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Public Health Microbiology

Strain Variation in Mycobacteriummarinum Fish Isolates

M. Ucko, A. Colorni, H. Kvitt, A. Diamant, A. Zlotkin, W. R. Knibb
M. Ucko
1Israel Oceanographic and Limnological Research Ltd., National Center for Mariculture, Eilat
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  • For correspondence: mucko@ocean.org.il
A. Colorni
1Israel Oceanographic and Limnological Research Ltd., National Center for Mariculture, Eilat
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H. Kvitt
1Israel Oceanographic and Limnological Research Ltd., National Center for Mariculture, Eilat
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A. Diamant
1Israel Oceanographic and Limnological Research Ltd., National Center for Mariculture, Eilat
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A. Zlotkin
2Department of Clinical Microbiology, The Hebrew University-Hadassah Medical School, Jerusalem 91120, Israel
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W. R. Knibb
1Israel Oceanographic and Limnological Research Ltd., National Center for Mariculture, Eilat
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DOI: 10.1128/AEM.68.11.5281-5287.2002
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  • FIG. 1.
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    FIG. 1.

    Phylogenetic relationships among Mycobacterium species derived from the 16S rRNA gene sequences, as constructed by maximum-parsimony analysis. The tree was rooted by using N. asteroides as the outgroup. Numbers on the branches indicate bootstrap proportions (100 replicates). Bar, 1.0 nucleotide replacement. Available GenBank accession numbers are shown.

  • FIG. 2.
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    FIG. 2.

    Phylogenetic relationships among Mycobacterium species derived from the hsp65 gene sequences (A) and the deduced sequences of the amino acids encoded by the Mycobacteriumhsp65 gene (B), as constructed by maximum-parsimony analysis. The tree was rooted by using N. asteroides as the outgroup. Numbers on the branches indicate bootstrap proportions (100 replicates). Bar, 1.0 nucleotide or amino acid replacement. Available GenBank accession numbers are shown.

  • FIG. 3.
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    FIG. 3.

    Enzyme restriction mapping. (A and B) A 614-bp PCR product from the 16S rRNA gene cleaved with restriction enzyme MslI. (A) Gel separation. (B) Restriction map. (C and D) A 796-bp product from the hsp65 gene cleaved with restriction enzyme DdeI. (C) Gel separation. (D) Restriction map. Lanes: M, 100-bp molecular size marker; 1, DL240490; 2, SR030597; 3, CC240299; 4, BB170200; 5, ATCC 927; 6, DLDK.

Tables

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  • TABLE 1.

    Mycobacterium isolates and their hosts

    IsolateaHostOriginbGroupcIdentity to GenBank accession no. for:
    16S rRNAhsp6516S rRNAhsp65
    ATCC 927Unspecified fishUnited States, marine, captiveCCIIAF456240AF456470
    DL240490European sea bass (Dicentrarchus labrax)Israel, marine (RS), culturedAA AF456238 AF456468
    DL150991European sea bass (D. labrax)Israel, marine (MS), culturedAA AF456238 AF456468
    DL045dEuropean sea bass (D. labrax)Greece, marine (MS), culturedAA AF456241 AF456471
    DL049dEuropean sea bass (D. labrax)Greece, marine (MS), culturedFF AF456242 AF456472
    CF030494Butterfly fish (Chaetodon fasciatus)Israel, marine (RS), captiveAA AF456238 AF456468
    PP241194eSea bream (Puntazzo puntazzo)Israel, marine (RS), cultured
    SO020195eRed drum (Sciaenops ocellatus)Israel, marine (RS), cultured
    SR250195Rabbit fish (Siganus rivulatus)Israel, marine (RS), culturedAA AF456238 AF456468
    DLDKfEuropean sea bass (D. labrax)Denmark, marine, culturedCCII AF456240 AF456470
    MC110595eGrey mullet (Mugil cephalus)Israel, marine (RS), cultured
    SA200695eGilthead sea bream (Sparus aurata)Israel, marine (RS), cultured
    Hybrid 270995gHybrid red sea bream (Pagrus major [female] × S. aurata [male])Israel, marine (RS), culturedA AF456238
    EA040995gWhite grouper (Epinephelus aeneus)Israel, marine (RS), culturedA AF456238
    SR300397gRabbit fish (S. rivulatus)Israel, marine (RS), wildA AF456238
    SR030597Rabbit fish (S. rivulatus)Israel, marine (RS), wildAA AF456238 AF456468
    EI100398gHawksbill sea turtle (Eretmochelys imbricata)Israel, marine (RS), captiveA AF456238
    CC240299Koi (Cyprinus carpio)Israel, freshwater, culturedBB AF456239 AF456469
    EA110499gWhite grouper (E. aeneus)Israel, marine (RS), culturedA AF456238
    BB170200Silver perch (Bidyanus bidyanus)Israel, freshwater, culturedBB AF456239 AF456469
    SV300500Lizard fish (Synodus variegatus)Israel, marine (RS), wildAA AF456238 AF456468
    DL041200European sea bass (D. labrax)Greece, marine (MS), culturedAA AF456238 AF456468
    185-408hGolden steaty (Acipenser baeri)Holland, freshwaterGG AF152558 j AF456473
    185-409hGolden steaty (A. baeri)Holland, freshwaterGG AF152558 j AF456473
    S4iSnakehead (Channa striatus)Thailand, freshwaterCCIII AF251565 j AF456474
    S267iSnakehead (C. striatus)Thailand, freshwaterCCIII AF251565 j AF456474
    • ↵ a Unless otherwise indicated, isolates were from Israel Oceanographic and Limnological Research Ltd., National Center for Mariculture, Eilat, Israel (the two first letters indicate the initials of the host genus and species; the digits indicate the date of isolation [daymonthyear]).

