Photobacterium damselae piscicida

Transcription

1 Aquaculture Sci Photobacterium damselae piscicida 1, Antimicrobial Susceptibility and Molecular Diversity of Photobacterium damselae subsp. piscicida Isolated in Japan from 2003 to 2009 Manabu FURUSHITA 1,, Tsubasa FUKUDA 1, Yutaka FUKUDA 2, Azumi YAMASHITA 3, Soetsu YANAGI 4, Kouji MAENO 4, Shinji TANAKA 5, Yukitaka SUGIHARA 6, Masaaki ABE 7, Taizo NAGANO 7 and Tsuneo SHIBA 1 Abstract: Susceptibility to 14 antimicrobials was examined for 247 Photobacterium damselae subsp. piscicida isolates from the several species of genus Seriola cultured in Japan from 2003 to The isolates showed resistance to at least one of 10 antimicrobials (ampicillin (ABPC), oxytetracycline (OTC), chloramphenicol (CP), kanamycin (KM), dihydrostreptomycin (DSM), flumequin (FMQ), oxolinic acid (OA), sulfamonomethoxine (SMMX), trimethoprim (TMP), fosfomycin (FOM)), whereas the multiple resistance to OTC, CP, KM, FMQ, OA and SMMX was shown by 55.5% of the isolates. All of the isolates were susceptible to florfenicol (FF), gentamycin (GM), bicozamycin (BCM) and enrofloxacin (ERFX). Random amplified polymorphic DNA (RAPD) patterns of the isolates were classified into 5 patterns (P2 P6). An emergence of a new predominant multiple resistance pattern (ABPC OTC DSM FMQ SMMX TMP FOM) was correlated with the change of the predominant RAPD pattern from P2 to P4. Key words: Seriola; Photobacterium damselae subsp. piscicida; Antimicrobial susceptibility; RAPD Department of Food Science and Technology, National Fisheries Univ., Nagata-honmachi, Shimonoseki, Yamaguchi , Japan 2 Fisheries Research Division, Oita Prefectural Agriculture, Forestry and Fisheries Research Center, Saiki, Oita , Japan 3 Center for Marine Studies, Ehime Prefectural Research Institute for Agriculture, Forestry and Fisheries, Shitaba, Uwajima, Ehime , Japan 4 Kagoshima Prefectural Fisheries Technology and Development Center, ,Ibusuki, Kagoshima , Japan 5 Mie Prefecture Fisheries Research Institute, Hamajima, Shima, Mie , Japan 6 Nagasaki Prefectural Institute of Fisheries, Taira, Nagasaki , Japan 7 Kagawa Prefectural Fisheries Experimental Station, Yashimahigashimachi Takamatsu, Kagawa , Japan Corresponding author : Tel, (+81) ; FAX, (+81) ; , furushita@fish-u.ac.jp M. Furushita

2 Photobacterium damselae subsp. piscicida 1963 white perch Morone americana striped bass M. saxatilis Snieszko et al sea bass Dicentrarchus labrax Balebona et al gilthead sea bream Sparus aurata Toranzo et al cobia Rachycentron canadum Lopez et al Seriola quinqueradiata 1970; 1972 Kusuda and Kawai % Aoki and Kitao 1985; Kim and Aoki 1993; Thyssen and Ollevier 2001; Bakopoulos et al. 2005; Kawanishi et al. 2006; Martínez-Manzanares et al P. damselae subsp. piscicida P. damselae subsp. piscicida Magariños et al P. damselae subsp. piscicida DNA RAPD Random amplified polymorphic DNA Magariños et al. 2000; Kawanishi et al. 2006; Kijima-Tanaka et al Seriola quinqueradiata, S. lalandi, S. dumerili P. damselae subsp. piscicida 247Table 1 1% NaCl Brain-Heart Infusion Agar BHIA, Beckton Dickinson 25Zappulli et al P. damselae subsp. piscicida PCR-RFLP 1% NaCl BHIA 1% NaCl Tryptic Soy Broth Beckton Dickinson25 NCCLSNational committee for clinical laboratory standards 2002, 2003MIC 1% NaCl Tryptic Soy Agar Beckton Dickinson 3 5 McFarland No MIC MIC 2% NaCl Muller-Hinton Agar Becton Dickinson ABPC BCM OTC FOM OAFMQ FF SMMX 8 CP KM DSM GM TMP EFRX 6 P. damselae subsp. piscicida ATCC51736, P. damselae subsp. piscicida ATCC17911, Escherichia coli ATCC25922, Pseudomonas aeruginosa ATCC27853, Staphylococcus aureus ATCC29231, Enterococcus faecalis ATCC29212, Aeromonas salmonicida subsp. salmonicida ATCC33658

