Molecular analysis of the tlyA gene in Campylobacter lari.

Full-length tlyA gene and its adjacent genetic loci from the urease-positive thermophilic Campylobacter (UPTC) CF89-12 [approximately 15,000 base pairs (bp) in length], as well as a reference strain Campylobacter lari RM2100 (approximately 9,000 bp), were analyzed. The possible open-reading frame of tlyA from UPTC CF89-12 was shown to have 720 bp with a calculated molecular mass of approximately 26.7 kDa. Using a primer pair designed in silico, a total of approximately 1.1 kbp consisting of putative promoter region, structural gene for tlyA, and its adjacent genetic loci were identified in all 17 C. lari isolates [n = 13 for UPTC; n = 4 for urease-negative (UN) C. lari]. Although sequence differences were demonstrated at approximately 20 loci within the 90 bp non-coding (NC) region, including the putative promoter structure candidates immediately upstream of the tlyA gene among the 18 isolates including C. lari RM2100, no sequence differences were identified within the NC region among the five UN C. lari isolates examined. A start codon ATG and a probable ribosome-binding site, AGGC(T)GG(A), for the tlyA gene were identified in all 18 isolates, including C. lari RM2100. The putative intrinsic ρ-independent transcriptional terminator structure candidate was also identified for the tlyA gene in both UPTC CF89-12 and C. lari RM2100. Additionally, the hemolysis assay was performed with some of the C. lari isolates. The tlyA gene nucleotide sequence data may possibly be useful for discrimination between UN C. lari and UPTC organisms, as well as for the differentiation among the four thermophilic Campylobacter species.

Prevalence of three campylobacter species, C. jejuni, C. coli, and C. lari, using multilocus sequence typing in wild birds of the Mid-Atlantic region, USA.

Campylobacter jejuni is responsible for the majority of bacterial foodborne gastroenteritis in the US, usually due to the consumption of undercooked poultry. Research on which avian species transmit the bacterium is limited, especially in the US. We sampled wild birds in three families-Anatidae, Scolopacidae, and Laridae-in eastern North America to determine the prevalence and specific strains of Campylobacter. The overall prevalence of Campylobacter spp. was 9.2% for all wild birds sampled (n = 781). Campylobacter jejuni was the most prevalent species (8.1%), while Campylobacter coli and Campylobacter lari prevalence estimates were low (1.4% and 0.3%, respectively). We used multilocus sequence typing PCR specific to C. jejuni to characterize clonal complexes and sequence types isolated from wild bird samples and detected 13 novel sequence types, along with a clonal complex previously only associated with human disease (ST-658). Wild birds share an increasing amount of habitat with humans as more landscapes become fragmented and developed for human needs. Wild birds are and will remain an important aspect of public health due to their ability to carry and disperse emerging zoonotic pathogens or their arthropod vectors. As basic information such as prevalence is limited or lacking from a majority of wild birds in the US, this study provides further insight into Campylobacter epidemiology, host preference, and strain characterization of C. jejuni.

Phylogenetic analysis of urease-positive thermophilic Campylobacter (UPTC) strains based on the molecular characterization of the flaA gene.

Molecular cloning, nucleotide sequencing, and characterization of the flaA gene from additional isolates of urease-positive thermophilic Campylobacter (UPTC) were performed. These isolates were obtained from the natural environment in Northern Ireland (n = 9 from mussels) and in England (n = 1 from sea water). All isolates carried the shorter flaA gene, [open reading frames (ORFs), 1,461 to 1,503 base pairs], without any internal termination codons, and did not carry any flaA pseudogenes. The UPTC isolates were well discriminated by the neighbor joining (NJ) phylogenetic tree constructed based on the putative flaA genes ORFs nucleotide sequence information. In addition, the NJ tree constructed based on the flaA-short variable region sequence information discriminated the Campylobacter lari isolates with a similar degree of discrimination power.

Reliability of nucleotide sequence information of full-length flagellin A gene (flaA) and flaA short variable region (SVR) for molecular discrimination of Campylobacter lari organisms.

We aimed to clarify if the thermophilic Campylobacter lari organisms including urease-negative (UN) C. lari and urease-positive thermophilic Campylobacter (UPTC) can be differentiated at the species and/or subspecies levels by employing the full-length flaA gene and flaA short variable region (SVR) nucleotide sequence information or not. Thermophilic Campylobacter isolates (n = 45) including UN C. lari (n = 17), UPTC (n = 18), and Campylobacter jejuni (n = 10) were well discriminated at the isolate level by the unweighted pair group method using arithmetic means analysis and neighbor joining procedures constructed based on the full-length flaA gene and flaA SVR nucleotide sequence information. Thus, these procedures may possibly be useful for epidemimological studies for C. lari and C. jejuni.

