Virulence Genes eaeA Research Articles

Biochemical characteristics of E. coli isolated from food of animal origin

Food of animal origin is a potential source of pathogenic E. coli that are dangerous to humans. While most strains are intestinal commensals, some can cause intestinal and extraintestinal infections. Their pathogenicity is associated with the presence of virulence genes, phylo-group and, in some strains, biochemical characteristics. In this study, a total of 61 E. coli isolates from meat, minced meat, meat preparations and carcass swabs from different animal species were analysed. The biochemical properties of the strains were determined using VITEK2 system and the data was compared with the presence of virulence genes from previous studies. The results showed a correlation between the presence of the eae virulence gene and the alkalinization of succinate. In addition, the presence of the virulence gene cnf1 was correlated with the enzyme tyrosine arylamidase, though the correlation between biochemical characteristics and the presence of virulence genes is questionable due to the small sample size and more strains need to be tested for further conclusions. Although no significant correlations were found between biochemical characteristics and the presence of virulence genes, this study describes the diversity of E. coli in the Republic of Croatia. The collected data also reveal new insights into biochemical characteristics of E. coli isolated from domestic and wild animals in the Republic of Croatia.

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

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

Extended Spectrum β-Lactamase-Producing Escherichia coli from Poultry and Wild Birds (Sparrow) in Djelfa (Algeria), with Frequent Detection of CTX-M-14 in Sparrow.

Antimicrobial resistance is a global threat that is spreading more and more in both human and animal niches. This study investigates the antimicrobial resistance and virulence threats of Escherichia coli isolates recovered from intestinal and fecal samples of 100 chickens, 60 turkeys, and 30 sparrows. Extended spectrum β-lactamase (ESBL) producing E. coli isolates were recovered in 12 of the animals tested, selecting one isolate per positive animal: sparrow (eight isolates, 26.7%), turkey (three isolates, 5%), and chicken (one isolate, 1%). The E. coli isolates were ascribed to B1 and D phylogenetic groups. The blaCTX-M-14 gene was detected in all ESBL-producing E. coli isolates from sparrow. The blaCTX-M-15 (two isolates) and blaCTX-M-14 genes (one isolate) were detected in the isolates of turkey, and the blaCTX-M-1 gene in one isolate from broiler. Three lineages were revealed among the tested isolates (ST/phylogenetic group/type of ESBL/origin): ST117/D/CTX-M-1/broiler, ST4492 (CC405)/D/CTX-M-15/turkey, and ST602/B1/CTX-M-14/sparrow. All isolates were negative for stx1, sxt2, and eae virulence genes. Our findings provide evidence that the sparrow could be a vector in the dissemination of ESBL-producing E. coli isolates to other environments. This study also reports, to our knowledge, the first detection of blaCTX-M-14 from sparrow at a global level and in turkey in Algeria.

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.

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.

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.

EHEC O111:H8 strain and norovirus GII.4 Sydney [P16] causing an outbreak in a daycare center, Brazil, 2019

BackgroundThis study describes the investigation of an outbreak of diarrhea, hemorrhagic colitis (HC), and hemolytic uremic syndrome (HUS) at a daycare center in southeastern Brazil, involving fourteen children, six staff members, six family members, and one nurse. All bacterial and viral pathogens detected were genetically characterized.ResultsTwo isolates of a strain of enterohemorrhagic Escherichia coli (EHEC) serotype O111:H8 were recovered, one implicated in a case of HUS and the other in a case of uncomplicated diarrhea. These isolates had a clonal relationship of 94% and carried the stx2a and eae virulence genes and the OI-122 pathogenicity island. The EHEC strain was determined to be a single-locus variant of sequence type (ST) 327. EHEC isolates were resistant to ofloxacin, doxycycline, tetracycline, ampicillin, and trimethoprim-sulfamethoxazole and intermediately resistant to levofloxacin and ciprofloxacin. Rotavirus was not detected in any samples, and norovirus was detected in 46.7% (14/30) of the stool samples, three of which were from asymptomatic staff members. The noroviruses were classified as the recombinant GII.4 Sydney [P16] by gene sequencing.ConclusionIn this outbreak, it was possible to identify an uncommon stx2a + EHEC O111:H8 strain, and the most recent pandemic norovirus strain GII.4 Sydney [P16]. Our findings reinforce the need for surveillance and diagnosis of multiple enteric pathogens by public health authorities, especially during outbreaks.

