Human Shiga Toxin-producing Escherichia Coli Infections Research Articles

Shiga Toxin-Producing Escherichia coli Strains from Romania: A Whole Genome-Based Description.

The zoonotic Shiga toxin-producing Escherichia coli (STEC) group is unanimously regarded as exceptionally hazardous for humans. This study aimed to provide a genomic perspective on the STEC recovered sporadically from humans and have a foundation of internationally comparable data. Fifty clinical STEC isolates, representing the culture-confirmed infections reported by the STEC Reference Laboratory between 2016 and 2023, were subjected to whole-genome sequencing (WGS) analysis and sequences were interpreted using both commercial and public free bioinformatics tools. The WGS analysis revealed a genetically diverse population of STEC dominated by non-O157 serogroups commonly reported in human STEC infections in the European Union. The O26:H11 strains of ST21 lineage played a major role in the clinical disease resulting in hospitalisation and cases of paediatric HUS in Romania surpassing the O157:H7 strains. The latter were all clade 7 and mostly ST1804. Notably, among the Romanian isolates was a stx2a-harbouring cryptic clade I strain associated with a HUS case, stx2f- and stx2e-positive strains, and hybrid strains displaying a mixture of intestinal and extraintestinal virulence genes were found. As a clearer picture emerges of the STEC strains responsible for infections in Romania, further surveillance efforts are needed to uncover their prevalence, sources, and reservoirs.

Characterization of Shiga Toxin-producing Escherichia coli Isolated from Cattle Around Ulaanbaatar City, Mongolia

Shiga toxin-producing Escherichia coli (STEC) are associated with severe infections including hemorrhagic colitis and hemolytic uremic syndrome in humans. Ruminants are known as reservoirs of STEC; however, no data are available on STEC in ruminants in Mongolia, where more than 5 million cattle and 25 million sheep are raised. To disclose the existence and characteristics of STEC in Mongolia, in this study, we isolated and characterized STEC from cattle in Mongolia. We collected 350 rectal swabs of cattle from 30 farms near Ulaanbaatar city and isolated 45 STEC from 21 farms. Rectal swabs were precultured with modified Escherichia coli broth and then inoculated to Cefixime-Tellurite Sorbitol MacConkey agar plate and/or CHROMagar STEC agar plate for the isolation of STEC. The isolation ratios in each farm were from 0% to 40%. Multiplex PCR for the estimation of O- and H-serotypes identified 12 O-genotypes (Og-types) and 11 H-genotypes (Hg-types) from 45 isolates; however, Og-types of 19 isolates could not be determined. Stx gene subtyping by PCR identified 2 stx1 subtypes (1a and 1c) and 4 stx2 subtypes (2a, 2c, 2d, and 2g). Forty-five isolates were divided into 21 different groups based on the Og- and Hg-types, stx gene subtypes and the existence of virulence factors, ehxA, eae, and saa, which includes several major serotypes associated with human illness such as O26:H11 and O157:H7. The most dominant isolate, OgUT:H19 [stx1a (+), stx2a (+), ehxA (+) and saa (+)], was isolated from eight farms. This is the first report on the characterization of STEC in cattle in Mongolia, and the results suggest the importance of further monitoring of STEC contamination in the food chains as well as STEC infection in humans.

Whole genome sequence-based characterisation of Shiga toxin-producing Escherichia coli isolated from game meat originating from several European countries

Game meat is becoming increasingly popular but may be contaminated with pathogenic bacteria such as Shiga toxin-producing Escherichia coli (STEC). STEC cause gastrointestinal illnesses including diarrhoea, haemorrhagic colitis (HC), and the haemolytic uremic syndrome (HUS). The aim of this study was to assess the occurrence of STEC in 92 meat samples from chamois (n = 2), red deer (n = 27), roe deer (n = 38), and wild boar (n = 25), from Switzerland and other European countries. After enrichment, Shiga-toxin encoding genes (stx) were detected by PCR in 78 (84%) of the samples and STEC were isolated from 23 (25%) of the same samples. Nine different serotypes and eight different sequence types (STs) were found, with O146:H28 ST738 (n = 10) and O110:H31 ST812 (n = 5) predominating. None of the STEC belonged to the so-called top-five serogroups O26, O103, O111, O145, and O157. Subtyping of stx identified stx1c (n = 9), stx2a (n = 1), stx2b (n = 19), stx2e (n = 2), and stx2g (n = 1). Additional virulence factors (VFs) comprised ehx (n = 12), iha (n = 21), sta1 (n = 1), and subAB (n = 19). None of the isolates contained the eae gene. Twenty-one STEC contained VFs associated with extra-intestinal pathogenic E. coli (ExPEC). Overall, the pathogenic potential of STEC in game meat is moderate, though the isolation of one STEC strain carrying stx2a, and of STEC/ExPEC hybrids suggests a role of game meat as a potential source of STEC infections in humans. Therefore, detailed knowledge of the safe handling and preparation of game meat is needed to prevent foodborne infections.

