Shiga Toxin-producing Escherichia Coli Serogroups Research Articles

A rapid on-site loop-mediated isothermal amplification technology as an early warning system for the detection of Shiga toxin-producing Escherichia coli in water.

Shiga toxin-producing Escherichia coli (STEC) is an important waterborne pathogen capable of causing serious gastrointestinal infections with potentially fatal complications, including haemolytic-uremic syndrome. All STEC serogroups harbour genes that encode at least one Shiga toxin (stx1 and/or stx2), which constitute the primary virulence factors of STEC. Loop-mediated isothermal amplification (LAMP) enables rapid real-time pathogen detection with a high degree of specificity and sensitivity. The aim of this study was to develop and validate an on-site portable diagnostics workstation employing LAMP technology to permit rapid real-time STEC detection in environmental water samples. Water samples (n=28) were collected from groundwater wells (n=13), rivers (n=12), a turlough (n=2) and an agricultural drain (n=1) from the Corrib catchment in Galway. Water samples (100 ml) were passed through a 0.22 µm filter, and buffer was added to elute captured cells. Following filtration, eluates were tested directly using LAMP assays targeting stx1, stx2 and E. coli phoA genes. The portable diagnostics workstation was used in field studies to demonstrate the on-site testing capabilities of the instrument. Real-time PCR assays targeting stx1 and stx2 genes were used to confirm the results. The limit of detection for stx1, stx2 and phoA LAMP assays were 2, 2 and 6 copies, respectively. Overall, stx1, stx2 and phoA genes were detected by LAMP in 15/28 (53.6 %), 9/28 (32.2 %) and 24/28 (85.7 %) samples, respectively. For confirmation, the LAMP results for stx1 and stx2 correlated perfectly (100 %) with those obtained using PCR. The portable diagnostics workstation exhibited high sensitivity throughout the on-site operation, and the average time from sample collection to final result was 40 min. We describe a simple, transferable and efficient diagnostic technology for on-site molecular analysis of various water sources. This method allows on-site testing of drinking water, enabling evidence-based decision-making by public health and water management authorities.

Evaluating the Effect of Temperature and Pooling in Detection and Isolation of the Major Non-O157 Shiga Toxin–Producing Escherichia coli Serogroups from Meat Samples with the Use of Alternative and Standard Methods

Pathogenic Shiga toxin–producing Escherichia coli (STEC) are an important cause of foodborne illness. The detection of STEC in finished products and during the manufacturing process has an important role as part of verification plans, to confirm that practices and procedures described in the food safety program are successfully applied to control STEC. The aim of this study is to evaluate the effect of temperature and pooling in detection and isolation of the major non-O157 STEC serogroups from meat samples with the use of alternative and standard methods. Bovine meat was experimentally inoculated with one of the “Top 6” non-O157 STEC strains (O26, O103, O111, O145, O45, and O121). Both ISO TS 13136:2012 and a novel alternative method were implemented to evaluate the impact of temperature and pooling. An increase of the enrichment temperature to 41.5 °C allowed the detection of the spiked strain in 10% more samples compared to enrichment at 37 °C. The use of 25- and 375-g sample tests demonstrated no statistically differences between both methods. And finally, this alternative method appears easy to use and time-saving for routine laboratory use.

Occurrence, antibiotic resistance and molecular characterisation of Shiga toxin-producing Escherichia coli isolated from broiler chickens in Tunisia

ABSTRACT 1. Shiga toxin-producing Escherichia coli (STEC) strains are associated with disease outbreaks which cause a public health problem. The aim of this study was to determine the frequency of STEC strains, their virulence factors, phylogenetic groups and antimicrobial resistance profiles in broiler chickens. 2. A total of 222 E.coli isolates were collected from the caecum of chickens intended to be slaughtered. Antibiotic susceptibility was tested against 21 antimicrobial agents and ESBL phenotype was assessed by double-disk synergy test. The presence of STEC virulence genes stx1, stx2,eaeA and ehxA was detected by PCR. The identification of STEC serogroups was realised by PCR amplification. Additive virulence genes, phylogenetic groups and integrons were examined among the STEC isolates. 3. Out of 222 E.coli isolates, 72 (32%) were identified as STEC strains and the most predominant serogroups were O103, O145 and O157. Shiga toxin gene 1 (stx1) was found in 84.7% (61/72) of the STEC strains, and eae and stx2 were detected in 38.8% and 13.8%, respectively. The ESBL phenotype was documented in 48.6% (35/72) of isolates. Most of the isolates (90.3%) carried class 1 integron with the gene cassette encoding resistance to trimethoprim (dfrA) and streptomycin (aadA) in 31.9% of the isolates. Class 2 integron was identified in 36.1% of isolates. 4. Broilers can be considered as a reservoir of STEC strains which have high virulence factors and integrons that might be transmitted to other chickens, environments and humans. It is important to undertake surveillance and efficient control measures in slaughterhouses and farms to control measures of STEC bacteria.

