Structural and Biochemical Characterization of Aminoglycoside Nucleotidyltransferase(6)-Ib From Campylobacter fetus subsp. fetus.

Aminoglycoside antibiotics have played a critical role in the treatment of both Gram-negative and Gram-positive bacterial infections. However, antibiotic resistance has severely compromised the efficacy of aminoglycosides. A leading cause of aminoglycoside resistance is mediated by bacterial enzymes that inactivate these drugs via chemical modification. Aminoglycoside nucleotidyltransferase-6 (ANT(6)) enzymes inactivate streptomycin by transferring an adenyl group from ATP to position 6 on the antibiotic. Despite the clinical significance of this activity, ANT(6) enzymes remain relatively uncharacterized. Here, we report the first high resolution x-ray crystallographic structure of ANT(6)-Ib from Campylobacter fetus subsp. fetus bound with streptomycin. Structural modeling and gel filtration chromatography experiments suggest that the enzyme exists as a dimer in which both subunits contribute to the active site. Moreover, superposition of the ANT(6)-Ib structure with the structurally related enzyme lincosamide nucleotidyltransferase B (LinB) permitted the identification of a putative nucleotide binding site. These data also suggest that residues D44 and D46 coordinate essential divalent metal ions and D102 functions as the catalytic base.

Antimicrobial susceptibilities of Campylobacter fetus: report from a reference laboratory.

Campylobacter fetus is known to cause human disease, particularly in elderly and immunocompromised hosts. There are limited published data for antimicrobial susceptibility patterns with this organism, and no interpretive criteria are available. We reviewed antimicrobial susceptibilities of C. fetus isolates tested at a tertiary care center and reference laboratory over an 11-year period. C. fetus isolates from patients treated at Mayo Clinic and those sent as referrals for identification and susceptibility were included. Antimicrobial susceptibility testing was performed using agar dilution for ciprofloxacin, doxycycline, erythromycin, gentamicin, meropenem, and tetracycline. Geographic distribution, culture source, organism minimal inhibitory concentration (MIC) distributions, and MIC50 and MIC90 were examined. Excluding duplicates, 105 unique isolates were identified from 110 positive cultures. Blood cultures represented the most common source, followed by body fluids, skin and soft tissue, and central nervous system. Gentamicin and meropenem had favorable MIC50 and MIC90 of 1 µg/mL. Ciprofloxacin demonstrated an MIC50 of 1 µg/mL; however, the MIC90 was >2 µg/mL. Erythromycin demonstrated MIC50 and MIC90 of 2 µg/mL. Tetracycline and doxycycline were tested on a limited number of isolates and showed a wide range of MICs. Gentamicin and meropenem demonstrated favorable MICs in C. fetus isolates. These may represent therapeutic options for consideration in serious C. fetus infections, pending susceptibility results. Ciprofloxacin, which showed variable results, may be more appropriate for use only after susceptibility testing. C. fetus interpretive criteria are needed to aid clinicians in selection of both empiric and definitive therapies. IMPORTANCE: Our findings contribute to the scant literature on Campylobacter fetus antimicrobial susceptibility test results. We used a reference test method of agar dilution and provide MICs for a large number of organisms and antimicrobial agents.

Design of a multi-epitope-based vaccine candidate against Bovine Genital Campylobacteriosis using a reverse vaccinology approach.

