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.

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).

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.

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 by fluorescence in situ hybridization (FISH).

Two new DNA FISH-probes for Campylobacter fetus were designed, in silico checked for cross-reactions and successfully evaluated in a multi-centric approach with 41 Campylobacter fetus isolates including isolates of all three know subspecies: Campylobacter fetus ssp. fetus, Campylobacter fetus ssp. venerealis, and Campylobacter fetus ssp. testudinum and 40 strains of five non-target Campylobacter species.

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.

Assessing the intra-species genetic variability in the clonal pathogen Campylobacter fetus: CRISPRs are highly polymorphic DNA markers.

Campylobacter fetus is a Gram-negative, microaerophilic bacterium that infects animals and humans. The subspecies Campylobacter fetus subsp. fetus (Cff) affects a broad range of vertebrate hosts and induces abortion in cows and sheep. Campylobacter fetus subsp. venerealis (Cfv) is restricted to cattle and causes the endemic disease bovine genital campylobacteriosis, which triggers reproductive problems and is responsible for major economic losses. Campylobacter fetus subsp. testudinum (Cft) has been isolated mostly from apparently healthy reptiles belonging to different species but also from ill snakes and humans. Genotypic differentiation of Cff and Cfv is difficult, and epidemiological information is scarce because there are few methods to study the genetic diversity of the strains. We analyze the efficacy of MLST, ribosomal sequences (23S gene and internal spacer region), and CRISPRs to assess the genetic variability of C. fetus in bovine and human isolates. Sequences retrieved from complete genomes were included in the analysis for comparative purposes. MLST and ribosomal sequences had scarce or null variability, while the CRISPR-cas system structure and the sequence of CRISPR1 locus showed remarkable diversity. None of the sequences here analyzed provided evidence of a genetic differentiation of Cff and Cfv in bovine isolates. Comparison of bovine and human isolates with Cft strains showed a striking divergence. Inter-host differences raise the possibility of determining the original host of human infections using CRISPR sequences. CRISPRs are the most variable sequences analyzed in C. fetus so far, and constitute excellent representatives of a dynamic fraction of the genome. CRISPR typing is a promising tool to characterize isolates and to track the source and transmission route of C. fetus infections.

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.

Effect of sample pooling and transport conditions on the clinical sensitivity of a real-time polymerase chain reaction assay for Campylobacter fetus subsp. venerealis in preputial samples from bulls.

The diagnosis of bovine genital campylobacteriosis (BGC) presents significant challenges, as traditional methods lack sensitivity when prolonged transport of samples is required. Assays of preputial samples by means of real-time polymerase chain reaction (PCR) provide good sensitivity and high throughput capabilities. However, there is limited information on the acceptable duration of transport and temperature during transport of samples. In addition, the use of pooled samples has proven to be a valuable strategy for the diagnosis of other venereal diseases in cattle. The objectives of the present study were to determine the effect of sample pooling and of transport time and temperature on the clinical sensitivity of a real-time quantitative PCR (qPCR) assay for Campylobacter fetus subsp. venerealis in preputial samples from beef bulls. Eight infected bulls and 176 virgin yearling bulls were used as the source of samples. The qPCR sensitivity was comparable for unpooled samples and pools of 5 samples, whereas sensitivity was decreased for pools of 10 samples. Sensitivity for the various pool sizes improved with repeated sampling. For shorter-term transport (2 and 48 h), sensitivity was greatest when the samples were stored at 4°C and 30°C, whereas for longer-term transport (96 h) sensitivity was greatest when the samples were stored at -20°C. The creation of pools of 5 samples is therefore a good option to decrease costs when screening bulls for BGC with the qPCR assay of direct preputial samples. Ideally the samples should be stored at 4°C and arrive at the laboratory within 48 h of collection, but when that is not possible freezing at -20°C could minimize the loss of sensitivity.

Le diagnostic de la campylobactériose génitale bovine (CGB) présente des défis significatifs, étant donné que les méthodes traditionnelles manquent de sensibilité lorsqu'un transport prolongé des échantillons est requis. Les épreuves utilisant des échantillons prépuciaux dans des épreuves de réaction d'amplification en chaine par la polymérase en temps réel (PCR) ont une bonne sensibilité et une capacité de rendement élevée. Toutefois, il y a peu d'information sur la durée acceptable du transport et de la température durant le transport des échantillons. De plus, l'utilisation d'échantillons regroupés s'est avéré être une stratégie valable pour le diagnostic d'autres maladies vénériennes chez les bovins. Les objectifs de la présente étude étaient de déterminer l'effet du regroupement d'échantillons et du temps de transport et de la température sur la sensibilité clinique d'une épreuve PCR quantitative en temps réel (qPCR) pour Campylobacter fetus ssp. venerealis dans des échantillons prépuciaux provenant de taureaux. Huit taureaux infectés et 176 bouvillons vierges ont été utilisés comme source des échantillons. La sensibilité du qPCR était comparable pour des échantillons non-regroupés et des regroupements de 5 échantillons, mais diminuée pour des regroupements de 10 échantillons. La sensibilité pour les différentes tailles de regroupement s'améliorait suite à des échantillonnages répétés. Pour des transport de courte durée (2 et 48 h), la sensibilité était plus élevée lorsque les échantillons étaient entreposés à 4 °C et 30 °C, alors que pour le transport de longue durée (96 h) la sensibilité était plus élevée lorsque les échantillons étaient entreposés à −20 °C. La création de regroupement de 5 échantillons est une bonne option pour diminuer les coûts lors du tamisage de taureaux pour CGB avec le qPCR effectué directement sur des échantillons prépuciaux. Idéalement, les échantillons devraient être entreposés à 4 °C et arriver au laboratoire au plus tard 48 h après le prélèvement, si ce n'est pas possible, la congélation à −20 °C pourrait minimiser la perte de sensibilité.(Traduit par Docteur Serge Messier).

