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Listeria monocytogeneshas been reported to cause various infectious diseases in both humans and animals. More rarely, ocular infections have been reported. To our knowledge, only two cases of Listeriakeratitis have been described in horses. We report kerato-conjunctivitis in four Norwegian horses associated with L. monocytogenes. Clinically, all cases were presented with recurrent unilateral kerato-conjunctivitis. L. monocytogenesbacteria were isolated from swab samples from all cases, and cytology carried out in 3 cases was indicative of L. monocytogenesinfection. The present report describes the first known cases in which L. monocytogeneshas been isolated from keratitic lesions in horses in Norway. A potential risk factor may be feeding of silage or haylage, but other sources of infection cannot be ruled out. The phenotypic features including antimicrobial susceptibility and serotype of the isolates are described. Laboratory detection of L. monocytogenesdemands extra caution since only low numbers of bacteria were detected in the eye-swabs, probably due to the low volume of sample material and the intracellular niche of the bacterium. A general poor response to treatment in all these cases indicates that clinicians should pay extra attention to intensity and duration of treatment if L. monocytogenesis identified in connection with equine kerato-conjunctivitis.

AbstractThe zebrafish (Danio rerio) is an increasingly used laboratory animal model in basic biology and biomedicine, novel drug development, and toxicology. The wide use has increased the demand for optimized husbandry protocols to ensure animal health care and welfare. The knowledge about the correlation between culturable zebrafish intestinal microbiota and health in relation to environmental factors and management procedures is very limited. A semi-quantitative level of growth of individual types of bacteria was determined and associated with sampling points. A total of 72 TAB line zebrafish from four laboratories (Labs A–D) in the Zebrafish Network Norway were used. Diagnostic was based on traditional bacterial culture methods and biochemical characterization using commercial kits, followed by 16S rDNA gene sequencing from pure subcultures. Also selected Gram-negative isolates were analyzed for antibiotic susceptibility to 8 different antibiotics. A total of 13 morphologically different bacterial species were the most prevalent: Aeromonas hydrophila, Aeromonas sobria, Vibrio parahaemolyticus, Photobacterium damselae, Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonas luteola, Comamonas testosteroni, Ochrobactrum anthropi, Staphylococcus cohnii, Staphylococcus epidermidis, Staphylococcus capitis, and Staphylococcus warneri.Only Lab B had significantly higher levels of total bacterial growth (OR=2.03), whereas numbers from Lab C (OR=1.01) and Lab D (OR=1.12) were found to be similar to the baseline Lab A. Sexually immature individuals had a significantly higher level of harvested total bacterial growth than mature fish (OR=0.82), no statistically significant differences were found between male and female fish (OR=1.01), and the posterior intestinal segment demonstrated a higher degree of culturable bacteria than the anterior segment (OR=4.1). Multiple antibiotic (>3) resistance was observed in 17% of the strains. We propose that a rapid conventional diagnostic bacteriological assay on the culturable microbiota profiles can be designed and used as quality measure of the husbandry routines of a zebrafish facility to ensure a bacterial standard safeguarding the zebrafish health and welfare.

ABSTRACTVibrio(Aliivibrio) salmonicidais the causal agent of cold-water vibriosis, a fatal bacterial septicemia primarily of farmed salmonid fish. The molecular mechanisms of invasion, colonization, and growth of V. salmonicidain the host are still largely unknown, and few virulence factors have been identified. Quorum sensing (QS) is a cell-to-cell communication system known to regulate virulence and other activities in several bacterial species. The genome of V. salmonicidaLFI1238 encodes products presumably involved in several QS systems. In this study, the gene encoding LitR, a homolog of the master regulator of QS in V. fischeri, was deleted. Compared to the parental strain, the litRmutant showed increased motility, adhesion, cell-to-cell aggregation, and biofilm formation. Furthermore, the litRmutant produced less cryptic bioluminescence, whereas production of acylhomoserine lactones was unaffected. Our results also indicate a salinity-sensitive regulation of LitR. Finally, reduced mortality was observed in Atlantic salmon infected with the litRmutant, implying that the fish were more susceptible to infection with the wild type than with the mutant strain. We hypothesize that LitR inhibits biofilm formation and favors planktonic growth, with the latter being more adapted for pathogenesis in the fish host.

