Shiga Toxin-producing Escherichia Coli O157 Research Articles

Assessment of Shiga Toxin-Producing Escherichia coli O157 Illnesses Prevented by Recalls of Beef Products.

Beef product recall data from 2005 through 2012 associated with Shiga toxin-producing Escherichia coli (STEC) O157 contamination were used to develop quantitative models to estimate the number of illnesses prevented by recalls. The number of illnesses prevented was based on the number of illnesses that occurred relative to the number of pounds consumed, then extrapolated to the number of pounds of recalled product recovered. A simulation using a Program Evaluation and Review Technique (PERT) probability distribution with illness-related recalls estimated 204 (95% credible interval, 117-333) prevented STEC O157 illnesses from 2005 through 2012. Recalls not associated with illnesses had more recalled product recovered and prevented an estimated 83 additional STEC O157 illnesses. Accounting for underdiagnosis resulted in an estimated total of 7500 STEC O157 illnesses prevented over 8 years. This study demonstrates that recalls, although reactive in nature, are an important tool for averting further exposure and illnesses.

Seasonal prevalence of potentially positive non-O157 Shiga toxin-producing Escherichia coli (STEC) bovine hides and carcasses in Costa Rica

The prevalence of potentially positive Shiga toxin-producing Escherichia coli (STEC) bovine hides and carcasses in three abattoirs in Costa Rica was estimated. Two export facilities (A and B) and one non-export establishment (C) were visited during the dry and rainy seasons of 2013. Swabs of hides pre-eviscerated and treated (180–220 peroxyacetic acid spray) carcasses were tested for the potential presence of STEC serogroups O26, O45, O103, O111, O121, and O145. The prevalence on hides during the rainy season was 86.7, 96.7 and 96.7% for facilities A, B, and C, respectively. During the dry season, the prevalence on hides was significantly lower in plants A and B (40% and 26.7%, respectively), but was marginally associated with the season in plant C (76.7%, P=0.0523). The prevalence of non-O157 STEC markers on treated carcasses was low (0 to 3.3%), suggesting that all plants were effective in minimizing the target non-O157 STEC in beef destined for export and for domestic consumption.

Characterization of Third-Generation-Cephalosporin-Resistant Shiga Toxin-Producing Strains of Escherichia coli O157:H7 in Japan

We isolated Shiga toxin-producing Escherichia coli O157:H7 strains resistant to third-generation cephalosporins. The resistant strains harbored blaCMY-2, a plasmid-mediated AmpC β-lactamase. Genotyping of isolates revealed the possible spread of this problematic bacterium. Results suggested the importance of the investigation and surveillance of enterobacteria with plasmids harboring blaCMY-2.

Complete Genome Sequence of Escherichia coli O145:NM Bacteriophage vB_EcoM_AYO145A, a New Member of O1-Like Phages.

Previously, bacteriophage vB_EcoM_AYO145A, which lyses Shiga toxin-producing Escherichia coli O145:NM, was classified as an O1-like virus of the Myoviridae family. Here, we report the complete genome sequence of this phage and a comparative genomic analysis with other known O1-like phages.

Genetically Marked Strains of Shiga Toxin–Producing O157:H7 and Non-O157 Escherichia Coli: Tools for Detection and Modeling

