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Staphylococcus Muscae, a New Species Isolated from Flies V

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Staphylococcus Muscae, a New Species Isolated from Flies V INTERNATIONALJOURNAL OF SYSTEMATICBACTERIOLOGY, Jan. 1992, p. 97-101 Vol. 42, No. 1 0020-7713/92/010097-05$02 .oo/o Copyright 0 1992, International Union of Microbiological Societies Staphylococcus muscae, a New Species Isolated from Flies V. HAJEK,l* W. LUDWIG,2 K. H. SCHLEIFER,2 N. SPRINGER,2 W. ZITZELSBERGER,2 R. M. KROPPENSTEDT,3 AND M. KOCUR4 Department of Microbiology, Faculty of Medicine, Palacky' University, 775 15 Olomouc, Czechoslovakia'; Lehrstuhl fur Mikrobiologie, Technische Universitat Miinchen, 0-8000 Munich, Germany2; Deutsche Sammlung von Mikroorganismen und Zellkulturen, 0-3300 Braunschweig, Germany3; and Czechoslovak Collection of Microorganisms, Masaryk University, Brno, Czechoslovakia4 A new coagulase-negative species of the genus Staphylococcus, Staphylococcus muscae, is described on the basis of the results of a study of four strains that were isolated from flies. 16s rRNA sequences of the type strains of S. muscae, Staphylococcus schleiferi, and staphylococcus sciuri were determined and used, together with the corresponding sequences of Staphylococcus aureus and Staphylococcus epidermidis, for a comparative analysis. The new species is characterized taxonomically; this species is differentiated from the other novobiocin-susceptiblestaphylococci by its physiological and biochemical activities, cell wall composition, and levels of genetic relatedness. The type strain of this species is strain MB4 (= CCM 4175). A total of 28 species are currently recognized in the genus were obtained from the Deutsche Sammlung von Mikroor- Staphylococcus; 19 species are listed in Bergey's Manual of ganismen (DSM), Braunschweig, Germany, the American Systematic Bacteriology (14), two species (Staphylococcus Type Culture Collection (ATCC), Rockville, Md., and the chromogenes and Staphylococcus lentus) have been ele- Czechoslovak Collection of Microorganisms (CCM), Brno, vated from the subspecies level (8, 19), and 7 species have Czechoslovakia. been described since 1984 (Staphylococcus arlettae, Staph- Phage typing was carried out by using the method of Blair ylococcus delphini, Staphylococcus equorum, Staphylococ- and Williams (1). We used 24 phages (25) for human Staph- cus felis, Staphylococcus kloosii, Staphylococcus lugdunen- ylococcus aureus strains, 12 phages (24) for bovine S. aureus sis, and Staphylococcus schleiferi) (5, 11, 21, 26). Most of strains, 4 experimental phages (2) for Staphylococcus inter- the new species were isolated from animals; the exceptions medius strains, and 4 phages (9) for Staphylococcus hyicus were S. lugdunensis and S. schleiferi, which were isolated strains. from humans. The descriptions of all 28 Staphylococcus We used the methods described by Schleifer and Kandler species were based on studies of strains that were isolated (20) and Schleifer (18) for cell wall preparation and determi- from mammals, birds, and foodstuffs of animal origin. nation. Cellular fatty acids were determined by gas chroma- In this paper, we describe a new coagulase-negative tography, using the method of Miller (17). DNA base com- species of staphylococci that was obtained