Eae Gene Research Articles

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.

Detection of eae Gene as A Marker Strain EPEC (Enteropathogenic Escherichia coli) in Chicken Meat Sold at Some Traditional Markets in Surabaya

This study aimed to detect the presence of eae gene in E. coli from chicken meat sold at the traditional market Surabaya. Identification of bacteria was carried out in several stages, inoculated on pre-enrichment media using Buffered Peptone Water (BPW), planting on Levine’s Eosin Methylene Blue Agar (L-EMB) media, suspected colonies on L-EMB were subjected to Triple Sugar Iron Agar (TSIA) and Indol test for the confirmation of E. coli. The confirmed E. coli isolates were tested by Polymerase chain reaction (PCR) to determine the presence of the eae gene. The results showed that E. coli could be identified in 28 out of 100 (28%) chicken meat samples, and eae gene was detected in three E. coli isolates (11%).

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.
 Study Design: Case-controlled study.
 Place and Duration of Study: Selected places in Port Harcourt, Rivers State, Nigeria, between November, 2020 to November, 2021.
 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 < 0.05 were considered statistically significant.
 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. 
 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.

Characterization of Escherichia coli O157:non-H7 isolated from retail food in China and first report of mcr-1/IncI2-carrying colistin-resistant E. coli O157:H26 and E. coli O157:H4

Escherichia coli O157 belongs to a diverse serogroup including different H serotypes. E. coli O157: H7 is the most common serotype that can cause acute gastroenteritis, hemorrhagic colitis (HC), and hemolytic-uremic syndrome (HUS) in humans. In recent years, some E. coli O157:non-H7 strains have been reported to cause sporadic cases and outbreaks of diarrheal diseases. However, the phenotypic and genotypic characteristics of E. coli O157:non-H7 are poorly understood. In this study, E. coli O157:non-H7 strains were isolated from retail food sold on markets in 13 cities in China during 2012–2016 and characterized systematically in terms of their H serotypes, virulence genes, antibiotic resistance, and genotypes. Six H serotypes (H26, H42, H11, H38, H4, and H5) were identified, of which, O157:H42 (31.4 %) and O157:H26 (28.6 %) were the most prevalent. Most of the isolates (82.9 %) carried virulence genes. Ten isolates (28.6 %) carried the eae gene and were identified as atypical enteropathogenic E. coli. Multilocus sequence typing showed that the E. coli O157:non-H7 strains demonstrated diverse sequence types with different evolutionary trends than E. coli O157:H7. All the isolates exhibited multidrug resistance. The isolates showed a high prevalence of resistance to AMC, AMP, CTX, CIP, T/S, TE, and FFC. The predominant antibiotic-resistance genes were TEM-1 (40.0 %), CTX-M-55 (34.3 %), aadA (74.3 %), sul2 (62.9 %), floR (91.4 %), tetA (85.7 %), qnrS (37.1 %), oqxA (62.9 %), and oqxB (62.9 %). For the first time, we identified IncI2 plasmid-mediated colistin-resistant strains (six O157:H26 and one O157:H4). These strains co-harbored plasmid-mediated mcr-1 gene, CTX-M-55, OXA-4, PMQR, and other resistant genes, which is of great concern. Colistin and cefotaxime are generally used as the last defense to treat complicated infections. The emergence of virulent and multidrug resistant strains in food poses a serious threat to human health. The strict monitoring and surveillance of multiple-drug resistant strains in food are needed to prevent their dissemination to humans.

