Flagellin Gene Sequence Research Articles

Genetic basis of quinolone resistance and epidemiology of resistant and susceptible isolates of porcine Campylobacter coli strains.

The aims of this study were to investigate the epidemiology of quinolone-resistant and -susceptible porcine isolates of Campylobacter coli and to characterize the genetic basis of quinolone resistance. Penner serotyping and flagellin gene sequence polymorphisms were used to investigate the epidemiology of the C. coli isolates. A total of 55 isolates were included, of which 30 were paired resistant and susceptible isolates from 15 pigs. Amplification of gyrA, gyrB and parC, followed by direct sequencing of amplicons was used to identify mutations in the targets of quinolones. Overall, 31 of the isolates were resistant to ciprofloxacin (minimum inhibitory concentrations (MIC), 2- >or = 32 microg x ml(-1)). Thirteen DdeI-flaA profiles were observed and resistant and susceptible strains were identified for nine profiles. The majority of resistant strains exhibited either profile 1 or 6. While profile 1 comprised susceptible and resistant strains, all of the strains with profile 6 were resistant to ciprofloxacin. The serogroup (O:24) of the profile 6 strains was identical. The only other serogroup to be uniformly associated with quinolone resistance was O:5. Strains with this phenotype comprised a number of genotypes, including profile 1. Only four of the paired isolates from individual pigs had the same profile. The genetic basis of quinolone resistance was investigated in two strains with ciprofloxacin MICs of 2 and > or = 32 miccrog x ml(-1), respectively. The amino acid substitution of isoleucine for threonine at position 86 was identified in the GyrA proteins from both strains. No mutations were identified in the GyrB proteins. There was an association between two of the genotypes, serotypes 5 and 24, and quinolone resistance. The association between genotype, serotype and resistance in C. coli isolates has not been reported previously. Only the mutation in GyrA associated with quinolone resistance was identified. No mutations in GyrB were identified. Amplification products of parC were not obtained and it may be that this gene is not present in some Campylobacter spp. This study provides data on the distribution of ciprofloxacin resistance between subtypes of C. coli.

Detection of Borrelia duttonii, a tick-borne relapsing fever agent in central Tanzania, within ticks by flagellin gene-based nested polymerase chain reaction.

Argasid ticks collected in the site near Mvumi Hospital, Dodoma, Tanzania, were subjected to flagellin gene-based nested polymerase chain reaction (PCR) amplification for examination of borrelial infections. Eight of 13 ticks gave a strong 350-bp signal; three had a weak signal at the same size, and the rest were negative. Sequence determination of eight of the positive samples resulted in three types of flagellin gene sequences. The first type of sequence (shown by three individuals) was identical to that of Borrelia duttonii strain Ly. The second type of sequence from PCR products from four individual ticks had only one base substitution without amino acid alteration in deduced protein sequence. The third type of sequence was different from that of any other Old World relapsing fever borreliae, and the tick was thought to be infected with an unknown Borrelia species. Ticks were also examined to determine the nucleotide sequence of the mitochondrial 16S rRNA gene. The partial sequence of approximately 470 bases was aligned for comparison with previously published sequences to identify the species. The sequences of 13 individual ticks were all identical, and the sequence similarity analysis revealed the ticks should be classified as members of the Ornithodoros porcinus species. The PCR method described in this report appears to be a reliable tool for the detection of borreliae and epidemiological study of tick-borne relapsing fever.

Borrelia sinica sp. nov., a lyme disease-related Borrelia species isolated in China.

