STEC Isolates Research Articles

Epidemiology, Virulence and Antimicrobial Resistance of Escherichia coli Isolated from Small Brazilian Farms Producers of Raw Milk Fresh Cheese.

This study aimed to identify contamination sources in raw milk and cheese on small farms in Brazil by isolating Escherichia coli at various stages of milk production and cheese manufacturing. The study targeted EAEC, EIEC, ETEC, EPEC, STEC, and ExPEC pathotypes, characterizing isolates for the presence of virulence genes, phylogroups, antimicrobial susceptibility, and phylogenetic relationships using PFGE and MLST. The presence of antimicrobial resistance genes and serogroups was also determined. Three categories of E. coli were identified: pathogenic, commensal, and ceftriaxone-resistant (ESBL) strains. Pathogenic EPEC, STEC, and ExPEC isolates were detected in milk and cheese samples. Most isolates belonged to phylogroups A and B1 and were resistant to antimicrobials such as nalidixic acid, ampicillin, kanamycin, streptomycin, sulfisoxazole, and tetracycline. Genetic analysis revealed that E. coli with identical virulence genes were present at different stages within the same farm. The most frequently identified serogroup was O18, and MLST identified ST131 associated with pathogenic isolates. The study concluded that E. coli was present at multiple points in milk collection and cheese production, with significant phylogroups and high antimicrobial resistance. These findings highlight the public health risk posed by contamination in raw milk and fresh cheese, emphasizing the need to adopt hygienic practices to control these microorganisms.

Occurrence, serotypes and virulence characteristics of Shiga toxin-producing and Enteropathogenic Escherichia coli isolates from dairy cattle in South Africa

Shiga toxin-producing and Enteropathogenic Escherichiacoli are foodborne pathogens commonly associated with diarrheal disease in humans. This study investigated the presence of STEC and EPEC in 771 dairy cattle fecal samples which were collected from 5 abattoirs and 9 dairy farms in South Africa. STEC and EPEC were detected, isolated and identified using culture and PCR. Furthermore, 339 STEC and 136 EPEC isolates were characterized by serotype and major virulence genes including stx1, stx2, eaeA and hlyA and the presence of eaeA and bfpA in EPEC. PCR screening of bacterial sweeps which were grown from fecal samples revealed that 42.2% and 23.3% were STEC and EPEC positive, respectively. PCR serotyping of 339 STEC and 136 EPEC isolates revealed 53 different STEC and 19 EPEC serotypes, respectively. The three most frequent STEC serotypes were O82:H8, OgX18:H2, and O157:H7. Only 10% of the isolates were classified as “Top 7” STEC serotypes: O26:H2, 0.3%; O26:H11, 3.2%; O103:H8, 0.6%; and O157:H7, 5.9%. The three most frequent EPEC serotypes were O10:H2, OgN9:H28, and O26:H11. The distribution of major virulence genes among the 339 STEC isolates was as follows: stx1, 72.9%; stx2, 85.7%; eaeA, 13.6% and hlyA, 69.9%. All the 136 EPEC isolates were eaeA-positive but bfpA-negative, while 46.5% carried hlyA. This study revealed that dairy cattle are a major reservoir of STEC and EPEC in South Africa. Further comparative studies of cattle and human STEC and EPEC isolates will be needed to determine the role played by dairy cattle STEC and EPEC in the occurrence of foodborne disease in humans.Please kindly check and confirm the country and city name in affiliation [6].This affiliation is correct.Please kindly check and confirm the affiliationsConfirmed. All Affiliations are accurate

Antibiotic Resistance and Disinfectant Resistance Among Escherichia coli Isolated During Red Meat Production

