Molecular Typing ofCampylobacterStrains Using thecmpGene Encoding the Major Outer Membrane Protein

Abstract

Thermophilic Campylobacter, particularly Campylobacter jejuni, is one of the major foodborne human pathogens of animal origin. Reliable and sensitive typing tools are required for understanding the epidemiology and ecology of this zoonotic bacteria agent. Currently, several molecular typing methods are available for differentiating Campylobacter strains, but each of them has limitations. Our previous study revealed that considerable sequence polymorphism exists in the cmp gene encoding the major outer membrane protein of Campylobacter and suggested that sequence variation of cmp may be utilized for discrimination of Campylobacter strains. In this study, we evaluated the feasibility of the cmp-based typing tool, using pulsed-field gel electrophoresis (PFGE) as the "gold" standard for comparison. The cmp alleles were sequenced from multiple Campylobacter strains, grouped, and compared with the PFGE profiles of these strains using Bionumerics. Results showed that 43 cmp sequence types and 43 PFGE types existed among the 60 Campylobacter isolates. Typeability of these strains is 100% using either the cmp-based method or PFGE. The discrimination indices are 0.973 for the cmp-based method and 0.969 for PFGE, respectively. The cmp sequence types are 77.6% congruent with the PFGE types. These results indicate that the cmp-based typing is a simple, yet highly discriminatory approach for molecular differentiation of C. jejuni strains.

Emergence of a Tetracycline-ResistantCampylobacter jejuniClone Associated with Outbreaks of Ovine Abortion in the United States

“…PFGE typing. PFGE analysis of the macrorestriction fragment patterns of C. jejuni genomic DNA was performed as described elsewhere (18). Briefly, fresh cultures of Campylobacter grown in Mueller-Hinton broth were embedded in chromosomal-grade agarose (Bio-Rad Laboratories, Inc., Hercules, CA) and treated with lysis buffer (10 mM Tris, 100 mM EDTA, pH 8.0, 1% N-lauroylsarcosine, 0.5 mg/ml proteinase K) overnight at 55°C in a shaker.…”

“…cmp typing of representative C. jejuni isolates. To confirm the results of PFGE macrorestriction typing, another molecular typing method established for C. jejuni also was included in this study (4,18). For this purpose, sequences of the cmp gene (encoding the MOMP protein) of representative Campylobacter abortion isolates, including PFGE subtypes I (IA-2, IA-3, SD-2, ID-2, and CA-1) and II (IA-1, SD-1, and ID-1), were determined (Fig.…”

Using Major Outer Membrane Protein Typing as an Epidemiological Tool To Investigate Outbreaks Caused by Milk-Borne Campylobacter jejuni Isolates in California

“…We speculated that the DNA sequences of hypervariable regions of the porA gene (also referred to as cmp), encoding the major outer membrane protein (MOMP) of C. jejuni, might be exploited for this purpose (14,15). As proof of concept, we developed a primer set that amplified a fragment of the porA gene (ϳ740 bp) and used phylogenetic analysis to compare the genetic relatedness of strains isolated from human stools with environ-mental strains isolated during two C. jejuni milk-borne outbreaks in California.…”

Comparison of Molecular Typing Methods Useful for Detecting Clusters of Campylobacter jejuni and C. coli Isolates through Routine Surveillance

“…flaA SVR typing was done according to the methods of Meinersmann et al (35). The sequence typing of the porA (cmp) gene was initially performed in the manner of Huang et al (28). However, since we had some difficulty obtaining full-length porA sequence using only the F3 and R3 primers for PCR amplification, PCR amplifications and sequencing reactions were done with the primers and methods used previously (6).…”

“…In this work, we have compared the results from the typing of Campylobacter isolates using PFGE; the Oxford MLST method; the Oxford flaA SVR typing method; combined MLST and flaA SVR typing (MLST ϩ flaA SVR); porA sequence typing (6,28) (http://pubmlst .org/campylobacter/); combined MLST, flaA SVR, and porA sequence typing (MLST ϩ flaA SVR ϩ porA); and comparative genomic fingerprinting (CGF), a newly developed multiplex PCR method designed to detect Campylobacter genes that were identified as having a high degree of intraspecies variability in comparative genomic hybridizations using DNA microarrays (33,51). Each method or combination of methods appeared to group isolates at different levels within the population and therefore was useful for a different purpose.…”