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Enterococcus Haemoperoxidus Sp. Nov. and Enterococcus Moraviensis Sp

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Enterococcus Haemoperoxidus Sp. Nov. and Enterococcus Moraviensis Sp International Journal of Systematic and Evolutionary Microbiology (2001), 51, 1567–1574 Printed in Great Britain Enterococcus haemoperoxidus sp. nov. and Enterococcus moraviensis sp. nov., isolated from water 1 Czech Collection of Pavel S) vec,1 Luc A. Devriese,2 Ivo Sedla! c) ek,1 Margo Baele,2 Microorganisms, Faculty of 3 2 3 4 Science, Masaryk Marc Vancanneyt, Freddy Haesebrouck, Jean Swings and Jir) ı! Dos) kar) University, Tvrde! ho 14, 602 00 Brno, Czech Republic Author for correspondence: Pavel S) vec. Tel: j420 5 43 24 72 31. Fax: j420 5 43 24 73 39. e-mail: mpavel!sci.muni.cz 2 Laboratory of Bacteriology, Faculty of Veterinary Medicine, Ghent University, A polyphasic taxonomic approach was used to study atypical enterococci Salisburylaan 133, isolated from surface waters. All strains were characterized by physiological B-9820 Ghent, Belgium and biochemical tests as well as by genotyping. The results of biochemical 3 BCCM/LMG Bacteria tests and tRNA intergenic length polymorphism analysis (tDNA-PCR) divided all Collection, Ghent studied strains uniformly into two groups. Because these groups were clearly University, K. L. Ledeganckstraat 35, separated from all enterococcal species described to date, 16S rDNA sequence B-9000 Ghent, Belgium analysis, DNA base composition analysis and DNA–DNA hybridization of 4 Department of Genetics representative strains were done to elucidate the taxonomic position of the and Molecular Biology, analysed groups. On the basis of the results obtained, the names Enterococcus Faculty of Science, Masaryk haemoperoxidus (type strain CCM 4851T l LMG 19487T) and Enterococcus University, Kotla! r) ska! 2, T T 611 37 Brno, moraviensis (type strain CCM 4856 l LMG 19486 ) are proposed for the two Czech Republic hitherto undescribed species. The type strains and reference cultures have been deposited in the Czech Collection of Microorganisms (CCM), Masaryk University, Brno, Czech Republic, and in the BCCM/LMG Culture Collection, Ghent University, Belgium. Keywords: Enterococcus haemoperoxidus sp. nov., Enterococcus moraviensis sp. nov., taxonomy, identification, water INTRODUCTION and their presence therein represents contamination from animals and plants (Deibel, 1964). The incidence Enterococci are Gram-positive cocci that are com- of enterococci in waters is generally considered to be monly isolated from human clinical specimens, some due to faecal contamination and these bacteria are kinds of food and the environment. Recent interest in monitored during microbiological quality testing of this bacterial genus has been encouraged by their water. In particular, E. faecalis, E. faecium, Entero- increasing clinical significance and acquired antibiotic coccus durans and Enterococcus hirae are considered to resistance (Facklam et al., 1999; Jett et al., 1994). be of faecal origin (Godfree et al., 1997). However, Therefore, most investigations relate to strains and enterococci of non-faecal origin may be also found in species isolated from clinical samples. Unfortunately, surface waters. Niemi et al. (1993) described E. little exact taxonomic information is available about casseliflavus as a typical species in forest industry the strains and species of enterococci occurring in the wastewaters in Finland, and they isolated atypical environment. Enterococci can be isolated from soil, enterococcal strains from pristine waters. plants, insects and wild animals as well as from water samples. Enterococcus faecalis, Enterococcus faecium, A series of atypical enterococci were isolated during an investigation into the occurrence of different entero- Enterococcus mundtii and Enterococcus casseliflavus ) are found associated with plants (Devriese et al., 1992). coccal species in surface waters (Svec & Sedla! c) ek, Soil is probably not the natural habitat of enterococci, 1999). All strains were characterized using morpho- logical, physiological as well as biochemical tests, but ................................................................................................................................................. their phenotypic characteristics did not correspond to The GenBank accession numbers for the 16S rRNA gene sequences of E. those of known enterococcal species. Because of their moraviensis CCM 4856T and E. haemoperoxidus CCM 4851T are AF286831 unclear taxonomic position, analysis of tRNA inter- and AF286832, respectively. genic length polymorphism (tDNA-PCR), 16S rDNA 01761 # 2001 IUMS 1567 P. S) vec and others sequencing, DNA base composition analysis and overnight on Columbia agar with 5% sheep blood was DNA–DNA hybridization were carried out. Our suspended in 20 µl lysis buffer (0n25% SDS, 