PHYLOTYPING, PATHOTYPING and PHENOTYPIC CHARACTERISTICS of Escherichia coli ISOLATED from VARIOUS STREET FOODS in BANGLADESH

The burden of street food contamination remains significantly higher among developing countries, making its consumers' health compromised. This study aimed at identifying Escherichia coli in local street foods, track their sources and characterize their virulence properties, antimicrobial susceptibility patterns, and incidence of recent fecal contamination. Twelve E. coli isolates isolated from 28 types of street foods sold at different locations of Dhaka metropolitan city were confirmed by both culture-based and molecular methods. Phylogroup B1 of environment origin was the most predominant (58%, n=12) among the isolates, followed by commensal phylogroup A (16%), phylogroup C (8%), D (8%), and E (8%). Alarmingly, 8% of the isolates were confirmed to be Enteropathogenic E. coli (EPEC), harboring hlyA gene, and 41% were due to recent fecal contamination cases confirmed by the presence of the eae gene. Isolates showed the highest resistance to ampicillin, followed by chloramphenicol. However, resistance could not be correlated to the presence of class 1 integron as isolates sensitive to these antibiotics also harbored this mobile genetic element. The findings of this study demonstrated the presence of antibiotic-resistant, potentially pathogenic E. coli in street food which emphasizes the health hazard associated with consumption of such foods.

Characterisation and epidemiological subtyping of Shiga toxin-producing Escherichia coli isolated from the beef production chain in Gauteng, South Africa

In South Africa, there is a shortage of epidemiologic data on Shiga toxin-producing Escherichia coli (STEC) in the beef production chain. This study was conducted to characterise STEC isolates originating from three studies conducted in a cattle feedlot, beef abattoirs and retail outlets in Gauteng province, South Africa. Polymerase chain reaction was used to detect virulence genes, the Epsilometer test to assess antimicrobial susceptibility, pulsed-field gel electrophoresis (PFGE) to investigate genetic relatedness of isolates, and conventional serotyping for phenotypic identification. Amongst the 86 STEC isolates, the eaeA gene was detected in 20 (23%), and 26 different serogroups were identified, including the clinically important O8, O174, O2, 020 and O117. The majority of the isolates (95%; 82/86) exhibited resistance to one or more antimicrobial agents, and 30 of the isolates (35%) exhibited multi-drug resistance (MDR), being resistant to at least three antimicrobial classes. The PFGE patterns showed a highly diverse but related STEC population, with 45 distinct patterns and evidence of horizontal transmission along the beef production chain. This is significant because it demonstrates continual environmental contamination and risk of contamination along the beef production chain and the food chain. To our knowledge, this is the first study that provides evidence of horizontal transmission of STEC along the beef production chain in South Africa. This epidemiological information could facilitate the development of a proactive strategy for reducing potential foodborne outbreaks and transmission of antimicrobial resistant pathogens in the food chain.

Multidrug-resistant Escherichia coli from free-living pigeons (Columba livia): Insights into antibiotic environmental contamination and detection of resistance genes.

Bacterial resistance is a public and one health problem. Free-living birds can be reservoirs of multidrug-resistant bacteria and resistance genes. This study aimed to characterize the antimicrobial resistance of Escherichia coli isolated from free-living urban pigeons (Columba livia) in South Brazil. Ninety-two animals were sampled, and one isolate was obtained from each one. The isolates were characterized, and the antimicrobial resistance profile and beta-lactam and colistin resistance genes were investigated. The isolates were classified as phylogroups B1 (35%), B2 (33%), A (16%) and D (16%), and 14% of the strains had the eae virulence gene. All isolates were resistant to at least one antimicrobial, and 63% of them were multidrug-resistant. Geographical location where the pigeons were captured and presence of the eae gene were associated with multidrug resistance. blaVIM and mcr-1 genes were detected in one and two isolates, respectively. This is the first report of these genes in E.coli of pigeons. The blaVIM -positive isolate was classified as Shiga toxin-producing E.coli, and the isolates with mcr-1 were classified as Enterohaemorrhagic E.coli and Enteropathogenic E.coli, which raise additional concerns related to public health since these are zoonotic pathotypes. The results reveal that pigeons carry multidrug-resistant pathogenic E.coli, which may interest public health. Nonetheless, further studies on whether these animals are sources of contamination for humans must be performed to understand their role in spreading antimicrobial resistance.

