non-STEC Strains Research Articles

Survival of Escherichia coli in Edible Land Snails: Implications for Heliciculture and Public Health.

Land snails are considered a delicacy in many countries in Europe and sub-Saharan Africa. However, the interaction of microbial pathogens with land snails may present a public health threat when handling and/or consuming snails. This study examines the survival of Escherichia coli in edible land snails in a model system. Well-studied Shigatoxigenic (STEC) and non-STEC strains were compared. Mature Helix spp. were experimentally fed with E. coli-inoculated oats for 48 h. The snail feces after inoculation were periodically sampled and cultured for a 30-day period and subjected to microbiological analyses. The average rate of decline of the non-STEC strain CSH-62 in the feces of live snails was significantly (p < 0.05) faster than that of STEC ERL 06-2503. In addition, the viable population of E. coli ERL 06-2503 significantly (p < 0.05) persisted for a longer time in the intestine of land snails than E. coli CSH-62. The results showed that the viable population of the E. coli strains examined demonstrated first-order kinetics, and their survival (CFU/mL) appeared significantly (p < 0.05) dependent on the E. coli pathotype. In addition, the continuous enumeration of E. coli in snail faeces indicated that land snails could serve as a mode of transmission of microbial pathogens to susceptible hosts, including humans. Further research is recommended to better quantify the direct and indirect health risks of pathogen transmission by edible snails to humans.

Impact of Shiga-toxin encoding gene transduction from O80:H2 Shiga toxigenic Escherichia coli (STEC) on non-STEC strains

Shiga toxin-producing Escherichia coli (STEC) are major foodborne pathogens that cause human diseases ranging from diarrhea to life-threatening complications including hemolytic–uremic syndrome. Virulence of STEC strains and their ability to cause severe diseases are associated with the activity of prophage-encoded Shiga toxins (Stxs). The first objective of this work was to isolate and characterize the Stx2d phage from STEC O80:H2 and to study the transfer of this phage in non-STEC strains. The second objective was to assess the survival of Galleria mellonella larvae inoculated with these transduced strains. Firstly, one bacteriophage isolated from a STEC O80:H2 strain was used to infect six non-STEC strains, resulting in the conversion of three strains. Then, stability assays were performed, showing that this phage was stable in the new STEC strains after three successive subculturing steps, as confirmed by a combination of short and long read genome sequencing approaches. This phage, vB_EcoS_ULI-O80_Stx2d, is resistant to moderate temperature and pH. It belongs to a currently unclassified genus and family within the Caudoviricetes class, shares 98% identity with Stx2_112808 phage and encodes several proteins involved in the lysogenic cycle. The yecE gene was identified at the insertion site. Finally, G. mellonella experiments showed that the transduced strains caused significantly higher mortality rates than the corresponding non-STEC strains. In conclusion, this study showed that stx2d gene from O80:H2 E. coli can be transferred to non-STEC strains and contributes to their virulence.

Characteristics of Bacteriophage Isolates and Expression of Shiga Toxin Genes Transferred to Non Shiga Toxin-Producing E. coli by Transduction.

A risk analysis of Shiga toxin (Stx)-encoding bacteriophage was carried out by confirming the transduction phage to non-Stx-producing Escherichia coli (STEC) and subsequent expression of the Shiga toxin genes. The virulence factor stx1 was identified in five phages, and both stx1 and stx2 were found in four phages from a total of 19 phage isolates with seven non-O157 STEC strains. The four phages, designated as φNOEC41, φNOEC46, φNOEC47, and φNOEC49, belonged morphologically to the Myoviridae family. The stabilities of these phages to temperature, pH, ethanol, and NaClO were high with some variabilities among the phages. The infection of five non-STEC strains by nine Stx-encoding phages occurred at a rate of approximately 40%. Non-STEC strains were transduced by Stx-encoding phage to become lysogenic strains, and seven convertant strains had stx1 and/or stx2 genes. Only the stx1 gene was transferred to the receptor strains without any deletion. Gene expression of a convertant having both stx1 and stx2 genes was confirmed to be up to 32 times higher for Stx1 in 6% NaCl osmotic media and twice for Stx2 in 4% NaCl media, compared with expression in low-salt environments. Therefore, a new risk might arise from the transfer of pathogenic genes from Stx-encoding phages to otherwise harmless hosts. Without adequate sterilization of food exposed to various environments, there is a possibility that the toxicity of the phages might increase.