Escherichia coli commonly found in the gastrointestinal tracts of food animals include Shiga toxin-producing E. coli (STEC, stx+, eae-), Enterohemorrhagic E. coli (EHEC, stx+, eae+), Enteropathogenic E. coli (EPEC, stx-, eae+), and “nondiarrheagenic” E. coli (NDEC, stx-, eae-). EHEC, EPEC, and STEC are associated with foodborne disease outbreaks. During meat processing, disinfectants are employed to control various bacteria, including human pathogens. Concerns exist that E. coli resistant to antibiotics are less susceptible to disinfectants used during meat processing. Since EHEC, EPEC, and STEC with reduced susceptibility to disinfectants are potential public health risks, the goal of this study was to evaluate the association of antibiotic resistant (ABR) E. coli with increased tolerance to 4% lactic acid (LA) and 150 ppm quaternary ammonium compounds (QACs). A pool of 3,367 E. coli isolated from beef cattle, veal calves, swine, and sheep at various processing stages was screened to identify ABR E. coli. Resistance to ≥1 of the six antibiotics examined was identified in 27.9%, 36.1%, 54.5%, and 28.7% among the NDEC (n = 579), EHEC (n = 693), EPEC (n = 787), and STEC (n = 1308) isolates evaluated, respectively. Disinfectant tolerance did not differ (P > 0.05) between ABR and antibiotic susceptible EHEC isolates. Comparable frequencies (P > 0.05) of biofilm formation or congo red binding were observed between ABR and antibiotic susceptible strains of E. coli. Understanding the frequencies of ABR and disinfectant tolerance among E. coli present in food-animal is a critically important component of meat safety.

Multiple-Drug Resistant Shiga Toxin-Producing E. coli in Raw Milk of Dairy Bovine.

Raw milk may contain pathogenic microorganisms harmful to humans, e.g., multidrug-resistant Escherichia coli non-O157:H7, which can cause severe colitis, hemolytic uremia, and meningitis in children. No studies are available on the prevalence of Shiga toxin-producing E. coli (STEC O157:H7) in sick or healthy dairy animals in the Khyber Pakhtunkhwa Province of Pakistan. This study aimed to isolate, characterize, and detect antibiotic resistance in STEC non-O157:H7 from unpasteurized milk of dairy bovines in this province. We collected raw milk samples (n = 800) from dairy farms, street vendors, and milk shops from different parts of the Khyber Pakhtunkhwa Province. E. coli was isolated from these samples followed by latex agglutination tests for serotyping. The detection of STEC was conducted phenotypically and confirmed by the detection of virulence genes genotypically. An antibiogram of STEC isolates was performed against 12 antibiotics using the disc diffusion method. A total of 321 (40.12%) samples were found to be positive for E. coli in this study. These samples were processed for the presence of four virulence genes (Stx1, Stx2, ehxA, eae). Forty samples (5.0%) were STEC-positive. Of these, 38%, 25%, 19%, and 18% were positive for Stx1, Stx2, ehxA, and eae, respectively. Genotypically, we found that 1.37% of STEC isolates produced extended-spectrum beta-lactamase (ESBL) and contained the blaCTX M gene. Resistance to various antibiotics ranged from 18% to 77%. This study highlights the risk of virulent and multidrug-resistant STEC non-O157:H7 in raw milk and the need for proper quality surveillance and assurance plans to mitigate the potential public health threat.

Clonal spread of non-O157 Shiga toxigenic Escherichia coli O21:H25 in raw water buffalo milks

Abstract Aims This study was conducted to investigate the presence of Shiga toxin-producing O157 and non-O157 E. coli in raw water buffalo milk, as well as to determine the virulence gene profiles, phylogroups, sequence types, and serotypes of the isolated strains. Methods and results A total of 200 hand-milked raw water buffalo milk samples were collected from 200 different water buffaloes over a period of three months from 20 different farms. Isolation of STEC was performed using CHROMagar STEC. Presence of stx1, stx2, and eaeA genes were investigated by mPCR. Phylogroups and sequence types of E. coli strains were determined by Clermont phylotyping and MLST. Serotyping was performed using PCR or WGS. According to the results, two milk samples obtained from two different farms were found as STEC-positive. All Stx-positive E. coli isolates belonged to phylogenetic group A and were assigned to ST10. WGS results indicated that serotype of two isolates was O21:H25 and average nucleotide identity was detected at 99.99%. Thirteen additional registered E. coli O21:H25 assembled WGS data were obtained from EnteroBase and a phylogenetic tree was constructed. Conclusions With this study, the presence of stx2 harboring E. coli O21:H25 in milk was identified for the first time. Although the identified serotype is considered a non-pathogen seropathotype, we conclude it could play an important role in the environmental circulation of Stx-phages and consequently contribute to the emergence of new STEC-related outbreaks.

