Molecular Based Identification and Typing of Campylobacter jejuni and C coli

Brigid Lucey, Fiona O'Halloran, and Seamus Fanning

1. Introduction

Thermophilic Campylobacter spp., mainly Campylobacter jejuni and to a lesser extent C. coli are recognized as the most common bacteriological causes of gastroenteritis in humans (1). As enteric infection with Campylobacter organisms cannot be distinguished from that caused by other enteric pathogens, a definitive diagnosis can only be made by isolating or detecting the organism from the feces. The epidemiology of Campylobacter enteritis has been complicated by the ubiquitous nature of the organism (commonly found as a commensal in the intestines of domestic animals, in milk, and in water). Furthermore, identification is carried out only to genus level by most clinical laboratories.

Because of the biochemical similarity known to exist between C. jejuni and C. coli, the hippurate hydrolysis test is often used as the only phenotypic test capable of differentiating the two species. This test, however, has some acknowledged technical limitations and is dependent on inoculum size; results can be difficult to interpret accurately (2). Furthermore, almost all C. jejuni isolates possess the hippuricase gene (3), fewer C. jejuni isolates express the hippuricase gene. For this reason, certain polymerase chain reaction (PCR)-based species identification methods, for both C. jejuni and C. coli, and for the other thermophilic species, provide more reliable identification; they also help to highlight mixed species cultures, should they occur. However, even with these methods, false negatives or nonspecifically amplified product(s) can occur in a minority of isolates tested owing to genomic anomalies. Thus a second molecular identification method may be required in these circumstances.

Gonzalez et al. (4) developed a species-specific PCR assay for the identification of C. jejuni and C. coli based on the ceuE gene, which is involved in siderophore transport. Using this method two primer sets are employed in separate PCR amplification reactions. Another method, developed by Eyers et al. (5), performs PCR amplification of 23S rRNA gene fragments, based on regions specific for C. jejuni, C. coli, C. lari,

From: Methods in Molecular Biology, vol. 268: Public Health Microbiology: Methods and Protocols Edited by: J. F. T. Spencer and A. L. Ragout de Spencer © Humana Press Inc., Totowa, NJ

and C. upsaliensis. In addition, Hani and Chan (6) developed a PCR assay that detected and amplified the hippuricase gene. This molecular approach may offer a more reliable means of identifying C. jejuni strains compared with the phenotypic hippurate hydrolysis test alone.

Epidemiological typing methods currently in use for Campylobacter spp. include phenotypic methods such as serotyping and genotypic methods including DNA amplification fingerprinting (DAF), pulsed-field gel electrophoresis (PFGE), and ribotyping and restriction fragment length polymorphism (RFLP) analysis of the flagellin A gene (flaA). Serotyping remains primarily a reference laboratory protocol because of the time and expense needed to maintain high-quality antisera. Furthermore, there are a high percentage of untypable strains, and the standard serotyping systems such as the Lior and Penner schemes are time-consuming and technically demanding (7).

DAF is a PCR-based typing method that typically uses randomly designed 10-mer primers, under conditions that allow some base-pair mismatches, to increase the number of primer binding sites. Primer binding throughout the genome generates an array of DNA amplicons with varying lengths and intensities, which typify a genomic fingerprint. Interstrain variation is dependent on the number, location, and degree of mismatches tolerated when the primer binds to the genome. Advantages of this technique include a high degree of typability, ease of performance, cost effectiveness, and ready availability of reagents and equipment. In a recent study of 378 Campylobacter isolates, from human and poultry sources, only one isolate proved untypable with DAF analysis (Lucey et al., unpublished observations), using the primer HLWL85 (8). DAF analysis has proved to be highly discriminatory when primers are carefully selected (see Note 1).

PFGE is a molecular typing technique that uses rare cutting restriction endonu-cleases to cleave bacterial genomes producing a small number of very large DNA fragments. These DNA fragments can only be resolved and visualized by PFGE methods. Despite some obvious technical considerations such as the labor-intensive process involved, PFGE is one method that offers a high degree of reproducibility and typability, with the production of highly discriminatory profiles and the possibility of interlaboratory comparisons if methods are standardized. Compared with DAF, PFGE requires specialized equipment and can be more expensive to run on a routine basis (7).

A study by M0ller Nielsen et al. (9) compared the effectiveness of six typing methods for use on a population of 90 Campylobacter isolates of human, cattle, and poultry origins (comprising outbreak and non-outbreak-related strains). The methods used were Penner heat-stable serotyping, automated ribotyping, DAF analysis, PFGE, RFLP of flaA, and denaturing gradient gel electrophoresis of flaA (flaA--DGGE). DAF and PFGE were shown to be the most discriminating methods, which the authors ascribed to their ability to determine polymorphisms across the entire bacterial genome. Serotyping was found to be the least discriminatory.

Bacterial resistance to antimicrobial agents often results from the acquisition of new genes, as well as from mutations. These new genes can be acquired by several mechanisms including genetic elements, such as resistance (R-)plasmids, transposons, and integrons. The latter group are a class of genetic elements involved in the dissemination of antimicrobial resistance-encoding genes. This is mediated by the integration of a gene cassette containing the resistance genes via a site-specific recombinational event (10). Integrons have been found to be associated with antimicrobial resistance in many Gram-negative species, including Salmonella, Pseudomonas, and Klebsiella. A recent report suggests that Campylobacter spp. also possess integron-like structures (11). When the plasticity of the Campylobacter genome is considered, this has interesting implications for the continued evolution of this species. Therefore, it is reasonable to suggest that complete characterization of these species should include investigations for the presence of unique integron structures.

