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).

The Campylobacter fetus S layer provides resistance to photoactivated zinc oxide nanoparticles.

The antimicrobial activity of metal-based compounds, including metal oxides, has resulted in numerous agricultural, industrial, and medical applications. Zinc oxide nanoparticles are toxic to Gram-positive and Gram-negative bacteria as well as to some fungi. In this study we assess the sensitivity of Campylobacter fetus, a Gram-negative bacterial pathogen of humans and animals, to ZnO nanoparticles and determine whether the S layer protects C. fetus from the antibacterial action of these nanoparticles. Broth and agar dilution assays revealed that ZnO nanoparticles at 100 µg/mL were bacteriocidal for C. fetus. Resazurin reduction assays confirmed the absence of metabolic activity, indicating that C. fetus cells had not entered into a viable but nonculturable state. Photoactivation of ZnO nanoparticles greatly enhanced their antibacterial activity, as evidenced by minimum bacteriocidal concentration (MBC) values decreasing to 16-62.5 µg/mL as a function of strain. MBC assays completed in the presence and absence of catalase revealed that H2O2, a product of ZnO nanoparticle photoactivation, contributed to C. fetus but not to C. jejuni cell death. S-layer-expressing C. fetus strains were more resistant to H2O2-mediated cell killing than were isogenic S-layer-deficient strains. These data indicate that C. fetus is sensitive to the antibacterial activity of ZnO nanoparticles and that the C. fetus S layer imparts protection against photoactivated nanoparticles.

Epidemiologic cutoff values to separate wild-type from non-wild-type Campylobacter fetus to ciprofloxacin.

The aim of the present study was to propose epidemiologic cutoffs that could be used in routine practice to separate wild-type from non-wild-type Campylobacter fetus to ciprofloxacin. A total of 123 C. fetus isolates obtained from human samples were used for this purpose. Based on the determination of inhibition zone diameter, minimum inhibitory concentration, and sequencing of the quinolone resistance determining region in the gyraseA gene, for all tested isolates, the following cutoffs were proposed: ciprofloxacin-wild type if the inhibition zone diameter was ≥22 mm or the minimum inhibitory concentration was ≤0.5 mg/L.

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.

Clinical and epidemiological analysis of Campylobacter fetus subsp. fetus infections in humans and comparative genetic analysis with strains isolated from cattle.

BACKGROUND: Campylobacter fetus subspecies fetus (CFF) is an important pathogen for both cattle and humans. We performed a systematic epidemiological and clinical study of patients and evaluated the genetic relatedness of 17 human and 17 bovine CFF isolates by using different genotyping methods. In addition, the serotype, the dissemination of the genomic island containing a type IV secretion system (T4SS) and resistance determinants for tetracycline and streptomycin were also evaluated. METHODS: The isolates from patients diagnosed with CFF infection as well as those from faecal samples of healthy calves were genotyped using pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), as well as single locus sequence typing (SLST) targeting cmp1 and cmp2 genes encoding two major outer membrane proteins in CFF. The presence of the genomic island and identification of serotype was determined by PCRs targeting genes of the T4SS and the sap locus, respectively. Tetracycline and streptomycin resistance phenotypes were determined by minimal inhibitory concentration. Clinical data obtained from medical records and laboratory data were supplemented by data obtained via telephone interviews with the patients and treating physicians. RESULTS: PFGE analysis defined two major clusters; cluster A containing 16 bovine (80 %) isolates and cluster B containing 13 human (92 %) isolates, suggesting a host preference. Further genotypic analysis using MLST, SLST as well as sap and T4SS PCR showed the presence of genotypically identical isolates in cattle and humans. The low diversity observed within the cmp alleles of CFF corroborates the clonal nature of this pathogen. The genomic island containing the tetracycline and streptomycin resistance determinants was found in 55 % of the isolates in cluster A and correlated with phenotypic antibiotic resistance. CONCLUSIONS: Most human and bovine isolates were separated on two phylogenetic clusters. However, several human and bovine isolates were identical by diverse genotyping methods, indicating a possible link between strains from these two hosts.

Presence of Campylobacter and Arcobacter species in in-line milk filters of farms authorized to produce and sell raw milk and of a water buffalo dairy farm in Italy.

