Routine identification of Campylobacter jejuni and Campylobacter coli from human stool samples

Correct identification of Campylobacter jejuni and Campylobacter coli isolates to the species or subspecies level is a cumbersome but nevertheless important task for a routine diagnostic laboratory. The widely used biochemical tests might be often misleading while more sophisticated phenotypic or genotypic methods are not generally available. This investigation was performed to assess the performance of common biochemical identification in comparison with species-specific PCR and gas liquid chromatography of whole cell fatty acid extracts (GLC). A total of 150 consecutive isolates from human stool samples were investigated (134 C. jejuni ssp. jejuni, 14 C. coli, two Helicobacter pullorum). From these 144, 145 and 149 isolates were correctly identified by biochemistry, GLC and PCR, respectively. Biochemical identification of all C. jejuni isolates was confirmed by PCR. GLC detected both H. pullorum strains but misidentified two C. coli strains as C. jejuni and one C. jejuni strain as C. coli. No single method can be defined as 'gold standard' for identification of C. jejuni and C. coli but a combination of techniques is needed. Therefore a stepwise identification scheme starting with biochemical reactions is suggested. All results other than C. jejuni should be confirmed by further methods. For indoxyl acetate-positive isolates species-specific PCR is recommended while GLC seems to be advantageous in indoxyl acetate-negative isolates.     

Genotyping of Campylobacter jejuni strains from Danish broiler chickens by restriction fragment length polymorphism of the LPS gene cluster

AIMS: To apply and evaluate LG (LPS genes) genotyping, which is a genotyping method based on a cluster of genes involved in the synthesis of surface lipopolysaccharides (LPS) in Campylobacter species, for typing of Campylobacter jejuni isolates obtained from Danish broiler chickens. Furthermore, the LG genotyping method was used to study the genetic stability of four C. jejuni strains after gastrointestinal passage through experimentally infected chickens. METHODS AND RESULTS: In the present study, the LG genotyping method was modified with respect to the restriction enzymes used. To validate the method, 63 Penner serotype reference strains and 107 C. jejuni chicken isolates, representing the most common Penner serotypes of C. jejuni in Danish poultry, were selected for typing. The method was successfully used for typing all isolates and the LG genotype profiles were reproducible. There were no changes in the LG genotype of the C. jejuni strains obtained after experimental passage through chickens. CONCLUSIONS: All C. jejuni strains obtained from broiler chickens were typeable by the LG genotyping method. Application of the RsaI restriction enzyme improved the method in terms of ease and consistency of analyses and increase of discriminatory power. SIGNIFICANCE AND IMPACT OF THE STUDY: The LG genotyping method is a valuable tool for typing C. jejuni isolates obtained from poultry. However, the association between Penner serotyping based on passive haemagglutination of heat-stable antigens and LG genotyping was low when applied to poultry isolates. This is in contrast to previous studies on isolates of human origin that reported a high correlation between results obtained by the two typing methods (Shi et al. 2002).     

Typings of Campylobacter jejuni isolated from patients of 2 outbreak cases by genotypic and phenotypic methods

We compared Campylobacter jejuni strains isolated from the patient stools associated with two food-borne diarrheal outbreak cases by the serotypic methods (Lior and Penner systems) and the genotypic methods (restriction fragment length polymorphism (RFLP) of flaA gene and pulsed-field gel electrophoresis (PFGE)). Fla-RFLP was based on the digestion of 410 bp DNA fragment by MboI restriction enzyme amplified from a 5' portion of C. jejuni flaA gene. Six distinctive fla-RFLP patterns were identified by examining 29 serotype reference strains and 58 strains isolated from the patients infected with C. jejuni independently. In the first outbreak case, 4 isolates were shown to be the same patterns each other by the fla-RFLP and PFGE, and by the Lior serotyping, except the Penner system that serotyped into 2 distinct types. On the other hand, in the second case, out of 10 isolates, 5 isolates were identical by the both genotypic and the both serotypic methods, and 4 isolates were not differentiated by the fla-RFLP and Penner system, but were separated into 4 types by PFGE in a little difference. The rest isolate was completely different from the other isolates by the all of methods used now. The findings suggest that the second case occurred by the infection of at least 3 different strains of C. jejuni.     