Prevalence, Molecular Characterization, and Antimicrobial Resistance Profiles of Shiga Toxin-Producing Escherichia coli Isolated from Raw Beef, Pork, and Chicken Meat in Vietnam.

Shiga toxin-producing Escherichia coli (STEC) is one of the most important foodborne pathogens, and the rise of antibiotic resistance to it is a significant threat to global public health. The purpose of this study is to investigate the prevalence, molecular characterization, and antibiotic resistance of STEC isolated from raw meat in Vietnam. The findings in this study showed that the prevalence of STEC in raw beef, pork, and chicken meat was 9.72% (7/72), 5.56% (4/72), and 1.39% (1/72), respectively. The STEC isolates were highly resistant to ampicillin (91.67%) and tetracycline (91.67%), followed by trimethoprim/sulfamethoxazole (83.33%), streptomycin (75%), and florfenicol (66.67%). The incidence of STEC virulence-associated genes, including stx1, stx2, eae, and ehxA, was 8.33% (1/12), 91.67% (11/12), 33.33% (4/12), and 58.33% (7/12), respectively. STEC serogroups O157, O26, and O111 were detected in 3 out of 12 STEC isolates. Two isolates were found to be ESBL producers carrying the blaCTX-M-55 gene, and three isolates were colistin-resistant strains harboring the mcr-1 gene. Notably, a STEC O111 isolate from chicken meat harbored both the blaCTX-M-55 and mcr-1 genes.

Characterization of Shiga toxin‐producing Escherichia coli bacteriophages isolated from agricultural environments and examination of their prevalence with bacterial hosts

AbstractIn the natural environment, ruminant livestock, including cattle, are the main reservoir of the outbreak–causing strains of Shiga toxin‐producing Escherichia coli (STEC), where bacteriophages sustainably thrive as well. This study focuses on the characterization of STEC‐specific bacteriophages isolated from cow manure samples in Maine farms and examines their prevalence with STEC hosts. Phenotypic features of representative isolates were characterized by using a transmission electron microscope. Similarly, host range, one‐step growth curve, thermal stability, lytic capability, and genomic analyses were performed to fully characterize selected representative isolates. Results showed that representative bacteriophage isolates belong to Myoviridae (S6P10 and S14P12) and Siphoviridae (S19). The most prevalent and common bacteriophages (46%) were specific to the O26 serogroup. The farm C sampling site had highly heterogenous bacteriophage populations that were specific to six STEC serogroups. The most prevalent bacteriophage isolate (S1P5, Escherichia phage vB_EcoM‐S1P5QW) was verified to have a double‐stranded DNA genome (166,102 bp) with 266 CDs of which 130 have known functions. The majority of the diverse bacteriophage isolates had strong lytic capabilities and a narrow host range that could withstand selected temperature conditions (−20, 37, and 62°C). Results of bacterial screening showed that STEC host strains were not detected in Farms A, C, and E, but were detected on Farms B and D. In conclusion, the highly‐diverse bacteriophage ecology found in cow manure samples may have been an important element in shaping the population of STEC serogroups, specifically in its natural environment, which can provide useful tools for potential antibiotic‐free therapeutics and diagnostic technologies.

