Rapid identification of Campylobacter spp. by melting peak analysis of biprobes in real-time PCR

We describe rapid PCR-biprobe identification of Campylobacter spp. This is based on real-time PCR with product analysis in the same system. The assay identifies enteropathogenic campylobacters to the species level on the basis of their degree of hybridization to three 16S ribosomal DNA (rDNA) biprobes. First-round symmetric PCR is performed with genus-specific primers which selectively target and amplify a portion of the 16S rRNA gene common to all Campylobacter species. Second-round asymmetric PCR is performed in a LightCycler in the presence of one of three biprobes; the identity of an amplified DNA-biprobe duplex is established after determination of the species-specific melting peak temperature. The biprobe specificities were determined by testing 37 reference strains of Campylobacter, Helicobacter, and Arcobacter spp. and 59 Penner serotype reference strains of Campylobacter jejuni and C. coli. From the combination of melting peak profiles for each probe, an identification scheme was devised which accurately detected the five taxa pathogenic for humans (C. jejuni/C. coli, C. lari, C. upsaliensis, C. hyointestinalis, and C. fetus), as well as C. helveticus and C. lanienae. The assay was evaluated with 110 blind-tested field isolates; when the code was broken their previous phenotypic species identification was confirmed in every case. The PCR-biprobe assay also identified campylobacters directly from fecal DNA. PCR-biprobe testing of stools from 38 diarrheic subjects was 100% concordant with PCR-enzyme-linked immunosorbent assay identification (13, 20) and thus more sensitive than phenotypic identification following microaerobic culture.     

PCR detection, identification to species level, and fingerprinting of Campylobacter jejuni and Campylobacter coli direct from diarrheic samples.

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.     

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

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.     

Ciprofloxacin resistance and its molecular mechanism in Campylobacter spp. isolated in Kuwait

Campylobacter spp. are an important cause of diarrhea in Kuwait. Because susceptibility data for ciprofloxacin and erythromycin, the two recommended drugs for treatment, are not available for this part of the world, 64 Campylobacter spp. isolates obtained from human diarrheal stools in Kuwait during 2000--2003 were studied for susceptibility to these antimicrobials by E-test. The utility of a simple mismatch amplification mutation assay (MAMA) PCR to detect base substitution in the gyrA gene mediating resistance to ciprofloxacin was also explored. Approximately, 53% (34/64) of the isolates were resistant to ciprofloxacin (MIC, 4-64 microg/ml) and 5% (3/64) to erythromycin (MIC>256 microg/ml). MAMA PCR showed a Thr-86-to-Ile mutation in gyrA gene of 23/26 ciprofloxacin-resistant C. jejuni, and in all resistant C. coli. Sequencing of PCR product showed that two resistant strains of C. coli studied had Thr-86-to-Ile (ACT--> ATT) gyrA mutation and three resistant strains of C. jejuni studied had Thr-86-to-Ile (ACA--> ATA) gyrA mutation. In addition, all the three C. jejuni strains had silent mutations. Thus, ciprofloxacin is of limited use for treatment in Kuwait and MAMA PCR is a useful assay to study gyrA mutation. Because Kuwait has a large expatriate population of workers, it can be a focus of spread of antimicrobial resistance.     

实时荧光PCR方法快速检测空肠弯曲菌方法的建立

目的建立实时荧光PCR快速检测空肠弯曲菌的方法。方法以空肠弯曲杆菌HipO基因的保守序列为模板设计特异性引物探针,建立一种能快速检测样本中空肠弯曲杆菌的实时荧光PCR方法;对方法的特异性和敏感性进行评价,并以正常人粪便为空白样本,添加一定量空肠弯曲菌标准株菌液进行检测,以对方法的检测效果进行初步评价。结果该实时荧光PCR方法只对空肠弯曲杆菌进行特异扩增,同种属的结肠弯曲菌及其他常见食源性病原菌均不能扩增;整个检测过程只需要80min,对空肠弯曲菌菌悬液可检测至5个细菌,对加标粪便样本可检测至10-100个细菌。结论本研究建立的实时荧光PCR检测空肠弯曲菌方法不仅能实现对空弯菌的快速检测,而且还为空弯菌的快速诊断及其引起的食源性疾病的监控溯源提供有意义的参考。     

Rapid detection and differentiation of pathogenic Campylobacter jejuni and Campylobacter coli by real-time PCR

