POLSKIE TOWARZYSTWO MIKROBIOLOG"W POLISH SOCIETY OF MICROBIOLOGIS TS

Abstract

AbstractA multiplex PCR system was developed for specific identification of genes encoding heat-labile (LTI) and heat-stable(STI and STII) toxins of enterotoxigenic Escherichia coli (ETEC) strains. In addition, primers specific for the E. coligene coding for 16S rRNA were used as an internal control of the DNA amplification. The specificity of the methodwas validated by single PCR tests performed with reference to E. coli strains as well as pig-isolated bacteria and 100%correlation was observed. The developed multiplex PCR allowed rapid and specific identification of enterotoxin-posi-tive E. coli and may be used as a sensitive and specific method for a direct determination of ETEC and to differentiatethem from other E. coli isolates.Key words:ETEC, m-PCR, LTI, STI, STII enterotoxins IntroductionMost of Escherichia coli strains are harmless commensals in the gut but some of them are importantenteric pathogen, causing diarrhoea in humans and animals (Holland, 1990; Nataro and Kaper, 1998). E. coliisolates associated with diarrhoea have been classified into six major groups, on the base of their distinctvirulence properties: enterotoxigenic (ETEC), enteropathogenic (EPEC), enterohemorrhagic (EHEC),enteroinvasive (EIEC), diffusely adherent (DAEC) and enteroaggregative (EAEC) (Nataro and Kaper, 1998).Among them, ETEC is a predominant bacterial agent of diarrhoea in young animals as well as in infants andin adults travelling to developing countries (Nataro and Kaper, 1998; Nagy and Fekete, 1999). Bacteriaof this group are defined as E. coli which are able to produce at least one enterotoxin: heat-labile I (LTI)heat-stable I (STI) or heat-stable II (STII) (Seriwatana et al., 1988; Celemin et. al., 1994; Searsand Kaper, 1996; Nair and Takeda, 1998). LTI and STI have been found in both human and animal ETECwhereas STII toxin is characteristic only for animal E. coli (Seriwatana et al., 1988; Celemin et al.,1994). ETEC have the ability to cause profuse, watery diarrhoea by releasing of either LT or ST entero-toxins or both. Differentiation between ETEC and other pathogenic and non-pathogenic E. coli bacteria aswell as other enteric isolates requires detection of enterotoxins or their genes and it is essential for properdetection and control of diarrhoeal diseases. Conventional techniques of E. coli diagnosis depend on growthin pure cultures and subsequent biochemical identification of relevant bacterial colonies. These proceduresare time-consuming and can take up to 3Œ5 days. Moreover, differentiation of pathogenic ETEC isolatesneeds additional toxin detection procedures (Ojeniyi et al., 1994). One of the approaches used for the toxindetermination is polymerase chain reaction (PCR) which is a very sensitive and highly specific molecularbiology tool widely used for diagnosis of ETEC. PCR performed with the use two or more primer pairsto amplify two or more target sequences, called multiplex PCR, has been applied for detection and differen-tiation of LTI, STI or LTI, STII and Shiga toxin genes (Nataro and Kaper, 1998; Tsen and Jian, 1998;Nagy and Fekete, 1999; Osek et al. 1999, Osek and Truszczyaeski, 2000, Osek 2001). However, these