Shiga toxigenic Escherichia coli (STEC) strains. This potentially lethal toxin may contribute to severe gastrointestinal and systemic disease in humans. In this study we have developed a trivalent PCR assay for the detection of the novel toxin A subunit gene subA, as well as stx1 and stx2. The three primer pairs used in the assay do not interfere with each other and generate amplification products of 556, 180, and 255 bp, respectively. The assay can be used for determining the toxin genotype of STEC isolates, as well as for direct detection of toxin genes in primary fecal culture extracts.

Isolation of shiga toxigenic Escherichia coli from raw beef in Palestine

Shiga toxigenic Escherichia coli (STEC) isolated from raw beef samples in northern Palestine during a 1-year period were characterized for virulence genes by a polymerase chain reaction (PCR) assay and screened for their antibiotic resistance. STEC was identified in 44 (14.7%) of 300 raw beef samples. Twelve (27.3%) of the STEC isolates were serotype O157. Nine of those were isolated during summer. The majority of STEC isolates (70.5%) harbored both stx 1 and stx 2 genes, while the others harbored either stx 1 or stx 2. High levels of resistance against different antimicrobial agents were detected. Resistance to at least three drugs was found in 55% of the isolates.

Presence of activatable Shiga toxin genotype (stx(2d)) in Shiga toxigenic Escherichia coli from livestock sources.

Stx2d is a recently described Shiga toxin whose cytotoxicity is activated 10- to 1000-fold by the elastase present in mouse or human intestinal mucus. We examined Shiga toxigenic Escherichia coli (STEC) strains isolated from food and livestock sources for the presence of activatable stx(2d). The stx(2) operons of STEC were first analyzed by PCR-restriction fragment length polymorphism (RFLP) analysis and categorized as stx(2), stx(2c vha), stx(2c vhb), or stx(2d EH250). Subsequently, the stx(2c vha) and stx(2c vhb) operons were screened for the absence of a PstI site in the stx(2A) subunit gene, a restriction site polymorphism which is a predictive indicator for the stx(2d) (activatable) genotype. Twelve STEC isolates carrying putative stx(2d) operons were identified, and nucleotide sequencing was used to confirm the identification of these operons as stx(2d). The complete nucleotide sequences of seven representative stx(2d) operons were determined. Shiga toxin expression in stx(2d) isolates was confirmed by immunoblotting. stx(2d) isolates were induced for the production of bacteriophages carrying stx. Two isolates were able to produce bacteriophages phi1662a and phi1720a carrying the stx(2d) operons. RFLP analysis of bacteriophage genomic DNA revealed that phi1662a and phi1720a were highly related to each other; however, the DNA sequences of these two stx(2d) operons were distinct. The STEC strains carrying these operons were isolated from retail ground beef. Surveillance for STEC strains expressing activatable Stx2d Shiga toxin among clinical cases may indicate the significance of this toxin subtype to human health.

Serotypes and virulence genes of bovine Shigatoxigenic Escherichia coli (STEC) isolated from a feedlot in Argentina

Grazing-fed cattle were previously demonstrated to be reservoir of non-O157 Shigatoxigenic Escherichia coli (STEC) serotypes in Argentina. The acid-resistance of some STEC strains makes it reasonable to assume the presence in feedlot of particular STEC serotypes. Fifty-nine animals were sampled every 2 weeks during 6 months by rectal swabs. Twenty-seven of 59 animals (45.8%) were shown to be Stx2 +; 3/59 (5.1%) carried Stx1 + and 7/59 (11.9%) were Stx1 + Stx2 +. Among 44 STEC isolates, 31 isolates were associated to 10 O serogroups (O2, O15, O25, O103, O145, O146, O157, O171, O174, O175) and 13 were considered non-typable (NT). Six H antigens (H2, H7, H8, H19, H21, H25) were distributed in 21 isolates whereas 23 were non-mobile (H–). Seventeen of 44 strains (38.6%) were eaeA + and 14 (31.8%) harbored the 60 MDa plasmid. The megaplasmid (Mp) and eaeA gene were simultaneously found in a limited number of serotypes belonging to the enterohaemorrhagic E. coli (EHEC). E. coli O157:H7 strains, isolated from four (6.8%) animals, corresponded to the Stx2 +, eaeA +, Mp + pattern. Three O157:H7 strains belonged to phage type 4 and the other strain was atypical. Many serotypes isolated from grain-fed cattle (O2:H25, O15:H21, O25:H19, O145:H–, O146:H–, O146:H21, O157:H7, O175:H8) also differed from those isolated by us previously from grazing animals. The serotypes O15:H21, O25:H19 and O175:H8 had not been identified at present as belonging to STEC. This work provides new data for the understanding of the ecology of STEC in grain-fed cattle and confirms that cattle are an important reservoir of STEC.

Isolation and characterisation of Shiga toxigenic Escherichia coli strains from northern Palestine.

Shiga toxigenic Escherichia coli (STEC) isolates from symptomatic and asymptomatic patients in northern Palestine in 1999 were screened for serotype O157 and characterised for virulence genes by multiplex PCR assay. Of the 176 STEC isolates, 124 (70.5%) were of serotype O157. All these isolates carried the gene for Shiga toxin type 1 (stx,) and 112 (90.3%) carried stx2. The intimin encoding gene locus eae was detected in 16 isolates (12.9%) and the enterohaemolysin encoding gene, hlyA, in 18 (14.5%). Statistical analysis showed a significant association between the presence of eaeA and hlyA, either alone or combined with stx1 and stx2 genes in O157 isolates from symptomatic infection. ERIC-PCR analysis of DNA from 80 serotype O157 isolates revealed three major clonal populations.