Phage tailspike modularity and horizontal gene transfer reveals specificity towards E. coli O-antigen serogroups

BackgroundThe interaction between bacteriophages and their hosts is intricate and highly specific. Receptor-binding proteins (RBPs) of phages such as tail fibers and tailspikes initiate the infection process. These RBPs bind to diverse outer membrane structures, including the O-antigen, which is a serogroup-specific sugar-based component of the outer lipopolysaccharide layer of Gram-negative bacteria. Among the most virulent Escherichia coli strains is the Shiga toxin-producing E. coli (STEC) pathotype dominated by a subset of O-antigen serogroups.MethodsExtensive phylogenetic and structural analyses were used to identify and validate specificity correlations between phage RBP subtypes and STEC O-antigen serogroups, relying on the principle of horizontal gene transfer as main driver for RBP evolution.ResultsWe identified O-antigen specific RBP subtypes for seven out of nine most prevalent STEC serogroups (O26, O45, O103, O104, O111, O145 and O157) and seven additional E. coli serogroups (O2, O8, O16, O18, 4s/O22, O77 and O78). Eight phage genera (Gamaleya-, Justusliebig-, Kaguna-, Kayfuna-, Kutter-, Lederberg-, Nouzilly- and Uetakeviruses) emerged for their high proportion of serogroup-specific RBPs. Additionally, we reveal sequence motifs in the RBP region, potentially serving as recombination hotspots between lytic phages.ConclusionThe results contribute to a better understanding of mosaicism of phage RBPs, but also demonstrate a method to identify and validate new RBP subtypes for current and future emerging serogroups.

Whole-Genome Sequencing of Shiga Toxin-Producing Escherichia coli for Characterization and Outbreak Investigation.

Shiga toxin-producing Escherichia coli (STEC) causes high frequencies of foodborne infections worldwide and has been linked to numerous outbreaks each year. Pulsed-field gel electrophoresis (PFGE) has been the gold standard for surveillance until the recent transition to whole-genome sequencing (WGS). To further understand the genetic diversity and relatedness of outbreak isolates, a retrospective analysis of 510 clinical STEC isolates was conducted. Among the 34 STEC serogroups represented, most (59.6%) belonged to the predominant six non-O157 serogroups. Core genome single nucleotide polymorphism (SNP) analysis differentiated clusters of isolates with similar PFGE patterns and multilocus sequence types (STs). One serogroup O26 outbreak strain and another non-typeable (NT) strain, for instance, were identical by PFGE and clustered together by MLST; however, both were distantly related in the SNP analysis. In contrast, six outbreak-associated serogroup O5 strains clustered with five ST-175 serogroup O5 isolates, which were not part of the same outbreak as determined by PFGE. The use of high-quality SNP analyses enhanced the discrimination of these O5 outbreak strains into a single cluster. In all, this study demonstrates how public health laboratories can more rapidly use WGS and phylogenetics to identify related strains during outbreak investigations while simultaneously uncovering important genetic attributes that can inform treatment practices.

Effect of potassium tellurite concentration in a chromogenic agar medium on isolation of tellurite-resistant “Top Seven” Shiga toxin-producing Escherichia coli from ground beef

The effect of potassium tellurite concentration in a chromogenic agar medium on the detection of tellurite-resistant “top seven” Shiga toxin-producing Escherichia coli (STEC) in beef was evaluated. Samples of ground beef were inoculated with tellurite-resistant STEC O26, O45, O103, O111, O121, O145, or O157 strains at geometric mean (±standard error of the mean) levels of 0, 49 (±1), 490 (±1), or 4900 (±1) CFU/10 g and enriched 1:10 (90 mL) in EC broth (40°C for 6 h). Following enrichment, aliquots of broth culture were treated by immunomagnetic separation with one of three pools of beads against STEC serogroups; pool I: O26, O45, and O121; pool II: O103, O111, and O145; and pool III: O157. After immunomagnetic separation, 50 μL of washed bead suspensions in buffered peptone water was spiral plated onto a modified Possé medium containing 0.5, 1.0, or 1.5 mg/L potassium tellurite, and incubated at 37°C for 18 h. Up to four isolated colonies were picked from each spiral plate based on expected colony phenotypes for STEC, and isolate identity was confirmed with an 11-plex PCR assay targeting the O serogroups and virulence genes. Overall, across all inoculum levels and strains, modified Possé media containing 0.5, 1.0, or 1.5 mg/L potassium tellurite each had a positive predictive value of 100%, and medium containing 0.5 mg/L potassium tellurite had numerically the highest sensitivity (100%) and negative predictive value (100%), which was significantly different from 1.5 mg/L (92.9% and 40.0%, respectively; P < 0.05). Similarly, there was an inverse relationship between potassium tellurite concentration and analytical specificity (number of colonies tested that were STEC-positive): 0.5 (1463 of 1482; 98.7%), 1.0 (1356 of 1411; 96.1%), and 1.5 mg/L (1187 of 1278; 92.9%; P < 0.05). These results suggest that 0.5 mg/L gives better performance than 1.0 or 1.5 mg/L of potassium tellurite in Possé medium for isolation of tellurite-resistant “top seven” STEC from ground beef.