Core Genome Sequencing Analysis of E. coli O157:H7 Unravelling Genetic Relatedness among Strains from Cattle, Beef, and Humans in Bishoftu, Ethiopia

E. coli O157:H7 is a known Shiga toxin-producing Escherichia coli (STEC), causing foodborne disease globally. Cattle are the main reservoir and consumption of beef and beef products contaminated with E. coli O157:H7 is an important source of STEC infections in humans. To emphasize the cattle-to-human transmission through the consumption of contaminated beef in Bishoftu, Ethiopia, whole-genome sequencing (WGS) was performed on E. coli O157 strains isolated from three sources (cattle, beef, and humans). Forty-four E. coli O157:H7 isolates originating from 23 cattle rectal contents, three cattle hides, five beef carcasses, seven beef cuts at retail shops, and six human stools in Bishoftu between June 2017 and May 2019 were included. This study identified six clusters of closely related E. coli O157:H7 isolates based on core genome multilocus sequence typing (cgMLST) by targeting 2513 loci. A genetic linkage was observed among the isolate genomes from the cattle rectal contents, cattle hides, beef carcasses at slaughterhouses, beef at retail shops, and human stool within a time frame of 20 months. All the strains carried practically the same repertoire of virulence genes except for the stx2 gene, which was present in all but eight of the closely related isolates. All the strains carried the mdfA gene, encoding for the MdfA multi-drug efflux pump. CgMLST analysis revealed genetically linked E. coli O157:H7 isolates circulating in the area, with a potential transmission from cattle to humans through the consumption of contaminated beef and beef products.

Serotype-dependent adhesion of Shiga toxin-producing Escherichia coli to bovine milk fat globule membrane proteins.

Shiga toxin-producing Escherichia coli (STEC) are food-borne pathogens that can cause severe symptoms for humans. Raw milk products are often incriminated as vehicule for human STEC infection. However, raw milk naturally contains molecules, such as the milk fat globule membrane and associated proteins, that could inhibit pathogen adhesion by acting as mimetic ligands. This study aimed to: (i) evaluate the capability of STEC cells to adhere to bovine milk fat globule membrane proteins (MFGMPs), (ii) highlight STEC surface proteins associated with adhesion and (iii) evaluate the variation between different STEC serotypes. We evaluated the physicochemical interactions between STEC and milk fat globules (MFGs) by analyzing hydrophobic properties and measuring the ζ-potential. We used a plate adhesion assay to assess adhesion between MFGMPs and 15 Escherichia coli strains belonging to three key serotypes (O157:H7, O26:H11, and O103:H2). A relative quantitative proteomic approach was conducted by mass spectrometry to identify STEC surface proteins that may be involved in STEC-MFG adhesion. The majority of E. coli strains showed a hydrophilic profile. The ζ-potential values were between -3.7 and - 2.9 mV for the strains and between -12.2 ± 0.14 mV for MFGs. Our results suggest that non-specific interactions are not strongly involved in STEC-MFG association and that molecular bonds could form between STEC and MFGs. Plate adhesion assays showed a weak adhesion of O157:H7 E. coli strains to MFGMPs. In contrast, O26:H11 and O103:H2 serotypes attached more to MFGMPs. Relative quantitative proteomic analysis showed that the O26:H11 str. 21,765 differentially expressed five outer membrane-associated proteins or lipoproteins compared with the O157:H7 str. EDL933. This analysis also found strain-specific differentially expressed proteins, including four O26:H11 str. 21,765-specific proteins/lipoproteins and eight O103:H2 str. PMK5-specific proteins. For the first time, we demonstrated STEC adhesion to MFGMPs and discovered a serotype effect. Several outer membrane proteins-OmpC and homologous proteins, intimin, Type 1 Fimbriae, and AIDA-I-that may be involved in STEC-MFG adhesion were highlighted. More research on STEC's ability to adhere to MFGMs in diverse biological environments, such as raw milk cheeses and the human gastrointestinal tract, is needed to confirm the anti-adhesion properties of the STEC-MFG complex.