Novel strategy of rapid typing of Shiga toxin-producing Escherichia coli using MALDI Biotyper and ClinProTools analysis

IntroductionRapid detection of Shiga toxin-producing Escherichia coli (STEC) is essential. Matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) allows rapid, accurate, and low-cost microbial identification. We aimed to examine the discrimination ability of MALDI Biotyper (Bruker Daltonics) between STEC and non-STEC. MethodsIn total, 234 strains (79 STEC strains and 155 non-STEC strains) isolated from stool between 2009 and 2014 were measured by MALDI Biotyper and mass spectra of 2000–20,000 m/z were analyzed with ClinProTools (Bruker Daltonics). Eighty-three strains were randomly extracted to produce STEC detection models using 3 algorithms, and 151 strains were used as validation samples to verify STEC detection performance and discrimination performance of Shiga toxins with the STEC detection models. ResultsThe STEC detection model with Quick Classifier (QC) algorithm was the most sensitive: sensitivity, 84.1% (37/44); specificity, 94.4% (101/107). Discrimination between STEC and non-STEC was excellent, but individual discrimination of Shiga toxins was not possible. ConclusionMALDI Biotyper may be a useful rapid diagnostic method for STEC infection.

The Validation of the SIMUL-qPCR Top 7 STEC Assay Collection: AOAC Performance Tested MethodSM 022001

The Simultaneous Multiplex Real Time PCR (SIMUL-qPCR) Top 7 STEC Assay Collection is a quick, reliable method for detecting Top 7 Shiga toxin-producing E. coli (STEC) in raw beef trim, raw ground beef, and beef carcass sampling sheets. Each assay multiplexes several targets in one run to identify E. coli O157: H7, O26, O45, O103, O111, O121, O145, Shiga toxin and intimin genes. This collection uses specifically optimized proprietary media for single-step recovery and enrichment of Enterohemorrhagic E. coli (EHEC). This report details the method validation study to validate raw beef trim, raw ground beef, and beef carcass sampling sheets. Matrix studies for raw beef trim, raw ground beef, and beef carcass sampling sheets, inclusivity/exclusivity, product consistency/stability, and robustness testing were conducted to assess the method's performance. 50 Top 7 STEC isolates were analyzed with the SIMUL-qPCR Assay. 32 isolates, including closely related non-E. coli species and E. coli non-STEC strains, were also tested. Inclusivity showed the collection detected the Shiga toxin (stx) gene, intimin (eae) gene, and Top 7 serogroups. None of the 32 exclusivity strains were detected. The candidate and reference methods' results had no statistically significant differences during matrix studies. Small variations in critical test parameters (enrichment time, extraction reagent volume, and extracted sample volume) didn't adversely affect the assay's performance, and stability testing indicated consistent results for at least one year. The data presented demonstrate that the SIMUL-qPCR Top 7 STEC Assay is a reliable method for detecting the Top 7 STEC.

Characterization of Non-O157 Escherichia coli from Cattle Faecal Samples in the North-West Province of South Africa.

