Stx2 Genes Research Articles

Extended-spectrum β-lactamase-producing E. coli from retail meat and workers: genetic diversity, virulotyping, pathotyping and the antimicrobial effect of silver nanoparticles

BackgroundThe spread of extended-spectrum β-lactamases (ESBL) producing E. coli from food animals and the environment to humans has become a significant public health concern. The objectives of this study were to determine the occurrence, pathotypes, virulotypes, genotypes, and antimicrobial resistance patterns of ESBL-producing E. coli in retail meat samples and workers in retail meat shops in Egypt and to evaluate the bactericidal efficacy of silver nanoparticles (AgNPs-H2O2) against multidrug resistant (MDR) ESBL-producing E. coli.ResultsA total of 250 retail meat samples and 100 human worker samples (hand swabs and stool) were examined for the presence of ESBL- producing E. coli. Duck meat and workers’ hand swabs were the highest proportion of ESBL- producing E. coli isolates (81.1%), followed by camel meat (61.5%). Pathotyping revealed that the isolates belonged to groups A and B1. Virulotyping showed that the most prevalent virulence gene was Shiga toxin 2 (stx2) associated gene (36.9%), while none of the isolates harbored stx1 gene. Genotyping of the identified isolates from human and meat sources by REP-PCR showed 100% similarity within the same cluster between human and meat isolates. All isolates were classified as MDR with an average multiple antibiotic resistance (MAR) index of 0.7. AgNPs-H2O2 at concentrations of 0.625, 1.25, 2.5 and 5 μg/mL showed complete bacterial growth inhibition.ConclusionsVirulent MDR ESBL-producing E. coli were identified in retail meat products in Egypt, posing significant public health threats. Regular monitoring of ESBL-producing E. coli frequency and antimicrobial resistance profile in retail meat products is crucial to enhance their safety. AgNPs-H2O2 is a promising alternative for treating MDR ESBL-producing E. coli infections and reducing antimicrobial resistance risks.

Escherichia coli isolates from meat and abattoirs environment in Egypt: molecular characterization and control by nanosilver particles

ABSTRACT Three hundred samples, including meat from the slaughtered carcass and water, air samples, and swabs from the floor, wall, and employees' hands, were collected from five municipal abattoirs spread across several Egyptian provinces. The Escherichia coli was isolated from floor swabs, meat, air, wall, hand, and water samples. Serotyping of the recovered isolates clarified the presence of various serotypes, including enterohemorrhagic serotypes (O111: H4, O128: H2, and O127: H6) and enterotoxigenic serotypes (O44: H18 and O125: H21). The isolates were resistant to cefotaxime (100%), amoxiclav (80%), then rifampin (66.7%). The stx1 gene, stx2 gene, eaeA gene, blaCMY2 gene and iss gene were detected in 10-80 % of the isolates. Nanosilver (AgNPs) showed that 12.5 ppm was the lowest concentration that prevented bacterial growth. It was observed that 12% of workers wore a clean white coat, only 24% washed their hands between activities during work, only 14% used soap for hand washing, and 42% utilized the same knife for meat and its offal.

Detection of Virulence Genes Stx1, Stx2, eae A and hly A in E.coli Isolated from Local Minced Meat

The current study included the investigation of E. coli bacteria in random samples of ground meat, the detection of virulence genes in isolates, and the study of isolates resistance to some widely used antibiotics. Twenty-four samples of minced meat were collected to isolate and identify E. coli bacteria by phenotypic, microscopic characteristics and traditional biochemical tests were done. Antibiotic susceptibility tests, genomes of isolates were extracted, the virulence factors Stx1, Stx2, eae A and hly A were investigated by multiplex PCR technique. 7 isolates showed Colonies with a metallic green luster on EMB medium, gram negative, rod shaped bacteria, facultative anaerobic bacteria, positive for indole, methyl red and catalase tests, and negative for Fox Proscore, urease and oxidase tests. All isolates were containing hly A gene, 6 isolates were containing the virulence gene Stx1, 5 isolates containing were eae A gene and 4 isolates were containing Stx2 gene. The results shown a high resistance of E. coli isolates to all types of antibiotics used, as all isolates were possessed multiple antibiotic resistance.