Ribotypes and AP-PCR fingerprints of thermophilic campylobacters from marine recreational waters

Thirty-two strains of thermophilic campylobacters isolated from marine recreational waters and seven reference strains were biotyped and analysed by chromosomal DNA Hae III ribopatterns and AP-PCR profiles based on a random 10-mer primer (5'-CAA TCG CCG T-3'). The majority of seawater isolates (90%) were Campylobacter coli , and three strains were Camp. jejuni. Southern blot hybridization analysis showed differences between the strains, and in a numerical analysis three main clusters were formed at the 45% similarity level, that corresponded to Camp. jejuni subsp. jejuni, Camp. coli , and a combination of Camp. coli and Camp. jejuni subsp. doylei. AP-PCR profiles also differentiated between the species but were less discriminatory than ribotyping because six strains (17%) could not be typed by this method. Numerical analysis gave four main clusters at the 45% similarity level, corresponding to Camp. jejuni subsp. jejuni, Camp. coli (two clusters) and Camp. lari. The study shows that strains within each species are diverse genomically. Both molecular methods were highly discriminatory, although some strains with identical ribotypes could be distinguished by AP-PCR, and they are valuable new alternatives to traditional typing in epidemiological studies of environmental campylobacters.     

Application of biochemical and polymerase chain reaction assays for identification of Campylobacter isolates from non-human primates

A multiplex polymerase chain reaction (PCR) assay was performed on 167 thermophilic campylobacters isolated from non-human primates. Samples were first identified by phenotypic methods resulting in 64 Campylobacter jejuni and 103 C. coli strains. Four strains identified biochemically as C. coli, were then determined to be C. jejuni by PCR. Comparison of methodologies showed that the main discrepancies were attributed to the hippurate hydrolysis test and sensitivity to cephalothin and nalidixic acid. Analysis of data showed that the application of phenotypic methods should be supplemented by a molecular method to offer a more reliable Campylobacter identification.     

The ability of Fla-typing schemes to discriminate between strains of Campylobacter jejuni

AIMS: The aim of this investigation was to compare the usefulness of two previously published flagellin PCR-RFLP typing (Fla-typing) techniques for the subtyping of Campylobacter jejuni strains, in terms of ease of use and discriminatory power. METHODS AND RESULTS: Six groups of isolates, which were epidemiologically unrelated but with similar Fla-types, and five groups of epidemiologically related poultry isolates, with similar PFGE profiles, were used in the comparison. The Fla-typing methods used varied in the number and length of fla-genes amplified and the restriction enzymes used. In addition, the use of separately amplified PCR fragments of both the flaA and flaB genes to generate RFLP profiles was investigated. CONCLUSION: The results clearly demonstrated that both previously published methods exhibit some advantages over the other. However, optimal discrimination was obtained by the use of separately amplified PCR fragments of both fla-genes. SIGNIFICANCE AND IMPACT OF THE STUDY: The subtyping of Camp. jejuni isolates is considered essential for epidemiological purposes. Genotyping methods are now more frequently used but have yet to be standardized. Fla-typing is a rapid and easy to use method with acceptable discriminatory power. However, the discriminatory power of the currently published Fla-typing techniques may be further improved by incorporating RFLP profiles of both fla-genes.     

Campylobacter insulaenigrae isolates from northern elephant seals (Mirounga angustirostris) in California

There are only two reports in the literature demonstrating the presence of Campylobacter spp. in marine mammals. One report describes the isolation of a new species, Campylobacter insulaenigrae sp. nov., from three harbor seals (Phoca vitulina) and a harbor porpoise (Phocoena phocoena) in Scotland, and the other describes the isolation of Campylobacter jejuni, Campylobacter lari, and an unknown Campylobacter species from northern elephant seals (Mirounga angustirostris) in California. In this study, 72 presumptive C. lari and unknown Campylobacter species strains were characterized using standard phenotypic methods, 16S rRNA PCR, and multilocus sequence typing (MLST). Phenotypic characterization of these isolates showed them to be variable in their ability to grow either at 42 degrees C or on agar containing 1% glycine and in their sensitivity to nalidixic acid and cephalothin. Based on both 16S rRNA PCR and MLST, all but 1 of the 72 isolates were C. insulaenigrae, with one isolate being similar to but distinct from both Campylobacter upsaliensis and Campylobacter helveticus. Phylogenetic analysis identified two C. insulaenigrae clades: the primary clade, containing exclusively California strains, and a secondary clade, containing some California strains and all of the original Scottish strains. This study demonstrates the inability of phenotypic characterization to correctly identify all Campylobacter species and emphasizes the importance of molecular characterization via 16S rRNA sequence analysis or MLST for the identification of Campylobacter isolates from marine mammals.     