Infectious Spondylitis in a Patient with Chronic Kidney Disease: Identification of Campylobacter fetus Subsp. testudinum by 16S Ribosomal RNA Sequencing.

We report the first case of spondylitis with bacteremia caused by Campylobacter fetus subsp. testudinum identified by 16S ribosomal ribonucleic acid (rRNA) gene sequencing. An 81-year-old man presented with fever and general weakness. His medical history included end-stage renal disease, hypertension, and type 2 diabetes. Despite empirical antibiotic treatment, his fever and back pain persisted. Magnetic resonance imaging with gadolinium enhancement showed a low-signal-intensity lesion in T1-weighted imaging and a high-signal-intensity lesion in T2-weighted imaging at the L3 vertebral body. C. fetus grew on 1 pair of blood cultures. C. fetus subsp. testudinum was identified via 16S rRNA sequencing of the cultivated organisms. The patient recovered uneventfully after 6 weeks of optimal antibiotic treatment, selected using susceptibility tests. C. fetus spondylitis is a very rare disease. In this unique case involving end-stage renal disease, the underlying pathogen was identified by 16S rRNA sequencing.

[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.

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.

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.

Pregnancy rates of beef cattle are not affected by Campylobacter fetus subsp. venerealis real-time PCR-positive breeding sires in New Zealand.

AIMS: Campylobacter fetus subspecies venerealis (C. fetus venerealis) is the causal agent of bovine genital campylobacteriosis, a venereal disease that is asymptomatic in bulls but responsible for reproductive wastage in female cattle. In New Zealand, a commercial real-time PCR assay was introduced in 2007 to identify the DNA of this pathogen in preputial scrapings; however, concerns were raised about the specificity of the test following anecdotal reports of a high number of test-positive bulls with no apparent relationship to reproductive performance. The objective of this study, therefore, was to examine the association between real-time PCR assay results from beef breeding bulls and pregnancy rates in beef herds using these bulls. METHODS: Veterinarians from four veterinary practices selected beef cattle herds with relatively high and low pregnancy rates between December 2008 and February 2009. Preputial scrapings were collected from bulls used for mating in those herds. Samples were tested using the real-time PCR assay under consideration. Bivariable and multivariable analyses were used to assess the relationship between pregnancy rates in each mob (15-month-old heifers, 27-month-old heifers and mixed-age cows) and the percentage of real-time PCR-positive bulls in each mob. RESULTS: Sixty-four (28.8%) of 222 bulls tested positive, 130 (58.6%) tested negative, and 28 (12.6%) returned an inconclusive result to the real-time PCR assay. The percentage of bulls testing real-time PCR-positive in these mobs was not associated with pregnancy rates (p=0.757) after controlling for mob, average body condition score of cows, cow to bull ratio, length of the mating period, and farm. CONCLUSION: Real-time PCR assay results were not associated with pregnancy rates, suggesting that the specificity of the real-time PCR assay was too low to be used to reliably detect C. fetus venerealis. This study adds to a growing body of evidence indicating that C. fetus venerealis strains are either absent from, or present at clinically insignificant levels of endemicity among, beef breeding herds in New Zealand. CLINICAL SIGNIFICANCE: The real-time PCR assay that was assessed in this study should not be used for the detection of C. fetus venerealis in bulls or for investigations of low conception rates in cattle in New Zealand. During the course of this survey, sequencing analysis of an apparent C. fetus venerealis isolate from the intestines of a Friesian bull turned out to be Campylobacter hyointestinalis. As a consequence, this real-time PCR assay for C. fetus venerealis is no longer being offered by diagnostic laboratories in New Zealand.

Comparative genome analysis of Campylobacter fetus subspecies revealed horizontally acquired genetic elements important for virulence and niche specificity.

Campylobacter fetus are important animal and human pathogens and the two major subspecies differ strikingly in pathogenicity. C. fetus subsp. venerealis is highly niche-adapted, mainly infecting the genital tract of cattle. C. fetus subsp. fetus has a wider host-range, colonizing the genital- and intestinal-tract of animals and humans. We report the complete genomic sequence of C. fetus subsp. venerealis 84-112 and comparisons to the genome of C. fetus subsp. fetus 82-40. Functional analysis of genes predicted to be involved in C. fetus virulence was performed. The two subspecies are highly syntenic with 92% sequence identity but C. fetus subsp. venerealis has a larger genome and an extra-chromosomal element. Aside from apparent gene transfer agents and hypothetical proteins, the unique genes in both subspecies comprise two known functional groups: lipopolysaccharide production, and type IV secretion machineries. Analyses of lipopolysaccharide-biosynthesis genes in C. fetus isolates showed linkage to particular pathotypes, and mutational inactivation demonstrated their roles in regulating virulence and host range. The comparative analysis presented here broadens knowledge of the genomic basis of C. fetus pathogenesis and host specificity. It further highlights the importance of surface-exposed structures to C. fetus pathogenicity and demonstrates how evolutionary forces optimize the fitness and host-adaptation of these pathogens.