Vibrio (Aliivibrio) salmonicida is the causal agent of cold-water vibriosis, a fatal bacterial septicemia primarily of farmed salmonid fish. The molecular mechanisms of invasion, colonization, and growth of V. salmonicida in the host are still largely unknown, and few virulence factors have been identified. Quorum sensing (QS) is a cell-to-cell communication system known to regulate virulence and other activities in several bacterial species. The genome of V. salmonicida LFI1238 encodes products presumably involved in several QS systems. In this study, the gene encoding LitR, a homolog of the master regulator of QS in V. fischeri, was deleted. Compared to the parental strain, the litR mutant showed increased motility, adhesion, cell-to-cell aggregation, and biofilm formation. Furthermore, the litR mutant produced less cryptic bioluminescence, whereas production of acylhomoserine lactones was unaffected. Our results also indicate a salinity-sensitive regulation of LitR. Finally, reduced mortality was observed in Atlantic salmon infected with the litR mutant, implying that the fish were more susceptible to infection with the wild type than with the mutant strain. We hypothesize that LitR inhibits biofilm formation and favors planktonic growth, with the latter being more adapted for pathogenesis in the fish host.

ABSTRACTA 45-kb R plasmid, pRAS1, that confers resistance to tetracyclines, trimethoprim, and sulfonamides was isolated in 1989 from an atypical strain of the fish pathogen Aeromonas salmonicida. This plasmid could be transferred by conjugation to Escherichia coliwith a high degree of efficiency (frequency, 0.48). The following year pRAS1 was isolated from A. salmonicidasubsp. salmonicidain the same area. Incompatibility group U plasmid pRAS1 contained a drug resistance-determining region of 12 kb consisting of a class 1 integron similar to In4 of Tn1696but with a dfrA16gene cassette inserted. Close to IS6100at the right end of Tn4 was a truncated Tn1721. Restriction enzyme analysis showed that R plasmid pAr-32, isolated from A. salmonicidain Japan in 1970, had the same backbone structure as pRAS1, while the drug resistance-determining region contained a complex class 1 integron with an aadA2cassette; the chloramphenicol resistance gene catA2, as in In6 of pSa; and a duplicate of the 3′ conserved segment of the integron.

Microbial resistance to antimicrobial agents continues to be a major problem. The frequent use and misuse of disinfectants based on quaternary ammonium compounds (QACs) in food-processing industries have imposed a selective pressure and may contribute to the emergence of disinfectant-resistant microorganisms. A total number of 1,325 Gram-negative isolates (Escherichia coli, other coliforms Vibrio spp., and Aeromonas spp.) and 500 Enterococcus spp. from food and food-processing industries and fish farming were screened for natural resistance to the QAC-based disinfectant benzalkonium chloride (BC). Of the 1,825 isolates, 16 strains, mainly from meat retail shops, showed low-level resistance to BC. None of the Enterococcus spp. from broiler, cattle, and pigs, the antibiotic-resistant E. coli from pig intestine and fish pathogens Vibrio spp. and Aeromonas spp. from the Norwegian fish farming industry were resistant to BC. The BC-resistant strains were examined for susceptibility to 15 different antibiotics, disinfectants, and dyes. No systematic cross-resistance between BC and any of the other antimicrobial agents tested was detected. Stable enhanced resistance in Enterobacter cloacae isolates was demonstrated by step-wise adaptation in increasing concentrations of BC. In conclusion, BC resistance among food-associated Gram-negative bacteria and Enterococcus spp. is not frequent, but resistance may develop to user concentrations after exposure to sublethal concentrations of BC.

Little is known about the occurrence of antimicrobial resistance determinants in staphylococci isolated from food and food processing industries. Quaternary ammonium compound (QAC)-resistant coagulase-negative staphylococci (CNS) isolated from food and food-processing industries were investigated for the presence of genetic determinants (qacA/B and qacC/smr) encoding resistance to the QAC benzalkonium chloride (BC), several antibiotic resistance genes, and staphylococcal insertion sequences IS257 and IS256. Six qacA/B-harboring strains were resistant to penicillin and hybridized to a blaZ probe. The qacA/B and blaZ probes hybridized to plasmids of similar size in three isolates. Molecular and genetic characterization of the 23-kb plasmid (pST6) of Staphylococcus epidermidis St.6 revealed the presence of qacB adjacent to an incomplete β-lactamase transposon Tn552 encoding the gene cluster blaZ, blaR, and blaI. Sequence analysis of flanking regions and the intergenic region between blaZ and qacB revealed the presence of IS257 downstream of blaZ as well as sin and binR between blaZ and qacB. In the three other BC and penicillin-resistant strains, the qacA/B and blaZ genes were located on separate plasmids. A qacC harboring S. epidermidis strain (St.17) also hybridized to tetK (tetracycline resistance) and ermB (erythromycin resistance) genes. The individual genes were located on separate plasmids, suggesting no linkage between QAC and antibiotic resistance determinants. Plasmid-free Staphylococcus aureus RN4220 allowed uptake of the pST6 plasmid DNA, indicating that the resistance genes could potentially be transferred to pathogens under selective stress. In conclusion, presence of both resistance determinants could lead to co-selection during antimicrobial therapy or disinfection in hospitals or in food industries.