from the body position was determined by thermal denaturation with a surfaces of flies. These organisms were found in about 7% of model 2600 spectrophotometer (Gilford Instruments Labo- the flies caught in certain cow sheds but not on flies caught ratories, Inc., Oberlin, Ohio) (3). DNA from Escherichia coli in human dwellings, stables, or piggeries (7). For this reason, K-12 was used as a standard (13). Radioactive labeling of this bacterium should be regarded as a transient rather than DNA and the DNA-DNA hybridization experiments were a resident on flies. carried out by using the filter method as described previously In this report we present phenetic and molecular taxo- (12). 16s rRNA genes (16s rDNA) were amplified in vitro by nomic data for a new species, Staphylococcus muscae. using the polymerase chain reaction as described previously by Ludwig et al. (16). The amplified 16s rDNA fragment was MATERIALS AND METHODS cloned in vector pBluescript (Stratagene, La Jolla, Calif.) and E. coli JM83. The cloned DNA was sequenced by using Bacterial strains. Four strains were isolated from flies the chain termination method in combination with site- trapped in cow sheds; strains MB4T, (T = type strain), specific primers (16). Sequences of 16s rRNA genes were MB21, and MB50 were isolated from Stomoxys calcitrans, aligned, similarities between sequence pairs were deter- and strain MB30 was isolated from Musca domestica (7). mined, and an unrooted phylogenetic tree was constructed The medium used for isolation was blood agar base no. 2 by using the method of Fitch and Margoliash (4) as described (Oxoid) supplemented with 5% defibrinated ovine blood. by Schleifer et al. (23). Nutrient agar (Oxoid) containing glucose (2%, wt/vol) and P agar (15) were used to propagate all of the isolates. Inocula RESULTS AND DISCUSSION for tests were prepared from 1-day cultures that were incubated at 37°C. All strains were stored as frozen suspen- The four strains which we investigated were gram-posi- sions in glycerol broth at -25°C (10) and also in a freeze- tive, catalase-positive cocci that occurred in irregular dried state. clumps, were facultatively anaerobic, were susceptible to Methods. The procedures used for determining morpho- furadantin, and were resistant to bacitracin. The DNA base logical, physiological, and biochemical characteristics have compositions of the four strains ranged from 40 to 41 mol% been described elsewhere (6). The type strains of the 14 guanine plus cytosine (strains MB4T and MB21, 40 mol%; novobiocin-susceptible Staphylococcus species (see Table 2) strains MB30 and MB50, 41 mol%); these values are some- what higher than the values obtained for the previously described staphylococci (30 to 39 mol%). The fatty acid * Corresponding author. compositions of three S. muscae strains are summarized in 97 98 HAJEK ET AL. INT. J. SYST.BACTERIOL. srms fiTGCMGTCGAGCGAACAGACGAGGTGCTTGCACCTCTGACGTTAGCGGCGGACGG 110 Ssch GATGAACGCTGGCGGCGTGCCTMTACATGCAAGTCGAGCGAACGGACGAGGAGCTTGCTCCTTTGAAGTTAGCGGCGGACGG Ssci PGATCAACGCTGGCGGCGTGCCTMTACATGCAAGTCGAGCGAACAGATGAGAAGCTTGCTTCTCTGATGTTAGCGGCGGACGG smus GTGAGTMCACGTGGGTMCCTACCTATMGACTGGAATMCTTCGGGAAACCGGAGCTMTGCCGGATMTATATTGAACCGCATGGTTCAATAGTGAAAGACGGTTTT 220 Ssch GTGAGTMCACGTGGGTMCCTACCTATMGACTGGAATMCTCCGGGAAACCGGGGCTMTGCCGGATMCATGTTGAACCGCATGGTTCAACAGTGAAAGACGGTCTT