A two-tube real-time assay, developed in a LightCycler, was used to detect, identify and differentiate Campylobacter jejuni and Campylobacter coli from all other pathogenic members of the family Campylobacteriaceae. In the first assay, continuous monitoring of the fluorescence resonance energy transfer (FRET) signal acquired from the hybridisation of two adjacent fluoroprobes, a specific FITC probe 5'-GTGCTAGCTTGCTAGAACTTAGAGA-FITC-3') and a universal downstream probe Cy5 (5'-Cy5-AGGTGITGCATGGITGTCGTTGTCG-PO(4)-3'), to the 681-base pair 16S rRNA gene amplicon target (Escherichia coli position 1024-1048 and 1050-1075, respectively) produced by the primer pair, F2 (ATCTAATGGCTTAACCATTAAAC, E. coli position 783) and Cam-Rev (AATACTAAACTAGTTACCGTC, E. coli position 1464), detected C. coli, C. lari and C. jejuni. As expected, a Tm of 65 degrees C was derived from the temperature-dependent probe DNA strand disassociation. In the second assay, an increase in fluorescence due to binding of the intercalating dye SYBR Green I to the DNA amplicons of the hippuricase gene (hipO) (produced by the primer pair hip2214F and hip2474R) was observed for C. jejuni but not for C. coli which lacks the hipO gene. A Tm of 85+/-0.5 and 56 degrees C determined from temperature-dependent dye-DNA disassociation identified C. jejuni and the non-specific PCR products, respectively, in line with our expectation. The two-tube assay was subsequently used to identify and differentiate the 169 Campylobacteriaceae isolates of animal, human, plant and bird origin held in our culture collection into C. coli (74 isolates), C. jejuni (86 isolates) and non-C. coli-C. jejuni (9 isolates). In addition, the method successfully detected C. jejuni, C. coli and C. lari from 24-h enrichment cultures initiated from 30 commercial chicken samples.     

Detection of mutations associated with macrolide resistance in thermophilic Campylobacter spp. by real-time PCR

Two point mutations (A2074C and A2075G) in the 23S rRNA gene of Campylobacter coli and C. jejuni are associated with erythromycin resistance. A real-time fluorescence resonance energy transfer PCR assay using a melting curve analysis was developed to identify these two point mutations and the wild-type genotype in thermophilic Campylobacter species such as C. coli, C. jejuni, and C. lari. Concerning these species, 141 strains were tested and a result obtained in 140 (99.3%). A single genotype was detected in 133 cases and two genotypes in seven cases. There was an agreement with the phenotypic methods except for one C. coli strain that was not amplified during the PCR assay. The A2075G mutation was mainly found among the 92 resistant strains tested (95.6%) and the A2074C mutation only twice (2.2%). One C. jejuni strain (1.1%) harbored both mutations on the same 23S rDNA copy. When compared to the phenotypic tests, the new real-time PCR assay was able to detect the correct genotype, in all cases except one (1.1%). This assay is more sensitive and more rapid than other PCR assays, the entire procedure taking less than 2 hr.     

Comparative performance of different PCR assays for the identification of Campylobacter jejuni and Campylobacter coli

The sensitivity and specificity of 7 PCR assays described for the identification of Campylobacter jejuni and Campylobacter coli were examined using alkaline cell lysates from a collection of 100 well characterized reference strains of C. jejuni, C. coli, Campylobacter lari and related Campylobacter, Helicobacter and Arcobacter species. Based on a preliminary evaluation, one multiplex test was excluded from further evaluation. The various assays differed considerably in sensitivity and specificity towards their target species. For C. coli, 4 of the 5 assays were 100% specific and sensitive, but for C. jejuni, none of the 5 assays were found to be 100% specific or sensitive. Subsequently, a statistically valid sample (n=263) was taken from a Belgian collection of 1906 human Campylobacter field isolates. This second collection was used to further evaluate two selected multiplex PCR assays. The present study indicates that PCR-based identification using each of the two selected multiplex PCR assays was highly reliable. The R-mPCR assay, followed by species-specific PCR assays or the ceu-oxr mPCR assay if necessary, is our current strategy of choice for the molecular identification of C. jejuni and C. coli. Results presented here should aid researchers in selecting a PCR assay suitable for their specific needs.     