The "Big Six": Hidden Emerging Foodborne Bacterial Pathogens.

Non-O157 Shiga toxin-producing Escherichia coli (STEC) are emerging serogroups that often result in diseases ranging from diarrhea to severe hemorrhagic colitis in humans. The most common non-O157 STEC are O26, O45, O103, O111, O121, and O145. These serogroups are known by the name "big six" because they cause severe illness and death in humans and the United States Department of Agriculture declared these serogroups as food contaminants. The lack of fast and efficient diagnostic methods exacerbates the public impact of the disease caused by these serogroups. Numerous outbreaks have been reported globally and most of these outbreaks were caused by ingestion of contaminated food or water as well as direct contact with reservoirs. Livestock harbor a variety of non-O157 STEC serovars that can contaminate meat and dairy products, or water sources when used for irrigation. Hence, effective control and prevention approaches are required to safeguard the public from infections. This review addresses the disease characteristics, reservoirs, the source of infections, the transmission of the disease, and major outbreaks associated with the six serogroups ("big six") of non-O157 STEC encountered all over the globe.

Shiga toxin-producing Escherichia coli clonal complex 32, including serotype O145:H28, in the UK and Ireland.

Introduction. Shiga toxin-producing Escherichia coli (STEC) O157:H7 has been the most clinically significant STEC serotype in the UK for over four decades. Over the last 10 years we have observed a decrease in STEC O157:H7 and an increase in non-O157 STEC serotypes, such as O145:H28.Gap Statement. Little is known about the microbiology and epidemiology of STEC belonging to CC32 (including O145:H28) in the UK. The aim of this study was to integrate genomic data with patient information to gain a better understanding of the virulence, disease severity, epidemic risk assessment and population structure of this clinically significant clonal complex.Methodology. Isolates of E. coli belonging to CC32 (n=309) in the archives of public health agencies in the UK and Ireland were whole-genome-sequenced, virulence-profiled and integrated with enhanced surveillance questionnaire (ESQ) data, including exposures and disease severity.Results. Overall, diagnoses of STEC belonging to CC32 (290/309, 94 %) in the UK have increased every year since 2014. Most cases were female (61 %), and the highest proportion of cases belonged to the 0-4 age group (53/211,25 %). The frequency of symptoms of diarrhoea (92 %), abdominal pain (84 %), blood in stool (71 %) and nausea (51 %) was similar to that reported in cases of STEC O157:H7, although cases of STEC CC32 were more frequently admitted to hospital (STEC CC32 48 % vs O157:H7 34 %) and/or developed haemolytic uraemic syndrome (HUS) (STEC CC32 9 % vs O157:H7 4 %).The majority of STEC isolates (268/290, 92 %) had the stx2a/eae virulence gene combination, most commonly associated with progression to STEC HUS. There was evidence of person-to-person transmission and small, temporally related, geographically dispersed outbreaks, characteristic of foodborne outbreaks linked to nationally distributed products.Conclusion. We recommend more widespread use of polymerase chain reaction (PCR) for the detection of all STEC serogroups, the development of consistent strategies for the follow-up testing of PCR-positive faecal specimens, the implementation of more comprehensive and standardized collection of epidemiological data, and routine sharing of sequencing data between public health agencies worldwide.

Serogroups and genetic diversity of diarrheagenic strains of Escherichia coli: a retrospective study.