3 Photobacterium damselae subsp. piscicida RAPD Magarinos et al DNA QuickGene DNA tissue kit S Fujifilm PCR Ready- To-Go RAPD Analysis Beads GE Healthcare Primer 5 5 -AACGCGCAAC-3 PCR % 251P. damselae subsp. piscicida 200 bp PCR BstUI 125 bp 76 bp 247 P. damselae subsp. piscicida P. damselae subsp. piscicida 247 Table 2 MIC 10 ABPC OTC CP KM DSM FMQ OA SMMX TMP FOM 80% FMQ 96.0% OTC 95.1% SMMX 82.6% ABPC 24.3% CP KM 61.9% 59.5% Table OTC CP KM FMQ OA SMMX % ABPC OTC DSM FMQ SMMX TMP FOM Table 1. The number of isolates of Photobacterium damselae subsp. piscicida Prefecture Nagasaki Oita Ehime Mie Kagoshima Kagawa Total Table 2. Minimum inhibitory concentration of antimicrobials against Photobacterium damselae subsp. piscicida Antibiotics MIC ( g/ml) Break Number of MIC50 MIC90 point resistant < >512 ( g/ml) ( g/ml) ( g/ml) isolates (%) ABPC (24.3) OTC (95.1) CP (61.9) FF KM >512 > (59.5) DSM (21.9) GM ERFX FMQ (96.0) OA (66.4) SMMX >512 > (82.6) TMP (19.4) BCM FOM (22.7)

4 Table 3. Relationship between antimicrobial resistance profile and RAPD pattern N Antimicrobial resistance profile RAPD Isolates in 2003 to 2009 pattern OA OTC ABPC SMMX SMMX OA SMMX KM SMMX OTC FMQ OTC FMQ OTC FMQ OA OTC FMQ SMMX OTC FMQ SMMX FMQ OA SMMX OTC CP FMQ SMMX OTC FMQ OA SMMX OTC CP FMQ OA SMMX OTC KM FMQ OA SMMX ABPC OTC CP FMQ SMMX ABPC OTC CP FMQ OA SMMX ABPC DSM FMQ SMMX TMP FOM ABPC OTC DSM FMQ SMMX FOM ABPC OTC FMQ SMMX TMP FOM ABPC OTC DSM FMQ TMP FOM ABPC OTC DSM FMQ TMP FOM OTC CP KM FMQ SMMX FOM OTC CP KM FMQ OA SMMX OTC CP KM FMQ OA SMMX OTC CP KM FMQ OA SMMX ABPC OTC CP KM FMQ OA SMMX ABPC OTC CP KM FMQ OA SMMX OTC CP KM DSM FMQ OA SMMX ABPC OTC DSM FMQ SMMX TMP FOM ABPC OTC DSM FMQ SMMX TMP FOM ABPC OTC DSM FMQ OA SMMX TMP FOM ABPC OTC DSM FMQ OA SMMX TMP FOM ABPC OTC CP DSM FMQ SMMX TMP FOM 4 1 N means the number of antimicrobials to which the isolates are resistant. Predominant antimicrobial resistance profiles are stained faint gray. DSM TMP P. damselae subsp. piscicida 247 RAPD DNA P1 P6 6 RAPD Fig. 1 P. damselae subsp. piscicida ATCC17911 P1 RAPD P2 6 P. damselae subsp. piscicida ATCC51736 P2 P1 P2 5

5 Photobacterium damselae subsp. piscicida Table 4. RAPD patterns of Photobacterium damselae subsp. piscicida Year RAPD pattern P1 P2 P3 P4 P5 P6 Total (6.7) (6.1) (5.9) 17 (17.6) 1 31 (12.9) (34.8) 1 (100) 76 (32.9) (25.0) 29 (27.6) 41 (26.8) Parentheses show the percentage of isolates which were resistant to 7 and more antimicrobials. Fig. 1. Electrophoresis of random amplified DNA of Photobacterium damselae subsp. piscicida. Lane M, 100bp DNA ladder; lane 1, P. damselae subsp. piscicida ATCC17911 (Pattern 1); lane 2, ATCC51736 (Pattern 2); lane 3, Strain (Pattern 3); Lane 4, pp (Pattern 4); Lane 5, MEP0701 (Pattern 5); lane 6, PP08-27 (Pattern 6). 500 bp RAPD RAPD Table 4RAPD P % RAPD P % RAPD P4 7 RAPD P RAPD P5 2007P P. damselae subsp. piscicida P. damselae subsp. piscicida Aoki and Kitao 1985; Takashima et al. 1985; 1988, 1990; Kim and Aoki 1993; Thyssen and Ollevier 2001 Kawanishi et al P. damselae subsp. piscicida NCCLS MIC ABPC KM OTC OA FMQ CP SMMX TMP Kawanishi et al FMQ SMMX 2002 ABPC KM OTC OA CP TMP MIC 2 DSM FOM DSM FOM FOM %1990FF ABPC P. damselae subsp. piscicida 1982 Takashima et al ABPC % Takashima et al. 1985; 1988, 1990; Kim and Aoki %