A propidium monoazide-polymerase spiral reaction (PMA-PSR) designed for direct detection of Escherichia coli O157:H7 viable cell

Escherichia coli O157:H7 is a major foodborne pathogen causing food poisoning. Standard detection method for foodborne E. coli is plate counting, which is time consuming and omits VBNC cells. PSR is an isothermal amplification technique identifying trace amount DNA. However, its effectiveness is influenced by residual DNA from dead cells. This study aimed at designing a PMA-PSR system for accurate determination of viable cells of E. coli O157:H7. Firstly, PSR was optimized in fluorescence indicator, betaine concentration and reaction time. The optimized system could successfully identify rfbE, stx1 and stx2 genes within 45 min at 65 °C. Secondly, sensitivity and specificity were tested in pure culture and different food samples. With 100% specificity, PSR could determine DNA amount as low as 1.12 pg/μL. Thirdly, a PMA-PSR system was designed for accurate determination of viable cells. Cells were treated with 5 μg/mL PMA to remove residual DNA from dead cells. Pretreated cells were adapted to PSR, thus viable cells were specifically detected. Fourthly, the system was applied in food samples with VBNC cells. Viable cells could be accurately determined in accordance with regular assay. In conclusion, the PMA-PSR system was an efficient tool for accurate determination of E. coli O157:H7 viable cells.

Study of Zoonotic Pathogens in Alien Population of Veiled Chameleons (Chamaeleo calyptratus) in the Canary Islands (Spain).

Veiled chameleons (Chamaeleo calyptratus) are native to the Arabian Peninsula that have been introduced as pets in many regions around the world, such as the Canary Islands (Spain). In this work, the gastrointestinal content from veiled chameleons of Gran Canaria island (Canary Islands) has been analyzed to determine the presence of zoonotic bacteria. Forty animals were analyzed using different selective culture media and PCR. The most isolated bacteria were Yersinia enterocolitica (52.4%), followed by Salmonella spp. (40.0%), with positive isolates for Salmonella Tyhpi and Salmonella Typhimurium. Pseudomonas spp. was found in 32.5% of the chameleons. More than half were positive for Pseudomonas aeruginosa. Antibiotic-resistant Staphylococcus spp. was detected in six animals plus one isolate of non-resistant Staphylococcus hominis. Multiple mycobacteria species belonging to both tuberculous and non-tuberculous complexes were identified as well as Escherichia coli carrying the stx1 and eae virulence genes with 12.5% and 7.5% prevalence, respectively. Listeria monocytogenes, Campylobacter spp., and Vibrio spp. were found in lower proportion (<5%). The results obtained indicate that veiled chameleons in Gran Canaria could be playing a role in the maintenance and dissemination of the pathogens detected, harming public health and biodiversity.

Comparison of Two Shiga Toxin-producing Escherichia coli (STEC) Isolation Protocols in Raw Cow’s Milk Cheese Enrichment Broths: Direct STEC Isolation Versus Techniques Based on Immuno-concentration

The presence of Shiga toxin-producing Escherichia coli (STEC) in dairy products made with raw milk is a major concern for food safety authorities and industries. Two approaches have been proposed to isolate STEC from food. In the IC-Protocol (immuno-concentration protocol), specific serogroups are identified in the enrichment broth after the detection of the stx and eae genes. An immuno-concentration of the targeted serogroups is performed before isolating them on specific media. In the DI-Protocol (direct isolation protocol), a direct isolation of all STEC present in the enrichment broth is carried out after the detection of stx genes. We compared the ability of these two methods to isolate STEC O26:H11, O103:H2, O111:H8, O145:H28, and O157:H7 after artificial inoculation in four different raw milk cheeses. Across all serogroups and cheese types, STEC were isolated in 83.3% of samples when using the IC-Protocol but only 53.3% of samples with the DI-Protocol. For two cheese types, the DI-Protocol failed to isolate STEC O157:H7 strains altogether. Our results suggest that IC-Protocol is a robust methodology to effectively isolate STEC across a range of cheese types.

