Stx2 Genes Research Articles

Detection of virulence genes and determination of the antimicrobial susceptibility of Escherichia coli isolates with mastitis in Mashhad, Iran – a short communication

The purpose of this study was to determine the virulence genes and antimicrobial resistance patterns of Escherichia coli isolated from milk samples of cows with bovine mastitis. Forty-seven E. coli isolates from clinical mastitis milk samples, from five dairy farms in Northeast of Iran, were subjected to multiplex PCR to determine virulence genes stx1, stx2, eaeA, hlyA, sta, F4, F17, fliC, and rfbE. In addition, antimicrobial susceptibility was assessed by applying disk diffusion methods. The eaeA and stx1 genes were most frequently detected in 42 (89.3%) and 34 (72.3%) isolates, respectively. However, the least frequent gene was F41 as it was found in only one isolate (2.1%). Furthermore, 9 out of 47 isolates were hlyA positive, and four isolates harbored the sta gene. The antimicrobial susceptibility demonstrated the highest resistance against lincomycin (100%) and neomycin (91.4%). Since these bacteria represent a high-risk pathogen on farms, the emergence of multiple antibiotic-resistant and pathogenic E. coli strains should be of great concern for public health.

Micro-injection Molded Droplet Generation System for Digital PCR Application.

Sensitive, effective, and quantitative analysis of infectious pathogens is an important task for the prevention of human health threats. Herein, we present an advanced approach to producing gene-encapsulated microdroplets for quantitative analysis using a micropatterned metal mold and injection molding technique with an automatically operated system. An injection molded microdroplet generation device was successfully fabricated with a minimum channel width of 30 μm and optimized to produce 100 μm diameter droplets. The optimized microchannel design and flow rate also enable the production of stable numbers of microdroplets (~ 16,000 droplets). To verify the applicability of our device and system to droplet-based digital PCR analysis, Escherichia coli (E. coli) O157:H7 was selected as a model bacterial pathogen, and the stx2 gene was amplified in the microdroplets. The generated microdroplets exhibit both chemical and mechanical stability, and our results are similar to those obtained by a commercially available method. Accordingly, the usefulness of the microdroplet generative device and system is confirmed as a simple, fast, and reliable tool for the quantitative molecular analysis of infectious diseases.

Molecular detection of zoonotic pathogens causing gastroenteritis in humans: Salmonella spp., Shigella spp. and Escherichia coli isolated from Rattus species inhabiting chicken farms in North West Province, South Africa.

Rodents are key carriers and reservoirs of various pathogens of public health importance to both human and animal diseases. This research was carried out in order to identify the selected pathogens, namely, Shigella spp., Salmonella spp. and Escherichia coli from rats that inhabit the poultry houses. A total of 154 samples from captured rats were examined for the zoonotic bacterial pathogens, of which 3.3%, 29.9% and 20.7% were harbouring Shigella spp., Salmonella spp., and E. coli, respectively. A total of 14 Shigella isolates expressed presence of ipaH gene, of which eight and five were positive for S. sonnei and S. boydii, respectively. For Salmonella, 68 isolates were positive for invA and other genes including spy with 26 (38%), sdfI 2 (18%), spvC 14 (20%), hilA 28 (41%), misL 43 (63%), orfL 31 (46%) and spiC 38 (56%). For E. coli, the aggR gene was the most prevalent (62 [42%]), followed by the eae gene, which was only detected in 21 (14%) isolates, while stx gene was not detected in any of the samples. This study shows that zoonotic pathogens with virulence genes are circulating in rodents from selected chicken farms in the North West Province of South Africa. Rodents must therefore be regarded as important carriers of zoonotic pathogens that can potentially infect both humans and animals.