The objectives of this study were to investigate the presence of Campylobacter spp. and Arcobacter spp. in dairy herds authorized for the production and sale of raw milk and in a water buffalo dairy farm, and to test the antimicrobial susceptibility of the isolates. A total of 196 in-line milk filters were collected from 14 dairy farms (13 bovine and 1 water buffalo) for detection of Campylobacter spp. and Arcobacter spp. by microbiological culture. For each farm investigated, 1 isolate for each Campylobacter and Arcobacter species isolated was tested using the Etest method (AB Biodisk, Solna, Sweden) to evaluate the susceptibility to ciprofloxacin, tetracycline, chloramphenicol, ampicillin, erythromycin, and gentamicin. A total of 52 isolates were detected in 49 milk filters in 12 farms (85.7%) out of 14 and the isolates were identified as Campylobacter jejuni (6), Campylobacter hyointestinalis ssp. hyointestinalis (8), Campylobacter concisus (1), Campylobacter fetus ssp. fetus (1), Arcobacter butzleri (22), and Arcobacter cryaerophilus (14). The small number of isolates tested for antimicrobial susceptibility precludes any epidemiological consideration but highlights that all Campylobacter isolates were susceptible to macrolides, which are the first-choice drugs for the treatment of campylobacteriosis, and that resistance to fluoroquinolones and tetracycline was detected; for Arcobacter isolates, resistance to ampicillin and chloramphenicol was detected. The sale of raw milk for human consumption by self-service automatic vending machines has been allowed in Italy since 2004 and the presence of C. jejuni in in-line milk filters confirms that raw milk consumption is a significant risk factor for human infection. The high occurrence of emerging Campylobacter spp. and Arcobacter spp. discovered in dairy farms authorized for production and sale of raw milk represents an emerging hazard for human health.

Antimicrobial susceptibility testing of German Campylobacter fetus subsp. venerealis isolates by agar disk diffusion method.

Campylobacter fetus subsp. venerealis is the causative agent of bovine genital campylobacteriosis and is transmitted by asymptomatic carrier bulls via contaminated semen during artificial insemination. The aim of the present study was to determine the in vitro susceptibility of Campylobacter fetus subsp. venerealis isolated from bovine specimens in the years from 2000 to 2009 in Germany to antibiotics generally used in semen treatment. The susceptibilities of 50 strains to spectinomycin (10 microg), gentamicin (10 microg), streptomycin (25 microg), penicillin (10 microg), lincomycin (10 microg), ciprofloxacin (5 microg), erythromycin (30 microg) and tetracycline (30 microg) were determined using a disk diffusion susceptibility test. All strains were susceptible to gentamicin. A considerably reduced susceptibility to one or more antimicrobial agents was detected in seven out of 50 isolates (14%) with the most frequent reduction in susceptibility to lincomycin and spectinomycin. Furthermore, strains with reduced susceptibility to more than one antimicrobial agent were always associated with reduced susceptibility to lincomycin. It is recommended to determine the antimicrobial susceptibility of Campylobacter fetus subsp. venerealis isolates in order to evaluate the efficacy of the generally used antibiotic treatment of bull semen and to detect possible resistances.

Two novel antibiotic resistance genes, tet(44) and ant(6)-Ib, are located within a transferable pathogenicity island in Campylobacter fetus subsp. fetus.

New tetracycline and streptomycin resistance genes, tet(44) and ant(6)-Ib, were identified in Campylobacter fetus subsp. fetus within a transferable pathogenicity island that is typically unique to Campylobacter fetus subsp. venerealis. The 640-amino-acid tetracycline resistance determinant, Tet 44, belongs to a class of proteins that confers resistance to tetracycline and minocycline by ribosomal protection. The 286-amino-acid streptomycin resistance determinant, ANT(6)-Ib, belongs to a family of aminoglycoside nucleotidyltransferases. The resistance phenotypes were demonstrated by gene inactivation and expression.

Anti-Campylobacter, anti-aerobic, and anti-oxidative effects of roselle calyx extract and protocatechuic acid in ground beef.

The inhibitory effect of roselle calyx extract and protocatechuic acid against susceptible and antibiotic-resistant Campylobacter jejuni, C. coli and C. fetus in agar plate and ground beef was examined. The minimal inhibitory concentrations of roselle calyx extract and protocatechuic acid against susceptible and antibiotic-resistant Campylobacter species were in the range of 96-152 and 20-44 microg/ml, respectively. Temperature treatments from 25 to 100 degrees C did not affect the anti-Campylobacter activity of protocatechuic acid. In ground beef stored at 15 degrees C for 6 days, roselle calyx extract and protocatechuic acid inhibited the survival and growth of aerobes, and susceptible and antibiotic-resistant Campylobacter species, in which protocatechuic acid exhibited dose-dependent effect. Both roselle calyx extract and protocatechuic acid decreased lipid oxidation levels in ground beef, in which protocatechuic acid also exhibited dose-dependent effect. The addition of roselle calyx extract or protocatechuic acid did not affect cooking loss, pH value, sensory attributes and content of fat, protein and moisture of beef samples during storage at 4 degrees C for 15 days. These data support that roselle calyx extract and protocatechuic acid may be used for muscle foods to prevent contamination from Campylobacter and aerobes, as well as delay lipid oxidation.