Characterization of cytolethal distending toxin of campylobacter species isolated from captive macaque monkeys

An association between certain Campylobacter species and enterocolitis in humans and nonhuman primates is well established, but the association between cytolethal distending toxin and disease is incompletely understood. The purpose of the present study was to examine Campylobacter species isolated from captive conventionally raised macaque monkeys for the presence of the cdtB gene and for cytolethal distending toxin activity. The identity of each isolate was confirmed on the basis of phenotypic and genotypic analyses. The presence of cytolethal distending toxin was confirmed on the basis of characteristic morphological changes in HeLa cells incubated with filter-sterilized whole-cell lysates of reference and monkey Campylobacter isolates and examinations by light microscopy, confocal microscopy, and flow cytometry. Although cdtB gene sequences were found in both Campylobacter jejuni and Campylobacter coli, the production of cytolethal distending toxin correlated positively (P < 0.0001) only with C. jejuni. We concluded that cytolethal distending toxin activity is a characteristic of C. jejuni. Our C. jejuni cdtB gene-specific PCR assay might be of assistance for differentiating toxigenic C. jejuni from C. coli in clinical laboratories.     

Microarray-based identification of thermophilic Campylobacter jejuni,C. coli,C. lari,and C. upsaliensis

DNA microarrays are an excellent potential tool for clinical microbiology, since this technology allows relatively rapid identification and characterization of microbial and viral pathogens. In the present study, an oligonucleotide microarray was developed and used for the analysis of thermophilic Campylobacter spp., the primary food-borne pathogen in the United States. We analyzed four Campylobacter species: Campylobacter jejuni, C. coli, C. lari, and C. upsaliensis. Our assay relies on the PCR amplification of specific regions in five target genes (fur, glyA, cdtABC, ceuB-C, and fliY) as a first step, followed by microarray-based analysis of amplified DNAs. Alleles of two genes, fur and glyA, which are found in all tested thermophilic Campylobacter spp., were used for identification and discrimination among four bacterial species, the ceuB-C gene was used for discrimination between C. jejuni and C. coli, and the fliY and cdt genes were used as additional genetic markers specific either for C. upsaliensis and C. lari or for C. jejuni. The array was developed and validated by using 51 previously characterized Campylobacter isolates. All isolates were unambiguously identified on the basis of hybridization patterns with 72 individual species-specific oligoprobes. Microarray identification of C. jejuni and C. coli was confirmed by PCR amplification of other genes used for identification (hipO and ask). Our results demonstrate that oligonucleotide microarrays are suitable for rapid and accurate simultaneous differentiation among C. jejuni, C. coli, C. lari, and C. upsaliensis.     

Identification of ciprofloxacin-resistant Campylobacter jejuni by use of a fluorogenic PCR assay

Fluoroquinolones are one class of antimicrobial agents commonly used to treat severe Campylobacter jejuni infection. C. jejuni strains resistant to high levels of the fluoroquinolone ciprofloxacin (MIC >/=16 microg/ml) have been predominantly characterized with a C-->T transition in codon 86 of gyrA. The gyrA gene encodes one subunit of DNA gyrase, which is a primary target for fluoroquinolone antibiotics. This study establishes a rapid PCR-based TaqMan method for identifying ciprofloxacin-resistant C. jejuni strains that carry the C-->T transition in codon 86 of gyrA. The assay uses real-time detection, eliminating the need for gel electrophoresis. Optimization of the assay parameters using purified Campylobacter DNA resulted in the ability to detect femtogram levels of DNA. The method should be useful for monitoring the development of ciprofloxacin resistance in C. jejuni. Compiled nucleotide sequence data on the quinolone resistance-determining region of gyrA in Campylobacter indicate that sequence comparison of this region is a useful method for tentative identification of Campylobacter isolates at the species level.     

Ciprofloxacin Resistance in Campylobacter jejuni Isolates: Detection of gyrA Resistance Mutations by Mismatch Amplification Mutation Assay PCR and DNA Sequence Analysis

The gyrA quinolone resistance determining region was sequenced from 13 ciprofloxacin-resistant and 20 ciprofloxacin-susceptible Campylobacter jejuni isolates. All isolates resistant to ciprofloxacin had Thr-86-to-Ile mutations, a mutation frequently associated with the acquisition of resistance to fluoroquinolones. A mismatch amplification mutation assay (MAMA) PCR protocol was developed that detects this gyrA mutation in quinolone-resistant isolates. The MAMA PCR provides a means for routine detection of the gyrA mutation without the need for sequencing the gyrA gene.     

