Jens Laurits Larsen

Occurrence of antimicrobial resistance in fish-pathogenic and environmental bacteria associated with four danish rainbow trout farms.

ABSTRACT Surveillance of bacterial susceptibility to five antimicrobial agents was performed during a 1-year period in and around four freshwater fish farms situated along a stream in western Denmark. Besides assessing the levels of antibiotic resistance among the culturable fraction of microorganisms in fish, water, and sediment samples, two major fish pathogens (88Flavobacterium psychrophilum isolates and 134Yersinia ruckeri isolates) and 313 motileAeromonas isolates, representing a group of ubiquitous aquatic bacteria, were isolated from the same samples. MICs were obtained applying a standardized agar dilution method. A markedly decreased susceptibility of F. psychrophilum isolates to most antimicrobial agents presently available for use in Danish aquaculture was detected, while the collected Y. ruckeriisolates remained largely sensitive to all therapeutic substances. Comparing the inlet and outlet samples, the increase of the antibiotic-resistant proportions observed among the culturable microflora was more pronounced and statistically significant among the motile aeromonads. High levels of individual and multiple antimicrobial resistances were demonstrated within the collected flavobacteria and aeromonads, thus indicating a substantial impact of fish farming on several groups of bacteria associated with aquacultural environments.

Incidence, Distribution, and Spread of Tetracycline Resistance Determinants and Integron-Associated Antibiotic Resistance Genes among Motile Aeromonads from a Fish Farming Environment

ABSTRACT A collection of 313 motile aeromonads isolated at Danish rainbow trout farms was analyzed to identify some of the genes involved in high levels of antimicrobial resistance found in a previous field trial (A. S. Schmidt, M. S. Bruun, I. Dalsgaard, K. Pedersen, and J. L. Larsen, Appl. Environ. Microbiol. 66:4908–4915, 2000), the predominant resistance phenotype (37%) being a combined oxytetracycline (OTC) and sulphadiazine/trimethoprim resistance. Combined sulphonamide/trimethoprim resistance (135 isolates) appeared closely related to the presence of a class 1 integron (141 strains). Among the isolates containing integrons, four different combinations of integrated resistance gene cassettes occurred, in all cases including a dihydrofolate reductase gene and a downstream aminoglycoside resistance insert (87 isolates) and occasionally an additional chloramphenicol resistance gene cassette (31 isolates). In addition, 23 isolates had “empty” integrons without inserted gene cassettes. As far as OTC resistance was concerned, only 66 (30%) out of 216 resistant aeromonads could be assigned to resistance determinant class A (19 isolates), D (n = 6), or E (n = 39); three isolates contained two tetracycline resistance determinants (AD, AE, and DE). Forty OTC-resistant isolates containing large plasmids were selected as donors in a conjugation assay, 27 of which also contained a class 1 integron. Out of 17 successful R-plasmid transfers to Escherichia coli recipients, the respective integrons were cotransferred along with the tetracycline resistance determinants in 15 matings. Transconjugants were predominantly tetApositive (10 of 17) and contained class 1 integrons with two or more inserted antibiotic resistance genes. While there appeared to be a positive correlation between conjugative R-plasmids andtetA among the OTC-resistant aeromonads, tetEand the unclassified OTC resistance genes as well as class 1 integrons were equally distributed among isolates with and without plasmids. These findings indicate the implication of other mechanisms of gene transfer besides plasmid transfer in the dissemination of antibiotic resistance among environmental motile aeromonads.

An inhibitor of bacterial quorum sensing reduces mortalities caused by Vibriosis in rainbow trout (Oncorhynchus mykiss, Walbaum).