The nutritional effect of probiotic Lactobacillus acidophilus on intestinal microbial flora and inhibition of Shiga toxin (Stx2) gene expression in zebrafish (Danio rerio) exposed to Escherichia coli O157:H7

The nutritional effect of probiotic Lactobacillus acidophilus on intestinal microbial flora and inhibition of Shiga toxin (Stx2) gene expression in zebrafish (Danio rerio) exposed to Escherichia coli O157:H7

Bacteriological Survey of Fresh Minced Beef on Sale at Retail Outlets in Scotland in 2019: Three Foodborne Pathogens, Hygiene Process Indicators, and Phenotypic Antimicrobial Resistance

The health and economic burden of foodborne illness is high, with approximately 2.4 million cases occurring annually in the United Kingdom. A survey to understand the baseline microbial quality and prevalence of food-related hazards of fresh beef mince on retail sale could inform risk assessment, management, and communication to ensure the safety of this commodity. In such a survey, a two-stage sampling design was used to reflect variations in population density and the market share of five categories of retail outlets in Scotland. From January to December 2019, 1,009 fresh minced beef samples were collected from 15 geographic areas. The microbial quality of each sample was assessed using aerobic colony count and Escherichia coli count. Samples were cultured for Campylobacter and Salmonella, and PCR was used to detect target genes (stx1 all variants, stx2 a to g, and rfbO157) for Shiga toxin-producing E. coli (STEC). The presence of viable E. coli O157 and STEC in samples with a positive PCR signal was confirmed via culture and isolation. Phenotypic antimicrobial sensitivity patterns of cultured pathogens and 100 E. coli isolates were determined, mostly via disk diffusion. The median aerobic colony count and E. coli counts were 6.4 × 105 (interquartile range, 6.9 × 104 to 9.6 × 106) and <10 CFU/g (interquartile range, <10 to 10) of minced beef, respectively. The prevalence was 0.1% (95% confidence interval [CI], 0 to 0.7%) for Campylobacter, 0.3% (95% CI, 0 to 1%) for Salmonella, 22% (95% CI, 20 to 25%) for PCR-positive STEC, and 4% (95% CI, 2 to 5%) for culture-positive STEC. The evidence for phenotypic antimicrobial resistance detected did not give cause for concern, mainly occurring in a few E. coli isolates as single nonsusceptibilities to first-line active substances. The low prevalence of pathogens and phenotypic antimicrobial resistance is encouraging, but ongoing consumer food safety education is necessary to mitigate the residual public health risk.

Multidrug-Resistant Shiga Toxin-Producing Escherichia coli Isolated from Philippine Native Swine (Sus scrofa L.)

The interest in the use of Philippine native swine (PNS) as a product alternative to the white hybrid has been growing in the Philippines. However, PNS raised in low-cost production systems are prone to infection by zoonotic pathogens, such as the Shiga toxin-producing Escherichia coli (STEC) that causes diarrhea and life-threatening complications. The multidrug-resistant (MDR) strains of STEC further aggravate the problem as they serve as reservoir of antibiotic resistance genes. This study aimed to detect the occurrence of MDR STEC in 57 fecal samples of weaning- and market-age PNS. In addition, the class 1 integrons of the MDR STEC were characterized due to their ability to capture antibiotic resistance genes and hitchhike in mobile genetic elements, thereby spreading resistance. Fifty-three out of fifty-seven PNS or 93% were found to be positive for the presence of STEC by amplifying the stx1 gene. Disk diffusion assay against 12 antibiotic agents showed that the STEC isolates are most resistant to tetracycline (73.58%), ampicillin (37.74%), trimethoprim/sulfamethoxazole (32.08%), and streptomycin (32.08%). Seventeen STEC isolates exhibited the MDR phenotype and contained class 1 integron (intI1). Four aadA (adenylyltransferase, streptomycin resistance) and dfrA (dihydrofolate reductase, trimethoprim resistance) gene variants were identified in the variable region of the class 1 integrons. The results of this study will be useful in devising effective management practices that would control the presence and spread of this pathogen in the farm.