Rapid detection and differentiation of Campylobacter jejuni, Campylobacter coli, and Campylobacter lari in food, using multiplex real-time PCR.

A multiplex real-time PCR assay based on four differently labeled TaqMan probes for detection and differentiation of the thermophilic Campylobacter species C. jejuni, C. coli, and C. lari was established and validated in food products. This assay combines two previously published PCR assays for C. jejuni and C. coli with a newly developed detection assay for C. lari and an internal amplification control system. The selectivity of the method was determined by analyzing 70 Campylobacter strains and 43 strains of other bacteria. The sensitivity was 50 fg of C. jejuni and C. lari DNA and 500 fg of C. coli DNA per PCR. It was possible to detect 1 to 10 CFU/25 g of food before preenrichment of all three species. More than 400 samples of various foods (poultry, seafood, and meat) were analyzed after 48 h of preenrichment parallel to the conventional diagnostic method of culture and biochemical identification. Using the established real-time PCR assay, 55.4% of the samples were recognized as positive for thermophilic Campylobacter species, whereas with the conventional method only 40.3% of the samples were positive. The real-time PCR assay also detected contaminations with two different Campylobacter species in 32.6% of the analyzed poultry samples, a finding of epidemiological interest. Compared with the original PCR method, which was established for the differentiation of bacterial isolates of C. jejuni and C. coli, this new method also detects and distinguishes C. lari, was validated as an analytical tool for food analysis, and provides reliable and extensive results within 2 days.

Prevalence of thermotolerant Campylobacter in partridges (Perdix perdix).

AIM: To estimate the prevalence of thermotolerant Campylobacter spp. in commercially reared partridges (Perdix perdix) in southern Italy. METHODS AND RESULTS: Cloacal swabs of partridges (n = 240), equally distributed between male and female birds, from a game bird farm located in the Southern Italy were examined for the prevalence of thermotolerant Campylobacter spp. The samples were processed in order to detect thermotolerant Campylobacter spp. by culture methods. The positive samples were then confirmed by multiplex polymerase chain reaction. Thermotolerant Campylobacter spp. were isolated from 118 (49.2%) of the 240 cloacal swabs examined. As proved by PCR, 100% of the strains were identified as Campylobacter coli (118/118), and 15 (12.7%) out of the 118 positive samples were also positive for Campylobacter jejuni. In contrast, Campylobacter lari was not identified. Adult partridges showed a significantly higher prevalence (P < 0.05) than younger ones. CONCLUSION: These results reinforce the assumption that game birds may be considered as potential carriers of Campylobacter spp. for human being and other animal species. SIGNIFICANCE AND IMPACT OF THE STUDY: Although an earlier 1986 publication described the prevalence of Campylobacter coli in commercially reared partridges, this is the first report to confirm the species of Campylobacter using a PCR test.

Novel Campylobacter lari-like bacteria from humans and molluscs: description of Campylobacter peloridis sp. nov., Campylobacter lari subsp. concheus subsp. nov. and Campylobacter lari subsp. lari subsp. nov.

A polyphasic study was undertaken to clarify the taxonomic position of Campylobacter lari-like strains isolated from shellfish and humans. The diversity within the strain collection was initially screened by means of fluorescent amplified fragment length polymorphism analysis and whole-cell protein electrophoresis, revealing the existence of two clusters distinct from C. lari and other Campylobacter species. The divergence of these clusters was confirmed by phenotypic analysis and by 16S rRNA and hsp60 gene sequence analysis. Phylogenetic analysis identified C. lari, Campylobacter jejuni, Campylobacter coli and Campylobacter insulaenigrae as the closest phylogenetic neighbours of both taxa. DNA-DNA hybridizations revealed that one cluster, comprising 10 strains, represented a novel Campylobacter species, for which the name Campylobacter peloridis sp. nov. is proposed, with 2314BVA(T) (=LMG 23910(T) =CCUG 55787(T)) as the type strain. The second cluster, comprising six strains, represents a novel subspecies within the species C. lari, for which the name Campylobacter lari subsp. concheus subsp. nov. is proposed, with 2897R(T) (=LMG 21009(T) =CCUG 55786(T)) as the type strain. The description of C. lari subsp. concheus has the effect of automatically creating the subspecies Campylobacter lari subsp. lari subsp. nov. (type strain LMG 8846(T)=NCTC 11352(T)).

Similarity of Campylobacter lari among human, animal, and water isolates in Norway.