ABSTRACTA rapid method for demonstration of gram-positive and gram-negative bacteria in milk is described. The technique is based on dilution of the sample in a medium, followed by filtration through a porous polysulfone membrane with a pore size retaining and concentrating bacteria from the sample. The bacteria concentrated on the surface of the membrane are stained with a cationic dye (toluidine blue) that can be visualized by the naked eye. After staining, the membrane is treated with ethanol-acetic acid (pH 2.8 to 3.0), which causes decolorization of gram-negative bacteria, whereas gram-positive bacteria retain the stain. The method does not require heat fixation, electrical power, microscopic examination, or specially trained personnel. The time needed to perform the test is approximately 5 min. The technique was applied to artificially infected milk and milk from cows with moderate or severe clinical mastitis for detection and differentiation of bacteria. The sensitivity of the filtration method was 92 and 100% for gram-positive and gram-negative bacteria, respectively, compared with traditional bacteriological culture of milk samples. The detection limit was 5 × 106CFU/ml for Staphylococcus aureusand 1 × 106CFU/ml forEscherichia coliin spiked milk samples. The overall specificity of the method was 86%. This diagnostic method can provide on-site guidance to the veterinarian to optimize use of antibiotics in mastitis therapy.

The presence of class 1 integrons was investigated in 38 sulfonamide-resistant strains of Aeromonas salmonicida subsp. salmonicida, atypical A. salmonicida and Escherichia coli conjugants with R plasmids originating from A. salmonicida. The strains originated from Finland, France, Japan, Norway, Scotland, Switzerland, and the United States. Additional resistance determinants in strains with class 1 integrons were also determined. Of 21 strains containing a class 1 integron, 19 had a single gene cassette, 1 strain had two cassettes, and 1 strain was found to lack an integrated gene cassette. In the integrons with single cassettes, aadA2 was present in eight strains, dfr16 in five strains, and aadA1 and dfrIIc in three strains each. In the integron with two cassettes, qacG and orfD were present. Tetracycline resistance was observed in 20 of the integron-positive strains, caused by the determinants Tet A and Tet E, in which Tet A frequently was associated with Tn1721. Class 1 integrons seem to be important in mediating antibiotic resistance also in the marine environment. The gene cassettes reported in this study are all described in bacteria associated with humans, and this demonstrates once more how the common gene pool is shared between organisms belonging to different environments.

The resistance genes and their surroundings on three self-transmissible plasmids found in Escherichia coli of the enteric normal flora of healthy pigs have been characterized. The resistance elements found are similar to those commonly found in clinical isolates, like the transposon Tn1721 including the Tet A tetracycline resistance determinant, Tn10 with the Tet B determinant, Tn21 including a class 1 integron with the aadA1a cassette inserted, sulII encoding sulfonamide resistance, and the strA-strB genes responsible for streptomycin resistance. The plasmids were able to mobilize into various recipients, including swine pathogens, zoonotic bacteria, and commensals when conjugation experiments were carried out. Transfer of plasmids did not require optimal conditions concerning nutrition and temperature as plasmids were transferred in 0.9% saline at room temperature, suggesting that in vivo transfer might be possible. This study shows that transferable resistance elements appearing in normal flora bacteria from animals are similar to those commonly found in clinical isolates of human origin. The results indicate a probable communication between pathogens and the normal flora with respect to exchange of resistance factors.

