Shiga Toxin-producing Escherichia Coli Virulence Research Articles

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.

Detection of Escherichia coli O157:H7 in Ground Beef Using Long-Read Sequencing.

Foodborne pathogens are a significant cause of illness, and infection with Shiga toxin-producing Escherichia coli (STEC) may lead to life-threatening complications. The current methods to identify STEC in meat involve culture-based, molecular, and proteomic assays and take at least four days to complete. This time could be reduced by using long-read whole-genome sequencing to identify foodborne pathogens. Therefore, the goal of this project was to evaluate the use of long-read sequencing to detect STEC in ground beef. The objectives of the project included establishing optimal sequencing parameters, determining the limit of detection of all STEC virulence genes of interest in pure cultures and spiked ground beef, and evaluating selective sequencing to enhance STEC detection in ground beef. Sequencing libraries were run on the Oxford Nanopore Technologies' MinION sequencer. Optimal sequencing output was obtained using the default parameters in MinKNOW, except for setting the minimum read length to 1 kb. All genes of interest (eae, stx1, stx2, fliC, wzx, wzy, and rrsC) were detected in DNA extracted from STEC pure cultures within 1 h of sequencing, and 30× coverage was obtained within 2 h. All virulence genes were confidently detected in STEC DNA quantities as low as 12.5 ng. In STEC-inoculated ground beef, software-controlled selective sequencing improved virulence gene detection; however, several virulence genes were not detected due to high bovine DNA concentrations in the samples. The growth enrichment of inoculated meat samples in mTSB resulted in a 100-fold increase in virulence gene detection as compared to the unenriched samples. The results of this project suggest that further development of long-read sequencing protocols may result in a faster, less labor-intensive method to detect STEC in ground beef.

First report of the prevalence of Shiga toxinproducing Escherichia coli in ground beef in Quindío, Colombia.

Shiga toxin-producing Escherichia coli (STEC) is a foodborne pathogen associated with clinical cases of diarrhea in humans. Its main virulence factors are the Shiga toxins (Stx1 and Stx2). Cattle are the main reservoir of STEC, and many outbreaks in humans have been related to the consumption of undercooked ground beef contaminated with this pathogen. To determine the prevalence of STEC in ground beef commercialized in all the butcher shops of a township in the department of Quindío and to characterize the virulence genes of the strains found. Thirty ground beef samples were taken in three different times; stx genes and other STEC virulence factors (eae, ehxA, saa) were detected by multiplex PCR. The overall prevalence of STEC was 33.33 % (10/30 positive samples). We isolated eight non-O157 (LEE-negative) strains with four different genetic profiles: stx2 / stx2-ehxA-saa / stx1-stx2-ehxA-saa / stx1-saa. This is the first report on the prevalence of STEC in ground beef in a township in the department of Quindío.

Genome engineering of Stx1-and Stx2-converting bacteriophages unveils the virulence of the dairy isolate Escherichia coli O174:H2 strain UC4224.

The past decade witnessed the emergence in Shiga toxin-producing Escherichia coli (STEC) infections linked to the consumption of unpasteurized milk and raw milk cheese. The virulence of STEC is primarily attributed to the presence of Shiga toxin genes (stx1 and stx2) carried by Stx-converting bacteriophages, along with the intimin gene eae. Most of the available information pertains to the "Top 7" serotypes associated with STEC infections. The objectives of this study were to characterize and investigate the pathogenicity potential of E. coli UC4224, a STEC O174:H2 strain isolated from semi-hard raw milk cheese and to develop surrogate strains with reduced virulence for use in food-related studies. Complete genome sequence analysis of E. coli UC4224 unveiled the presence of a Stx1a bacteriophage, a Stx2a bacteriophage, the Locus of Adhesion and Autoaggregation (LAA) pathogenicity island, plasmid-encoded virulence genes, and other colonization facilitators. In the Galleria mellonella animal model, E. coli UC4224 demonstrated high pathogenicity potential with an LD50 of 6 CFU/10 μL. Upon engineering E. coli UC4224 to generate single and double mutant derivatives by inactivating stx1a and/or stx2a genes, the LD50 increased by approximately 1 Log-dose in the single mutants and 2 Log-doses in the double mutants. However, infectivity was not completely abolished, suggesting the involvement of other virulence factors contributing to the pathogenicity of STEC O174:H2. Considering the possibility of raw milk cheese serving as a reservoir for STEC, cheesemaking model was developed to evaluate the survival of UC4224 and the adequacy of the respective mutants as reduced-virulence surrogates. All tested strains exhibited the ability to survive the curd cooking step at 48°C and multiplied (3.4 Log CFU) in cheese within the subsequent 24 h. These findings indicate that genomic engineering did not exert any unintended effect on the double stx1-stx2 mutant behaviour, making it as a suitable less-virulent surrogate for conducting studies during food processing.