Hetero-Pathogenic O181:H4 EAHEC Strain of Sequence Type ST678 Associated with Hemolytic-Uremic Syndrome in Schoolchildren in Russia.

In the last decade, the importance of hetero-pathogenic enteroaggregative Shiga-toxin-producing E. coli for public health has increased. Recently, we described the genetic background of the EAHEC O181:H4 strain of ST678 carrying the stx2 gene in prophage and five plasmids, including the plasmid-carrying aggR and aaiC genes. Here, we present the morphological and enzymatic characteristics of this strain, as well as susceptibility to antimicrobials, biofilm formation, etc. Methods: Bacterial morphology was studied using an electron microscope. Susceptibility to antimicrobials was determined using the microdilution method. Cytotoxicity was estimated in Vero cells. Virulence was studied on mice. The morphological and enzymatic properties of the hetero-pathogenic EAHEC strain were typical for E. coli; electron microscopy revealed the specific flagella. The strain was susceptible to most antibiotics and disinfectants but resistant to ampicillin and ciprofloxacin and showed a high degree of biofilm formation. Cytotoxicity towards Vero cells was estimated as 80%. The emergence of a new O181:H4 EAHEC strain poses a potential threat to humans because of the virulence potential that must be taken into account in the epidemiological analysis of outbreaks and sporadic cases of foodborne infections associated with hemolytic-uremic syndrome.

Genetic diversity of Escherichia coli harboring virulence gene Stx1 and Stx2 isolated from common carp fish in Nineveh Governorate using ERIC-PCR

Genetic diversity of Escherichia coli harboring virulence gene Stx1 and Stx2 isolated from common carp fish in Nineveh Governorate using ERIC-PCR

Detection and Characterization of hly,cdtI and stxI genes, in MDR uropathogenic and intestinal E.coli isolates, Hilla city, Iraq.

Background:. Escherichia coli infections represent a considerable burden in human. Illness caused by E. coli have a significant burden on patients and the healthcare system, so prompt recognition, and appropriate treatment are necessary. Escherichia coli is the first bacterial pathogen in humans responsible for deaths associated with antibiotic resistance&#x0D; Aim of study: detection of hly, cdtI and stxI genes through molecular analysis in MDR E.coli isolates.&#x0D; Methods: From November 2021 to May 2022, atotal of 150 urine samples were collected from patients suffering from urinary tract infections were admitted and visit Maternity and Children Babylon Hospital in Al-Hilla city. 100 stool samples were (as control). The identification and antibiotic susceptibility profile of E.coli were done by vitek2 compact system.The isolates were subjected to PCR assays with specific primers for hly , cdtI and stxI .&#x0D; Results: Only 50(45.5%) E.coli isolates were recovered from the urine samples. In contrast, 60(60%) E.coli isolates from the stool samples. A high rate of MDR was detected among E. coli isolates, where it was( 60%) and (53.3%) in the uropathogenic and intestinal isolates, respectively.&#x0D; The PCR results were showed that MDR uropathogenic E. coli and intestinal E.coli were hardbourd hly gene (46.7%)and (90.6%), respectively. The cdtI gene was detected in (43.4%) of uropathogenic E. coli, and (40.6%) of intestinal E.coli. The stx gene was detected in (16.7%) of uropathogenic E. coli, and (43.7%) of intestinal E.coli.&#x0D; Conclusion: The presence of a high percentage of toxin in intestinal E.coli is a serious warning because of the possibility of their becoming virulent if the bacteria change their natural habitat or are exposed to a certain stress.

Shiga Toxin-Producing Escherichia coli (STEC) Associated with Calf Mortality in Uruguay.

