Search Results (17)

Irrigation water can serve as a reservoir and transmission route for pathogenic Escherichia coli, posing a threat to food safety and public health. This study builds upon a previous survey conducted in Hermosillo, Sonora (Mexico), where 445 samples were collected from a local Honeydew melon farm and associated packing facilities. Among the 32 E. coli strains recovered, two strains, A34 and A51, were isolated from irrigation water and selected for further molecular characterization by PCR, due to their high pathogenic potential. Both strains were identified as hybrid aEPEC/STEC pathotypes carrying bfpA and stx1 virulence genes. Adhesion assays in HeLa cells revealed aggregative and diffuse patterns, suggesting enhanced colonization capacity. Phylogenetic analysis classified A34 within group B2 as associated with extraintestinal pathogenicity and antimicrobial resistance, while A51 was unassigned to any known phylogroup. Serotyping revealed somatic antigens shared with Shigella boydii 16, suggesting possible horizontal gene transfer or antigenic convergence. Antibiotic susceptibility testing showed resistance to multiple β-lactam antibiotics, including cephalosporins, linked to the presence of bla_CTX-M-151 and bla_CTX-M-9. Although no plasmid-mediated quinolone resistance genes were detected, resistance may involve efflux pumps or mutations in gyrA and parC. These findings are consistent with previous reports of E. coli adaptability in agricultural environments, suggesting potential genetic adaptability. While our data support the presence of virulence and resistance markers, further studies would be required to demonstrate mechanisms such as horizontal gene transfer or adaptive evolution. Full article

Background/Objective. Toxin-producing strains of Clostridioides difficile (C. diff) are the most commonly identified cause of healthcare-associated infection in the elderly. Risk factors include advanced age, hospitalization, prior or concomitant systemic antibacterial therapy, chemotherapy, and gastrointestinal surgery. Patients with unspecified and new-onset diarrhea with ≥3 unformed stools in 24 h are the target population for C. diff infection (CDI) testing. To present data on the risks of laboratory misdiagnosis in managing CDI. Materials. In two general hospitals, we examined 116 clinical stool specimens from hospitalized patients with acute diarrhea suspected of nosocomial or antibiotic-associated diarrhea (AAD) due to C. diff. Enzyme immunoassay (EIA) tests for the detection of C. diff toxins A (cdtA) and B (cdtB) in stool, automated CLIA assay for the detection of C. diff GDH antigen and qualitative determination of cdtA and B in human feces and anaerobic stool culture were applied for CDI laboratory diagnosis. MALDI-TOF (Bruker) was used to identify the presumptive anaerobic bacterial colonies. The following methods were used as confirmatory diagnostics: the LAMP method for the detection of Salmonella spp. and simultaneous detection of C. jejuni and C. coli, an E. coli Typing RT-PCR detection kit (ETEC, EHEC, STEC, EPEC, and EIEC), API 20E and aerobic stool culture methods. Results. A total of 40 toxigenic strains of C. diff were isolated from all 116 tested diarrheal stool samples, of which 38/40 produced toxin B and 2/40 strains were positive for both cdtA and cdtB. Of the stool samples positive for cdtA (6/50) and/or cdtB (44/50) by EIA, 33 were negative for C. diff culture but positive for the following diarrheal agents: Salmonella enterica subsp. arizonae (1/33, LAMP, culture, API 20E); C. jejuni (2/33, LAMP, culture, MALDI TOF); ETEC O142 (1/33), STEC O145 and O138 (2/33, E. coli RT-PCR detection kit, culture); C. perfringens (2/33, anaerobic culture, MALDI TOF); hypermycotic enterotoxigenic K. pneumonia (2/33) and enterotoxigenic P. mirabilis (2/33, culture; PCR encoding LT-toxin). Two of the sixty-six cdtB-positive samples (2/66) showed a similar misdiagnosis when analyzed using the CLIA method. However, the PCR analysis showed that they were cdtB-negative. In contrast, the LAMP method identified a positive result for C. jejuni in one sample, and another was STEC positive (stx1+/stx2+) by RT-PCR. We found an additional discrepancy in the CDI test results: EPEC O86 (RT-PCR eae+) was isolated from a fecal sample positive for GHA enzyme (CLIA) and negative for cdtA and cdtB (CLIA and PCR). However, the culture of C. diff was negative. These findings support the hypothesis that certain human bacterial pathogens that produce enterotoxins other than C. diff, as well as intestinal commensal microorganisms, including Klebsiella sp. and Proteus sp., contribute to false-positive EIA card tests for C. diff toxins A and B, which are the most widely used laboratory tests for CDI. Conclusions. CDI presents a significant challenge to clinical practice in terms of laboratory diagnostic management. It is recommended that toxin-only EIA tests should not be used as the sole diagnostic tool for CDI but should be limited to detecting toxins A and B. Accurate diagnosis of CDI requires a combination of laboratory diagnostic methods on which proper infection management depends. Full article