BACKGROUND: Bovine Genital Campylobacteriosis (BGC), a worldwide distributed venereal disease caused by Campylobacter fetus subsp. venerealis (Cfv), has a relevant negative economic impact in cattle herds. The control of BGC is hampered by the inexistence of globally available effective vaccines. The present in silico study aimed to develop a multi-epitope vaccine candidate against Cfv through reverse vaccinology. RESULTS: The analysis of Cfv strain NCTC 10354 proteome allowed the identification of 9 proteins suitable for vaccine development. From these, an outer membrane protein, OmpA, and a flagellar protein, FliK, were selected for prediction of B-cell and T-cell epitopes. The top-ranked epitopes conservancy was assessed in 31 Cfv strains. The selected epitopes were integrated to form a multi-epitope fragment of 241 amino acids, which included 2 epitopes from OmpA and 13 epitopes from FliK linked by GPGPG linkers and connected to the cholera toxin subunit B by an EAAAK linker. The vaccine candidate was predicted to be antigenic, non-toxic, non-allergenic, and soluble upon overexpression. The protein structure was predicted and optimized, and the sequence was successfully cloned in silico into a plasmid vector. Additionally, immunological simulations demonstrated the vaccine candidate's ability to stimulate an immune response. CONCLUSIONS: This study developed a novel vaccine candidate suitable for further in vitro and in vivo experimental validation, which may become a useful tool for the control of BGC.

Biochemical and molecular characterization of Campylobacter fetus isolates from bulls subjected to bovine genital campylobacteriosis diagnosis in Spain.

BACKGROUND: Bovine genital campylobacteriosis (BGC) is caused by Campylobacter fetus subsp. venerealis (Cfv) including its biovar intermedius (Cfvi). This sexually transmitted disease induces early reproductive failure causing considerable economic losses in the cattle industry. Using a collection of well-characterized isolates (n = 13), C. fetus field isolates (n = 64) and saprophytic isolates resembling Campylobacter (n = 75) obtained from smegma samples of breeding bulls, this study evaluated the concordance of the most used phenotypic (H2S production in cysteine medium and 1% glycine tolerance) and molecular (PCR) methods for the diagnosis of BGC and assessed possible cross-reactions in the molecular diagnostic methods. RESULTS: Characterization at the subspecies level (fetus vs. venerealis) of C. fetus isolated from bull preputial samples using phenotypic and molecular (PCR targeting nahE and ISCfe1) methods showed moderate concordance (κ = 0.462; CI: 0.256-0.669). No cross-reactions were observed with other saprophytic microaerophilic species or with other Campylobacter species that can be present in preputial samples. Whole genome sequencing (WGS) of discrepant isolates showed 100% agreement with PCR identification. For the differentiation of Cfv biovars, comparison of the H2S test (at 72 h and 5 days of incubation) and a PCR targeting the L-cysteine transporter genes showed higher concordance when H2S production was assessed after 5 days (72 h; κ = 0.553, 0.329-0.778 CI vs. 5 days; κ = 0.881, 0.631-1 CI), evidencing the efficacy of a longer incubation time. CONCLUSIONS: This study confirmed the limitations of biochemical tests to correctly identify C. fetus subspecies and biovars. However, in the case of biovars, when extended incubation times for the H2S test (5 days) were used, phenotypic identification results were significantly improved, although PCR-based methods produced more accurate results. Perfect agreement of WGS with the PCR results and absence of cross-reactions with non-C. fetus saprophytic bacteria from the smegma demonstrated the usefulness of these methods. Nevertheless, the identification of new C. fetus subspecies-specific genes would help to improve BGC diagnosis.

Campylobacter fetus Invasive Infections and Risks for Death, France, 2000-2021.

Campylobacter fetus accounts for 1% of Campylobacter spp. infections, but prevalence of bacteremia and risk for death are high. To determine clinical features of C. fetus infections and risks for death, we conducted a retrospective observational study of all adult inpatients with a confirmed C. fetus infection in Nord Franche-Comté Hospital, Trevenans, France, during January 2000-December 2021. Among 991 patients with isolated Campylobacter spp. strains, we identified 39 (4%) with culture-positive C. fetus infections, of which 33 had complete records and underwent further analysis; 21 had documented bacteremia and 12 did not. Secondary localizations were reported for 7 (33%) patients with C. fetus bacteremia, of which 5 exhibited a predilection for vascular infections (including 3 with mycotic aneurysm). Another 7 (33%) patients with C. fetus bacteremia died within 30 days. Significant risk factors associated with death within 30 days were dyspnea, quick sequential organ failure assessment score >2 at admission, and septic shock.

