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PCR detection, identification to species level, and fingerprinting of Campylobacter jejuni and Campylobacter coli
D Linton1, A J Lawson, R J Owen
1Virus Reference Division, Central Public Health Laboratory, London, United Kingdom.
Insights
New PCR assays rapidly identify Campylobacter jejuni and Campylobacter coli directly from stool samples. These methods enable precise species identification and strain typing, improving diagnosis of enteropathogenic Campylobacter infections.
Area of Science:
- Microbiology
- Molecular Biology
- Infectious Diseases
Background:
- Campylobacter jejuni and Campylobacter coli are leading causes of bacterial gastroenteritis worldwide.
- Accurate and rapid species identification is crucial for effective diagnosis and treatment.
- Existing methods for Campylobacter identification can be time-consuming and may not detect all strains.
Purpose of the Study:
- To develop and validate novel PCR assays for the rapid and accurate detection and differentiation of Campylobacter jejuni and Campylobacter coli.
- To enable direct identification of these bacteria from clinical fecal samples.
- To assess the utility of PCR for Campylobacter species incidence and flaA genotyping in clinical settings.
Main Methods:
- Design of three distinct PCR assays targeting 16S rRNA, hippuricase gene, and aspartokinase gene sequences.
- Development of a DNA purification protocol for direct PCR analysis from fecal samples.
- Validation of PCR assays using reference strains and clinical stool samples, with comparison to culture and phenotypic methods.
- Strain typing using PCR-restriction fragment length polymorphism of the flagellin (flaA) gene.
Main Results:
- The developed PCR assays successfully identified Campylobacter jejuni and Campylobacter coli based on specific gene sequences.
- Direct detection of Campylobacter species from fecal samples was achieved with high concordance to culture methods.
- Analysis of 20 clinical samples revealed C. jejuni in 17, C. coli in 2, and a coinfection in 1 sample.
- flaA genotyping of fecal DNA matched that of corresponding isolates, confirming the accuracy of direct detection.
Conclusions:
- The developed PCR assays provide a rapid, sensitive, and specific method for identifying Campylobacter jejuni and Campylobacter coli directly from stool.
- These assays facilitate accurate species-level diagnosis and enable strain typing for epidemiological investigations.
- The methodology offers a valuable tool for clinical laboratories to improve the diagnosis of enteropathogenic Campylobacter infections.
Abstract:
Three sets of primers were designed for PCR detection and differentiation of Campylobacter jejuni and Campylobacter coli. The first PCR assay was designed to coidentify C. jejuni and C. coli based on their 16S rRNA gene sequences. The second PCR assay, based on the hippuricase gene sequence, identified all tested reference strains of C. jejuni and also strains of that species which lack detectable hippuricase activity. The third PCR assay, based on the sequence of a cloned (putative) aspartokinase gene and the downstream open reading frame, identified all tested reference strains of C. coli. The assays will find immediate application in the rapid identification to species level of isolates. The assays combine with a protocol for purification of total DNA from fecal samples to allow reproducible PCR identification of campylobacters directly from stools. Of 20 clinical samples from which campylobacters had been cultured, we detected C. jejuni in 17, C. coli in 2, and coinfection of C. jejuni and Campylobacter hyointestinalis in 1. These results were concordant with culture and phenotypic identification to species level. Strain typing by PCR-restriction fragment length polymorphism of the flagellin (flaA) gene detected identical flaA types in fecal DNA and the corresponding campylobacter isolate. Twenty-five Campylobacter-negative stool samples gave no reaction with the PCR assays. These PCR assays can rapidly define the occurrence, species incidence, and flaA genotypes of enteropathogenic campylobacters.