Molecular Characterization of Shiga Toxin Produced by Escherichia coli Isolated from Milk Samples in Baghdad City

Raw milk is a nutrient-rich food that is considered a high-quality nutritional medium for many microorganism, including Escherichia coli. The aim of the present work was the diagnosis, by molecular methods, of Shiga toxins produced by E. coli strains isolated from cow milk samples collected from different farms in Al-Mahmmodia, Al yoosifya, Al lattiffya, Al howasha, and Arab Jboor in the government of Baghdad during the summer season. Milk samples were incubated in media for bacterial isolation. Isolates were identified using Gram staining and biochemical tests. Out of 50 samples, 15 (30%) showed the presence of E. coli. To confirm the identity of the isolates, their 16S rRNA genes were amplified using specific primers. The results showed that all isolates were E. coli. Shiga toxin-producing E. coli (STEC) were detected among the samples. The prevalence of stx1 genes was higher than that of stx2 among them. No STECs were found among six of the sample isolates, and none of these isolates was positive for stx1 and stx2. SDS-PAGE was used to determine the molecular weight of the toxin, and four selected E. coli bacteria producing Shiga-like toxins showed a clear band of approximately 70 kDa.

Prevalence, virulence genes, and antimicrobial profiles of Escherichia coli O157:H7 isolated from healthy cattle in Tunisia.

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). 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. 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. 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.

Antimicrobial resistance, virulence genes, and phylogenetic characteristics of pathogenic Escherichia coli isolated from patients and swine suffering from diarrhea

BackgroundEscherichia (E.) coli causes colibacillosis in swine and humans, and is frequently associated with antimicrobial resistance. In this study we aimed to compare antimicrobial resistance, O-serogroups, virulence genes, and multi-locus sequence type of E. coli between isolates from pigs and patients suffering from diarrhea, and the most prevalent pathogenic E. coli strain from swine isolates in Korea.MethodsWe tested 64 and 50 E. coli strains from pigs and patients suffering from diarrhea for antimicrobial susceptibility test, virulence genes, O-serogroups, and multi-locus sequence typing.ResultsWe confirmed that isolates from swine showed significantly higher resistance than from those from patients, especially to fluoroquinolone (ciprofloxacin: 37.5 and 10.0%; norfloxacin: 29.7 and 8.0%, respectively). Stx1 (46.0%) was most frequently detected in patients followed by stx2 (38.0%). There was no significant difference in stx2 (swine: 23.4%, patients: 38.0%). In isolates from patients, O157 (12.0%) was the most prevalent O-serogroup, and two isolates (3.1%) from pigs were confirmed to have O157. Additionally, sequence type (ST) 10 (swine: 6 isolates, patients: 2 isolates) and ST 88 (swine: 2 isolates, patients: 1 isolate) were simultaneously detected.ConclusionsWe found that both isolates from swine and human had the stx2 gene, which could cause severe disease. Moreover, antimicrobial resistance was significantly higher in pigs than in patients. These results suggest that pig could act as a reservoir in human infection and antimicrobial resistance could be transferred to human from pigs.

Genetic Diversity and Virulence Profiling of Multi-Drug Resistant Escherichia coli of Human, Animal, and Environmental Origins.

Rapid urbanization has increased human-animal interaction and consequently enhanced the chances to acquire zoonotic diseases. The current investigation is focused to uncover the genetic diversity of multidrug-resistant E. coli strains between different ecologies (i.e., humans, livestock, and environment) at the molecular level by employing antimicrobial resistance profiling, virulence genes profiling, and microbial typing approach using ERIC PCR. Based on multiple antibiotic resistance, overall, 19 antibiotic resistance patterns (R1–R19) were observed. Most of the strains (49/60) were detected to have the combinations of stx, eaeA, and hlyA genes and considered STEC/EPEC/EHEC. A total of 18 unique genetic profiles were identified based on ERIC-PCR fingerprints and most of the strains (13) belong to P1 whereas the least number of strains were showing profiles P7 and P8-P11 (one member each profile). The calculated values for Shannon index (H) for human, animal, and environment are 1.70, 1.82, and 1.78, respectively revealing the highest genetic diversity among the E. coli strains of animal origin. The study revealed that drug-resistant pathogenic E. coli strains could be transmitted bidirectionally among the environment, humans, and animals.

Virulence Genes of Pathogenic Escherichia coli in Wild Red Foxes (Vulpes vulpes).