​Prevalence of Shigatoxigenic, Enteropathogenic and Antimicrobial Drug Resistant Escherichia coli from Captive Wild Animals of Alipore Zoo, Kolkata, India

Background: Escherichia coli is one of the intestinal microbiota in wildlife as in domestic animals and humans. The prevalent serogroups, pathogenic strains like STEC, EPEC and antimicrobial resistance is less frequently studied in captive wildlife of India. The present study was aimed to study the prevalence of STEC, EPEC and antimicrobial resistant E. coli from captive wild animals in a zoo in Kolkata, India. Methods: This study was done during March 2019 to June 2019 for E. coli from faecal samples of captive wild birds and mammals. The pathotypes STEC and EPEC were detected for the presence of stx1, stx2 and eae virulence genes, respectively. Extended spectrum beta- lactamases, ampC beta-lactamases production and antimicrobial susceptibility were detected by standard phenotypic methods. Result: In this study, some E.coli isolates were found harbouring genes for ETEC and EPEC. Extended spectrum beta- lactamases and ampC beta-lactamases were produced by some isolates. Most E. coli isolates were detected multidrug-resistant but were mostly sensitive to gentamicin and amikacin. This study demonstrated that captive wild animals harbour potentially pathogenic E. coli that were resistant for many antimicrobials.

Presence and Virulence Characteristics of Shiga Toxin Escherichia coli and Non-Shiga Toxin-Producing Escherichia coli O157 in Products from Animal Protein Supply Chain Enterprises in South Africa.

Consumption of food that is contaminated with Shiga toxin–producing Escherichia coli (STEC) has been linked to serious foodborne disease outbreaks. Our aim was to provide a descriptive study on the presence and virulence factors of STEC and non-STEC O157 isolates recovered from 2017 diverse meat and meat product samples from all provinces of South Africa (n = 1758) and imported meat from South Africa's major ports of entry (n = 259). A cross-sectional study was undertaken to analyze raw intact meat, raw processed (nonintact) meat, and ready-to-eat (RTE) meat from cattle, game, sheep, pork, and poultry. Isolation was performed using International Organization for Standardization-based microbiological techniques, while detection and characterization were performed using real-time PCR (RT-PCR) and conventional PCR targeting the stx1, stx2, eae, and ehxA genes. A total of 28 of 1758 (1.59%; confidence interval [CI] 1.1–2) samples from the domestic market tested positive (n = 10 Escherichia coli O157:H7; n = 14 Escherichia coli O157: non-H7; and n = 4 non-O157 STEC), while 4/259 (1.54%; CI 0.4–4) samples from ports of entry tested positive for Escherichia coli O157:H7 based on RT-PCR. On average, diverse samples from domestic meat and meat products from cattle showed the highest number of positive samples (22/1758; 1.3%; CI 0.8–2). RT-PCR detected more positive samples (n = 32) compared with culture (n = 17). Sixteen different virulence factor combinations were observed. Our findings demonstrate a relatively low presence of diverse STEC strains along the meat value chain. To our knowledge, this is the first extensive report in South Africa to analyze STEC and non-STEC O157 from local and imported samples from many animal species. This is important as it reveals virulence factors in STEC strains circulating in meat and meat products in South Africa, which contribute to the risk of infection.

Genetic and phylogenetic characterization of Shiga toxin-producing Escherichia coli and enteropathogenic E. coli from livestock in Jiangsu by using whole-genome sequencing.

