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Shiga toxin (Stx) is one of the most potent bacterial toxins known to cause serious gastrointestinal disease in humans. However, the high costs in terms of time, equipment, and operators hinder their further application in timely diagnosis. In this work, we provide a method of coating globotriaosylceramide (Gb3), an Stx receptor, on a glass chip to detect food toxins in real time and a portable fluorescence detection system that can diagnose Stx after loading the chip. The total size of the system with an organic light-emitting diode (OLED) display is about 8.8 × 6.2 × 15 cm3 and its weight is about 375.5 g with a lithium-ion battery. The novel solvent-assisted lipid bilayer method was utilized to coat a bilayer of receptors on the glass substrate instead of placing a bi-lipid membrane and inserting receptors into the membrane, thereby increasing the fluorescence intensity within the chip and being detected by the system. The optimal conditions for coating receptors were verified and the flow rate of 3 μL/min and flow time of 10 min for the process of solvent exchange showed relatively good efficiency. The limit of detection was 250 pg/mL of Stx1B and the system successfully detected Stx in the lettuce eluate sample. The stability of the chip remained highly reliable within 3 days of storage, but after 14 days, the chip lost most of its detection ability due to delamination of the coated receptor bilayer. [ABSTRACT FROM AUTHOR]

ABSTRACT Shiga toxin–producing Escherichia coli (STEC) are major foodborne pathogens that result in thousands of hospitalizations each year in the United States. Cattle, the natural reservoir, harbor STEC asymptomatically at the recto-anal junction (RAJ). The molecular mechanisms that allow STEC and non-STEC E. coli to adhere to the RAJ are not fully understood, in part because most adherence studies utilize human cell culture models. To identify a set of bovine-specific E. coli adherence factors, we used the primary RAJ squamous epithelial (RSE) cell-adherence assay to coculture RSE cells from healthy Holstein cattle with diverse E. coli strains from bovine and nonbovine sources. We hypothesized that a comparative genomic analysis of the strains would reveal factors associated with RSE adherence. After performing adherence assays with historical strains from the E. coli Reference Center (n = 62) and strains newly isolated from the RAJ (n = 15), we used the bioinformatic tool Roary to create a pangenome of this collection. We classified strains as either low or high adherence and using the Scoary program compiled a list of accessory genes correlated with the “high adherence” strains. While none of the correlations were statistically significant, several gene clusters were associated with the high-adherence phenotype, including two that encode uncharacterized proteins. We also demonstrated that non-STEC E. coli strains from the RAJ are more adherent than other isolates and can outcompete STEC in coculture with RSEs. Further analysis of adherence-associated gene clusters may lead to an improved understanding of the molecular mechanisms of RSE adherence and may help develop probiotics targeting STEC in cattle.IMPORTANCEE. coli strains that produce Shiga toxin cause foodborne illness in humans but colonize cattle asymptomatically. The molecular mechanisms that E. coli uses to adhere to cattle cells are largely unknown. Various strategies are used to control E. coli in livestock and limit the risk of outbreaks. These include vaccinating animals against common E. coli strains and supplementing their feed with probiotics to reduce the carriage of pathogens. No strategy is completely effective, and probiotics often fail to colonize the animals. We sought to clarify the genes required for E. coli adherence in cattle by quantifying the attachment to bovine cells in a diverse set of bacteria. We also isolated nonpathogenic E. coli from healthy cows and showed that a representative isolate could outcompete pathogenic strains in cocultures. We propose that the focused study of these strains and their adherence factors will better inform the design of probiotics and vaccines for livestock.

Shiga toxin-producing Escherichia coli (STEC) causes a wide spectrum of diseases including hemorrhagic colitis and hemolytic uremic syndrome (HUS). Previously, we developed a rapid, sensitive, and potentially portable assay that identified STEC by detecting Shiga toxin (Stx) using a B-cell based biosensor platform. We applied this assay to detect Stx2 present in food samples that have been implicated in previous STEC foodborne outbreaks (milk, lettuce, and beef). The STEC enrichment medium, modified Tryptone Soy Broth (mTSB), inhibited the biosensor assay, but dilution with the assay buffer relieved this effect. Results with Stx2a toxoid-spiked food samples indicated an estimated limit of detection (LOD) of ≈4 ng/mL. When this assay was applied to food samples inoculated with STEC, it was able to detect 0.4 CFU/g or 0.4 CFU/mL of STEC at 16 h post incubation (hpi) in an enrichment medium containing mitomycin C. Importantly, this assay was even able to detect STEC strains that were high expressors of Stx2 at 8 hpi. These results indicate that the STEC CANARY biosensor assay is a rapid and sensitive assay applicable for detection of STEC contamination in food with minimal sample processing that can complement the current Food Safety Inspection Service (US) methodologies for STEC. [ABSTRACT FROM AUTHOR]

ABSTRACT Escherichia coli O157:H7-adulterated food products are associated with disease outbreaks in humans. Although cattle feces are a source for E. coli O157:H7 contamination, it is unclear if human-associated outbreak isolates differentially colonize and shed in the feces of cattle from that of non-outbreak isolates. It is also unclear if phenotypes, such as biofilm formation, cell attachment, or toxin production, differentiate environmental E. coli O157:H7 isolates from those associated with human illness. The objective of this study was to compare the genotypes and phenotypes of a diverse set of E. coli O157:H7 isolates, with the intent of identifying differences that could inform cattle colonization and fecal shedding, along with virulence potential in humans. Isolates differed in attachment phenotypes on human Caco-2 cells and bovine-derived recto-anal junction squamous epithelial cells, with curli having a strong impact on attachment to the human-derived cell line. The prototypical E. coli O157 isolate EDL933 had the greatest expression of the adhesin gene iha, yet it had decreased expression of the virulence genes stx2, eae, and ehxA compared the lineage I/II isolates RM6067W and/or FRIK1989. Strong or weak biofilm production was not associated with significant differences in cattle colonization or shedding, suggesting biofilms may not play a major role in cattle colonization. No significant differences in cattle colonization and fecal shedding were detected, despite genomic and in vitro phenotypic differences. The outbreak isolate associated with the greatest incidence of hemolytic uremic syndrome, RM6067W, induced the greatest Vero cell cytotoxicity and had the greatest stx2 gene expression.IMPORTANCEFoodborne illness has major impacts on global health and imposes financial hardships on food industries. Escherichia coli serotype O157:H7 is associated with foodborne illness. Cattle feces are a source of E. coli O157:H7, and routine surveillance has led to an abundance of E. coli O157:H7 genomic data. The relationship between E. coli O157:H7 genome and phenotype is not clearly discerned for cattle colonization/shedding and improved understanding could lead to additional strategies to limit E. coli O157:H7 in the food chain. The goal of the research was to evaluate genomic and phenotypic attributes of E. coli O157:H7 associated with cattle colonization and shedding, environmental persistence, and human illness. Our results indicate variations in biofilm formation and in vitro cellular adherence was not associated with differences in cattle colonization or shedding. Overall, processes involved in cattle colonization and various phenotypes in relation to genotype are complex and remain not well understood.