Emergence of a tetracycline-resistant Campylobacter jejuni clone associated with outbreaks of ovine abortion in the United States.

Campylobacter infection is one of the major causes of ovine abortions worldwide. Historically, Campylobacter fetus subsp. fetus was the major cause of Campylobacter-associated abortion in sheep; however, Campylobacter jejuni is increasingly associated with sheep abortions. We examined the species distribution, genotypes, and antimicrobial susceptibilities of abortion-associated Campylobacter isolates obtained from multiple lambing seasons on different farms in Iowa, Idaho, South Dakota, and California. We found that C. jejuni has replaced C. fetus as the predominant Campylobacter species causing sheep abortion in the United States. Most strikingly, the vast majority (66 of 71) of the C. jejuni isolates associated with sheep abortion belong to a single genetic clone, as determined by pulsed-field gel electrophoresis, multilocus sequence typing, and cmp gene (encoding the major outer membrane protein) sequence typing. The in vitro antimicrobial susceptibilities of these isolates to the antibiotics that are routinely used in food animal production were determined using the agar dilution test. All of the 74 isolates were susceptible to tilmicosin, florfenicol, tulathromycin, and enrofloxacin, and 97% were sensitive to tylosin. However, all were resistant to tetracyclines, the only antibiotics currently approved in the United States for the treatment of Campylobacter abortion in sheep. This finding suggests that feeding tetracycline for the prevention of Campylobacter abortions is ineffective and that other antibiotics should be used for the treatment of sheep abortions in the United States. Together, these results indicate that a single tetracycline-resistant C. jejuni clone has emerged as the major cause of Campylobacter-associated sheep abortion in the United States.

Temporal prevalence of antimicrobial resistance in Campylobacter spp. from beef cattle in Alberta feedlots.

Antimicrobial resistance (AMR) was temporally assessed in campylobacters isolated from beef cattle (7,738 fecal samples from 2,622 animals) in four commercial feedlots in Alberta. All calves were administered chlortetracycline and oxytetracycline in feed, and a majority of the animals (93%) were injected with long-acting oxytetracycline upon arrival at the feedlot. Fecal samples from individual animals were collected upon arrival (i.e., entry sample), 69 days (standard deviation [SD] = 3 days) after arrival (i.e., interim sample), and 189 days (SD = 33 days) after arrival (i.e., exit sample) at the feedlot. In total, 1,586 Campylobacter isolates consisting of Campylobacter coli (n = 154), Campylobacter fetus (n = 994), Campylobacter jejuni (n = 431), Campylobacter hyointestinalis (n = 4), and Campylobacter lanienae (n = 3) were recovered and characterized. The administration of antimicrobials did not decrease carriage rates of campylobacters, and minimal resistance (< or =4%) to azithromycin, ciprofloxacin, enrofloxacin, gentamicin, and meropenem was observed. In contrast, substantive increases in the prevalence of isolates resistant to tetracycline and doxycycline (56 to 89%) for C. coli, C. fetus, and C. jejuni, as well as in the number of animals (7 to 42%) from which resistant isolates were recovered, were observed during the feedlot period. Increased resistance to erythromycin (total isolates and carriages rates) was also observed in isolates of C. coli over the three isolation times. The majority of C. fetus isolates recovered were resistant to nalidixic acid, but this was independent of when they were isolated. A relatively limited number of multidrug-resistant isolates were recovered and consisted primarily of C. coli resistant to tetracyclines and erythromycin (10% of isolates). Over the course of the feedlot period, considerable increases in antimicrobial resistance were observed in C. coli, C. fetus, and C. jejuni, but with the exception of erythromycin resistance in C. coli, the administration of antimicrobial agents to beef cattle was found to have a minimal impact on resistance to macrolides and fluoroquinolones, the two classes of antimicrobials used to treat campylobacteriosis in humans. However, the widespread use of antimicrobial agents in beef production and the possible horizontal transfer of mobile genetic elements with antimicrobial resistance determinants among Campylobacter and other bacterial taxa emphasize the need to monitor AMR development in bacteria from beef cattle.