There are knowledge gaps regarding STEC and EPEC strains in livestock in Jiangsu, China. This study aimed to evaluate the potential public health significance of STEC and EPEC strains isolated from livestock by determining the serotypes, virulence profiles, and genetic relationship with international STEC strains. A total of 68 STEC and 37 EPEC strains were obtained from 231 faecal sheep samples and 70 faecal cattle samples. By using whole-genome sequencing (WGS) analysis, all STEC belonged to 15 O: H serotypes, and the most prevalent serotypes were O6:H10 (19.1%), O155:H21 (14.7%), and O21:H25 (10.3%). The main Shiga toxin gene subtypes detected were stx1c (41.2%), stx1a (26.5%), stx2b (14.7%), and stx2k (14.7%). Only the STEC from cattle carried eae gene. Other adherence-associated or toxin-related genes, including lpfA (70.6%), iha (48.5%), subA (54.4%), and ehxA (33.8%), were found in STEC. All EPEC strains were bfpA-negative, and the predominant eae variants were eae-β1 (62.2%), eae-ζ (21.6%), and eae-θ (8.1%). The core-genome multi-locus sequence typing (cgMLST) analysis revealed nine scattered clusters in STEC and one dominant cluster in EPEC. The strains with the same serotypes, including O22:H8 and O43:H2 in the two towns, possessed a closely genomic distance. The core genome single-nucleotide polymorphism (cgSNP) showed that part of STEC strains in this study was clustered with isolates possessing the same serotypes from the Netherlands, Sweden, and Xinjiang of China. Five serotypes of STEC isolates were associated with the clinical STEC strains from databases. This study provided the diverse serotypes and the virulence genes profiles in STEC and EPEC strains. Local strains possessed widely diverse and scattered clusters by cgMLST. Closely genomic correlation with clinical isolates displayed that part of the STEC strains may threaten to public health. Non-O157 STEC strains act as important pathogens for human infections. This study supports the increased surveillance work of non-O157 STEC rather than just O157 STEC in this region.

The prevalence of Campylobacter spp., Listeria monocytogenes and Shiga toxin-producing Escherichia coli in Norwegian dairy cattle farms: A comparison between free stall and tie stall housing systems.

AimsThis study explored how dairy farm operating systems with free‐stall or tie‐stall housing and cow hygiene score influence the occurrence of zoonotic bacteria in raw milk.Methods and ResultsSamples from bulk tank milk (BTM), milk filters, faeces, feed, teats and teat milk were collected from 11 farms with loose housing and seven farms with tie‐stall housing every second month over a period of 11 months and analysed for the presence of STEC by culturing combined with polymerase chain reaction and for Campylobacter spp. and L. monocytogenes by culturing only. Campylobacter spp., L. monocytogenes and STEC were present in samples from the farm environment and were also detected in 4%, 13% and 7% of the milk filters, respectively, and in 3%, 0% and 1% of BTM samples. Four STEC isolates carried the eae gene, which is linked to the capacity to cause severe human disease. L. monocytogenes were detected more frequently in loose housing herds compared with tie‐stalled herds in faeces (p = 0.02) and feed (p = 0.03), and Campylobacter spp. were detected more frequently in loose housing herds in faeces (p < 0.01) and teat swabs (p = 0.03). An association between cow hygiene score and detection of Campylobacter spp. in teat milk was observed (p = 0.03).ConclusionSince some samples collected from loose housing systems revealed a significantly higher (p < 0.05) content of L. monocytogenes and Campylobacter spp. than samples collected from tie‐stalled herds, the current study suggests that the type of housing system may influence the food safety of raw milk.Significance and Impact of the StudyThis study highlights that zoonotic bacteria can be present in raw milk independent of hygienic conditions at the farm and what housing system is used. Altogether, this study provides important knowledge for evaluating the risk of drinking unpasteurized milk.

Molecular Epidemiology of Antibiotic Resistance Genes and Virulence Factors in Multidrug-Resistant Escherichia coli Isolated from Rodents, Humans, Chicken, and Household Soils in Karatu, Northern Tanzania.