In Uruguay, the mortality of dairy calves due to infectious diseases is high. Escherichia coli is a natural inhabitant of the intestinal microbiota, but can cause several infections. The aim of the work was to characterize E. coli isolates from intestinal and extraintestinal origin of dead newborn calves. Using PCR, virulence gene characteristics of pathogenic E. coli were searched. The pathogenic E. coli were molecularly characterized and the phylogroup, serogroup and the Stx subtype were determined. Antibiotic susceptibility was determined using the Kirby-Bauer disk diffusion method and plasmid-mediated quinolone resistance (PMQR) genes with PCR. Finally, clonal relationships were inferred using PFGE. Gene characteristics of the Shiga toxin-producing E. coli (STEC), Enteropathogenic E. coli (EPEC) and Necrotoxigenic E. coli (NTEC) were identified. The prevalence of the iucD, afa8E, f17, papC, stx1, eae and ehxA genes was high and no f5, f41, saa, sfaDE, cdtIV, lt, sta or stx2 were detected. The prevalence of STEC gene stx1 in the dead calves stood out and was higher compared with previous studies conducted in live calves, and STEC LEE+ (Enterohemorrhagic E. coli (EHEC)) isolates with stx1/eae/ehxA genotypes were more frequently identified in the intestinal than in the extraintestinal environment. E. coli isolates were assigned to phylogroups A, B1, D and E, and some belonged to the O111 serogroup. stx1a and stx1c subtypes were determined in STEC. A high prevalence of multi-resistance among STEC and qnrB genes was determined. The PFGE showed a high diversity of pathogenic strains with similar genetic profiles. It can be speculated that EHEC (stx1/eae/ehxA) could play an important role in mortality. The afa8E, f17G1 and papC genes could also have a role in calf mortality. Multidrug resistance defies disease treatment and increases the risk of death, while the potential transmissibility of genes to other species constitutes a threat to public health.

Rapid detection of Shiga-toxin-producing Escherichia coli O157:H7 based on a colorimetric loop-mediated isothermal amplification (cLAMP) assay using a molecular beacon paired with HRPzyme.

Contamination by Escherichia coli O157:H7 is considered a threat in the livestock and food industries. Therefore, it is necessary to develop methods for the convenient and rapid detection of Shiga-toxin-producing E. coli O157:H7. This study aimed to develop a colorimetric loop-mediated isothermal amplification (cLAMP) assay using a molecular beacon to rapidly detect E. coli O157:H7. Primers and a molecular beacon were designed for targeting the Shiga-toxin-producing virulence genes (stx1 and stx2) as molecular markers. Additionally, Bst polymerase concentration and amplification conditions for bacterial detection were optimized. The sensitivity and specificity of the assay were also investigated and validated on artificially tainted (100-104 CFU/g) Korean beef samples. The cLAMP assay could detect 1 × 101 CFU/g at 65 °C for both genes, and the assay was confirmed to be specific for E. coli O157:H7. The cLAMP takes about an hour and does not require expensive devices (e.g., thermal cycler and detector). Hence, the cLAMP assay proposed herein can be used in the meat industry as a fast and simple way to detect E. coli O157:H7.

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.

Investigation of virulence factors of shiga toxin-producing Escherichia coli strains isolated from sheep

All over the world, Escherichia coli (STEC), which produces Shiga toxin, is recognized as an important zoonotic pathogen. Ruminants, particularly cattle, are their main reservoirs, but the role of small ruminants in the epidemiology of human infections has not been extensively evaluated in many countries. In this study, it was aimed to determine the Shiga toxin subtypes of Stx1, Stx2, eae and ehxa genes of Shiga toxin producing isolates within Escherichia coli strains in sheep and to determine the comparative effect of variables such as race and sex on them. In this study, virulence genes of STEC strains and Shiga toxin subtypes were identified in 215 E. coli isolates isolated from 272 rectal swab from 12 different sheep herds in and around Ankara. Of the 215 isolates, only 19 (8.84%) Stx1, 3 (1.4%) Stx2, 5 (2.33%) eae and 9 (4.19%) ehxa genes, 13 (6%, 05) Stx1-Stx2-ehxa, 5 (2.33%) Stx1-eae -ehxa, 3 (1.4%) Stx2-eae-ehxa together and 1 (0.47%) Stx1- eae, 30 (13.95%) Stx1- ehxa, 1 (0.47%) Stx2- eae, 15 (6.98%) Stx2-ehxa and 10 (4.65%) eae – ehxa were detected together. Of the 101 (46.98%) isolates, none of the Stx1, Stx2, eae and ehxa genes were detected. In stx1 subtyping in sheep, a high rate (82.35%) of stx1c was isolated. In stx2 subtyping stx2f (%62,86), stx2c (%54,29), stx2b (%45,71), stx2d (%22,86), stx2h (%22,86) subtypes are more common in different combinations. Detected at a high rate. These data indicate the presence of STEC in sheep and their potential to cause serious infection in humans