Pathogenic potential of Escherichia coli O157 and O26 isolated from young Belgian dairy calves by recto-anal mucosal swab culturing.

The purpose of this study was to investigate the occurrence of Escherichia coli O157 and O26 on Belgian dairy cattle farms, the presence of virulence genes in the confirmed isolates and the association of E. coli O26 presence with calf diarrhoea. In total, 233 recto-anal mucosal swabs (RAMS) were obtained from healthy and diarrheic dairy calves on three farms, each alternately visited three consecutive times. RAMS were analysed for presence of E. coli O157 and O26, and stx1, stx2 and eae virulence genes. Overall, 19% of RAMS tested positive for E. coli O157, while 31% tested positive for E. coli O26. The majority of isolates possessed both stx and eae, denoting a high pathogenic potential to humans. While both serogroups persisted at farm level, persistence within the same animal over time appeared to be relatively rare. Interestingly, E. coli O26 was already abundantly present at a younger age compared to E. coli O157. Calf diarrhoea could not be associated with presence of E. coli O26. Young dairy calves are important on-farm reservoirs of potentially pathogenic E. coli O157 and O26. A role of E. coli O26 in calf diarrhoea could not be confirmed. O157 and O26 are responsible for the majority of human STEC infections. Gaining more epidemiological information regarding their occurrence and persistence on cattle farms will contribute to a better understanding of STEC ecology and risk of human transmission.

Molecular characterisation of antimicrobial resistance and virulence genes in Escherichia coli strains isolated from diarrhoeic and healthy rabbits in Tunisia

The purpose of this study was to identify &lt;em&gt;Escherichia coli&lt;/em&gt; isolates in diarrhoeic and healthy rabbits in Tunisia and characterise their virulence and antibiotic resistance genes. In the 2014-2015 period, 60 faecal samples from diarrhoeic and healthy rabbits were collected from different breeding farms in Tunisia. Susceptibility to 14 antimicrobial agents was tested by disc diffusion method and the mechanisms of gene resistance were evaluated using polymerase chain reaction and sequencing methods. Forty &lt;em&gt;E. coli&lt;/em&gt; isolates were recovered in selective media. High frequency of resistance to tetracycline (95%) was detected, followed by different levels of resistance to sulphonamide (72.5%), streptomycin (62.5%), trimethoprim-sulfamethoxazole (60%), nalidixic acid (32.5%), ampicillin (37.5%) and ticarcillin (35%). &lt;em&gt;E. coli&lt;/em&gt; strains were susceptible to cefotaxime, ceftazidime and imipenem. Different variants of bla&lt;sub&gt;TEM&lt;/sub&gt;, &lt;em&gt;tet&lt;/em&gt;, &lt;em&gt;sul&lt;/em&gt; genes were detected in most of the strains resistant to ampicillin, tetracycline and sulphonamide, respectively. The presence of class 1 integron was studied in 29 sulphonamide-resistant &lt;em&gt;E. coli&lt;/em&gt; strains from which 15 harboured class 1 integron with four different arrangements of gene cassettes, &lt;em&gt;dfrA17&lt;/em&gt;+&lt;em&gt;aadA5&lt;/em&gt; (n=9), &lt;em&gt;dfrA1&lt;/em&gt; + &lt;em&gt;aadA1&lt;/em&gt; (n=4), &lt;em&gt;dfrA12&lt;/em&gt; + &lt;em&gt;addA2&lt;/em&gt; (n=1), &lt;em&gt;dfrA12&lt;/em&gt;+&lt;em&gt;orf&lt;/em&gt;+&lt;em&gt;addA2&lt;/em&gt; (n=1). The &lt;em&gt;qnrB&lt;/em&gt; gene was detected in six strains out of 13 quinolone-resistant &lt;em&gt;E. coli&lt;/em&gt; strains. Seventeen &lt;em&gt;E. coli&lt;/em&gt; isolates from diarrhoeic rabbits harboured the enteropathogenic eae genes associated with different virulence genes tested (&lt;em&gt;fimA&lt;/em&gt;, &lt;em&gt;cnf1&lt;/em&gt;, &lt;em&gt;aer&lt;/em&gt;), and affiliated to B2 (n=8) and D (n=9) phylogroups. Isolated &lt;em&gt;E. coli&lt;/em&gt; strains from healthy rabbit were harbouring &lt;em&gt;fim A&lt;/em&gt; and/or &lt;em&gt;cnf1&lt;/em&gt; genes and affiliated to A and B1 phylogroups. This study showed that &lt;em&gt;E. coli&lt;/em&gt; strains from the intestinal tract of rabbits are resistant to the widely prescribed antibiotics in medicine. Therefore, they constitute a reservoir of antimicrobial-resistant genes, which may play a significant role in the spread of antimicrobial resistance. In addition, the eae virulence gene seemed to be implicated in diarrhoea in breeder rabbits in Tunisia.