Diverse serogroups of Escherichia coli cause sporadic cases and outbreaks of diarrhea among children. Our study aimed to evaluate the serogroups of diarrheagenic strains of E. coli that cause diarrheal disease in children under two years old, and clarify if the cases were sporadic or outbreaks. The retrospective study included 130 strains of pathogenic E. coli, isolated from children who were less than two years of age, and had diarrheal disease, between May 2016 and July 2019. The study was conducted in the Bacteriology Laboratory (County Clinical Hospital, Mureș, Romania). The 130 strains were sero-grouped using polyvalent and monovalent O antisera. Enterobacterial Repetitive Intergenic Consensus-PCR (ERIC-PCR) was performed to evaluate the similarity between different E. coli strains, and a simplex polymerase chain reaction (PCR) was performed to detect the presence of the hlyA gene that is specific to the enterohemorrhagic strains. After agglutination with polyvalent O antisera, slightly more than half of the strains (50.77%) were sero-grouped as Shiga toxin-producing E. coli (STEC), and the rest of the strains belonged to the Enteropathogenic Escherichia coli (EPEC) serogroups. Serogroup O157 was the most frequently identified (16.51% of the total number of typeable strains), and one strain was positive for hlyA. ERIC-PCR revealed a high diversity of strains, with an overall 50% similarity. STEC serogroups were the most common strains causing diarrheal disease, and O-157 was the dominant serogroup identified. The strains included in our study presented high genetic diversity, suggesting that most of the cases were sporadic.

A systematized review and qualitative synthesis of potential risk factors associated with the occurrence of non-O157 Shiga toxin-producing Escherichia coli (STEC) in the primary production of cattle.

Human infection with Shiga toxin-producing Escherichia coli (STEC) causes an estimated 2.8 million cases of acute illness worldwide each year. Serogroup O157 is the most commonly diagnosed STEC in humans, but cases linked to non-O157 STEC serogroups have increased recently due to increased surveillance and improvements to detection methods. Cattle are an important reservoir for STEC O157 and the same may be true for non-O157 STEC; therefore, reducing the occurrence of these pathogens in cattle could mitigate human infection risk. A systematized literature review of articles published within the Scopus database since 2010 (employing a partially systematic approach) was therefore conducted followed by qualitative synthesis of evidence to provide a structured overview of potential risk factors for non-O157 STEC in primary cattle production. Overall, few relevant studies were identified (n=22), highlighting that more studies are needed. Consistently significant associations were only identified with respect to cattle age (broadly higher rate of isolation from young animals compared to adults) and season of sampling (generally increased isolation of non-O157 STEC in summer). However, wide variation in study designs, including notable differences in laboratory detection methods, means drawing more general conclusions is currently not possible based on the results of this review. However, it is likely that the development of more sensitive methods for non-O157 STEC detection in potential livestock reservoirs and increased standardization across statistically sound epidemiological investigations are required to identify pertinent risk factors.

Population Analysis of O26 Shiga Toxin-Producing Escherichia coli Causing Hemolytic Uremic Syndrome in Italy, 1989-2020, Through Whole Genome Sequencing.

Shiga toxin-producing Escherichia coli (STEC) belonging to the O26 serogroup represent an important cause of Hemolitic Uremic Syndrome (HUS) in children worldwide. The localization of STEC virulence genes on mobile genetic elements allowed the emergence of clones showing different assets of this accessory genomic fraction. A novel O26 STEC clone belonging to Sequence Type (ST) 29 and harboring stx2a, ehxA and etpD plasmid-borne genes has emerged and spread in Europe since the mid-1990s, while another ST29 clone positive for stx2d and lacking plasmid-borne virulence genes was recently described as emerging in France. In Italy, O26 has been the most frequently detected STEC serogroup from HUS cases since the late 1990s. In this study we describe the genomic characterization and population structure of 144 O26 STEC strains isolated from human sources in Italy in the period 1989-2020. A total of 89 strains belonged to ST21, 52 to ST29, two to ST396 and one to ST4944. ST29 strains started to be isolated from 1999. 24 strains were shown to harbour stx1a, alone (n=20) or in combination with stx2a (n=4). The majority of the strains (n=118) harbored stx2a genes only and the two ST396 strains harbored stx2d. A Hierarchical Clustering on Principal Components (HCPC) analysis, based on the detection of accessory virulence genes, antimicrobial resistance (AMR) genes and plasmid replicons, classified the strains in seven clusters identified with numbers from 1 to 7, containing two, 13, 39, 63, 16, 10 and one strain, respectively. The majority of the genetic features defining the clusters corresponded to plasmid-borne virulence genes, AMR genes and plasmid replicons, highlighting specific assets of plasmid-borne features associated with different clusters. Core genome Multi Locus Sequence Typing grouped ST21 and ST29 strains in three clades each, with each ST29 clade exactly corresponding to one HCPC cluster. Our results showed high conservation of either the core or the accessory genomic fraction in populations of ST29 O26 STEC, differently from what observed in ST21 strains, suggesting that a different selective pressure could drive the evolution of different populations of these pathogens possibly involving different ecological niches.

