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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.

Acyltransferases↗

[Antagonistic activity of Lactobacillus bacteria strains against anaerobic gastrointestinal tract pathogens (Helicobacter pylori, Campylobacter coli, Campylobacter jejuni, Clostridium difficile)].

Antagonistic activity of Lactobacillus strains has been known for some time. This property is connected with production of many active substances by lactobacilli e.g., organic acids and bacteriocin-like substances which interfere with other indigenous microorganisms inhabiting the same ecological niche, including also anaerobic gastrointestinal tract pathogens. Growing interest of clinical medicine in finding new approaches to treatment and prevention of common inflammatory infections of the digestive tract resulted in studies on a possible usage of lactic acid bacteria. Last years, several in vitro and in vivo experiments on antagonism of different Lactobacillus strains against Helicobacter pylori and Clostridium difficile were performed. These observations had been done on already established, well known probiotic Lactobacillus strains. We tested antibacterial activities of Lactobacillus strains isolated from human digestive tract. As indicator bacteria, four species known as anaerobic bacterial etiologic agents of gastroenteric infections: Helicobacter pylori, Campylobacter jejuni, C. coli and Clostridium difficile were used. Some of them were obtained from international collections, others were clinical isolates from specimens taken from patients with different defined gastrointestinal infections. We used a slab method of testing inhibitory activity described in details previously. Following conclusions were drawn from our study: All tested human Lactobacillus strains were able to inhibit the growth of all strains of anaerobic human gastrointestinal pathogens used in this study. Inhibitory activities of tested Lactobacillus strains against Helicobacter pylori, Campylobacter spp., and Clostridium difficile as measured by comparing mean diameters of the inhibition zones were similar. Differences in susceptibility of individual indicator strains of Campylobacter spp. and Clostridium difficile to inhibitory activity of Lactobacillus strains were small. A similar mechanism of inhibition of anaerobic bacteria by lactobacilli is postulated.

Campylobacter coli↗

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.

Australia↗

Molecular discrimination between Campylobacter jejuni, Campylobacter coli, Campylobacter lari and Campylobacter upsaliensis by polymerase chain reaction based on a novel putative GTPase gene.

Polymerase chain reaction (PCR) mediated DNA fingerprinting has resulted in the identification of a novel Campylobacter jejuni gene, encoding a GTPase protein. The gene, consisting of 383 amino acids contained semi-conserved GTP-binding sites (designated G-1 to G-4), that are characteristic for members of the GTPase protein superfamily. Remarkably, this gene from C. Jejuni appears to encode a member of a novel family of GTP-binding proteins, containing two separate putative GTP-binding domains, each comprising a series of semi-conserved GTP-binding motifs. Spacing between these motifs is highly conserved. Based on this novel gene, a general PCR strategy for the identification of C. jejuni, C. coli, C. lari and C. upsaliensis was developed. PCR primers were deduced from GTP-binding motifs G-1 and G-3 of the first GTP-binding domain. These GTP-binding sites flank a variable region of precisely 117 bp in the four Campylobacter spp. that allowed the development of species-specific probes. This PCR-hybridization assay offers a novel tool for rapid molecular detection and specific identification of the thermophilic Campylobacter spp.

Amino Acid Sequence↗

Cloning, sequencing, and characterization of the lipopolysaccharide biosynthetic enzyme heptosyltransferase I gene (waaC) from Campylobacter jejuni and Campylobacter coli.

Campylobacter jejuni and Campylobacter coli are common causes of gastrointestinal disease and a proportion of C. jejuni infections have been shown to be associated with the Guillain-Barré syndrome. The waaC gene from Campylobacter coli, involved in lipopolysaccharide core biosynthesis, was cloned by complementation of a heptose-deficient strain of Salmonella typhimurium, as judged by novobiocin sensitivity, lipopolysaccharide (LPS)-specific phage sensitivity, and polyacrylamide-resolved lipopolysaccharide profiles. The C. jejuni waaC gene was subsequently cloned using the waaC gene isolated from C. coli as a probe. The C. jejuni and C. coli waaC genes are capable of encoding proteins of 342 amino acids with calculated molecular masses of 39381Da and 39317Da, respectively. Sequence and in-vitro analyses suggested that the C. coli waaC gene may be transcribed from its own promoter. Translation of the C. coli waaC gene in a cell-free system yielded a protein with a Mr of 39000. The waaC gene was detected in every C. jejuni and C. coli isolate tested as judged by dot-blot hybridization analysis. Southern hybridization analysis indicated that both Campylobacter species contain a single copy of the waaC gene. Unlike Escherichia coli and S. typhimurium isolates, the waaC gene in C. jejuni and C. coli isolates does not appear to be linked to the waaF (rfaF) gene.