A total of 49 isolates of Campylobacter lari from human, poultry, ducks, pigs, and water were genetically characterized. The species were identified by biotyping and multiplex polymerase chain reaction (PCR). Automatic riboprints were performed with the PstI restriction enzyme and RiboPrinter. The identification of the isolates was predicted when the corresponding pattern matched one of the patterns of the DuPont identification (DUP-ID) library and was then assigned an identification number. Thirty-five (71.4%) of the isolates were given a DUP-ID number. The isolates from water and animals showed a high degree of similarity to the human strains represented by DUP-PST1-1010, DUP-PST1-1166, DUP-PST1-1178, and DUP-PST1-1081. Some profiles (i.e., DUP-PST1-2021 and DUP-PST1-1184) were found only among the human isolates. Dendrogram analysis using BioNumerics grouped isolates into three main clusters. One of those clusters contained DUP-PST1-2021, DUP-PST1-1184, and DUP-PST1-1081, which was found in both humans and ducks. A second cluster generated DUP-PST1-1010, found in both humans and poultry, and DUP-PST1-1079, found in water. The third cluster consisted of two strains, DUP-PST1-1066 and DUP-PST1-1078, originating in humans, animals, and water. Three human strains and two poultry strains were diverse and formed their own clusters and could not be assigned a DUP-ID number. Because of the similarity of C. lari isolated from humans, poultry, ducks, pigs, and water, as well as the limited knowledge of environmental survival and its virulence factors, special hygienic precautions should be taken to avoid the risk of transmitting Campylobacter.

Amplified fragment-length polymorphism and protein profiling for identification of Campylobacter lari subgroups.

Amplified fragment-length polymorphism analysis (AFLP) has been shown to be a suitable method for subtyping of bacteria belonging to the genus Campylobacter. Campylobacter lari is a phenotypically and genotypically diverse species that comprises the classical nalidixic acid-resistant thermophilic campylobacters and the biochemical C. lari variants, urease-positive, nalidixic acid-susceptible, and urease producing nalidixic acid-susceptible strains. AFLP profiling and whole-cell protein profile analysis are suitable methods for studying the taxonomic and epidemiological relationships among strains of the C. lari variants. Numerical analysis of AFLP profiles and of partial protein profiles allows the discrimination of distinct C. lari genogroups. No correlation of these genogroups with different sources of the strains has been identified until now.

Speciation of Campylobacter coli, C. jejuni, C. helveticus, C. lari, C. sputorum, and C. upsaliensis by matrix-assisted laser desorption ionization-time of flight mass spectrometry.

Multiple strains of Campylobacter coli, C. jejuni, C. helveticus, C. lari, C. sputorum, and C. upsaliensis isolated from animal, clinical, or food samples have been analyzed by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Whole bacterial cells were harvested from colonies or confluent growth on agar and transferred directly into solvent and then to a spot of dried 3-methoxy-4-hydroxycinnamic acid (matrix). Multiple ions in the 5,000- to 15,000-Da mass range were evident in spectra for each strain; one or two ions in the 9,500- to 11,000-Da range were consistently high intensity. "Species-identifying" biomarker ions (SIBIs) were evident from analyses of multiple reference strains for each of the six species, including the genome strains C. jejuni NCTC 11168 and C. jejuni RM1221. Strains grown on nine different combinations of media and atmospheres yielded SIBI masses within +/-5 Da with external instrument calibration. The highest-intensity C. jejuni SIBIs were cytosolic proteins, including GroES, HU/HCj, and RplL. Multiple intraspecies SIBIs, corresponding probably to nonsynonymous nucleotide polymorphisms, also provided some intraspecies strain differentiation. MALDI-TOF MS analysis of 75 additional Campylobacter strains isolated from humans, poultry, swine, dogs, and cats revealed (i) associations of SIBI type with source, (ii) strains previously speciated incorrectly, and (iii) "strains" composed of more than one species. MALDI-TOF MS provides an accurate, sensitive, and rapid method for identification of multiple Campylobacter species relevant to public health and food safety.

A newly constructed primer pair for the PCR amplification, cloning and sequencing of the flagellin (flaA) gene from isolatesof urease-negative Campylobacter lari.