Improved Genomic Identification, Clustering, and Serotyping of Shiga Toxin-Producing Escherichia coli Using Cluster/Serotype-Specific Gene Markers.

Shiga toxin-producing Escherichia coli (STEC) have more than 470 serotypes. The well-known STEC O157:H7 serotype is a leading cause of STEC infections in humans. However, the incidence of non-O157:H7 STEC serotypes associated with foodborne outbreaks and human infections has increased in recent years. Current detection and serotyping assays are focusing on O157 and top six (“Big six”) non-O157 STEC serogroups. In this study, we performed phylogenetic analysis of nearly 41,000 publicly available STEC genomes representing 460 different STEC serotypes and identified 19 major and 229 minor STEC clusters. STEC cluster-specific gene markers were then identified through comparative genomic analysis. We further identified serotype-specific gene markers for the top 10 most frequent non-O157:H7 STEC serotypes. The cluster or serotype specific gene markers had 99.54% accuracy and more than 97.25% specificity when tested using 38,534 STEC and 14,216 non-STEC E. coli genomes, respectively. In addition, we developed a freely available in silico serotyping pipeline named STECFinder that combined these robust gene markers with established E. coli serotype specific O and H antigen genes and stx genes for accurate identification, cluster determination and serotyping of STEC. STECFinder can assign 99.85% and 99.83% of 38,534 STEC isolates to STEC clusters using assembled genomes and Illumina reads respectively and can simultaneously predict stx subtypes and STEC serotypes. Using shotgun metagenomic sequencing reads of STEC spiked food samples from a published study, we demonstrated that STECFinder can detect the spiked STEC serotypes, accurately. The cluster/serotype-specific gene markers could also be adapted for culture independent typing, facilitating rapid STEC typing. STECFinder is available as an installable package (https://github.com/LanLab/STECFinder) and will be useful for in silico STEC cluster identification and serotyping using genome data.

High prevalence and pathogenic potential of Shiga toxin-producing Escherichia coli strains in raw mutton and beef in Shandong, China

Shiga toxin-producing Escherichia coli (STEC) is a foodborne pathogen that can cause severe human diseases such as hemolytic uremic syndrome (HUS). Human STEC infections are frequently caused through consumption of contaminated foods, especially raw meats. This study aimed to investigate the prevalence of STEC in raw meats and to characterize the meat-derived STEC strains using whole genome sequencing. Our study showed that 26.6% of raw mutton, and 7.5% of raw beef samples were culture-positive for STEC. Thirteen serotypes were identified in 22 meat-derived isolates in this study, including the virulent serotypes O157:H7 and O26:H11. Seven Shiga toxin (Stx) subtypes were found in 22 isolates, of these, stx1c and stx1c + stx2b were predominant. The recently-reported stx2k subtype was found in three mutton-sourced isolates. A number of other virulence genes such as genes encoding intimin (eae), enterohemorrhagic E. coli (EHEC) hemolysin (ehxA), EHEC factor for adherence (efa1), heat-stable enterotoxin 1 (astA), type III secretion system effectors, were detected in meat-derived STEC strains. One mutton-sourced isolate was resistant to three antibiotics, i.e., tetracycline, chloramphenicol, and trimethoprim-sulfamethoxazole. Whole-genome phylogeny indicated the genomic diversity of meat-derived strains in this study. O157:H7 and O26:H11 isolates in this study were phylogenetically grouped together with strains from HUS patients, suggesting their pathogenic potential. To conclude, our study reported high STEC contaminations in retail raw meats, particularly raw mutton, genomic characterization indicated pathogenic potential of meat-derived STEC strains. These findings highlight the critical need for increased monitoring of STEC in retail raw meats in China.

Added Value of Genomic Surveillance of Virulence Factors in Shiga Toxin-Producing Escherichia coli in New South Wales, Australia.

The disease caused by Shiga toxin-producing Escherichia coli (STEC) remains a significant public health challenge globally, but the incidence of human STEC infections in Australia remains relatively low. This study examined the virulence characteristics and diversity of STEC isolates in the state of New South Wales between December 2017 and May 2020. Utilisation of both whole and core genome multi-locus sequence typing (MLST) allowed for the inference of genomic diversity and detection of isolates that were likely to be epidemiologically linked. The most common STEC serotype and stx subtype detected in this study were O157:H7 and stx1a, respectively. A genomic scan of other virulence factors present in STEC suggested interplay between iron uptake system and virulence factors that mediate either iron release or countermeasures against host defence that could result in a reduction of stx1a expression. This reduced expression of the dominant stx genotype could contribute to the reduced incidence of STEC-related illness in Australia. Genomic surveillance of STEC becomes an important part of public health response and ongoing interrogation of virulence factors in STEC offers additional insights for the public health risk assessment.