A survey was performed for Lyme disease borrelia in the southern part of China, in Zhejiang, Sichuan and Anhui provinces, along the Yangtze River valley, in May of 1997 and 1998. Twenty isolates from Ixodes granulatus, Ixodes ovatus, Apodemus agrarius and Niviventer confucianus were obtained. These isolates were characterized by RFLP of the 5S-23S rDNA intergenic spacer, sequence analysis of the intergenic spacer, 16S rDNA and flagellin gene, DNA-DNA hybridization analysis, SDS-PAGE and Western blotting with mAbs. Six isolates from A. agrarius, five from I. granulatus collected in Zhejiang province and one from N. confucianus in Sichuan province were highly similar to strains 10MT and 5MT isolated in Korea and classified as Borrelia valaisiana. Four isolates from A. agrarius and I. granulatus collected in Zhejiang province generated unique RFLP patterns and phylogenetic analysis of the 16S rDNA and flagellin gene sequences suggested that the isolates should be classified as B. valaisiana. Furthermore, three isolates (CMN1a, CNM2, CMN3T) from N. confucianus captured in Sichuan province and one (CWO1) from I. ovatus in Anhui province showed lower 165 rDNA sequence similarity (less than 99.0%) to sequences of previously described Lyme disease-related Borrelia species. DNA-DNA hybridization results revealed that strains CMN3T and CMN1a were clearly distinct from all other known Lyme disease Borrelia species. Electron microscope observation showed the spirochaetes to be morphologically similar to those of Borrelia, but the cells contained only four periplasmic flagella inserted at each end of the spirochaetes. Based on these results, a new Borrelia species, Borrelia sinica sp. nov., is proposed. Strain CMN3T is the type strain of this new species.

Routine diagnosis of Borrelia burgdorferi (sensu lato) infections using a real-time PCR assay

To establish a one-tube fluorogenic real-time PCR assay for routine detection of Borrelia burgdorferi (sensu lato) DNA in various clinical specimens. A fragment of the flagellin gene sequence was amplified with the TaqMan chemistry using primers and a probe common to Borrelia burgdorferi sensu stricto, Borrelia afzelii, Borrelia garinii and Borrelia valaisiana. A recombinant plasmid containing the chromosomal gene coding for the flagellin protein was used as standard. The specificity of the assay was documented with 48 different clinically relevant Borrelia burgdorferi strains. No cross-reaction occurred with unrelated bacteria, viruses and fungi. At an analytic sensitivity of 10 copies, excellent precision within runs and between runs was observed. The potential presence of inhibitors of the Taq DNA polymerase was monitored by spiking aliquots of each sample with a plasmid containing the target sequence. Among 56 cerebrospinal fluid samples taken from 54 patients with clinical suspicion of neuroborreliosis, one (1.8%) tested positive for Borrelia burgdorferi sensu lato DNA. Borrelia burgdorferi DNA was also detected in five (17.9%) of 28 synovial fluid specimens and in one (20%) of five synovial membrane biopsies obtained from 31 patients with arthropathies. In order to test for the absence of false-positive results, 84 samples from 83 patients without evidence of Lyme disease were investigated. None of these samples showed measurable amounts of Borrelia burgdorferi DNA. By its established features, such as speed, reliability, sensitivity, specificity, the inclusion of carryover prevention and the monitoring of inhibitors in individual test tubes, this real-time PCR assay has proved to be a potent tool for the detection of Borrelia burgdorferi DNA under routine conditions in diagnostic laboratories.

Borrelia lonestari DNA in adult Amblyomma americanum ticks, Alabama.

Polymerase chain reaction analysis of 204 Amblyomma americanum and 28 A. maculatum ticks collected in August 1999 near the homes of patients with southern tick-associated rash illness and in control areas in Choctaw County, Alabama, showed Borrelia lonestari flagellin gene sequence from two adult A. americanum. The presence of B. lonestari in A. americanum ticks from Alabama suggests that this suspected pathogen may be widespread in the southeastern United States.

Variation in Bordetella bronchiseptica flaA does not correlate with typing by macro-restriction analysis by pulsed-field gel electrophoresis.