ABSTRACT Shiga toxin (Stx) is produced by some pathogenic strains of Escherichia coli. To study the impact of Stx on the human intestine, we utilized human intestinal organoids and human intestinal enteroids grown as human intestinal enteroid monolayers (HIEMs) in transwells. To establish optimal experimental conditions, HIEMs were grown with or without mesenchymal cells added to the basolateral wells to recapitulate the interactions between the intestinal epithelium and the underlying mesenchyme. Monolayer barrier integrity was determined through transepithelial electrical resistance (TEER) readings. Apical saline was used on the apical surface since growth medium caused uneven development of the TEER. The medium used for epithelial cells contains added growth factors, while the mesenchymal medium lacks these growth factors. We have shown that mesenchymal cells can maintain the epithelial monolayer in the medium lacking growth factors, suggesting they produce these factors. Furthermore, growth factors produced by mesenchymal cells need to build up in the medium over time, since daily medium changes were not as effective as medium changes performed every 3 days. We have also shown that addition of growth factors is toxic to mesenchymal cells. Epithelial cells were more resistant to Stx2 than the mesenchymal cells, and mesenchymal cells contributed to epithelial cell death. Epithelial cells tolerated luminal exposure better than basolateral exposure. These studies demonstrate the importance of understanding tissue interactions in a disease state when using in vitro and in vitro models.IMPORTANCEThese studies have cemented the need for complex cell culture models when studying host-pathogen interactions. Common animal models such as mice are resistant to E. coli O157:H7 infections and intestinal delivery of Stx2, while humans appear to be sensitive to both. It has been proposed that in humans, shiga toxin-producing E. coli-mediated intestinal damage destroys the intestinal barrier and allows basolateral access to Stx2. In mice, there is no epithelial damage; therefore, they are resistant to epithelial delivery of Stx2 while remaining sensitive to Stx2 injection. Our studies show that like mice, the human epithelial layer is quite resistant to Stx2, and it is the sensitivity of the mesenchymal cells that kills the epithelial cells. We have shown that Stx2 is transported through the intact epithelium without causing damage to the resistant epithelial layer. Understanding tissue interactions during infections is therefore critical in determining the effects of pathogens on human tissues.

Shiga-toxin-producing Escherichia coli (STEC) causes a wide spectrum of diseases including hemorrhagic colitis and hemolytic uremic syndrome (HUS). The current Food Safety Inspection Service (FSIS) testing methods for STEC use the Food and Drug Administration (FDA) Bacteriological Analytical Manual (BAM) protocol, which includes enrichment, cell plating, and genomic sequencing and takes time to complete, thus delaying diagnosis and treatment. We wanted to develop a rapid, sensitive, and potentially portable assay that can identify STEC by detecting Shiga toxin (Stx) using the CANARY (Cellular Analysis and Notification of Antigen Risks and Yields) B-cell based biosensor technology. Five potential biosensor cell lines were evaluated for their ability to detect Stx2. The results using the best biosensor cell line (T5) indicated that this biosensor was stable after reconstitution with assay buffer covered in foil at 4 °C for up to 10 days with an estimated limit of detection (LOD) of ≈0.1–0.2 ng/mL for days up to day 5 and ≈0.4 ng/mL on day 10. The assay detected a broad range of Stx2 subtypes, including Stx2a, Stx2b, Stx2c, Stx2d, and Stx2g but did not cross-react with closely related Stx1, abrin, or ricin. Additionally, this assay was able to detect Stx2 in culture supernatants of STEC grown in media with mitomycin C at 8 and 24 h post-inoculation. These results indicate that the STEC CANARY biosensor developed in this study is sensitive, reproducible, specific, rapid (≈3 min), and may be applicable for surveillance of the environment and food to protect public health. [ABSTRACT FROM AUTHOR]

Background: The pathogens E. coli and S. enterica that caused substantial health problems and financial losses were believed to have originated primarily from Egypt's dairy farms. Aim: The purpose of this study was to ascertain the occurrence of E. coli and S. enterica in three large dairy farms located in the Egyptian governorate of Sharkia. Furthermore, biochemical and serological characteristics of the isolated isolates were described. Further analysis revealed that several E. coli serovars had the genes stx1, stx2, eaeA, and hylA, while invA, stn, and hilA genes were found in several S. enterica serotypes using a multi-plex PCR. Methods: A total of 540 samples of fresh raw cow milk, water, feedstuffs, feces, (108 each), as well as swabs from feeders, milker hands and cattle crushes (36 each ), were gathered and analyzed. Results: The recovery of E. coli from various sampling sources was shown to have an overall prevalence of 62.2% (336/540) in the results. Fecal samples had isolated S. enterica, with a frequency of 0.74% (4/540). The existence of various groups of serovars, such as O26, O44, O55, O78 and O111 for E. coli and S. enteritidis, S. typhimurium and S. inganda for S. enterica was revealed by serological identification of the two species. However, it was discovered that a number of E. coli serovars had much higher percentages of the eaeA and hylA genes as well as shiga-toxin types 1 and 2 (stx1 and stx2). The presence of the invA gene, a diagnostic marker for S. enterica was 100% across all serovars. Salmonella enteritidis possessed both the enterotoxin gene (stn) and the hyper-invasive locus gene (hilA). Salmonella typhimurium had the hilA gene, whereas Sal. inganda had the stn gene. Conclusion: E. coli and S. enterica recovered in this study have significant genetic risk factors for high pathogenicity and virulence, posing a real threat to dairy population productivity and health, which could spread to the general public through milk. [Open Vet J 2024; 14(1.000): 214-224]