The fish pathogen Vibrio anguillarum produces quorum sensing signal molecules, N-acyl homoserine lactones (AHLs), which in several Gram-negative human and plant pathogenic bacteria regulate virulence factors. Expression of these factors can be blocked using specific quorum-sensing inhibitors (QSIs). The purpose of this study was to investigate the effect of a QSI, furanone C-30, on mortality of rainbow trout during challenge with V. anguillarum. Addition of 0.01 or 0.1 microM furanone C-30 to rainbow trout infected by cohabitation caused a significant reduction in accumulated mortality from 80-100% in challenge controls to 4-40% in treated groups. Furanone C-30 had no effect in an immersion challenge system, probably due to a very high water exchange and a rapid dilution of furanone C-30. Growth and survival of V. anguillarum were not affected by the concentrations of furanone C-30 used in the challenge experiments, thus avoiding selection for resistance. To elucidate the mechanism of disease control by furanone C-30, we determined its effect on the bacterial proteome, motility, and respiration. No effects were seen of furanone C-30 in any of these experiments. Although no cytotoxic effect on HeLa cells were observed, exposure to 1 microM (or higher) concentrations of furanone C-30 had detrimental effects on the rainbow trout. Our results indicate that QSIs can be used in non-antibiotic based control of fish diseases. However, they also underline the need for development of novel, less toxic QSI compounds and the need for understanding the exact mechanism(s) of action.

Taxonomic evidence that Vibrio carchariae Grimes et al. 1985 is a junior synonym of Vibrio harveyi (Johnson and Shunk 1936) Baumann et al, 1981.

A collection of 94 Vibrio isolates closely related to Vibrio harveyi, together with named reference and type strains, were investigated for phenotypic and genotypic properties. Using amplified fragment length polymorphism (AFLP), nine clusters were recognized. The largest cluster (n = 36), considered to be the bona fide V. harveyi group, contained the type strains of V. harveyi and Vibrio carchariae and most of the strains isolated from fish. The type strains of all other species, including Vibrio alginolyticus, Vibrio parahaemolyticus, Vibrio campbellii and Vibrio natriegens, clustered outside this group. By ribotyping, V. harveyi and V. carchariae patterns were very similar, insofar as they shared most bands. The V. campbellii type strain had several bands in common with the type strains of both V. harveyi and V. carchariae, whereas the other species were clearly distinct from these three species. DNA-DNA hybridization experiments showed 88% DNA binding between the type strains of V. harveyi and V. carchariae, whereas the DNA binding between V. harveyi and V. campbellii was 40%. Although the delineation of the species V. harveyi is still uncertain, the authors propose, on the basis of a number of tests, to delineate a core of V. harveyi strains which contained the type strains of both V. harveyi and V. carchariae. It is concluded that V. carchariae is the junior synonym of V. harveyi.

Extended Serotyping Scheme for Vibrio anguillarum with the Definition and Characterization of Seven Provisional O-Serogroups

Abstract. The present paper summarizes the serotyping scheme of the fish pathogenic bacterium Vibrio anguillarum and defines seven additional O-serogroups. Strains, collected in our laboratories that were nontypable with antisera against the previously defined 16 O-serotypes, were used for generating new antisera and were characterized further by means of LPS profiles, Western blots, and serological reactions. On the basis of the results, it is suggested that the seven new O-serogroups are to be included in the existing serotyping system as serotypes O17–O23. However, the existence of further V. anguillarum strains that were not typable with any of the 23 O-antisera suggested the existence of additional O-serotypes within this species. The relevance of the description of additional O-serotypes for the species V. anguillarum is discussed.

Involvement of a sialic acid-binding lectin with hemagglutination and hydrophobicity of Flavobacterium psychrophilum.

ABSTRACT Strains of Flavobacterium psychrophilum were studied for their ability to adhere and cause agglutination of erythrocytes and yeast cells. Strains of the serotype Th showed low or no hemagglutinating (HA) properties toward human, avian, bovine, and rainbow trout erythrocytes, whereas strains of serotype Fd and FpT exhibited distinct HA properties. None of the strains was able to cause agglutination of yeast cells. Greater adherence specificity toward rainbow trout blood cells was seen for the HA-positive strains. Growth at 5°C, compared to that at 15°C, induced an increase in the hemagglutination of some strains. HA activities of F. psychrophilum were inhibited only by sialic acid (N-acetyl-neuraminic acid), heat treatment at 65°C, and proteinase K treatment and not by any of seven other carbohydrates, periodate oxidation, or treatment with trypsin. The supernatant from washed bacterial cells also showed some HA properties. All strains were shown to be highly hydrophobic by the hydrophobic interaction chromatography test, although some contradictions to the results of the salt aggregation test (showing some strains as less hydrophobic) were seen. These results indicate that the aggregation of F. psychrophilum and erythrocytes depend on a lectin present on the surface of HA-positive F. psychrophilum strains and absent on HA-negative strains. This lectin reacts specifically with sialic acid. The adhesion differences observed for F. psychrophilum strains do not appear to correlate with the virulence but still provide insights into the interaction of F. psychrophilum and rainbow trout.

