Virulence Factors Stx1 Research Articles

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

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

Research and molecular characteristic of Shiga toxin-producing Escherichia coli isolated from sheep carcasses.

Domestic ruminants are regarded as the major reservoir of Shiga toxin-producing Escherichia coli (STEC) closely related to human infection. A total of 363 ovine carcasses were swabbed in an Algiers city slaughterhouse for research on STEC. First of all, screening of the STECs was carried out by a multiplex PCR searching for the genes coding for the virulence factors stx1 , stx2 and eae. This step was followed by STEC isolation and serotyping. The presence of stx+ /stx+ eae+ genes was shown in 116 sheep carcasses (31·95%). From the 116 positive samples, 20 bacterial strains (17·24%) were isolated. Nineteen strains belonged to the species E.coli (STEC), and 1 belonged to Citrobacter braakii (eae+ stx1 + ). During this study, the presence of potentially pathogenic STEC for humans on the surface of sheep carcasses was confirmed. Corrective measures should be considered at the slaughterhouse level to avoid outbreaks of STEC in Algeria. SIGNIFICANCE AND IMPACT OF THE STUDY: PCR screening revealed the significant presence of the genetic markers of Shiga toxin-producing Escherichia coli (STEC) (stx+ /stx+ eae+ ) on the surfaces of sheep carcasses. Citrobacter braakii (stx1 + eae+ ) was isolated for the first time in this study. The risk of foodborne diseases due to STEC must be taken into account in Algeria. To prevent the emergence of epidemic outbreaks among children and older by people, preventive measures should be taken.

Identification of Virulence Factors of Escherichia coli Isolates from Fecal Samples of Calves in Southern Brazil

Background: Escherichia coli (E. coli) is an enteropathogen that commonly causes diarrhea in calves. However, not all E. coli isolates are pathogenic. The aim of this study was to identify E. coli virulence factors derived from fecal samples collected in the southern region of Rio Grande do Sul state (RS) from calves with and without diarrhea, as well as investigate the antimicrobial susceptibility of E. coli isolates from calves with diarrhea.Materials, Methods & Results: Forty stool samples were collected in 12 farms, each one from calves having one day to six months of age, with and without diarrhea. The total DNA of from these isolates was extracted and a PCR using primers specific for the virulence factors Stx1, Eae, F41, F5 and STa was conducted. The susceptibility testing used the disk diffusion method and the susceptibility profile was evaluated against the following antimicrobials: ampicillin, penicillin, chloramphenicol, enrofloxacin, gentamicin, trimethoprim, sulfonamide, tetracycline and streptomycin. From all calves, 15 (15/40, 37.5%) had diarrheal stools and 25 (25/40, 62.5%) had normal or semi-liquid stools. Twelve (12/40; 30%) E. coli isolates showed at least one virulence factor. These factors were found in four isolates (4/15; 26.6%) from diarrheal stools and eight isolates (8/25; 28.5%) from normal stool. The Stx1 factor was identified in five isolates (5/40; 12.5%), and the Eae and the Sta factors in one (1/40; 0.2%) and in atypical associations between Stx1 and Eae and also between Eae and F41 in two isolates (2/40; 0.5%). Also, the Eae and Sta factors were identified in one isolate (1/40; 0.2%). The susceptibility test showed resistance to penicillin and tetracycline in 93% and 80% of the tested isolates, respectively.Discussion: The identification of virulence factors is necessary because E. coli is an enterobacterium present in calves gastrointestinal tract, to prove its pathogenicity. The virulence factor most commonly found in E. coli isolates derived from feces of calves with and without diarrhea in the southern region of the RS was the Stx1 (Shiga toxin-producing E. coli STEC). It is likely that the highest occurrence of E. coli isolates positive for the Stx1 virulence factor was due to the fact that cattle were the main reservoirs of this type of bacteria. The occurrence of enterohemorrhagic E. coli (EHEC) in animals of nine and 34 days of life, respectively, is highlighted. Studies have shown that contamination of animal foods with EHEC can cause enteric disorders, hemorrhagic colitis, and uremic hemolytic syndrome (UHS) in humans. Although in the present study the identification of the Stx2 factor was not performed, authors describe that the presence of the genes encoding Stx2 and Eae is determinant in the occurrence of UHS. In the susceptibility test, it was observed that E. coli isolates from diarrheal stools showed resistance to antimicrobials penicillin (10 mg) and tetracycline (30 mg) [93% and 80%, respectively], ampicillin (10 mg) [47%], streptomycin (10 mg) [47%], trimethoprim (5 mg) [47%] and sulfonamide (300 mg) [53%]. Although the percentage of antimicrobial resistance varies among studies, it is believed that the indiscriminate use of antimicrobial therapies as a common practice among rural properties contributes to bacterial resistance to these drugs. The sensitivity profile to antimicrobials showed that the analyzed Escherichia coli isolates are resistant to the antimicrobials commonly used for diarrhea treatment in the southern region of the Rio Grande do Sul state, Southern Brazil.

