Staphylococcus condimenti sp. nov., from soy sauce mash, and Staphylococcus carnosus (Schleifer and Fischer 1982) subsp. utilis subsp. nov

Staphylococcus condimenti sp. nov., from soy sauce mash, and Staphylococcus carnosus (Schleifer and Fischer 1982) subsp. utilis subsp. nov
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  Downloaded from www.microbiologyresearch.org byIP: Tue, 16 Feb 2016 18:52:00 International Journal of Systematic Bacteriology (1 998), 48, 651-658 zyxwvu rinted in Great Britain Staphylococcus condimenti sp. nov., f rom soy sauce mash, and Staphylococcus carnosus (Schleifer and Fischer 1982) subsp. utilis SI nov. zyx ndreas J. Probst,' Christian Hertel, Lothar Richter,' Lars Wassil Wolfgang Ludwig2 and Walter P. Hammes' Author for correspondence: Christian Hertel. Tel: zyxw 49 711 459 4255. Fax: 49 711 459 4199 e-mail : hertel@uni-hohenheim.de rbsp. 2 1 lnstitut far Le bensm tteltech no og e, Universitat Hohenheim, Garbenstr. 28, 70593 Stuttgart, Germany Mikrobiologie, Technische Universitat Munchen, 80290 Munich, Germany 2 Lehrstuhl fur ~ Based on the sequence data of 235 rRNA of Staphylococcus carnosus Staphylococcus piscifermentans Staphylococcus aureus and Staphylococcus epidermidis species-specif c probes were constructed. Their application revealed a heterogeneity within 18 strains previously identified as S. carnosus. Strains of this group were selected, and their 235 rRNA sequence was determined. It was revealed that the strains of 5. carnosus can be placed in at least three sub-groups. This grouping was supported by physiological data and DNA-DNA similarity studies. Based on these results, we propose the new species staphylococcus condimenti sp. nov. The type strain is S. condimenti F-ZT (= DSM 116743. The phylogenetic position of the new species within the radiation of other staphylococcal strains is reflected by a 16s rRNA-based tree. Furthermore, it is proposed to designate the new subspecies of Staphylococcus carnosus Schleifer and Fischer 1982, Staphylococcus carnosus subsp. utilis subsp. nov. The type strain of 5. carnosus subsp. utilis is SK lT = DSM 116763. Keywords: zyxwvuts taphylococcus condimenti sp. nov., Staphylococcus carnosus subsp. utilis subsp. nov., soy mash INTRODUCTION well as 16s and 23s rDNA sequence analysis in combination with physiological data. Moreover, 23s Strains of the species Staphylococcus carnosus were srcinally isolated from fermenting sausages (1 7). It has been shown that they exert positive effects on the formation of flavour and the reddening reaction and therefore strains of this species are used as common components in starter cultures for the production of fermented sausage and cured ham (3). In more recent rDNA-targeted oligonucleotide probes were con- structed for the rapid identification of these species and other food-relevant staphylococci such as Staphyl- ococcus zyxwv ureus and Staphylococcus epidermidis. METHODS studies (20, 21), it was that strains Of Organisms and The bacterial strains investigated and the strains used to construct and evaluate articipate in the Of fish and in Asia. A closely group Of strains the specificity of the oligonucleotide probes are those isolated from fermenting fish sauce was characterized and described as the new species StaPhYlococcus Piscifermentans 2 1). In this communication, we Pro- pose an emended classification within the species S. carnosus based on DNA-DNA similarity studies as ............................... ................................................................................................................. The EMBL accession numbers for the 165 and 235 rRNA sequences reported in this paper are Y 1 5754 and Y 1 5751 5. piscifermentans SK 033 and Y 15750 and Y15755 5. condimenti), and ~15752 or the partial 23s rRNA sequence 5. epidermidis DSM 200443. in Table 1 Staphylococci, Kocuria varians and Escherichia coli were grown at 37 C on Standard I medium (Merck). Lactic acid bacteria were grown anaerobically at 30 zyxw   on MRS medium (2), with the