Shiga toxin-producing E. coli (STEC) is an important group of foodborne pathogens in the United States and worldwide. Nearly half of STEC-induced diarrheal disease in the United States is caused by serotype O157:H7, while non-O157 STEC account for the remaining illnesses. Thus, the U.S. Department of Agriculture (USDA) Food Safety and Inspection Service has instituted regulatory testing of beef products and has a zero-tolerance policy for regulatory samples that test positive for STEC O157:H7 and six other non-O157 STEC (serogroups O26, O45, O103, O111, O121, and O145). In this study, positive control (PC) strains for the detection of STEC O157:H7 and the six USDA-regulated non-O157 STEC were constructed. To ensure that the food testing samples are not cross-contaminated by the PC sample, it is important that the STEC-PC strains are distinguishable from STEC isolated from test samples. The PC strains were constructed by integrating a unique DNA target sequence and a gene for spectinomycin (Sp) resistance into the chromosomes of the seven STEC strains. End-point and real-time PCR assays were developed for the specific detection of the PC strains and were tested using 93 strains of E. coli (38 STEC O157:H7, at least 6 strains of each of the USDA-regulated non-O157 STEC, and 2 commensal E. coli) and 51 strains of other bacteria (30 species from 20 genera). The PCR assays demonstrated high specificity for the unique target sequence. The target sequence was detectable by PCR after 10 culture passages (∼100 generations), demonstrating the stability of the integrated target sequence. In addition, the strains were tested for their potential use in modeling the growth of STEC. Plating the PC strains mixed with ground beef flora on modified rainbow agar containing Sp eliminated the growth of the background flora that grew on modified rainbow agar without Sp. Thus, these strains could be used to enumerate and model the growth of STEC in the presence of foodborne background flora.

Molecular Subtyping of Shiga Toxin–Producing Escherichia coli Using a Commercial Repetitive Sequence–Based PCR Assay

PCR-based typing methods, such as repetitive sequence-based PCR (rep-PCR), may facilitate the identification of Shiga toxin-producing Escherichia coli (STEC) by serving as screening methods to reduce the number of isolates to be processed for further confirmation. In this study, we used a commercial rep-PCR typing system to generate DNA fingerprint profiles for STEC O157 (n = 60) and non-O157 (n = 91) isolates from human, food, and animal samples and then compared the results with those obtained from pulsed-field gel electrophoresis (PFGE). Fifteen serogroups were analyzed using the Kullback Leibler or extended Jaccard statistical method, and the unweighted pair group method of averages algorithm was used to create dendrograms. Among the 151 STEC isolates tested, all were typeable by rep-PCR. Among the non-O157 isolates, rep-PCR clustered 79 (88.8%) of 89 isolates according to serogroup status, with peak differences ranging from 1 (96.4% similarity) to 12 (58.7% similarity). The genetic relatedness of the non-O157 serogroups mirrored the branching of distinct clonal groups elucidated by other investigators. Although the discriminatory power of rep-PCR (Simpson's index of diversity [SID] = 0.954) for the O157 isolates was less than that of PFGE (SID = 0.993), rep-PCR was able to identify 29 pattern types, suggesting that this method can be used for strain typing, although not to the same level as PFGE. Similar results were obtained from analysis of the non-O157 isolates. With rep-PCR, we assigned non-O157 isolates to 46 pattern types with a SID of 0.977. By PFGE, non-O157 STEC strains were divided into 77 pattern types with a SID of 0.996. Together, these results indicate the ability of the rep-PCR typing system to distinguish between and within O157 and non-O157 STEC groups. Rapid PCR-based typing methods could be invaluable tools for use in outbreak investigations by excluding unrelated STEC isolates within 24 h.

Feces of feedlot cattle contain a diversity of bacteriophages that lyse non-O157 Shiga toxin-producing Escherichia coli.

This study aimed to isolate and characterize bacteriophages that lyse non-O157 Shiga toxin-producing Escherichia coli (STEC) from cattle feces. Of 37 non-O157 STEC-infecting phages isolated, those targeting O26 (AXO26A, AYO26A, AYO26B), O103 (AXO103A, AYO103A), O111 (AXO111A, AYO111A), O121 (AXO121A, AXO121B), and O145 (AYO145A, AYO145B) were further characterized. Transmission electron microscopy showed that the 11 isolates belonged to 3 families and 6 genera: the families Myoviridae (types rV5, T4, ViI, O1), Siphoviridae (type T5), and Podoviridae (type T7). Genome size of the phages as determined by pulsed-field gel electrophoresis ranged from 38 to 177 kb. Excluding phages AXO26A, AYO103A, AYO145A, and AYO145B, all other phages were capable of lysing more than 1 clinically important strain from serogroups of O26, O91, O103, O111, O113, O121, and O128, but none exhibited infectivity across all serogroups. Moreover, phages AYO26A, AXO121A, and AXO121B were also able to lyse 4 common phage types of STEC O157:H7. Our findings show that a diversity of non-O157 STEC-infecting phages are harbored in bovine feces. Phages AYO26A, AYO26B, AXO103A, AXO111A, AYO111A, AXO121A, and AXO121B exhibited a broad host range against a number of serogroups of STEC and have potential for the biocontrol of STEC in the environment.