Background: The pathogens Escherichia coli and Salmonella enterica that caused substantial health problems and financial losses were believed to have originated primarily from Egypt’s dairy farms. Aim: The purpose of this study was to ascertain the occurrence of E. coli and S. enterica in three large dairy farms located in the Egyptian governorate of Sharkia. Furthermore, biochemical and serological characteristics of the isolated isolates were described. Further analysis revealed that several E. coli serovars had the genes stx1, stx2, eaeA, and hylA, while invA, stn, and hilA genes were found in several S. enterica serotypes using a multi-plex PCR. Methods: A total of 540 samples of fresh raw cow milk, water, feedstuffs, feces, (108 each), as well as swabs from feeders, milker hands and cattle crushes (36 each ), were gathered and analyzed. Results: The recovery of E. coli from various sampling sources was shown to have an overall prevalence of 62.2% (336/540) in the results. Fecal samples had isolated S. enterica, with a frequency of 0.74% (4/540). The existence of various groups of serovars, such as O26, O44, O55, O78 and O111 for E. coli and Salmonella enteritidis, Salmonella typhimurium and Salmonella inganda for S. enterica was revealed by serological identification of the two species. However, it was discovered that a number of E. coli serovars had much higher percentages of the eaeA and hylA genes as well as shiga-toxin types 1 and 2 (stx1 and stx2). The presence of the invA gene, a diagnostic marker for S. enterica was 100% across all serovars. Salmonella enteritidis possessed both the enterotoxin gene (stn) and the hyper-invasive locus gene (hilA). Salmonella typhimurium had the hilA gene, whereas S. inganda had the stn gene. Conclusion: Escherichia coli and S. enterica recovered in this study have significant genetic risk factors for high pathogenicity and virulence, posing a real threat to dairy population productivity and health, which could spread to the general public through milk. [ABSTRACT FROM AUTHOR]

Trends in the incidence of O157 and non-O157 serogroups of Shiga toxin-producing Escherichia coli (STEC) infections have markedly diverged. Here, we estimate the extent to which STEC serogroups share the same transmission routes and risk factors, potentially explaining these trends. With 3048 STEC cases reported in Minnesota from 2010 to 2019, we used lasso penalized regression to estimate pooled odds ratios (pOR) for the association between STEC risk factors and specific STEC serogroups and Shiga toxin gene profiles. We used random forests as a confirmatory analysis. Across an extended period of time, we found evidence for person-to-person transmission associated with the O26 serogroup, relative to other serogroups (pOR = 1.32 for contact with an individual with diarrhea). Rurality was less associated with non-O157 serogroups than O157 (pOR = 1.21 for each increasing level of rurality). We also found an association between unpasteurized juice and strains carrying only stx1 (pOR = 1.41). Collectively, these results show differences in risk factors across STEC types, which suggest differences in the most effective routes of transmission. Serogroup-specific disease control strategies should be explored. Specifically, preventative measures for non-O157 STEC need to extend beyond those we have employed for O157 STEC. • Person-to-person transmission risk factors are associated with O26 STEC. • Non-O157 STEC are less associated with rural environments than O157 STEC. • STEC with only stx1 are associated with unpasteurized juice consumption. • Strategies to prevent O157 transmission may not be as effective for non-O157 STEC. [ABSTRACT FROM AUTHOR]

Shiga toxin-producing Escherichia coli (STEC) causes a wide spectrum of diseases including hemorrhagic colitis and hemolytic uremic syndrome (HUS). Previously, we developed a rapid, sensitive, and potentially portable assay that identified STEC by detecting Shiga toxin (Stx) using a B-cell based biosensor platform. We applied this assay to detect Stx2 present in food samples that have been implicated in previous STEC foodborne outbreaks (milk, lettuce, and beef). The STEC enrichment medium, modified Tryptone Soy Broth (mTSB), inhibited the biosensor assay, but dilution with the assay buffer relieved this effect. Results with Stx2a toxoid-spiked food samples indicated an estimated limit of detection (LOD) of ≈4 ng/mL. When this assay was applied to food samples inoculated with STEC, it was able to detect 0.4 CFU/g or 0.4 CFU/mL of STEC at 16 h post incubation (hpi) in an enrichment medium containing mitomycin C. Importantly, this assay was even able to detect STEC strains that were high expressors of Stx2 at 8 hpi. These results indicate that the STEC CANARY biosensor assay is a rapid and sensitive assay applicable for detection of STEC contamination in food with minimal sample processing that can complement the current Food Safety Inspection Service (US) methodologies for STEC.

Shiga toxin-producing Escherichia coli (STEC) consists of diverse strains differing in genetic make-up and virulence potential. To better understand the pathogenicity potential of STEC carried by the wildlife, three STEC and one E. coli strains isolated from wild birds near a major agricultural region in California were selected for comparative pathogenomic analyses. Three American crow (Corvus brachyrhynchos) strains, RM9088, RM9513, and RM10410, belonging to phylogroup A with serotypes O109:H48, O9:H30, and O113:H4, respectively, and a red-winged blackbird (Agelaius phoeniceus) strain RM14516 in phylogroup D with serotype O17:H18, were examined. Shiga toxin genes were identified in RM9088 (stx1a), RM10410 (stx1a C stx2d), and RM14516 (stx2a). Unlike STEC O157:H7 strain EDL933, none of the avian STEC strains harbored the pathogenicity islands OI-122, OI-57, and the locus of enterocyte effacement, therefore the type III secretion system biogenesis genes and related effector genes were absent in the three avian STEC genomes. Interestingly, all avian STEC strains exhibited greater (RM9088 and RM14516) or comparable (RM10410) cytotoxicity levels compared with EDL933. Comparative pathogenomic analyses revealed that RM9088 harbored numerous genes encoding toxins, toxins delivery systems, and adherence factors, including heat-labile enterotoxin, serine protease autotransporter toxin Pic, type VI secretion systems, protein adhesin Paa, fimbrial adhesin K88, and colonization factor antigen I. RM9088 also harbored a 36-Kb high pathogenicity island, which is related to iron acquisition and pathogenicity in Yersinia spp. Strain RM14516 carried an acid fitness island like the one in EDL933, containing a nine gene cluster involved in iron acquisition. Genes encoding extracellular serine protease EspP, subtilase cytotoxin, F1C fimbriae, and inverse autotransporter adhesin IatC were only detected in RM14516, and genes encoding serine protease autotransporter EspI and P fimbriae were only identified in RM10410. Although all curli genes were present in avian STEC strains, production of curli fimbriae was only detected for RM9088 and RM14516. Consistently, strong, moderate, and little biofilms were observed for RM9088, RM14516, and RM10410, respectively. Our study revealed novel combinations of virulence factors in two avian strains, which exhibited high level of cytotoxicity and strong biofilm formation. Comparative pathogenomics is powerful in assessing pathogenicity and health risk of STEC strains. [ABSTRACT FROM AUTHOR]