Detection of hemolytic Shiga toxin-producing Escherichia coli in fresh vegetables and efficiency of phytogenically synthesized silver nanoparticles by Syzygium aromaticum extract and gamma radiation against isolated pathogens

BackgroundShiga toxin-producing E. coli (STEC) is a major cause of foodborne diseases accompanied by several clinical illnesses in humans. This research aimed to isolate, identify, and combat STEC using novel alternative treatments, researchers have lately investigated using plant extract to produce nanoparticles in an environmentally acceptable way. At various gamma-ray doses, gamma irradiation is used to optimize the conditions for the biogenically synthesized silver nanoparticles (Ag NPs) using an aqueous extract of clove as a reducing and stabilizing agent.MethodsOn a specific medium, 120 vegetable samples were screened to isolate STEC and molecularly identified using real-time PCR. Moreover, the antibacterial and antibiofilm activities of biogenically synthesized Ag NPs against the isolated STEC were examined.ResultsTwenty-five out of 120 samples of eight types of fresh vegetables tested positive for E. coli, as confirmed by 16S rRNA, of which three were positive for the presence of Stx-coding genes, and six were partially hemolytic. Seven antibiotic disks were used to determine antibiotic susceptibility; the results indicated that isolate STX2EC had the highest antibiotic resistance. The results demonstrated that Ag NPs were highly effective against the STEC isolates, particularly the isolate with the highest drug resistance, with inhibition zones recorded as 19 mm for STX2EC, 11 mm for STX1EC1, and 10 mm for STX1EC2 at a concentration of 108 µg/mL. MICs of the isolates STX1EC1, and STX1EC2 were 13.5 µg/mL whereas it was detected as 6.75 µg/mL for STX2EC. The percentages of biofilm inhibition for STX1EC2, STX1EC1, and STX2EC, were 78.7%, 76.9%, and 71.19%, respectively.ConclusionThese findings suggest that the biogenic Ag NPs can be utilized as a new promising antibacterial agent to combat biofouling on surfaces.

Virulence and antimicrobial resistance of Shiga toxin-producing Escherichia coli from dairy goat and sheep farms in The Netherlands.

The aim of our study was to investigate the virulence and resistance of STEC from small ruminants farms in The Netherlands. Moreover, the potential transmission of STEC between animals and humans on farms was evaluated. From 182 farms, in total, 287 unique STEC isolates were successfully recovered from animal samples. In addition, STEC was isolated from eight out of 144 human samples. The most detected serotype was O146:H21; however, among other serotypes also O26:H11, O157:H7, and O182:H25 isolates were present. Whole genome sequencing covering all human isolates and 50 of the animal isolates revealed a diversity of stx1, stx2, and eae sub-types and an additional 57 virulence factors. The assessed antimicrobial resistance phenotype, as determined by microdilution, was concordant with the genetic profiles identified by WGS. WGS also showed that three of the human isolates could be linked to an animal isolate from the same farm. The obtained STEC isolates showed great diversity in serotype, virulence, and resistance factors. Further analysis by WGS allowed for an in-depth assessment of the virulence and resistance factors present and to determine the relatedness of human and animal isolates.

