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.

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.

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.

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.

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.

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.

Jejunal infection with Campylobacter.

A patient had common variable immunodeficiency, chronic malabsorption, and Campylobacter jejuni infection. Infection was diagnosed by jejunal aspiration. A follow-up jejunal aspirate was culture positive at the same time that a stool culture was negative. Infection resulted in worsening of chronic diarrhea, but it was not associated with clinical features of colitis or proctitis. The duration of infection was prolonged and initial antimicrobial therapy was ineffective. Single drug therapy with erythromycin ethylsuccinate and then chloramphenicol led to the emergence of resistant organisms. After five months of bacterial excretion, combination therapy with metronidazole and neomycin sulfate eliminated the organism. This case emphasizes that the clinical manifestations and response to therapy of C jejuni infection can be altered in immunodeficient patients.

Persistence of multiply antibiotic-resistant Campylobacter jejuni in a patient with the acquired immune deficiency syndrome.

Chronic diarrhea due to Campylobacter jejuni has been described in patients with hypogammaglobulinemia. A patient with the acquired immune deficiency syndrome (AIDS) and normal total serum immunoglobulins had persistent diarrhea and C. jejuni on stool culture for seven and a half months despite repeated antibiotic therapy. Antibiotic sensitivity studies revealed the C. jejuni to be multiply antibiotic-resistant. Evaluation of the mechanism of resistance showed the organism harbored a conjugative plasmid capable of transferring resistance to tetracycline, but not to other antibiotics. It is concluded that C. jejuni infection may rarely result in chronic diarrhea in patients with AIDS. The combination of an immune deficiency state including abnormal B cell function previously described in AIDS and multiple antibiotic resistance may have contributed to the persistence of the organism in this case.

Gram-negative bacillary endocarditis. Interpretation of the serum bactericial test.

Although the serum bactericidal test is commonly used in the management of infective endocarditis, little has been written about its validity or limitations. We report three cases of gram-negative bacillary endocarditis (Pseudomonas aeruginosa, Vibrio fetus and Serratia marcescens) encountered in 1 year at a Veterans Administration hospital. Serum bactericidal titers were considered necessary to identify inadequate antibiotic regimens or to avoid unnecessary drug toxicity. The limitations of the test, particularly those pertaining to gram-negative infections, are reviewed. Misleading results during treatment with aminoglycoside antibiotics could be due to the tendency of serum to become alkaline on standing. A detailed study of the interaction of the complement-dependent bactericidal system of serum with eight antibiotics is presented. In the context of the serum bactericidal test, the interaction was additive or synergistic in 15 of 16 determinations, indicating the need to include a control study of serum sensitivity of the infecting microorganism in each case.

Adherence of Campylobacter jejuni and Campylobacter coli to porcine intestinal brush border membranes.

The adhesion of Campylobacter jejuni and C. coli to isolated porcine intestinal brush border membranes was studied by phase-contrast and electron microscopy. Approximately 45% of the cell population adhered to the brush borders, possibly in a specific manner. Pretreatment of the brush borders with trypsin or pronase, and competitive inhibition with L-rhamnose caused a slight reduction of the adhesion. Different forms of pretreatment of the bacterial cells reduced their ability to adhere, but also their motility.

In-vitro effect of edta-tris-lysozyme solutions on selected pathogenic bacteria.

The in-vitro effect of EDTA-Tris-lysozyme solution on 16 pathogenic bacteria of medical or veterinary importance was determined. Marked decreases in bacterial count occurred with Pseudomonas aeruginosa, Escherichia coli, Moraxella osloensis and Campylobacter fetus, and smaller decreses with Salmonella typhimurium, Shigella boydii, Aeromonas hydrophila, proteus mirabilis, Listeria monocytogenes and Erysipelothrix insidiosa. The test solution had no effect on Klebsiella ozaenae, Brucella canis, Cornynebacterium pyogenes, Coryne, renale, Streptococcus equi and staphylococcus aureus.