Simple SummaryEscherichia coli is a commensal of the intestinal tract of humans and animals, but some pathotypes can cause severe infections. Enteropathogenic E. coli (EPEC), Shiga toxin-producing E. coli (STEC), and enterohemorrhagic E. coli (EHEC) are the pathotypes most frequently involved in enteric disorders observed in people and domestic animals. Wildlife may harbor and excrete these pathotypes too, therefore, they may be source of infections for humans and domestic animals. Vulpes vulpes seem to be involved in the epidemiology of pathogenic E. coli strains, and thus they could be a relevant threat mainly when they invade human settlements in rural and urban areas.Different pathotypes of Escherichia coli can cause severe diseases in animals and humans. Wildlife may contribute to the circulation of pathogenic pathotypes, including enteropathogenic E. coli (EPEC), Shiga toxin-producing E. coli (STEC), and enterohemorrhagic E. coli (EHEC). This study analyzed 109 DNA samples previously extracted from fecal specimens collected from red foxes (Vulpes vulpes) to detect E. coli virulence genes eaeA, hlyA, stx1, and stx2, that characterize the EPEC, STEC, and EHEC strains. Thirty-one (28.4%) samples were positive for at least one investigated virulence gene: eaeA gene was detected in 21 (19.2%) samples, hlyA in 10 (9.1%), stx1 in 6 (5.5%), and stx2 in 4 (3.6%). Nine DNA samples resulted positive for two or three virulence genes: five (4.6%) samples were positive for eaeA and hlyA genes, two (1.8%) for eaeA and stx1, one (0.9%) for hlyA and stx1, one (0.9%) for eaeA, hlyA and stx2. Red foxes seem to be involved in the epidemiology of these infections and their role could be relevant because they may be source of pathogenic E. coli for other wild animals, as well as domestic animals and humans.

Virulence and antimicrobial-resistance of shiga toxin-producing E. coli (STEC) Isolated from edible shellfish and its public health significance

Shiga toxin-producing E. coli (STEC) are an important cause of foodborne illness in humans with infections ranging from mild non-bloody diarrhea to bloody diarrhea (BD) and hemolytic uremic syndrome (HUS). This study aimed to investigate the distribution of STEC in shellfish from coastal shores of Lake Timsah in Ismailia Governorate, Egypt and its probable hazard to seafood consumers. Samples from the external surface and tissues of shrimp (n = 45), crabs (n = 45), and oysters (n = 45) batches were examined bacteriologically for the presence of STEC and tested for their antibiotic sensitivity. Moreover, occurrence of virulence genes was determined via detection of stx1, stx2 and eaeA genes using PCR. Overall, E. coli and presumptive STEC isolates (from CHROMagar) were identified from the surface (55.6 and 5.9%) and tissues (42.2 and 8.9%) of the examined shellfish batches, respectively. Five STEC isolates had been confirmed and found belonging to O26:H11, O125:H6, O146:H21, and O159 serogroups, those were 4 isolates from tissues of the three shellfish species and one isolate from the crab surface. The STEC isolates were multi-drug resistant, showing complete resistance to; penicillins, amoxycillin/clavulanic acid, colistin, fosfomycin, ceftriaxone, ciprofloxacin, and tetracycline, however, they were sensitive to gentamycin except O159 serogroup. The current study revealed low level of contamination of shellfish from coastal shores of Lake Timsah with STEC, however, it also highlights the extreme level of antimicrobial resistance exhibited by the presumptive and confirmed STEC isolates which is very hazardous for seafood consumers in the study area.