exception of Tetra- genococcus halophilus DSM 20339' and strains of Carno- bacterium sp. These strains were cultivated on MRS medium containing 6.5 NaCl and CASO-yeast medium (Merck), respectively. Physiological characterization. Physiological characteristics were determined with the aid of the ID 32 STAPH system ~~~~~ ~ 00728 1998 IUMS 65 1  Downloaded from www.microbiologyresearch.org byIP: Tue, 16 Feb 2016 18:52:00 A. J Probst and others Table 1. zyxwvutsrq trains and sources of micro-organisms T = Type strain. The following strains were used for the evaluation of the probes: zyxw arnobacterium divergens (DSM 20623T), Carnobacterium piscicola (DSM 20730T), Enterococcus faecalis (DSM 20478T), Eschevichia coli LTH 1288, Kocuria varians (DSM 20033T), Lactobacillus curvatus LTH 1432, Lactobacillus pentosus (DSM 203 14T), Lactobacillus plantarum (DSM 201 74T), Lactobacillus sakei LTH 677, Leuconostoc carnosum (DSM 5576T), Pediococcus acidilactici (DSM 20284T), Pediococcus pentosaceus (DSM 20336T), Staphylococcus arlettae (DSM 20672T), Staphylococcus auricularis (DSM 20609T), Staphylococcus capitis (DSM 20326T), Staphylococcus caprae (DSM 2060flT), Staphylococcus caseolyticus (DSM 20597T), Staphylococcus chromogenes (DSM 20454T), Staphylococcus cohnii (DSM 20260T), Staphylococcus equorum (DSM 20674T), Staphylococcus gallinarum (DSM 206 loT), Staphylococcus haemolyticus (DSM 20263T), Staphylococcus hominis (DSM 20328T), Staphylococcus hyicus (DSM 20459T), Staphylococcus intermedius (DSM 20373T), Staphylococcus kloosii (DSM 20676T), Staphylococcus lentus (DSM 20352T), Staphylococcus saprophyticus (DSM 20229T), Staphylococcus simulans (DSM 20322T), Staphylococcus warneri (DSM 203 1 6T), Staphylococcus xylosus (DSM 20266T), Tetragenococcus halophilus (DSM 20339T). DSM, DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen; LTH, strain collection of the Institut fur Lebensmitteltechnologie, Universitat Hohenheim, Germany. Species Strain Source zyxwv S. carnosics SK 361T (DSM 20501T) Schleifer & Fischer (17) 833 (LTH 3840) 836 (LTH 3841) F-2T LTH 3734T) SK 06 (LTH 3723) SK 07 (LTH 3724) SK 08 (LTH 3728) SK 09 (LTH 3726) SK 10 (LTH 3727) SK llT LTH 3728T) SK 12 (LTH 3729) SK 13 (LTH 3730) LTH 18 Meat starter strains LTH 53 LTH 175 LTH 1574 LTH 2102 Monte1 et al. (15) Tanasupawat et al. (20) F-8 (LTH 3735) S. piscifermentans (10 strains) Tanasupawat et al. (21) S. aureus (10 strains) Type strain and strains S. epidermidis (8 strains) Type strain and strains of from various foods various srcin (bioMerieux). Acid production from additional carbo- hydrates @-galactose, D-melezitose, D-sorbitol) and glycerol was studied as described by Kloos et al. (8). Catalase activity was detected according to Knauf et al. (9). The sensitivity to lysozyme (400 pg ml-l and 1.6 mg ml-l) and lysostaphin (200 pg ml-') was determined on agar plates (8). Tolerance to NaCl was examined by incubating for 24 h on P-agar (8) containing NaCl at concentrations of 0.5, 5, 10 and 15%. Relation to temperature was determined on P-agar at 8, 15, 25, 34, 42 and 45°C. The effect of pH on growth was investigated under aerobic conditions in P-medium con- taining 20 mM ammonium citrate adjusted to an initial pH of 5.0. zyxwvutsr solation of genomic DNA. Cells of a 5 ml overnight culture were harvested by centrifugation, washed in 2 ml 5 x TE buffer (16) and resuspended in 100 pl 5 x TE buffer con- taining 100 pg RNase ml-l. Staphylococci were lysed by adding 50 pl 10 mM Tris/HCl (pH 7.6) containing 5 mg lysozyme ml-l (Serva) and 0.5 mg lysostaphin ml-l (Sigma). All other organisms were lysed by the addition of 50 p1 of 10 mM Tris/HCl (pH 7.6) containing 10 mg lysozyme ml-'. Cell suspensions were incubated at 37 C until they became viscous. Thereafter, 150 p12 h SDS and 50 pl proteinase K (18 mg ml-l; Boehringer Mannheim) were added. The mixtures were incubated for 30 min at 55 C. The DNA was purified by repeated phenol-chloroform extractions, precipi- tated with ethanol and dissolved in 100 p1 of 1 x TE buffer. rDNA sequencing. Overlapping