Genetic screen technology has been applied to study the mechanism of action of bacterial toxins—a special class of virulence factors that contribute to the pathogenesis caused by bacterial infections. These screens aim to identify host factors that directly or indirectly facilitate toxin intoxication. Additionally, specific properties of certain toxins, such as membrane interaction, retrograde trafficking, and carbohydrate binding, provide robust probes to comprehensively investigate the lipid biosynthesis, membrane vesicle transport, and glycosylation pathways, respectively. This review specifically focuses on recent representative toxin-based genetic screens that have identified new players involved in and provided new insights into fundamental biological pathways, such as glycosphingolipid biosynthesis, protein glycosylation, and membrane vesicle trafficking pathways. Functionally characterizing these newly identified factors not only expands our current understanding of toxin biology but also enables a deeper comprehension of fundamental biological questions. Consequently, it stimulates the development of new therapeutic approaches targeting both bacterial infectious diseases and genetic disorders with defects in these factors and pathways. [ABSTRACT FROM AUTHOR]

Shiga toxin-producing strains represent pathogenic group that is of concern in food production. The present study evaluated forty-eight E. coli isolates (11 with intact stx gene, while remaining isolates presented only stx-fragments) for Shiga toxin production. The four most expressive stx-producers (O26, O103, O145, and O157) were selected to evaluate effects of pH (3.5, 4.5, and 7) and temperature (35, 40, and 50°C). After determining acid stress effects in media on Stx-induction, we mimicked "in natura" conditions using milk, apple, and orange juices. Only isolates that showed the presence of intact stx gene (11/48) produced Shiga toxin. In addition, acid pH had a role in down-regulating the production of Shiga toxin, in both lactic acid and juices. In contrast, non-lethal heating (40°C), when in neutral pH and milk was a favorable environment to induce Shiga toxin. Lastly, two isolates (O26 and O103) showed a higher capacity to produce Shiga toxin and were included in a genomic cluster with other E. coli involved in worldwide foodborne outbreaks. The induction of this toxin when subjected to 40°C may represent a potential risk to the consumer, since the pathogenic effect of oral ingestion of Shiga toxin has already been proved in an animal model. [ABSTRACT FROM AUTHOR]

Weather affects key aspects of bacterial behavior on plants but has not been extensively investigated as a tool to assess risk of crop contamination with human foodborne pathogens. A novel mechanistic model informed by weather factors and bacterial state was developed to predict population dynamics on leafy vegetables and tested against published data tracking Escherichia coli O157:H7 (EcO157) and Salmonella enterica populations on lettuce and cilantro plants. The model utilizes temperature, radiation, and dew point depression to characterize pathogen growth and decay rates. Additionally, the model incorporates the population level effect of bacterial physiological state dynamics in the phyllosphere in terms of the duration and frequency of specific weather parameters. The model accurately predicted EcO157 and S. enterica population sizes on lettuce and cilantro leaves in the laboratory under various conditions of temperature, relative humidity, light intensity, and cycles of leaf wetness and dryness. Importantly, the model successfully predicted EcO157 population dynamics on 4-week-old romaine lettuce plants under variable weather conditions in nearly all field trials. Prediction of initial EcO157 population decay rates after inoculation of 6-week-old romaine plants in the same field study was better than that of long-term survival. This suggests that future augmentation of the model should consider plant age and species morphology by including additional physical parameters. Our results highlight the potential of a comprehensive weatherbased model in predicting contamination risk in the field. Such a modeling approach would additionally be valuable for timing field sampling in quality control to ensure the microbial safety of produce. [ABSTRACT FROM AUTHOR]

Cattle can harbour enterohaemorrhagic Escherichia coli O157:H7 serotype in their faecal matters. This study aimed to isolate E. coli O157:H7 from the intestinal digesta, cattle dungs and water; and assess the antibiotic susceptibility and production of shiga toxins by the isolates. The counts of viable bacteria and faecal coliforms in 13 each of intestinal digesta and cattle dung and the 12 water samples were determined using nutrient agar and Eosin methylene blue agar respectively. Sorbitol MacConkey agar (SMAC) was used to screen for E. coli O157:H7 among the 38 E. coli isolates. PCR amplification of uidA genes was used to authenticate the isolates as enterohaemorrhagic E. coli O157:H7 serotype while amplification of stx1 and stx2 showed the production of shiga toxins. The antibiotic susceptibility patterns of the isolates were determined using standard disk diffusion method. The count of viable bacteria and faecal coliform was highest in the intestinal digesta followed by cattle dungs. There was 100% susceptibility to ofloxacin coupled with 100% resistance to augmentin by all the 8 E. coli O157 and 10 non-O157 isolates. The E. coli O157 isolates were more susceptible to ciprofloxacin, gentamicin, ampicillin and cefuroxime than non-O157 isolates but were less susceptible to nitrofurantoin and ceftazidime than non-O157. Eight (44.4%) out of the 18 presumptive E. coli O157 on SMAC amplified uidA genes and were confirmed as E. coli O157. They were isolated only from intestinal digesta and cattle dung. The prevalence of stx1 and stx2 genes among the E. coli O157 was 37.5% and 12.5% respectively. It is concluded from this study that intestinal digesta and cattle dung harboured E. coli O157: H7 some of which possessed shiga toxin. [ABSTRACT FROM AUTHOR]

Shiga toxin-producing Escherichia coli (STEC) consists of diverse strains differing in genetic make-up and virulence potential. To better understand the pathogenicity potential of STEC carried by the wildlife, three STEC and one E. coli strains isolated from wild birds near a major agricultural region in California were selected for comparative pathogenomic analyses. Three American crow (Corvus brachyrhynchos) strains, RM9088, RM9513, and RM10410, belonging to phylogroup A with serotypes O109:H48, O9:H30, and O113:H4, respectively, and a red-winged blackbird (Agelaius phoeniceus) strain RM14516 in phylogroup D with serotype O17:H18, were examined. Shiga toxin genes were identified in RM9088 (stx1a), RM10410 (stx1a + stx2d), and RM14516 (stx2a). Unlike STEC O157:H7 strain EDL933, none of the avian STEC strains harbored the pathogenicity islands OI-122, OI-57, and the locus of enterocyte effacement, therefore the type III secretion system biogenesis genes and related effector genes were absent in the three avian STEC genomes. Interestingly, all avian STEC strains exhibited greater (RM9088 and RM14516) or comparable (RM10410) cytotoxicity levels compared with EDL933. Comparative pathogenomic analyses revealed that RM9088 harbored numerous genes encoding toxins, toxins delivery systems, and adherence factors, including heat-labile enterotoxin, serine protease autotransporter toxin Pic, type VI secretion systems, protein adhesin Paa, fimbrial adhesin K88, and colonization factor antigen I. RM9088 also harbored a 36-Kb high pathogenicity island, which is related to iron acquisition and pathogenicity in Yersinia spp. Strain RM14516 carried an acid fitness island like the one in EDL933, containing a nine gene cluster involved in iron acquisition. Genes encoding extracellular serine protease EspP, subtilase cytotoxin, F1C fimbriae, and inverse autotransporter adhesin IatC were only detected in RM14516, and genes encoding serine protease autotransporter EspI and P fimbriae were only identified in RM10410. Although all curli genes were present in avian STEC strains, production of curli fimbriae was only detected for RM9088 and RM14516. Consistently, strong, moderate, and little biofilms were observed for RM9088, RM14516, and RM10410, respectively. Our study revealed novel combinations of virulence factors in two avian strains, which exhibited high level of cytotoxicity and strong biofilm formation. Comparative pathogenomics is powerful in assessing pathogenicity and health risk of STEC strains.