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

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

Occurrence and Reduction of Shiga Toxin-Producing Escherichia coli in Wastewaters in the Kathmandu Valley, Nepal

Inadequately treated effluents discharged from wastewater treatment plants (WWTPs) severely affect the environment and the surrounding population. This study analyzed the presence of the Shiga toxin-producing Escherichia coli (STEC) genes, stx1, and stx2, and the E. coli gene, sfmD, in municipal WWTP A (n = 11) and B (n = 11) where the reductions were also evaluated; hospitals (n = 17), sewage treatment plants (STPs) (n = 4) and non-functional WWTPs (not-working WWTPs) (n = 5) in the Kathmandu Valley, Nepal. The sfmD gene was detected in 100% of the samples in WWTPs, hospitals, and not-working WWTPs and 50% of STP samples. The highest detection of stx1 and stx2 was shown in the WWTP influents, followed by WWTP effluents, not-working WWTP wastewater, hospital wastewater, and STP wastewater. Log10 reduction values of sfmD, stx1, and stx2 in WWTP A were 1.7 log10, 1.7 log10, 1.4 log10, whereas those in WWTP B were 0.5 log10, 0.6 log10, 0.5 log10, respectively, suggesting the ineffective treatment of STEC in the wastewater in the Kathmandu Valley. The high concentrations of the stx genes in the wastewaters suggest the increasing presence of aggressive STEC in the Kathmandu Valley, which should be a major public health concern.

Detection of Verocytotoxin-Producing Escherichia Coli in Raw and Fermented (Nono) Milk in Sokoto Metropolis, Sokoto State, Nigeria.

Verocytotoxin-producing Escherichia coli (VTEC) also called Shiga toxin producing Escherichia coli (STEC), are food borne organisms which cause fatal disease in human. The bacteria are frequently found in cattle gastrointestinal tract with high potential of contaminating animal products such as milk, meat, and cheese. A cross-sectional study was conducted to investigate the prevalence of VTEC and to detect the presence of stx1, stx2, and eaeA genes in raw and fermented milk sold within sokoto metropolis.Milk samples were analysed using bacterial culture, biochemical test and PCR for molecular identification. Bacteriological culture and biochemical characterization produced Escherichia coli with detection rate 0f 16.0% (38/238). Molecular identification of isolates by Polymerase chain reaction revealed 47.4% (18/38) detection rate of VTEC. The total prevalence of VTEC in the study was 7.6% (18/238), with proportion of raw and fermented milk were 12.5% (13/104) and 3.7% (5/134). There was no association (P≥ 0.05) between VTEC and different milk types. Amplification of isolate by mPCR using specific primers stx1, stx2, and eaeA gene confirmed that (94.4% (17) harboured stx2 and 1 (5.6%) harboured stx1 and eaeA genes.The study established prevalence of VTEC in raw and fermented milk consumed within sokoto metropolis, and presence of stx1, stx2 and eaeA gene. These findings indicate potential faecal contamination of the milk with VTEC in raw and fermented milk. Veterinary services in the state should educate farmers on the importance of farm hygiene and enlighten the Hausa/Fulani nomad and public on the danger associated with the consumption of contaminated milk.

Effect of encapsulation and natural polyphenolic compounds on bacteriophage stability and activity on Escherichia coli in Lactuca sativa L. var. longifolia

AbstractThe goals of the present study were to characterize the phages isolated by magnetic nanocomplex, to determine the effect of natural polyphenols on phage activity against E. coli and the stability of free and encapsulated bacteriophages under UV irradiation, and their efficacy against E. coli on romaine lettuce (Lactuca sativa L. var. longifolia) as a fresh food model. The morphology of the two magnetically isolated phages were characterized using the transmission electron microscope (TEM). All isolated phages were classified in a morphological family of tailed phages of the order Caudovirales, Myoviridae family. The PCR results showed that the stx1 and cI genes were not on any phage. Assays were carried out to determine the effect of four polyphenolic natural extracts on phages under UV radiation. The highest activity and stability of phages expressed in phage titer (PFU/ml) were observed in the presence of polyphenols from tarbush leaves (Flourensia cernua DC). Coliphages were encapsulated in alginate/chitosan and alginate/PVA carriers with and without polyphenols from tarbush leaves. Encapsulation led to a higher phage stability under UV radiation. All encapsulated formulations and free coliphages were applied on the surfaces of romaine lettuce containing E. coli cells. An effective reduction of 2 log of E. coli titers from lettuce was observed. Encapsulated phage formulations with and without polyphenols from tarbush leaves could be considered for future application in fresh food preservation.