Genomic Epidemiology of Shiga Toxin-Producing Escherichia coli Isolated from the Livestock-Food-Human Interface in South America.

Shiga toxin-producing Escherichia coli (STEC) are zoonotic pathogens responsible for causing food-borne diseases in humans. While South America has the highest incidence of human STEC infections, information about the genomic characteristics of the circulating strains is scarce. The aim of this study was to analyze genomic data of STEC strains isolated in South America from cattle, beef, and humans; predicting the antibiotic resistome, serotypes, sequence types (STs), clonal complexes (CCs) and phylogenomic backgrounds. A total of 130 whole genome sequences of STEC strains were analyzed, where 39.2% were isolated from cattle, 36.9% from beef, and 23.8% from humans. The ST11 was the most predicted (20.8%) and included O-:H7 (10.8%) and O157:H7 (10%) serotypes. The successful expansion of non-O157 clones such as ST16/CC29-O111:H8 and ST21/CC29-O26:H11 is highlighted, suggesting multilateral trade and travel. Virulome analyses showed that the predominant stx subtype was stx2a (54.6%); most strains carried ehaA (96.2%), iha (91.5%) and lpfA (77.7%) genes. We present genomic data that can be used to support the surveillance of STEC strains circulating at the livestock-food-human interface in South America, in order to control the spread of critical clones "from farm to table".

Transmission Dynamics of Shiga Toxin-Producing Escherichia coli in New Zealand Cattle from Farm to Slaughter.

Cattle are asymptomatic carriers of Shiga toxin-producing Escherichiacoli (STEC) strains that can cause serious illness or death in humans. In New Zealand, contact with cattle feces and living near cattle populations are known risk factors for human STEC infection. Contamination of fresh meat with STEC strains also leads to the potential for rejection of consignments by importing countries. We used a combination of PCR/matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF) and whole-genome sequencing (WGS) to evaluate the presence and transmission of STEC on farms and in processing plants to better understand the potential pathways for human exposure and thus mitigate risk. Animal and environmental samples (n = 2,580) were collected from six farms and three meat processing plants in New Zealand during multiple sampling sessions in spring of 2015 and 2016. PCR/MALDI-TOF analysis revealed that 6.2% were positive for "Top 7" STEC. Top 7 STEC strains were identified in all sample sources (n = 17) tested. A marked increase in Top 7 STEC prevalence was observed between calf hides on farm (6.3% prevalence) and calf hides at processing plants (25.1% prevalence). Whole-genome sequencing was performed on Top 7 STEC bacterial isolates (n = 40). Analysis of STEC O26 (n = 25 isolates) revealed relatively low genetic diversity on individual farms, consistent with the presence of a resident strain disseminated within the farm environment. Public health efforts should focus on minimizing human contact with fecal material on farms and during handling, transport, and slaughter of calves. Meat processing plants should focus on minimizing cross-contamination between the hides of calves in a cohort during transport, lairage, and slaughter.IMPORTANCE Cattle are asymptomatic carriers of Shiga toxin-producing E. coli (STEC) strains, which can cause serious illness or death in humans. Contact with cattle feces and living near cattle are known risk factors for human STEC infection. This study evaluated STEC carriage in young calves and the farm environment with an in-depth evaluation of six farms and three meat processing plants over 2 years. An advanced molecular detection method and whole-genome sequencing were used to provide a detailed evaluation of the transmission of STEC both within and between farms. The study revealed widespread STEC contamination within the farm environment, but no evidence of recent spread between farms. Contamination of young dairy calf hides increased following transport and holding at meat processing plants. The elimination of STEC in farm environments may be very difficult given the multiple transmission routes; interventions should be targeted at decreasing fecal contamination of calf hides during transport, lairage, and processing.