Shiga-toxin-producing Escherichia coli (STEC) causes a wide spectrum of diseases including hemorrhagic colitis and hemolytic uremic syndrome (HUS). The current Food Safety Inspection Service (FSIS) testing methods for STEC use the Food and Drug Administration (FDA) Bacteriological Analytical Manual (BAM) protocol, which includes enrichment, cell plating, and genomic sequencing and takes time to complete, thus delaying diagnosis and treatment. We wanted to develop a rapid, sensitive, and potentially portable assay that can identify STEC by detecting Shiga toxin (Stx) using the CANARY (Cellular Analysis and Notification of Antigen Risks and Yields) B-cell based biosensor technology. Five potential biosensor cell lines were evaluated for their ability to detect Stx2. The results using the best biosensor cell line (T5) indicated that this biosensor was stable after reconstitution with assay buffer covered in foil at 4 °C for up to 10 days with an estimated limit of detection (LOD) of ≈0.1–0.2 ng/mL for days up to day 5 and ≈0.4 ng/mL on day 10. The assay detected a broad range of Stx2 subtypes, including Stx2a, Stx2b, Stx2c, Stx2d, and Stx2g but did not cross-react with closely related Stx1, abrin, or ricin. Additionally, this assay was able to detect Stx2 in culture supernatants of STEC grown in media with mitomycin C at 8 and 24 h post-inoculation. These results indicate that the STEC CANARY biosensor developed in this study is sensitive, reproducible, specific, rapid (≈3 min), and may be applicable for surveillance of the environment and food to protect public health.

Shiga toxin-producing strains represent pathogenic group that is of concern in food production. The present study evaluated forty-eight E. coli isolates (11 with intact stx gene, while remaining isolates presented only stx-fragments) for Shiga toxin production. The four most expressive stx-producers (O26, O103, O145, and O157) were selected to evaluate effects of pH (3.5, 4.5, and 7) and temperature (35, 40, and 50°C). After determining acid stress effects in media on Stx-induction, we mimicked “in natura” conditions using milk, apple, and orange juices. Only isolates that showed the presence of intact stx gene (11/48) produced Shiga toxin. In addition, acid pH had a role in down-regulating the production of Shiga toxin, in both lactic acid and juices. In contrast, non-lethal heating (40°C), when in neutral pH and milk was a favorable environment to induce Shiga toxin. Lastly, two isolates (O26 and O103) showed a higher capacity to produce Shiga toxin and were included in a genomic cluster with other E. coli involved in worldwide foodborne outbreaks. The induction of this toxin when subjected to 40°C may represent a potential risk to the consumer, since the pathogenic effect of oral ingestion of Shiga toxin has already been proved in an animal model.

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is a foodborne pathogen producing Shiga toxins (Stx1 and Stx2), which can cause hemorrhagic diarrhea and life-threatening infections. O157:H7 strain EDL933 carries prophages CP-933V and BP-933W, which encode Shiga toxin genes (stx1 and stx2, respectively). The aim of this work was to investigate the mechanisms of adaptive resistance of EHEC strain EDL933 to a typically lethal dose of gamma irradiation (1.5 kGy). Adaptive selection through six passages of exposure to 1.5 kGy resulted in the loss of CP-933V and BP-933W prophages from the genome and mutations within three genes: wrbA, rpoA, and Wt_02639 (molY). Three selected EHEC clones that became irradiation adapted to the 1.5-kGy dose (C1, C2, and C3) demonstrated increased resistance to oxidative stress, sensitivity to acid pH, and decreased cytotoxicity to Vero cells. To confirm that loss of prophages plays a role in increased radioresistance, clones C1 and C2 were exposed to bacteriophage-containing lysates. Although phage BP-933W could lysogenize C1, C2, and E. coli K-12 strain MG1655, it was not found to have integrated into the bacterial chromosome in C1-U and C2-U lysogens. Interestingly, for the E. coli K-12 lysogen (K-12-U), BP-933W DNA had integrated at the wrbA gene (K-12-U). Both C1-U and C2-U lysogens regained sensitivity to oxidative stress, were more effectively killed by a 1.5-kGy gamma irradiation dose, and had regained cytotoxicity and acid resistance phenotypes. Further, the K-12-U lysogen became cytotoxic, more sensitive to gamma irradiation and oxidative stress, and slightly more acid resistant. IMPORTANCE: Gamma irradiation of food products can provide an effective means of eliminating bacterial pathogens such as enterohemorrhagic Escherichia coli (EHEC) O157:H7, a significant foodborne pathogen that can cause severe disease due to the production of Stx. To decipher the mechanisms of adaptive resistance of the O157:H7 strain EDL933, we evolved clones of this bacterium resistant to a lethal dose of gamma irradiation by repeatedly exposing bacterial cells to irradiation following a growth restoration over six successive passages. Our findings provide evidence that adaptive selection involved modifications in the bacterial genome, including deletion of the CP-933V and BP-933W prophages. These mutations in EHEC O157:H7 resulted in loss of stx1 and stx2, loss of cytotoxicity to epithelial cells, and decreased resistance to acidity, critical virulence determinants of EHEC, concomitant with increased resistance to lethal irradiation and oxidative stress. These findings demonstrate that the potential adaptation of EHEC to high doses of radiation would involve elimination of the Stx-encoding phages and likely lead to a substantial attenuation of virulence. [ABSTRACT FROM AUTHOR]