Characterization of the Shiga toxin-producing Escherichia coli O26 isolated from human in Poland between 1996 and 2014.

Shiga toxin-producing Escherichia coli (STEC) O26 infections can be comparable with STEC O157 infections in severity of the acute haemolytic-uremic syndrome HUS and long-term sequelae. Among O26 STEC isolates, highly virulent clone O26:H11/H- Sequence Type 29 (ST 29) emerged in Germany in mid-1990s and spread to European countries. However, up to date, no STEC O26:H11/H- belonging to ST29 has been documented in Poland. In this study, we determined the relationship and clonal structure, stx genotypes, plasmid gene profiles and antimicrobial resistance of nine human STEC O26:H11/H- strains from human patients in Poland between 1996 and 2014. Of the 9 human STEC O26:H11/H- strains, two belonged to ST29 and were isolated from two children with HUS and renal failure with sepsis respectively. These strains showed the molecular characteristics of the emerging human-pathogenic ST29 clone (stx1-, stx2a+, eae+, ehxA+, etpD+, katP-, espP-). The remaining STEC O26:H11/H- strains examined in this study, belonged to ST21, with plasmid genes profiles frequently reported in ST21 strains in Europe. STEC O26 infections with serious human health consequences highlight the need of continuous surveillance of non-O157 STEC and implementation of the diagnostic approaches focused on their detection. Significance and impact of the study: These study provides the first data on the occurrence of emerging Shiga toxin-producing Escherichia coli O26:H11 ST 29 clone in human patients in Poland. Those strains show the molecular characteristics of highly virulent new ST29 pathotype (stx1-, stx2a+, eae+ ehxA+, etpD+, katP-, espP-). These results demonstrated prompt efforts to implement diagnostic approaches detection of those pathogen in the European countries.

Genome sequencing and comparative genomics provides insights on the evolutionary dynamics and pathogenic potential of different H-serotypes of Shiga toxin-producing Escherichia coli O104.

BackgroundVarious H-serotypes of the Shiga toxin-producing Escherichia coli (STEC) O104, including H4, H7, H21, and H¯, have been associated with sporadic cases of illness and have caused food-borne outbreaks globally. In the U.S., STEC O104:H21 caused an outbreak associated with milk in 1994. However, there is little known on the evolutionary origins of STEC O104 strains, and how genotypic diversity contributes to pathogenic potential of various O104 H-antigen serotypes isolated from different ecological niches and/or geographical regions.ResultsTwo STEC O104:H21 (milk outbreak strain) and O104:H7 (cattle isolate) strains were shot-gun sequenced, and the genomes were closed. The intimin (eae) gene, involved in the attaching-effacing phenotype of diarrheagenic E. coli, was not found in either strain. Examining various O104 genome sequences, we found that two “complete” left and right end portions of the locus of enterocyte effacement (LEE) pathogenicity island were present in 13 O104 strains; however, the central portion of LEE was missing, where the eae gene is located. In O104:H4 strains, the missing central portion of the LEE locus was replaced by a pathogenicity island carrying the aidA (adhesin involved in diffuse adherence) gene and antibiotic resistance genes commonly carried on plasmids. Enteroaggregative E. coli-specific virulence genes and European outbreak O104:H4-specific stx2-encoding Escherichia P13374 or Escherichia TL-2011c bacteriophages were missing in some of the O104:H4 genome sequences available from public databases. Most of the genomic variations in the strains examined were due to the presence of different mobile genetic elements, including prophages and genomic island regions. The presence of plasmids carrying virulence-associated genes may play a role in the pathogenic potential of O104 strains.ConclusionsThe two strains sequenced in this study (O104:H21 and O104:H7) are genetically more similar to each other than to the O104:H4 strains that caused an outbreak in Germany in 2011 and strains found in Central Africa. A hypothesis on strain evolution and pathogenic potential of various H-serotypes of E. coli O104 strains is proposed.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-015-0413-9) contains supplementary material, which is available to authorized users.