Antibiotic resistant shiga toxin producing Escherichia coli isolates from milk and milk products

Totally 450 milk and milk products (50 each of raw buffalo & cow milk; Kareish, Tallaga and Roquefort cheeses; yoghurt; rayeb; condensed milk and sour cream) samples were randomly purchased from various markets in Kafr El-Sheikh for isolation and identification of STEC. Results showed that 60%, 80%, 30%, 10%, 12%, 20%, 6% and 22% of buffalo & cow milk, Kareish, Roquefort, Tallaga, yoghurt, rayeb and sour cream respectively, contained E.coli, but negative in condensed milk. Fifty suspected isolates were serologically identified using E.coli O157:H7 kits, and confirmed in 69.2%, 53.8%, 60%, 16.7% and 20% isolates of buffalo & cow milk, Kareish, Tallaga and yoghurt, while was negative in Roqufort, rayeb, and sour cream. Using PCR assay, 21 confirmed isolates were examined, 44.4, 71.4, 66.7, 100 and 100% isolates of buffalo & cow milk, Kareish, Tallaga and yoghurt were positive for rfbE gene; while 55.6, 42.8, 66.7, 100 and 100 % isolates of buffalo & cow milk, Kareish, Tallaga and yoghurt were positive for blaTEM, but all isolates were negative for blaCMY2. Consequently, raw milk and most dairy products including fermented products were possible source of E.coli O157:H7food poisoning, so usage of accurate hygienic measures through manufacture and retail of milk products be essential.

Occurrence, Serotypes and Virulence Characteristics of Shiga-Toxin-Producing Escherichia coli Isolates from Goats on Communal Rangeland in South Africa.

Shiga-toxin-producing Escherichia coli is a foodborne pathogen commonly associated with human disease characterized by mild or bloody diarrhea hemorrhagic colitis and hemolytic uremic syndrome. This study investigated the occurrence of STEC in fecal samples of 289 goats in South Africa using microbiological culture and PCR. Furthermore, 628 goat STEC isolates were characterized by serotype (O:H) and major virulence factors by PCR. STEC was found in 80.2% (232/289) of goat fecal samples. Serotyping of 628 STEC isolates revealed 63 distinct serotypes including four of the major top seven STEC serogroups which were detected in 12.1% (35/289) of goats: O157:H7, 2.7% (8/289); O157:H8, 0.3%, (1/289); O157:H29, 0.3% (1/289); O103:H8, 7.6% (22/289); O103:H56, 0.3% (1/289); O26:H2, 0.3% (1/289); O111:H8, 0.3% (1/289) and 59 non-O157 STEC serotypes. Twenty-four of the sixty-three serotypes were previously associated with human disease. Virulence genes were distributed as follows: stx1, 60.6% (381/628); stx2, 72.7% (457/628); eaeA, 22.1% (139/628) and hlyA, 78.0% (490/628). Both stx1 and stx2 were found in 33.4% (210/628) of isolates. In conclusion, goats in South Africa are a reservoir and potential source of diverse STEC serotypes that are potentially virulent for humans. Further molecular characterization will be needed to fully assess the virulence potential of goat STEC isolates and their capacity to cause disease in humans.

Isolation and characterization of bacteriophage Ib_pec2 against Shigatoxigenic E.coli

The current study aimed to isolate and characterize bacteriophage against drug resistant, Shiga toxigenic E.coli, one of the zoonotic, food-borne organisms associated with ruminants, mainly cattle. STEC were isolated (n=35) from neonatal calves, dairy workers and the surrounding environment and their antimicrobial resistance pattern was studied. Out of the 35 isolates tested, 17 isolates were found to be multi-drug resistant to important antibiotics like as ampicillin, amoxicillin-clavulanate, ciprofloxacin, streptomycin and tetracycline. Bacteriophage namely Ib_pec2 was isolated against NM—18-040 isolate and its morphology, genetic and proteomic characterization was done. Morphological analysis by TEM revealed bacteriophage belonging to myoviridae family. The genetic characterization of g23 gene revealed that the bacteriophage belonged to Tequatrovirus of myoviridae family. SDS-PAGE analysis of structural proteins followed by LC-MS/MS analysis could able to identify five proteins identical to Tequatrovirus of myoviridae family. One step growth curve experiments revealed a latency period of 40 mins and a burst size of 893 PFU/ bacteria. Temperature and pH ranging from 40-50°C, pH 7-8, respectively were ideal for phage survival and multiplication. Phage was able to lyse 22 out of 35 STEC isolates. Thus, the study could able to characterize Ib_pec2 which could be used in control of STEC in the near future. STEC is a commensal organism in the gastrointestinal tract of ruminants but pathogenic in humans. Bacteriophages can be used as alternatives to antibiotics to control its growth in ruminants and prevent its further spillage in the environment.