L-Arabinose inhibits Shiga toxin type 2-converting bacteriophage induction in Escherichia coli O157:H7

ABSTRACT The pathogenicity of Escherichia coli (E. coli) O157:H7 is predominantly associated with Shiga toxin 2 (Stx2) that poses a huge threat to human and animal intestinal health. Production of Stx2 requires expression of stx2 gene, which is located in the genome of lambdoid Stx2 prophage. Growing evidence has implicated that many commonly consumed foods participate in the regulation of prophage induction. In this study, we aimed to explore whether specific dietary functional sugars could inhibit Stx2 prophage induction in E. coli O157:H7, thereby preventing Stx2 production and promoting intestinal health. We demonstrated that Stx2 prophage induction in E. coli O157:H7 was strongly inhibited by L-arabinose both in vitro and in a mouse model. Mechanistically, L-arabinose at doses of 9, 12, or 15 mM diminished RecA protein levels, a master mediator of the SOS response, contributing to reduced Stx2-converting phage induction. L-Arabinose inhibited quorum sensing and oxidative stress response, which are known as positive regulators of the SOS response and subsequent Stx2 phage production. Furthermore, L-arabinose impaired E. coli O157:H7 arginine transport and metabolism that were involved in producing Stx2 phage. Collectively, our results suggest that L-arabinose may be exploited as a novel Stx2 prophage induction inhibitor against E. coli O157:H7 infection.

Evaluation of Larger Test Portion Sizes for Escherichia coli Shiga Toxin Producer (STEC) on the Detection by Immunomagnetic Separation and Real-Time PCR in Meat and Vegetables

The aims of this study were (i) to evaluate the performance of the Assurance® GDS method combining immunomagnetic separation and real-time PCR for STEC detection in pooled samples (up to 375g) of vegetables and meat, and (ii) to compare its performances to that of the reference method ISO/TS-13136:2012 (25 g sample size) in artificially contaminated samples. The alternative Assurance® GDS method includes enrichment in proprietary broth at 41.5°C for 10 h followed by primary screening of TOP 7 STEC containing eae, stx genes and O157:H7 markers using MPX-Top 7 (IMS+PCR), secondary screening for serogroup identification using MPX-ID/EHEC-ID (IMS+PCR). And finally, cultural confirmation from same IMS beads on two selective agars is set up. For reference method, the enrichment was performed in BPW at 41.5°C for 18 h. A total of 120 samples of meat and vegetables, including 20 uninoculated and 100 samples spiked with stressed cells (<12 CFU/25 or 375g) of STEC, were analyzed using both methods. Our results showed that the Assurance® GDS method LOD50 ranged from 0.12 CFU/375 to 0.522 CFU/375 g and the LOD95 from 0.518 CFU/375 to 2.257 CFU/375 g. These data are similar to the LOD50 data of the reference method.

The role of PhoP/PhoQ system in regulating stress adaptation response in Escherichia coli O157:H7.

The development of acid tolerance response (ATR) as a result of low pH in Escherichia coli O157:H7 (E. coli O157:H7) contaminating beef during processing is considered a major food safety concern. Thus, in order to explore the formation and molecular mechanisms of the tolerance response of E. coli O157:H7 in a simulated beef processing environment, the resistance of a wild-type (WT) strain and its corresponding ΔphoP mutant to acid, heat, and osmotic pressure was evaluated. Strains were pre-adapted under different conditions of pH (5.4 and 7.0), temperature (37°C and 10°C), and culture medium (meat extract and Luria-Bertani broth media). In addition, the expression of genes related to stress response and virulence was also investigated among WT and ΔphoP strains under the tested conditions. Pre-acid adaptation increased the resistance of E. coli O157:H7 to acid and heat treatment while resistance to osmotic pressure decreased. Moreover, acid adaptation in meat extract medium simulating slaughter environment increased ATR, whereas pre-adaptation at 10°C reduced the ATR. Furthermore, it was shown that mildly acidic conditions (pH=5.4) and the PhoP/PhoQ two-component system (TCS) acted synergistically to enhance acid and heat tolerance in E. coli O157:H7. Additionally, the expression of genes related to arginine and lysine metabolism, heat shock, and invasiveness was up-regulated, which revealed that the mechanism of acid resistance and cross-protection under mildly acidic conditions was mediated by the PhoP/PhoQ TCS. Both acid adaptation and phoP gene knockout reduced the relative expression of stx1 and stx2 genes which were considered as critical pathogenic factors. Collectively, the current findings indicated that ATR could occur in E. coli O157:H7 during beef processing. Thus, there is an increased food safety risk due to the persistence of tolerance response in the following processing conditions. The present study provides a more comprehensive basis for the effective application of hurdle technology in beef processing.