Rapid identification of diverse Campylobacter lari strains isolated from mussels and oysters using a reverse hybridization line probe assay

Campylobacters isolated from mussels and oysters in The Netherlands were analysed by a novel assay, based on DNA amplification with primers, based on semiconserved GTP-binding sites of a putative GTPase gene. Polymerase chain reaction (PCR) was followed by a single step reverse hybridization line probe assay (PCR-LiPA). This permits identification of Campylobacter jejuni, C. coli, C. lari and C. upsaliensis . Among a group of 44 isolates, three C. jejuni , one C. upsaliensis , one double infection with C. jejuni and C. coli , and 38 C. lari strains were identified. These results were in complete agreement with conventional identification methods and whole cell protein analysis. One C. hyointestinalis isolate was not identified by the PCR-LiPA, since the reverse hybridization assay does not comprise specific probes for this particular species. PCR products from 36 C. lari isolates were sequenced and phylogenetic analysis revealed the presence of two major C. lari subgroups: one comprised 11 highly homologous sequences, whereas the 25 sequences in the other subgroup were more heterogeneous. This confirmed earlier findings that C. lari isolates comprise a more heterogeneous group of isolates as compared with C. jejuni , C. coli and C. upsaliensis . Based on the sequence information, a novel PCR-LiPA was developed that permits specific and rapid detection of the different C. lari variants.     

Phenotypic and Genotypic Characterizations of Campylobacter jejuni Isolated from the Broiler Meat Production Process

A set of C. jejuni isolates of different origins and flaA-genotypes obtained throughout the broiler meat production chain was tested in this study for a possible correlation of their origin, phylogenetic relationship, and phenotypic properties. Interestingly, the results showed a correlation of the origin and the phylogenetic relationship between the C. jejuni isolates and their ability to form biofilm, but not in their ability to survive at -18, 5, 20, and 48?°C. Two strains, a broiler cloacae isolate and a broiler fillet isolate, were unable to develop biofilm, while most of the C. jejuni isolates originating from meat and surfaces of the slaughterhouse readily formed biofilms after both 24, 48, and 72?h. Interestingly, these biofilm-forming strains were closely related. Furthermore, two strains that were isolated after disinfection developed significantly more biofilms after 24?h of incubation than the remaining strains. A comparative genomic analysis using DNA microarrays showed that the gene contents of strains that efficiently formed biofilms were different from those that did not. The study suggests that biofilm formation might be a lineage specific property, allowing C. jejuni to both survive environmental stress at the slaughterhouse and to attach to the surface of meat.     

High-resolution genomic fingerprinting of Campylobacter jejuni and Campylobacter coli by analysis of amplified fragment length polymorphisms

A method for high-resolution genomic fingerprinting of the enteric pathogens Campylobacter jejuni and Campylobacter coli, based on the determination of amplified fragment length polymorphism, is described. The potential of this method for molecular epidemiological studies of these species is evaluated with 50 type, reference, and well-characterised field strains. Amplified fragment length polymorphism fingerprints comprised over 60 bands detected in the size range 35-500 bp. Groups of outbreak strains, replicate subcultures, and 'genetically identical' strains from humans, poultry and cattle, proved indistinguishable by amplified fragment length polymorphism fingerprinting, but were differentiated from unrelated isolates. Previously unknown relationships between three hippurate-negative C. jejuni strains, and two C. coli var. hyoilei strains, were identified. These relationships corresponded to available epidemiological data. We conclude that this amplified fragment length polymorphism fingerprinting method may be a highly effective tool for molecular epidemiological studies of Campylobacter spp.     