Molecular and phenotypic characterization of Escherichia coli from calves in an important meat-producing region in Brazil.

Escherichia coli strains that lead to enteritis are considered an important cause of diarrhea in calves. For correct identification, these microorganisms must be differentiated from non-pathogenic members of the intestinal microbiota. The aim of the present work was to characterize E. coli isolates in calves regarding the presence of virulence genes that cause enteritis and evaluate the sensitivity of the isolates to different antimicrobials. One hundred forty-nine samples from beef cattle and 27 samples from dairy cattle were evaluated. All samples were submitted to microbiological identification and the disk diffusion antibiogram test. The polymerase chain reaction method was used to detect virulence genes. A hundred seventy-six samples were biochemically identified as E. coli and antibiograms were determined. The samples were then submitted to PCR; 35 were positive for the eae gene (19.88%), 135 (76.70%) for the stx1 gene, 62 (35.22%) for the stx2 gene, 159 (90.34%) for the sta gene and 35 (19.88%) for the ltII gene. No samples were positive for the cnf gene. Based on these results, the E. coli isolates were classified into pathotypes: enteropathogenic (n = 3), enterohemorrhagic (n = 32), Shiga toxin-producing (n = 122) and enterotoxigenic (n = 163). The antimicrobial sensitivity tests revealed that 77.2% of the isolates were resistant to three or more pharmacological groups, characterizing these isolates as multidrug resistant. Enterotoxigenic E. coli was the predominant pathotype. Moreover, the prevalence of multidrug-resistant isolates was very high, accounting for the vast majority of isolates.

Antimicrobial Sensitivity of Shiga toxin-producing Escherichia coli (STEC) and Virulence Genes of Representative Isolates in Port Harcourt, Nigeria

Aim: To assess the antimicrobial susceptibility, Shiga toxin-producing Escherichia coli (STEC) and virulence genes of representative isolates in some selected sites of Port Harcourt, Nigeria.&#x0D; Study Design: Case-controlled study.&#x0D; Place and Duration of Study: Selected places in Port Harcourt, Rivers State, Nigeria, between November, 2020 to November, 2021.&#x0D; Methodology: Three hundred and forty-nine (349) samples were analyzed, 80 meat and 63 waste waters from five abattoirs cited in the city, 46 meat samples from five selected roadside butchers sites in the city, 109 patients stool samples, 30 stool samples from food sellers, 20 stool samples from healthy subjects and 1 commercial bottled water which served as control samples. A combination of methods was employed: conventional culture using Tryptone soya broth as an enrichment media, chromagar STEC and serology with O157 latex reagents. Multiplex polymerase chain reaction analysis was used to screen for the presence of specific virulence genes in representative isolates and 16SrRNA sequence data was used to confirm the identity of isolates. GraphPad Prism 2.01 was used to perform the statistical analysis and p values &lt; 0.05 were considered statistically significant.&#x0D; Results: The results showed that 20 (100%) of the selected isolates profiled for virulence gene harbored a combination of stx1 + stx2, while 45% carried the eaeA gene. The culture and molecular methods used in STEC detection showed a good level of agreement comparatively. The result of isolates sequenced for identification by targeting the 16S gene and compared with publicly available sequences on NCBI for phylogenetic analysis showed 100% identities to the ribosomal RNA gene of seven uncultured Escherichia coli strains with Gene Accession numbers (CP049290, MN208208, KY780343, CP058233 MK641327, CP082357, CP082129, MH346270). Isolates were resistant to Ceftazidime 98.9%, Cefuroxime 100%, Cefixime 100%, Augmentin 100% and sensitive to the quinolones and nitrofuratoin. The virulence gene combination pattern, the antibiogram pattern and the phylogenetic analysis result indicated genetic relatedness of isolates. &#x0D; Conclusion: The representative isolates harbored stx1 and stx2 combination, only 45% harbored stx1, stx2 and eaeA genes combination. Phylogenetic analysis of the representative isolates confirmed the genetic similarity of isolates.

