Genotyping and antibiotic resistance patterns of Campylobacter fetus subsp.venerealis from cattle farms in India.

Bovine genital campylobacteriosis caused by Campylobacter fetus subsp. venerealis (Cfv) is of considerable economic importance to the cattle industry worldwide. Cfv causes syndrome of temporary infertility in female cattle, early embryonic mortality, aberrant oestrus cycles, delayed conception, abortions and poor calving rates. In the present study, a total of 200 samples obtained from vaginal swabs, cervicovaginal mucous (CVM), preputial washes and semen straws were investigated that were obtained from organized cattle farm of MLRI, Manasbal and unorganized sectors. Out of a total of 200 samples, 49 (47·57%) vaginal swabs, 1 (3·33%) preputial wash and 8 (25%) carried out CVM samples were positive for Cfv, whereas none of the semen straws were positive for Cfv. A total of eleven isolates of Cfv were recovered. PFGE (Pulse field gel electrophoresis) analysis revealed four different pulsotypes (I-IV) circulating in the screened farms. A common pulsotype circulating among farms could not be established. Insertion element (ISCfe1), a 233 bp amplicon of Cfv, was sequenced and the sequence was deposited in GenBank (accession no: MK475662).

Campylobacter fetus Cluster Among Men Who Have Sex With Men, Montreal, Quebec, Canada, 2014-2016.

From March 2014 to December 2016, a cluster of 13 cases of Campylobacter fetus intestinal and extraintestinal infections, including 2 patients with an aortic mycotic aneurysm, caused significant morbidity. The cluster likely resulted from sexual transmission between men having sex with men living in the greater Montreal area, Quebec, Canada.

Whole genome sequence analysis indicates recent diversification of mammal-associated Campylobacter fetus and implicates a genetic factor associated with H2S production.

BACKGROUND: Campylobacter fetus (C. fetus) can cause disease in both humans and animals. C. fetus has been divided into three subspecies: C. fetus subsp. fetus (Cff), C. fetus subsp. venerealis (Cfv) and C. fetus subsp. testudinum (Cft). Subspecies identification of mammal-associated C. fetus strains is crucial in the control of Bovine Genital Campylobacteriosis (BGC), a syndrome associated with Cfv. The prescribed methods for subspecies identification of the Cff and Cfv isolates are: tolerance to 1 % glycine and H2S production. RESULTS: In this study, we observed the deletion of a putative cysteine transporter in the Cfv strains, which are not able to produce H2S from L-cysteine. Phylogenetic reconstruction of the core genome single nucleotide polymorphisms (SNPs) within Cff and Cfv strains divided these strains into five different clades and showed that the Cfv clade and a Cff clade evolved from a single Cff ancestor. CONCLUSIONS: Multiple C. fetus clades were observed, which were not consistent with the biochemical differentiation of the strains. This suggests the need for a closer evaluation of the current C. fetus subspecies differentiation, considering that the phenotypic differentiation is still applied in BGC control programs.

A rural worker infected with a bovine-prevalent genotype of Campylobacter fetus subsp. fetus supports zoonotic transmission and inconsistency of MLST and whole-genome typing.

Whole-genome characterisation in clinical microbiology enables to detect trends in infection dynamics and disease transmission. Here, we report a case of bacteraemia due to Campylobacter fetus subsp. fetus in a rural worker under cancer treatment that was diagnosed with cellulitis; the patient was treated with antibiotics and recovered. The routine typing methods were not able to identify the microorganism causing the infection, so it was further analysed by molecular methods and whole-genome sequencing. The multi-locus sequence typing (MLST) revealed the presence of the bovine-associated ST-4 genotype. Whole-genome comparisons with other C. fetus strains revealed an inconsistent phylogenetic position based on the core genome, discordant with previous ST-4 strains. To the best of our knowledge, this is the first C. fetus subsp. fetus carrying the ST-4 isolated from humans and represents a probable case of zoonotic transmission from cattle.

[Efficiency of bacteriological culture and the immunofluorescent assay to detect Campylobacter fetus in bovine genital fluids]. / Eficiencia del cultivo bacteriológico y de la inmunofluorescencia en la detección de Campylobacter fetus en fluidos genitales bovinos.