Ssci GTGAGTMCACGTGGGTMCCTACCTATMGACTGGGATMCTCCGGGAAACCGGGGCTMTACCGGATMTATTTTGAACCGCATGGTTCAATAGTGAAAGACGGTTTC smus G-CTGTCACTTATAGATGGACCCGCGCCGTATTAGCTAGTTGGTAAGGTMCGGCTTACCAAGGCGACGATACGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGAAC 330 Ssch G-CTGTCACTTATAGATGGACCCGCGCCGTATTAGCTAGTTGGTGGGGTMCGGCCTACCMGGCGACGATACGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGAAC Ssc i GGCTGTCACTTATAGATGGACCCGCGCCGTATTAGCTAGTTGGTMGGTMCGGCTTACCAAGGCGACGATACGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGAAC smus TGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCMTGGGCCAAAGCCTGACGGAGCMCGCCGCGTGAGTGATGAAGGTCTTCGGATCGTAAA 440 Ssch TGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTAGGG Ssci TGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCMTGGGCGAAAGCCTGACGGAGCMCGCCGCGTGAGTGATGAAGGTCTTCGGATCGTAAA smus GCTCTGTTATTAGGCAAGAACAAACGTGTMGTMCTGTGCACGTCTTGACGGTACCTMTCACAAAGCCACGGCTMCTACGTGCCAGCAGCCGCGGTAATACGTAGGT 550 Ssch GCTCTGTTGTTAGGGAAGAACAAATGTGTMGTAACTGTGCACGTCTTGACGGCACCTMCCAGAAAGCCACGGCTMCTACGTGC~GCAGCCGCGGTAATACGTAGGT Ssc i ACTCTGTTGTTAGGGAAGAACAAATTTGTTAGTMCTGAACAAGTCTTGACGGTACCTMCCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGT smus GGCAAGCGTTATCCGGAATTATTGGGCGTAAAGCGCGCGTAGGCGGTTTTTTAAGTCTGATGT~GCCCACGGCTCAACCGTGGAGGGTCATTGGCTG~CT660 Ssch GGCMGCGTTATCCGGAATTATTGGGCGTAAAGCACGCGTAGGCGGTTTTTTMGTCTGATGTGAAAGCCCACGGCTCMCCGTGGAGGGTCATTG~CTG~CT Ssci GGCMGCGTTATCCGGAATTATTGGGCGTAAACCGCGCGCGTAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCACGGCTCAACCGTG~GGGTCATTGGAAACTGG~CT smus TGAGTGCAGAAGAGGAMGTGCAATTCCATGTGTAGCGGT~TGCGCAGAGATATGGAGGAACACCAGTGGCGMGGCGGCTTTCTGGTCTGCAACTGACGCTGATGT 770 Ssch TGAGTGCAGAAGAGGAAAGTGGAATTCCATGTGTAGCGGT~TGCGCAGAGATATGGAG~CACCAGTGGCGAAGGCGGCTTTCTGGTCTGCAACTGACGCTGATGT Ssc i TGAGTGCAGAAGAGCAGAGTGGAATTCCATGTGTAGCGGT~TGCGCAGAGATATGGAGGAACACCAGTGGCGAAGGCGGCTCTCTGGTCTGTAACTGACGCTGATGT smus GCGAAAGCGTGGGGATCAAACAGGATTAGATACCCTGGTAGTCCACGCCGT~CGATGAGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATT 880 Ssch GCCAAAGCGTGGGGATCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATT Ssc i GCGAAAGCGTGGGGATCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATT smus AAGCACTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGACCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGCCT 990 Ssch AAGCACTCCGCCTGGGGAGTACGGTCGCMGACTGAAACTCAAAGCAATTGACGGGGACCCGCACMGCGGTGGAGCATGTGGTTTAATTCG~GCAACGCGAAGAACCT Ssci AAGCACTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGACCCGCACMGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCT smus TACCAAATCTTGACATCCTTTGACCGCACTAGAGATAGTGTTTTCCTCTTCGGAGGACAAAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGG 1100 Ssch TACCAAATCKTGACATCCTTTGACCGCTCTAGAGATACACTGTTGGG Ssc i TACCAAATCPTGACATCCTTTGACCGCTCTAGAGATAGAGTCTTCCCCTTCGGGGGACAAAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGA~TGTTGGG smus TTMGTCCCGUACCAGCGCCCCTTGAGCTTAGTTGCCATCATTMGTTGGGCACTCTMGTTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTC~TC1210 Ssch TTMGTCCCGUACCAGCGCACCCTTGAGCTTAGTTGCCATCATTMGTTGGGCACTCTMGTTGACTGCCGGTGACAAACCGGAG~GGTGGGGATGACGTC~TC Ssci TTMGTCCCGCAACGAGCGCMCCCTTMGCTTAGTTGCCATCATTAAGTTGGGCACTCTAGGTTGACTGCCGGTGACAAACCGGAGG~GGTGGGGAT~CGTCAAATC smus ATCATGCCCCTTATGATTTGGGCTACACACGTGCTACMTGGACATTACAAAGGGCAGCGAAACCGCGAGGTCAAGCAAATCCCAT~GTTGTTCTCAGTTCG~TTGT1320
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