Identification, Shiga toxin subtypes and prevalence of minor serogroups of Shiga toxin-producing Escherichia coli in feedlot cattle feces

Shiga toxin-producing Escherichia coli (STEC) are foodborne pathogens that cause illnesses in humans ranging from mild to hemorrhagic enteritis with complications of hemolytic uremic syndrome and even death. Cattle are a major reservoir of STEC, which reside in the hindgut and are shed in the feces, a major source of food and water contaminations. Seven serogroups, O26, O45, O103, O111, O121, O145 and O157, called ‘top-7’, are responsible for the majority of human STEC infections in North America. Additionally, 151 serogroups of E. coli are known to carry Shiga toxin genes (stx). Not much is known about fecal shedding and prevalence and virulence potential of STEC other than the top-7. Our primary objectives were to identify serogroups of STEC strains, other than the top-7, isolated from cattle feces and subtype stx genes to assess their virulence potential. Additional objective was to develop and validate a novel multiplex PCR assay to detect and determine prevalence of six serogroups, O2, O74, O109, O131, O168, and O171, in cattle feces. A total of 351 strains, positive for stx gene and negative for the top-7 serogroups, isolated from feedlot cattle feces were used in the study. Of the 351 strains, 291 belonged to 16 serogroups and 60 could not be serogrouped. Among the 351 strains, 63 (17.9%) carried stx1 gene and 300 (82.1%) carried stx2, including 12 strains positive for both. The majority of the stx1 and stx2 were of stx1a (47/63; 74.6%) and stx2a subtypes (234/300; 78%), respectively, which are often associated with human infections. A novel multiplex PCR assay developed and validated to detect six serogroups, O2, O74, O109, O131, O168, and O171, which accounted for 86.9% of the STEC strains identified, was utilized to determine their prevalence in fecal samples (n = 576) collected from a commercial feedlot. Four serogroups, O2, O109, O168, and O171 were identified as the dominant serogroups prevalent in cattle feces. In conclusion, cattle shed in the feces a number of STEC serogroups, other than the top-7, and the majority of the strains isolated possessed stx2, particularly of the subtype 2a, suggesting their potential risk to cause human infections.

Distribution of Novel Og Types in Shiga Toxin-Producing Escherichia coli Isolated from Healthy Cattle.

Shiga toxin-producing Escherichia coli (STEC) is an important foodborne pathogen. Although most cases of STEC infection in humans are due to O157 and non-O157 serogroups, there are also reports of infection with STEC strains that cannot be serologically classified into any O serogroup (O-serogroup untypeable [OUT]). Recently, it has become clear that even OUT strains can be subclassified based on the diversity of O-antigen biosynthesis gene cluster (O-AGC) sequences. Cattle are thought to be a major reservoir of STEC strains belonging to various serotypes; however, the internal composition of OUT STEC strains in cattle remains unknown. In this study, we screened 366 STEC strains isolated from healthy cattle by using multiplex PCR kits including primers that targeted novel O-AGC types (Og types) found in OUT E. coli and Shigella strains in previous studies. Interestingly, 94 (25.7%) of these strains could be classified into 13 novel Og types. Genomic analysis revealed that the results of the in silico serotyping of novel Og-type strains were perfectly consistent with those of the PCR experiment. In addition, it was revealed that a dual Og8+OgSB17-type strain carried two types of O-AGCs from E. coli O8 and Shigella boydii type 17 tandemly inserted at the locus, with both antigens expressed on the cell surface. The results of this comprehensive analysis of cattle-derived STEC strains may help improve our understanding of the strains circulating in the environment. Additionally, the DNA-based serotyping systems used in this study could be used in future epidemiological studies and risk assessments of other STEC strains.

A cluster of Shiga Toxin-producing Escherichia coli O157:H7 highlights raw pet food as an emerging potential source of infection in humans.

In August 2017, a cluster of four persons infected with genetically related strains of Shiga toxin-producing Escherichia coli (STEC) O157:H7 was identified. These strains possessed the Shiga toxin (stx) subtype stx2a, a toxin type known to be associated with severe clinical outcome. One person died after developing haemolytic uraemic syndrome. Interviews with cases revealed that three of the cases had been exposed to dogs fed on a raw meat-based diet (RMBD), specifically tripe. In two cases, the tripe had been purchased from the same supplier. Sampling and microbiological screening of raw pet food was undertaken and indicated the presence of STEC in the products. STEC was isolated from one sample of raw tripe but was different from the strain causing illness in humans. Nevertheless, the detection of STEC in the tripe provided evidence that raw pet food was a potential source of human STEC infection during this outbreak. This adds to the evidence of raw pet food as a risk factor for zoonotic transmission of gastrointestinal pathogens, which is widely accepted for Salmonella, Listeria and Campylobacter spp. Feeding RMBD to companion animals has recently increased in popularity due to the belief that they provide health benefits to animals. Although still rare, an increase in STEC cases reporting exposure to RMBDs was detected in 2017. There has also been an increased frequency of raw pet food incidents in 2017, suggesting an increasing trend in potential risk to humans from raw pet food. Recommendations to reduce the risk of infection included improved awareness of risk and promotion of good hygiene practices among the public when handling raw pet food.