Escherichia coli are commensal bacteria in the gastrointestinal tract of mammals, but some strains have acquired Shiga-toxins and can cause enterohemorrhagic diarrhoea and kidney failure in humans. Shiga-toxigenic E. coli (STEC) strains such as E. coli O157:H7 and some non-O157 strains also contain other virulence traits, some of which contribute to their ability to form biofilms. This study characterized non-O157 E. coli from South African cattle faecal samples for their virulence potential, antimicrobial resistance (AMR), biofilm-forming ability, and genetic relatedness using culture-based methods, pulsed-field gel electrophoresis (PFGE), and whole genome sequencing (WGS). Of 80 isolates screened, 77.5% (62/80) possessed Shiga-toxins genes. Of 18 antimicrobials tested, phenotypic resistance was detected against seven antimicrobials. Resistance ranged from 1.3% (1/80) for ampicillin-sulbactam to 20% (16/80) for tetracycline. Antimicrobial resistance genes were infrequently detected except for tetA, which was found in 31.3% (25/80) and tetB detected in 11.3% (9/80) of isolates. Eight biofilm-forming associated genes were detected in STEC isolates (n = 62) and two non-STEC strains. Prevalence of biofilm genes ranged from 31.3% (20/64) for ehaAβ passenger to 100% for curli structural subunit (csgA) and curli regulators (csgA and crl). Of the 64 STEC and multi-drug resistant isolates, 70.3% (45/64) and 37.5% (24/64) formed strong biofilms on polystyrene at 22 and 37 °C, respectively. Of 59 isolates screened by PFGE, 37 showed unique patterns and the remaining isolates were grouped into five clusters with a ≥90% relatedness. In silico serotyping following WGS on a subset of 24 non-O157 STEC isolates predicted 20 serotypes comprising three novel serotypes, indicating their diversity as potential pathogens. These findings show that North West South African cattle harbour genetically diverse, virulent, antimicrobial-resistant and biofilm-forming non-O157 E. coli. Biofilm-forming ability may increase the likelihood of persistence of these pathogens in the environment and facilitate their dissemination, increasing the risk of cross contamination or establishment of infections in hosts.

Molecular Mechanisms Governing "Hair-Trigger" Induction of Shiga Toxin-Encoding Prophages.

Shiga toxin (Stx)-encoding E. coli (STEC) strains are responsible for sporadic outbreaks of food poisoning dating to 1982, when the first STEC strain, E. coli O157:H7, was isolated. Regardless of STEC serotype, the primary symptoms of STEC infections are caused by Stx that is synthesized from genes resident on lambdoid prophage present in STEC. Despite similar etiology, the severity of STEC-mediated disease varies by outbreak. However, it is unclear what modulates the severity of STEC-mediated disease. Stx production and release is controlled by lytic growth of the Stx-encoding bacteriophage, which in turn, is controlled by the phage repressor. Here, we confirm our earlier suggestion that the higher spontaneous induction frequency of Stx-encoding prophage is a consequence, in part, of lower intracellular repressor levels in STEC strains versus non-STEC strains. We also show that this lowered intracellular repressor concentration is a consequence of the utilization of alternative binding/regulatory strategies by the phage repressor. We suggest that a higher spontaneous induction frequency would lead to increased virulence.

Genetic Analysis of Virulence Potential of Escherichia coli O104 Serotypes Isolated From Cattle Feces Using Whole Genome Sequencing

Escherichia coli O104:H4, a Shiga toxin-producing hybrid pathotype that was implicated in a major foodborne outbreak in Germany in 2011, has not been detected in cattle. However, serotypes of O104, other than O104:H4, have been isolated from cattle feces, with O104:H7 being the most predominant. In this study, we investigated, based on whole genome sequence analyses, the virulence potential of E. coli O104 strains isolated from cattle feces, since cattle are asymptomatic carriers of E. coli O104. The genomes of ten bovine E. coli O104 strains (six O104:H7, one O104:H8, one O104:H12, and two O104:H23) and five O104:H7 isolated from human clinical cases were sequenced. Of all the bovine O104 serotypes (H7, H8, H12, and H23) that were included in the study, only E. coli O104:H7 serotype possessed Shiga toxins. Four of the six bovine O104:H7 strains and one of the five human strains carried stx1c. Three human O104 strains carried stx2, two were of subtype 2a, and one was 2d. Genomes of stx carrying bovine O104:H7 strains were larger than the stx-negative strains of O104:H7 or other serotypes. The genome sizes were proportional to the number of genes carried on the mobile genetic elements (phages, prophages, transposable elements and plasmids). Both bovine and human strains were negative for intimin and other genes associated with the type III secretory system and non-LEE encoded effectors. Plasmid-encoded virulence genes (ehxA, epeA, espP, katP) were also present in bovine and human strains. All O104 strains were negative for antimicrobial resistance genes, except one human strain. Phylogenetic analysis indicated that bovine E. coli O104 strains carrying the same flagellar antigen clustered together and STEC strains clustered separately from non-STEC strains. One of the human O104:H7 strains was phylogenetically closely related to and belonged to the same sequence type (ST-1817) as the bovine O104:H7 STEC strains. This suggests that the bovine feces could be a source of human illness caused by E. coli O104:H7 serotype. Because bovine O104:H7 strains carried virulence genes similar to human clinical strains and one of the human clinical strains was phylogenetically related to bovine strains, the serotype has the potential to be a diarrheagenic pathogen in humans.