Detection of Shiga Toxin-Producing Escherichia coli (STEC) in the Endocervix of Asymptomatic Pregnant Women. Can STEC Be a Risk Factor for Adverse Pregnancy Outcomes?

The presence of Escherichia coli in the vaginal microbiome has been associated with pregnancy complications. In previous works, we demonstrated that Shiga toxin-producing Escherichia coli (STEC) can produce abortion and premature delivery in rats and that Shiga toxin type 2 (Stx2) can impair human trophoblast cell lines. The hypothesis of this work was that STEC may colonize the lower female reproductive tract and be responsible for adverse pregnancy outcomes. Thus, the aim of this work was to evaluate the presence and prevalence of virulence factor genes from STEC in the endocervix of asymptomatic pregnant women. For that purpose, endocervical swabs were collected from pregnant women during their prenatal examination. Swab samples were enriched in a differential medium to select Enterobacteria. Then, positive samples were analyzed by PCR to detect genes characteristic of Escherichia sp. (such as uidA and yaiO), genes specific for portions of the rfb (O-antigen-encoding) regions of STEC O157 (rfb O157), and STEC virulence factor genes (such as stx1, stx2, eae, lpfA O113, hcpA, iha, sab, subAB). The cytotoxic effects of stx2-positive supernatants from E. coli recovered from the endocervix were evaluated in Vero cells. Our results showed that 11.7% of the endocervical samples were positive for E. coli. Additionally, we found samples positive for stx2 and other virulence factors for STEC. The bacterial supernatant from an isolate identified as E. coli O113:NT, carrying the stx2 gene, exhibited cytotoxic activity in Vero, Swan 71 and Hela cells. Our results open a new perspective regarding the presence of STEC during pregnancy.

Comparative Genomics of Shiga Toxin-Producing Escherichia coli Strains Isolated from Pediatric Patients with and without Hemolytic Uremic Syndrome from 2000 to 2016 in Finland.

ABSTRACTShiga toxin-producing Escherichia coli (STEC) infection can cause mild to severe illness, such as nonbloody or bloody diarrhea, and the fatal hemolytic uremic syndrome (HUS). The molecular mechanism underlying the variable pathogenicity of STEC infection is not fully defined so far. Here, we performed a comparative genomics study on a large collection of clinical STEC strains collected from STEC-infected pediatric patients with and without HUS in Finland over a 16-year period, aiming to identify the bacterial genetic factors that can predict the risk to cause HUS and poor renal outcome. Of 240 STEC strains included in this study, 52 (21.7%) were from pediatric patients with HUS. Serotype O157:H7 was the main cause of HUS, and Shiga toxin gene subtype stx2a was significantly associated with HUS. Comparative genomics and pangenome-wide association studies identified a number of virulence and accessory genes overrepresented in HUS-associated STEC compared to non-HUS STEC strains, including genes encoding cytolethal distending toxins, type III secretion system effectors, adherence factors, etc. No virulence or accessory gene was significantly associated with risk factors for poor renal outcome among HUS patients assessed in this study, including need for and duration of dialysis, presence and duration of anuria, and leukocyte counts. Whole-genome phylogeny and multiple-correspondence analysis of pangenomes could not separate HUS STEC from non-HUS STEC strains, suggesting that STEC strains with diverse genetic backgrounds may independently acquire genetic elements that determine their varied pathogenicity. Our findings indicate that nonbacterial factors, i.e., characteristics of the host immunity, might affect STEC virulence and clinical outcomes.IMPORTANCE Shiga toxin-producing Escherichia coli (STEC) is a serious public health burden worldwide which causes outbreaks of gastrointestinal diseases and the fatal hemolytic uremic syndrome (HUS) characterized by the triad of mechanical hemolytic anemia, thrombocytopenia, and acute renal failure. Understanding the mechanism underlying the disease severity and patient outcome is of high importance. Using comparative genomics on a large collection of clinical STEC strains from STEC-infected patients with and without HUS, our study provides a reference of STEC genetic factors/variants that can be used as predictors of the development of HUS, which will aid risk assessment at the early stage of STEC infection. Additionally, our findings suggest that nonbacterial factors may play a primary role in the renal outcome in STEC-infected patients with HUS; further studies are needed to validate this.