Rapid Identification of Thermotolerant Campylobacter jejuni, Campylobacter coli, Campylobacter lari, and Campylobacter upsaliensis from Various Geographic Locations by a GTPase-Based PCR-Reverse Hybridization Assay

Recently, a gene from Campylobacter jejuni encoding a putative GTPase was identified. Based on two semiconserved GTP-binding sites encoded within this gene, PCR primers were selected that allow amplification of a 153-bp fragment from C. jejuni, C. coli, C. lari, and C. upsaliensis. Sequence analysis of these PCR products revealed consistent interspecies variation, which allowed the definition of species-specific probes for each of the four thermotolerant Campylobacter species. Multiple probes were used to develop a line probe assay (LiPA) that permits analysis of PCR products by a single reverse hybridization step. A total of 320 reference strains and clinical isolates from various geographic origins were tested by the GTP-based PCR-LiPA. The PCR-LiPA is highly specific in comparison with conventional identification methods, including biochemical and whole-cell protein analyses. In conclusion, a simple method has been developed for rapid and highly specific identification of thermotolerant Campylobacter species.     

Evaluation of 11 PCR assays for species-level identification of Campylobacter jejuni and Campylobacter coli

We examined the sensitivity and specificity of 11 PCR assays described for the species identification of Campylobacter jejuni and Campylobacter coli by using 111 type, reference, and field strains of C. jejuni, C. coli, and Campylobacter lari. For six assays, an additional 21 type strains representing related Campylobacter, Arcobacter, and Helicobacter species were also included. PCR tests were initially established in the laboratory by optimizing conditions with respect to five type and reference strains of C. jejuni, C. coli, and C. lari. One PCR test for C. coli failed to give appropriate results during this initial setup phase and was not evaluated further. The remaining 10 assays were used to examine heated lysate and purified DNA templates as appropriate of well-characterized type, reference, and field strains of C. jejuni (n = 62), C. coli (n = 34), and C. lari (n = 15). The tests varied considerably in their sensitivity and specificity for their respective target species. No assay was found to be 100% sensitive and/or specific for all C. jejuni strains tested, but four assays for C. coli gave appropriate responses for all strains examined. Between one and six strains of C. jejuni gave amplicons in four of seven C. jejuni PCR tests only where purified DNA was used as the template; corresponding results were seen with one strain of C. coli in each of three assays for the latter species. Our findings indicate that a polyphasic strategy for PCR-based identification should be used to identify C. jejuni and C. coli strains. The data may assist laboratories in selecting assays suited for their needs and in designing evaluations of future PCR tests aimed to identify these species.     

Uneven distribution of the luxS gene within the genus Campylobacter

Polymerase chain reaction (PCR) amplification was performed on 20 isolates of five Campylobacter species using a degenerate primer pair designed in silico to generate a product of the luxS gene or its homologue from Campylobacter organisms. Although the primer pair successfully amplified products of approximately 500 base pairs (bp) with the eight isolates of C. jejuni and C. coli and some of C. upsaliensis and C. fetus, it failed to amplify fragments with all four isolates of C. lari (two urease-negative C. lari; two urease-positive thermophilic campylobacters). When Southern blot hybridisation analysis was carried using the mixed luxS gene fragments prepared from the C. jejuni, C. coli, C. upsaliensis and C. fetus strains as a probe, all C. jejuni, C. coli, C. upsaliensis and C. fetus isolates gave positive signals, but no positive signal was detected with any C. lari isolate. These results clearly indicate that C. jejuni, C. coli, C. upsaliensis and C. fetus carry the luxS gene or its homologue. However, no luxS gene or its homologue was identified to occur in the C. lari genome. Although autoinducer-2 assays were positive in C. jejuni, C. coli, C. upsaliensis and C. fetus isolates, it was negative with all the C. lari isolates examined. In addition, a biofilm formation assay demonstrated that biofilm formation in the C. lari species does not appear to correlate with the occurrence of the luxS gene because biofilm formation occurred among some isolates of C. lari.     

Specific detection of Campylobacter jejuni and Campylobacter coli by using polymerase chain reaction.

Development of a routine detection assay for Campylobacter jejuni and Campylobacter coli in clinical specimens was undertaken by using the polymerase chain reaction (PCR). An oligonucleotide primer pair from a conserved 5' region of the flaA gene of C. coli VC167 was used to amplify a 450-bp region by PCR. The primer pair specifically detected 4 strains of C. coli and 47 strains of C. jejuni; but it did not detect strains of Campylobacter fetus, Campylobacter lari, Campylobacter upsaliensis, Campylobacter cryaerophila, Campylobacter butzleri, Campylobacter hyointestinalis, Wolinella recta, Helicobacter pylori, Escherichia coli, Shigella spp., Salmonella spp., Vibrio cholerae, Citrobacter freundii, or Aeromonas spp. By using a nonradioactively labeled probe internal to the PCR product, the assay could detect as little as 0.0062 pg of purified C. coli DNA, or the equivalent of four bacteria. In stools seeded with C. coli cells, the probe could detect between 30 and 60 bacteria per PCR assay. The assay was also successfully used to detect C. coli in rectal swab specimens from experimentally infected rabbits and C. jejuni in human stool samples.     