Base Sequence↗

Genomic rearrangements associated with antigenic variation in Campylobacter coli.

Campylobacter coli and Campylobacter jejuni share a limited number of highly conserved DNA sequences with members of the family Enterobacteriaceae. One of these sequences was cloned from C. coli VC167, and the region of homology to the enteric sequences was determined to be confined to a 700-base-pair region. The DNA represented in this clone undergoes a programmed, reversible rearrangement in VC167 that is associated with flagellar antigenic variation.

Antigenic Variation↗

Discrimination of enterobacterial repetitive intergenic consensus PCR types of Campylobacter coli and Campylobacter jejuni by Fourier transform infrared spectroscopy.

Fourier transform infrared spectroscopy (FT-IR) has been used together with pattern recognition methodology to study isolates belonging to the species Campylobacter coli and Campylobacter jejuni and to compare FT-IR typing schemes with established genomic profiles based on enterobacterial repetitive intergenic consensus PCR (ERIC-PCR). Seventeen isolates were cultivated under standardized conditions for 2, 3, and 4 days to study variability and improve reproducibility. ERIC-PCR profiles and FT-IR spectra were obtained from strains belonging to the species Campylobacter coli and C. jejuni, normalized, and explored by hierarchical clustering and stepwise discriminant analysis. Strains could be differentiated by using mainly the first-derivative FT-IR spectral range, 1,200 to 900 cm(-1) (described as the carbohydrate region). The reproducibility index varied depending on the ages of the cultures and on the spectral ranges investigated. Classification obtained by FT-IR spectroscopy provided valuable taxonomic information and was mostly in agreement with data from the genotypic method, ERIC-PCR. The classification functions obtained from the discriminant analysis allowed the identification of 98.72% of isolates from the validation set. FT-IR can serve as a valuable tool in the classification, identification, and typing of thermophilic Campylobacter isolates, and a number of types can be differentiated by means of FT-IR spectroscopy.

Animals↗

Role of an inducible single-domain hemoglobin in mediating resistance to nitric oxide and nitrosative stress in Campylobacter jejuni and Campylobacter coli.

Campylobacter jejuni expresses two hemoglobins, each of which exhibits a heme pocket and structural signatures in common with vertebrate and plant globins. One of these, designated Cgb, is homologous to Vgb from Vitreoscilla stercoraria and does not possess the reductase domain seen in the flavohemoglobins. A Cgb-deficient mutant of C. jejuni was hypersensitive to nitrosating agents (S-nitrosoglutathione [GSNO] or sodium nitroprusside) and a nitric oxide-releasing compound (spermine NONOate). The sensitivity of the Cgb-deficient mutant to methyl viologen, hydrogen peroxide, and organic peroxides, however, was the same as for the wild type. Consistent with the protective role of Cgb against NO-related stress, cgb expression was minimal in standard laboratory media but strongly and specifically induced after exposure to nitrosative stress. In contrast, the expression of Cgb was independent of aeration and the presence of superoxide. In the absence of preinduction by exposure to nitrosative stress, no difference was seen in the degree of respiratory inhibition by NO or the half-life of the NO signal when cells of the wild type and the cgb mutant were compared. However, cells expressing GSNO-upregulated levels of Cgb exhibited robust NO consumption and respiration that was relatively NO insensitive compared to the respiration of the cgb mutant. Based on similar studies in Campylobacter coli, we also propose an identical role for Cgb in this closely related species. We conclude that, unlike the archetypal single-domain globin Vgb, Cgb forms a specific and inducible defense against NO and nitrosating agents.

Adaptation, Physiological↗

Advantages of peptide nucleic acid oligonucleotides for sensitive site directed 16S rRNA fluorescence in situ hybridization (FISH) detection of Campylobacter jejuni, Campylobacter coli and Campylobacter lari.

Traditionally fluorescence in situ hybridization (FISH) has been performed with labeled DNA oligonucleotide probes. Here we present for the first time a high affinity peptide nucleic acid (PNA) oligonucleotide sequence for detecting thermotolerant Campylobacter spp. using FISH. Thermotolerant Campylobacter spp, including the species Campylobacter coli, Campylobacter jejuni and Campylobacter lari, are important food and water borne pathogens. The designed PNA probe (CJE195) bound with higher affinity to a previously reported low affinity site on the 16S rRNA than the corresponding DNA probe. PNA also overcame the problem of the lack of affinity due to the location of the binding site and the variation of the target sequence within species. The PNA probe specificity was tested with several bacterial species, including other Campylobacter spp. and their close relatives. All tested C. coli, C. jejuni and C. lari strains were hybridized successfully. Aging of the Campylobacter cultures caused the formation of coccoid forms, which did not hybridize as well as bacteria in the active growth phase, indicating that the probe could be used to assess the physiological status of targeted cells. The PNA FISH methodology detected C. coli by membrane filtration method from C. coli spiked drinking water samples.