A genotyping method based on PCR-RFLP analysis of the flagellin gene (flaA) was applied to 30 mainly feline isolates of Bordetella bronchiseptica. These isolates were separated into three PCR-RFLP groups with the restriction endonucleases HaeIII, MspI, MboI and RsaI. flaA nucleotide sequences representing each of the three groups differed from each other by 11-13%. One of the groups exhibited far greater flaA sequence identity with the cryptic flagellin gene sequence of B. pertussis (>97%) than with flaA sequences from representatives of the other B. bronchiseptica PCR-RFLP groups. Amongst the 30 isolates were at least 10 representing each of the two major genotypes (A and B) identified in a previous study by macro-restriction analysis and pulsed-field gel electrophoresis (PFGE), as well as representatives of other less common genotypes. Each of the major PFGE genotypes contained strains representing more than one flagellin genotype. Indeed, there was no correlation between the two molecular typing methods. PFGE analysis may identify differences due to genomic re-arrangements rather than genuine variations in gene content. If so, relationships inferred on the basis of PFGE or other molecular methods for whole genome comparison should be treated with caution.

Rapid detection and identification of Clostridium chauvoei by PCR based on flagellin gene sequence

We developed a one-step polymerase chain reaction (PCR) system that specifically detects Clostridium chauvoei. Oligonucleotide primers were designed to amplify a 516-bp fragment of the structural flagellin gene. The specificity of the PCR was investigated by analyzing 59 strains of clostridia, and seven strain of other genera. A 516-bp fragment could be amplified from all the C. chauvoei strains tested, and no amplification was observed by using DNAs from the other strains tested, including Clostridium septicum. Similarly, this PCR-based method specifically detected C. chauvoei DNA sequences in samples of muscle and exudate of obtained from mice within 12 h of inoculation. In tests using samples of muscle or liver, the limit of detection was about 200 organisms per reaction. These results suggest that the one-step PCR system may be useful for direct detection and identification of C. chauvoei in clinical specimens.

Flagellin Gene Sequence Variation in the genus Pseudomonas

Flagellin gene (fliC) sequences from 18 strains of Pseudomonas sensu stricto representing 8 different species, and 9 representative fliC sequences from other members of the gamma sub-division of proteobacteria, were compared. Analysis was performed on N-terminal, C-terminal and whole fliC sequences. The fliC analyses confirmed the inferred relationship between P. mendocina, P. oleovorans and P. aeruginosa based on 16S rRNA sequence comparisons. In addition, the analyses indicated that P. putida PRS2000 was closely related to P. fluorescens SBW25 and P. fluorescens NCIMB 9046T, but suggested that P. putida PaW8 and P. putida PRS2000 were more closely related to other Pseudomonas spp. than they were to each other. There were a number of inconsistencies in inferred evolutionary relationships between strains, depending on the analysis performed. In particular, whole flagellin gene comparisons often differed from those obtained using N- and C-terminal sequences. However, there were also inconsistencies between the terminal region analyses, suggesting that phylogenetic relationships inferred on the basis of fliC sequence should be treated with caution. Although the central domain of fliC is highly variable between Pseudomonas strains, there was evidence of sequence similarities between the central domains of different Pseudomonas fliC sequences. This indicates the possibility of recombination in the central domain of fliC genes within Pseudomonas species, and between these genes and those from other bacteria.

Characterization and identification of Borrelia isolates as Borrelia valaisiana in Taiwan and Kinmen Islands.

Eleven pure cultures of Borrelia from 3 species of wild rodents (Apodemus agrarius, Mus formosanus, Rattus losea) captured in Taichung, located in the center of Taiwan island, and on Kinmen Island were characterized. Five isolates showed restriction fragment length polymorphism (RFLP) patterns of 5S-23S rRNA gene intergenic spacer sequences identical to those of strains 5MT and 10MT, identified as Borrelia valaisiana, which were isolated in the southern tip of South Korea. Although the remaining six isolates showed novel RFLP patterns, these isolates showed more similarity to members of B. valaisiana from Korea, Japan and Europe based on 16S rRNA gene and flagellin gene sequences. This led us to speculate that transmission and proliferation of this type of borrelia occurred between Taiwan and the southern part of South Korea.