Flooding adds pathogenic Escherichia coli strains to the water sources in southern Khyber Pakhtunkhwa, Pakistan

Purpose: Seasonal rains in Pakistan result in heavy floods across the country, whereby faecal contaminants will be added to the water bodies and cause numerous food-borne outbreaks. The present study was aimed to determine the prevalence of diarrheagenic Escherichia coli (DEC) strains in the water sources. Materials and Methods: Two hundred water samples collected during (2011–2012) were processed for the isolation of E. coli (EC) strains. EC strains were further analysed for antibiotic susceptibility patterns, and pathogroups-specific virulence factors stx1, stx2, stx2c, eae, tir, hlyA, bfpA, estA and eltA were detected using multiplex polymerase chain reaction. Results: Thirty-three percent of the water samples were contaminated with EC pathotypes. Fifty percent (33/66) of the DEC pathotypes were identified as enterotoxigenic EC (ETEC). Seventy-two percent (13/18) of the enteropathogenic EC (EPEC) strains were identified as typical EPEC and 28% (5/18) as atypical EPEC. Eleven percent (7/66) of the Shiga toxin EC (STEC) isolates carried a combination of stx1 and stx2 genes. Summer was found as a peak season with 47% (31/66) for EC pathogroups’ activities. Eighty-nine percent of the strains showed resistance against tetracycline. Conclusion: ETEC and EPEC are the primary causes of water contamination in southern regions of Khyber Pakhtunkhwa province, Pakistan. Firm adherence to the prescribed drugs can decrease trends in antibiotic resistance.

Aislamiento de Escherichia coli enteropatógeno O157:H16 de un caso de diarrea infantil y sus contactos familiares en La Pampa, Argentina

Enteropathogenic Escherichia coli (EPEC) is a major causative agent of acute diarrhea in children in developing countries. This pathotype is divided into typical EPEC (tEPEC) and atypical EPEC (aEPEC), based on the presence of the bfp virulence factor associated with adhesion, encoded in the pEAF plasmid. In the present study, the isolation of aEPEC O157:H16 from a bloody diarrhea case in a child and his household contacts (mother, father and sister) is described. The strain was characterized as E. coli O157:H16 eae-ɛ-positive, sorbitol fermenter with β-glucuronidase activity, susceptible to all antimicrobials tested, and negative for virulence factors stx1, stx2, ehxA and bfp. XbaI-PFGE performed on all isolates showed the AREXHX01.1040 macrorestriction pattern, with 100% similarity. These results highlight the importance of epidemiological surveillance of E. coli O157-associated diarrhea cases identified in children and their family contacts, as well as the incorporation of molecular techniques that allow the detection of the different E. coli pathotypes.

Detection of five Shiga toxin-producing Escherichia coli genes with multiplex PCR

Escherichia coli serogroup O157 is the pathogen most commonly associated with foodborne disease outbreaks, but epidemiological studies suggest that non-O157 Shiga toxin-producing E. coli (STEC) is a major player as well. The ten most clinically relevant STECs belong to serogroups O26, O103, O111, O145, O157, O91, O113, O128, O45, and O121; but emerging strains, such as O104:H4 that was identified with the 2011 German outbreak, could become more prevalent in the future. A 75-min conventional multiplex PCR assay, IS-5P, targeting the four virulence factors stx1, stx2, eae, and ehxA plus the O157:H7-specific +93 uidA single nucleotide polymorphism was developed to better assess the potential pathogenicity of STEC isolates. All 212 STEC DNAs showed one to five amplification products, while the non-E. coli DNA did not react to this multiplex PCR assay. Enrichment broths obtained from baby spinach, alfalfa sprouts, and cilantro artificially inoculated with O26, O103, and O121 STECs reacted positively to the multiplex assay. Unlike the current FDA BAM 5P PCR, designed for the specific detection of O157:H7, IS-5P will identify potentially harmful O157:H7 and non-O157 STECs so they can be removed from the nation's food supply.

Method for the detection of priority Shiga toxin-producing Escherichia coli in beef trim.