Porcine edema disease (ED), which causes enormous economic losses in pig farms, is caused by Shiga toxin type 2e (Stx2e) Escherichia coli (STEC), which frequently occurs in young piglets. In this study, we aimed to express a fused Stx2e peptide on a phage surface to generate an innovative sandwich ELISA for the detection of STEC antigen in field pig farming samples. The amino acid sequences at positions 241–319 were selected for capture antibody (T1D2) production. T1D2 was selected after the third round of biopanning, and it showed a high yield with no major impurities. T1D2-ELISA can detect recombinant modified Stx2e antigen, and the detection limit of the antigen was approximately below 20 pg/mL. The sensitivity of T1D2-ELISA was determined using five different stool samples, with a total of 25 stool samples. Positive Stx2e antigen samples were detected only in one of the 25 samples using T1D2-ELISA. The ELISA values of positive stool samples were >300 pg and <600 pg. In conclusion, we developed an innovative ELISA for the detection of STEC antigens in field pig farming samples. It can also be used to easily detect STEC antigens in porcine stool samples. We anticipate that our novel T1D2-ELISA method will enable the effective monitoring of STEC antigen content during industrial vaccine production. By leveraging this approach, we aimed to enhance production efficiency and ensure high-quality vaccines. Full article

We used whole genome sequencing (WGS) as an epidemiologic surveillance tool to elucidate the transmission dynamics of Shiga toxin-producing Escherichia coli (STEC) strains along the beef production chain in South Africa. Isolates were obtained from a cattle farm, abattoirs and retail outlets. Isolates were analysed using WGS on a MiSeq platform (Illumina, San Diego, CA, USA) and phylogenetic analysis was carried out. Of the 85 isolates, 39% (33) carried the stx gene and 61% (52) had lost the stx gene. The prevalence of stx subtypes was as follows; stx_1a 55% (18/33), stx_1b 52% (17/33), stx_2a 55% (18/33), stx_2b 27% (9/33), stx_2dB 30% (10/33) and stx_2d1A 15% (5/33). Thirty-five different serogenotypes were detected, of which 65% (56) were flagellar H-antigens and 34% (29) were both O-antigens and flagellar H-antigens. We identified 50 different sequence types (STs), and only nine of the isolates were assigned to three different clonal complexes. Core genome phylogenetic analysis revealed genetic relatedness, and isolates clustered mainly according to their STs and serogenotypes regardless of stx subtypes. This study provides evidence of horizontal transmission and recirculation of STEC strains in Gauteng province and demonstrates that every stage of the beef production chain plays a significant role in STEC entry into the food chain. Full article

Shiga toxin-producing Escherichia coli (STEC) poses a significant public health risk due to its zoonotic potential and association with severe human diseases, such as hemorrhagic colitis and hemolytic uremic syndrome. Ruminants are recognized as primary reservoirs for STEC, but swine also contribute to the epidemiology of this pathogen, highlighting the need for effective prevention strategies across species. Notably, a subgroup of STEC that produces Shiga toxin type 2e (Stx2e) causes edema disease (ED) in newborn piglets, economically affecting pig production. This study evaluates the immunogenicity of a chimeric protein-based vaccine candidate against STEC in pregnant sows and the subsequent transfer of immunity to their offspring. This vaccine candidate, which includes chimeric proteins displaying selected epitopes from the proteins Cah, OmpT, and Hes, was previously proven to be immunogenic in pregnant cows. Our analysis revealed a broad diversity of STEC serotypes within swine populations, with the cah and ompT genes being prevalent, validating them as suitable antigens for vaccine development. Although the hes gene was detected less frequently, the presence of at least one of these three genes in a significant proportion of STEC suggests the potential of this vaccine to target a wide range of strains. The vaccination of pregnant sows led to an increase in specific IgG and IgA antibodies against the chimeric proteins, indicating successful immunization. Additionally, our results demonstrated the effective passive transfer of maternal antibodies to piglets, providing them with immediate, albeit temporary, humoral immunity against STEC. These humoral responses demonstrate the immunogenicity of the vaccine candidate and are preliminary indicators of its potential efficacy. However, further research is needed to conclusively evaluate its impact on STEC colonization and shedding. This study highlights the potential of maternal vaccination to protect piglets from ED and contributes to the development of vaccination strategies to reduce the prevalence of STEC in various animal reservoirs. Full article