Stable marker on flagellin gene sequences related to arabinose non-assimilating pathogenic Burkholderia pseudomallei.

Using PCR-based isolation and sequence analysis of the flagellin gene from two distinct biotypes of Burkholderia pseudomallei, a 15-bp deletion was found within the variable domain of the gene in isolates capable of assimilating arabinose (Ara+). This finding led to the development of a PCR-based method in order to differentiate and identify pathogenic B. pseudomallei for epidemiological study. A pair of specific primers was designed covering the 15-bp deletion region at the variable domain. PCR-amplification products of 176 and 191 bp in size were detected from 41 Ara+ isolates and 39 Ara - isolates of B. pseudomallei, respectively. Moreover, flagellin gene fragments of other bacterial species tested in this study were not amplified using these primers. The results suggest that the flagellin gene sequences of both B. pseudomallei biotypes in this region are stable and distinct. This method can be applied and useful for the epidemiological study of B. pseudomallei.

Determination of members of a Borrelia afzelii-related group isolated from Ixodes nipponensis in Korea as Borrelia valaisiana.

The 16S rRNA sequences of the Korean Borrelia strains 5MT and 9MT, isolated from Ixodes nipponensis, showed identities of 99.0-99.1% to that of B. afzelii. The strains were tentatively classified as belonging to the B. afzelii-related group. In this study, Korean isolates, including these strains, were characterized further and compared with recently described new species. These strains generated a RFLP pattern that has not been found previously in RFLP analysis of the 5S-23S rRNA intergenic spacer and the flagellin gene. When phylogenetic trees were constructed, based on the 5S-23S rRNA intergenic spacer, flagellin gene and 16S rRNA sequences, these Korean isolates formed a cluster with the Borrelia strain Am501 isolated from Ixodes columnae in Japan and Borrelia valaisiana strains VS116T and UK isolated from Ixodes ricinus in Europe and were distinguishable from the other species. However, these three groups of strains were divergent from each other in the molecular masses of the putative outer surface protein A (OspA) and in the sequences of the ospA gene. These findings suggest that these Korean isolates and one Japanese isolate are members of B. valaisiana and that OspA of this species is divergent, as is that of Borrelia garinii. This led to the speculation that B. valaisiana strains are adapted to the vector ticks found in each locality.

Standardisation of polymerase chain reaction for the detection of Salmonella typhi in typhoid fever.

To improve the diagnosis of Salmonella typhi infection, a polymerase chain reaction (PCR) assay was developed for the amplification of the dH flagellin gene of S typhi. Primers were designed...

Competence of a migratory bird, red-bellied thrush ( Turdus chrysolaus), as an avian reservoir for the Lyme disease spirochetes in Japan

To evaluate the competence of migratory birds as reservoirs for the Lyme disease spirochetes, we examined two species of migrants, Red-bellied thrush ( Turdus chrysolaus) and Black-faced bunting ( Emberiza spodocephala) in Nemuro, the northern part of Japan. Spirochetes were found in four individual birds out of 11 T. chrysolaus, three isolates were detected from the skins and the other one was obtained from the liver. No spirochete was found to be infected in 20 E. spodocephala. As far as we know, this is the first record of direct detection of the spirochetes from migratory birds in Japan. The spirochetes were also isolated from immature ixodid ticks, Ixodes persulcatus, fed on those species of birds. The spirochetes were transmitted trans stadially to the next stage, when infected ticks molted. All of the isolates from birds and ticks were identified as Borrelia garinii by our ribotyping and flagellin gene sequence analyses. Our results strongly suggest that the migratory birds are reservoirs in the transmission of the Lyme disease spirochetes in Japan.

The flagellin gene as a stable marker for detection of Pseudomonas fluorescens SBW25.