Polymerase Chain Reaction-Based Prevalence of Serogroups of Escherichia coli Known to Carry Shiga Toxin Genes in Feces of Finisher Pigs.

Shiga toxin-producing Escherichia coli (STEC) are major foodborne pathogens and seven serogroups, O26, O45, O103, O111, O121, O145, and O157, that account for the majority of the STEC-associated illness in humans. Similar to cattle, swine also harbor STEC and shed them in the feces and can be a source of human STEC infections. Information on the prevalence of STEC in swine feces is limited. Therefore, our objective was to utilize polymerase chain reaction (PCR) assays to determine prevalence of major virulence genes and serogroups of STEC. Fecal samples (n = 598), collected from finisher pigs within 3 weeks before marketing in 10 pig flows located in 8 states, were included in the study. Samples enriched in E. coli broth were subjected to a real-time PCR assay targeting three virulence genes, Shiga toxin 1 (stx1), Shiga toxin 2 (stx2), and intimin (eae), which encode for Shiga toxins 1 and 2, and intimin, respectively. A novel PCR assay was designed and validated to detect serogroups, O8, O20, O59, O86, O91, O100, O120, and O174, previously reported to be commonly present in swine feces. In addition, enriched fecal samples positive for Shiga toxin genes were subjected to a multiplex PCR assay targeting O26, O45, O103, O104, O111, O121, O145, and O157 serogroups implicated in human clinical infections. Of the 598 fecal samples tested by real-time PCR, 25.9%, 65.1%, and 67% were positive for stx1, stx2, and eae, respectively. The novel eight-plex PCR assay indicated the predominant prevalence of O8 (88.6%), O86 (35.5%), O174 (24.1%), O100 (20.2%), and O91 (15.6%) serogroups. Among the seven serogroups relevant to human infections, three serogroups, O121 (17.6%), O157 (14%), and O26 (11%) were predominant. PCR-based detection indicated high prevalence of Shiga toxin genes and serogroups that are known to carry Shiga toxin genes, including serogroups commonly prevalent in cattle feces and implicated in human infections and in edema disease in swine.

Phenotypic and Genotypic Antimicrobial Resistance in Non-O157 Shiga Toxin-Producing Escherichia coli Isolated From Cattle and Swine in Chile.

Non-O157 Shiga toxin-producing Escherichia coli (STEC) is a zoonotic pathogen that causes bloody diarrhea and hemolytic-uremic syndrome in humans, and a major cause of foodborne disease. Despite antibiotic treatment of STEC infections in humans is not recommended, the presence of antimicrobial-resistant bacteria in animals and food constitutes a risk to public health, as the pool of genes from which pathogenic bacteria can acquire antibiotic resistance has increased. Additionally, in Chile there is no information on the antimicrobial resistance of this pathogen in livestock. Thus, the aim of this study was to characterize the phenotypic and genotypic antimicrobial resistance of STEC strains isolated from cattle and swine in the Metropolitan region, Chile, to contribute relevant data to antimicrobial resistance surveillance programs at national and international level. We assessed the minimal inhibitory concentration of 18 antimicrobials, and the distribution of 12 antimicrobial resistance genes and class 1 and 2 integrons in 54 STEC strains. All strains were phenotypically resistant to at least one antimicrobial drug, with a 100% of resistance to cefalexin, followed by colistin (81.5%), chloramphenicol (14.8%), ampicillin and enrofloxacin (5.6% each), doxycycline (3.7%), and cefovecin (1.9%). Most detected antibiotic resistance genes were dfrA1 and tetA (100%), followed by tetB (94.4%), blaTEM−1 (90.7%), aac(6)-Ib (88.9%), blaAmpC (81.5%), cat1 (61.1%), and aac(3)-IIa (11.1%). Integrons were detected only in strains of swine origin. Therefore, this study provides further evidence that non-O157 STEC strains present in livestock in the Metropolitan region of Chile exhibit phenotypic and genotypic resistance against antimicrobials that are critical for human and veterinary medicine, representing a major threat for public health. Additionally, these strains could have a competitive advantage in the presence of antimicrobial selective pressure, leading to an increase in food contamination. This study highlights the need for coordinated local and global actions regarding the use of antimicrobials in animal food production.