stretches of 23s rDNA were amplified with Pwo polymerase (Boehringer Mannheim) in a GeneAmp 2400 PCR system (Perkin-Elmer). PCR frag- 652 In terna i0 na zyx o urna of Systematic Bacteriology 48  Downloaded from www.microbiologyresearch.org byIP: Tue, 16 Feb 2016 18:52:00 Emended description of Staphylococcus carnosus ments were cloned using the pCR-Script Amp zyxwvu K(+) cloning kit (Stratagene). Plasmid DNA was isolated from E. coli with the aid of Qiagen-tip 100 columns (Qiagen). Sequencing was performed using an AutoRead sequencing kit (Pharmacia) as recommended by the supplier. Sequences were read by an ALF DNA sequencer (Pharmacia) and analysed with DNASIS (Hitachi Europe). 16s rDNA was amplified in vitro and sequenced directly as described previously (1 8). zyxwvutsr equence data analysis. The rRNA sequence data were added to alignments of deposited complete primary struc- tures of 16s and 23s rRNA. Phylogenetic analyses were performed by applying maximum-parsimony and maximum-likelihood approaches on data sets varying with respect to the selection of reference sequences as well as sequence positions. The corresponding tools of the ARB program package (1 1) were used for alignment, selection of positions according to variability, calculation of similarities as well as tree reconstruction, evaluation and drawing. Design of species-specific probes. A comparative analysis of aligned 23s rDNA sequences of different staphylococci species revealed regions that were used as target sites for species-specific oligonucleotide probes. To optimize the specificity of the probes, target sites were chosen with respect to the most destabilizing effect of mismatches on the oligonucleotide-DNA hybrid (7, 13). Probe hybridizations. For the application of species-specific oligonucleotide probes, 5 pg DNA from each strain was denatured in 0.2 M NaOH/2 zyxwvuts   SSC (16) at 37 C for 5 min and transferred to a nylon membrane (Qiabrane; Qiagen) using a dot-blot apparatus (Schleicher & Schuell). Sub- sequently, the DNA was immobilized by incubating at 80 C for 1 h. Oligonucleotides were labelled with the 3'-digoxi- genin (DIG) oligolabelling kit (Boehringer Mannheim). Hybridizations were carried out as described by the supplier. The membranes were washed twice for 10min in 2 x SSC/O-l zyxwvutsrq O SDS. The temperatures used for hybridization and washing are shown in Table 3. Detection of hybrids was performed using a DIG luminescent detection kit (Boehringer Mannheim). For further hybridizations, oligonucleotides and antibodies were removed by washing the membranes twice in 0.2 M NaOH/O.5% SDS at 37 C for 10 min. Subsequently, the membranes were washed five times in 2 x SSC to avoid carry-over of NaOH. DNA-DNA hybridization. DNA similarity was determined using radioactively labelled genomic DNA of strain F-2T as a probe. DNA zyxwvuts 5 pg) from selected type strains was transferred to and immobilized on a nylon membrane as described above. To determine the accessibility of DNA, hybridization with the radioactively labelled 16s rDNA bacterial probe EUB338 (1) was carried out as described previously (6). Radioactive-labelling of probe EUB338 was done with the aid of Ready-To-Go T4 polynucleotide kinase (Pharmacia) and Redivue [Y-~~P]ATP Amersham). Genomic DNA of strain F-2T was labelled with a Ready-To- Go DNA-labelling kit and Redivue [E-~~PI~ATP. ubse- quent hybridization was performed at 68 C for 24 h. The membrane was washed twice at 70 C for 15 min in 2 x SSC/O.l% SDS and once in 0.1 x SSC/O.5 YO SDS to ensure stringent conditions (23). Autoradiograms were digitized with a flatbed scanner (Hewlett-Packard). The DNA simi- larities were calculated using WinCam software (Cybertech), taking into consideration the accessibility of immobilized DNA for hybridization (10). RESULTS Design and application of oligonucleotide probes To construct species-specific probes, the complete 16s and 23s rRNA sequences of strain S. pisciferrnentans SK 03T and parts of the 23s rRNA sequence of S. epidermidis (DSM 20044T) were determined by sequencing of in vitro amplified or cloned rDNA. The 16s and 23s rRNA sequences of S. carnosus and S. aureus have been described previously (14). The comparison of variable regions of these 23s rRNA sequences permitted the construction of specific oligo- nucleotide probes. The application revealed that probes Stpis2, Staur and Stepi were specific for S. piscifermentans, S. aureus and S. epidermidis, respect- ively, while probe Stcar2 hybridized with the DNA of only a few strains of S. carnosus. Therefore, 16s and 23s rRNA sequences of strain F-2T as well as parts of the 23s rRNA sequences of strains F-8, SK 06 and SK 12 were analysed, and the results were used to construct probes F2son2 and Stcar4. By applying probes Stcar2, Stcar4 and F2son2, the strains formerly classified as S. carnosus could be allotted to groups F, A and B (Table 2). The specificities of all probes were evaluated with DNA isolated from the staphylococci and type strains of species relevant in fermenting high-protein food substrates. The sequences of all probes and the temperatures used for hybridization and washing are given in Table 3. DNA similarity Quantitative DNA-DNA reassociation studies were performed with DNA of the strains of group F and the type strains of S. carnosus and S. piscifermentans. As shown in Table 4, strain F-2T exhibited a high level of DNA relatedness with strain F-8, but only low levels with S. carnosus SK 361T 58 YO) nd S. pisciferrnentans SK 03T (51 ). Phylogenetic implications It was calculated that the 16s as well as the 23s rRNA sequences of strain F-2T, S. piscifermentans SK 03T and the type strain of S. carnosus (14) share more than 99.9 and 98.9 YO overall sequence similarity, respect- ively. For the small-subunit rRNAs, only one to three base changes were found. On the other hand, 13-29 base changes were detected in the 23s rRNA of these strains, confirming the higher overall fraction of variable residues within large-subunit rRNA sequences (12, 14). A 16s rRNA-based phylogenetic tree reflecting the position of the strains within the radiation of other staphylococcal strains is shown in Fig. 1. Cultural and morphological properties To support the grouping depicted in Table 2, the strains were studied in more detail. All strains grew well on P-agar between 15 and 42 C. None of the International Journal of Systerna ic Bacteriology 48 zyxwvut 53  Downloaded from www.microbiologyresearch.org byIP: Tue, 16 Feb 2016 18:52:00 A. J. Probst and others Table 2. zyxwvutsr pecificity of oligonucleotide probes F2son2, Stcar2, Stcar4, Stpis2, Staur and Stepi Species Strain Reaction with probe* : F2son2 Stcar2 Stcar4 Stpis2 Staur Stepi Group F F-2T(LTH 3734) F-8 (LTH 3735) Group A SK 361Tt (DSM 20501) SK 07 (LTH 3724) SK 13 (LTH 3730) 833 (LTH 3840) 836 (LTH 3841) LTH 18 LTH 53 LTH 175 LTH 1574 LTH 2102 SK 06 (LTH 3723) SK 08 (LTH 3728) SK 09 (LTH 3726) SK 10 (LTH 3727) Group B ND ND ND ND ND SK llT LTH 3728) SK 12 (LTH 3729)t zyx . piscifermentans (1 0 strains) S. aureus (1 0 strains) S. epidermidis (8 strains) ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND, Not determined. * No hybrids were obtained with DNA of other strains listed in Table 1 t Strain used for construction of specific probe. Table 3. Description of specific probes for the strains of groups F, A and B as well as of zy . zyx iscifermentans S. aureus and 5. epidermidis Target organism Probe Sequence (5’-3’) Temperature (“C) used for: Hybridization Washing Group F F2son2 CGCCATTCTCAAGGT 43 45 Group A Stcar2 ACCTTGAGAATAGCG 43 46 Group B Stcar4 ACCTTGTGAATAGCG 43 46 zyx . piscifermentans Stpis2 CGCCATTCATAAGGT 43 45 S. aureus Staur AGCCTTAACGAGTACCGG 50 53 S. epidermidis Stepi CGGCACTCATAAGGCTG 50 52 Bacteria EUB338* GCTGCCTCCCGTAGGAGT 43 45 * Targeted against 16s rRNA (1). strains grew at 8 “C. After 48 h incubation, colonies (0.5-2 mm diameter) appeared circular, smooth, slightly raised and cream-coloured or orange (strains SK 07 and SK 13). All strains tolerated concentrations of NaCl