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.

Potential Zoonotic Pathovars of Diarrheagenic Escherichia coli Detected in Lambs for Human Consumption from Tierra del Fuego, Argentina.

Diarrheagenic Escherichia coli (DEC) pathovars impact childhood health. The southern region of Argentina shows the highest incidence of hemolytic uremic syndrome (HUS) in children of the country. The big island of Tierra del Fuego (TDF) in Argentina registered an incidence of five cases/100,000 inhabitants of HUS in 2019. This work aimed to establish the prevalence of STEC, EPEC, and EAEC in lambs slaughtered in abattoirs from TDF as well as to characterize the phenotypes and the genotypes of the isolated pathogens. The prevalence was 26.6% for stx+, 5.7% for eae+, and 0.27% for aagR+/aaiC+. Twelve STEC isolates were obtained and belonged to the following serotypes: O70:HNT, O81:H21, O81:HNT, O102:H6, O128ab:H2, O174:H8, and O174:HNT. Their genotypic profiles were stx1c (2), stx1c/ehxA (3), stx2b/ehxA (1), stx1c/stx2b (2), and stx1c/stx2/ehxA (4). Six EPEC isolates were obtained and corresponded to five serotypes: O2:H40, O32:H8, O56:H6, O108:H21, and O177:H25. All the EPEC isolates were bfpA- and two were ehxA+. By XbaI-PFGE of 17 isolates, two clusters were identified. By antimicrobial susceptibility tests, 8/12 STEC and 5/6 EPEC were resistant to at least one antibiotic. This work provides new data to understand the ecology of DEC in TDF and confirms that ovine are an important carrier of these pathogens in the region.

Characterization of antimicrobial susceptibility, extended-spectrum \u03b2-lactamase genes and phylogenetic groups of Shigatoxin producing Escherichia coli isolated from patients with diarrhea in Iran

BackgroundShiga toxin‐producing Escherichia coli (STEC) are among common foodborne bacterial pathogens and healthy livestock are the main source of this bacterium. Severe diseases attribute to two types of cytotoxin Stx1 and Stx2, which are also called Shiga toxin (Stx). Infection of humans with STEC may result in Acute diarrhea with or without bleeding, hemorrhagic colitis (HC) and the hemolytic uremic syndrome (HUS). As antibiotic resistance is increasingly being reported among STEC isolates obtained from livestock and patients worldwide, in this study the pattern of antibiotic resistance in clinical isolates was determined.MethodsStool samples were collected from patients with diarrhea. All samples were cultured and identified by biochemical and molecular tests. Antimicrobial susceptibility test and assessment of extended-spectrum β-lactamase (ESBL)-related genes were conducted. Moreover, phylogenetic groups were analyzed using quadruplex PCR, and DNA analysis assessed multi-locus sequence types (MLST).ResultsOut of 340 E. coli samples, 174 were identified as STEC by PCR. Antimicrobial susceptibility test results showed that, 99.4%, 96% and 93.1% of isolates were susceptible to imipenem/ertapenem, piperacillin–tazobactam and amikacin, respectively. The highest resistance was towards ampicillin (68.4%), followed by trimethoprim–sulfamethoxazole (59.8%), and tetracycline (57.5%). A total of 106 (60.9%) isolates were multidrug resistance (MDR) and 40.8% of isolates were determined to be extended spectrum β-lactamase producers. In 94.4% of isolates, genes responsible for ESBL production could be detected, and blaTEM was the most prevalent, followed by blaCTX-M9. Furthermore, phylogenetic grouping revealed that majority of STEC strains belonged to Group C, followed by Groups E, B2 and A. MLST unveiled diverse ST types.ConclusionA periodical surveillance studies and thorough understanding of antibiotic resistant profiles in STEC isolates could help select effective antibiotic treatment for patients and develop strategies to effectively manage food contamination and human infections.