Regional differences in presence of Shiga toxin-producing Escherichia coli virulence-associated genes in the environment in the North West and East Anglian regions of England.

Shiga toxin-producing Escherichia coli is carried in the intestine of ruminant animals, and outbreaks have occurred after contact with ruminant animals or their environment. The presence of STEC virulence genes in the environment was investigated along recreational walking paths in the North West and East Anglia regions of England. In all, 720 boot sock samples from walkers' shoes were collected between April 2013 and July 2014. Multiplex PCR was used to detect E. coli based on the amplification of the uidA gene and investigate STEC-associated virulence genes eaeA, stx1 and stx2. The eaeA virulence gene was detected in 45·5% of the samples, where stx1 and/or stx2 was detected in 12·4% of samples. There was a difference between the two regions sampled, with the North West exhibiting a higher proportion of positive boot socks for stx compared to East Anglia. In univariate analysis, ground conditions, river flow and temperature were associated with positive boot socks. The detection of stx genes in the soil samples suggests that STEC is present in the English countryside and individuals may be at risk for infection after outdoor activities even if there is no direct contact with animals. SIGNIFICANCE AND IMPACT OF THE STUDY: Several outbreaks within the UK have highlighted the danger of contracting Shiga toxin-producing Escherichia coli from contact with areas recently vacated by livestock. This is more likely to occur for STEC infections compared to other zoonotic bacteria given the low infectious dose required. While studies have determined the prevalence of STEC within farms and petting zoos, determining the risk to individuals enjoying recreational outdoor activities that occur near where livestock may be present is less researched. This study describes the prevalence with which stx genes, indicative of STEC bacteria, were found in the environment in the English countryside.

Survey of Intact and Nonintact Raw Pork Collected at Retail Stores in the Mid-Atlantic Region of the United States for the Seven Regulated Serogroups of Shiga Toxin–Producing Escherichia coli

A total of 514 raw pork samples (395 ground or nonintact and 119 intact samples) were purchased at retail stores in Pennsylvania, Delaware, and New Jersey between July and December 2017. All raw pork samples were screened for serogroup O26, O45, O103, O111, O121, O145, or O157:H7 cells of Shiga toxin-producing Escherichia coli (STEC-7) using standard microbiological and molecular methods. In short, 21 (5.3%) of the 395 ground or nonintact pork samples and 3 (3.4%) of the 119 intact pork samples tested positive via the BAX system real-time PCR assay for the stx and eae virulence genes and for the somatic O antigens for at least one of the STEC-7 serogroups. However, none of these 24 presumptive-positive pork samples subsequently yielded a viable isolate of STEC displaying a STEC-7 serogroup-specific surface antigen in combination with the stx and eae genes. These data suggest that cells of STEC serogroups O26, O45, O103, O111, O121, O145, or O157:H7 are not common in retail raw pork samples in the mid-Atlantic region of the United States.

The epidemiology of Shiga toxin-producing Escherichia coli infections in the South East of England: November 2013–March 2017 and significance for clinical and public health