A study of Campylobacter fetus subsp. jejuni strains isolated from the stools of diarrhoeic children in Sri Lanka.

Nine strains of Campylobacter fetus subsp. jejuni were isolated from the stools of diarrhoeic children in Colombo (Sri Lanka). The morphology of most of the strains was typical but three strains produced a spreading film of growth. No discrete domed colonies were seen. All were able to grow in a candle jar, and in a medium containing 1% glycine. All produced hydrogen sulphide. All were susceptible to ampicillin, carbenicillin, tetracycline, chloramphenicol, neomycin, erythromycin, gentamicin and furoxone .

The use of membrane filters applied directly to the surface of agar plates for the isolation of Campylobacter jejuni from feces.

Cellulose triacetate membrane filters applied directly to the surface of non-selective blood agar plates were found to be as effective as the use of antibiotic media in isolating Campylobacter jejuni from patients with diarrhea. This method was used in parallel with selective media in the examination of 1000 specimens of feces. Campylobacters were isolated from 56 specimens using all methods. The membrane filter method detected 50 (89%), 45 of which were C. jejuni, and selective media 45 strains of C. jejuni (80%). Membrane filters used in this way can result in the detection of most cases of campylobacter enteric infection and can be used by small laboratories with limited access to selective media. They may also facilitate the isolation of antibiotic sensitive campylobacters.

In vitro antibacterial activity of enoxacin (CI-919).

Antibacterial activity of enoxacin was evaluated against more than 3,700 clinical isolates using the agar-dilution method and an inoculum of 10(4)-10(5) cells per site. For comparison other antibiotics appropriate for each species were also included. For most enterobacteria and for Staphylococcus aureus, Staphylococcus epidermidis and Pseudomonas aeruginosa, the MIC90 of enoxacin was below 2 mg/l. Serratia marcescens was more resistant; the MIC90 being 4 mg/ml. Enoxacin also showed high activity against Campylobacter jejuni and Neisseria gonorrhoeae. Streptococci were comparatively resistant, 32 mg/l to 64 mg/l of the compound being required to inhibit 90% of strains.

Antibacterial effect of the glucose oxidase-glucose system on food-poisoning organisms.

The antibacterial effect of the glucose oxidase-glucose system was studied on food-poisoning organisms including Staphylococcus aureus, Salmonella infantis, Clostridium perfringens, Bacillus cereus, Campylobacter jejuni, Listeria monocytogenes and Yersinia enterocolitica using automated turbidometry. The bacteria were grown in sterile-filtered meat medium which was either raw or heat-denaturated. The results showed a clear growth inhibition with combinations of 0.5-1.0 mg/ml glucose and 0.5-1.0 IU/ml glucose oxidase. The growth inhibition was more effective in the heat-denaturated meat medium. The most resistant pathogens were Campylobacter jejuni and Listeria monocytogenes, however growth inhibition was still evident. The possible application of the glucose oxidase-glucose system in food products inhibiting the growth of pathogens and spoilage organisms is discussed.

Studies on inactivation of pathogenic microorganisms in culture media and in bovine semen by photosensitive agents.

The application of three photosensitive agents for disinfection of bovine semen was investigated. Bovine microbial pathogens suspended in tissue culture medium and/or PBS and also added to bovine semen were exposed to the photosensitive agents followed by irradiation. Hematoporphyrin, hematoporphyrin derivative and thiopyronine were effective against bovine herpes virus-1, bovine viral diarrhoea virus, Mycoplasma bovigenitalium, Mycoplasma canadense, and Ureaplasma diversum in culture media. In addition, thiopyronine was effective against Leptospira pomona. Similar treatments were not effective against Leptospira hardjo, Mycoplasma bovis, or Campylobacter fetus subsp. venerealis. When microorganisms were added to bovine semen, only bovine herpes virus-1 was controlled by the photosensitive agents used at concentrations which did not appear harmful to sperm cells.