Detection of virulence gene of Shiga toxin-producing Escherichia coli (STEC) strains from animals with diarrhoea and water samples in the North-West Province, South Africa

BackgroundShiga toxin-producing Escherichia coli (STEC) strains have the potential to produce Shiga-toxins, which may lead to enterohemorrhagic diarrhoea and kidney failure in individuals. Moreover, virulence genes of E. coli correlate to their pathogenic nature causing severe morbidity and mortality in humans and animals. Hemolytic-uremic syndrome (HUS), hemorrhagic colitis, might lead to life-threatening hemolytic anemia, thrombocytopenia, and acute renal failure. Escherichia coli (E. coli) are well-known as gastrointestinal bacteria of humans and warm-blooded animals. The current paper was aimed at molecularly identifying virulent STEC strains from “animal with diarrhoea” and water samples from farms in the North-West Province, South Africa. MethodsA total of 25 samples (water, animals with diarrhoea such as pig, piglet, sheep, and rabbit) from two farms were used to isolate E. coli using standard microbiological methods. Molecular method was used to detect the virulent gene. Gram staining and biochemical assays were utilized for presumptive identification of E. coli and the results supported our findings. ResultsOut of 25 samples, 23 isolates tested positive for E. coli using molecular techniques targeting the uidA specific primers at 556 bp. Prevalence of E. coli was 92%. Virulence genes were detected, specifically stx2, hlyA, eaeA, was present in 48% (11/23), 43% (10/23) and 52% (12/23) respectively. Shiga toxin type 1 was not investigated since shiga toxin type 2 is more potent. ConclusionWe conclude that the animals suffering from diarrhoea are carriers and/or source of pathogenic E. coli, specifically STEC. There is an urgent need to identify and characterize the isolates that were found to be responsible for the wide distribution of STEC virulence genes detected showed the potential pathogenic nature of the E. coli stains and serves a massive healthcare concern.

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.

Shiga Toxin–Producing Escherichia coli (STEC) and STEC-Associated Virulence Genes in Raw Ground Pork in Canada

Shiga toxin-producing Escherichia coli (STEC) O157:H7/nonmotile and some non-O157 STEC strains are foodborne pathogens. In response to pork-associated O157 STEC outbreaks in Canada, we investigated the occurrence of STEC in Canadian retail raw ground pork during the period of 1 November 2014 to 31 March 2016. Isolated STEC strains were characterized to determine the Shiga toxin gene (stx) subtype and the presence of virulence genes encoding intimin (eae) and enterohemorrhagic E. coli hemolysin (hlyA). O157 STEC and non-O157 STEC strains were isolated from 1 (0.11%) of 879 and 13 (2.24%) of 580 pork samples, respectively. STEC virulence gene profiles containing both eae and hlyA were found only in the O157 STEC (stx2a, eae, hlyA) isolate. The eae gene was absent from all non-O157 STEC isolates. Of the 13 non-O157 STEC isolates, two virulence genes of stx1a and hlyA were found in four (30.8%) O91:H14 STEC isolates, whereas one virulence gene of stx2e, stx1a, and stx2a was identified in five (38.5%), two (15.4%), and one (7.7%) STEC isolates, respectively, of various serotypes. The remaining non-O157 STEC isolate carried stx2, but the subtype is unknown because this isolate could not be recovered for sequencing. O91:H14 STEC (stx1a, hlyA) was previously reported in association with diarrheal illnesses, whereas the other non-O157 STEC isolates identified in this study are not known to be associated with severe human illnesses. Virulence gene profiles identified in this study indicate that the occurrence of non-O157 STEC capable of causing severe human illness is rare in Canadian retail pork. However, O157 STEC in ground pork can occasionally occur; therefore, education regarding the potential risks associated with STEC contamination of pork would be beneficial for the public and those in the food industry to help reduce foodborne illnesses.