Development of Recombinase Aided Amplification (RAA)-Exo-Probe Assay for the Rapid Detection of Shiga Toxin-Producing Escherichia coli.

Shiga toxin-producing Escherichia coli (STEC) is a significant cause of foodborne illness causing various gastrointestinal diseases including hemolytic uremic syndrome (HUS), the most severe form, which can lead to kidney failure or even death. Here, we report the development of recombinase aided amplification (RAA)-exo-probe assays targeting the stx1 and stx2 genes for the rapid detection of STEC in food samples. Primers and exo-probes were designed and optimized for the detection of stx1 and stx2 using RAA technology. The optimal STEC RAA-exo-probe assays were then tested for specificity and sensitivity, and validated in both spiked and real food samples. These assays were found to be 100% specific to STEC strains and were also highly sensitive with a detection limit of 1.6 × 103 CFU/mL or 32 copies/reaction. Importantly, the assays were able to successfully detect STEC in spiked and real food samples (beef, mutton, and pork), with a detection limit as low as 0.35 CFU/25g in beef samples after an overnight enrichment step. Overall, the RAA assay reactions completed within ∼20 min and were less dependent on expensive equipment, suggesting they can be easily adopted for in-field testing requiring only a fluorescent reader. As such, we have developed two rapid, sensitive, and specific assays that can be used for the routine monitoring of STEC contamination in food samples, particularly in the field or in poorly equipped labs.

Detection of multidrug-resistant Shiga toxin-producing Escherichia coli in some food products and cattle faeces in Al-Sharkia, Egypt: one health menace

BackgroundShiga toxin-producing Escherichia coli (STEC) is a zoonotic pathogen, that is transmitted from a variety of animals, especially cattle to humans via contaminated food, water, feaces or contact with infected environment or animals. The ability of STEC strains to cause gastrointestinal complications in human is due to the production of Shiga toxins (sxt). However, the transmission of multidrug-resistance STEC strains are linked with a severity of disease outcomes and horizontal spread of resistance genes in other pathogens. The result of this has emerged as a significant threat to public health, animal health, food safety, and the environment. Therefore, the purpose of this study is to investigate the antibiogram profile of enteric E. coli O157 isolated from food products and cattle faeces samples in Zagazig City, Al-Sharkia, Egypt, and to reveal the presence of Shiga toxin genes stx1 and stx2 as virulence factors in multidrug-resistant isolates. In addition to this, the partial 16S rRNA sequencing was used for the identification and genetic recoding of the obtained STEC isolates.ResultsThere was a total of sixty-five samples collected from different geographical regions at Zagazig City, Al-Sharkia—Egypt, which were divided into: 15 chicken meat (C), 10 luncheon (L), 10 hamburgers (H), and 30 cattle faeces (CF). From the sixty-five samples, only 10 samples (one from H, and 9 from CF) were identified as suspicious E. coli O157 with colourless colonies on sorbitol MacConkey agar media with Cefixime- Telurite supplement at the last step of most probable number (MPN) technique. Eight isolates (all from CF) were identified as multidrug-resistant (MDR) as they showed resistance to three antibiotics with multiple antibiotic resistance (MAR) index ≥ 0.23, which were assessed by standard Kirby-Bauer disc diffusion method. These eight isolates demonstrated complete resistance (100%) against amoxicillin/clavulanic acid, and high frequencies of resistance (90%, 70%, 60%,60%, and 40%) against cefoxitin, polymixin, erythromycin, ceftazidime, and piperacillin, respectively. Those eight MDR E. coli O157 underwent serological assay to confirm their serotype. Only two isolates (CF8, and CF13), both from CF, were showed strong agglutination with antisera O157 and H7, as well as resistance against 8 out of 13 of the used antibiotics with the highest MAR index (0.62). The presence of virulence genes Shiga toxins (stx1 and stx2) was assessed by PCR technique. CF8 was confirmed for carrying stx2, while CF13 was carrying both genes stx1, and stx2. Both isolates were identified by partial molecular 16S rRNA sequencing and have an accession number (Acc. No.) of LC666912, and LC666913 on gene bank. Phylogenetic analysis showed that CF8, and CF13 were highly homologous (98%) to E. coli H7 strain, and (100%) to E. coli DH7, respectively.ConclusionThe results of this study provides evidence for the occurrence of E. coli O157:H7 that carries Shiga toxins stx1 and/or stx2, with a high frequency of resistance to antibiotics commonly used in human and veterinary medicine, in Zagazig City, Al-Sharkia, Egypt. This has a high extent of public health risk posed by animal reservoirs and food products with respect to easy transmission causing outbreaks and transfer resistance genes to other pathogens in animal, human, and plants. Therefore, environmental, animal husbandry, and food product surveillance, as well as, clinical infection control, must be strengthened to avoid the extra spread of MDR pathogens, especially MDR STEC strains.

