Phylogenetic relationships of Campylobacter jejuni based on porA sequences.

Campylobacter porins are the dominant major outer membrane protein (MOMP) of these bacteria. They are composed of hypervariable, surface-exposed, peptide loops and membrane-embedded, conserved peptide regions. Porins are functionally important and may also be useful for molecular subtyping methods but have not yet been well characterized. We therefore sequenced the porA gene from 39 Campylobacter isolates, including multilocus sequence type (MLST) reference strains, isolates from patients with the Guillain-Barré syndrome, other clinical isolates, and serotyping reference strains. These were compared with additional sequences available from GenBank. Three distinct porA lineages were observed after phylogenetic analysis. Both Campylobacter coli and Campylobacter jejuni were found with group 3 porA sequences, and this was the only group showing any evidence of recombination among porA genes. There was no recombination between porA genes from C. jejuni groups 1 and 2, suggesting there may be functional constraints on changes at this locus. Most of the amino acid differences among the three groups were present in surface-exposed loops, and dissimilar substitutions were found when groups 1 and 2 MOMP were compared. Different MOMP sequence groups may have different biological or antigenic properties, which in turn may be associated with survival in different environments, host adaptation, or virulence.

Temperate bacteriophages affect pulsed-field gel electrophoresis patterns of Campylobacter jejuni.

The recently sequenced genome of Campylobacter jejuni RM1221 revealed the presence of three integrated bacteriophage-like elements. In this study, genes from the first element, a Mu-like bacteriophage, were amplified by PCR and used to probe pulsed-field gels of clinical C. jejuni strains obtained from a waterborne outbreak (Ontario, Canada, 2000). These highly similar strains differed only by their pulsed-field gel electrophoresis (PFGE) patterns due to an apparent insertion or deletion of a 40-kb fragment. Bacteriophage probes hybridized to these different bands in Southern blot analysis, indicating that homologues of bacteriophage genes were present in the outbreak strains. Investigation of the bacteriophage insertion sites in these isolates suggested that bacteriophage acquisition, loss, or transposition was responsible for the PFGE pattern variation. The bacteriophage gene sequences were similar, but not identical, in the outbreak strains and RM1221, indicating that differences may exist between the bacteriophages.

Colony multiplex PCR assay for identification and differentiation of Campylobacter jejuni, C. coli, C. lari, C. upsaliensis, and C. fetus subsp. fetus.

A multiplex PCR assay was used to simultaneously detect genes from the five major clinically relevant Campylobacter spp. Those genes selected were hipO and 23S rRNA from Campylobacter jejuni; glyA from each of C. coli, C. lari, and C. upsaliensis; and sapB2 from C. fetus subsp. fetus. The assay was evaluated with 137 clinical and environmental isolates and was found to be rapid and easy to perform and had a high sensitivity and specificity for characterizing isolates, even in mixed cultures.

Bacterial DNA decontamination for reverse transcription polymerase chain reaction (RT-PCR).

For RT-PCR, removal of contaminating genomic DNA in RNA samples using manganese sulfate was more effective than magnesium. DNA contamination was removed in 3 microg of nucleic acid using 10 U of RNase-free DNase I in 10-microl reaction volumes. The digestion procedure was compatible with commercial RNA extraction kits and was suitable for RT-PCR assay.