Microbiological Survey of Wheat Flour Sold at Retail in Canada, 2018 to 2019

Following two O121 Shiga toxin-producing Escherichia coli (STEC) outbreaks linked to wheat flour, this study was conducted to gain baseline information on the occurrence of bacterial pathogens and levels of indicator organisms in wheat flour in Canada. A total of 347 prepackaged wheat flour samples were analyzed for Salmonella species, STEC, Listeria monocytogenes, aerobic colony count (ACC), total coliforms, and Escherichia coli. Salmonella spp. and O157 STEC were not detected in any of the samples. L. monocytogenes was identified in two samples (0.6%) at levels below the limit of detection (<0.7 log CFU/g). Non-O157 STEC were isolated from six samples (1.7%) and were characterized for the presence of STEC virulence genes: stx1, stx2, and their subtypes, eae, hlyA, and aggR. One O103:H25 STEC isolate carried virulence genes (stx1a+eae) that are known to be capable of causing diarrhea and/or bloody diarrhea in humans. Of the five remaining non-O157 STEC isolates, four carried single stx2a or stx2c genes and were considered to have the potential of causing diarrhea. The remaining non-O157 STEC isolate (stx2), while not a priority non-O157 STEC, was not available for sequencing; thus, its potential to cause illness is unknown. ACC, total coliforms, and E. coli were detected (≥0.48 log CFU/g) in 98.8, 72.6, and 0.6% of the flour samples. The mean counts of ACC were greater in whole wheat flour compared with the other flour types tested (P < 0.001). The results of this study suggest that the occurrence of O157 STEC and Salmonella is low but that the occurrence of non-O157 STEC in wheat flour with the potential to cause human illness of diarrhea is relatively common. Therefore, the consumption of raw flour could increase the likelihood of STEC infections. Further research is merited for potential risk mitigation strategies within the food production system and with consumers.

Choice of library preparation affects sequence quality, genome assembly, and precise in silico prediction of virulence genes in shiga toxin-producing Escherichia coli.

Whole genome sequencing (WGS) provides essential public health information and is used worldwide for pathogen surveillance, epidemiology, and source tracking. Foodborne pathogens are often sequenced using rapid library preparation chemistries based on transposon technology; however, this method may miss random segments of genomes that can be important for accurate downstream analyses. As new technologies become available, it may become possible to achieve better overall coverage. Here we compare the sequence quality obtained using libraries prepared from the Nextera XT and Nextera DNA Prep (Illumina, San Diego, CA) chemistries for 31 Shiga toxin-producing Escherichia coli (STEC) O121:H19 strains, which had been isolated from flour during a 2016 outbreak. The Nextera DNA Prep gave superior performance metrics including sequence quality, assembly quality, uniformity of genome coverage, and virulence gene identification, among other metrics. Comprehensive detection of virulence genes is essential for making educated assessments of STECs virulence potential. The phylogenetic SNP analysis did not show any differences in the variants detected by either library preparation method which allows isolates prepared from either library method to be analysed together. Our comprehensive comparison of these chemistries should assist researchers wishing to improve their sequencing workflow for STECs and other genomic risk assessments.

Hemolytic uremic syndrome associated with Shiga toxin-producing Escherichia coli infection in Argentina: update of serotypes and genotypes and their relationship with severity of the disease.