Bovine genital campylobacteriosis is a reproductive disease that affects cattle production. It is caused by Campylobacter fetus subspecies, C. fetus fetus (Cff) and C. fetus venerealis (Cfv). The aim of this study was to identify the presence of C. fetus in genital fluids by bacteriological culture and direct immunofluorescence (DIF) and to compare the results. Two groups of 6 heifers and 5 bulls, one infected with Cff (Cff group) and the other with Cfv (Cfv group) were formed. Two heifers and 2 bulls, all of them uninfected, made up the control group. Samples of cervicovaginal mucus and preputial fluid were processed by culture and DIF. In the Cff group, 100% of the heifers and 80% of the bulls were infected, while in the Cfv group, 50% of the heifers and 60% of the bulls were infected. The degree of agreement (Kappa values) from benchmarking diagnostic techniques were 0.57 for heifers in the Cff group and 0.52 for heifers in the Cfv group, whereas the values for bulls were 0.17 and 0.27, respectively. Heifers yielded more positive results in the DIF assay than in the culture, exhibiting 5.6% increase in the Cff group and 7.4% in the Cfv group. The lowest percentage of positive results for DIF in bulls, 40% less for the Cff group and 5.2% for the Cfv group, could be due to improper sampling. Kappa values showed moderate agreement for the heifers and low for the bulls.

Inconsistency of phenotypic and genomic characteristics of Campylobacter fetus subspecies requires reevaluation of current diagnostics.

Classifications of the Campylobacter fetus subspecies fetus and venerealis were first described in 1959 and were based on the source of isolation (intestinal versus genital) and the ability of the strains to proliferate in the genital tract of cows. Two phenotypic assays (1% glycine tolerance and H2S production) were described to differentiate the subspecies. Multiple molecular assays have been applied to differentiate the C. fetus subspecies, but none of these tests is consistent with the phenotypic identification methods. In this study, we defined the core genome and accessory genes of C. fetus, which are based on the closed genomes of five C. fetus strains. Phylogenetic analysis of the core genomes of 23 C. fetus strains of the two subspecies showed a division into two clusters. The phylogenetic core genome clusters were not consistent with the phenotypic classifications of the C. fetus subspecies. However, they were consistent with the molecular characteristics of the strains, which were determined by multilocus sequence typing, sap typing, and the presence/absence of insertion sequences and a type I restriction modification system. The similarity of the genome characteristics of three of the phenotypically defined C. fetus subsp. fetus strains to C. fetus subsp. venerealis strains, when considering the core genome and accessory genes, requires a critical evaluation of the clinical relevance of C. fetus subspecies identification by phenotypic assays.

Campylobacter fetus subsp. testudinum subsp. nov., isolated from humans and reptiles.

A polyphasic study was undertaken to determine the taxonomic position of 13 Campylobacter fetus-like strains from humans (n = 8) and reptiles (n = 5). The results of matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) MS and genomic data from sap analysis, 16S rRNA gene and hsp60 sequence comparison, pulsed-field gel electrophoresis, amplified fragment length polymorphism analysis, DNA-DNA hybridization and whole genome sequencing demonstrated that these strains are closely related to C. fetus but clearly differentiated from recognized subspecies of C. fetus. Therefore, this unique cluster of 13 strains represents a novel subspecies within the species C. fetus, for which the name Campylobacter fetus subsp. testudinum subsp. nov. is proposed, with strain 03-427(T) ( = ATCC BAA-2539(T) = LMG 27499(T)) as the type strain. Although this novel taxon could not be differentiated from C. fetus subsp. fetus and C. fetus subsp. venerealis using conventional phenotypic tests, MALDI-TOF MS revealed the presence of multiple phenotypic biomarkers which distinguish Campylobacter fetus subsp. testudinum subsp. nov. from recognized subspecies of C. fetus.

Characterization of a Campylobacter fetus-like strain isolated from the faeces of a sick leopard tortoise (Stigmochelys pardalis) using matrix-assisted laser desorption/ionization time of flight as an alternative to bacterial 16S rDNA phylogeny.

UNLABELLED: This article describes the isolation and characterization of a Campylobacter-like isolate originating from the faeces of a sick leopard tortoise. Molecular as well as matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) characterization suggests that it could correspond to a new Campylobacter species. SIGNIFICANCE AND IMPACT OF THE STUDY: The major impact of this work is the demonstration that proteomics and especially MALDI-TOF typing can be used as an alternative method to 16S rDNA sequencing for phylogeny and can lead to the discovery of new Campylobacters.

Meningitis and endocarditis caused by Campylobacter fetus after raw-liver ingestion.

We report Campylobacter fetus meningitis associated with endocarditis in a 75-year-old diabetic man after he consumed raw liver. C. fetus was isolated from blood samples and cerebrospinal fluid. Cure was obtained with combined intravenous imipenem-gentamicin for 4 weeks; no relapse occurred after 6 months of follow-up.

Application of a new diagnostic approach to a bovine genital campylobacteriosis outbreak in a Saskatchewan beef herd.

A new real-time quantitative polymerase chain reaction (qPCR) test was used to diagnose Campylobacter fetus subsp. venerealis infection associated with dramatic reproductive losses in a commercial cow-calf herd. The results were verified with repeated culture, phenotypic characterization of the organism and DNA sequencing. This case demonstrates the need for a practical field test for C. fetus subsp. venerealis and the importance of considering this organism as a potential cause of pregnancy failure in beef herds.