Identification of Campylobacter fetus subspecies by phenotypic differentiation and PCR.

The species Campylobacter fetus is divided into the subspecies C. fetus subsp. venerealis and C. fetus subsp. fetus, which differ in their epidemiologies and clinical importance. The differences between these subspecies make accurate distinction between the two essential. First, the value of seven key tests for the traditional differentiation of C. fetus was investigated. Afterwards, the results of the phenotypic differentiation and PCR were compared to address the question of the reliability of this PCR assay. Altogether, 103 C. fetus isolates were investigated, including the type strains of C. fetus subsp. fetus and C. fetus subsp. venerealis. Depending on the result of the glycine tolerance test, the isolates could be separated into 81 C. fetus subsp. venerealis isolates (glycine intolerant) and 22 C. fetus subsp. fetus isolates (glycine tolerant). For all C. fetus subsp. venerealis strains tested, the results of the selenite reduction assay and sensitivity to metronidazole and cefoperazone completely agreed with the results of the glycine tolerance test (correspondence, 100%). Seventy-three C. fetus subsp. venerealis isolates did not grow at 42 degrees C (correspondence, 90.1%), but eight isolates showed a faintly discernible, flat, dark gray growth. For 22 C. fetus subsp. fetus isolates, the results of additional phenotypic tests only partly agreed with the results of the glycine tolerance test. For C. fetus subsp. fetus the results of the glycine tolerance test showed a relatively good correspondence with those of the selenite reduction assay (correspondence, 81.8%), assays for cefoperazone resistance (correspondence, 86.4%), and assays for growth at 42 degrees C (correspondence, 81.8%). The results of the glycine tolerance test and PCR completely agreed for the 103 C. fetus isolates tested. We conclude that at present the traditional phenotypic characterization of C. fetus subspecies under strongly defined conditions remains indispensable, but this PCR assay constitutes a valuable adjunctive technique for the confirmation of phenotypic results.

Campylobacter fetus of reptile origin as a human pathogen.

A Campylobacter species was isolated from blood from a febrile patient with precursor T-cell acute lymphoblastic leukemia, and after antibiotic treatment, a similar bacterium was isolated from blood 37 days later. Although phenotypic testing did not definitively identify the organisms, molecular analysis indicated that they were the same strain of Campylobacter fetus subsp. fetus and were of reptile origin.

Campylobacter fetus meningitis in a diabetic adult cured by imipenem.

Reported here is the successful management of a severe case of Campylobacter fetus subspecies fetus meningitis, complicated by septic shock, in a patient without overt immunosuppression who was cured by imipenem. Meningitis caused by C. fetus has rarely been reported in adults, and only exceptionally in non-immunocompromised patients, and septic shock has not previously been reported as a complication of such meningitis. The best antibiotic regimen for treating this condition remains to be determined. Imipenem has displayed high in vitro activity against C. fetus but has been used rarely in clinical practice. It was administered in this case with good results.

Epidemiology and antimicrobial susceptibilities of 111 Campylobacter fetus subsp. fetus strains isolated in Québec, Canada, from 1983 to 2000.

The epidemiology and antimicrobial susceptibilities of 111 Campylobacter fetus subsp. fetus strains isolated from 103 patients from 1983 to 2000 in Québec, Canada, were determined. The median number of patients infected annually with this bacteria was seven, with an incidence of 0.1 per 100,000 population. The male-to-female ratio was 1.1 to 1.0. The patients originated from 13 of the 18 Québec socioeconomic regions. The age range of the patients was 6 months to 90 years old, 53% being > or = 70 years old and 2% being <20 years old. The isolation site was blood for 69% of the patients, stools for 20%, and other body fluids for 11% of them. Three patients suffered a relapse, with the same strain being isolated from the same site at different times as confirmed by pulse-field gel electrophoresis. All isolates were susceptible to ampicillin, gentamicin, meropenem, and imipenem, with 90% minimal inhibitory concentrations of 4, 1, 0.12, and < or = 0.06 microg/ml, respectively. Three percent and two percent of the strains were, respectively, resistant and intermediate to ciprofloxacin. Thirty-four percent of the strains were resistant to tetracycline. There was a nonsignificant increase in resistance to ciprofloxacin (P = 0.27) and to tetracycline (P = 0.65) in recent years. The percentages of intermediate and resistant MICs were, respectively, 12 and 1% for cefotaxime and 71 and 0% for erythromycin. All strains were beta-lactamase negative.