Shiga toxin-producing Escherichia coli (STEC) infection is the most common cause of hemolytic uremic syndrome (HUS). Only few studies correlated serotypes and stx genotypes with disease severity. This study aimed to update STEC serotypes, stx genotypes, and virulence factors (eae and ehxA) in a cohort of patients with STEC-HUS and investigate whether they influence the severity of disease. In this multicentric study, children hospitalized between 2005 and 2016 with STEC-HUS confirmed by the National Reference Laboratory were included. Serotypes (O157, O145, O121, and others), stx genotypes (stx1a, stx2a, stx2c, stx2d, and others), and virulence factors were analyzed, and their association with dialysis requirement (>10 days); severe neurological, cardiovascular, and/or bowel involvement; and death was assessed. The records of 280 patients were reviewed; 160 females, median age 21 months (IQR18m). STEC O157 was isolated in 206 (73.6%) patients, O145 in 47 (16.8%), O121 in 15 (5.4%), and other serotypes in 12 (4.2%). The stx2a/2c genotype was carried by 179 (63.9%) strains, stx2a by 94 (33.6%), stx1a/stx2a by five (1.8%), and stx1a only by two (0.7%). All strains except six harbored eae and ehxA genes. Fifty-nine (21.1%) patients had severe neurological involvement, 29 (10.4%) severe bowel injury, 14 (5%) cardiovascular involvement, 53 (18.9%) required > 10 days of dialysis, and 12 (4.3%) died. Neither serotypes nor stx genotypes detected were significantly linked to severity. Serotype O157 and virulence stx2a/2c, eae, ehxA genotype are prevalent in Argentina, and no relationship was found between severity and serotypes and genotypes of STEC detected.

Differential induction of Shiga toxin in environmental Escherichia coli O145:H28 strains carrying the same genotype as the outbreak strains

Shiga toxin-producing Escherichia coli (STEC) O145 is a major serotype associated with severe human disease. Production of Shiga toxins (Stxs), especially Stx2a, is thought to be correlated with STEC virulence. Since stx genes are located in prophages genomes, induction of prophages is required for effective Stxs production. Here, we investigated the production of Stxs in 12 environmental STEC O145:H28 strains under stresses STEC encounter in natural habitats and performed comparative analysis with two O145:H28 clinical strains, one linked to a 2010 U.S. lettuce-associated outbreak (RM13514) and the other linked to a 2007 Belgium ice cream-associated outbreak (RM13516). Similar to the outbreak strains, all environmental strains belong to Sequence Type (ST)-78 using the EcMLST typing scheme. Although all Stx1a-prophages were grouped together, variations in Stx1a production were observed prior to or following the inductions. Among all stx2a positive environmental strains, only the Stx2a-prophage in cattle isolate RM9154-C1 was clustered with the Stx2a-prophages in RM13514, the Stx2a-phage induced from a STEC O104:H4 strain linked to the 2011 outbreak of enterohemorrhagic infection in Germany, and the Stx2a-prophage in STEC O157:H7 strain EDL933, a prototype of enterohemorrhagic E. coli. Furthermore, the Stx2a-prophage in RM9154-C1 shared the same chromosomal insertion site and carried the same antiterminator Q gene and the late promoter PR' as the Stx2a-prophage in RM13514. Following mitomycin C or enrofloxacin treatment, the production of Stx2a in RM9154-C1 was the highest among all environmental strains tested. In contrast, following acid challenge and recovery, the production of Stx2a in RM9154-C1 was the lowest among all the environmental strains tested, at a level comparable to the clinical strains. A significant increase in Stx2a production was detected in all strains when exposed to H2O2, although the induction fold was much lower than those by other inducers. This low-efficiency induction of Stx-prophages by H2O2, a natural inducer of Stx-prophages, supports the hypothesis of bacterial altruism in controlling Stxs production, a strategy that assures the survival of the STEC population as a whole by sacrificing a small fraction of cells for Stxs production and release. Differential induction of Stxs among strains carrying nearly identical Stx-prophages suggests a role of host bacteria in regulating Stxs production. Our study revealed diverse Stx-prophages in STEC O145:H28 strains that were genotypically indistinguishable. Identification of a cattle isolate harboring a Stx2a-prophage associated with high virulence supports the premise that cattle, a natural reservoir of STEC, serve as a source of hypervirulent STEC strains.