Flagellin gene central regions from 111 isolates of Pseudomonas fluorescens SBW25 obtained from soil during a field release experiment were analysed using a combined PCR/RFLP technique to look for variation. In addition, a 858 bp flagellin gene sequence from the original strain and the last isolate obtained from the release site were compared. There was no variation in flagellin gene sequences indicating that the gene was stable over the period of the release, and that the flagellin gene is a suitable marker for use in the detection of bacteria in release experiments. A comparison of Ps. fluorescens SBW25 flagellin with other sequenced flagellins revealed closest homology to the flagellin of Ps. putida PRS2000.

Detection of Campylobacter jejuni and Camp. coli in chicken faecal samples by PCR

PCR primers were selected from the flagellin gene sequences flaA and flaB of Campylobacter coli to amplify DNA from Camp. jejuni and Camp. coli. When the PCR products were analysed by hybridization to an internal probe immobilized in microtitre wells, positive reactions were observed only for strains of Camp. jejuni and Camp. coli. The assay was used to analyse 31 chicken faecal samples. Full correspondence was found between the PCR assay conducted on the enriched cultures and the standard culture method. When analysing the transport medium prior to enrichment, the PCR assay detected nine of 11 culture positive samples.

Phylogenetic Analysis of Borrelia Species Based on Flagellin Gene Sequences and Its Application for Molecular Typing of Lyme Disease Borreliae

We determined almost complete flagellin gene sequences of various Borrelia species and aligned them with previously published sequences. A neighbor-joining phylogenetic analysis showed that the genus Borrelia was divided into the following three major clusters: New World relapsing fever borreliae (Borrelia turicatae, Borrelia parkeri, and Borrelia hermsii), Old World relapsing fever borreliae (Borrelia crocidurae, Borrelia duttonii, and Borrelia hispanica), and Lyme disease borreliae (Borrelia burgdorferi sensu stricto, Borrelia garinii, and Borrelia afzelii). Agents of animal spirochetosis (Borrelia coriaceae and Borrelia anserina) and species of unknown pathogenicity (Borrelia miyamotoi and Borrelia lonestari) were related to relapsing fever borreliae. Although the Lyme disease borreliae, two related species (Borrelia japonica and Borrelia andersonii), and some newly described genomic groups (groups PotiB2, VS116, DN127, Hk501, and Ya501) were closely related to each other, each taxon formed an independent branch on the phylogenetic tree. The data obtained in this study indicate that the flagellin genes are useful in Borrelia taxonomy. To distinguish the Lyme disease borreliae from related organisms easily, we designed an oligonucleotide primer set for the flagellin gene and performed a PCR-restriction fragment length polymorphism (PCR-RFLP) analysis. The primer set amplified an approximately 580-bp DNA fragment that included species-specific restriction sites, and HapII, HhaI, CelII, HincII, or DdeI digestion of the product resulted in distinctively different PCR-RFLP patterns. The PCR-RFLP typing method which we developed should facilitate rapid identification of Lyme disease borreliae and related organisms obtained from biological and clinical specimens.

Flagellin gene and protein variation amongst clinical isolates of Pseudomonas aeruginosa.

Flagellin gene sequences from 64 clinical isolates of the opportunistic pathogen Pseudomonas aeruginosa were amplified by PCR and subjected to RFLP analysis by using seven restriction enzymes to digest the amplified products. Using this approach the isolates were assigned to one of 13 groups. The method was rapid, reproducible and applicable to all isolates. In contrast, serotyping failed to satisfactorily resolve 49% of the strains tested. The vast majority of clinical isolates generated amplified products of 1.02 kb (type a) or 1.25 kb (type b). Electron microscopical analysis revealed evidence fax some. flagellar structural variation between P. aeruginosa strains. This study provides further evidence that the flagellin gene is a widely applicable and useful genetic marker for studying genetic variation within populations of closely related bacteria.

Single-tube nested polymerase chain reaction assay based on Flagellin gene sequences for detection of Borrelia burgdorferi sensu lato.