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. Full article

Edema disease (ED) is a severe and lethal infectious ailment in swine, stemming from Shiga-toxin-producing Escherichia coli (STEC). An efficient, user-friendly, and safe vaccine against ED is urgently required to improve animal welfare and decrease antibiotic consumption. Recombinant attenuated Salmonella vaccines (RASV) administered orally induce both humoral and mucosal immune responses to the immunizing antigen. Their potential for inducing protective immunity against ED is significant through the delivery of STEC antigens. rSC0016 represents an enhanced recombinant attenuated vaccine vector designed for Salmonella enterica serotype Choleraesuis. It combines sopB mutations with a regulated delay system to strike a well-balanced equilibrium between host safety and immunogenicity. We generated recombinant vaccine strains, namely rSC0016 (pS-FedF) and rSC0016 (pS-rStx2eA), and assessed their safety and immunogenicity in vivo. The findings demonstrated that the mouse models immunized with rSC0016 (pS-FedF) and rSC0016 (pS-rStx2eA) generated substantial IgG antibody responses to FedF and rStx2eA, while also provoking robust mucosal and cellular immune responses against both FedF and rStx2eA. The protective impact of rSC0016 (pS-FedF) against Shiga-toxin-producing Escherichia coli surpassed that of rSC0016 (pS-rStx2eA), with percentages of 83.3%. These findings underscore that FedF has greater suitability for vaccine delivery via recombinant attenuated Salmonella vaccines (RASVs). Overall, this study provides a promising candidate vaccine for infection with STEC. Full article

Atypical hemolytic uremic syndrome (HUS) develops as a result of damage to the endothelium of microvasculature vessels by Shiga toxin produced by enterohemorrhagic Escherichia coli (STEC-HUS). STEC-HUS remains the leading cause of acute kidney injury (AKI) in children aged 6 months to 5 years. The pathomorphological essence of the disease is the development of thrombotic microangiopathy (TMA). One of the key causes of TMA is an imbalance in the ADAMTS13–von Willebrand factor (vWF)–platelet system. The goal of the work was to clarify the role of a moderate decrease in ADAMTS13 activity in the pathogenesis of STEC-HUS. The activity of ADAMTS13 was determined in 138 children (4 months–14.7 years) in the acute period of STEC-HUS and the features of the course of the disease in these patients were analyzed. The study revealed a decrease in the activity and concentration of ADAMTS13 in 79.8% and 90.6% of patients, respectively. Measurements of von Willebrand factor antigen content and the activity of von Willebrand factor in the blood plasma of part of these patients were carried out. In 48.6% and 34.4% of cases, there was an increase in the antigen concentration and the activity of the Willebrand factor, respectively. Thrombocytopenia was diagnosed in 97.8% of children. We have demonstrated that moderately reduced ADAMTS13 activity correlates with the risk of severe manifestations of STEC-HUS in children; the rate of developing multiple organ failure, cerebral disorders, pulmonary edema, and acute kidney injury with the need for dialysis increases. It is assumed that reduction in ADAMTS13 activity may serve as a predictor of disease severity. Full article

Shiga-toxin-producing Escherichia coli (STEC) is typically detected on food products mainly due to cross-contamination with faecal matter. The serotype O157:H7 has been of major public health concern due to the severity of illness caused, prevalence, and management. In the food chain, the main methods of controlling contamination by foodborne pathogens often involve the application of antimicrobial agents, which are now becoming less efficient. There is a growing need for the development of new approaches to combat these pathogens, especially those that harbour antimicrobial resistant and virulent determinants. Strategies to also limit their presence on food contact surfaces and food matrices are needed to prevent their transmission. Recent studies have revealed that bacteriophages are useful non-antibiotic options for biocontrol of E. coli O157:H7 in both animals and humans. Phage biocontrol can significantly reduce E. coli O157:H7, thereby improving food safety. However, before being certified as potential biocontrol agents, the safety of the phage candidates must be resolved to satisfy regulatory standards, particularly regarding phage resistance, antigenic properties, and toxigenic properties. In this review, we provide a general description of the main virulence elements of E. coli O157:H7 and present detailed reports that support the proposals that phages infecting E. coli O157:H7 are potential biocontrol agents. This paper also outlines the mechanism of E. coli O157:H7 resistance to phages and the safety concerns associated with the use of phages as a biocontrol. Full article