Large-Scale Phylogenetic Analysis Reveals a New Genetic Clade among Escherichia coli O26 Strains.

ABSTRACTShiga toxin-producing Escherichia coli (STEC) O26 is the predominant non-O157 serogroup causing hemolytic uremic syndrome worldwide. Moreover, the serogroup is highly dynamic and harbors several pathogenic clones. Here, we investigated the phylogenetic relationship of STEC O26 at a global level based on 1,367 strains from 20 countries deposited in NCBI and Enterobase databases. The whole-genome-based analysis identified a new genetic clade, called ST29C4. The new clade was unique in terms of multilocus sequence type (ST29), CRISPR (group Ia), and dominant plasmid gene profile (ehxA+/katP-/espP-/etpD-). Moreover, the combination of multiple typing methods (core genome single nucleotide polymorphism [SNP] typing, CRISPR typing, and virulence genes analysis) demonstrated that this new lineage ST29C4 was in the intermediate phylogenetic position between ST29C3 and other non-ST29C3 strains. Besides, we observed that ST29C4 harbored extraintestinal pathogenic E. coli (ExPEC)-related virulence gene (VG), tsh, and STEC-associated VG, stx2a, suggesting the emergence of a hybrid pathogen. The ST29C4 strains also exhibited high similarity in stx2a-prophage and integrase with the O104:H4 strain, further demonstrating its potential risk to human health. Collectively, the large-scale phylogenetic analysis extends the understanding of the clonal structure of O26 strains and provides new insights for O26 strain microevolution.IMPORTANCE Shiga toxin-producing Escherichia coli (STEC) O26 is the second prevalent STEC serogroup only to O157, which can cause a series of diseases ranging from mild diarrhea to life-threatening hemolytic uremic syndrome (HUS). The serogroup is highly diverse and multiple clones are characterized, including ST29C1-C3 and ST21C1-C2. However, the phylogenetic relationship of these clones remains fully unclear. In this study, we revealed a new genetic clade among O26 strains, ST29C4, which was unique in terms of CRISPR, multilocus sequence type (MLST), and plasmid gene profile (PGP). Moreover, the combination of multiple typing methods demonstrated that this new clone was located in the intermediate phylogenetic position between ST29C3 and other non-ST29C3 strains (i.e., ST29C1-C2 and ST21C1-C2). Overall, the large-scale phylogenetic analysis extends our current understanding of O26 microevolution.

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.

Risk factors of Shiga toxin-producing Escherichia coli in livestock raised on diversified small-scale farms in California.

The increasing number of diversified small-scale farms (DSSF) that raise outdoor-based livestock in the USA reflects growing consumer demand for sustainably produced food. Diversified farms are small scale and raise a combination of multiple livestock species and numerous produce varieties. This 2015-2016 cross-sectional study aimed to describe the unique characteristics of DSSF in California, estimate the prevalence of Shiga toxin-producing Escherichia coli (STEC) in livestock and evaluate the association between risk factors and the presence of STEC in livestock, using generalised linear mixed models. STEC prevalence was 13.62% (76/558). Significant variables in the mixed-effect logistic regression model included daily maximum temperature (OR 0.95; CI95% 0.91-0.98), livestock sample source (cattle (OR 4.61; CI95% 1.64-12.96) and sheep (OR 5.29; CI95% 1.80-15.51)), multiple species sharing the same barn (OR 6.23; CI95% 1.84-21.15) and livestock having contact with wild areas (OR 3.63; CI95% 1.37-9.62). Identification of STEC serogroups of public health concern (e.g. O157:H7, O26, O103) in this study indicated the need for mitigation strategies to ensure food safety by evaluating risk factors and management practices that contribute to the spread and prevalence of foodborne pathogens in a pre-harvest environment on DSSF.