An interlaboratory study on the detection methods for enterotoxigenic Escherichia coli in vegetables using enterotoxin gene screening and selective agars for ETEC-specific isolation

Enterotoxigenic Escherichia coli (ETEC) causes acute diarrhea and is transmitted through contaminated food and water; however, systematic procedures for its specific detection in foods have not been established. To establish an efficient detection method for ETEC in food, an interlaboratory study using ETEC O148 and O159 as representative serogroups was first conducted with 13 participating laboratories. A series of tests including enrichment, real-time PCR assays, plating on selective agars, and concentration by immunomagnetic separation followed by plating onto selective agar (IMS-plating methods) were employed. This study particularly focused on the detection efficiencies of real-time PCR assays for enterotoxin genes (sth, stp, and lt), IMS-plating methods, and direct plating onto sorbitol MacConkey agar and CHROMagar STEC medium, supplemented with tobramycin, which is a novel modification in the preparation of a selective agar. Cucumber and leek samples inoculated with ETEC O148 and O159, either at 4–7 CFU/25 g (low levels) or at 21–37 CFU/25 g (high levels) were used as samples with uninoculated samples used as controls. At high inoculation levels, the sensitivities of sth, stp, and lt detection, direct-plating, and IMS-plating methods in cucumber inoculated with O148 and in both foods inoculated with O159 were 100%. In leek inoculated with high levels of O148, the sensitivities of sth, stp, and lt detection, direct-plating, and the IMS-plating method were 76.9%, 64.1%, and 74.4%, respectively. At low inoculation levels, the sensitivities of sth, stp, and lt detection, direct plating, and IMS-plating method in cucumber inoculated with O148 and in both foods inoculated with O159 were in the range of 87.2–97.4%. In leek inoculated with low levels of O148, the sensitivities of sth, stp, and lt detection, direct plating, and the IMS-plating method were 59.0%, 33.3%, and 38.5%, respectively. Thus, ETEC in food contaminated with more than 21 CFU/25 g were detected at high rate (over 74%) using real-time PCR assays and IMS-plating onto selective agar. Therefore, screening sth, stp, and lt genes followed by isolation of STEC using the IMS-plating method may be an efficient method for ETEC detection.

Occurrence, virulence genes, and antimicrobial profiles of Escherichia coli O157 isolated from ruminants slaughtered in Al Ain, United Arab Emirates

BackgroundShiga toxin-producing Escherichia coli (STEC) is a major source of food-borne illness around the world. E. coli O157 has been widely reported as the most common STEC serogroup and has emerged as an important enteric pathogen. Cattle, in particular have been identified as a major E. coli O157:H7 reservoir of human infections; however, the prevalence of this organism in camels, sheep, and goats is less understood. The aim of this study was to evaluate the occurrence and concentration of E. coli serotype O157 in the feces of healthy camels (n = 140), cattle (n = 137), sheep (n = 141) and goats (n = 150) slaughtered in United Arab Emirates (UAE) for meat consumption between September 2017 and August 2018. We used immunomagnetic separation coupled with a culture-plating method to detect E. coli O157. Non-sorbitol fermenting colonies were assessed via latex-agglutination testing, and positive cultures were analyzed by performing polymerase chain reactions to detect genes encoding attaching and effacing protein (eaeA), hemolysin A (hlyA, also known as ehxA) and Shiga toxin (stx1 and stx2), and E. coli O157:H7 specific genes (rfb O157, uidA, and fliC). All E. coli O157 isolates were analyzed for their susceptibility to 20 selected antimicrobials.ResultsE. coli O157 was observed in camels, goats, and cattle fecal samples at abundances of 4.3, 2, and 1.46%, respectively, but it was undetectable in sheep feces. The most prevalent E. coli O157 gene in all STEC isolates was stx2;, whereas, stx1 was not detected in any of the samples. The fecal samples from camels, goats, and cattle harbored E. coli O157 isolates that were 100% susceptible to cefotaxime, chloramphenicol, ciprofloxacin, norfloxacin, and polymyxin B.ConclusionTo our knowledge, this is the first report on the occurrence of E. coli O157 in slaughter animals in the UAE. Our results clearly demonstrate the presence of E. coli O157 in slaughtered animals, which could possibly contaminate meat products intended for human consumption.