The interaction of rodents with humans and chicken in the household environment can facilitate transmission of multidrug-resistant (MDR) Escherichia coli (E. coli), causing infections that are difficult to treat. We investigated the presence of genes encoded for carbapenem, extended spectrum beta-lactamases (ESBL), tetracycline and quinolones resistance, and virulence among 50 MDR E. coli isolated from human (n = 14), chicken (n = 12), rodent (n = 10), and soil (n = 14) samples using multiplex polymerase chain reaction (PCR). Overall, the antimicrobial resistance genes (ARGs) detected were: blaTEM 23/50 (46%), blaCTX-M 13/50 (26%), tetA 23/50 (46%), tetB 7/50 (14%), qnrA 12/50 (24%), qnrB 4/50 (8%), blaOXA-48 6/50 (12%), and blaKPC 3/50 (6%), while blaIMP, blaVIM, and blaNDM-1 were not found. The virulence genes (VGs) found were: ompA 36/50 (72%), traT 13/50 (26%), east 9/50 (18%), bfp 5/50 (10%), eae 1/50 (2%), and stx-1 2/50 (4%), while hlyA and cnf genes were not detected. Resistance (blaTEM, blaCTX-M, blaSHV, tetA, tetB, and qnrA) and virulence (traT) genes were found in all sample sources while stx-1 and eae were only found in chicken and rodent isolates, respectively. Tetracycline resistance phenotypes correlated with genotypes tetA (r = 0.94), tetB (r = 0.90), blaKPC (r = 0.90; blaOXA-48 (r = 0.89), and qnrA (r = 0.96). ESBL resistance was correlated with genotypes blaKPC (r = 0.93), blaOXA-48 (r = 0.90), and qnrA (r = 0.96) resistance. Positive correlations were observed between resistance and virulence genes: qnrB and bfp (r = 0.63) also blaTEM, and traT (r = 0.51). Principal component analysis (PCA) indicated that tetA, tetB, blaTEM, blaCTX-M, qnrA, and qnrB genes contributed to tetracycline, cefotaxime, and quinolone resistance, respectively. While traT stx-1, bfp, ompA, east, and eae genes contributed to virulence of MDR E. coli isolates. The PCA ellipses show that isolates from rodents had more ARGs and virulence genes compared to those isolated from chicken, soil, and humans.

Phylogenetic Groups/B2 Subgroup Distributions, Serogrouping and Identification of Virulence Factors in Extended-Spectrum Cephalosporin-Resistant Escherichia coli Strains Isolated from the Stool of Healthy Children Under 10 Years Old

Background: Segregation of Escherichia coli (E. coli) into the phylogenetic groups was observed in the experiments so that group B2 contained the enteropathogenic E. coli (EPEC) strains and extraintestinal pathogenic E. coli (ExPEC). Objectives: This study aimed to identify B2 phylogenetic groups in the extended-spectrum Cephalosporins resistant E. coli isolated from the stool of healthy children under 10 years old. Methods: One hundred E. coli resistant to broad-spectrum Cephalosporins were collected from the feces of healthy children under 10. Subsequently, we grouped phylogenetic via PCR based on the genes yjaA, chuA, arpA, as well, as TspE4.C2. Then, according to Clermont et al.’s study, we used two individual multiplex PCRs for identifying B2 sub-groups (I-X subgroups). Serogroup typing with the 12 O-antigen was analyzed via PCR, and finally, 10 virulence genes (cnf1, papG, ibeA, malX, usp, cdt, eae, bfp, and afa-Dr) were identified with PCR. Results: The age range of the healthy children was between 1 and 10 years. The B2 and unknown phylogroups were the most common strains in this study. The most common B2 subgroups were I (2%), IX (1%), V (8%), IV, V, VII (1%), IX, V (3%), IX, V, III, I (1%), IX, V, III, VII, I (%1), V, I (6%), V, III, I (3%), and V, III, VII (1%), with each subgroup carrying distinctive sets of ExPEC virulence markers. The results also showed that 29% of E. coli in the healthy children had malX and 23% had papGII. It was also found that 32% of the strains isolated from the healthy children had antigens O2 and 36% were unknown. Conclusions: In this study, 27% of the strains belonged to B2 phylogroup and 6% to B1 phylogroup. Moreover, serogroups O2, O16, and O25 were predominant and belonged to B2 phylogroup. Moreover, malX, papGII, usp, papGIII, aggR, and eae virulence genes also had the highest to lowest supply among the tested strains, respectively. Moreover, B2 isolates were shown to have further virulence-related genes in comparison to the non B2 isolates.