The present study aimed to explore for the first time the occurrence and the antimicrobial resistance profiles of E. coli O157:H7 and O55:H7 isolates in camel meat in Egypt. Among the 110 camel meat samples examined using standardized microbiological techniques, 10 (9.1%) and 32 (29.1%) were positive for E. coli O157:H7 and E. coli O55:H7, respectively. In total, 24 isolates were verified as E. coli O157:H7, while 102 isolates were confirmed serologically as E. coli O55:H7. Multiplex PCR revealed the existence of eaeA, stx1, stx2, and EHEC-hlyA among E. coli O157:H7 and O55:H7 isolates (n = 126) at various percentages. According to their resistance against 14 antibiotics, 16.7% and 83.3% of O157:H7 isolates and 8.6% and 76.5% of O55:H7 isolates were classified into extensively drug-resistant and multi-drug-resistant, respectively, whereas 29.4% and 22.2% of E. coli isolates were resistant to cefotaxime and ciprofloxacin, respectively. The study results emphasize that camel meat may be a vehicle for multi- and extensively drug-resistant E. coli O157:H7 and O55:H7 strains, indicating a potential threat to public health. Further studies based on the molecular evidence of the antimicrobial resistance genes and enrolling a larger number of samples are recommended for a better understanding of the antimicrobial resistance phenomenon of camel-meat-originating pathogenic E. coli strains. [ABSTRACT FROM AUTHOR]

Shiga toxin-producing Escherichia coli (STEC) O121 is among the top six nonO157 serogroups that are most frequently associated with severe disease in humans. While O121:H19 is predominant, other O121 serotypes have been frequently isolated from environmental samples, but their virulence repertoire is poorly characterized. Here, we sequenced the complete genomes of two animal isolates belonging to O121:H7 and O121:H10 and performed comparative genomic analysis with O121:H19 to assess their virulence potential. Both O121:H7 and O121:H10 strains carry a genome comparable in size with the O121:H19 genomes and belong to phylogroup B1. However, both strains appear to have evolved from a different lineage than the O121:H19 strains according to the core genes-based phylogeny and Multi Locus Sequence Typing. A systematic search of over 300 E. coli virulence genes listed in the Virulence Factor DataBase revealed a total of 73 and 71 in O121:H7 and O121:H10 strains, respectively, in comparison with an average of 135 in the O121:H19 strains. This variation in the virulence genes repertoire was mainly attributed to the reduction in the number of genes related to the Type III Secretion System in the O121:H7 and O121:H10 strains. Compared to the O121: H19 strains, the O121:H7 strain carries more adherence and toxin genes while the O121:H10 strain carries more genes related to the Type VI Secretion System. Although both O121:H7 and O121:H10 strains carry the large virulence plasmid pEHEC, they do not harbor all pEHEC virulence genes in O121:H19. Furthermore, unlike the O121:H19 strains, neither the O121:H7 nor O121:H10 strain carried the Locus of Enterocyte Effacement, OI-122, nor the tellurite resistance island. Although an incomplete Locus of Adhesion and Autoaggregation (LAA) was identified in the O121:H7 and O121:H10 strains, a limited number of virulence genes were present. Consistently, both O121:H7 and O121:H10 strains displayed significant reduced cytotoxicity than either the O157:H7 strain EDL933 or the O121:H19 strain RM8352. In fact, the O121:H7 strain RM8082 appeared to cause minimal cytotoxicity to Vero cells. Our study demonstrated distinct evolutionary lineages among the strains of serotypes O121:H19, O121:H10, and O121:H7 and suggested reduced virulence potentials in STEC strains of O121:H10 and O121:H7. [ABSTRACT FROM AUTHOR]

Shiga toxins (Stxs), produced by Shigella dysenteriae serotype 1 and certain Escherichia coli pathotypes, cause hemorrhagic colitis, which can progress to hemolytic uremic syndrome (HUS) and central nervous system (CNS) pathology. The underlying mechanisms of toxin-induced inflammation remain unclear. The p38 mitogen-activated protein kinase (MAPK) and its downstream target, MAPKAPK2 (MK2), play key roles in various cellular responses. We identified Tristetraprolin (TTP) as a novel substrate of MK2 in Stx-intoxicated cells. Western blot analysis showed that Stxs induce phosphorylation of MK2 (Thr334) and TTP in globotriaosylceramide (Gb 3 )-positive cells, including D-THP-1 macrophage-like cells and HK-2 renal epithelial cells, but not in Gb 3 -negative T84 colon carcinoma cells. After treatment with wild-type Stx, the activity of phosphorylated MK2 and TTP persists for up to 8 h, while Stx2a mut , which lacks N-glycosidase activity, causes transient MK2/TTP phosphorylation. This suggests that Stxs selectively mediate MK2 and TTP activation in a Gb 3 -dependent manner. Knockdown of TTP in Stx2a-treated D-THP-1 cells upregulates proinflammatory cytokines such as TNF-α, IL-1β, IL-6, IL-8, MCP-1, and MIP-1α. The MK2 inhibitor PF-3644022 significantly reduces TTP phosphorylation and blocks the production of IL-6, IL-8, MCP-1, and MIP-1α in Stx2a-stimulated HK-2 cells. In conclusion, the MK2-TTP signaling pathway regulates the inflammatory response induced by Stxs in toxin-sensitive cells.

Hemolytic uremic syndrome (HUS) is a systemic sequelae from gastrointestinal infection with Shiga toxin (Stx) producing Escherichia coli (STEC) that can result in acute kidney injury, lasting renal disease, and death. Despite a window for intervention between hemorrhagic diarrhea and onset of HUS, no specific therapies exist to prevent or treat HUS following STEC infection. Furthermore, there is no way to predict which patients with STEC will develop HUS or any rapid way to determine which Stx variant is present. To address this, we have broadened the therpay to neutralize additional toxin variants. It contains a multimer of nanobodies derived from camelid heavy chain antibody fragments (VHHs). An improved V HH-based n eutralizing a gent (VNA2) is delivered intramuscularly as RNA combined with LION nanoparticles rather than mRNA, that replicates on administration (repRNA), resulting in a rapidly circulating VNA that can bind systemic toxin. The RNA/VNA2-Stx administered intramuscularly prevents toxicity and death in a mouse model of acute Stx toxicity., Competing Interests: The authors declare no conflict of interest.