PCR-based restriction fragment length polymorphism (RFLP) analysis and serotyping of Campylobacter jejuni isolates from diarrheic patients in China and Japan

Abstract A molecular typing approach for Campylobacter jejuni was applied with restriction fragment length polymorphism (RFLP) analysis of a 702-bp PCR-amplified portion of the flagellin-A ( flaA ) gene. We analyzed a total of 179 strains, including 69 independent clinical isolates from diarrheic patients in Japan, 85 isolates in China, and 25 heat-stable (HS) serotype strains by Penner and Hennessy ((1980) J. Clin. Microbiol. 12, 732–737). Six Afa I, seven Mbo I, and five Hae III RFLPs were found in the 702-bp flaA segment from the 179 strains. Using a combination of these three enzymes, 25 separate RFLP groups were recognized. While 59 of 154 (38.3%) strains obtained in Japan and China were nontypeable by the HS antigenic scheme, all but two of 154 (98.7%) could be typed by RFLP typing. All 11 isolates of HS-19 strains, which are frequently isolated from Guillain-Barré syndrome (GBS) patients, showed an identical RFLP pattern (Cj-1), and Cj-1 consisted only of HS-19 strains. This suggests that the HS-19:Cj-l strain is distinct among C. jejuni strains. This molecular typing method provides a rapid and reliable typing scheme for epidemiological studies of C. jejuni , and may also be useful for the analysis of C. jejuni subtypes from GBS patients.     

Application of single-strand conformation polymorphism and denaturing gradient gel electrophoresis for fla sequence typing of Campylobacter jejuni

Campylobacter jejuni is a frequent cause of bacterial gastroenteritis in humans all over the world. Several molecular typing methods are used to study the epidemiology of Campylobacter spp. infections. The aim of the present study was to investigate the application of single-strand conformation polymorphism (SSCP) and denaturing gradient gel electrophoresis (DGGE) analysis as rapid primary subtyping methods for C. jejuni. A variable fragment from the 3' end of the flaA to the 3' end of the intergenic region, separating the flaA and flaB genes, was subjected to SSCP and DGGE analysis. A total of 48 clinical C. jejuni isolates, 49 C. jejuni strains isolated from poultry, 2 strains isolated from ducks and 1 strain isolated from a pheasant were assigned to 24 distinct SSCP patterns. Sequence analysis of the respective DNA fragments revealed that every different fla sequence type could be distinguished by SSCP. DGGE proved to be equally discriminatory. Both methods can be applied as primary subtyping methods, because pulsed-field gel electrophoresis (PFGE) and amplified fragment length polymorphism (AFLP) analysis further differentiated isolates belonging to the same fla sequence types.     

Differentiation of campylobacter populations as demonstrated by flagellin short variable region sequences

The DNA sequence of the flaA short variable region (SVR) was used to analyze a random population of Campylobacter isolates to investigate the weakly clonal population structure of members of the genus. The SVR sequence from 197 strains of C. jejuni and C. coli isolated from humans, bovine, swine, and chickens identified a group of 43 strains containing disparate short variable region sequences compared to the rest of the population. This group contains both C. jejuni and C. coli strains but disproportionately consisted of bovine isolates. Relative synonymous codon usage analysis of the sequences identified two groups: one group typified C. jejuni, and the second group was characteristic for C. coli and the disparate alleles were not clustered. The data show that there is significant differentiation of Campylobacter populations according to the source of the isolate even without considering the disparate isolates. Even though there is significant differentiation of chicken and bovine isolates, the bovine isolates did not show any difference in ability to colonize chickens. It is possible that disparate sequences were obtained through the lateral transfer of DNA from Campylobacter species other than C. jejuni and C. coli. It is evident that recombination within the flaA SVR occurs rapidly. However, the rate of migration between populations appears to limit the distribution of sequences and results in a weakly clonal population structure.     