This study describes the epidemiology of Shiga toxin-producing Escherichia coli (STEC) infections in a population in the South East of England. From 1 November 2013 to 31 March 2017 participating diagnostic laboratories reported Shiga toxin gene (stx) positive real-time PCR results to local public health teams. Stx positive faecal samples/isolates were referred to the Gastrointestinal Bacteria Reference Unit (GBRU) for confirmation by culture and typing by whole genome sequencing (WGS). Key clinical information was collected by public health teams.Results/Key findings. Altogether, 548 faecal specimens (420 were non-travel associated) were stx positive locally, 535 were submitted to the GBRU. STEC were isolated from 42 %, confirmed by stx PCR in 21 % and 37 % were PCR negative. The most common non-travel associated STEC serogroups were O157, O26, O146 and O91. The annualized incidence of confirmed STEC infections (PCR or culture) was 5.8 per 100 000. The ratio of O157 to non-O157 STEC serogroups was 1:7. The annualized incidence of non-O157 haemolytic uraemic syndrome-associated Escherichia coli (HUSEC) strains was 0.4 per 100 000. Bloody diarrhoea was reported by 58 % of cases infected with E. coli O157, 33 % of cases infected with non-O157 HUSEC strains and 12 % of other lower risk non-O157 strains. Overall, 76 % of non-O157 HUSEC isolates possessed the eae virulence gene. HUSEC including serogroup O157 were uncommon and more likely to cause bloody diarrhoea than other STEC. The routine use of stx PCR testing can influence clinical management. Understanding the local epidemiology facilitates a proportionate public health response to STEC, based on clinical and microbiological characteristics including stx subtype(s).

Prevalence of Shiga toxin-producing Escherichia coli in pasture-based dairy herds.

Shiga toxin-producing Escherichia coli strains (STEC) are food-borne pathogens. While E.coli O157:H7 is commonly associated with cattle, less is known about the prevalence of non-O157 STEC serogroups in bovines. This study evaluated the prevalence and virulence status of O157:H7 and six E.coli O-serogroups (O26, O103, O45, O145, O121, O111) in New Zealand dairy farms using molecular as well as culture-based methods. Fresh farm dairy effluent (FDE) (n=36) and composite calf faeces (n=12) were collected over three samplings from 12 dairy farms. All seven target serogroups were detected through molecular techniques. Of the 202 isolates which were serologically confirmed following traditional culturing and immunomagnetic separation (IMS), O103, O26, O45 and O121 were the most common serogroups, being found in 81, 47, 42 and 32% of the FDE and in 17, 33, 25 and 9% of the calf faeces respectively. The majority (157/202) of the isolates were negative for stx and eae virulence genes. The prevalence of the seven target STEC was low, and only nine O26 isolates (4%) were recovered from four of the farms. The study has highlighted the need for improving the isolation of Top 7 STEC from the stx-negative populations present in fresh dairy effluent and calf faeces. SIGNIFICANCE AND IMPACT OF THE STUDY: Shiga toxin-producing Escherichia coli (STEC) are important food-borne pathogens that can cause severe illness in humans. Cattle are asymptomatic reservoirs for STEC, and transmission to humans can be by consumption of food products or water contaminated with cattle faeces. Our study investigated the prevalence of O157:H7 and six E.coli serogroups of STEC (O26, O103, O45, O145, O121, O111) over time in the dairy reservoir and increases the knowledge and understanding of these pathogens on pasture-based farms. Such information is required to develop risk-assessment models aiming at limiting transmission of these STEC to human.

Use of pulsed field gel electrophoresis genetic typing for tracing contamination with virulent Escherichia coli O157:H7 in beef-cattle producing farms

E. coli O157:H7 is an important causative agent of a variety of foodborne infections including hemorrhagic colitis (HC), hemolytic uremic syndrome (HUS) and thrombotic thrombocytopenic purpura (TTP) that may lead to renal failure in humans. The main source of infection is contaminated food of animal origin and a number of animal species especially cattle have been associated with this pathogen. Contamination usually occurs when faeces of animals get into contact with carcass during slaughtering. The aim of the present study was to determine the virulence profiles and genetic relationships of E. coli O157:H7 strains isolated from cattle some commercial farms in the North West Province, South Africa. A total of 260 faecal samples were collected from eight farms over a six-month period and assessed for the presence of E. coli O157:H7 through PCR analysis. Bacteria 16S rRNA gene fragments were amplified as an internal control while the rfbO157 and flicH7 gene PCR were used to confirm identities of isolates. STEC virulence genes stx1, stx2, eaeA and the hlyA were detected using PCR and the genetic relatedness of the isolates was determined using Pulse Field Gel electrophoresis (PFGE). A total of 69 (26.5%) E. coli O157:H7 isolates were detected in samples obtained from all the eight farms. A large proportions of the E. coli isolates possessed the stx1 (66.7%) and stx2 (97.1%) genes respectively. In addition, other accessory virulence genes eaeA and hlyA were detected in 30 (43.5%) and 43 (62.3%) of the E. coli isolates respectively. Pulsed-Filed Gel Electrophoresis (PFGE) analysis grouped these isolates into 6 clusters and the largest cluster contained 17 isolates originating from different farms. These results provide valid evidence that cattle harbour virulent and genetically similar E. coli strains particularly those belonging to the serotype O157 and therefore these animals may serve as possible source for zoonotic transfer of these pathogens to humans. The implication is that these isolates may have severe public health consequences on consumers.