0n05 M NaOH) results showed that the enterococcal strains analysed and heated at 95 mC for 5 min. Subsequently, 180 µl sterile represent two new species, for which the names distilled water was added and the suspension was centrifuged Enterococcus haemoperoxidus sp. nov. and Entero- at 13000 r.p.m. for 5 min. The supernatant was used directly for tDNA-PCR as well as for 16S rDNA sequencing. coccus moraviensis sp. nov. are proposed. tRNA intergenic length polymorphism analysis (tDNA-PCR). tDNA-PCR was done using the consensus primers METHODS T5A (5h-AGTCCGGTGCTCTAACCAACTGAG-3h) and fluorescent-labelled T3B (5h-AGGTCGCGGGTTCGAAT- Bacterial strains. The strains analysed in this work were CC-3h) described by Welsh & McClelland (1992). Capillary isolated from surface waters by membrane filtration and electrophoresis of PCR products was done on an ABI subsequent incubation of the filters on Slanetz–Bartley agar PRISM 310 Genetic Analyzer (Applied Biosystems) as plates at the 37 mC during a routine analysis of water quality, described previously (Baele et al., 2000). Cluster analysis of ) as described previously (Svec & Sedla! c) ek, 1999). All strains tDNA-PCR fingerprints was done using the UPGMA were isolated from different sampling sites in the region of algorithm with the software (Felsenstein, 1989) and North Moravia in the Czech Republic (see Table 1). the dendrogram was visualized using the program Phenotypic studies. Isolated strains were cultivated on described by Page (1996). Columbia agar with 5% sheep blood. Consequently, growth 16S rRNA sequence analysis. The 16S rRNA gene was was checked and characterized on Slanetz–Bartley agar, amplified using the primers αβ-NOT (5h-TCAAACTAGG- kanamycin\aesculin\azide agar, BBL Enterococcosel agar, ACCGAGTC-3h) and ωMB (5h-TACCTTGTTACTTCACC- Edwards agar, aesculin\bile agar and skim milk medium. CCA-3h) and the Taq Mastermix (Qiagen). The subsequent Growth in a 5% CO# atmosphere and in a normal sequencing reactions were done using the BigDye Ter- atmosphere was compared on blood agar at 37 mC. Tests for minator sequencing kit (Applied Biosystems) and forward growth at various temperatures as well as in 6n5% NaCl sequencing primers *Gamma, *PD, *O, *3, *R and reverse were performed in brain\heart infusion (BHI) broth and primers Gamma, PD and 3 described by Coenye et al. bacteria were cultivated for up to 5 d. Catalase tests were (1999). The sequences were determined on an ABI PRISM done by dispersing growth from Columbia agar with and 310 Genetic Analyzer (Applied Biosystems). Cluster analysis without blood in 3% hydrogen peroxide. Motility was tested was done using program (Applied Maths). Pair- on semi-solid medium according to Facklam & Wilkinson wise alignment homologies were calculated and a dendro- (1981). Group D antigen was tested using a Streptococcal gram was constructed using the neighbour-joining method grouping kit (Oxoid). Amylase production was looked for with 100% open gap penalty and 0% unit gap penalty on Columbia agar base containing 0n1% starch. Biochemical values. tests were performed using API galleries 20 STREP and 50 CH (bioMe! rieux) as well as the CRYSTAL Gram-positive DNA base composition. DNA was prepared as described ID kit (Becton Dickinson) according to the manufacturers’ elsewhere (Vancanneyt et al., 2001). The enzymic degra- instructions. dation of DNA into nucleosides was done as described by Mesbah et al. (1989). The nucleotide mixture was then DNA isolation. One loop of a bacterial culture grown separated by HPLC using a Waters SymmetryShield C8 Table 1. Source and locality of isolation of the strains Strain Source and locality Enterococcus haemoperoxidus sp. nov. 25 ( l CCM 4855) River Ols) e, Tr) inec 60 ( l CCM 4853), 152, 313, Drinking water, Fry! dek-Mı!stek 504 (l CCM 4886), 616 382 Drinking water, C) eladna! 434 Spring Ha! jek, Fry! dek-Mı!stek 435, 457, 473 Drinking water, Tr) inec 440T (l CCM 4851T l LMG 19487T) Service water, Paskov 450 (l CCM 4854) Swimming pool, Fry! dek-Mı!stek 466 Swimming pool, Tr) inec 501 (l CCM 4852) Drinking water, Vı!tkovice 562 Drinking water, Lhotka Enterococcus moraviensis sp. nov. 206 (l CCM 4857) Drinking water, C) eladna! 330T (l CCM 4856T l LMG 19486T) Spring Ha! jek, Fry! dek-Mı!stek 430 (l CCM 4859), 494, 568 (l CCM 4885), Drinking water, Fry! dek-Mı!stek 570 (l CCM 4858) 531 (l CCM 4860) Drinking water, Fryc) ovice 1568 International Journal of Systematic and Evolutionary Microbiology 51 Two new species of Enterococcus from water ..................................................................................................... Fig. 1. Dendrogram based on tDNA-PCR fingerprint patterns demonstrating the relatedness between E. haemoperoxidus sp. nov., E. moraviensis sp. nov. and other enterococcal species. Numbers of strains analysed are indicated in parentheses. column thermostatted
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