Public Health Investigation of Two Outbreaks of Shiga Toxin-Producing Escherichia coli O157 Associated with Consumption of Watercress.

An increase in the number of cases of Shiga toxin-producing Escherichia coli (STEC) O157 phage type 2 (PT2) in England in September 2013 was epidemiologically linked to watercress consumption. Whole-genome sequencing (WGS) identified a phylogenetically related cluster of 22 cases (outbreak 1). The isolates comprising this cluster were not closely related to any other United Kingdom strain in the Public Health England WGS database, suggesting a possible imported source. A second outbreak of STEC O157 PT2 (outbreak 2) was identified epidemiologically following the detection of outbreak 1. Isolates associated with outbreak 2 were phylogenetically distinct from those in outbreak 1. Epidemiologically unrelated isolates on the same branch as the outbreak 2 cluster included those from human cases in England with domestically acquired infection and United Kingdom domestic cattle. Environmental sampling using PCR resulted in the isolation of STEC O157 PT2 from irrigation water at one implicated watercress farm, and WGS showed this isolate belonged to the same phylogenetic cluster as outbreak 2 isolates. Cattle were in close proximity to the watercress bed and were potentially the source of the second outbreak. Transfer of STEC from the field to the watercress bed may have occurred through wildlife entering the watercress farm or via runoff water. During this complex outbreak investigation, epidemiological studies, comprehensive testing of environmental samples, and the use of novel molecular methods proved invaluable in demonstrating that two simultaneous outbreaks of STEC O157 PT2 were both linked to the consumption of watercress but were associated with different sources of contamination.

Diversity of CRISPR loci and virulence genes in pathogenic Escherichia coli isolates from various sources

Shiga toxin-producing Escherichia coli (STEC) strains, including those of O157:H7 and the “big six” serogroups (i.e., O26, O45, O103, O111, O121, and O145) are food-borne pathogens that pose a serious health threat to humans. Ruminants, especially cattle, are a major reservoir for O157 and non-O157 STEC. In the present study, 115 E. coli strains isolated from small and very small beef processing plants were screened for virulence genes (stx1, stx2, eae) using a multiplex polymerase chain reaction (PCR). Thirteen (11.3%) of the 115 isolates tested positive for stx1, stx2, or eae genes, but only 4 (3.5%) tested positive for either stx1 or stx2. A multiplex PCR reaction targeting eight O-serogroups (O26, O45, O103, O111, O113, O121, O145, O157) identified 12 isolates as O26, O103, O111, or O145, with E. coli O26 being the most predominant serogroup (61.5%). The thirteen isolates were further analyzed using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) subtyping. Consistent with previous studies, CRISPR alleles from strains of the same serogroup were similar in their spacer content and order, regardless of the isolation source. A completely different CRISPR allele was observed in one isolate (“7-J”) which exhibited a different O-serogroup (O78). Our results confirmed previous findings that CRISPR loci are conserved among phylogenetically-related strains. In addition, 8 E. coli O26 isolates and a collection of 42 E. coli O26 isolates were screened for 12 enterohemorrhagic E. coli-specific genes. Seven genes (ECs848—Hypothetical Protein, ECs2226—Hypothetical Protein, ECs3857—nleB, ECs3858—Hypothetical Protein, ECs4552—escF, ECs4553—Hypothetical Protein, and ECs4557—sepL) were found in all 50 isolates. An additional 5 genes (ECs1322—ureA urease subunit γ, ECs1323—ureB urease subunit β, ECs1326—ureF, ECs1561—Hypothetical Protein, and ECs1568—Hypothetical Protein) were found to be highly prevalent in isolates from human sources, while lower in isolates from beef processing plants, cattle, and other sources. This finding indicates the possible role of these genes in virulence of human O26 strains.