Targeted Amplicon Sequencing for Single-Nucleotide-Polymorphism Genotyping of Attaching and Effacing Escherichia coli O26:H11 Cattle Strains via a High-Throughput Library Preparation Technique.

Enterohemorrhagic Escherichia coli (EHEC) O26:H11, a serotype within Shiga toxin-producing E. coli (STEC) that causes severe human disease, has been considered to have evolved from attaching and effacing E. coli (AEEC) O26:H11 through the acquisition of a Shiga toxin-encoding gene. Targeted amplicon sequencing using next-generation sequencing technology of 48 phylogenetically informative single-nucleotide polymorphisms (SNPs) and three SNPs differentiating Shiga toxin-positive (stx-positive) strains from Shiga toxin-negative (stx-negative) strains were used to infer the phylogenetic relationships of 178 E. coli O26:H11 strains (6 stx-positive strains and 172 stx-negative AEEC strains) from cattle feces to 7 publically available genomes of human clinical strains. The AEEC cattle strains displayed synonymous SNP genotypes with stx2-positive sequence type 29 (ST29) human O26:H11 strains, while stx1 ST21 human and cattle strains clustered separately, demonstrating the close phylogenetic relatedness of these Shiga toxin-negative AEEC cattle strains and human clinical strains. With the exception of seven stx-negative strains, five of which contained espK, three stx-related SNPs differentiated the STEC strains from non-STEC strains, supporting the hypothesis that these AEEC cattle strains could serve as a potential reservoir for new or existing pathogenic human strains. Our results support the idea that targeted amplicon sequencing for SNP genotyping expedites strain identification and genetic characterization of E. coli O26:H11, which is important for food safety and public health.

Development and validation of two SYBR green PCR assays and a multiplex real-time PCR for the detection of Shiga toxin-producing Escherichia coli in meat

Shiga toxin-producing Escherichia coli (STEC) are recognized as food-borne pathogens. We developed and validated two SYBR green PCR (SYBR-PCR) and a real-time multiplex PCR (RT-PCR) to detect stx1 and stx2 genes in meat samples, and compared these techniques in ground beef samples from retail stores. One set of primers and one hydrolysis probe were designed for each stx gene. For RT-PCR, an internal amplification control (IAC) was used. All PCR intra-laboratory validations were performed using pure strains and artificially contaminated ground beef samples. A total of 50 STEC and 30 non-STEC strains were used. Naturally contaminated ground beef samples (n=103) were obtained from retail stores and screened with SYBR-PCR and RT-PCR, and stx-positive samples were processed for STEC isolation. In the intra-laboratory validation, each PCR obtained a 1×102CFUmL−1 limit of detection and 100% inclusivity and exclusivity. The same results were obtained when different laboratory analysts in alternate days performed the assay. The level of agreement obtained with SYBR-PCR and RT-PCR was kappa=0.758 and 0.801 (P<0.001) for stx1 and stx2 gene detection, respectively. Two PCR strategies were developed and validated, and excellent performance with artificially contaminated ground beef samples was obtained. However, the efforts made to isolate STEC from retail store samples were not enough. Only 11 STEC strains were isolated from 35 stx-positive ground beef samples identically detected by all PCRs. The combination of molecular approaches based on the identification of a virulence genotypic profile of STEC must be considered to improve isolation.