Application d'une nouvelle approche diagnostique lors d'une éclosion de campylobactériose génitale bovine dans un troupeau bovin de la Saskatchewan. Un nouveau test quantitatif d'amplification en chaîne par la polymérase en temps réel (qACP) a été utilisé pour diagnostiquer une infection par Campylobacter fetus sous-espèce venerealis associée à une baisse spectaculaire de la reproduction dans un troupeau commercial de vaches-veaux. Les résultats ont été vérifiés à l'aide de cultures répétées, d'une caractérisation phénotypique de l'organisme et du séquençage de l'ADN. Ce cas démontre le besoin d'un test sur le terrain pratique pour C. fetus sous-espèce venerealis et l'importance de considérer cet organisme comme une cause potentielle d'échec de la gestation dans les troupeaux bovins.(Traduit par Isabelle Vallières).

Comparative in silico analysis of chemotaxis system of Campylobacter fetus.

Chemoreceptor and chemotaxis signal transduction cascade genes of C. fetus subsp. fetus 82-40 show high level of similarity to that in C. jejuni and appears to include sixteen diverse transducer-like protein (tlp) genes that appear similar to nine of the twelve tlp genes in the C. jejuni NCTC 11168 with a percent identity ranging from 15 to 50%. Sixteen putative C. fetus 82-40 tlp genes belong to three classes: A, B, and C, as well as an aerotaxis gene, based on their predicted structure. C. fetus subsp. fetus 82-40 chemoreceptor and chemotaxis signal transduction pathway genes have close phylogenetic relationship of chemotaxis genes between Campylobacteraceae and Helicobacteraceae.

Phenotypic and genetic analyses of two Campylobacter fetus isolates from a patient with relapsed prosthetic valve endocarditis.

Campylobacter fetus can cause intestinal and systemic disease in humans and are well-established veterinary and economic pathogens. We report the complete genomic sequences of two C. fetus subsp. fetus (Cff) isolates recovered in 2017 (CITCf01) and 2018 (CITCf02) from a case of recurrent prosthetic valve endocarditis. Both were capable of growth aerobically. Their genomes were found to be highly conserved and syntenic with 99.97% average nucleotide identity (ANI) while differences in their respective sap loci defined the temporal separation of their genomes. Based on core genome phylogeny and ANI of 83 Cff genomes belonging to the previously described human-associated Cff lineage, CITCf01 and CITCf02 grouped in a clade of 11 sequence type (ST)3 Cff (including the Cff type strain NCTC 10842T). CITCf01 and CITCf02 were marked for their lack of unique genomic features when compared to isolates within the subspecies and the type strain in particular. We identified point mutations in oxidative stress response genes, among others, that may contribute to aerobiosis. We report a case of Cff causing relapsed prosthetic valve endocarditis and we highlight the sap island as a polymorphic site within the genetically stable ST3 lineage, central to pathogenicity.

Genetic relationships among reptilian and mammalian Campylobacter fetus strains determined by multilocus sequence typing.

Reptile Campylobacter fetus isolates and closely related strains causing human disease were characterized by multilocus sequence typing. They shared approximately 90% nucleotide sequence identity with classical mammalian C. fetus, and there was evidence of recombination among members of these two groups. The reptile group represents a possible separate genomospecies capable of infecting humans.

Evaluation of PCR assays for the detection of Campylobacter fetus in bovine preputial scrapings and the identification of subspecies in South African field isolates.

As a result of the high lability and slow growth of Campylobacter fetus subspecies, the laboratory diagnosis of bovine genital campylobacteriosis has always been difficult. This is especially true under South African conditions, where farms are far apart, laboratories are only present in major centres and there are high ambient temperatures. In order to overcome the shortcomings associated with traditional diagnostic methods, the implementation of a molecular assay was sought. This work describes how a previously published PCR assay (MG3F/ MG4R primers) was adapted, optimised and applied in the diagnostic laboratory to test preputial samples directly for the presence of Campylobacter fetus. Field evaluation of the assay revealed an analytical sensitivity and specificity of 85.7% and 99%, respectively. Subsequent genotyping and phenotyping of a diverse collection of South African field isolates revealed that South Africa has an unexpected and previously unreported high incidence of Campylobacter fetus subsp. venerealis biovar intermedius strains. These strains were not identified correctly by the subspecies-specific primer set evaluated. Until such time that cost- effective genotyping methods are available to diagnostic laboratories in South Africa, and other countries with these atypical Campylobacter fetus subsp. venerealis strains, the need for bacterial culture will persist. Identification to subspecies level of isolates at present remains dependent upon a single phenotypic criterion, namely tolerance to 1% glycine.

Genomic analysis of Campylobacter fetus subspecies: identification of candidate virulence determinants and diagnostic assay targets.