Abstract: The recent discovery that the fish pathogen Vibrio salmonicida is closely related to the luminous bacteria Vibrio fischeri and Vibrio logei suggested that V. salmonicida might also be capable of bioluminescence. Interestingly, cells of V. salmonicida were found to produce light in culture, but only when exposed to either an aliphatic aldehyde and/or the major V. fischeri autoinducer N-(3-oxo-hexanoyl)-l-homoserine lactone, a transcriptional activator of the luminescence (lux) genes. An extract of spent medium of V. salmonicida that should contain any V. salmonicida acyl-homoserine lactone autoinducer, when added to V. fischeri cells, led to an induction of their luminescence. These results show that V. salmonicida is a newly recognized luminous bacterial species that apparently both produces an autoinducer activity and responds to exogenous V. fischeri autoinducer.

Yersinia ruckeriproduces an antibacterial factor which inhibits the growth of a wide spectrum of Gram-negative and Gram-positive bacteria, though not other strains of Y. ruckeri.The antibacterial factor was produced at low temperatures (4–20°C), but not at 37°C. The activity was lost after treatment of the supernatant with chloroform, UV-light and after boiling of the supernatant. One did not succeed in obtaining the antibacterial factor in a sterile solution.

Vibrio salmonicidacultures held in dialysis tubing containers were implanted in the peritoneal cavity of rainbow trout (Oncorhynchus mykiss) for periods of between 2 and 8 days. Implants were inoculated with 1×109cfu. Bacteria within the implants were found to replicate rapidly during the initial implantation period (1·05×1010cfu after 4 days), stabilise in number, then decrease (minimum 4·8×107cfu after 8 days). Escaping bacteria resulted in septicaemic conditions in those fish implanted for 7 days or longer. Bacterial outer membrane protein expression was compared before and after implantation using SDS-PAGE. Expression of three proteins of approximately 99, 90 and 78kDa, was up-regulated underin vivoconditions byV. salmonicida. Ultrastructural studies using transmission electron microscopy and ruthinium red containing fixatives suggest thatV. salmonicidadoes not produce a capsulein vivo. The results are discussed in relation to vaccine development.

Objectives: The aim of this study was to determine the occurrence of fusidic acid resistance, mediated by the fusB gene, among coagulase-negative staphylococci (CoNS) isolated from bovine mastitis.Methods: A total of 113 CoNS isolates were screened for susceptibility to fusidic acid by using a disc diffusion method. The fusB gene was detected by using PCR and subsequent DNA sequencing. The localization of fusB was determined by hybridization.Results: The fusB gene was detected in 3 of 11 fusidic acid-resistant bovine CoNS isolates. The organization of the fusB downstream region on a 40 kb plasmid in a Staphylococcus haemolyticus isolate (288/96) was highly similar to the previously reported organization of fusB on plasmid pUB101 from a Staphylococcus aureus isolate of human origin. The fusB gene was chromosomally located in the remaining two isolates.Conclusions: Fusidic acid resistance mediated by fusB is not the dominant resistance mechanism in fusidic acid-resistant CoNS studied in this work. The similarity between the organization of the fusB downstream region in S. haemolyticus (isolate 288/96) and on plasmid pUB101 from an S. aureus isolate of human origin indicates a common ancestral origin of these genes.

Antibiotic resistance in bacteria is an emerging problem globally. Resistant bacteria are found in human and animal microbiota, as well as in the environment. Wastewater receives bacteria from all these sources and thus can provide a measurement of abundance and diversity of antibiotic-resistant bacteria circulating in communities. In this study, water samples were collected from a wastewater pump station in a Norwegian suburban community over a period of 15 months.

In spite of yearly vaccination, outbreaks of canine infectious respiratory disease are periodically seen amongst domestic dogs. These infections compromise host defense mechanisms, and, when combined with other stressful events, allow opportunistic pathogens like Streptococcus equisubsp. zooepidemicusto create serious disease. Early recognition and treatment are tremendously important for a successful outcome in these cases. A polyvalent vaccine was given to 22 racing dogs three days after a competition, followed by two days of rest, and then the dogs were returned to regular training. Coughing was noticed among the dogs four days after immunisation. Three days after this outbreak one of the dogs was unusually silent and was found dead the next morning. Simultaneously two other dogs developed haemorrhagic expectorate, depression and dyspnea and were brought in to the veterinary hospital. Streptococcus equisubsp. zooepidemicuswas isolated in pure culture from all three cases. They were treated and rehabilitated successfully, and won a sledge race three months later. This paper discusses the necropsy results, treatment regime, rehabilitation and the chronology of vaccination, stressful events and disease.