up to 15%. zyxwvut hysiological characterization The study of physiological characteristics supported the genotypic grouping of the strains. As shown in Table 5, strains of groups F, A and B exhibited 654 International Journal of Systematic Bacteriology 8  Downloaded from www.microbiologyresearch.org byIP: Tue, 16 Feb 2016 18:52:00 Emended description of Staphylococcus carnosus Table 4. zyxwvutsr NA similarities zyxwvutsr f strains F-2T, F-8, zyxwvuts . carnosus S 361T and S. piscifermentans SK 03T using 32P-labelled DNA of strain F-ZT as a probe zyxwvut Strain EUB338 F-2T probe Calculated DNA (relative signal) (relative signal) homology ( ) F-2T 100 F-8 99 SK 361T 102 SK 03T 83 100 97 59 42 100 98 58 51 5. gallinarum 5. saprophyticus / 5. cohnii . xylosusy/ - , , simulans 5. auricularis 5. carnosus 5. condimenti 5. piscifermentans 5. muscae - 5. hyicus S. intermedius zyxwv I z   . sciuri \ ‘ . schleiferi 5. chromogenes S. ielis 5. Ientus 1 Fig. 7. 165 rRNA-based tree reflecting the phylogenetic relationships of staphylococci. The tree is based on a maximum-likelihood tree and a data set containing all available almost complete 165 rRNA sequences from staphylococci and selected reference organisms from other phylogenetic groups. For the calculations, only those alignment positions sharing identical residues in at least 50 of all staphylococcal sequences were used. The tree topology was corrected according to the results of distance matrix as well as maximum-parsimony analyses. Multifurcations indicate that a common branching order was not supported by applying alternative treeing methods. The bar indicates 1 estimated sequence divergence. catalase and arginine dihydrolase activity but no p glucuronidase activity. They reduced nitrate to nitrite (except strain SK 09), and nitrite was also reduced as described previously (5). Acid was produced from glucose, fructose, N-acetylglucosamine and glycerol. All strains were susceptible to novobiocin. None of these strains produced acid from raffinose, ribose, cellobiose, arabinose and turanose. Strains assigned to group A exhibited phosphatase activity and produced acid from D-mannose and D-sorbitol. None of these characteristics was present in the strains of group B. Strains F-2T and F-8 were allotted to group F and exhibited urease as well as high lipolytic activity. DISCUSSION Staphylococci in food, such as S. carnosus and S. piscifermentans, may exert desirable effects as com- ponents of a fermentation flora, whereas other species, for example zyxwvutsr . aureus and S. epidermidis are known as food poisoning organisms or potential pathogens. To develop a system for their identification and differen- tiation, oligonucleotide probes were designed by the comparative analysis of the large subunit rRNA primary structures of the type strains. The evaluation of the specificity of these probes revealed a genetic heterogeneity of the S. carnosus strains studied. Further sequence analyses of 23s rRNA genes led to the design of probes that, in combination with the type strain-specific probe, allowed the detection of all strains formerly classified as S. carnosus. The strains could be divided into three sub-groups (F, A and B). The strains of group F were srcinally found in soy sauce mash and described as the new species ‘Tetra- coccus soyae’ by Ueno & Omata (22). However, this species was never validated. In more recent studies, these strains have been identified as S. carnosus (20), mainly because of their DNA-DNA similarity level of 69-72 YO. However, the strains exhibited certain specific physiological characteristics, and the results of our DNA-DNA hybridization studies (5 1-58 YO imi- larity) are also indicative of their separation from the species S. carnosus. On the other hand, the overall similarities of rRNA sequences of strains F-2T, S. piscifermentans SK 03T and the corresponding sequences of strain S. carnosus SK 361T were rather International Journal of Systematic Bacteriology 48 655
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