Improved detection of Escherichia coli and coliform bacteria by multiplex PCR

Abstract

Background: The presence of coliform bacteria is routinely assessed to establish the microbiological safety of water supplies and raw or processed foods. Coliforms are a group of lactose-fermenting Enterobacteriaceae, which most likely acquired the lacZ gene by horizontal transfer and therefore constitute a polyphyletic group. Among this group of bacteria is Escherichia coli, the pathogen that is most frequently associated with foodborne disease outbreaks and is often identified by β-glucuronidase enzymatic activity or by the redundant detection of uidA by PCR. Because a significant fraction of essential E. coli genes are preserved throughout the bacterial kingdom, alternative oligonucleotide primers for specific E. coli detection are not easily identified.

Results: In this manuscript, two strategies were used to design oligonucleotide primers with differing levels of specificity for the simultaneous detection of total coliforms and E. coli by multiplex PCR. A consensus sequence of lacZ and the orphan gene yaiO were chosen as targets for amplification, yielding 234 bp and 115 bp PCR products, respectively.

Conclusions: The assay designed in this work demonstrated superior detection ability when tested with lab collection and dairy isolated lactose-fermenting strains. While lacZ amplicons were found in a wide range of coliforms, yaiO amplification was highly specific for E. coli. Additionally, yaiO detection is non-redundant with enzymatic methods.

Fig. 1

Alignment of lacZ sequences and designing of lacZ3 oligonucleotide primers. a Clusters of DNA sequences hits selected after Blast using lacZ from E. coli K-12 strain MG1655 as the query sequence. 195 hits, with a minimum pairwise identity of 64 % with the query sequence, from different enterobacteria were aligned. The total number of sequences corresponding to each lineage is shown on the right in brackets. The area of each dot correlates with the number of hits from each lineage with the same identity and bit-score. b The consensus sequence of a lacZ fragment (derived from sequences outlined in panel a) was used to design LacZ3-F and LacZ3-R oligonucleotide primers. Consensus and primer sequences were obtained using ClustalW and Primer 3 software, respectively. The degree of conservation of each position in the logo sequence is shown by the relative height of each base. The lacZ3R primer binding site overlaps with 3’ end of the lacZB (Bej et al., [25, 26]) primer (indicated by a pink arrow)

Fig. 2

Comparison of lacZ3-yaiO and lacZB-uidA primer sets for E. coli and coliform identification. a Agarose gels (1.5 %) electrophoresis showing representative multiplex PCR amplified products from bacterial DNA. Lanes: 1, Klebsiella pneumoniae; 2, Klebsiella oxytoca; 3,Enterobacter aerogenes; 4, Enterobacter intermedius; 5, Enterobacter cloacae; 6, Shigella sonnei; 7, Serratia marcenses; 8, Yersinia enterocolitica; 9, Salmonella typhymurium; 10, Citrobacter youngae; 11; Citrobacter freundii; 12, Hafnia alvei; M, molecular weight marker (1Kb Plus DNA ladder); 13, Escherichia coli K-12; 14, E. coli B; 15, E. coli B/r; 16, E. coli C; 17, E. coli W (Waskman); 18, E. coli W (Stoke); 19, E. coli RT1. The oligonucleotide primer pairs used are indicated on the left or below each picture. For size comparison, the locations of 100 and 200 bp bands are shown when the marker is omitted. b In vitro and in silico comparison of lacZ3-yaiO and lacZB-uidA multiplex PCR amplicons. In silico analysis (see Methods) is indicated by color shading. Cyan: positive amplification. Light red: no amplification. White: Not tested. Each PCR reaction was carried out four times. In vitro PCR products are shown by signs indicating the percentage of positive amplifications obtained. “+”; “+ − “; and “-“ represent 100 %, 50 % and 0 % positive results, respectively. No other values, (i.e., neither 75 % nor 25 % positive amplifications) were obtained. “≠“ indicates a different sized PCR product. c The 3’ end of the lacZB-R primer binds to a zone of low conservation. From top to bottom (arbitrary scale), each panel depicts the binding sites of the lacZ primers, the consensus sequence of lacZ, its coverage considering all the sequences aligned, sequence logo, and % identity