Comparative Transcriptomics of Shiga Toxin-Producing and Commensal Escherichia coli and Cytokine Responses in Colonic Epithelial Cell Culture Infections.

Ingestion of Shiga toxin-producing Escherichia coli (STEC) can result in a range of illness severity from asymptomatic to hemorrhagic colitis and death; thus risk assessment of STEC strains for human pathogenicity is important in the area of food safety. Illness severity depends in part on the combination of virulence genes carried in the genome, which can vary between strains even of identical serotype. To better understand how core genes are regulated differently among strains and to identify possible novel STEC virulence gene candidates that could be added to the risk assessment repertoire, we used comparative transcriptomics to investigate global gene expression differences between two STEC strains associated with severe illness and a commensal E. coli strain during in vitro intestinal epithelial cell (IEC) infections. Additionally, we compared a wide array of concomitant cytokine levels produced by the IECs. The cytokine expression levels were examined for a pattern representing STEC pathogenicity; however, while one STEC strain appeared to elicit a proinflammatory response, infection by the other strain produced a pattern comparable to the commensal E. coli. This result may be explained by the significant differences in gene content and expression observed between the STEC strains. RNA-Seq analysis revealed considerable disparity in expression of genes in the arginine and tryptophan biosynthesis/import pathways between the STEC strains and the commensal E. coli strain, highlighting the important role some amino acids play in STEC colonization and survival. Contrasting differential expression patterns were observed for genes involved in respiration among the three strains suggesting that metabolic diversity is a strategy utilized to compete with resident microflora for successful colonization. Similar temporal expression results for known and putative virulence genes were observed in the STEC strains, revealing strategies used for survival prior to and after initial adherence to IECs. Additionally, three genes encoding hypothetical proteins located in mobile genetic elements were, after interrogation of a large set of E. coli genomes, determined to likely represent novel STEC virulence factors.

Differential expression of the virulence gene nleB among Shiga toxin-producing Escherichia coli strains

Shiga toxin-producing Escherichia coli (STEC) is a zoonotic foodborne pathogen associated with hemolytic uremic syndrome (HUS) that vary in their ability to cause disease in humans. STEC represents a serious problem for public health and Argentina is the country with the highest HUS incidence worldwide. Non-LEE effector (nle) genes, present on pathogenicity islands (PAIs), encode translocated substrates of the type III secretion system (T3SS), which could have an important role in STEC virulence. Particularly, nleB is one of the main effector genes proposed as a virulence marker that is involved in the action of T3SS during the STEC infection. NleB inhibits the inflammatory response of the host cell allowing the bacteria to persist in the first stage of the infection. In order to identify the potential risk of STEC strains for public health, the aim of this study was to evaluate and compare basal nleB transcription of 24 STEC strains belonging to 10 serotypes isolated from cattle, food and patients. The results showed differences in nleB transcription among strains. Some non-O157:H7 strains presented transcription levels above the control, an O157:H7 HUS-producing strain. On the other hand, no significant differences were found in basal transcription levels associated with origin or serotype but differences were found between HUS and non-HUS strains. These differences in nleB transcription may be of importance in STEC pathogenesis and could help to differentiate high and low virulence STEC strains.

Molecular Detection and Antimicrobial Resistance Patterns of Shiga Toxigenic Escherichia coli Isolated from Bovine Subclinical Mastitis Milk Samples in Kurdistan, Iran.