Promising Nucleic Acid Lateral Flow Assay Plus PCR for Shiga Toxin-ProducingEscherichia coli

Shiga toxin (Stx)-producing Escherichia coli (STEC) is a frequent cause of foodborne infections, and methods for rapid and reliable detection of STEC are needed. A nucleic acid lateral flow assay (NALFA) plus PCR was evaluated for detecting STEC after enrichment. When cell suspensions of 45 STEC strains, 14 non-STEC strains, and 13 non-E. coli strains were tested with the NALFA plus PCR, all of the STEC strains yielded positive results, and all of the non-STEC and non-E. coli strains yielded negative results. The lower detection limit for the STEC strains ranged from 0.1 to 1 pg of genomic DNA (about 20 to 200 CFU) per test, and the NALFA plus PCR was able to detect Stx1- and Stx2-producing E. coli strains with similar sensitivities. The ability of the NALFA plus PCR to detect STEC in enrichment cultures of radish sprouts, tomato, raw ground beef, and beef liver inoculated with 10-fold serially diluted STEC cultures was comparable to that of a real-time PCR assay (at a level of 100 to 100,000 CFU/ml in enrichment culture). The bacterial inoculation test in raw ground beef revealed that the lower detection limit of the NALFA plus PCR was also comparable to that obtained with a real-time PCR assay that followed the U.S. Department of Agriculture guidelines. Although further evaluation is required, these results suggest that the NALFA plus PCR is a specific and sensitive method for detecting STEC in a food manufacturing plant.

Comparison of the RAPID-B® flow cytometer and the BAX® system for the detection of non-O157 shiga toxin-producing Escherichia coli (STEC) in beef products

Abstract The beef industry continues to be interested in reliable rapid detection technologies for shiga toxin-producing Escherichia coli (STEC). Current rapid technologies require several hours of pre-enrichment and additional time on the rapid technology instrument. A flow cytometer-based system (RAPID-B®) has been shown to improve the turn-around for results with a more rapid pre-enrichment requiring only 6.5 h pre-enrichment for a 25 g and 8.5 h for a 375 g sample, followed by an additional 30 min time to achieve final results using the screening technology. The purpose of this study was to validate the RAPID-B® technology for non-O157 STEC detection as compared to the USDA-FSIS reference method which utilizes the BAX® system. A total of 180 STEC isolates from various sources and 20 non-STEC strains were used to evaluate specificity and sensitivity using the RAPID-B® flow cytometer. Also, three different weights (25, 325 and 375 g) of beef trim and ground beef samples were spiked with each STEC to verify detection sensitivity of BAX® system and RAPID-B® flow cytometer. For both methods, samples were confirmed by culturing using the USDA-FSIS reference method regardless of the screening result. The RAPID-B® flow cytometer showed that 180 isolates were all positive and the 20 non-STEC strains were all negative. For spiked beef samples, overall detection sensitivity was the same for both the BAX® system and RAPID-B® flow cytometer. When detection sensitivity was based on sample weight, there was no differences in 25 and 375 g samples between RAPID-B® flow cytometer and USDA-FSIS reference method. The RAPID-B® system yielded the same sensitivity as the reference method with a decrease of over 10 h of pre-enrichment time and 3 h of rapid screening detection time. In conclusion, the RAPID-B® flow cytometer based on whole cell detection generated similar results as BAX® system therefore the RAPID-B® flow cytometer system could be a valuable rapid method for the detection of non-O157 STEC in beef products.

Prevalence of hemolysin genes and comparison of ehxA subtype patterns in Shiga toxin-producing Escherichia coli (STEC) and non-STEC strains from clinical, food, and animal sources.