Most cases of clinical Campylobacter enteritis are sufficiently mild or self-limiting not to require antimicrobial chemotherapy (12). Nevertheless, in severe or recurrent cases for which antibiotics are required, susceptibility testing is important to ensure appropriate and timely treatment. Macrolides remain the agents of choice for such cases, and resistance rates remain comparatively low (13). Since the 1980s, however, the development of the fluoroquinolones, which are effective against most enteric pathogens, offered an effective therapy to treat acute bacterial diarrhea; ciprofloxacin becoming used extensively as prophylaxis for travellers (14). The emergence of resistance to these agents, however, has since then made their efficacy less certain. Resistance was reported to develop among patients after treatment with fluoroquinolones (15) and was also found to coincide with the introduction of these agents in veterinary medicine (16). In Campylobacter and other Gram-negative bacteria, fluoroquinolones operate by interfering with the type II topoisomerase (DNA gyrase) and topoisomerase IV (17). The predominant mechanism of resistance to ciprofloxacin in C. jejuni and C. coli has been shown to result from a mutation in the gyrA gene, whereby all isolates tested demonstrated a Thr-86-Ile substitution in the A-subunit of DNA gyrase (18,19). A specific PCR assay, the mismatch amplification mutation assay (MAMA-PCR), has been demonstrated to be a useful screening method for ciprofloxacin resistance among each of these isolates (18,19). This method uses a conserved, forward primer and a reverse, diagnostic primer, which together generate a 265-bp product that is a positive indication of the presence of the Thr-86-Ile amino acid substituion consistent with resistance to ciprofloxacin.

2. Materials

All materials marked with an asterisk (*) were autoclaved at 121°C for 15 min prior to use.

2.1. DNA Extraction

This protocol is a modified version of a purification method for Campylobacter spp. (20) to yield purified DNA. Cultures were treated prior to DNA extraction with formaldehyde to inhibit any DNAse activity (see Note 2), according to the method of Gibson et al. (21).

1. Preston agar Campylobacter Agar Base, Oxoid CM689, containing Modified Campylobacter Selective Supplement Sr204E and 5% (v/v) lysed horse blood. Prepare according to the manufacturer's instructions.

2. Distilled water*.

4. Formaldehyde solution (37-40% v/v).

5. 5X TE solution*: 50 mM Tris-HCl, pH 8.0, 50 mM EDTA.

6. Lysozyme: obtained lyophilized from Sigma (Poole, UK), reconstituted in 5X TE solution to a final concentration of 5 mg/mL, aliquot, and store at -20°C.

7. 20% (w/v) Sodium dodecyl sulfate (SDS), heat to 35-45°C to dissolve.

8. Proteinase K: obtained lyophilized from Sigma, reconstituted in 5X TE solution to a final concentration of 10 mg/mL, aliquot, and store at -20°C.

9. Phenol:chloroform:isoamylalcohol (25:24:1) solution (Sigma), saturated with 10 mM Tris-HCl, pH 8.0, 1 mM EDTA.

10. 3 M Ammonium acetate. Filter sterilize and store at room temperature.

12. 1X TE solution*: 10 mM Tris-HCl, pH 8.0, 1 mM EDTA.

2.2. PCR Amplification Methods

1. Sterile water.

2. Deoxyribonucleoside triphosphates (dNTPs; Promega, Madison WI). Provided at a concentration of 100 mM each dNTP (dATP, dCTP, dGTP, dTTP). Prepare a working solution of 1.25 mM dNTP by adding 2.5 ^L of each dNTP to 190 ^L of sterile H2O. Store at -20°C until required.

3. Taq DNA polymerase (Promega; 5 U/^L). Supplied with 25 mM MgCl2 and 10X reaction buffer (100 mM Tris-HCl, pH 9.0, 500 mM KCl, 1% [w/v] Triton X-100).

4. Oligonucleotide primers diluted to optimal working concentration in sterile H2O. All primers used for speciation, DAF analysis, integron analysis, and ciprofloxacin resistance-determining MAMA-PCR were purchased from Oswel DNA Services (Southampton, UK) and were purified by high-performance liquid chromatography (HPLC) prior to use. Primer sequences along with their relevant characteristics are listed in Table 1.

2.3. Agarose Gel Electrophoresis

1. Conventional agarose gel electrophoresis was used to characterize PCR amplicons, and a list of the reagents used is given in Chapter 2 of this volume.

2. Molecular weight markers: Three DNA molecular weight marker mixtures were employed to analyze PCR products. These included DNA marker III (generates fragments from 0.12 to 21.2 kbp, DNA marker V(generates fragments from 8 to 587 bp), and DNA markerXIV (generates fragments from 100 to 1500 bp in length). All markers were supplied by Roche Diagnostics (East Sussex, UK).

2.4. Pulsed-Field Gel Electrophoresis

2.4.1. Preparation of Agarose Plugs and Restriction Endonuclease Digestion

1. Preston agar (Campylobacter Agar Base, Oxoid CM689, containing Modified Campylobacter Selective Supplement Sr204E and 5% [v/v] Lysed Horse Blood), prepared according to the manufacturer's instructions.

3. Formaldehyde solution (37-40% v/v).

4. 1-mL Sterile plastic syringes with the nozzle removed.

5. Sterile microcentrifuge tubes (1.5 and 2.0 mL).

6. Sterile clear polystyrene capped 17 x 120-mm (Sarstedt) tubes (or equivalent).

Table 1

Sequence and Characteristics of Oligonucleotide Primers Used for PCR

Sequence and Characteristics of Oligonucleotide Primers Used for PCR

Table 1

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