Sequence Variation in the Outer Membrane Protein-Encoding Gene cmeC, Conferring Multidrug Resistance among Campylobacter jejuni and Campylobacter coli Strains Isolated from Different Hosts

Abstract

A novel PCR primer pair was used to detect the presence of cmeC in 131 Campylobacter jejuni and C. coli strains isolated from various hosts (cattle, turkeys, humans, and pigs). DNA sequence analysis revealed a high degree of genetic variation between the two species, while extremely limited genetic variation among isolates of the same species was detected.

Campylobacter spp. are considered to be among the major food-borne pathogens in the United States and are reported to cause more cases of illness than Salmonella and Shigella species combined (1), about 2.5 million cases annually (11). Campylobacter uses efflux pumps to promote antimicrobial resistance (3, 8, 13) by extruding structurally diverse antimicrobial agents from the bacterial cells (14). In C. jejuni, a multidrug efflux pump designated CmeABC has been characterized as being encoded by a chromosomally located operon comprising three genes (cmeA, cmeB, and cmeC) that code for a periplasmic fusion protein, an inner membrane efflux transporter, and an outer membrane protein, respectively (8, 13). This pump was reported to have a critical role in bile resistance and in vivo colonization by C. jejuni (9) and is regulated by cmeR, located upstream of the operon (10). A cmeABC operon in a C. coli isolate was also characterized previously and showed significant homology to the operon of C. jejuni (5).

Using PCR, Olah et al. (12) recently examined the prevalence of the cmeABC operon in Campylobacter strains from turkeys. The study concluded that the CmeABC efflux system appeared to be more prevalent in C. jejuni than C. coli isolates and that not all the isolates tested—particularly among the C. coli strains—possessed all three components of the operon. This observation led us to expect that there may be sequence divergence between C. jejuni and C. coli and that the primers used were not specific for C. coli, thus giving false-negative results. The aim of the present study was to design one pair of PCR primers that is able to amplify the cmeC gene from both C. jejuni and C. coli and to explore the genetic variation in its DNA sequence.

A total of 77 C. jejuni and 64 C. coli strains isolated from various hosts, including cattle (36 C. jejuni and 30 C. coli isolates), turkeys (20 C. jejuni and 13 C. coli isolates), humans (11 C. jejuni isolates), and pigs (21 C. coli isolates), were examined. Species identification was confirmed using PCR (4). DNA was extracted from all isolates by the boiling method (6). An aliquot of 3 μl of the extract was used as a DNA template in PCR. The PCR primers used to amplify the internal fragments from the cmeC gene were as follows: forward, 5′-AGATGAAGCTTTTGTAAATT-3′, and reverse, 5′-TATAAGCAATTTTATCATTT-3′. The same primers were used for both PCR amplification and DNA sequencing. PCR amplification was performed using the Taq PCR master mix kit (QIAGEN Inc., Valencia, CA). Conditions for cmeC amplification were 94°C for 5 min, followed by 30 amplification cycles (95°C for 1 min, 48°C for 1 min, and 72°C for 1 min), and then a final extension at 72°C for 10 min. PCR products were purified using the QIAquick PCR purification kit (QIAGEN Inc., Valencia, CA).

DNA sequencing of the amplified fragments in both directions was performed at the Iowa State University DNA Sequencing and Synthesis Facility, Ames. Sequence assembly was performed using the Lasergene 7 software (DNASTAR, Inc.). All sequences were aligned and compared using the BioNumerics software (Applied Maths, Inc., Austin, TX). BLAST searching was performed through the NCBI website.

In the present study, the newly designed PCR primer pair successfully detected the presence of the cmeC gene in all 131 C. jejuni and C. coli isolates tested regardless of the host species of origin. The PCR primer pair was designed to amplify a fragment of approximately 500 bp within the cmeC gene by annealing to a DNA sequence conserved in the two species. This finding indicates that cmeC is present among all strains of C. jejuni and C. coli tested and is not common only among C. jejuni isolates as previously reported (12). DNA sequence analysis of the amplified cmeC fragments revealed a high degree of genetic variation (83% identity) between C. jejuni and C. coli regardless of the isolates' host species. Figure 1 shows a comparison of the cmeC DNA sequences from C. jejuni strain 110H and C. coli strain 037C, which represent the most common sequence types among the tested isolates of each species. The genetic variation between the two Campylobacter species is made evident by the mismatched bases. It is also noteworthy that the C. jejuni DNA sequences have a deletion of 6 bases that are present in the C. coli sequences. The sequenced region in all the C. jejuni isolates screened was 454 bp, while it was 460 bp in the C. coli isolates.

FIG. 1.

Comparison of the cmeC DNA sequences of C. jejuni strain 110H, representing sequence type ST2, and C. coli strain 037C, representing sequence type ST8. Mismatches are shown in red font, and deletions are indicated by red dashes. DNA sequences of the amplified fragments within the cmeC genes showed 83% identity between the two species. Numbers above sequences indicate nucleotide positions.

A dendrogram generated using the BioNumerics software perfectly separated isolates of the two species into individual groups with 83% similarity regardless of the host species (Fig. 2). Extremely limited genetic variation (over 98% similarity) among isolates of the same species was detected. The limited degree of variation was higher among the C. jejuni isolates (seven sequence types) than among the C. coli isolates (only two sequence types) (Fig. 2). There was no strong correlation between the sequence types and the isolates' host backgrounds. The level of genetic variation among the human isolates was higher (six sequence types) than those among the isolates from cattle (four sequence types) and from turkeys (two sequence types) (data not shown). Cagliero et al. (2) recently reported a similarly high degree of genetic variation in the cmeB gene in C. jejuni and C. coli. Campylobacter in general is known for its high level of genetic diversity because of its ability to undergo genetic recombination and its natural ability to acquire exogenous DNA through natural transformation (7).

FIG. 2.

Dengrogram generated using the BioNumerics software showing the perfect separation of C. jejuni and C. coli strains into two distinct groups at 83% similarity according to their cmeC DNA sequences. A representative strain of each sequence (seq.) type is shown. The total number of strains belonging to each sequence type regardless of the host species is also shown. Clustering was generated by the unweighted-pair group method with arithmetic means.

When subjected to a BLAST search against sequences in GenBank, the C. jejuni cmeC DNA sequences determined in the present study were more than 98% homologous to the published sequences from C. jejuni. The homology of the cmeC sequences from the C. coli isolates to the published sequences from C. coli was greater than 99%, with the exception of the sequence from C. coli CIT-382 (5), which showed only 85% identity. In our study (Fig. 2), of the 64 C. coli strains, 62 showed the same cmeC sequencing type (ST8) and only 2 isolates showed a different sequencing type (ST9), with only one mismatched base (data not shown). We also noticed that the C. coli CIT-382 sequence (5) showed 100% identity to our most common C. jejuni sequencing type (ST2), which has 100% identity to the sequences from C. jejuni subsp. jejuni NCTC 11168 and C. jejuni subsp. jejuni 81-176. Based on our results, C. coli CIT-382 seems to act as C. jejuni rather than as C. coli and may be misidentified.

To our knowledge, the present study is the first to determine partial sequences of the cmeC genes from a large number of C. jejuni and C. coli isolates (131 strains) from various hosts and to document the genetic variation in these DNA sequences. This genetic variation can be used to differentiate between C. jejuni and C. coli.

Nucleotide sequence accession numbers.

The partial cmeC gene DNA sequences determined in this study for representative isolates of each sequencing type were deposited in GenBank under accession numbers EF460800 to EF460816 (17 total).