An Overview of the Elusive Passenger in the Gastrointestinal Tract of Cattle: The Shiga Toxin Producing Escherichia coli.

For approximately 10,000 years, cattle have been our major source of meat and dairy. However, cattle are also a major reservoir for dangerous foodborne pathogens that belong to the Shiga toxin-producing Escherichia coli (STEC) group. Even though STEC infections in humans are rare, they are often lethal, as treatment options are limited. In cattle, STEC infections are typically asymptomatic and STEC is able to survive and persist in the cattle GIT by escaping the immune defenses of the host. Interactions with members of the native gut microbiota can favor or inhibit its persistence in cattle, but research in this direction is still in its infancy. Diet, temperature and season but also industrialized animal husbandry practices have a profound effect on STEC prevalence and the native gut microbiota composition. Thus, exploring the native cattle gut microbiota in depth, its interactions with STEC and the factors that affect them could offer viable solutions against STEC carriage in cattle.

Feces of Finisher Pigs Have a Low Prevalence of Shiga Toxin-Producing Escherichia coli that are of Public Health Importance

Shiga toxin-producing Escherichia coli (STEC) are major food pathogens that cause mild to bloody diarrhea, including complications of kidney damage and even death, particularly in children and elderly. Seven serogroups of STEC, O26, O45, O103, O111, O121, O145, and O157, called top-7 STEC, are responsible for the majority of STEC infections in the US. Shiga toxins, which are proteins secreted by the bacteria, are major virulence factors contributing to the disease. There are two Shiga toxin types, 1 and 2, encoded stx1 and stx2 gene, respectively and each type has several subtypes. Another major virulence factor, intimin, a protein on the bacterial cell surface encoded by the eae gene, mediates attachment of the bacterial cell to the intestinal epithelial cells. The severity of STEC infections in humans is dependent on the Shiga toxin type and subtype. Cattle are a major reservoir of STEC and carry the bacteria in the hindgut and shed them in the feces, which is a source of contamination of food and water. Swine have also been shown to harbor STEC in the gut and shed in the feces, and a few outbreaks of STEC infections in humans have been linked to pork and pork products. The STEC does not cause infections in cattle, but in swine, particularly in weaned piglets, it causes edema disease. We conducted a study that utilized molecular (polymerase chain reaction [PCR]) and culture methods to determine prevalence and characteristics of top-7 STEC in the feces of finisher pigs collected from ten pig flows in eight states. A total of 598 fecal samples were collected and analyzed. The overall preva­lence of Shiga toxin genes, stx1 or stx2, was 70.1%, and eae was detected in 66.7% of the samples. Based on the PCR method, among the top-7 STEC, O26 (14.4%), O121 (22.9%) and O157 (18.5%) were the predominant serogroups detected. None of the E. coli O157 isolated, a serogroup implicated in pork-linked outbreaks, contained Shiga toxin genes. Although a number of fecal samples were positive for the top-7 STEC serogroups, culture method identified one strain each of stx1-positive O26 (0.2%) and O103 (0.2%), and 23 strains of stx2-positive O121 (3.9%). Serogroups O26 and O103 possessed stx1a subtype and eae, which have the potential to cause serious infections in humans. Serogroup O121 carried the stx2e subtype, which is involved in causing edema disease in swine and rarely implicated in human infections. Our results indicated that

Pathogenicity assessment of Shiga toxin‐producing Escherichia coli (STEC) and the public health risk posed by contamination of food with STEC