Use of the omp50 gene for identification of Campylobacter species by PCR

We studied the prevalence of the omp50 gene and the Omp50 protein in Campylobacter strains. Immunodetection assays and DNA-DNA hybridizations showed that most C. coli strains tested were negative and most C. jejuni and C. lari strains tested were positive. A PCR assay was developed, using the omp50 gene as a species-specific target. We propose a combination of a hippurate test and an omp50 assay to perform identification of Campylobacter species.     

Identification of thermotolerant campylobacter species by fluorescence in situ hybridization

Thermotolerant Campylobacter spp. (Campylobacter jejuni, C. coli, C. lari, and C. upsaliensis) are leading causes of food-borne diarrhea in humans. In this study, the usefulness of fluorescence in situ hybridization (FISH) for the identification of Campylobacter isolates was investigated. A hierarchical FISH probe set that included six group-, genus-, and species-specific probes was developed and evaluated with 12 reference strains and 94 clinical isolates of Campylobacter, Arcobacter, and Helicobacter. FISH correctly identified all isolates to the genus level and detected all thermotolerant Campylobacter isolates. The assay showed high degrees of sensitivity for the identification of C. jejuni (90%), C. coli (97%), C. lari (81%), and C. upsaliensis (100%) to the species level.     

Correct identification and discrimination between Campylobacter jejuni and C. coli by a standardized hippurate test and species-specific polymerase chain reaction

Hippurate hydrolysis test results of 240 Campylobacter strains were compared with those of two multiplex polymerase chain reaction (PCR) assays. Of the 152 strains identified in Finnish clinical microbiology routine laboratories as C. coli (hippurate-negative), 11% were C. jejuni (hippurate-positive) by standardized hippurate test and 39% by PCR in the reference laboratory. Two of the 81 hippurate-positive strains were identified as C. coli. Standardizing the hippurate test by determining minimum and maximum turbidity limits (McFarland 6 and McFarland 10, OD(450) values 0.8 and 1.4, respectively) for the bacterial cell suspension eliminated the false-positive results, but 32% of the 145 hippurate-negative strains were still identified as C. jejuni by PCR. The species identification of Campylobacter isolates in Finland could be improved by using a standardized hippurate hydrolysis test to identify hippurate-positive C. jejuni and testing hippurate-negative strains by molecular methods. This would also improve the epidemiological data on this important zoonotic pathogen.     

Pet dogs and chicken meat as reservoirs of Campylobacter spp. in Barbados

Campylobacter spp. are the second most common pathogen isolated from stools of patients with gastroenteritis in Barbados. The aim of this study was to identify reservoirs of Campylobacter and the likely source(s) of human infection. Fecal specimens from 596 animals and 311 samples of animal food products were analyzed for the presence of Campylobacter spp. by standard culture techniques. Isolates were characterized by conventional phenotypic tests, confirmed by latex agglutination and PCR with genus-specific primers, and identified by the use of species-specific primers. High isolation rates were obtained for chickens (94.2%), pigs (90.5%), dogs (46.9%), cats (37.3%), and wild birds (39.3%). Campylobacter was also recovered from monkeys (17.1%) and sheep (4.2%) but not from cows. Chicken meat was frequently contaminated with Campylobacter (58.4%), but its recovery from other animal food products was rare. Campylobacter jejuni was the most commonly identified species in humans (63.6%), chickens (86.6%), dogs (51.5%), and chicken meat (79.8%). Porcine isolates were predominantly C. coli (98.4%), while cats harbored mainly C. upsaliensis and C. helveticus. Wild birds alone carried urease-positive thermophilic campylobacters. C. jejuni and C. coli isolates from different sources were compared with isolates from humans by randomly amplified polymorphic DNA typing with the primers OPA 11 and HLWL 85. Genotyping revealed similarities between isolates from chicken meat and those from humans and could not distinguish between two clinical isolates and four canine strains. Our results suggest that dogs are significant reservoirs of Campylobacter and contribute to human enteric infections and that chicken meat is a likely vehicle for the transmission of campylobacters to humans.