Phenotypic and genotypic identification of yeasts from cheese

Eighty-five yeast strains isolated from different cheeses of Austria, Denmark, France, Germany, and Italy were identified using physiological methods and genotypically using random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) analysis. Good congruence was found between the phenotypic and genotypic data for 39 of the isolates. However, 26 isolates of Geotrichum could only be identified to the species level using the genotypic methods and 7 isolates were correctly identified to the genus level only using phenotypic identification methods. The phenotypic identification did not agree with the genotypic data for 14 yeast isolates. Using ubiquinone analysis, yeast cell wall sugars and the diazonium blue B test 5 incorrectly identified isolates with phenotypic methods could be identified genotypically. In addition the 7 isolates identified only to the genus level by the phenotypic methods and the 26 Geotrichum strains were identified to the species level using the polyphasic molecular approach mentioned above. Eleven strains remained unidentified. The 76 identified yeast isolates were assigned to 39 species, the most frequent assignments were made to Debaryomyces hansenii, Geotrichum candidum, Issatchenkia orientalis, Kluyveromyces lactis, K. marxianus, Saccharomyces cerevisiae, Yarrowia lipolytica, andCandida catenulata. It is proposed that Debaryomyces hansenii (Zopf) Lodder et Kreger-van Rij and Debaryomyces fabryi Ota should be reinstated. The RAPD-PCR data reinforced the view that the species Galactomyces geotrichum is heterogeneous with all of the Geotrichum isolates from cheese products being assigned G. geotrichum group A sensu M.T. Smith. It is suggested that the name Geotrichum candidum be conserved for this rather common species.     

Campylobacter jejuni strains compete for colonization in broiler chicks

Campylobacter jejuni isolates possess multiple adhesive proteins termed adhesins, which promote the organism's attachment to epithelial cells. Based on the proposal that one or more adhesins are shared among C. jejuni isolates, we hypothesized that C. jejuni strains would compete for intestinal and cecal colonization in broiler chicks. To test this hypothesis, we selected two C. jejuni strains with unique SmaI pulsed-field gel electrophoresis macrorestriction profiles and generated one nalidixic acid-resistant strain (the F38011 Nal(r) strain) and one streptomycin-resistant strain (the 02-833L Str(r) strain). In vitro binding assays revealed that the C. jejuni F38011 Nal(r) and 02-833L Str(r) strains adhered to LMH chicken hepatocellular carcinoma epithelial cells and that neither strain influenced the binding potential of the other strain at low inoculation doses. However, an increase in the dose of the C. jejuni 02-833L Str(r) strain relative to that of the C. jejuni F38011 Nal(r) strain competitively inhibited the binding of the C. jejuni F38011 Nal(r) strain to LMH cells in a dose-dependent fashion. Similarly, the C. jejuni 02-833L Str(r) strain was found to significantly reduce the efficiency of intestinal and cecal colonization by the C. jejuni F38011 Nal(r) strain in broiler chickens. Based on the number of bacteria recovered from the ceca, the maximum number of bacteria that can colonize the digestive tracts of chickens may be limited by host constraints. Collectively, these data support the hypothesis that C. jejuni strains compete for colonization in chicks and suggest that it may be possible to design novel intervention strategies for reducing the level at which C. jejuni colonizes the cecum.     

Characterization of cross-reacting serotypes of Campylobacter jejuni

Some strains of Campylobacter jejuni react with more than one reference antiserum from the serotyping scheme based on heat-stable lipopolysaccharide antigens. To investigate the molecular basis of these cross-reactions, lipopolysaccharides from the reference strains for serotypes 4, 13, 16, 43, and 50 and isolates recovered during two different outbreaks of C. jejuni enteritis were analyzed by passive haemagglutination and sodium dodecyl sulphate-polyacrylamide gel electrophoresis coupled with silver staining or immunoblotting. The results showed that lipopolysaccharides from the reference strains and the isolates reacted with antisera prepared against heterologous strains in various combinations and that both silver-stainable, low Mr and non-silver-stainable, high Mr lipopolysaccharide components provided the antigenic determinants associated with the cross-reactions. There were strain-to-strain differences in the structural and antigenic properties of these macromolecules and shared antigenic determinants were not always provided by a common structure. Analysis of the silver-stained lipopolysaccharide profiles, outer membrane protein patterns, and chromosomal DNA restriction patterns indicated that strains with the same lipopolysaccharide profile could have the same outer membrane protein pattern and the same DNA restriction pattern. These results provided evidence for the presence of clones within this antigenic complex and implicated antigenic variation in some strains as the phenomenon responsible for the multiplicity of cross-reactions.     