Animals↗

Isolation and antimicrobial susceptibility of Campylobacter coli and Campylobacter jejuni from slaughter hogs.

Cultural examination of cecal contents from 109 market weight hogs slaughtered in Prince Edward Island during May-July 1988 yielded 62 isolates of Campylobacter coli and seven Campylobacter jejuni. A commercial latex agglutination test helped to confirm the identification of Campylobacter. When tested against four drugs: erythromycin, tetracycline, kanamycin and ampicillin, 11 isolates showed multiple resistance. Resistance to erythromycin was seen in 19% and 28.6% of Campylobacter coli and Campylobacter jejuni respectively. All the isolates were susceptible to nitrofurans, gentamicin and chloramphenicol.

Ampicillin Resistance↗

Experimental infection of the mouse mammary gland with Campylobacter coli.

Campylobacter coli strains of bovine and avian origin were inoculated into the mammary gland of mice. A bovine strain isolated from a case of mastitis produced gross and histological changes in most of the glands; one bovine and one avian faecal isolate did not. Histologically, lesions were characterised by neutrophil infiltration in the alveolar spaces and necrosis and oedema in the interalveolar tissue. On bacteriological examination, the bovine mastitis strain could be isolated from most of the glands, but neither of the faecal strains. The mouse, therefore, appears to provide a convenient model for studying campylobacter mastitis.

Animals↗

Genomic diversity of Campylobacter coli and Campylobacter jejuni isolates recovered from free-range broiler farms and comparison with isolates of various origins.

In many industrialized countries, the incidence of campylobacteriosis exceeds that of salmonellosis. Campylobacter bacteria are transmitted to humans mainly in food, especially poultry meat products. Total prevention of Campylobacter colonization in broiler flocks is the best way to reduce (or eliminate) the contamination of poultry products. The aim of this study was to establish the sources and routes of contamination of broilers at the farm level. Molecular typing methods (DNA macrorestriction pulsed-field gel electrophoresis and analysis of gene polymorphism by PCR-restriction fragment length polymorphism) were used to characterize isolates collected from seven broiler farms. The relative genomic diversity of Campylobacter coli and Campylobacter jejuni was determined. Analysis of the similarity among 116 defined genotypes was used to determine clusters within the two species. Furthermore, evidence of recombination suggested that there were genomic rearrangements within the Campylobacter populations. Recovery of related clusters from different broiler farms showed that some Campylobacter strains might be specifically adapted to poultry. Analysis of the Campylobacter cluster distribution on three broiler farms showed that soil in the area around the poultry house was a potential source of Campylobacter contamination. The broilers were infected by Campylobacter spp. between days 15 and 36 during rearing, and the type of contamination changed during the rearing period. A study of the effect of sanitary barriers showed that the chickens stayed Campylobacter spp. free until they had access to the open area. They were then rapidly colonized by the Campylobacter strains isolated from the soil.

Animal Husbandry↗

A combined polymerase chain reaction and restriction endonuclease enzyme assay for discriminating between Campylobacter coli and Campylobacter jejuni.

A combined polymerase chain reaction and restriction endonuclease (RE) enzyme assay was developed to discriminate between Campylobacter coli and Campylobacter jejuni. Amplimers of the FlaA gene obtained by PCR were digested with AluI and HinfI to distinguish C. coli from C. jejuni. With AluI digestion C. jejuni-specific bands were observed at 110, 140 and 160 bp and C. coli-specific bands at 293 and 147 bp. C. jejuni-specific bands of 349 and 109 bp were found by HinfI digestion but HinfI did not digest the FlaA amplimer of C. coli. This combined technique is fast and easy to perform, and distinguishes the two campylobacters unequivocally.

Campylobacter coli↗

Multiplex PCR for identification of Campylobacter coli and Campylobacter jejuni from pure cultures and directly on stool samples.