Campylobacter fetus seeding of a cavernous malformation resulting in brain abscess: case report and literature review.

Microbial seeding of a cerebral cavernous malformation is an extremely rare occurrence with only 3 cases reported in the literature thus far. Campylobacter fetus is an opportunistic pathogen that rarely causes neurological infection with only 3 cases of C. fetus cerebral abscesses and 38 cases of C. fetus meningitis reported in the literature. There have been no cases of cerebral cavernous malformation seeding by C. fetus reported to date. We report the first case of cerebral cavernous malformation seeding by C. fetus, a case occurring in a previously healthy 16-year-old female who presented with suspected left cerebellar cavernous malformation with subacute hemorrhage. She underwent a suboccipital craniectomy for the resection of the cavernous malformation with additional intraoperative findings suggestive of cerebral abscess. Following positive blood and CSF cultures and surgical pathology results, the patient was diagnosed with C. fetus meningoencephalitis with co-infected left cerebellar cavernous malformation. This is the fourth reported case of microbial seeding of a cerebral cavernous malformation, and to our knowledge, the first case of a C. fetus-infected cavernous malformation. Compared to previous reports, the clinical events of this case strongly support the presence of a preexisting lesion that was secondarily seeded versus de novo formation as a result of prior infection.

[Prevalence and geographical distribution of bovine sexually transmitted diseases in the province of Formosa, Argentina]. / Prevalencia y distribución geográfica de las enfermedades de transmisión sexual de los bovinos en la provincia de Formosa, Argentina.

Bovine genital campylobacteriosis (BGC) and bovine trichomonosis (BT) are sexually transmitted diseases (STDs) that affect bovine breeding herds, decreasing their reproductive efficiency. The objective of this work was to estimate the prevalence of these diseases and their temporal-spatial distribution in the province of Formosa, Argentina. The cross-sectional study conducted between 2018 and 2021 included a total of 15,571 bulls, inter-herd prevalence being 29.62% and 17.23% for BGC and BT, respectively. The prevalence of positive animals was 2.05% for BGC and 0.43% for BT. The temporal-spatial analysis of BGC showed two distinct spatial groupings, one group had a low risk of contracting the disease (RR = 0.13; p < 0.001; 2018-2021) while the other group had a high risk (RR = 2.84; p < 0.001; 2020-2021). BT had a high-risk group for the disease (RR = 35.24; p < 0.001; 2019). This study shows that STDs are endemic in the region, providing updated and valuable information as a tool for the health management of these diseases.

Genome analysis of clinical isolate of Campylobacter fetus subspecies fetus MMM01 from India reveals genetic determinants of pathogenesis and adaptation.

In this study we report the whole genome sequencing (WGS) based analysis of blood-borne Campylobacter fetus subsp. fetus MMM01 isolated from a diabetic patient to obtain deeper insights in to the virulence and host adaptability. The sequenced genome of C. fetus subsp. fetus MMM01 along with reference genomes retrieved from NCBI was subjected to various in-silico analysis including JSpecies, MLST server, PATRIC server, VFanalyzer, CARD, PHASTER to understand their phylogenetic relation, virulence and antimicrobial resistance profile. The genome had a size of 1,788,790 bp, with a GC content of 33.09%, nearly identical to the reference strain C. fetus subsp. fetus 82-40. The MLST based phylogenetic tree constructed revealed the polyphyletic branching and MMM01 (ST25) was found to be closely related to ST11, both belong to the sap-A serotype which are more common in human infections. VFanalyzer identified 88 protein-coding genes coding for several virulence factors including Campylobacter adhesion to fibronectin, flagellar apparatus, cytolethal distending toxin operons and Campylobacter invasion antigen proteins which enhance the virulence of bacteria along with resistance genes against antibiotics including fluoroquinolone, chloramphenicol, tetracycline, and aminoglycoside in MMM01, which points to enhanced survival and pathogenicity of this zoonotic pathogen. It was interesting to find that MMM01 lacked FGI-II island found in most of the clinical isolates, which encoded CRISPR Cas and prophage II regions. More details about the complexity and evolution of this zoonotic pathogen could be learned from future studies that concentrate on comparative genome analysis using larger genome datasets.