Vibrio vulnificus produces quorum sensing signals of the AHL-class

Vibrio vulnificus is an aquatic pathogenic bacterium that can cause vibriosis in humans and fish. The species is subdivided into three biotypes with the fish-virulent strains belonging to biotype 2. The quorum sensing (QS) phenomenon mediated by furanosyl borate diester or autoinducer 2 (AI-2) has been described in human strains of biotype 1, and here we show that the luxS gene which encodes AI-2 is present in all strains of V. vulnificus regardless of origin, biotype or serovar. In this study, we also demonstrate that V. vulnificus produces QS signals of the acylated homoserine lactone (AHL) class (AI-1). AHLs were detected in strains of biotype 1 and 2 from water, fish and human wound infections but not in strains isolated from human septicaemic cases. The AHL compound was identified as N-butanoyl-homoserine-lactone (C(4)-HL) by both reporter strains and by HPLC-high-resolution MS. C(4)-HL was detected when AHL-positive strains were grown in low-nutrient medium [modified sea water yeast extract (MSWYE)] but not in rich media (tryptic soy broth or brain-heart infusion) and its production was enhanced when blood factors were added to MSWYE. C(4)-HL was detected in vivo, in eels infected with AHL-positive biotype 2 strains. No known AHL-related gene was detected by PCR or Southern blot suggesting that AHL-related genes in V. vulnificus are different from those found in other Gram-negative bacteria.

Conjugal Transfer of Large Plasmids Conferring Oxytetracycline (OTC) Resistance: Transfer between Environmental Aeromonads, Fish-Pathogenic Bacteria, and Escherichia coli

Abstract The ability of environmental, motile Aeromonas species and fish-pathogenic bacteria to transfer oxytetracycline resistance to various bacterial species was assessed in vitro and in situ. Oxytetracycline-resistant motile aeromonads and A. salmonicida with large plasmids were used as donors; the recipients were a rifampicin-resistant Escherichia coli, the fish pathogens Yersinia ruckeri and Flavobacterium psychrophilum, a Pseudomonas putida strain, and motile aeromonads originating from aquatic environments. Transfers of oxytetracycline resistance were seen in both laboratory and environmental settings. In vitro transfers from motile Aeromonas spp. to E. coli using filter mating yielded transfer frequencies ranging from 1.4 × 10−9 to 6.8 × 10−5 transconjugants per recipient. Tetracycline resistance determinants A and D and unidentified tetracycline resistance determinants were transferred, usually on plasmids of approximately 150 kilo-base-pairs in size. In vitro transfer from motile Aeromonas spp....

Immunological reactivity of Vibrio anguillarum sero-subgroups O2a and O2b, and comparison of their lipopolysaccharide profiles.

Abstract. One hundred and twenty-nine Vibrio anguillarum serogroup O2 strains were compared by slide agglutination and Western blotting for their lipopolysaccharide (LPS) structure. The strains showed six different LPS profiles, four different reaction patterns in Western blotting, and four different kinds of reaction in slide agglutination, when both unabsorbed and absorbed anti-O2a and anti-O2b sera were used. All in all, nine different groups were detected when the combination of these three methods was applied. The two serological methods gave corresponding results for almost all strains (96%). Most of these strains (84%) belonged to sero-subgroup O2a, while 12% of the strains belonged to sero-subgroup O2b. The remaining six strains had varying reactions in the used serological methods; therefore, their sero-subgroups could not be determined. These results suggest the existence of additional sero-subgroups within serogroup O2.

Ribotypes ofVibrio anguillarum O1 from Italy and Greece

Thirty-one strains ofVibrio anguillarum serogroup O1 isolated in Italy and Greece from dead fish were subjected to rRNA gene restriction pattern analysis. WithHindIII as the restriction enzyme, two different ribotypes were detected, of which one profile was dominant. One profile was found in all strains except one isolated from sea bass, sea bream, and mullet, while the second profile was found in all three strains isolated from rainbow trout and in one strain from a sea bass. WithEcoRI only one profile was found.