6 % % 29.8% ABPC 2007 RAPD P2 P4 RAPD P ABPC ABPC ABPC ABPC MIC g/ml Takashima et al. 1985; Kim and Aoki g/ml OTC CP KM FMQ OA SMMX2002 OTC CP KM FMQ OA SMMX ABPC OTC DSM FMQ SMMX TMP FOM DSM TMP RAPD Table % % ABPC RAPD P RAPD P % 6 22 ABPC OTC DSM FMQ SMMX TMP FOM 7 ABPC Photobacterium damselae subsp. piscicida ABPC OTC CP KMDSM FMQ OA SMMX TMP FOM 55.5% 6 OTC CP KM FMQ OA SMMX RAPD Random amplified polymorphic DNA5 RAPD P2 P RAPD P2 P Aoki, T. and T. Kitao (1985) Detection of transferable R plasmids in strains of the fish pathogenic bacterium, Pasteurella piscicida. J. Fish Dis., 8, Bakopoulos, V., A. Adams and R. H. Richards (2005) Some biochemical properties and antibiotic sensitivities of Pasteurella piscicida isolated in Greece and comparison with strains from Japan, France and Italy. J. Fish Dis., 18, 1-7. Balebona, M. C., M. A. Morinigo, J. Sedano, E. Martinez- Mazanares, A. Vidaurreta, J. J. Borrego and A. E. Toranzo (1992) Isolation of Pasteurella piscicida from sea bass in southwestern Spain. Bull. Eur. Ass. Fish Pathol., 12, Kawanishi, M., M. Kijima, A. Kojima, K. Ishihara, H. Esaki, K. Yagyu, T. Takahashi, S. Suzuki and Y. Tamura (2006) Drug resistance and random amplified polymorphic DNA analysis of Photobacterium damselae subsp. piscicida isolates from cultured Seriola (yellowtail, amberjack and kingfish) in Japan. Lett. Appl. Microbiol., 42, Kijima-Tanaka, M., M. Kawanishi, Y. Fukuda, S. Suzuki and K.

7 Photobacterium damselae subsp. piscicida Yagyu (2007) Molecular diversity of Photobacterium damselae subsp. piscicida from cultured amberjacks (Seriola spp.) in Japan by pulsed-field gel electrophoresis and plasmid profiles. J. Appl. Microbiol., 103, Kim, E. H. and T. Aoki (1993) Drug resistance and broad geographical distribution of identical R plasmids of Pasteurella piscicida isolated from cultured yellowtail in Japan. Microbiol. Immunol., 37, I I Kusuda, R. and K. Kawai (1998) Bacterial diseases of cultured marine fish in Japan. Fish Pathol., 33, Pasteurella piscicida Pasteurella piscicida Lopez, C., P. R. Rajan, J. H. Y. Lin, T. Y. Kuo and H. L. Yang (2002) Disease outbreak in seafarmed cobia (Rachycentron canadum) associated with Vibrio spp., Photobacterium damselae subsp. piscicida, monogenean and myxosporean parasites. Bull. Eur. Ass. Fish Pathol., 22, Magariños, B., J. L. Romalde, I. Bandín, B. Fouz and A. E. Toranzo (1992) Phenotypic, antigenic, and molecular characterization of Pasteurella piscicida strains isolated from fish. Appl. Environ. Microbiol., 58, Magariños, B., A. E. Toranzo, J. L. Barja and J. L. Romalde (2000) Existence of two geographically-linked clonal lineages in the bacterial fish pathogen Photobacterium damselae subsp. piscicida evidenced by random amplified polymorphic DNA analysis. Epidemiol. Infect., 125, Martínez-Manzanares, E., S. T. Tapia-Paniagua, P. Díaz-Rosales, M. Chabrillón and M. A. Moriñigo (2008) Susceptibility of Photobacterium damselae subsp. piscicida strains isolated from Senegalese sole, Solea senegalensis Kaup, and gilthead seabream, Sparus aurata L., to several antibacterial agents. J. Fish Dis., 3, National committee for clinical laboratory standards (2002) Performance standards for antimicrobial disk and dilution susceptibility tests for bacteria isolated from animals. Approved standard M31-A2. Wayne, PA: National committee for clinical laboratory standards, 86 pp. National committee for clinical laboratory standards (2006) Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically. Approved standard M7-A7. Wayne, PA: National committee for clinical laboratory standards, 49 pp pp Snieszko S. F., G. L. Bullock, E. Hollis and J. G. Boone (1964) Pasteurella sp. from an epizootic of white perch (Roccus americanus) in Chesapeake bay tidewater areas. J. Bacteriol., 88, Takashima. N., T. Aoki and T. Kitao (1985) Epidemiological surveillance of drug-resistant strains of Pasteurella piscicida. Fish Pathol., 20, Thyssen, A. and F. Ollevier (2001) In vitro antimicrobial susceptibility of Photobacterium damselae subsp. piscicida to 15 different antimicrobial agents. Aquaculture, 200, Toranzo, A. E., S. Barreifo, J. F. Casal, A. Figueras, B. Magarinos and J. L. Barja (1991) Pasteurellosis in cultured gilthead seabream (Sparus aurata): first report in Spain. Aquaculture, 99, Zappulli, V., T. Patarnello, P. Patarnello, F. Frassineti, R. Franch, A. Manfrin, M. Castagnaro and L. Bargelloni (2005) Direct identification of Photobacterium damselae subspecies piscicida by PCR-RFLP analysis. Dis. Aquat. Org., 65,