A newly constructed primer pair (lari-Af/lari-Ar) designed to generate a product of the flagellin (flaA) gene for urease-negative Campylobacter lari produced a PCR amplicon of about 1700 bp for 16 isolates from 7 seagulls, 5 humans, 3 food animals and one mussel in Japan and Northern Ireland. Nucleotide sequencing and alignments of the flaA amplicons from these isolates demonstrated that the deduced amino acid sequences of the possible open reading frame were 564-572 amino acid residues in length with calculated molecular weights of 58,804 to 59,463. The deduced amino acid sequence similarity analysis strongly suggested that the ORF of the flaA from the 16 isolates showed 70-75% sequence similarities to those of Campylobacter jejuni isolates. The approximate Mr of the flagellin purified from some of the isolates of urease-negative C. lari was estimated to range from 59.6 to 61.8 kDa. Thus, flagellin from the isolates of urease-negative C. lari was shown for the first time to have a molecular size similar to those of C. jejuni and Campylobacter coli isolates, but to be different from the shorter flaA and smaller flagellin of urease-positive thermophilic Campylobacter (UPTC) isolates. Flagellins from C. lari spp., consisting of the two representative taxa of urease-negative C. lari and UPTC, thus show genotypic and phenotypic diversity.

Extended multilocus sequence typing system for Campylobacter coli, C. lari, C. upsaliensis, and C. helveticus.

A multilocus sequence typing (MLST) system has been reported previously for Campylobacter jejuni to both differentiate strains and identify clonal lineages. However, sequence variation at the MLST loci prevents its use for closely related Campylobacter species. We describe herein an expanded MLST method to include three clinically relevant Campylobacter species, C. coli, C. lari, and C. upsaliensis, and a fourth Campylobacter species, C. helveticus. The C. coli and C. helveticus methods use the same seven C. jejuni loci (aspA, atpA, glnA, gltA, glyA, pgm, and tkt); however, adk and pgi were substituted for aspA and gltA in C. lari and for gltA and pgm in C. upsaliensis. Multiple C. coli (n = 57), C. lari (n = 20), C. upsaliensis (n = 78), and C. helveticus (n = 9) isolates, representing both clinical and environmental sources, were typed. All four species were genetically diverse: the majority (> 80%) of the isolates had unique sequence types (STs). Using this method, mixed C. lari, C. upsaliensis, and C. helveticus isolates were identified; upon separation, each isolate was shown to contain two strains of the same species with distinct STs. Additionally, the expanded MLST method was able to detect potential lateral transfer events between C. jejuni and either C. coli or C. lari and between C. upsaliensis and C. helveticus. Thus, the expanded MLST method will prove useful in differentiating strains of five Campylobacter species, identifying mixed Campylobacter cultures, and detecting genetic exchange within the genus.

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.

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 distinct Campylobacter lari genogroups by amplified fragment length polymorphism and protein electrophoretic profiles.

Campylobacter lari is a phenotypically and genotypically diverse species that comprises the classical nalidixic acid-resistant thermophilic campylobacters (NARTC) and the biochemical C. lari variants, including the urease-positive campylobacters (UPTC), the nalidixic acid-susceptible campylobacters (NASC), and the urease-producing nalidixic acid-susceptible campylobacters. To study the taxonomic and epidemiological relationships among strains of the C. lari variants, amplified fragment length polymorphism (AFLP) profiling and whole-cell protein profile analysis were performed with 55 C. lari strains. Great genetic heterogeneity in AFLP and protein profiles was observed. Numerical analysis of AFLP profiles and of partial protein profiles allowed discrimination of four distinct genogroups. AFLP cluster I included nearly homogeneous patterns for C. lari NARTC strains (genogroup I). UPTC strains together with non-urease-producing NASC strains produced highly diverse patterns and were placed in genogroup II. The genogroup III strains had the NASC phenotype and produced more homogeneous patterns. Finally, genogroup IV strains had the classical NARTC phenotype and produced AFLP patterns that were very distinct from those of other genogroups. One UPTC strain had aberrant patterns and clustered separately, which may indicate that there is an additional genogroup. Preliminary DNA-DNA hybridization experiments suggested that genogroups I and III represent a single genomic species and that genogroup IV represents a distinct species. The detection of moderate levels of DNA-DNA hybridization between a genogroup II reference strain and genogroup I and III reference strains highlights the need for further DNA-DNA hybridization experiments to clarify the taxonomic status of the former group. No correlation of genogroups with different sources of strains was identified. These data show that UPTC strains are genetically diverse and distinct from NARTC strains. In addition, they indicate that the classical NARTC phenotype encompasses at least two genogroups.

Toward an international standard for PCR-based detection of food-borne thermotolerant Campylobacters: validation in a multicenter collaborative trial.

As part of a European research project, the performance of a PCR assay to detect food-borne thermotolerant campylobacters (Campylobacter jejuni, C. coli, and C. lari) was evaluated through an international collaborative trial involving 12 participating laboratories. DNA from 10 target and 8 nontarget strains was tested, and the results were reported as the presence of a positive signal after gel electrophoresis. The overall inclusivity (sensitivity) was 93.7%, and the exclusivity (specificity) was 100%. The results indicate that the assay can become an international standard and can be confidently applied in microbiological laboratories.