Molecular epidemiology, clinical features and significance of Shiga toxin detection from routine testing of gastroenteritis specimens

After introduction of faecal multiplex PCR that includes targets for stx1 and stx2 genes, we found stx genes were detected in 120 specimens from 111 patients over a 31-month period from 2018-2020 from a total of 14,179 separate tests performed. The proportion of stx1 only vs stx2 only vs stx1 and stx2 was 35%, 22% and 42%, respectively. There were 54 specimens which were culture positive, with 33 different serotypes identified, the predominant serotype being O157:H7 (19%). Eighty-two patients had clinical data available; we found a high rate of fever (35%), bloody diarrhoea (34%), acute kidney injury (27%), hospital admission (80%) and detection of faecal co-pathogens (23%). Only one patient developed haemolytic uraemic syndrome. We found no significant association with stx genotype and any particular symptom or complication. We found a significant association of serotypes O157:H7 and O26:H11 with bloody stool, but no significant association with any other symptom or complication.

Shiga toxin-producing Escherichia coli (STEC) and atypical enteropathogenic E. coli (aEPEC) in Swedish retail wheat flour.

Wheat flour has been identified as the source of multiple outbreaks of gastrointestinal disease caused by shiga toxin-producing Escherichia coli (STEC). We have investigated the presence and genomic characteristics of STEC and related atypical enteropathogenic E. coli (aEPEC) in 200 bags of Swedish-produced retail wheat flour, representing 87 products and 25 brands. Samples were enriched in modified tryptone soya broth (mTSB) and screened with real-time PCR targeting stx1, stx2 and eae, and the serogroups O157, O121 and O26. Isolation was performed by immunomagnetic separation (IMS) for suspected STEC/aEPEC O157, O121 and O26, and by screening pools of colonies for other STEC. Real-time PCR after enrichment revealed 12 % of samples to be positive for shiga toxin genes (stx1 and/or stx2) and 11 % to be positive for intimin (eae). Organic production, small-scale production or whole grain did not significantly influence shiga toxin gene presence or absence in a generalized linear mixed model analysis. Eight isolates of STEC were recovered, all of which were intimin-negative. Multiple serotype/sequence type/shiga toxin subtype combinations that have also been found in flour samples in other European countries were recovered. Most STEC types recovered were associated with sporadic cases of STEC among humans in Sweden, but no types known to have caused outbreaks or severe cases of disease (i.e. haemolytic uraemic syndrome) were found. The most common finding was O187:H28 ST200 with stx2g, with possible links to cervid hosts. Wildlife associated with crop damage is a plausible explanation for at least some of the surprisingly high frequency of STEC in wheat flour.