Placentitis and abortion caused by a multidrug resistant strain of Campylobacter fetus subspecies fetus in a sheep in Uruguay.

Campylobacter fetusfetus (Cff) is a major infectious cause of abortion in sheep worldwide, and an opportunistic human pathogen. Information on Cff as an ovine abortifacient in South America is limited. We describe a case of abortion caused by a multidrug resistant strain of Cff in a sheep in Uruguay. In August 2017, 3/57 pregnant ewes (5.3%) aborted whithin one week. Histopathologic examination of the placenta of an aborted ewe revealed severe neutrophilic and fibrinonecrotizing placentitis with vasculitis and thrombosis of the chorionic arterioles. Cff was isolated on microaerobic culture in Skirrow agar, and further confirmed by 16S rDNA PCR amplification and sequencing, and endpoint and real time PCR assays. Antimicrobial sensitivity testing revealed resistance to tetracyclines, nalidixic acid, telithromycin and clindamycin. Other abortifacients were not detected. Further studies are necessary to determine the geographic distribution, ecology, epidemiology, economic impact, and antimicrobial resistance of Cff in sheep flocks in Uruguay.

Campylobacter fetus bacteremia and meningitis in an acute lymphoblastic leukemia patient undergoing maintenance therapy: a case report.

BACKGROUND: Campylobacter fetus is an uncommon Campylobacter species, and its infections mainly cause infective endocarditis, aortic aneurysm, and meningitis rather than enteritis. It is more likely to be detected in blood than Campylobacter jejuni or Campylobacter coli, specifically reported in 53% of patients. In our case, C. fetus was detected in both blood and cerebrospinal fluid (CSF) cultures. CASE PRESENTATION: A 33-year-old woman, who was on maintenance chemotherapy for acute lymphoblastic leukemia (ALL), presented to our clinic with chief complaints of severe headache and nausea. Blood and CSF cultures revealed C. fetus. We administrated meropenem 2 g intravenously (IV) every 8 h for 3 weeks, and she was discharged without neurological sequelae. CONCLUSION: We encountered a case of C. fetus meningitis without gastrointestinal symptoms, neck stiffness or jolt accentuation in a patient with ALL. Undercooked beef was considered the source of C. fetus infection in this case, suggesting that the need for a neutropenic diet and safe food handling be considered.

Campylobacter fetus subspecies venerealis meningitis associated with a companion dog in a young adult: a case report.

BACKGROUND: Campylobacter spp., common commensals in the gastrointestinal tract of animals, especially poultry, can cause acute gastrointestinal illness in humans through animal-to-human transmission. Although Campylobacter fetus, especially subspecies fetus, rarely leads to systemic infections such as bacteremia in immunocompromised patients, it is unclear whether Campylobacter fetus subspecies venerealis (Cfv) causes infectious diseases in humans. CASE PRESENTATION: A 28-year-old man with a history of chronic alcoholism visited the emergency department with weakness of the left extremities. The patient was clinically diagnosed with community-acquired bacterial meningitis. The organism from the blood culture was subsequently identified as Campylobacter fetus. On phylogenetic analysis, the 16S rRNA sequence showed 99.93% similarity with other Cfv 16S rRNA sequences. The patient had no exposure to identifiable sources except for close contact with a companion dog, which could have been a possible source of transmission. CONCLUSIONS: This case suggests that Cfv could lead to human systemic infections such as meningitis and that companion animals, in addition to well-known animal hosts, could be sources of transmission.

Sequential breast implant infections due to Campylobacter fetus subsp. fetus.