"FAMILY CLUSTER ASSOCIATED WITH SHIGA TOXIN-PRODUCING ESCHERICHIA COLI O26 SEROGROUP"

"Introduction: Shiga toxin-producing Escherichia coli (STEC) can cause severe human infections especially in the pediatric population who are at risk of progressing to life threatening systemic complications such as hemolytic uremic syndrome (HUS). In Romania, enhanced monitoring of STEC-associated HUS was implemented in year 2016 after a cluster of infections caused by STEC O26:H11 serotype heightened public awareness of their consequences. Objectives: This study describes the microbiological investigation initiated to identify a STEC-associated HUS pediatric case and determine additional infections among his household contacts. Methods: Fecal specimens collected from a small child hospitalized with HUS and his asymptomatic parents and brother were investigated using a culture-based approach which targeted STEC isolation, identification, and typing. PCR-based assays for key virulence genes (i.e., stx1 and stx2 types/subtypes, eae, and ehxA) were combined with traditional methods such as serogroup determination by slide agglutination. Also, multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE) were used for strain genotyping. Results: STEC was identified only in the stool samples of the siblings, one who developed HUS and the other who remained asymptomatic. Both strains belonged to O26 E. coli serogroup and displayed an identical virulence gene profile (presence of stx2a, eae, and ehxA genes). Molecular typing by PFGE showed the STEC strains as highly related genetically and MLST assigned them to sequence type ST21 lineage. Conclusion: A family cluster of O26 STEC infections was detected by using a culture- based molecular approach. Further analysis at whole genome sequencing resolution level is needed for a detailed characterization of such pathogens of significant public health importance. Keywords: HUS, Shiga toxin-producing Escherichia coli, virulence genes, PCR, molecular typing"

Virulence determinant and antimicrobial resistance traits of Emerging MDR Shiga toxigenic E.coli in diarrheic dogs

Shiga-toxigenic Escherichiacoli (STEC) is incriminated in severe hemorrhagic enteritis in dogs, which is considered a veterinary and public health alarm. To investigate the prevalence, antimicrobial resistance patterns, virulence determinants, and distribution of antimicrobial resistance genes in STEC strains isolated from dogs: 80 fecal samples were obtained from diseased dogs suffering from hemorrhagic diarrhea from pet animal clinics in Ismailia governorate, Egypt. The obtained samples were examined bacteriologically. Moreover, the retrieved isolates were tested for serogrouping, Congo-red binding, antimicrobial resistance, and PCR-based determination of virulence and antimicrobial resistance genes. The prevalence of E.coli in the examined diseased dogs was 23.75% (19/80). The serogrouping of the recovered isolates revealed that 84.2% of the tested isolates were distributed into three serogroups: O146 (36.8%), O111 (31.5%), and O26 (15.7%). Meanwhile, three isolates were untypable (15.8%). Moreover, all the tested E.coli serovars were positive for CR-binding. PCR revealed that the prevalence of stx1, eaeA, hlyA, and stx2 virulence genes was 100%, 100%, 100%, and 47.3%, respectively. Our findings revealed that 31.5% of the recovered isolates showed MDR to five antimicrobial classes and harbored blaTEM, blaCTX-M, tetA, tetB, and sul1 genes. Alarmingly, three isolates were carbapenem-resistant. Two strains harbored the blaKPC gene, while one strain carried the blaNDM-1 gene. Concisely, as far as we know, this is the first study that reported the existence of MDR-STEC in dogs in Egypt. The stx1 gene is the most predominant Shiga toxin gene that accompanied the STEC isolated from hemorrhagic enteritis in dogs. The emerging MDR-STEC in dogs commonly harbors blaTEM, blaCTX-M, sul1, tetA, tetB, and qnrA resistance genes. Meropenem, levofloxacin, and tigecycline exhibited talented antimicrobial activity against MDR-STEC isolated from dogs.