    • ↵ b RS, Red Sea; MS, Mediterranean Sea.

    • ↵ c According to the phylogenetic analysis as it appears in Fig. 1 and 2A.

    • ↵ d Kindly provided by H. Nousias, Oceanos, Fish Health Center, Preveza, Greece.

    • ↵ e Not included in the 16S rRNA and hsp65 gene sequence study.

    • ↵ f Kindly provided by I. Dalsgaard, Fish Disease Laboratory, Danish Institute for Fisheries and Marine Research, Frederiksberg, Denmark.

    • g Not included in the hsp65 gene sequence study.

    • ↵ h Kindly provided by O. L. M. Haenen and D. Bakker, Laboratory of Fish Diseases, Institute for Animal Science and Health, Lelystad, Holland.

    • ↵ i Kindly provided by A. Adams, Institute of Aquaculture, University of Stirling, Stirling, Scotland.

    • ↵ j Accession numbers are already available from GenBank.

  • TABLE 2.

    PCR primers

    GenePrimerPositionaSequenceb
    16S rRNA246c1-20F 5′-AGA GTT TGA TCC TGG CTC AG-3′
    My161-78F 5′-GGA AAG GTC TCT TCG GAG-3′
    414c200-216R 5′-CAT CCC ACA CCG CWA AAG-3′
    266c593-612R 5′-CAC GCY CAC AGT TAA GCY GT-3′
    614593-614F 5′-CTT AAC TGT GAG CGT GCG-3′
    1522c1503-1522R 5′-AAG GAG GTG ATC CAG CCG CA-3′
    hsp65 HSF11-21F 5′-GAT CCG GAG GAA TCA CTT CGC-3′
    TB11d167-186F 5′-ACC AAC GAT GGT GTG TCC AT-3′
    TB12d588-607R 5′-CTT GTC GAA CCG CAT ACC CT-3′
    HS12F588-607F 5′-AGG GTA TGC GGT TCG ACA AG-3′
    HS4F1079-1099F 5′-ACA GCG ACT CCG ACT ACG ACC-3′
    M2e1367-1383R 5′-TTG AAG GCG ATC TGC TT-3′
    HSR11628-1648R 5′-TCA GAA ATC CAT GCC ACC CAT-3′
    • ↵ a According to GenBank accession no. AF456238 for the 16S rRNA gene and accession no. AF456468 for the hsp65 gene.

    • ↵ b F, forward, R, reverse. Bold letters in primers sequences represent the following degeneracy: W, A or T; Y, C or T.

    • ↵ c Designed by Böddinghaus et al. (2).

    • d Designed by Telenti et al. (31).

    • ↵ e Designed by Plikaytis et al. (21).

  • TABLE 3.

    Mycobacterium sp. sequences available in the GenBank database compared to the sequences of our isolates

    Gene studyIsolateOriginClusteraGenBank accession no.
    16S rRNA M. marinum Unknownb; identical to strain ATCC 927C X52920
    M. marinum Rainbow fish (Melanotaenia praecox); TaiwanC AF251565
    Mycobacterium sp. strain M175Striped bass (Morone saxatilis); United StatesC AY005147
    M. ulcerans HumanC X88926
    M. bovis Strain BCGD M20940
    M. tuberculosis Human; strain H37RvD Z83862
    M. avium HumanE AF306455
    M. avium subsp. paratuberculosisStrain ATCC 19698E X52934
    M. chlorophenolicum Freshwater lake sedimentF X79094
    Mycobacterium sp. strain Fuerth 1999HumanG AF152558
    N. asteroidesc Strain ATCC 3306H X57949
    hsp65 M. ulcerans Human; Australia (isolate 93147172)CI AF456475 d
    M. avium subsp. paratuberculosisRuminantE X74518
    M. avium UnknownbE AF281650
    M. tuberculosis Human; strain H37RvD AL021932
    M. bovis Strain BCG P3D M17705
    N. asteroides c Strain ATCC 3306H AF352019
    • ↵ a According to the phylogenetic analysis as it appears in Fig. 1 and 2A.

    • ↵ b Unspecified source.

    • ↵ c Used as an outgroup.

    • ↵ d Isolate was kindly provided by A. Sievers, Mycobacterium Reference Laboratory, Fairfield Hospital, Fairfield, Victoria, Australia. It was sequenced and submitted by us to GenBank.

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Strain Variation in Mycobacteriummarinum Fish Isolates
M. Ucko, A. Colorni, H. Kvitt, A. Diamant, A. Zlotkin, W. R. Knibb
Applied and Environmental Microbiology Nov 2002, 68 (11) 5281-5287; DOI: 10.1128/AEM.68.11.5281-5287.2002

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Strain Variation in Mycobacteriummarinum Fish Isolates
M. Ucko, A. Colorni, H. Kvitt, A. Diamant, A. Zlotkin, W. R. Knibb
Applied and Environmental Microbiology Nov 2002, 68 (11) 5281-5287; DOI: 10.1128/AEM.68.11.5281-5287.2002
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KEYWORDS

Bacterial Proteins
Chaperonins
fishes
Mycobacterium marinum
RNA, Ribosomal, 16S

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