Characterization of verotoxigenic E.coli and enteropathogenic E.coli isolated from infants with diarrhea in combination with antimicrobial resistance pattern in Minia, Egypt

Diarrheal disease is considered a significant health problem, principally in developing countries as it is one of the leading causes of morbidity and mortality especially in infants. Studies found that STES and EPEC strains are among the most predominant causative agents in acute diarrhea. The aim of the study was to investigate the presence of certain virulence factors and antibiotic resistances of STEC and EPEC originating from infants with diarrhea in Egypt. A total of 200 infants of different ages suffering from diarrhea between August 2017 and February 2019 were registered in this study. Standard cultural and biochemical methods were used to isolate E. coli species while; PCR was applied for the detection of virulence genes (stx1, stx2, eae and pfb A) for STEC and EPEC isolates, respectively. Eighty nine (44.5%) E. coli strains were isolated from 200 stool samples. A total of 40 (44.9%) strains were identified as STEC, and 1(1.12%) strain was atypical EPEC. Fourteen of the STEC serotypes were O157:H7. Susceptibilities of 40 STEC and 1 aEPEC isolates were determined for ten antimicrobial drugs. STEC O157:H7 strains and STEC Non-O157:H7 exhibited the highest resistance against cefazolin and no resistance against imipnem and Amikacin, respectively while, the isolated strain of aEPEC was sensitive to all the studied antibiotics except cefazolin. Results showed that STEC is one of the major causes of diarrhea that showed resistant to commonly used antibiotics so representatives must pay great attention to this matter in order to increase the health of the community.

Comprehensive evaluation and implementation of improvement actions in bovine abattoirs to reduce pathogens exposure

The slaughter process plays an important role in animal welfare, meat quality, safety and public health through the meat production chain. In this study, we performed a three-stage evaluation: I) comprehensive evaluation, II) implementation of improvement actions and III) verification of the success of the actions implemented in three abattoirs from Argentina during 2016-2018. Risk was estimated using two checklists, quantified on a 1–100 scale and classified as high (1–40), moderate (41–70) and low (71–100). In stages I and III, Salmonella spp., E. coli O157:H7 and non-O157 STEC were detected and isolated in samples from carcasses (n = 252), the environment (n = 252); head meat (n = 21) and viscera washing and chilling water (n = 105). Carcass samples were analyzed for mesophilic aerobic organisms, coliforms and E. coli enumeration. Of 201 water samples taken, 42.0–75.6 % were non-potable quality. After the implementation of improvement actions in stage II (building, processes, systems for water purification and training), the estimation of risk of contamination was reduced from high to moderate in all three abattoirs, the count of indicator microorganisms decreased in two abattoirs, and the presence of pathogens significantly decreased. Salmonella spp. was not isolated from any of the samples collected in two abattoirs. Isolation of E. coli O157:H7 decreased in carcass and was not isolated from viscera washing and chilling water. Isolation of non-O157 STEC decreased in carcass but not in environmental samples. Finally, 75.0–95.0 % of water samples were of potable quality. Although this was only the first step in the process of change and improvement of abattoirs, the assessment of the situation and the proposal of solutions to correct deviations in a joint effort with the health authorities helped to implement a work model for enhancing food safety before meat reaches consumers.

