Development of Multiplex PCR Assays for Detection of Enterotoxigenic Escherichia coli Colonization Factors and Toxins

Abstract

Four multiplex PCR assays for detection of 19 enterotoxigenic Escherichia coli (ETEC) colonization factorsand an improved ETEC toxin multiplex PCR were developed and tested on Bangladeshi and Bolivian ETEC strain collections. The assays will be useful for surveillance of ETEC infections in diagnostic laboratories that have access to PCR.Infection with enterotoxigenic Escherichia coli (ETEC) is one of the main causes of childhood diarrhea in developing countries and in travelers to these areas (7). ETEC strains express one or both of two different enterotoxins, the heatstable toxins (STh and STp) and a heat-labile toxin (LT), and more than 23 different colonization factors (CFs) that are named CFA/I and CS1 to CS22 (2). The LT enterotoxin and the CF antigens are immunogenic, and one or both of these components are included in most vaccines that are being developed against ETEC diarrhea (14,15). To evaluate the putative impact of different vaccine compositions on ETEC diarrhea, it is important to characterize clinical ETEC isolates from different parts of the world with regard to toxin and CF profiles, since they have been observed to vary from one geographic region to another (4,7,8,10,12).In order to simplify and accelerate the detection of the different CFs and toxins by PCR, we have established a set of multiplex PCR assays for detection of the most-prevalent CFs and the toxins using previously established primers (11), as well as new primers when appropriate. The CF primers were assembled into four panels designed to amplify 19 of the most common CFs, and a new set of STh primers was included in the previously established toxin multiplex PCR (Table 1).To establish the multiplex assays for the CFs and toxins, 10-to 100-ng amounts of DNA from ETEC reference strains boiled in water (Table 1) were mixed with master mix as described previously (11), except that 400 nM deoxynucleoside triphosphates and 8 to 10 forward and reverse primers at a final concentration of 200 nM each were added to the mix. The PCRs were amplified by an initial denaturation at 94°C (1 min), followed by 35 cycles of amplification (94°C for 30 s, 52°C for 30 s, and 72°C for 1 min), and finally, 5 min at 72°C. The products were run on 3% agarose gels or 3% MetaPhor gels (MetaPhor agarose; Cambrex Bio Science Rockland, Inc.) for better resolution and visualized under UV light (Fig. 1). Only the correct CF and toxin PCR products were amplified, and by mixing several strains and performing multiplex PCR on the mixed DNA, we verified that the primers in each panel could detect the presence of several CFs in one reaction.To evaluate the new multiplex PCR methods for ETEC toxins and CFs, 106 clinical ETEC isolates were analyzed by multiplex PCR and the results were compared with results obtained by using the previously established ETEC toxin multiplex PCR and with the dot blot method using available monoclonal antibodies (11). We used two different sets of ETEC strains that were isolated in two geographically different developing countries: 65 Boli...

Comparison of virulence markers and antibiotic resistance in enterotoxigenic Escherichia coli isolated ten years apart in Tehran

“…Classification of the CFs by antibody typing is time-consuming and requires reagents that are not readily available except in reference laboratories; additionally, the phenotypic expression is easily [8,[12][13][14][15][16]. Although the PCR method is easier and faster to perform, it is intolerant of sequence variation, whereas the DNA-based hybridization is largely tolerant of minor base pair differences between alleles of the same genes.…”

“…To date more than 20 CFs have been characterized with the diversity being due to differences in the primary amino acid sequences of the fimbrial structural subunits [6]. These proteins have been grouped into families or as distinct fimbriae on the basis of the underlying genetic diversity and are named colonization factor antigen I (CFA/I) and coli surface antigen 1 (CS1) to coli surface antigen 22 (CS22) [7,8]. All of the genetically related class 5 family of ETEC fimbriae which includes CFA/I, CS1, CS2, CS4, CS14, CS17, CS19 and putative CF O71 (PCFO71) as well as CS3 and CS20 are regulated by the transcriptional activator Rns or its functional homolog CfaD [7,9,10].…”

“…Moreover, despite the important role that CFs play in the pathogenesis of ETEC, in approximately 30-50% of the isolates worldwide, no CF is detected [11]. ETEC fimbriae have generally been identified by agglutination or immunoblotting with poly or monoclonal antibodies, but these reagents are not widely available, leading to the publication of various genotypic methods, including mono-and multiplex PCRs for the identification of CFs [8,[12][13][14][15][16].…”

Escherichiacoli DERIVED FROM DIFFERENT SOURCES SHARE ANTIGENIC CHARACTERISTICS WITH Shigellaboydii 18AND VIRULENCE FACTORS WITH ENTEROTOXIGENIC E. coli

“…For the pair of primers lacY and ipaH, a duplex PCR was used with the following parameters: 35 amplification cycles with initial denaturationat 94°C for 30 sec, annealingat 60°C for 25 sec, extension at 72°C for 30 sec and final extension at 72°C for 5 mins. In addition, the primers were used to detect the ltA, sth, stp, cfaI, cs1, cs3 y cs21 genes from ETEC employing the previously described conditions (Bekal, 2003;Rodas, 2009;Mazariego-Espinosa, 2010;Chattopadhyay, 2012).…”

“…For the pair of primers lacY and ipaH, a duplex PCR was used with the following parameters: 35 amplification cycles with initial denaturationat 94°C for 30 sec, annealingat 60°C for 25 sec, extension at 72°C for 30 sec and final extension at 72°C for 5 mins. In addition, the primers were used to detect the ltA, sth, stp, cfaI, cs1, cs3 y cs21 genes from ETEC employing the previously described conditions (Bekal, 2003;Rodas, 2009;Mazariego-Espinosa, 2010;Chattopadhyay, 2012).The same PCR technique was used to determine the presence of the wzx (flippase) and wxy (polymerase) genes, which relate to the biosynthesis of the somatic antigen of S. boydii 18. The nucleotide sequences of the wzx and wzy primers (Table 1) were obtained from the complete S. boydii 18 deposited in the GenBank with access number AY948196 (Feng, 2005).…”