Shiga toxin - producing Escherichia coli (STEC) are major foodborne pathogens that result in thousands of hospitalizations each year in the United States. Cattle, the natural reservoir, harbor STEC asymptomatically at the recto-anal junction (RAJ). The molecular mechanisms that allow STEC and non-STEC E. coli to adhere to the RAJ are not fully understood, in part because most adherence studies utilize human cell culture models. To identify a set of bovine-specific E. coli adherence factors, we used the primary RAJ squamous epithelial (RSE) cell-adherence assay to coculture RSE cells from healthy Holstein cattle with diverse E. coli strains from bovine and nonbovine sources. We hypothesized that a comparative genomic analysis of the strains would reveal factors associated with RSE adherence. After performing adherence assays with historical strains from the E. coli Reference Center ( n = 62) and strains newly isolated from the RAJ ( n = 15), we used the bioinformatic tool Roary to create a pangenome of this collection. We classified strains as either low or high adherence and using the Scoary program compiled a list of accessory genes correlated with the "high adherence" strains. While none of the correlations were statistically significant, several gene clusters were associated with the high-adherence phenotype, including two that encode uncharacterized proteins. We also demonstrated that non-STEC E. coli strains from the RAJ are more adherent than other isolates and can outcompete STEC in coculture with RSEs. Further analysis of adherence-associated gene clusters may lead to an improved understanding of the molecular mechanisms of RSE adherence and may help develop probiotics targeting STEC in cattle., Importance: E. coli strains that produce Shiga toxin cause foodborne illness in humans but colonize cattle asymptomatically. The molecular mechanisms that E. coli uses to adhere to cattle cells are largely unknown. Various strategies are used to control E. coli in livestock and limit the risk of outbreaks. These include vaccinating animals against common E. coli strains and supplementing their feed with probiotics to reduce the carriage of pathogens. No strategy is completely effective, and probiotics often fail to colonize the animals. We sought to clarify the genes required for E. coli adherence in cattle by quantifying the attachment to bovine cells in a diverse set of bacteria. We also isolated nonpathogenic E. coli from healthy cows and showed that a representative isolate could outcompete pathogenic strains in cocultures. We propose that the focused study of these strains and their adherence factors will better inform the design of probiotics and vaccines for livestock., Competing Interests: The authors declare no conflict of interest.

Wildlife plays significant roles in the dissemination and zoonotic transmission of pathogens. The enterohaemorrhagic Escherichia coli (EHEC) are associated with complicated cases of food‑borne illnesses. This study investigated the presence of EHEC serogroups (O26, O45, O103, O145, O91, O111, O128, O121 and O157) in wildlife species: cane rats (Thryonomys swinderianus), royal antelope (Neotragus pygmaeus), African giant rats (Cricetomys gambianus) and waterbuck (Kobus ellipsiprymnus). EHEC and non‑EHEC isolates from these wildlife sources were tested for susceptibility to antimicrobial agents. Overall, 127 (83.0 %) out of 153 samples yielded E. coli. Nine (5.9%) samples were positive for EHEC belonging to three serogroups as follows: O26 (n = 2), O111 (n = 2) and O103 (n = 5). The EHEC isolates were from cane rats (n = 6) and royal antelope (n = 3) and possessed virulence‑associated genes stx1 (77.8%), stx2 (100.0%), eaeA (100.0%) and hlyA (100.0%). Overall, 127 E. coli isolates showed resistance to ampicillin (99.2%), ceftiofur (90.6%), tetracycline (90.0%), cephalexin (87.4%), cefotaxime (50.4%), streptomycin 42.5%, ceftazidime (41.7%), nalidixic acid (37.0%), ciprofloxacin (43.6%), amoxicillin/clavulanic acid (32.3%), gentamicin (27.6%), sulphamethoxazole/trimethoprim (25.2%), norfloxacin (17.3%) and chloramphenicol (11.0%). The role of wildlife in the dissemination and transmission of antimicrobial resistant and zoonotic bacteria should not be neglected for effective preventive and control strategies.

Shiga toxin-producing Escherichia coli (STEC) O121 is among the top six non-O157 serogroups that are most frequently associated with severe disease in humans. While O121:H19 is predominant, other O121 serotypes have been frequently isolated from environmental samples, but their virulence repertoire is poorly characterized. Here, we sequenced the complete genomes of two animal isolates belonging to O121:H7 and O121:H10 and performed comparative genomic analysis with O121:H19 to assess their virulence potential. Both O121:H7 and O121:H10 strains carry a genome comparable in size with the O121:H19 genomes and belong to phylogroup B1. However, both strains appear to have evolved from a different lineage than the O121:H19 strains according to the core genes-based phylogeny and Multi Locus Sequence Typing. A systematic search of over 300 E. coli virulence genes listed in the Virulence Factor DataBase revealed a total of 73 and 71 in O121:H7 and O121:H10 strains, respectively, in comparison with an average of 135 in the O121:H19 strains. This variation in the virulence genes repertoire was mainly attributed to the reduction in the number of genes related to the Type III Secretion System in the O121:H7 and O121:H10 strains. Compared to the O121:H19 strains, the O121:H7 strain carries more adherence and toxin genes while the O121:H10 strain carries more genes related to the Type VI Secretion System. Although both O121:H7 and O121:H10 strains carry the large virulence plasmid pEHEC, they do not harbor all pEHEC virulence genes in O121:H19. Furthermore, unlike the O121:H19 strains, neither the O121:H7 nor O121:H10 strain carried the Locus of Enterocyte Effacement, OI-122, nor the tellurite resistance island. Although an incomplete Locus of Adhesion and Autoaggregation (LAA) was identified in the O121:H7 and O121:H10 strains, a limited number of virulence genes were present. Consistently, both O121:H7 and O121:H10 strains displayed significant reduced cytotoxicity than either the O157:H7 strain EDL933 or the O121:H19 strain RM8352. In fact, the O121:H7 strain RM8082 appeared to cause minimal cytotoxicity to Vero cells. Our study demonstrated distinct evolutionary lineages among the strains of serotypes O121:H19, O121:H10, and O121:H7 and suggested reduced virulence potentials in STEC strains of O121:H10 and O121:H7.