Growth characteristics of Shiga toxin-producing Escherichia coli (STEC) stressed by chlorine, sodium chloride, acid, and starvation on lettuce and cantaloupe

Shiga toxin-producing Escherichia coli (STEC) is a major foodborne pathogen causing serious illnesses and hospitalizations in the United States. Bacteria that are exposed to environmental stresses during food processing may exhibit different growth patterns in the subsequent growth environment. The purpose of this study was to examine the effect of environmental stresses on the growth of O15H and non-O157 STEC in lettuce or cantaloupe. Strains of O157:H7 and non-O157 STEC (O26:H11, O103:H1, O104:H4, and O145:NM) were subjected to four selected stresses including 2 ppm of chlorine, aw of 0.97 (osmotic stress), and pH 5 (acid stress) at 22 °C for 24 h, or starvation (lack of nutrients) at 22 °C for 15 d. A cocktail mix of stressed or non-stressed (control) O157 or non-O157 at 3 log CFU/g (control or stressed) was inoculated on lettuce or cantaloupe and incubated at 10 and 22 °C for four weeks. While there were significant differences (p < 0.05) in the growth of stressed and unstressed cells of non-O157 STEC, no difference was observed in the growth of stressed and unstressed O157 STEC cells. Osmotic-stressed non-O157 STEC had significantly higher cell populations than control with 2 log difference (9.0 vs. 6.8 log CFU/g) at 10 °C on lettuce and 1 log difference (9.3 vs. 8.3 log CFU/g) at 22 °C on cantaloupe after 4 weeks. Acid-stressed non-O157 STEC had significantly higher cell populations than control at 10 °C after 4 weeks with >1 log difference (7.7 vs. 6.3 log CFU/ml) on cantaloupe. Starvation-stressed non-O157 STEC showed significantly higher cell populations than control with 1 log difference (8.4 vs. 7.2 log CFU/g) at 22 °C on cantaloupe after 4 weeks. The results indicated that osmotic, acid, or starvation stress may enhance the growth of non-O157 STEC on lettuce or cantaloupe and lead to a greater safety risk.

Survival of Shiga toxin-producing Escherichia coli O157:H7 in Minas frescal cheese

Shiga toxin-producing Escherichia coli (STEC) O157:H7 strains (isolated by cattle’s faeces and a reference strain, EDL933), were inoculated into pasteurized milk (102 and 103 cells.mL–1) to prepare the Minas frescal cheese. As control was used uninfected milk. Physicochemical and microbiological analyses were performed to milk and elaborated cheese. The O157:H7 strains were quantified in the stages of cheese processing and during 0, 2, 4, 5, 7, 10 and 15 storage days at 8 °C onto Sorbitol MacConkey Agar supplemented with potassium tellurite and cefixime (CT-SMAC). O157:H7 was not present in the pasteurised milk prior to the artificial inoculation. At the end of the processing the cheese had 10 to 100 times more STEC O157:H7 than the initial inoculum. During the storage, the Minas frescal cheese exhibited the largest population increase on the 4th and 5th day when inoculated with 102 and 103 cells.mL–1, respectively. Additionally, viable cells were found up to the 10th and 15th day, according to the amount of initial inoculum. This number of cells is able to cause infection in humans, and therefore, Minas frescal cheese, even when stored under refrigeration, is a potential vehicle of disease caused by STEC O157:H7.

Insight into Shiga toxin genes encoded by Escherichia coli O157 from whole genome sequencing.