Campylobacter jejuni and Campylobacter coli are the leading causes of bacterial intestinal infections worldwide, while Campylobacter fetus subsp. fetus (C. fetus) has been reported to cause extraintestinal infections, including medical device implant infections. However, breast implant infections have rarely been reported. We describe the case of a 64-year-old woman with breast implant infection and vertebral osteomyelitis due to C. fetus. The patient recovered by surgical removal of the infected left implant and was treated with antibiotics for 6 weeks. However, two weeks after the completion of antibiotics, she experienced an infection in the right implant due to C. fetus, which had developed quinolone resistance with a G91T mutation during the treatment course. This case showed that C. fetus can cause breast implant infections, and although the infection may appear to be unilateral initially, the possibility of sequential contralateral infection should be considered.

Performance of bovine genital campylobacteriosis diagnostic tests in bulls from Uruguay: a Bayesian latent class model approach.

The sensitivity (Se) and specificity (Sp) of three diagnostic tests for the detection of Campylobacter fetus venerealis (Cfv) using field samples were estimated using a Bayesian latent class model (BLCM), accounting for the absence of a gold standard. The tests included in this study were direct immunofluorescence antibody test (IFAT), polymerase chain reaction (PCR), and real-time PCR (RT-PCR). Twelve farms from two different populations were selected and bull prepuce samples were collected. The IFAT was performed according to the OIE Manual. The conventional PCR was performed as multiplex, targeting the gene nahE for C. fetus species identification and insertion element ISCfe1 for Cfv identification. The RT-PCR was performed as uniplex: one targeting the gene nahE for C. fetus and the other targeting the insertion ISCfe1 (ISC2) for Cfv. Results from the BLCM showed a median Se of 11.7% (Bayesian credibility interval (BCI): 1.93-29.79%), 53.7% (BCI: 23.1-95.0%), and 36.1% (BCI: 14.5-71.7%) for IFAT, PCR, and RT-PCR respectively. The Sp were 94.5% (BCI: 90.1-97.9%), 97.0% (BCI: 92.9-99.3%), and 98.4% (BCI: 95.3-99.7%) for IFAT, PCR, and RT-PCR respectively. The correlation between PCR and RT-PCR was positive and low in samples from both sampled population (0.63% vs 8.47%). These results suggest that diagnostic sensitivity of the studied tests is lower using field samples than using pure Cfv strains.

Polyclonal Campylobacter fetus Infections Among Unrelated Patients, Montevideo, Uruguay, 2013-2018.

In Montevideo (2013-2018), 8 Campylobacter fetus extraintestinal infections were reported. The polyclonal nature of strains revealed by whole-genome sequencing and the apparent lack of epidemiological links was incompatible with a single contamination source, supporting alternative routes of transmission.

Effects of Campylobacter fetus on bull sperm quality.

Campylobacter fetus is a gram-negative, motile, spiral or S-shaped bacterium, which induces campylobacteriosis. This disease causes decrease productivity of cattle. Although considerable research has been done on the role of C. fetus on female fertility, little is known about the effect on bulls. The aim of this work was to evaluate the effect of C. fetus subsp. fetus (Cff) and C. fetus subsp. venerealis (Cfv) on bull sperm quality. Samples of frozen semen (n = 29 straws) were each distributed into three groups: two of them incubated with the microorganism (Cff, Cfv) and a control group. The proportions of live spermatozoa, with functional membrane and true acrosomal reaction in control group were significantly (P < 0.01) greater than those observed in Cff and Cfv groups. However, no significant differences (P > 0.05) were found in sperm chromatin structure among treatments. In adhesion assay, proportions of spermatozoa with adhered Campylobacter were similar for both subspecies. Results confirm that Cff and Cfv have the same ability to bind in an irreversible way to bull spermatozoa and to affect sperm quality. It is proposed that adherence could be considered as the main cause of sperm alterations, and also an important step of pathogenesis and venereal transmission.

Assessment of Campylobacter fetus subsp. venerealis molecular diagnosis using clinical samples of bulls.