Shiga toxin (stx) is the principal virulence factor of the foodborne pathogen, Shiga toxin-producing Escherichia coli (STEC) O157:H7 and is associated with various lambdoid bacterio (phages). A comparative genomic analysis was performed on STEC O157 isolates from cattle (n = 125) and clinical (n = 127) samples to characterize virulence genes, stx-phage insertion sites and antimicrobial resistance genes that may segregate strains circulating in the same geographic region. In silico analyses revealed that O157 isolates harboured the toxin subtypes stx1a and stx2a. Most cattle (76.0%) and clinical (76.4%) isolates carried the virulence gene combination of stx1, stx2, eae and hlyA. Characterization of stx1 and stx2-carrying phages in assembled contigs revealed that they were associated with mlrA and wrbA insertion sites, respectively. In cattle isolates, mlrA and wrbA insertion sites were occupied more often (77% and 79% isolates respectively) than in clinical isolates (38% and 1.6% isolates, respectively). Profiling of antimicrobial resistance genes (ARGs) in the assembled contigs revealed that 8.8% of cattle (11/125) and 8.7% of clinical (11/127) isolates harboured ARGs. Eight antimicrobial resistance genes cassettes (ARCs) were identified in 14 isolates (cattle, n = 8 and clinical, n = 6) with streptomycin (aadA1, aadA2, ant(3")-Ia and aph(3")-Ib) being the most prevalent gene in ARCs. The profound disparity between the cattle and clinical strains in occupancy of the wrbA locus suggests that this trait may serve to differentiate cattle from human clinical STEC O157:H7. These findings are important for stx screening and stx-phage insertion site genotyping as well as monitoring ARGs in isolates from cattle and clinical samples. [ABSTRACT FROM AUTHOR]

Introduction: Shiga toxin-producing Escherichia coli (STEC) O157:H7 is associated with intestinal infection in humans and is considered an important cause of food-borne diseases. The aim of the study was to assess the incidence of E. coli O157:H7 in fecal samples of healthy cattle collected in slaughterhouses (n = 160) and from five farms (n = 100). Methodology: E. coli isolates were detected on MacConkey agar. A total of 236 E. coli isolates were recovered from fecal samples of healthy cattle. We used sorbitol MacConkey medium to detect non-sorbitol fermenting colonies. These bacteria were examined for the presence of O157:H7 antigen by latex agglutination. The isolation of E. coli O157:H7 has been confirmed with PCR amplification of rfbEO157 and fliCH7 specific genes for serogroup O157 and with multiplex PCR of stx1, stx2, eaeA, and ehxA. All isolates were examined for their susceptibility to 21 antibiotics by the disc diffusion method. Results: Of the 236 E. coli isolates, 4.2% (10/236) were positive for STEC O157:H7. Shiga toxin gene (stx2) and ehxA were present in 70% of isolates, stx1 and eae were confirmed in 60% of the isolates. Other virulence factors screened (fimH, sfa/focDE, cdt3, traT, iutA, and hlyA) were present among the 10 isolates. All E. coli O157:H7 isolates were sensitive to amoxicillin/clavulanic acid, cefotaxime, cefepime, aztreonam, colistin, and sulfamethoxazole/trimethoprim. All isolates belong to the phylo-group E. Conclusions: This is the first study of the incidence of E. coli O157:H7 in cattle in Tunisia. Our finding proves the existence of STEC O157:H7 in healthy animals producing food for human consumption which could be a source of food-borne disease. [ABSTRACT FROM AUTHOR]

Shiga toxin-producing E. coli hemolytic-uremic syndrome (STEC-HUS) is characterized by acute renal failure associated with thrombocytopenia and mciroangiopathic hemolytic anemia following exposure to STEC. It may present with spectrum of multi-organ dysfunction syndrome (MODS). Pathogenesis of STEC-HUS is partially understood. Proposed is two sequential molecular pathogeneses. First path is from Shiga toxins to endotheliopathy as a result of organotropism and endothelial heterogeneity. Shiga toxins binding to Gb3-endowed ECs, leads to endotheliopathy in the kidneys and other organs. Also, Shiga toxins downregulating CD59 provoke unprotected activation of complement, in which C5b-9 causes channel formation and promotes additional endotheliopathy. Second path is from endotheliopathy to microthrombogenesis. Endotheliopathy triggers activation of two independent molecular pathways (i.e., inflammatory and microthrombotic), promoting inflammation and formation of “microthrombi” composed of platelet and unusually large von Willebrand factor multimers (ULVWF) complexes. Microthrombogenesis is initiated by the activated platelet and endothelial exocytosis of ULVWF. If ADAMTS13 is insufficient due to excessive production of ULVWF, uncleaved ULVWF are anchored to ECs. ULVWF anchored to Gb3-endowed and CD59-disendowed ECs in situ are long elongated strings and recruit the activated platelets to assemble microthrombi. Microthrombi cause disseminated intravascular microthrombosis (DIT), leading to vascular microthrombotic disease (VMTD). The result is organ phenotype syndromes of MODS such as HUS, encephalopathy, acute respiratory distress syndrome (ARDS), etc. DIT/VMTD triggers thrombotic thrombocytopenic purpura (TTP)-like syndrome. Current understanding of Gb3 and CD59 roles and experience with complement inhibition therapy support the complex molecular pathogenesis of STEC-HUS is due to heterogeneity-associated endotheliopathy, leading to impaired ADAMTS13 function.

Genetic screen technology has been applied to study the mechanism of action of bacterial toxins—a special class of virulence factors that contribute to the pathogenesis caused by bacterial infections. These screens aim to identify host factors that directly or indirectly facilitate toxin intoxication. Additionally, specific properties of certain toxins, such as membrane interaction, retrograde trafficking, and carbohydrate binding, provide robust probes to comprehensively investigate the lipid biosynthesis, membrane vesicle transport, and glycosylation pathways, respectively. This review specifically focuses on recent representative toxin-based genetic screens that have identified new players involved in and provided new insights into fundamental biological pathways, such as glycosphingolipid biosynthesis, protein glycosylation, and membrane vesicle trafficking pathways. Functionally characterizing these newly identified factors not only expands our current understanding of toxin biology but also enables a deeper comprehension of fundamental biological questions. Consequently, it stimulates the development of new therapeutic approaches targeting both bacterial infectious diseases and genetic disorders with defects in these factors and pathways.

The main objective of this study is to determine the PCR quantification levels required for detecting enterohemorrhagic Escherichia coli O157 by examining its most virulent genes: stx1, stx2, and rfbE. The PCR’s sensitivity results show a detection limit between 0,011 ng µL-1 of DNA and 0,033 ng µL-1 of DNA for stx1 and stx2 and of 0,0037 ng µL-1 DNA for rfbE. The lowest cut-off limit ranged between 0,033 ng µL-1 of DNA and 0,099 ng µL-1 of DNA for stx1 and stx2, while for rfbE it is 0,033 ng μL-1 of DNA. The selectivity (inclusivity and exclusivity) and the robustness are 100% for the three genes examined, showing the PCR’s reproducibility for distinguishing E. coli O157 from the other serotypes. It is concluded that conventional PCR is a technique that allows monitoring the presence of E. coli O157 by targeting its most virulent genes (stx1, stx2, and rfbE). [ABSTRACT FROM AUTHOR]