The provisional molecular approach, proposed by EFSA in 2013, for the pathogenicity assessment of Shiga toxin-producing Escherichia coli (STEC) has been reviewed. Analysis of the confirmed reported human STEC infections in the EU/EEA (2012–2017) demonstrated that isolates positive for any of the reported Shiga toxin (Stx) subtypes (and encoding stx gene subtypes) may be associated with severe illness (defined as bloody diarrhoea (BD), haemolytic uraemic syndrome (HUS) and/or hospitalisation). Although strains positive for stx2a gene showed the highest rates, strains with all other stx subtypes, or combinations thereof, were also associated with at least one human case with a severe clinical outcome. Serogroup cannot be used as a predictor of clinical outcome and the presence of the intimin gene (eae) is not essential for severe illness. These findings are supported by the published literature, a review of which suggested there was no single or combination of virulence markers associated exclusively with severe illness. Based on available evidence, it was concluded that all STEC strains are pathogenic in humans, capable of causing at least diarrhoea and that all STEC subtypes may be associated with severe illness. Source attribution analysis, based on ‘strong evidence’ outbreak data in the EU/EEA (2012–2017), suggests that ‘bovine meat and products thereof’, ‘milk and dairy products’, ‘tap water including well water’ and ‘vegetables, fruit and products thereof’ are the main sources of STEC infections in the EU/EEA, but a ranking between these categories cannot be made as the data are insufficient. Other food commodities are also potentially associated with STEC infections but rank lower. Data gaps are identified, and are primarily caused by the lack of harmonisation in sampling strategies, sampling methods, detection and characterisation methods, data collation and reporting within the EU.

Whole Genome Sequencing Characterization of Shiga Toxin–Producing Escherichia coli Isolated from Flour from Swiss Retail Markets

Shiga toxin-producing Escherichia coli (STEC) strains are often found in food and cause human infections. Although STEC O157:H7 is most often responsible for human disease, various non-O157 subtypes have caused individual human infections or outbreaks. The importance of STEC serogroup typing is decreasing while detection of virulence gene patterns has become more relevant. Whole genome sequencing (WGS) reveals the entire spectrum of pathogen information, such as toxin variant, serotype, sequence type, and virulence factors. Flour has not been considered as a vector for STEC; however, this product has been associated with several STEC outbreaks in the last decade. Flour is a natural product, and milling does not include a germ-reducing step. Flour is rarely eaten raw, but the risks associated with the consumption of unbaked dough are probably underestimated. The aim of this study was to determine the prevalence of STEC in flour samples (n = 93) collected from Swiss markets and to fully characterize the isolates by PCR assay and WGS. The prevalence of STEC in these flour samples was 10.8% as indicated by PCR, and a total of 10 STEC strains were isolated (two flour samples were positive for two STEC subtypes). We found one stx2-positve STEC isolate belonging to the classic serogroups frequently associated with outbreaks that could potentially cause severe disease. However, we also found several other common or less common STEC subtypes with diverse virulence patterns. Our results reveal the benefits of WGS as a characterization tool and that flour is a potentially and probably underestimated source for STEC infections in humans.

DNA Microarray-Based Genomic Characterization of the Pathotypes of Escherichia coli O26, O45, O103, O111, and O145 Isolated from Feces of Feedlot Cattle

Shiga toxin-producing Escherichia coli (STEC) serogroups O26, O45, O103, O111, O121, and O145, referred to as the top six non-O157 serogroups, are responsible for more than 70% of human non-O157 STEC infections in North America. Cattle harbor non-O157 strains in the hindgut and shed them in the feces. The objective of this study was to use the U.S. Food and Drug Administration (FDA) E. coli identification (ECID) DNA microarray to identify the serotype, assess the virulence potential of each, and determine the phylogenetic relationships among five of the six non-O157 E. coli serogroups isolated from feedlot cattle feces. Forty-four strains of STEC, enterohemorrhagic E. coli (EHEC), enteropathogenic E. coli (EPEC), or putative nonpathotype E. coli (NPEC) of cattle origin and five human clinical strains of EHEC were assayed with the FDA-ECID DNA microarray. The cattle strains harbored diverse flagellar genes. The bovine and human strains belonging to serogroups O26, O45, and O103 carried stx1 only, O111 carried both stx1 and stx2, and O145 carried either stx1 or stx2. The strains were also positive for various subtypes of intimin and other adhesins (IrgA homologue adhesin, long polar fimbriae, mannose-specific adhesin, and curli). Both human and cattle strains were positive for LEE-encoded type III secretory system genes and non-LEE-encoded effector genes. SplitsTree4, a program used to determine the phylogenetic relationship among the strains, revealed that the strains within each serogroup clustered according to their pathotype. In addition to genes encoding Shiga toxins, bovine non-O157 E. coli strains possessed other major virulence genes, including those for adhesins, type III secretory system proteins, and plasmid-borne virulence genes, similar to human clinical strains. Because virulence factors encoded by these genes are involved in the pathogenesis of various pathotypes of E. coli, the bovine non-O157 strains could cause human illness. The FDA-ECID DNA microarray assay rapidly provided a profile of the virulence genes for assessment of the virulence potential of each strain.