Prevalence of Shiga Toxin-Producing Escherichia coli O157 and Non-O157 Serogroups Isolated from Fresh Raw Beef Meat Samples in an Industrial Slaughterhouse.

Background The aims of the current study are the identification of O157 and non-O157 Shiga Toxin-Producing Escherichia coli (STEC) serogroups isolated from fresh raw beef meat samples in an industrial slaughterhouse, determination of antimicrobial resistance patterns, and genetic linkage of STEC isolates. Materials and Methods A total of 110 beef samples were collected from the depth of the rump of cattle slaughtered at Hamadan industrial slaughterhouse. After detection of E. coli isolates, STEC strains were identified according to PCR for stx1, stx2, eaeA, and hlyA virulence genes, and STEC serogroups (O157 and non-O157) were identified by PCR. The genetic linkage of STEC isolates was analyzed by the ERIC- (Enterobacterial Repetitive Intergenic Consensus-) PCR method. The antimicrobial susceptibility of STEC isolates was detected by the disk diffusion method according to CLSI guidelines. Results Among 110 collected beef samples, 77 (70%) were positive for E. coli. The prevalence of STEC in E. coli isolates was 8 (10.4%). The overall prevalence of O157 and non-O157 STEC isolates was 12.5% (one isolate) and 87.5% (7 isolates), respectively. The hemolysin gene was detected in 25% (2 isolates) of STEC strains. Evaluation of antibiotic resistance indicated that 100% of STEC isolates were resistant to ampicillin, ampicillin-sulbactam, amoxicillin-clavulanic acid, and cefazolin. Resistance to tetracycline and ciprofloxacin was detected in 62.5% and 12.5% of isolates, respectively. The analysis of the ERIC-PCR results showed five different ERIC types among the STEC isolates. Conclusion The isolation of different clones STECs from beef and the presence of antibiotic-resistant isolates indicate that more attention should be paid to the hygiene of slaughterhouses.

Distribution of virulence factors, antimicrobial resistance genes and phylogenetic relatedness among Shiga toxin-producing Escherichia coli serogroup O91 from human infections

Shiga toxin-producing Escherichia coli (STEC) belonging to the serogroup O91 are among the most common non-O157 STEC serogroups associated with human illness in Europe. This study aimed to analyse the virulence factors, antimicrobial resistance genes and phylogenetic relatedness among 48 clinical STEC O91 isolates collected during 2003–2019 in Switzerland. The isolates were subjected to whole genome sequencing using short-read sequencing technologies and a subset of isolates additionally to long-read sequencing. They belonged to O91:H10 (n=6), O91:H14 (n=40), and O91:H21 (n=2). Multilocus sequence typing showed that the O91:H10 isolates all belonged to sequence type (ST)641, while the O91:H14 isolates were assigned to ST33, ST9700, or were non-typeable. Both O91:H21 isolates belonged to ST442. Shiga toxin gene stx1a was the most common Shiga toxin gene subtype among the isolates, followed by stx2b, stx2d and stx2a. All isolates were LEE-negative and carried one or two copies of the IrgA adhesin gene iha. In a subset of long-read sequenced isolates, modules of the Locus of Adhesion and Autoaggregation pathogenicity island (LAA-PAI) carrying iha and other genes such as hes, lesP or agn43 were identified. A large proportion of STEC O91:H14 carried the subtilase cytotoxin gene subA, colicin genes (cba, cea, cib and cma) or microcin genes (mcmA, mchB, mchC and mchF). STEC O91:H14 were further distinguished from STEC O91:H10/H21 by one or more virulence factors found in extraintestinal pathogenic E. coli (ExPEC), including hlyF, iucC/iutA, kpsE and traT. The hlyF gene was identified on a novel mosaic plasmid that was unrelated to hlyF+ plasmids described previously in STEC. Core genome phylogenetic analysis revealed that STEC O91:H10 and STEC O91:H21 were clonally conserved, whereas STEC O91:H14 were clonally diverse. Among three STEC O91:H14 isolates, a number of resistance genes were identified, including genes that mediate resistance to aminoglycosides (aadA, aadA2, aadA9, aadA23, aph(3'')-Ib and aph(6)-Id), chloramphenicol (cmlA), sulphonamides (sul2 and sul3), and trimethoprim (drfA12). Our data contribute to understanding the genetic diversity and differing levels of virulence potential within the STEC O91 serogroup.