Pathogenic E. coli from Cattle as a Reservoir of Resistance Genes to Various Groups of Antibiotics

Antimicrobial resistance has become a worldwide concern in all public health domains and reducing the spread has become a global priority. Pathogenic E. coli is responsible for a number of illnesses in humans and outbreaks in the past have been correlated with the consumption of contaminated bovine products. This is why surveillance in all the steps of production is essential. This study focused on identifying the pathogenic strains of E. coli in two large bovine abattoirs from Romania and France, and on associating them with the antimicrobial resistance patterns. A total of 250 samples from intestinal content were aseptically collected during the evisceration step of the cattle slaughtering process, from which 242 E. coli strains were isolated. Seventeen percent of all samples tested positive to at least one E. coli isolate carrying eaeA, stx1 and stx2 genes. The most prevalent genetic profile found in the E. coli strains tested was Stx1-positive and Stx2/eaeA-negative. More than 68% of the pathogenic E. coli isolated in Romania showed multi-drug resistance (MDR) and in France, the percentage was significantly lower (38%). The MDR profiles showed a high gene diversity for antibiotic resistance, which represents a great risk for environmental spread and human health. Our results indicate that in Romania, bovines can represent a reservoir for MDR E. coli and, hence, a surveillance system for antimicrobials usage in farm animals is highly needed.

A237 VIRULENCE POTENTIAL OF BACTERIA ISOLATED FROM NON-INFLAMED COLON IN PATIENTS WITH INFLAMMATORY BOWEL DISEASES

BackgroundAs inflammation can impact microbes, our lab has focused on non-inflamed bowel sections of patients with inflammatory bowel diseases (IBD) and found bacterial alterations in the terminal ileum. We are also interested in the appendix given the involvement of the peri-appendicular region and effects of appendectomy in ulcerative colitis.AimsIn this study, we aimed to identify and evaluate microbes from the appendix and non-inflamed regions of the colon in IBD patients. We hypothesized that microbes originating from the non-inflamed sections of the colon or appendix are more invasive and could trigger inflammation in IBD patients.Methods16S DNA Sanger sequencing was performed on bacteria, aerobically and anaerobically cultured from tissues of the appendix, peri-appendicular region, cecum, and ascending colon, collected from the resected colons of 5 pediatric IBD patients. The invasive capacity of the identified bacteria was evaluated by gentamicin protection assays on Caco-2 intestinal epithelial cells in vitro. Presence of select invasive or adhesive genes in the bacteria was assessed by PCR.Results Escherichia coli was the most frequently cultured species in the aerobic cultures; additional aerobic species included Enterococcus and Klebsiella. Bifidobacterium and Erysipelatoclostridium were identified among the anaerobic cultures. Gentamicin protection assays indicated that Klebsiella isolated from the peri-appendicular region was significantly more invasive (mean ~3000 CFU/ml) than HB101 (~1000 CFU/ml; non-invasive control; P<0.05). Enterococcus avium and Enterococcus faecalis were also more invasive than HB101 in the peri-appendicular region (~10000 CFU/ml) and cecum (~4000 CFU/ml; P<0.05), respectively. E. coli isolated from the appendix showed a higher invasive potential (~3000 CFU/ml) than E. coli isolated from other sections. Additionally, PCR showed that E. coli obtained from different sections, except the ascending colon of one the patients, had the fimH gene while the other types of bacteria did not. None of the isolated bacteria had Hemolysin ( hlyA) or attaching and effacing ( eaeA) genes.ConclusionsBacteria, especially E. coli, from non-inflamed bowel in IBD (including the appendix) appear to have increased invasive potential. This suggests that the microenvironment in these regions could be altered, resulting in increased invasion of bacteria or the gut harboring more invasive microbial populations in IBD patients. As a result, inflammation could be triggered or exacerbated through this reservoir of pathobionts.Funding AgenciesCIHR