BACKGROUND: Campylobacter fetus subsp. venerealis (Cfv) is the pathogen responsible for Bovine Genital Campylobacteriosis (BGC), a venereal disease of cattle associated with impaired reproductive performance. Although several PCR assays were developed to identify this pathogen, most of them are still poorly evaluated in clinical samples. This study evaluated real-time PCR assays for Cfv detection in preputial samples of bulls (n = 308). RESULTS: The detection at the subspecies level (Cfv) compared four assays: two targeting ISCfe1 and two targeting parA gene. The detection at the species level (C. fetus) considered an assay targeting the nahE gene and a commercial kit for C. fetus identification. At the subspecies level, assays directed either to different targets (parA and ISCfe1), or to the same target (ISCfe1 or parA), showed a high percentage of disagreeing results. All samples positive at the subspecies level (n = 169) were negative in C. fetus detection assays, which strongly suggests the horizontal gene transfer of ISCfe1 and parA to other bacterial species. This was confirmed by microbiological isolation of three Campylobacter portucalensis strains responsible for false positive results. Sequences with a high level of identity with ISCfe1 and parA gene of Cfv were identified in C. portucalensis genome. CONCLUSIONS: Overall, this study reveals that PCR assays solely directed to a subspecies target originate a high rate of false positive results, due to the presence of parA and ISCfe1 homologous sequences in other bacterial species, namely of the genus Campylobacter. Although the specificity of these methods may be higher if applied to bulls from herds with clinical features of BGC or in other geographical regions, current PCR diagnosis should couple subspecies and species targets, and further research must be envisaged to identify Cfv specific molecular targets.

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).

Versatile nano-platform for tailored immuno-magnetic carriers.

Custom immuno-magnetic devices are desirable tools for biomedical and biotechnological applications. Herein, surface active maghemite nanoparticles (SAMNs) are proposed as a versatile platform for developing tailored immuno-magnetic nano-carriers by simple wet reactions. Two examples for conjugating native and biotinylated antibodies were presented along with their successful applications in the recognition of specific foodborne pathogens. Nanoparticles were functionalized with rhodamine B isothiocyanate (RITC), leading to a fluorescent nano-conjugate, and used for binding anti-Campylobacter fetus antibodies (SAMN@RITC@Anti-Cf). The microorganism was selectively captured in the presence of two other Campylobacter species (C. jejuni and C. coli), as verified by PCR. Alternatively, SAMNs were modified with avidin, forming a biotin-specific magnetic nano-carrier and used for the immobilization of biotinylated anti-Listeria monocytogenes antibodies (SAMN@avidin@Anti-Lm). This immuno-magnetic carrier was integrated in piezoelectric quartz crystal microbalance (QCM) sensor for the detection of L. monocytogenes in milk, showing a detection limit of 3 bacterial cells. The present work presents a new category of customized immuno-magnetic nano-carriers as a competitive option for suiting specific applications. Graphical abstract ᅟ.

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.

Pericarditis caused by Campylobacter fetus subspecies fetus associated with ingestion of raw beef liver.

Campylobacter fetus is an organism residing primarily in the gastrointestinal tracts of cattle and sheep and transmitting to humans through ingestion of contaminated food products or surface water. The organism has caused various extraintestinal infections but, to date, purulent pericarditis due to the organism has rarely been described. We report a case of purulent pericarditis due to C. fetus subsp. fetus, occurring in a patient having several predisposing conditions, including receiving hemodialysis therapy, recent surgery for cecal cancer, and administration of esomeprazole. The patient mentioned having eaten homemade raw beef liver two weeks before the onset, suggesting that the ingested food product was contaminated with C. fetus and the organism transmitted to the pericardium through the bloodstream although blood culture was negative. The causative organism, recovered from the pericardial effusion, was unidentifiable with commercial systems but determinable with molecular methods at the subspecies level. The patient fully improved with pericardiocentesis and subsequent administration of ciprofloxacin, to which the organism was considered susceptible, for a total of four weeks. This is the first case of C. fetus pericarditis in which a history of ingesting a raw food product was clearly mentioned.