Shiga-toxin producing Escherichia coli detection in samples from Abruzzo and Molise regions, Italy

Abstract Background Top five serogroups (O157, O111, O26, O103 and O145) of Shiga-toxin producing Escherichia coli (STEC) represent the third most important increasing zoonosis reported in European Union. The ISO methods of analysis, used in foods and environmental samples, quite often does not confirm the molecular detection of STEC genes (stx1 and stx2) in enrichment broth. The aim of this study was to summarize the results of the tests carried out at Istituto Zooprofilattico Sperimentale dell'Abruzzo e del Molise in the last six years (2016-2021), comparing genes detection in enrichment broth and in colonies grown on agar plates. Methods Along the 2016-2021, a total of 1716 foods and environmental samples from Abruzzo and Molise regions were tested, according to ISO/TS 13136:2012. Results Out of 1716 samples, 16 (0.9%) shown presumptive positive presence of STEC (stx1 and/or stx2) in broth. In spite of that, the presence of STEC (positive colonies for stx1 and/or stx2) was confirmed only in 4 out of 16 samples (one sample of cheese and 3 of minced meat). Then, eae gene was found in 54 samples in broth and only in 3 samples from colonies, during a foodborne outbreak investigation. Serogroup specific targets were detected in 6 broth samples (O26, O145 and O157) and confirmed in colonies only in 2 minced meat samples (O157). Conclusions STEC are a heavy hazard for the consumers and the number of cases is slightly increasing since 2015. Our results highlight a low number of presumptive positive samples and even lower number of STEC detection obtained with the ISO classical method. Furthermore, detection of the target genes in broth was rarely confirmed after plating, highlighting a weak point of the analytical method that needs to be investigated. Efforts are necessary to improve the method and the STEC confirmation from colonies. Immunomagnetic beads targeting the top five serogroups and digital PCR could help in increasing the recovery rate of cultivable STEC. Key messages The presumptive positive samples for STEC genes in foodstuffs from the Abruzzo and Molise regions are quite rare, even lower is the number of confirmed colonies by agar plate isolation. Specific immunomagnetic beads for the top five STEC serogroups could improve the method and the STEC colonies detection, increasing the recovery rate of cultivable strains.

Antibiotic Susceptibility Profiles and Frequency of Resistance Genes in Clinical Shiga Toxin-Producing Escherichia coli Isolates from Michigan over a 14-Year Period.

Shiga toxin-producing Escherichia coli (STEC) is an important foodborne pathogen that contributes to over 250,000 infections in the United States each year. Because antibiotics are not recommended for STEC infections, resistance in STEC has not been widely researched despite an increased likelihood for the transfer of resistance genes from STEC to opportunistic pathogens residing within the same microbial community. From 2001 to 2014, 969 STEC isolates were collected from Michigan patients. Antibiotic susceptibility profiles to clinically relevant antibiotics were determined using disc diffusion, while epidemiological data were used to identify factors associated with resistance. Whole-genome sequencing was used for serotyping, examining genetic relatedness, and identifying genetic determinants and mechanisms of resistance in the non-O157 isolates. Increasing frequencies of resistance to at least one antibiotic were observed over the 14 years (P = 0.01). While the non-O157 serogroups were more commonly resistant than O157 (odds ratio, 2.4; 95% confidence interval,1.43 to 4.05), the frequency of ampicillin resistance among O157 isolates was significantly higher in Michigan than the national average (P = 0.03). Genomic analysis of 321 non-O157 isolates uncovered 32 distinct antibiotic resistance genes (ARGs). Although mutations in genes encoding resistance to ciprofloxacin and ampicillin were detected in four isolates, most of the horizontally acquired ARGs conferred resistance to aminoglycosides, β-lactams, sulfonamides, and/or tetracycline. This study provides insight into the mechanisms of resistance in a large collection of clinical non-O157 STEC isolates and demonstrates that antibiotic resistance among all STEC serogroups has increased over time, prompting the need for enhanced surveillance.