Nishibuchi, Mitsuaki

Radu, Son

Rusul, Gulam

Universiti Putra Malaysia Institutional Repository

UPM Research Report 1998: Volume 2PREVALENCE AND MOLECULARCHARACTERISATION OF ESCHERICHIA COLI0157: H7 AND NON-0157Gulam Rusul, Son Radu and Mitsuaki NishibuchiFaculty of Food Science and BiotechnologyUniversiti Putra Malaysia, 43400 UPM, Serdang, Selangor,Malaysia, and IDivision of Human Environment, Center forSoutheast Asian Studies, Kyoto University, JapanKeywords: Escherichia coli 0157: H7 and non-0157,antibiotic, plasmid RAPD, PFGE.IntroductionMost E. coli strains are harmless commensals in the humanguts. However, within this species, there are pathogenicstrains that are associated with intestinal infection. Thesestrains produced toxin, which causes diarrhoea in humans.Enterotoxigenic (ETEC) and enterohemorrhagic (EHEC) areamong the most frequent enteric pathogen. ETEC strainsproduce toxin known as ST and LT, whereas EHEC strainsproduce toxin known as shiga-like toxin (SLT-I) and SLT-II,because of their similarity to the toxins produced by Shigelladysenteriae (Karmali, 1989). E. coli 0157:H7 causes a spec-trum of illness ranging from haemorrhagic colitis to hemo-lytic-uraemic syndrome and thrombocytopenic purpura. Themajority of infections are associated with raw meat products,untreated and pasteurised milk and water, and more novelfoods have become contaminated and implicated as a sourceof infection (Feng, 1995). Thus the food industry and publichealth microbiologists therefore need to screen high riskfoods for E. coli 0157: H7. The objectives of the presentstudy were to isolate and characterise E. coli 0157:H7 andnon-0157 from clinical, environment and food sources bytheir plasmid profiles and antibiotic susceptibility, as well asto determine the genetic transfer of their plasmid and antibi-otic resistance phenotypes. They were further typed by ran-dom amplified polymorphic DNA (RAPD) and pulsed fieldgel electrophoresis (PFGE).Materials and MethodsE. coli strains isolated from clinical, environmental and foodsources were identified by standard biochemical tests, andwere tested for presence of the H7 antigen by using antise-rum as described by the manufacturer. Isolates were screenedfor resistance to selected antibiotics by the disc diffusiontechnique. Plasmid DNA isolation were performed by the al-kaline lysis method. Molecular typing of the E. coli strainsby PFGE and RAPD techniques were performed as describedpreviously (Son et al. 1998a). Detection of the presence ofvirulence factors (ST, SLT-I and SLT-II) were conducted byradioactively labelled DNA probe or by biotinylated DNAprobe in hybridisation assay. Conjugation studies were con-ducted to determine the transferability of their plasmid andresistance phenotypes as described previously (Son et al.1997).Results and DiscussionA total of 586 strains of E. coli were isolated from beef(274), raw milk (64), duck intestines (64), poultry (76),aquatic (25), animals (30) and clinical sources (33). E. coli3120157:H7 strains were isolated from beef samples (77 of 274)and clinical sources (3 of 33), but not from the other sourcesexamined. The antimicrobial susceptibility analysis revealedthat all the E. coli strains tested demonstrated resistance toone or more of the antimicrobial agents tested. Though re-sistance is not considered a virulence factor, the multiple re-sistance phenotypes of the E. coli strains may assist in theestablishment and persistence of the organism in the host.The multiple antibiotic resistance index (MAR) of the E. colistrains ranged from 0.1 to 1.0, and are considered to haveoriginated from high risk sources of contamination like hu-mans, commercial poultry farms, swine and dairy cattlewhere antibiotics are often used. It is a well accepted notionthat when plasmids are present in an organism, some selec-tive advantage is operative even though plasmids, undernormal circumstances, are not essential for the viability ofthe host. Determination of the plasmid profiles among the E.coli strains revealed that plasmids of 1.4 to >60 megadalton(MDa) were detected. Selected strains of E. coli 0157:H7and non-0157 were used for genetic analysis to performmatings with E. coli K12 as recipient to screen for transfer ofantibiotic resistance. Enbloc transfer of the donor's resistancephenotypes and the concomitant transfer of plasmids rangingin sizes from 1.8 to 60 MDa were observed. In DNA hybridi-zation assay, using radioactively labeled and biotinylatedDNA probes, both clinical and beef isolates of E. coli0157:H7 and non-0157 were positive for virulence factors(SLT-I, SLT-II and ST, respectively) (Son et al. 1997).Twelve strains of E. coli 0157:H7 isolated from beef werecharacterized by RAPD and PFGE analysis. The results ob-tained showed that the 12 strains were genetically distinct.An important outcome of this study is the first report on theisolation of E. coli 0157:H7 from beef meat marketed inMalaysia (Son et al. 1998b).ConclusionsE. coli 0157:H7 and non-0157 strains possessing importantvirulence traits were shown to be distributed at a consider-able frequency. The differences in antibiogram, plasmid pro-files, RAPD and PFGE among the E. coli strains examinedsuggest that the strains may have originated from diversesources.ReferencesFeng, P. 1995. Escherichia coli 0157:H7: novel vehicles of infectionand emergence of phenotypic variants. Emerging Infectious Dis-eases. 1: 47-52.Karmali, M.A. 1989. Infection by Vero cytotoxin-producing Es-cherichia coli. Clinical Microbiology Review. 2: 15-38.Son, R., Rusul, G., Karim, M.I.A. and Yusoff, K. 1997. Detection ofenterotoxigenic Escherichia coli associated with diarrhoea by bi-otinylated heat-stable (ST) DNA probe. Malaysian Applied Biol-ogy. 26: 19-23.Son, R., Zainuri, A., Rusul, G., Zaiton, H., Sahilah, A.M., Morigaki,T. and Nishibuchi, M. 1998a. Molecular typing of Escherichiacoli 0157:H7 by pulsed field gel electrophoresis. Malaysian Ap-plied Biology. 27: 135-138.Son, R., Sahilah, A.M., Rusul, G., Raha, A.R., Rozila, A. and Sam-uel. L. 1998b. Beef as a source multi-resistant Escherichia coli0157 harbouring transferable plasmid and resistance phenotype.Journal of Bioscience. 9: 20-27.Supported by IRPA Grant 06-02-04-0006

Prevalence and molecular characterisation of Escherichia coli O157: H7 and non-O157

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Prevalence and molecular characterisation of Escherichia coli O157: H7 and non-O157

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