Bovine subclinical mastitis is regarded as a devastating disease due to the economic costs imposed on dairy husbandry. Moreover, it is a hazard in the public sector in the cases of zoonotic bacteria because of the potential role of unpasteurized milk and dairy products to propagate the infectious agent to the human food chain. The present study aimed to evaluate the frequency, virulence content, and antimicrobial resistance profile of Shiga toxin-producing Escherichia coli (STEC) strains isolated from bovine subclinical mastitis in Kurdistan Province, West of Iran. A total of 400 bovine subclinical mastitis milk samples recognized in the California Mastitis Test were collected aseptically and analyzed for the presence of E. coli phenotypically and molecularly. The isolates were genotypically screened for stx1, stx2, and eae genes. Furthermore, O157:H7 STEC strain was searched among the isolates in a duplex polymerase chain reaction. The antimicrobial resistance scheme of the isolates was determined using the agar disk diffusion method. In general, 173 (43.25%) E. coli isolates were detected among which 39 (22.54%) isolates were STEC. The frequency of STEC virulence genotypes was stx2 (25 isolates, 64.10%), stx2+eae (6 isolates, 15.38%), stx1+stx2 (6 isolates, 15.38%), and stx1+stx2+eae (2 isolates, 5.12%). In addition, three O157: H7 strains were identified with the genetic content of stx1+stx2+eae (2 isolates) and stx1+stx2 (1 isolate). The most prevalent antimicrobial resistance was observed against streptomycin, tetracycline, and ampicillin. Gentamycin, amoxicillin-clavulanic acid, and trimethoprim-sulfadiazine were the most effective antibiotics against O157 strains, whereas gentamycin, ciprofloxacin, and nitrofurantoin were effective against non-O157 strains. The results revealed the significant role of STEC in bovine subclinical mastitis in the studied region. In addition, the distribution of O157:H7 strain and high prevalence of multidrug resistance among the isolates is a matter of concern. Therefore, there is a potential threat of human infection following the consumption of contaminated milk with STEC in Kurdistan Province, Iran.

Shiga Toxin–Producing Escherichia coli Contamination of Raw Beef and Beef-Based Ready-to-Eat Products at Retail Outlets in Pretoria, South Africa

A cross-sectional study was conducted to determine the prevalence of and factors associated with Shiga toxin-producing Escherichia coli (STEC) in raw beef and ready-to-eat (RTE) beef products sold in 31 retail outlets in Pretoria, South Africa, and nearby areas. A total of 463 beef and RTE samples were screened for four STEC virulence genes (stx1, stx2, eaeA, and hlyA) and seven O-serogroups (O113, O157, O26, O91, O145, O111, and O103) with a multiplex PCR assay. The total aerobic plate count (TAPC) per gram was also determined. A total of 38 STEC isolates were recovered and characterized by conventional PCR assay and serotyping. The overall prevalence of STEC in the beef and RTE samples tested was 16.4% (76 of 463 samples; 95% confidence interval, 13 to 20%). The prevalence of STEC differed significantly by product type (P < 0.0001), with the highest prevalence (35%) detected in boerewors (spicy sausage). The STEC prevalences in minced beef, brisket, RTE cold beef, and biltong were 18, 13, 9, and 5%, respectively. The most frequently detected stx gene was stx2 (13%), and STEC serogroups from recovered isolates were detected at the following prevalences: O2, 15%; O8, 12%; O13, 15%; O20, 8%; O24, 3%; O39, 3%; O41, 8%; O71, 3%; O76, 3%; O150, 12%; and O174, 3%. A high proportion (77%) of the samples had TAPCs that exceeded the South African microbiological standards for meat export (5.0 log CFU/g). The prevalence of O157 STEC (16%) and the diversity of non-O157 STEC serogroups found in five common beef-based products from retail outlets in South Africa suggest exposure of raw beef and beef products to multiple contamination sources during carcass processing and/or cutting and handling at retail outlets. These data provide direct estimates of the potential health risk to consumers from undercooked contaminated products and indicate the need to improve sanitary practices during slaughter and processing of beef and beef-based RTE products. A risk-based surveillance system for STEC may be needed.

Shiga Toxin-Associated Hemolytic Uremic Syndrome: A Narrative Review.