A method has been developed for the detection in beef trim of priority Shiga toxin-producing E. coli (STEC) strains, defined as E. coli possessing the virulence factors stx1 and/or stx2 and intimin (eae), with O serogroups O26, O45, O103, O111, O121, O145, or O157. The method is based on recovery of the target bacteria by overnight enrichment in a broth optimized for recovery of O157 and non-O157 STEC, followed by screening using multiplex PCR techniques targeting (i) stx1, stx2, and eae (STE PCR) and (ii) gene sequences associated with the seven priority O serogroups (Poly O PCR), and then direct plating of broth samples positive in both STE and Poly O PCR onto Rainbow agar. Colonies on agar media were screened batchwise for STEC by the STE PCR, and presumptive isolates were characterized using a multiplex PCR and cloth-based hybridization array system targeting key virulence and O serogroup-specific markers. Using one representative strain of each priority O serogroup individually inoculated in beef trim samples, the method exhibited a limit of detection approaching 1 to 2 viable STEC cells per 65 g. None of the uninoculated trim samples produced positive results with either of the screening PCR procedures or on analysis of colonies recovered on plating media. STEC-negative samples were readily identified by screening PCR within 24 h, with a turnaround time of fewer than 4 days for confirmation of positives. The inclusivity and exclusivity characteristics of the screening PCR techniques were verified using a total of 65 different priority STEC strains: 24 nonpriority STEC, 15 non-STEC bacteria, and only those strains bearing the targeted characteristics produced screening PCR-positive results.

PCR MULTIPLEX PARA DETERMINAÇÃO DE MARCADORES DE VIRULÊNCIA EM Escherichia coli ISOLADAS DE BOVINOS COM DIARRÉIA EM MINAS GERAIS

Diarrhea is one of the most common diseases in calves and leads to substantial economic loss due to treatment costs, increased mortality and morbidity rates, and growth constraints. Enterotoxigenic Escherichia coli (ETEC), attaching and effacing E. coli (AEEC) and Shiga toxin-producing E. coli (STEC) have been identified as causes of diarrhea and dysentery in calves. This study investigates virulence factors of E. coli strains isolated from calves by multiplex PCR. The animals were up to 60 days old and were raised in Minas Gerais State, Brazil. Samples were obtained from semi-intensive dairy farms in the center-west and Alto Paranaiba regions of Minas Gerais. Feces were collected and processed routinely. The DNA templates of E. coli strains were subjected to multiplex PCR for the detection of the genes encoding the virulence factors Stx1, Stx2, Int, F41, K99, and STa. Out of 156 isolates, 51 (32,7%) were positive, whereas 105 (67,3%) did not present any of the virulence factors investigated. Results show that E. coli is an important cause of diarrhea in calves in Minas Gerais and that multiplex PCR test is a useful tool for the identification and characterization of pathogenic E. coli strains.

Antimicrobial Resistance and Virulence Factorsstx1,stx2, andeaein GenericEscherichia coliIsolates from Calves in Western Canadian Cow-Calf Herds

The objective of this study was to evaluate the association between antimicrobial resistance (AMR) in 106 fecal generic Escherichia coli isolates from calves in cow-calf herds, based on either phenotype or the presence of resistance genes, and the occurrence of virulence factors stx1, stx2, and eae. Three virulence genes and 23 AMR genes for six antimicrobial families were examined using DNA hybridization and PCR. Antimicrobial susceptibility of the isolates was tested using microbroth dilution (Sensititre, TREK Diagnostic Systems, Cleveland, OH) and the 2002 National Antimicrobial Monitoring System (NARMS) panel. The 106 isolates examined in this study were a stratified random subset from a larger study of AMR in cow-calf herds; 88.7% of the selected isolates were resistant to at least one antimicrobial, and 89.6% of the selected isolates were positive for at least one resistance gene. At least one virulence factor was identified in 48.1% (95%CI, 37.7-58.7) of the isolates. The most common virulence gene detected was stx2 followed by eae. Neither AMR measured phenotypically nor the presence of AMR genes were associated with the presence of above Shiga-toxigenic E. coli virulence factors in this population of healthy beef calves.

Antibiotic resistance and molecular epidemiology of Escherichia coli O26, O103 and O145 shed by two cohorts of Scottish beef cattle

The aim of this study was to identify the profile of antibiotic resistance among E. coli O26, O103 and O145 in two cohorts of Scottish beef cattle on two farms and to determine whether there is an association between resistant phenotypes and the genotypic PFGE patterns to suggest clonality among resistant strains. MICs of 11 antibiotics for 297 E. coli O26, 152 E. coli O103 and 13 E. coli O145 were determined. Isolates were screened for the presence integrons 1 and 2 and the virulence factors stx1, stx2, eaeA and ehxA by PCR with specific primers. PFGE subtyping was performed after digestion with XbaI endonuclease. Among E. coli O26, O103 and O145 there were four, four and one isolates, respectively, that harboured a class 1 integron. A class 2 integron was detected in only one O145 isolate. Diversity in PFGE patterns was higher among E. coli O103 and O145 strains compared with the O26 serotype; and PFGE demonstrated 13, 27 and 6 different patterns among O26, O103 and O145 isolates, respectively. Selective PFGE types that harboured virulence factors were widespread among the cattle population throughout the sampling period. There were multiply resistant isolates that were of similar PFGE patterns. The dissemination and persistence of certain PFGE genotypes among the cattle population was evident in this study. Certain resistance phenotypes, especially among E. coli O26 isolates, were associated with distinct PFGE clones.