Shiga toxin-producing Escherichia coli (STEC) belonging to certain serogroups (e.g., O157 and O26) can cause serious conditions like hemolytic-uremic syndrome (HUS), but other strains might be equally pathogenic. While virulence factors, like stx and eae, have been well studied, little is known about the prevalence of the E. coli hemolysin genes (hlyA, ehxA, e-hlyA, and sheA) in association with these factors. Hemolysins are potential virulence factors, and ehxA and hlyA have been associated with human illness, but the significance of sheA is unknown. Hence, 435 E. coli strains belonging to 62 different O serogroups were characterized to investigate gene presence and phenotypic expression of hemolysis. We further investigated ehxA subtype patterns in E. coli isolates from clinical, animal, and food sources. While sheA and ehxA were widely distributed, e-hlyA and hlyA were rarely found. Most strains (86.7%) were hemolytic, and significantly more hemolytic (95%) than nonhemolytic strains (49%) carried stx and/or eae (P < 0.0001). ehxA subtyping, as performed by using PCR in combination with restriction fragment length polymorphism analysis, resulted in six closely related subtypes (>94.2%), with subtypes A/D being eae-negative STECs and subtypes B, C, E, and F eae positive. Unexpectedly, ehxA subtype patterns differed significantly between isolates collected from different sources (P < 0.0001), suggesting that simple linear models of exposure and transmission need modification; animal isolates carried mostly subtypes A/C (39.3%/42.9%), food isolates carried mainly subtype A (81.9%), and clinical isolates carried mainly subtype C (66.4%). Certain O serogroups correlated with particular ehxA subtypes: subtype A with O104, O113, and O8; B exclusively with O157; C with O26, O111, and O121.

The Microcosm Mediates the Persistence of Shiga Toxin-Producing Escherichia coli in Freshwater Ecosystems

Water is a major route for infection of humans by exotoxin-producing bacteria, including Shiga toxin-producing Escherichia coli (STEC). While STEC has the potential to be present in nearly every type of water source, its distribution is sporadic, and an understanding of factors that govern its emergence and persistence within water is lacking. In this study, we examined the influence of microbe content on STEC persistence in freshwater. We found that depletion of microbes in the water leads to a considerable increase in the persistence of STEC, an effect that can be mitigated by adding grazing protists to the water. STEC strains appear to be more resistant to the impact of grazing protists than E. coli strains that lack the Shiga toxin (stx) gene. Our results demonstrate that the microcosm can dramatically influence the persistence of STEC in aquatic ecosystems and that the overall impact by microbes on STEC strains is fundamentally different from that of non-STEC strains of bacteria. Overall, these results provide insight into why STEC and possibly other exotoxin-producing bacterial pathogens display such variability in abundance, distribution, and persistence in aquatic ecosystems.

Association of Nucleotide Polymorphisms within the O-Antigen Gene Cluster of Escherichia coli O26, O45, O103, O111, O121, and O145 with Serogroups and Genetic Subtypes

Shiga toxin-producing Escherichia coli (STEC) strains are important food-borne pathogens capable of causing hemolytic-uremic syndrome. STEC O157:H7 strains cause the majority of severe disease in the United States; however, there is a growing concern for the amount and severity of illness attributable to non-O157 STEC. Recently, the Food Safety and Inspection Service (FSIS) published the intent to regulate the presence of STEC belonging to serogroups O26, O45, O103, O111, O121, and O145 in nonintact beef products. To ensure the effective control of these bacteria, sensitive and specific tests for their detection will be needed. In this study, we identified single nucleotide polymorphisms (SNPs) in the O-antigen gene cluster that could be used to detect STEC strains of the above-described serogroups. Using comparative DNA sequence analysis, we identified 22 potentially informative SNPs among 164 STEC and non-STEC strains of the above-described serogroups and designed matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF) assays to test the STEC allele frequencies in an independent panel of bacterial strains. We found at least one SNP that was specific to each serogroup and also differentiated between STEC and non-STEC strains. Differences in the DNA sequence of the O-antigen gene cluster corresponded well with differences in the virulence gene profiles and provided evidence of different lineages for STEC and non-STEC strains. The SNPs discovered in this study can be used to develop tests that will not only accurately identify O26, O45, O103, O111, O121, and O145 strains but also predict whether strains detected in the above-described serogroups contain Shiga toxin-encoding genes.