The severity of human infection by one of the many Shiga toxin-producing Escherichia coli (STEC) is determined by a number of factors: the bacterial genome, the capacity of human societies to prevent foodborne epidemics, the medical condition of infected patients (in particular their hydration status, often compromised by severe diarrhea), and by our capacity to devise new therapeutic approaches, most specifically to combat the bacterial virulence factors, as opposed to our current strategies that essentially aim to palliate organ deficiencies. The last major outbreak in 2011 in Germany, which killed more than 50 people in Europe, was evidence that an effective treatment was still lacking. Herein, we review the current knowledge of STEC virulence, how societies organize the prevention of human disease, and how physicians treat (and, hopefully, will treat) its potentially fatal complications. In particular, we focus on STEC-induced hemolytic and uremic syndrome (HUS), where the intrusion of toxins inside endothelial cells results in massive cell death, activation of the coagulation within capillaries, and eventually organ failure.

Evaluation of chromogenic selective agar (CHROMagar STEC) for the direct detection of Shiga toxin-producing Escherichia coli from faecal specimens

Shiga toxin-producing Escherichia coli (STEC) are zoonotic pathogens that cause symptoms of severe gastrointestinal disease, including haemolytic uraemic syndrome (HUS), in humans. Currently in England, STEC serotypes other than O157:H7 are not cultured at the local hospital laboratories. The aim of this study was to evaluate the utility of CHROMagar STEC for the direct detection of STEC from faecal specimens in a diagnostic setting, compared to the current reference laboratory method using PCR targeting the Shiga-toxin gene (stx) to test multiple colonies cultured on MacConkey agar. Of the 292 consecutive faecal specimens submitted to the Gastrointestinal Bacterial Reference Unit that tested positive for stx by PCR, STEC could not be cultured on MacConkey agar or CHROMagar STEC from 87/292 (29.8 %). Of the 205 that were cultured, 106 (51.7 %) were detected on both MacConkey agar and CHROMagar STEC and 99 (48.3 %) were detected on MacConkey agar only. All 106 (100 %) isolates that grew on CHROMagar STEC had the ter gene cassette, known to be associated with resistance to tellurite, compared to 13/99 (13.1 %) that were not detected on CHROMagar STEC. CHROMagar STEC supported the growth of 36/40 (90 %) isolates harbouring stx2a or stx2d, the subtypes most frequently associated with progression to HUS. Of the 92 isolates harbouring eae, an important STEC virulence marker, 77 (83.7 %) grew on CHROMagar STEC. CHROMagar STEC is a useful selective media for the rapid, near-patient detection of STEC that have the potential to cause HUS.

Occurrence of Pathogenic and Potentially Pathogenic Bacteria in Microgreens, Sprouts, and Sprouted Seeds on Retail Market in Riga, Latvia.

Microgreens and sprouts have been used for raw consumption for a long time and are generally viewed as a healthy food. However, several serious outbreaks of foodborne illness have been recorded in European countries, Japan, and North America. Many companies in Latvia nowadays are producing this type of products. The aim of this study was to characterize the incidence of Shiga toxin-producing Escherichia coli (STEC), Salmonella spp., and Listeria spp. in microgreens, sprouts, and seeds intended for domestic production of microgreens on retail market in Riga, Latvia, from January to April 2019. The background microflora was identified as well. A total of 45 samples were purchased, including fresh and processed sprouts, microgreens, baby greens, as well as seeds intended for domestic production of microgreens and sprouts. The samples were processed according to the methods set by the International Organization for Standardization (ISO)-ISO/TS 13136:2012 for STEC, ISO 6579-1:2017 for Salmonella spp., and ISO 11290-1:2017 for Listeria spp. Molecular detection of Salmonella spp. was also performed using real-time polymerase chain reaction. The typical and atypical colonies isolated from selective plates were identified with matrix-assisted laser desorption and ionization time-of-flight mass spectrometry. Listeria monocytogenes was not detected in any of the tested samples. However, the presence of Listeria innocua was detected in two (4.4%) of the samples. Three (6.7%) samples of dried sprouts were positive for the STEC virulence genes. Salmonella spp. was detected in one (2.2%) sample of common sunflower seeds. Altogether, 46 different background bacterial species were identified. The majority were environmental bacteria characteristic to soil, water, and plants, including coliform bacteria. The results provide evidence that microgreens and seeds available for Latvian consumers are generally safe, however, attention has to be paid to dried sprouts.