Validation of the 3M™ Molecular Detection Assay 2-STEC Gene Screen (stx and eae) for Detection of Shiga Toxin Gene (stx1 and/or stx2) and Intimin Gene (eae) in Dried Cannabis Flower and Dried Hemp Flower: AOAC Performance Tested MethodSM 071902.

The 3M™ Molecular Detection Assay 2-STEC Gene Screen (stx and eae) method is based on gene amplification by the use of real time loop-mediated isothermal amplification when used with the 3M Molecular Detection System for the rapid and specific detection of Shiga toxin gene (stx1 and/or stx2) and intimin gene (eae) from Shiga toxin-producing Escherichia coli (STEC) in enriched products. The 3M Molecular Detection Assay 2-STEC Gene Screen (stx and eae) was approved as AOAC Performance Tested MethodSM Certificate No. 071902. This matrix extension study evaluated the 3M Molecular Detection Assay 2-STEC Gene Screen (stx and eae) method for detection of STECs in dried cannabis flower [>0.3% delta 9-tetrahydrocannabinol (THC)] and dried hemp flower (≤0.3% THC) at a 10 g test portion size. Testing followed procedures outlined in 3M Molecular Detection Assay 2-STEC Gene Screen (stx and eae) product instructions and Standard Method Performance Requirements (SMPR®) for Detection of Shiga Toxin-Producing Escherichia coli in Cannabis and Cannabis Products (AOAC SMPR 2020.012). The method was evaluated at low, high, and non-inoculated levels. Results showed no statistically significant difference between the presumptive positive 3M Molecular Detection Assay 2-STEC Gene Screen (stx and eae) results and the SMPR 2020.012 recommended cultural confirmations. This study provides data that demonstrate the 3M Molecular Detection Assay 2-STEC Gene Screen (stx and eae) is a reliable method for the rapid and specific detection of STEC organisms in dried cannabis flower and dried hemp flower. The 3M Molecular Detection Assay 2-STEC Gene Screen (stx and eae) method is suitable for the rapid and specific detection of STEC organisms in dried cannabis flower and dried hemp flower.

Validation of the 3M™ Molecular Detection Assay 2-STEC Gene Screen (stx) for Detection of Shiga Toxin Gene (stx1 and/or stx2) in Dried Cannabis Flower and Dried Hemp Flower: AOAC Performance Tested MethodSM 071903.

The 3M™ Molecular Detection Assay 2-STEC Gene Screen (stx) method is based on gene amplification by the use of real-time loop-mediated isothermal amplification when used with the 3M Molecular Detection System for the rapid and specific detection of Shiga toxin gene (stx1 and/or stx2) from Shiga toxin-producing Escherichia coli (STEC) in enriched products. The 3M Molecular Detection Assay 2-STEC Gene Screen (stx) was approved as AOAC Performance Tested MethodSM Certificate No. 071903. This matrix extension study evaluated the 3M Molecular Detection Assay 2-STEC Gene Screen (stx) method for detection of STECs in dried cannabis flower [>0.3% delta 9-tetrahydrocannabinol (THC)] and dried hemp flower (≤0.3% THC) at a 10 g test portion size. Testing followed procedures outlined in 3M Molecular Detection Assay 2-STEC Gene Screen (stx) product instructions and Standard Method Performance Requirement (SMPR®) for Detection of Shiga Toxin-Producing Escherichia coli in Cannabis and Cannabis Products (AOAC SMPR 2020.012). The method was evaluated at low, high, and non-inoculated levels. Results showed no statistically significant difference between the presumptive positive 3M Molecular Detection Assay 2-STEC Gene Screen (stx) results and the SMPR 2020.012 recommended cultural confirmations. This study provides data that demonstrate the 3M Molecular Detection Assay 2-STEC Gene Screen (stx) is a reliable method for the rapid and specific detection of STECs in dried cannabis flower and dried hemp flower. The 3M Molecular Detection Assay 2-STEC Gene Screen (stx) method is suitable for the rapid and specific detection of STECs in dried cannabis flower and dried hemp flower.