Distribution of Virulence Genes and Their Association of Serotypes in Pathogenic Escherichia coli Isolates From Diarrheal Patients in Korea

ObjectivesTo characterise the genetic and serological diversity of pathogenic Escherichia coli, we tested 111 E coli strains isolated from diarrhoeal patients in Korea between 2003 and 2006. MethodsThe isolates were tested through polymerase chain reaction (PCR) and slide agglutination method for the detection of virulence genes and serotypes, respectively. To compare the expression of Shiga toxin (stx)-1 and stx2 genes, real-time quantitative reverse-transcriptase PCR and rapid exprssion assay, reversed-passive latex agglutination, were performed. ResultsForty-nine Shiga toxin-producing E coli (STEC) strains and 62 non-STEC strains, including 20 enteropathogenic E coli, 20 enterotoxigenic E coli, 20 enteroaggregative E coli, and 2 enteroinvasive E coli were randomly chosen from the strains isolated from diarrhoeal patients in Korea between 2003 and 2006. PCR analysis indicated that locus of enterocyte effacement pathogenicity island, that is, eaeA, espADB, and tir genes were present in STEC, enteropathogenic E coli, and enteroinvasive E coli. Quorum sensing-related gene luxS was detected in most of pathogenic E coli strains. Major serotypes of the STEC strains were O157 (26%) and O26 (20%), whereas the non-STEC strains possessed various serotypes. Especially, all the strains with serotype O157 carried stx2 and the tested virulence factors. Of the STEC strains, the data of real-time quantitative reverse-transcriptase PCR and reversed-passive latex agglutination tests showed that messenger RNA- and protein expression of stx2 gene were higher than those of stx1 gene. ConclusionOur results provide the epidemiological information regarding the trend of STEC and non-STEC infections in the general population and show the fundamental data in association of serotypes with virulence genes in diarrhoeagenic E coli strains from Korea.

Factors affecting survival of Shigatoxin-producing Escherichia coli on abiotic surfaces

Shigatoxin-producing Escherichia coli (STEC) causes severe infections, and has been the cause of a number of foodborne outbreaks. Knowledge on the survival of STEC is crucial in order to limit the risk of cross contamination and transfer of STEC to food during processing. In this study survival of STEC and non-STEC on surfaces under various humidities, temperatures and in the presence of different types of soil was investigated. A model system with controlled relative humidity and temperature was established by using saturated salt solutions. All the 12 STEC strains had a reduction in viable count during incubation at 70% RH at 12 °C. The reduction was 2–3.5 log and 4.5–5.5 log after 1 and 7 days of incubation, respectively. Surviving cells were observed after 19 days of incubation. The STEC strains were more resistant to desiccation than non-STEC strains. STEC survived better at 12 °C, compared to 20 °C. The survival of STEC was much lower than the survival of a Staphylococcus simulans strain tested, which showed less than 1 log reduction until day 7 at 70% RH at 12 °C, while several STEC strains had comparable survival to a Salmonella Agona strain. The survival of two STEC strains tested was highest at 98% RH. The lowest survival was observed at 85% RH, with better survival at drier conditions. Presence of proteins and glucose protected the cells at dry conditions. Two commercial disinfectants tested at in-use concentration had limited effect (0.8–2.5 log reduction) against STEC on stainless steel, especially for cells incubated at high relative humidity (98% RH). STEC surviving on surfaces in the food industry may impose a risk for cross contamination. Cleaning and use of suitable disinfectants will reduce the survival of STEC, but surfaces should be allowed to dry completely since humid conditions will promote the survival and growth of STEC.

Serogroups, Atypical Biochemical Characters, Colicinogeny and Antibiotic Resistance Pattern of Shiga Toxin‐producingEscherichia coliIsolated from Diarrhoeic Calves in Gujarat, India