BACKGROUND: Campylobacter fetus subspecies venerealis is the causative agent of bovine genital campylobacteriosis, asymptomatic in bulls the disease is spread to female cattle causing extensive reproductive loss. The microbiological and molecular differentiation of C. fetus subsp. venerealis from C. fetus subsp. fetus is extremely difficult. This study describes the analysis of the available C. fetus subsp. venerealis AZUL-94 strain genome (approximately 75-80%) to identify elements exclusively found in C. fetus subsp. venerealis strains as potential diagnostic targets and the characterisation of subspecies virulence genes. RESULTS: Eighty Kb of genomic sequence (22 contigs) was identified as unique to C. fetus subsp. venerealis AZUL-94 and consisted of type IV secretory pathway components, putative plasmid genes and hypothetical proteins. Of the 9 PCR assays developed to target C. fetus subsp. venerealis type IV secretion system genes, 4 of these were specific for C. fetus subsp. venerealis biovar venerealis and did not detect C. fetus subsp. venerealis biovar intermedius. Two assays were specific for C. fetus subsp. venerealis AZUL-94 strain, with a further single assay specific for the AZUL-94 strain and C. fetus subsp. venerealis biovar intermedius (and not the remaining C. fetus subsp. venerealis biovar venerealis strains tested). C. fetus subsp. fetus and C. fetus subsp. venerealis were found to share most common Campylobacter virulence factors such as SAP, chemotaxis, flagellar biosynthesis, 2-component systems and cytolethal distending toxin subunits (A, B, C). We did not however, identify in C. fetus the full complement of bacterial adherence candidates commonly found in other Campylobacter spp. CONCLUSION: The comparison of the available C. fetus subsp. venerealis genome sequence with the C. fetus subsp. fetus genome identified 80 kb of unique C. fetus subsp. venerealis AZUL94 sequence, with subsequent PCR confirmation demonstrating inconsistent amplification of these targets in all other C. fetus subsp. venerealis strains and biovars tested. The assays developed here highlight the complexity of targeting strain specific virulence genes for field studies for the molecular identification and epidemiology of C. fetus.

[Isolation of Campylobacter fetus from persons with obstetric-gynecological infections].

Campylobacter fetus subspecies fetus is a cause of different obstetric-gynecological diseases. It is a first time when rate of infection with Campylobacter was studied and connection between the infection and development of chronic gynecologic diseases and pathology of labor was established. Bacteria were isolated and identified in 36.0% +/- 0.7 of studied women admitted to inpatient clinics. It was established that Campylobacter fetus subspecies fetus can cause abnormalities in placenta functions as well as different inflammatory processes during pregnancy.

Clinical and microbiological characteristics of patients with bacteremia caused by Campylobacter species with an emphasis on the subspecies of C. fetus.

OBJECTIVES: This study was intended to investigate the clinical and microbiological characteristics of patients with bacteremia caused by Campylobacter species. METHODS: From April 1998 to May 2014, 56 adults with bacteremia caused by Campylobacter species were evaluated. These Campylobacter species isolates were confirmed to the species level using 16S rRNA gene sequencing (all isolates) and multiplex PCR analysis (for C. fetus only). The performance of identification for Campylobacter species by the Bruker Biotyper MALDI-TOF MS was evaluated. The genetic relatedness of C. fetus isolates was analyzed by multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE). RESULTS: The leading underlying medical conditions of these patients were malignancy (46.4%), hypertension (35.7%), and liver cirrhosis (23.2%). The overall 30-day mortality rate was 5.4%. Using 16S rRNA sequencing analysis, 26 isolates of C. coli, 11 of C. jejuni, and 19 of C. fetus, including 15 C. fetus subsp. fetus and five C. fetus subsp. venerealis, were identified. Among the five C. fetus subsp. venerealis isolates recognized by 16S rRNA gene sequencing, only two isolates were C. fetus subsp. venerealis by multiplex PCR method. The Bruker Biotyper MALDI-TOF MS failed to correctly identify C. fetus subsp. venerealis isolates. MLST analysis of C. fetus isolates revealed three STs: ST20 (n = 12), ST11 (n = 5), and ST57 (n = 2), which were compatible with three major PFGE clusters. CONCLUSION: Database expansion of MALDI-TOF MS for the correct identification of C. fetus to subspecies levels is needed. A novel clone of ST57-PFGE Cluster C of C. fetus subsp. venerealis was noted.

Identification of Campylobacter fetus subsp. venerealis virulence genes in cervical mucus from cows.

We used the polymerase chain reaction to identify virulence genes in cervico-vaginal mucus samples from cows positive for Campylobacter fetus subsp. venerealis. There was positivity for the pldA, racR, dnaJ, cdtA, and cdtB genes. No samples showed the cdtC, ciaB, cadF, wlaN, and virB11 genes.