The origin of human and calf infections by Shigatoxigenic (STEC) and enteropathogenic (EPEC) Escherichia coli O80:H2 is still unknown. The aim of this study was to identify E. coli O80 in healthy cattle with an emphasis on melibiose non-fermenting E. coli O80:H2. Faecal materials collected from 149 bulls at 1 slaughterhouse and 194 cows on 9 farms were tested with O80 antigen-encoding gene PCR after overnight growth in enrichment broths. The 53 O80 PCR-positive broths were streaked on different (semi-)selective agar plates. Five E. coli colonies from 3 bulls and 11 from 2 cows tested positive with the O80 PCR, but no melibiose non-fermenting E. coli was isolated. However, these 16 E. coli O80 were negative with PCR targeting the fliC_H2, eae, stx1, stx2 and hlyF genes and were identified by WGS to serotypes and sequence types O80:H6/ST8619 and O80:H45/ST4175. They were phylogenetically related to E. coli O80:H6 and O80:H45 isolated from different animal species in different countries, respectively, but neither to STEC and EPEC O80:H2/ST301, nor to other serotypes of the clonal complex 165. As a conclusion, healthy adult cattle were not identified as a source of contamination of humans and calves by STEC or EPEC O80:H2. Full article

Background: Shiga toxin-producing Escherichia coli (STEC) O157:H7 and O104:H4 strains are important causative agents of food-borne diseases such as hemorrhagic colitis and hemolytic–uremic syndrome, which is the leading cause of kidney failure and death in children under 5 years as well as in the elderly. Methods: the native E. coli O157:H7 and O104:H4 lipopolysaccharides (LPS) were partially deacylated under alkaline conditions to obtain apyrogenic S-LPS with domination of tri-acylated lipid A species—Ac₃-S-LPS. Results: intraperitoneal immunization of BALB/c mice with Ac₃-S-LPS antigens from E. coli O157:H7 and O104:H4 or combination thereof (di-vaccine) at single doses ranging from 25 to 250 µg induced high titers of serum O-specific IgG (mainly IgG1), protected animals against intraperitoneal challenge with lethal doses of homologous STEC strains (60–100% survival rate) and reduced the E. coli O157:H7 and O104:H4 intestinal colonization under an in vivo murine model (6–8-fold for monovalent Ac₃-S-LPS and 10-fold for di-vaccine). Conclusions: Di-vaccine induced both systemic and intestinal anti-colonization immunity in mice simultaneously against two highly virulent human STEC strains. The possibility of creating a multivalent STEC vaccine based on safe Ac₃-S-LPS seems to be especially promising due to a vast serotype diversity of pathogenic E. coli. Full article

The serogroup O55 of E. coli is composed of strains whose mechanisms of virulence are different from each other. Since the O55 polysaccharides are present in all E. coli O55 strains, and so are the polymers that compose the capsule of O55 atypical enteropathogenic E. coli (aEPEC), it was investigated whether anti-O55 antibodies were able to help the innate immune system to eliminate capsulated aEPEC and Shiga toxin-producing E. coli (STEC) belonging to the serogroup O55. The results demonstrate that the capsule of EPEC was able to inhibit the deposition of C3b on the bacterial surface and, as a consequence, their lysis by the alternative pathway of the complement system. However, in the presence of antibodies, the ability of the complement to lyse these pathogens was restored. It was also observed that macrophages were able to ingest EPEC and STEC, but they were only able to kill the ingested pathogens in the presence of antibodies. Anti-O55 antibodies were also able to inhibit aEPEC and STEC O55 adherence to human epithelial cells. In summary, the results demonstrated that the O55 polysaccharides have the potential to induce an effective humoral immune response against STEC and EPEC, indicating that they are good antigen targets to be used in vaccine formulations against these pathogens. Full article