This study was designed to investigate the antibiotic resistance, colicinogeny, serotyping and atypical biochemical characteristics of 41 Shiga toxin-producing Escherichia coli (STEC) strains detected using polymerase chain reaction from 90 E. coli strains isolated from 46 diarrhoeic calves. The STEC strains belonged to 14 different serogroups. Seventeen per cent of the STEC strains carried the eaeA gene while 14.28% of the 49 non-STEC strains were eaeA positive. Twenty eight (68.29%) of the 41 STEC strains were rhamnose non-fermentors. All the STEC strains revealed resistance to at least three of the antibiotics tested. 100% resistance was found against kanamycin and cephalexin followed by cephaloridine, enrofloxacin, amikacin, ampicillin, tetracycline, ceftiofur, ciprofloxacin, colistin and co-trimoxazole. Eighteen (44%) of the STEC strains produced colicin and all these colicinogenic strains were resistant to three or more antibiotics. Eleven STEC strains (26.82%) showed urease activity. The results of this study suggest that diarrhoeic calves are an important reservoir of STEC strains that are potentially pathogenic for farm animals and humans. Moreover, rhamnose fermentation, colicinogeny and atypical biochemical behaviour, such as urease activity, may serve as important markers or diagnostic tools for epidemiological surveys to trace the source of infection in disease outbreaks.

Development of a Simple Latex Agglutination Assay for Detection of Shiga Toxin-Producing Escherichia coli (STEC) by Using Polyclonal Antibody against STEC

Rabbit antiserum raised against the whole-cell antigen of Shiga toxin-producing Escherichia coli (STEC) strain VT3 (stx1+ stx2+ eae+) was repeatedly adsorbed with heat-killed cells of different non-STEC strains and other enteric bacteria. Thus, the antiserum obtained was designated VT3 antiserum. VT3 antiserum reacted with intimin type gamma. We assessed the reactivity of VT3 antiserum to whole-cell lysates of 87 strains of E. coli and other enteric bacteria by immunoblotting. The antiserum recognized the 97-kDa protein in whole-cell lysate from strain VT3, and 36 (83.7%) of the 43 STEC strains were positive for the STEC antigen. None of the non-STEC strains or strains of other species examined tested positive by immunoblotting. Based on this result, we developed a latex agglutination assay for the detection of STEC strains. Thirty-five (81.4%) of the 43 STEC strains tested positive for the STEC antigen by the latex agglutination assay. One (3.3%) of the 30 non-STEC strains and none of the strains of the other enteric bacteria included in this study tested positive by the latex agglutination assay. The corresponding specificity of the latex agglutination assay was approximately 98%. Results of this study showed the production of STEC antiserum and the generation of a simple, cost-effective, sensitive, and specific latex agglutination assay for establishing an etiological diagnosis of STEC.

A comparative study of biofilm formation by Shiga toxigenic Escherichia coli using epifluorescence microscopy on stainless steel and a microtitre plate method

Attachment of Shiga toxigenic Escherichia coli (STEC) to surfaces and the formation of biofilms may enhance persistence in a food processing environment and present a risk of contaminating products. Seven strains of STEC and three non-STEC strains were selected to compare two biofilm quantification methods; epifluorescence microscopy on stainless steel (SS) and a microtitre plate assay. The influence of prior growth in planktonic (nutrient broth) and sessile (nutrient agar) culture on biofilm production, as well as expression of surface structures and the possession of antigen 43 (encoded by agn43) on biofilm formation were also investigated. Biofilms were produced in diluted nutrient broth at 25 °C for 24 and 48 h. Curli expression was determined using congo red indicator agar, while the presence of agn43 was determined using polymerase chain reaction. No correlation was found between counts for epifluorescence microscopy on SS and the absorbance values obtained with the microtitre plate method for planktonic and sessile grown cultures. Different abilities of individual STEC strains to attach to SS and microtitre plates were found with some strains attaching better to each surface following growth in either planktonic or sessile culture. All O157 STEC strains had low biofilm counts on SS for planktonic and sessile grown cultures; however, one STEC O157:H− strain (EC516) had significantly greater ( p < 0.05) biofilm production on microtitre plates compared to the other O157 STEC strains. EC516 and other STEC (O174:H21 and O91:H21) strains expressing curli fimbriae were found to produce significantly greater ( p < 0.05) biofilms on microtitre plates compared to the non-curli expressing strains. No relationship was found between the production of type-I fimbriae, motility, agn43 and bacterial physicochemical properties (previously determined) and biofilm formation on SS or microtitre plates. Variations between the two biofilm determination methods may suggest that the biofilm production on microtitre plates may not be appropriate to represent other surfaces such as SS and that caution should be taken when selecting a method to quantify biofilm production on a surface.