Molecular Approach in Detection of Enterovirulent Escherichia coli Strains from Traditional Shrimp Farms of Bangladesh

DOI: 10.20286/ajlsr-040288 This study was conducted to detect the presence of enterovirulent Escherichia coli strains using PCR technique in some selected unhygienic and hygienic traditional shrimp farms in Bangladesh. The target genes chosen for this investigation included: the phoa housekeeping gene (present in all E. coli ); the lt1, lt2 and st1 genes of ETEC; the vtverotoxin, and eae virulence genes of EHEC and EPEC, respectively. Six pairs of oligonucleotide primers were designed to amplify internal fragments of these genes by PCR to generate PCR products that were analyzed by gel electrophoresis. The presence of E. coli strains were determined by visualization of strain specific band comparing with a 100 bp DNA size marker in UV transilluminator. The results revealed that water of both hygienic and unhygienic farms were highly contaminated with E. coli followed by sediment and shrimp. Whereas, only water, sediment and shrimp of unhygienic farms were found to be contaminated with virulent E. coli strains. Among the virulent E. coli strains, ETEC and EPEC strains were detected in unhygienic farms, but no EHEC strain was detected. However, no contamination with virulent E. coli strains was found in hygienic farms.

Identification of Shiga toxin-producing (STEC) and enteropathogenic (EPEC) Escherichia coli in diarrhoeic calves and comparative genomics of O5 bovine and human STEC

Escherichia coli producing Shiga toxins (Stx) and the attaching-effacing (AE) lesion (AE-STEC) are responsible for (bloody) diarrhoea in humans and calves while the enteropathogenic E. coli (EPEC) producing the AE lesion only cause non-bloody diarrhoea in all mammals. The purpose of this study was (i) to identify the pathotypes of enterohaemolysin-producing E. coli isolated between 2009 and 2013 on EHLY agar from less than 2 month-old diarrhoeic calves with a triplex PCR targeting the stx1, stx2, eae virulence genes; (ii) to serotype the positive isolates with PCR targeting the genes coding for ten most frequent and pathogenic human and calf STEC O serogroups; and (iii) to compare the MLSTypes and virulotypes of calf and human O5 AE-STEC after Whole Genome Sequencing using two server databases (www.genomicepidemiology.org). Of 233 isolates, 206 were triplex PCR-positive: 119 AE-STEC (58%), 78 EPEC (38%) and 9 STEC (4%); and the stx1+eae+ AE-STEC (49.5%) were the most frequent. Of them, 120 isolates (84% of AE-STEC, 23% of EPEC, 22% of STEC) tested positive with one O serogroup PCR: 57 for O26 (47.5%), 36 for O111 (30%), 10 for O103 (8%) and 8 for O5 (7%) serogroups. The analysis of the draft sequences of 15 O5 AE-STEC could not identify any difference correlated to the host. As a conclusion, (i) the AE-STEC associated with diarrhoea in young calves still belong to the same serogroups as previously (O5, O26, O111) but the O103 serogroup may be emerging, (ii) the O5 AE-STEC from calves and humans are genetically similar.

Studies on E. Coli Isolated from Newly Weaned Rabbits in Ismailia Governorate

This study was planned to gain more about E. coli causing diarrhea in newly weaned rabbits in Ismailia governorate. A total of (192) rectal swab was collected from newly weaned dirrhoiec rabbits at different seasonal period extended from (April 2014 –March 2015) to examine the presence of E. coli. These isolates were further characterized by biochemical tests and polymerase chain reaction. E. coli was isolated in 84 of the 192 samples (43.75%). Seasonal prevalence of Escherichia coli infection revealed that the highest incidence occurred in summer season 29/84 (65.9%). Followed by spring 21/84(40.4%), then in winter 15/84 (32.6%), and finally in autumn 19/84 (38%). The isolated E. coli strain were found to belong to O stereotypes in order of frequency O158, O128, O125, O27, O18, O20, O148 and Untypable (20%, 13.33%, 13.33 13.33%, 6.67%, 6.67%, 6.67%, and 20% isolates respectively). Based on PCR, All examined E. coli were positive100 %( 7/7) for eae virulence gene, while 87.8 %( 6/7) of the tested E. coli isolates were positive to tsh gene. It was concluded that Combination of genotypic with phenotypic analysis of E. coli isolates is more valuable as an epidemiological tool for differentiation and identification of the isolates than one test alone.

