Differentiation of Campylobacter coli , Campylobacter jejuni , Campylobacter lari , and Campylobacter upsaliensis by a Multiplex PCR Developed from the Nucleotide Sequence of the Lipid A Gene lpxA

Abstract

We describe a multiplex PCR assay to identify and discriminate between isolates of Campylobacter coli, Campylobacter jejuni, Campylobacter lari, and Campylobacter upsaliensis. The C. jejuni isolate F38011 lpxA gene, encoding a UDP-N-acetylglucosamine acyltransferase, was identified by sequence analysis of an expression plasmid that restored wild-type lipopolysaccharide levels in Escherichia coli strain SM105 [lpxA(Ts)]. With oligonucleotide primers developed to the C. jejuni lpxA gene, nearly full-length lpxA amplicons were amplified from an additional 11 isolates of C. jejuni, 20 isolates of C. coli, 16 isolates of C. lari, and five isolates of C. upsaliensis. The nucleotide sequence of each amplicon was determined, and sequence alignment revealed a high level of species discrimination. Oligonucleotide primers were constructed to exploit species differences, and a multiplex PCR assay was developed to positively identify isolates of C. coli, C. jejuni, C. lari, and C. upsaliensis. We characterized an additional set of 41 thermotolerant isolates by partial nucleotide sequence analysis to further demonstrate the uniqueness of each species-specific region. The multiplex PCR assay was validated with 105 genetically defined isolates of C. coli, C. jejuni, C. lari, and C. upsaliensis, 34 strains representing 12 additional Campylobacter species, and 24 strains representing 19 non-Campylobacter species. Application of the multiplex PCR method to whole-cell lysates obtained from 108 clinical and environmental thermotolerant Campylobacter isolates resulted in 100% correlation with biochemical typing methods.

Identification of host-associated alleles by multilocus sequence typing of Campylobacter coli strains from food animals

“…As with RRC026 (ST-21), RRC050 (ST-38), NCSU064 (ST-1162) and RRC043 (ST-1244), the species designations of strains comprising the divergent STs in the secondary cluster were examined further with additional PCR primer sets. Amplification using cadF (Konkel et al, 1999), hipO and lpxA (Klena et al, 2004) primer sets was in agreement with the previously determined C. coli status of these strains.…”

“…Finally, the cattle strain RRC043 possessed the novel ST-1244 ST, consisting of six alleles identified previously in C. jejuni or with a high similarity to existing C. jejuni alleles, as well as the C. coli allele uncA17. Even though these strains had been classified originally as C. coli, results typical for C. jejuni were obtained for these three strains with the hipO and lpxA (Klena et al, 2004) primer sets (data not shown). In addition to the alleles within the ST-21, ST-38, ST-1162 and ST-1244 STs, four other divergent alleles were identified.…”

“…The hipO gene was amplified using the hipF (59-ATG ATG GCT TCT TCG GAT AG-39) and hipR (59-GCT CCT ATG CTT ACA ACT GC-39) primer set. For lpxA, multiplex PCR reactions were performed using the following primers: cj0121 (59-ACA ACT TGG TGA CGA TGT TGT A-39), cc0120 (59-AGA CAA ATA AGA GAG AAT CAG-39) and KK2 (59-CAA TCA TGW GCN ATA TGR CAA TAN GCC-39) (Klena et al, 2004). PCR reactions were performed on an MJ Research Tetrad thermocycler with the following settings: 30 s at 94 uC, 30 s at 50 uC, 2 min at 72 uC (35 cycles).…”

Evaluation of a Cytolethal Distending Toxin (cdt) Gene‐Based Species‐Specific Multiplex PCR Assay for the Identification of Campylobacter Strains Isolated from Poultry in Thailand

“…Owing to very weak hippuricase activity of some C. jejuni strains, hippurate-negative strains of C. jejuni are also well recognized (32). Several PCR-based assays have been developed to facilitate the differentiation of C. jejuni from C. coli (4,5,9,15,18,24,33), however the ability of these assays to accurately identify Campylobacter species has not yet been established.…”

Gastroenteritis caused by Campylobacter concisus

“…Molecular methods using PCR or other nucleic acid techniques have been reported (Klena et al, 2004;Maher et al, 2003;Zhang et al, 2009). For reliable species determination, sequenced PCR products should be compared to published genome sequences of type strains or reference strains from bacterial culture collections.…”