Molecular Typing of Avian Escherichia coli Isolates by Random Amplification of Polymorphic DNA

Abstract

Escherichia coli is a common inhabitant of the gastrointestinal tract of most animals. Like most pathogenic E. coli, avian isolates cannot be distinguished biochemically from the normal commensals inhabiting the gastrointestinal tract of birds. Using a molecular approach, we were able to identify genetic differences among avian E. coli isolates by restriction fragment length polymorphism (RFLP) and random amplification of polymorphic DNA (RAPD) by the polymerase chain reaction (PCR). Several different RFLPs were observed among avian E. coli isolates using DNA probes for 16S ribosomal RNA genes (rrn) and insertion sequence elements (IS2). We were also able to observe differences in DNA banding patterns generated by RAPD analysis. Similarities and differences among avian E. coli were discernible using RFLPs and RAPD analysis, whereas conventional bacteriological methods failed to differentiate these isolates. Based on RAPD patterns, avian E. coli appear to be genetically diverse. Of 16 different RAPD types (RT) encountered, 84% of E. coli fell into seven major RTs. One RT was present in clinical isolates but absent from the commensals isolated in this study. Many of these different E. coli RTs were not geographically restricted to northern Georgia but were also observed in other southern states in the United States. Resistance to various antibiotics was randomly associated with different E. coli RTs. Sarafloxacin resistance was present among different E. coli RTs, suggesting that antibiotic usage is not selecting for a clonal population in avian E. coli. RAPD provides a rapid and powerful tool to study the epidemiology of avian E. coli.

Avian pathogenicEscherichia colitransmission from broiler breeders to their progeny in an integrated poultry production chain

“…length polymorphism typing (Maurer et al ., 1998). The RAPD technique is simple, cheap, fast, requires only nanogram amounts of DNA and easily handles large numbers of samples (Ramasoota et al ., 2000;Chansiripornchai et al ., 2001).…”

“…(2000) found that RAPD analysis for typing E. coli isolates has the highest discriminatory capacity compared with serotyping and ribotyping. RAPD has also been used successfully to discriminate E. coli strains isolated from humans (Pacheco et al ., 1997;Hopkins & Hilton, 2001), cattle, swine (Ramasoota et al ., 2000;Aslam et al ., 2004) and avian species (Maurer et al ., 1998;Chansiripornchai et al ., 2001); Ramasoota et al . (2001) differentiated different clones inside the same E. coli serotype using RAPD.…”

“…Twenty nanograms of bacterial DNA were used as a template in the random amplified polymorphic DNA (RAPD) kit containing room-temperature stable dried Ready-to-Go beads (Amersham Biosciences, Little Chalfont, UK). The kit was used as described by the supplier with the decamer primer 1290 (Maurer et al ., 1998) and primer 6 (Chansiripornchai et al ., 2001) (Table 1). Amplification products were resolved by electrophoresis on 2% agarose gel and detected by ethidium bromide staining; in the same gel, a molecular marker 100 base pair ladder (Invitrogen) was run.…”

Molecular tools for the characterisation of antibiotic-resistant bacteria

“…They are mosaic repetitive elements composed of various combinations of three subunit sequences referred to as boxA, boxB and boxC, with molecular lengths of 59, 45 and 50 nucleotides, respectively. Most of the fingerprinting techniques mentioned above were described by Louws et al [35] and have been applied for the analysis of antibiotic resistance genes [8,11,40]. RAPD and ERIC-PCR are generally much more discriminative than PFGE, distinguishing different genotypes among isolates considered as belonging to the same clone by PFGE.…”

Genotypic Characterization of Avian Escherichia coli by Random Amplification of Polymorphic DNA

“…The most commonly used molecular genetic fingerprinting technique is a restriction fragment length polymorphism (RFLP) analysis. Maurer et al, (1998) claimed that fingerprinting by RAPD revealed more genetic differences among avian E. coli strains than RFLP analysis. Akopyanz et al (1992); Makino et al (1994) and Pacheco et al (1996) observed that oligos with high GC content (>60%) resulted in a greater and better reproducible number of strain specific bands in enterotoxigenic E. coli.…”