The ability of Shiga toxin-producing Escherichia coli (STEC) to cause severe illness in humans is determined by multiple host factors and bacterial characteristics, including Shiga toxin (Stx) subtype. Given the link between Stx2a subtype and disease severity, we sought to identify the stx subtypes present in whole genome sequences (WGS) of 444 isolates of STEC O157. Difficulties in assembling the stx genes in some strains were overcome by using two complementary bioinformatics methods: mapping and de novo assembly. We compared the WGS analysis with the results obtained using a PCR approach and investigated the diversity within and between the subtypes. All strains of STEC O157 in this study had stx1a, stx2a or stx2c or a combination of these three genes. There was over 99% (442/444) concordance between PCR and WGS. When common source strains were excluded, 236/349 strains of STEC O157 had multiple copies of different Stx subtypes and 54 had multiple copies of the same Stx subtype. Of those strains harbouring multiple copies of the same Stx subtype, 33 had variants between the alleles while 21 had identical copies. Strains harbouring Stx2a only were most commonly found to have multiple alleles of the same subtype (42%). Both the PCR and WGS approach to stx subtyping provided a good level of sensitivity and specificity. In addition, the WGS data also showed there were a significant proportion of strains harbouring multiple alleles of the same Stx subtype associated with clinical disease in England.

High Genotypic and Phenotypic Similarity Among Shiga Toxin–ProducingEscherichia coliO111 Environmental and Outbreak Strains

Escherichia coli serogroup O111 is among the six most commonly reported non-O157:H7 Shiga toxin-producing E. coli (STEC), which are emerging as important foodborne pathogens. We have assembled a collection of environmental and clinical strains of E. coli O111 from diverse sources and investigated various genotypic and phenotypic characteristics of these strains to gain a better understanding of the epidemiology and biology of this serogroup. Sixty-three percent of the strains (24/38) were of H-type 8, which dominated the environmental- and outbreak-strains group, whereas the sporadic-case strains were more heterogeneous in H-type. All of the environmental and outbreak strains harbored the Shiga toxin 1 gene (stx1), eae, and ehx, and a subset of these also carried the Shiga toxin 2 gene (stx2). Only 9 of 16 sporadic-case strains produced stx1 and/or stx2, and these were mostly of H-type 8 and 10. Pulsed-field gel electrophoresis analysis revealed a cluster of environmental, outbreak, and sporadic illness strains with high phylogenetic similarity. Strains in this pulsogroup were all of the H8 type and STEC pathotype, and carried eae and ehx. Smaller clusters of highly similar STEC O111 strains included outbreak and sporadic illness strains isolated during different time periods or from different geographical locations. A distinct aggregative behavior was observed in the cultures of all environmental and outbreak STEC O111 strains, but not in those of sporadic-case strains. Among environmental and outbreaks strains, aggregation was positively correlated with production of curli fimbriae and RpoS function, and negatively with cellulose synthesis, while the nonaggregative behavior of sporadic-case strains correlated (positively) only with cellulose production. Our results indicate that STEC O111 strains sharing high genotypic similarity and important phenotypic traits with STEC O111 outbreak strains are present in the agricultural environment and may contribute to the burden of foodborne disease.

Shiga toxin-producing Escherichia coli O157, O26 and O111 in cattle faeces and hides in Italy

IntroductionRuminants are regarded as the natural reservoir for Shiga toxin-producing Escherichia coli (STEC), especially of serogroup O157.Materials and methodsDuring 2011 and 2012, 320 samples (160 faecal samples from the rectum...

Viabilidade de Escherichia coli O153:H25, O113:H21 e O111:H8 (STEC não-O157) produtoras de toxina Shiga em queijo minas frescal

A existência de um reservatório animal é de grande importância na transmissão de Escherichia coli, produtora de toxina shiga (STEC) aos humanos. Epidemiologicamente, o sorotipo O157:H7 tem sido o mais envolvido em surtos de doença humana causada por STEC, porém surtos envolvendo STEC não pertencentes ao sorogrupo O157 (STEC não-O157) têm sido descritos. Inúmeros trabalhos constatam uma elevada ocorrência destes microrganismos em fezes de bovinos no Brasil, entretanto, pouco se sabe sobre a transmissão destes aos produtos de origem animal em nosso país. Neste trabalho, foi avaliada a viabilidade de E.coli O153:H25; O113:H21 e O111:H8 em Queijo Minas Frescal (QMF), produzido com inóculos de STEC não O157: H7 e armazenados a 8ºC. Realizaram-se contagens de E. coli e psicrotróficos totais após o processamento do queijo e com intervalos de sete e quinze dias. Foi observado aumento nas contagens de E. coli STEC não O157: H7 e psicrotróficos totais logo após o processamento do QMF, bem como durante o armazenamento a 8ºC, temperatura máxima recomendada pela legislação brasileira. Demonstra-se que, caso haja contaminação da matéria-prima com STEC não O157: H7 (deste estudo), o processamento do QMF não elimina os microrganismos e a temperatura máxima recomendada pela legislação não inibe a multiplicação bacteriana, mantendo-se o risco à população. Reforça-se, portanto, a atenção à qualidade da matéria-prima, das ferramentas de qualidade no campo e na indústria de alimentos para garantir a inocuidade do produto final