Microbiological quality of water from the rivers of Curitiba, Paraná State, Brazil, and the susceptibility to antimicrobial drugs and pathogenicity of Escherichia coli.

Water safety is determined by several markers, and Escherichia coli is one of the most important indicators of water quality. The objective of this study was to evaluate the microbiological parameters in environmental samples of fresh water from rivers of Curitiba and its metropolitan area in Paraná State, Brazil. In addition, we evaluated the pathogenicity and susceptibility to antimicrobial drugs in E. coli. These evaluations were performed by quantitative and qualitative methods employing selective media for isolating thermotolerant coliforms and biochemical tests for identifying E. coli. Pathogenic strains of E. coli were detected by PCR multiplex using specific primers. From the water samples, 494 thermotolerant coliforms were obtained, of which 96 (19.43%) isolates were characterized as E. coli. Three isolates were identified as enteroaggregative E. coli, one as enterotoxigenic E. coli, one as enteropathogenic E. coli, and two carried the Eae virulence gene. E. coli susceptibility to commonly employed antimicrobial drugs was analyzed by the disc diffusion method. The results showed 49 (51.04%) isolates resistant to all the drugs assayed, 16 (16.67%) with an intermediate resistance to all drugs, and 31 (32.29%) intermediately or fully resistant to one or more drugs tested. The highest rate of resistance was observed for tetracycline 30 μg, streptomycin 10 μg, and ceftazidime 30 μg. Detection of E. coli is associated with water contamination by fecal material from humans and warm-blooded animals. The occurrence of resistant strains can be the result of the indiscriminate use of antimicrobial drugs and poor sanitation in the areas assayed.

Evaluation of a Multiplex Real-Time PCR Method for Detecting Shiga Toxin–Producing Escherichia coli in Beef and Comparison to the U.S. Department of Agriculture Food Safety and Inspection Service Microbiology Laboratory Guidebook Method

The "top-six" non-O157 Shiga toxin-producing Escherichia coli (STEC) serogroups (O26, O45, O103, O111, O121, and O145) most frequently associated with outbreaks and cases of foodborne illnesses have been declared as adulterants in beef by the U.S. Department of Agriculture Food Safety and Inspection Service (FSIS). Regulatory testing in beef began in June 2012. The purpose of this study was to evaluate the DuPont BAX System method for detecting these top six STEC strains and strains of E. coli O157:H7. For STEC, the BAX System real-time STEC suite was evaluated, including a screening assay for the stx and eae virulence genes and two panel assays to identify the target serogroups: panel 1 detects O26, O111, and O121, and panel 2 detects O45, O103, O145. For E. coli O157:H7, the BAX System real-time PCR assay for this specific serotype was used. Sensitivity of each assay for the PCR targets was ≥1.23 × 10(3) CFU/ml in pure culture. Each assay was 100% inclusive for the strains tested (20 to 50 per assay), and no cross-reactivity with closely related strains was observed in any of the assays. The performance of the BAX System methods was compared with that of the FSIS Microbiology Laboratory Guidebook (MLG) methods for detection of the top six STEC and E. coli O157:H7 strains in ground beef and beef trim. Generally, results of the BAX System method were similar to those of the MLG methods for detecting non-O157 STEC and E. coli O157:H7. Reducing or eliminating novobiocin in modified tryptic soy broth (mTSB) may improve the detection of STEC O111 strains; one beef trim sample inoculated with STEC O111 produced a negative result when enriched in mTSB with 8 mg/liter novobiocin but was positive when enriched in mTSB without novobiocin. The results of this study indicate the feasibility of deploying a panel of real-time PCR assay configurations for the detection and monitoring of the top six STEC and E. coli O157:H7 strains in beef. The approach could easily be adapted for additional multiplex assays should regulations expand to include other O serogroups or virulence genes.