Genomic variations define divergence of water/wildlife-associated Campylobacter jejuni niche specialists from common clonal complexes

Although the major food-borne pathogen Campylobacter jejuni has been isolated from diverse animal, human and environmental sources, our knowledge of genomic diversity in C. jejuni is based exclusively on human or human food-chain-associated isolates. Studies employing multilocus sequence typing have indicated that some clonal complexes are more commonly associated with particular sources. Using comparative genomic hybridization on a collection of 80 isolates representing diverse sources and clonal complexes, we identified a separate clade comprising a group of water/wildlife isolates of C. jejuni with multilocus sequence types uncharacteristic of human food-chain-associated isolates. By genome sequencing one representative of this diverse group (C. jejuni 1336), and a representative of the bank-vole niche specialist ST-3704 (C. jejuni 414), we identified deletions of genomic regions normally carried by human food-chain-associated C. jejuni. Several of the deleted regions included genes implicated in chicken colonization or in virulence. Novel genomic insertions contributing to the accessory genomes of strains 1336 and 414 were identified. Comparative analysis using PCR assays indicated that novel regions were common but not ubiquitous among the water/wildlife group of isolates, indicating further genomic diversity among this group, whereas all ST-3704 isolates carried the same novel accessory regions. While strain 1336 was able to colonize chicks, strain 414 was not, suggesting that regions specifically absent from the genome of strain 414 may play an important role in this common route of Campylobacter infection of humans. We suggest that the genomic divergence observed constitutes evidence of adaptation leading to niche specialization.     

Phenotypic diversity of Campylobacter isolates from sporadic cases of human enteritis in the UK

AIMS: The aim of this study was to identify and subtype a large collection of isolates of Campylobacter spp. to quantify diversity among strains causing human disease from geographically diverse sources in the United Kingdom. METHODS AND RESULTS: Isolates were characterized by the Penner serotyping scheme, Preston phage typing and biotyping methods. The diversity index calculated from the combined results of all three methods was 0.997 and indicated that isolates from sporadic cases of infection are very diverse. Strong associations between common phagetypes (PG52, PG121 and PG55) and the three most common serotypes (HS1, HS2 and HS4) found in the study were evident. CONCLUSIONS: Strains of C. jejuni causing human infections in the United Kingdom are very phenotypically diverse. Individual strains characterized by serotype, phagetype and biotype were detected throughout the 7-month study period and from geographically distinct sources, indicating an unrecognized outbreak or other epidemiologically significant source of human infection. SIGNIFICANCE AND IMPACT OF THE STUDY: The low frequency incidence of most C. jejuni strains should enable easy recognition of outbreaks by strain type surveillance at local, regional and national level in the United Kingdom. The characterization of common strain profiles in this study by simple phenotypic methods could provide the basis for strain specific epidemiological studies for reservoirs of infection and transmission routes for human infection.     

烟草可培养内生细菌的分离及多样性分析

采用稀释平板法, 从健康烟草的根、茎、叶组织中分离到267株内生细菌。利用细菌菌落表征性状和16S rRNA序列对这些分离物进行了多样性分析。通过数值分析比较了8个菌落形态表征性状, 以类平均连锁聚类法的方式进行聚类分析, 在2.15的水平上可分成5个聚类群和56个亚群。16S rDNA序列系统发育分析表明267株分离物可分为21个类群。研究表明同一菌落形态类型的菌株在系统发育树中不一定聚为一类, 菌落形态分类与分子生物学方法分类结果不完全一致。经克隆测序分析表明, 这267株分离物分别与GenBank中6类细菌中的21个已知种相似性达到98%?99%。其中芽孢杆菌属(Bacillus)细菌是烟草可培养内生细菌的优势种群。     

Classification of Brucella strains isolated from marine mammals by infrequent restriction site-PCR and development of specific PCR identification tests

Brucella strains have been isolated since the 1990s from a wide variety of marine mammals and represent potential zoonotic pathogens. They have distinctive phenotypic and molecular characteristics from the terrestrial mammal Brucella species, and two new species names have been previously proposed based on DNA polymorphism at the omp2 locus and their preferential host, i.e. Brucella cetaceae for cetacean isolates and Brucella pinnipediae for pinniped isolates. The results presented in this study on characterization of these strains by infrequent restriction site-PCR (IRS-PCR), taking into account the higher number of IS711 elements in their genome compared to terrestrial mammal Brucella species, supports this classification. The nucleotide sequences of specific DNA fragments detected by IRS-PCR were determined and used to develop PCR identification tests for either B. cetaceae or B. pinnipediae.