The mosaic genome structure and phylogeny of Shiga toxin-producing Escherichia coli O104:H4 is driven by short-term adaptation

Shiga toxin-producing Escherichia coli (STEC) O104:H4 emerged as an important pathogen when it caused a large outbreak in Germany in 2011. Little is known about the evolutionary history and genomic diversity of the bacterium. The current communication describes a comprehensive analysis of STEC O104:H4 genomes from the 2011 outbreak and other non-outbreak-related isolates. Outbreak-related isolates formed a tight cluster that shared a monophyletic relation with two non-outbreak clusters, suggesting that all three clusters originated from a common ancestor. Eight single nucleotide polymorphisms, seven of which were non-synonymous, distinguished outbreak from non-outbreak isolates. Lineage-specific markers indicated that recent partitions were driven by selective pressures associated with niche adaptation. Based on the results, an evolutionary model for STEC O104:H4 is proposed. Our analysis provides the evolutionary context at population level and describes the emergence of clones with novel properties, which is necessary for developing comprehensive approaches to early warning and control.

Detection of shiga-toxin producing E. coli (STEC) in leafy greens sold at local retail markets in Alexandria, Egypt

Leafy green vegetables, a popular and an indispensable ingredient of the daily menus of Egyptians' diets, currently presents a great concern in terms of microbiological hazards. To the best of our knowledge, this is the first report that provides scientific evidence for prevalence of shiga-toxigenic Escherichia coli (STEC) in leafy greens sold at open air local retail markets and superstores in the Egyptian environment. A total of 486 conventional and organic leafy green samples that are eaten raw were collected from different areas in Alexandria, evaluated for total E. coli counts (ECCs), and screened for E. coli O157:H7 using conventional and molecular methods. Recovery of E. coli (≥102CFU/g) from all studied types of leafy greens was indicative of fecal contamination. Total ECCs in conventional samples ranged from 5.47 to 2.56logCFU/g. Based on their inability to ferment sorbitol on CT-SMAC media, 26 presumptive E. coli O157 isolates were detected in 71.4% (270/378) of the studied conventional samples. From all studied organic samples, only 2 types (organic cabbage and parsley, 16.7%) were contaminated with presumptive E. coli O157. All 28 isolates were further serotyped as E. coli O157 by latex agglutination test, and biochemically confirmed as E. coli. Multiplex PCR assays confirmed the ability of 21.4% (6/28) of the E. coli O157 strains to produce shiga-toxins (Stxs), and their virulence markers were as follows: stx1, 66.6% (4/6); stx2, 50% (3/6); stx1/stx2, 16.7% (1/6); eaeA, 83.3% (5/6); and hlyA, 16.7% (1/6). Only 2 strains recovered from conventional and organic parsley could possibly be classified as E. coli O157:H7 based on the presence of stx-genes (either stx1 or stx2 or both). Results of the present research highlight that high E. coli loads, together with recovery of STEC O157 isolates could pose serious health risks to the produce consumers. This emphasizes the urgent need for health authorities to value and utilize the existing knowledge to identify strategies that reduce microbiological risks due to fecal contamination of agricultural products, and implement control measures at all stages of the food chain to specifically eliminate the presence of STEC O157 on the leafy green category.

Molecular typing of VTEC strains isolated from food, feed and animals

Molecular typing of VTEC strains isolated from food, feed and animals