O-serogroups, virulence genes, antimicrobial susceptibility, and MLST genotypes of Shiga toxin-producing Escherichia coli from swine and cattle in Central China

BackgroundShiga toxin-producing Escherichia coli (STEC) is a leading cause of worldwide food-borne and waterborne infections. Despite an increase in the number of STEC outbreaks, there is a lack of data on prevalence of STEC at the farm level, distribution of serogroups, and virulence factors.ResultsIn the present study, a total of 91 (6.16%) STEC strains were isolated from 1477 samples including pig intestines, pig feces, cattle feces, milk, and water from dairy farms. The isolation rates of STEC strains from pig intestines, pig feces, and cattle feces were 7.41% (32/432), 4.38% (21/480), and 9.57% (38/397), respectively. No STEC was isolated from the fresh milk and water samples. By O-serotyping methods, a total of 30 types of O-antigens were determined, and the main types were O100, O97, O91, O149, O26, O92, O102, O157, and O34. Detection of selected virulence genes (stx1, stx2, eae, ehxA, saa) revealed that over 94.51% (86/91) of the isolates carried more than two types of virulence associated genes, and approximately 71.43% (65/91) of the isolates carried both stx1 and stx2, simultaneously. Antimicrobial susceptibility tests showed that most of the STEC isolates were susceptible to ofloxacin and norfloxacin, but showed resistance to tetracycline, kanamycin, trimethoprim-sulfamethoxazole, streptomycin, amoxicillin, and ampicillin. MLST determined 13 categories of sequence types (STs), and ST297 (31.87%; 29/91) was the most dominant clone. This clone displayed a close relationship to virulent strains STEC ST678 (O104: H4). The prevalence of ST297 clones should receive more attentions.ConclusionsOur preliminary data revealed that a heterogeneous group of STEC is present, but the non-O157 serogroups and some ST clones such as ST297 should receive more attentions.

High prevalence of non-O157 Shiga toxin-producing Escherichia coli in beef cattle detected by combining four selective agars

BackgroundShiga toxin-producing Escherichia coli (STEC) are emerging foodborne pathogens that are public health concern. Cattle have been identified as the major STEC reservoir. In the present study, we investigated the prevalence and characteristics of STEC strains in beef cattle from a commercial farm in Sichuan province, China.ResultsAmong 120 beef cattle fecal samples, stx genes were positive in 90% of samples, as assessed using TaqMan real-time PCR, and 87 (72.5%) samples were confirmed to yield at least one STEC isolate by culture using four selective agars, MacConkey, CHROMagar™ ECC, modified Rainbow® Agar O157, and CHROMagar™ STEC, from which 31, 32, 91, and 73 STEC strains were recovered, respectively. A total of 126 STEC isolates were selected and further characterized. Seventeen different O:H serotypes were identified, all of which belonged to the non-O157 serotypes. One stx1 subtype (stx1a) and three stx2 subtypes (stx2a, stx2c, and stx2d) were present among these isolates. The intimin encoding gene eae, and other adherence-associated genes (iha, saa, and paa) were present in 37, 125, 74, and 30 STEC isolates, respectively. Twenty-three isolates carried the virulence gene subA, and only one harbored both cnf1 and cnf2 genes. Three plasmid-origin virulence genes (ehxA, espP, and katP) were present in 111, 111, and 7 isolates, respectively. The 126 STEC isolates were divided into 49 pulsed-field gel electrophoresis (PFGE) patterns.ConclusionsOur study showed that the joint use of the selective MacConkey and modified Rainbow® Agar O157 agars increased the recovery frequency of non-O157 STEC strains in animal feces, which could be applied to other samples and in regular STEC surveillance. Moreover, the results revealed high genetic diversity of non-O157 STEC strains in beef cattle, some of which might have the potential to cause human diseases.