An inherent drawback of nested PCR systems to increase sensitivity of PCR-based assays is that tubes must be opened after the first round of amplification in order to transfer template molecules to the second amplification reaction; this procedure introduces the risk of carry-over contamination of negative specimens. To obviate this disadvantage, a nested PCR assay for detection of Borrelia burgdorferi in which both amplifications are performed in a single tube that remains closed throughout the entire process was devised. The assay is based on flagellin gene sequences with previously determined species-wide and species-specific properties. The nested PCR system proved to be 1000 times more sensitive than the conventional assay. Using the nested PCR system, ten spirochaetes could be routinely detected by agarose gel electrophoresis alone, whereas the conventional PCR system could detect only 10(4) spirochaetes under these conditions. After Southern transfer on amplification products and hybridization with 320- or chemiluminescent-labeled probes, the nested PCR system could easily detect a single spirochaete by both means, whereas the sensitivity of the conventional PCR assay varied from 10(1) (32P) to 10(3) (chemiluminescence) spirochaetes. This single-tube nested PCR system should be a useful addition to the current range of diagnostic assays for Lyme borreliosis.

A phylogenetic analysis of Borrelia burgdorferi sensu lato isolates associated with Lyme disease in Japan by flagellin gene sequence determination.

We determined nearly complete flagellin gene sequences for Borrelia burdgdorferi sensu lato isolates (11 isolates obtained from Ixodes persulcatus ticks and patients in Hokkaido, Japan, and 1 European isolate) representing six different restriction fragment length polymorphism (RFLP) ribotype groups following cloning of the PCR-amplified genes. These sequences were aligned with those of representatives of the three Borrelia species associated with Lyme disease, and a phylogenetic tree was constructed by the Clustal method. On the Lyme disease borrelia portion of the tree, the species were clearly delineated into three different phylogenetic groups, in complete agreement with the division of B. burgdorferi sensu lato into three species. A phylogenetic analysis revealed that the representatives of RFLP ribotype groups IV, V, and VI clustered tightly with each other and belonged on the same branch as Borrelia garinii. We used the criteria that are currently used to delineate bacterial species and determined the levels of DNA relatedness for these Borrelia isolates. For the RFLP ribotype group IV, V, and VI isolates, the levels of DNA relatedness ranged from 79 to 88%, and the levels of relatedness to the reference strain of B. garinii ranged from 70 to 80%. The levels of DNA relatedness of the RFLP ribotype group IV, V, and VI isolates to the representatives of other species associated with Lyme disease ranged from 53 to 66%. All of these findings indicate that the RFLP ribotype group IV, V, and VI isolates should be included in the species B. garinii.

Flagellin gene profiling of Campylobacter jejuni heat-stable serotype 1 and 4 complex

Flagellin gene ( flaA) sequence polymorphisms were used to discriminate amongst 167 strains of Campylobacter jejuni serotype HS1 and the HS4 complex. Direct PCR of cell suspensions provided a rapid method for analysing DNase-negative strains, whereas purified DNA was necessary for the DNase-positive strains. Nine different PCR-RFLP patterns (genotypes) were identified by analysis with HinΛ and 12 with Ddel, giving a total of 19 combined flaA profile types. The most common combined fla types were H1D1 (35%) and H1D2 (20%) for serotype HS1, and H1D2 (23%) and H4D7 (43%) for serotype HS4. Comparison of flaA typing with other key subtyping methods for C. jejuni showed it to be less discriminatory than pulsed field gel electrophoretic (PFGE) profiling, but more so than ribotyping. Fla types provided a useful indication of strain diversity, but as some were conserved across different serotypes, ribotypes and PFGE types, the same fla type could not be used as the sole basis for grouping strains. We provide evidence for several distinct subgroups based on conserved multiple genomic criteria within the HS1 and HS4 strains, and conclude that monitoring of such subgroups could provide a novel basis for future epidemiological surveillance of C. jejuni.