MOLECULAR DETECTION OF SHIGA TOXIN (stx1 and stx2) AND INTIMIN (eaeA) GENES IN ESCHERICHIA COLI ISOLATED FROM FECAL SAMPLES OF CATTLE, SHEEP, AND HUMAN IN BASRAH GOVERNORATE

The present study aims to isolate and identify Escherichia coli from fecal samples of farmanimals and human, also, it aims to molecular detection of shigatoxin and intimin genes inisolates. A total of (264) fecal samples and swabs were collected from different parts of Basrahin the period extending from September 2018 to January 2019. These samples were composed of(85) samples from cows, (94) samples from humanand (85) samples from sheep. Differenttechniques were used in this study to detect the presence of E. coli; these techniques includedconventional microbiological assays and molecular techniques (amplification of uidA gene byusing polymerase chain reaction).The results of these techniques indicated 50 (18.9%) were E. coli from the tested samples.These isolates were subjected to PCR to detect Shiga toxins and intimin genes (stx1, stx2, andeaeA). The results of PCR confirmed all (50) isolates were harbor at least one virulence gene.Out of 50 isolates 20 (40%) carried stx2 gene alone, the percentages of the carrier were (66.7 %,41.7% and 23.5%) from human, sheep and cattle samples, respectively. The genes (stx1 andstx2) were detected together in 9/50 (18%), represent (52.9%) of cattle isolates. The intimin gene(eaeA) alone was detected in 2/50 (4%), represent (11.8%) of cattle isolates. (28%) of isolatesharbor (stx2 and eaeA) genes, the isolates belong to human and sheep isolates (33.3%) and(45.8%), respectively. Presence of the genes (stx1, stx2, and eaeA) were discovered in (10%)of isolates, (11.8%) of cattle and (12.5%) of sheep. The isolates were resistant to ampicillin, tetracycline (92%, 74%), respectively. However, theisolates were susceptible to imipenem, gentamycin, chloramphenicol, ciprofloxacin, andcefotaxime with a ratio of 100%, 92%, 78%, 68%, and 58%, respectively.

Molecular Characterization of Resistance and Virulence Genes in Escherichia coli Isolated from Bats (Eidolon helvum) Faeces in Osun State, Nigeria

Escherichia coli is one of the primary intestinal commensal organisms found in endothermic animals and hence, it is widely disseminated in the environment. A total of 36 (10.68%) Escherichia coli was isolated from 101 faecal samples collected from straw coloured fruit bats (Eidolon helvum) faeces from three different major cities (Ile-Ife, Osogbo and Ilesa) in Osun State, Nigeria. The E. coli isolates showed a higher percentage antibiotic resistance to augumentin (83.33%), followed by cefuroxime (69.44%), ceftazidime (55.55%), amoxicillin (38.88%), and cefotaxime (33.33%), but relatively low to eterpenem, meropenem, and tetracycline (11.11%) and also nitrofurantoin (8.33%). Multiple antibiotic resistance to three or more antibiotics was recorded among the isolates in all the study locations. Escherichia coli (16.7%) were multiple antibiotic-resistant. Six (100%) of the multiple antibiotic-resistant E. coli posesses MultiDHA gene while 3 (50%) were positive for TEM gene. The virulence gene eaeA had the highest prevalence of 83.3% while the least was observed in PAPC (16.7%) and ISS (16.7%) but no biofilm production was observed in all the isolates. The resistance found across the three locations indicated that resistance genes can be transmitted to other animal and human through direct and or indirect contact. The antibiotic resistance profile and patterns as well as the antibiotic resistance genes detected indicated the possibility of cross transmission and spread of the resistance trait among the organisms with great consequences in therapeutic management of infections resulting from such a source. The dendrogram shows that the isolates were genetically related across the three locations.