Even if serotyping based on O antigens is still routinely used by most laboratories for the detection of diarrheagenic Escherichia coli, this method can provide false-positive reactions, due to the high diversity of O antigens. Molecular methods represent a valuable tool that clarifies these situations. In the Bacteriology Laboratory of Mureș County Hospital, between May 2016 and July 2019, 160 diarrheagenic E. coli strains were isolated from children under 2 years old with diarrheic disease. The strains were identified as Shiga toxin-producing E. coli (STEC)/enteropathogenic Escherichia coli (EPEC) via agglutination with polyvalent sera. STEC strains were serotyped using monovalent sera for serogroup O157. Simplex PCR was performed on the strains to determine the presence of the hlyA gene, and, for the positive ones, the hemolytic activity was tested. Antibiotic susceptibility of the identified diarrheagenic E. coli strains was also investigated. STEC strains were the most frequently identified (49.1%), followed by EPEC (40.2%). The hlyA gene was identified in 12 cases, representing 18.2% of the STEC strains. Even if the extended-spectrum β-lactamase (ESBL)-producing strains represented only 10%, a relevant percentage of multidrug-resistant (MDR) strains (24%) was identified. Full article

Shiga toxin-producing Escherichia coli (STEC) causes outbreaks and sporadic cases of gastroenteritis. STEC O157:H7 is the most clinically relevant serotype in the world. The major virulence determinants of STEC O157:H7 are the Shiga toxins and the locus of enterocyte effacement. However, several accessory virulence factors, mainly outer membrane proteins (OMPs) that interact with the host cells may contribute to the virulence of this pathogen. Previously, the elongation factor thermo unstable (EF-Tu), l-asparaginase II and OmpT proteins were identified as antigens in OMP extracts of STEC. The known subcellular location of EF-Tu and l-asparaginase II are the cytoplasm and periplasm, respectively. Therefore, we investigate whether these two proteins may localize on the surface of STEC and, if so, what roles they have at this site. On the other hand, the OmpT protein, a well characterized protease, has been described as participating in the adhesion of extraintestinal pathogenic E. coli strains. Thus, we investigate whether OmpT has this role in STEC. Our results show that the EF-Tu and l-asparaginase II are secreted by O157:H7 and may also localize on the surface of this bacterium. EF-Tu was identified in outer membrane vesicles (OMVs), suggesting it as a possible export mechanism for this protein. Notably, we found that l-asparaginase II secreted by O157:H7 inhibits T-lymphocyte proliferation, but the role of EF-Tu at the surface of this bacterium remains to be elucidated. In the case of OmpT, we show its participation in the adhesion of O157:H7 to human epithelial cells. Thus, this study extends the knowledge of the pathogenic mechanisms of STEC. Full article

Non-O157 Shiga toxin-producing Escherichia coli (STEC) E. coli are emerging pathotypes that are frequently associated with diseases in humans around the world. The consequences of these serogroups for public health is a concern given the lack of effective prevention and treatment measures. In this study, ten bacteriophages (phages; SA20RB, SA79RD, SA126VB, SA30RD, SA32RD, SA35RD, SA21RB, SA80RD, SA12KD and SA91KD) isolated from cattle faeces collected in the North-West of South Africa were characterized. Activity of these phages against non-O157 STEC isolates served as hosts for these phages. All of the phages except SA80RD displayed lytic against non-O157 E. coli isolates. Of 22 non-O157 E. coli isolates, 14 were sensitive to 9 of the 10 phages tested. Phage SA35RD was able to lyse 13 isolates representing a diverse group of non-O157 E. coli serotypes including a novel O-antigen Shiga toxigenic (wzx-Onovel5:H19) strain. However, non-O157 E. coli serotypes O76:H34, O99:H9, O129:H23 and O136:H30 were insensitive to all phages. Based on transmission electron microscopy, the non-O157 STEC phages were placed into Myoviridae (n = 5) and Siphoviridae (n = 5). Genome of the phage ranged from 44 to 184.3 kb. All but three phages (SA91KD, SA80RD and SA126VB) were insensitive to EcoRI-HF and HindIII nucleases. This is the first study illustrating that cattle from North-West South Africa harbour phages with lytic potentials that could potentially be exploited for biocontrol against a diverse group of non-O157 STEC isolated from the same region. Full article