A multiplex-PCR method, specifically designed for application in routine diagnostic laboratories, was developed for the detection of Campylobacter coli and Campylobacter jejuni. Primers were directed towards the following loci: the hippuricase gene (hipO) characteristic of C. jejuni, a sequence partly covering an aspartokinase gene characteristic of C. coli, and a universal 16S rDNA gene sequence serving as an internal positive control for the PCR. The method was tested on 47 C. coli strains and 88 C. jejuni strains, and found to be almost 100% in concordance with biochemical analyses (all except for one C. coli strain), regardless of whether the DNA was prepared from colonies by a simple boiling procedure or by DNeasy Tissue Kit. Pure cultures of C. coli and C. jejuni were identified at 10-100 cells per PCR. When the multiplex-PCR method was used on spiked human stool samples, both strains were identified at 10(5) cells per ml stool. This sensitivity limit was the same whether the DNA was purified by the method of KingFisher mL or QIAamp DNA Stool Kit. When the same spiked stools were grown on modified charcoal cefoperazone deoxycholate agar (mCCDA) plates before PCR, the sensitivity was 100 cells per ml stool, indicating that culturing of campylobacters on mCCDA plates is superior to direct DNA extraction at least when fresh stool samples are analysed by PCR.

Amidohydrolases↗

Aminoglycoside-phosphotransferases APH(3')-IV and APH(3") synthesized by a strain of Campylobacter coli.

Campylobacter coli strain 981 of animal origin was resistant to erythromycin, tetracycline, streptomycin, kanamycin, ribostamycin, neomycin, paromomycin, lividomycin, and butirosin. Resistance to aminoglycosides of strain 981 was mediated by phosphotransferases APH (3') type-IV and APH (3"). C. coli 981 harboured three plasmids of 24, 34, and 40 Megadaltons respectively. None of these plasmids were transferable to Escherichia coli K-12 by conjugation.

Aminoglycosides↗

Improved biotyping schemes for Campylobacter jejuni and Campylobacter coli.

Campylobacter jejuni (20 strains) and Campylobacter coli (12 strains) were assigned to four biovars for each species based on phenotypic tests that were easy to perform and interpret. The resulting biotyping schemes offer a greater degree of distinction among C. jejuni and C. coli strains than any of the other biotyping schemes previously described for these organisms.

Alkaline Phosphatase↗

Inhibition of Campylobacter coli and Campylobacter jejuni by antibiotics used in selective growth media.

The ability of Campylobacter coli and Campylobacter jejuni to grow in the presence of antibiotics used in selective growth media was compared. MIC data for C. coli indicated that some strains were more susceptible to the antibiotics than were the C. jejuni strains tested. A reduction of greater than 1 log cycle in the numbers of cells growing on plates containing antibiotics was considered to be a marked level of inhibition. Only one of nine of the antibiotic combinations studied did not markedly inhibit most of the C. coli strains tested. Although one C. coli strain was not inhibited by any of the antibiotic combinations, the other six strains were inhibited for up to 7 log cycles. The addition of blood or growth supplements reduced but did not eliminate the inhibitory effect. The inhibition of laboratory strains of C. coli on media developed for the isolation of Campylobacter spp. indicates that the incidence of C. coli may be underestimated.

Anti-Bacterial Agents↗

Development of species-specific DNA probes for Campylobacter jejuni, Campylobacter coli, and Campylobacter lari by polymerase chain reaction fingerprinting.

The application of polymerase chain reaction (PCR) fingerprinting assays enables discrimination between species and strains of microorganisms. PCR primers aiming at arbitrary sequences in combination with primers directed against the repetitive extragenic palindrome (REP) or enterobacterial repetitive intergenic consensus (ERIC) motifs generate isolate-specific DNA banding patterns. Analysis of these PCR fingerprints obtained for 33 isolates of Campylobacter jejuni, 30 isolates of Campylobacter coli, and 8 isolates of Campylobacter lari revealed that besides generation of isolate-specific fragments, species-specific DNA fragments of identical size were synthesized. It appeared that these DNA fragments could be used as species-specific probes, since they are unique for the pattern which they are deriving from. The probes do not cross-react with amplified DNA originating from a large panel of nonrelated microorganisms. Moreover, these probes displayed species specificity, as they reacted with a single restriction fragment on Southern blots containing DNA from C. jejuni, C. coli, and C. lari and other Campylobacter species. This combination of PCR fingerprinting and probe hybridization results in a highly specific identification assay and provides an example of specific test development without the prior need for DNA sequence information. The principle of the procedure holds great promise for the rapid isolation of DNA probes which, in combination with a general PCR assay, may lead to efficient typing and detection procedures for a multitude of medically important nonviral microorganisms.

Base Sequence↗