International Journal of Systematic and Evolutionary Microbiology (2001), 51, 1949–1957 Printed in Great Britain Legionella gresilensis sp. nov. and Legionella beliardensis sp. nov., isolated from water in France 1 Centre National de Franc: ois Lo Presti,1‡ Serge Riffard,1 He! le' ne Meugnier,1 Re! fe! rence des Legionella 1 2 3 UPRES EA1655, Faculte! de Monique Reyrolle, Yves Lasne, † Patrick A. D. Grimont, Me! decine RTH Laennec, Francine Grimont,3 Robert F. Benson,4 Don J. Brenner,4 Rue Guillaume Paradin, 4 1 1 69372 Lyon cedex 08, Arnold G. Steigerwalt, Jerome Etienne and Jean Freney France 2 Laboratoire des Author for correspondence: Franc: ois Lo Presti. Tel: j33 0 169 79 79 60. Fax: j33 0 169 79 79 20. Radioisotopes et de e-mail: Francois.Lo-Presti!sanofi-synthelabo.com Biochimie mole! culaire, Ho# pital Edouard Herriot, ! ! 69437 Lyon cedex 03, Novel Legionella-like isolates, strains Montbeliard A1T and Greoux 11 D13T, France isolated from two different French water sources, were studied taxonomically 3 Unite! des Ente! robacte! ries, and phylogenetically. Morphological and biochemical characterization revealed Institut Pasteur, 75724 Paris cedex 15, France that they were Gram-negative, aerobic, non-spore-forming bacilli with a cut- glass appearance that grew only on L-cysteine-supplemented buffered charcoal 4 Respiratory Diseases Branch, Meningitis and yeast extract agar. Phenotypic characterization using fatty acid and ubiquinone Special Pathogens Branch, profiles and SDS-PAGE analysis confirmed that they were closely related, but National Center for distinct from, other species of the genus Legionella, since serotyping could not Infectious Diseases, Centers for Disease Control and relate them to any existing serogroup. Genotypic profiles generated by Prevention, Atlanta, randomly amplified polymorphic DNA and 16S–23S rDNA spacer region PCR GA 30333, USA analyses were unique for each of these isolates. DNA–DNA relatedness values ! ! of strains Montbeliard A1T and Greoux 11 D13T to each other and to other Legionella type strains were less than 25%. Phylogenetic affiliation of these organisms obtained by 16S rDNA sequence comparisons confirmed that they were distinct from any other known Legionella species. All the above results confirm that these strains constitute two novel species for which the names ! Legionella gresilensis sp. nov. (type strain Greoux 11 D13T ATCC 700509T CIP l ! l 106631T) and Legionella beliardensis sp. nov. (type strain Montbeliard A1T l ATCC 700512T l CIP 106632T) are proposed. Keywords: Legionella gresilensis sp. nov., Legionella beliardensis sp. nov., taxonomy, identification INTRODUCTION subsequently named Legionella. In 1979, this genus was composed of a single species, Legionella pneumo- In 1976, a severe outbreak of pneumonia among phila (Brenner et al., 1979). Since then, the genus has veterans of the American Legion, in the city of become very large, with 43 species currently recognized Philadelphia, led to the discovery of a new genus (Lo Presti et al., 1999; Franzin & Gioannini, 2000). These bacteria have been isolated from a large diversity ................................................................................................................................................. of samples: human clinical specimens, animals (Fabbi † Deceased 11 February 2001. et al., 1998), natural and man-made water samples ‡ Present address: Sanofi-Synthelabo Recherche, Centre de Chilly- (Fliermans, 1996; Fliermans et al., 1981; Stout et al., Mazarin, Ge! nomique Cardiovasculaire/Thrombose, 1 av. Pierre Brossolette, 1985), and protozoa (Newsome et al., 1998). However, 91385 Chilly-Mazarin cedex, France. some species, such as Legionella lytica and other Abbreviations: DFA, direct immunofluorescence assay; LLAPs, Legionella- Legionella-like amoebal pathogens (LLAPs), remain like amoebal pathogens; RAPD, randomly amplified polymorphic DNA; SAT, slide agglutination test. unculturable on the buffered charcoal\yeast extract The GenBank accession numbers for the 16S rDNA sequences of strains (BCYE) medium normally used to grow legionellae Gre! oux 11 D13T, Montbe! liard A1T, ATCC 33737T and ATCC 51914T are and on all other media tested (Adeleke et al., 1996; AF122883–AF122886, respectively. Hookey et al., 1996). 01664 # 2001 IUMS 1949 F. Lo Presti and others Routine identification of these bacteria is based on on blood agar and on BCYEα without both -cysteine and phenotypic properties (biochemical tests, serotyping), iron. The plates were incubated at 37 mCinan80%H#O but molecular biology techniques have resulted in the atmosphere, with or without 2n5% CO#. development of new methods giving a more reliable Bacterial strains. The eight atypical Legionella isolates characterization of Legionella species. Genotyping (Table 1) were compared with the species and serogroup methods such as ribotyping (Grimont et al., 1989), type strains of 42 Legionella species and 65 serogroups 16S–23S intergenic spacer region PCR (Riffard et al., (Table 3). Included in this study were some potentially novel 1998), 16S or 5S rRNA gene sequencing (Fry et al., species. The as yet not formally described ‘Legionella 1991; Adeleke et al., 1996; Birtles et al., 1996; Hookey donaldsonii’ (strain ‘Glasgow’ 86\35784; a gift from T. G. et al., 1996; Murdoch et al., 1999), macrophage Harrison, Colindale, UK) and strain IB V no. 2 were also mip compared with our strains. As amoebal cultures were not infectivity potentiator ( ) gene sequencing (Ratcliff available, one validly published species, Legionella lytica, et al., 1998) and randomly amplified polymorphic could not be used for the DNA hybridization studies DNA (RAPD) (Lo Presti et al., 1998) have been shown (Hookey et al., 1996). to be useful in classifying isolated strains to the species level. These methods are used to supplement DNA Cultural and biochemical tests. Each strain tested was relatedness studies, which are used to confirm the cultured onto BCYEα or GVC medium when required. Requirement for cysteine on subculture was tested by its designation of strains as novel species. Strains that omission when preparing the BCYEα media. Auto- cannot be recovered onto BCYE can be first charac- fluorescence of the strains was tested by examining cultures terized by 16S rDNA sequencing, which allows them under Wood’s light (366 nm). Tests for catalase, oxidase, β- to be classified as legionellae. lactamase, hippurate hydrolysis, urease, glucose utilization, Routine investigations of French water from different gelatinase, nitrate reduction and presence of flagella were origins (hospital and thermal spa) frequently lead to done as previously described (Brenner et al., 1985). Brown- ing of tyrosine-supplemented agar was considered positive if the isolation of untypable isolates. After phenotypic the browning produced around the colonies was larger on characterization, isolates remaining unidentified have tyrosine-supplemented agar than any browning produced been completely typed by genotypic methods. These around the colonies of the same strain on tyrosine-free agar experiments have allowed identification of two novel after 3 d growth (Vickers & Yu, 1984). The colour of the species, Legionella beliardensis sp. nov. and Legionella colonies was also noted on plates containing DGVP agar, a gresilensis sp. nov., isolated from different French GVP medium modified by the addition of dyes (bromocresol water samples. purple and bromothymol blue) allowing a visual differ- entiation between Legionella (Tatlockia) micdadei, Legion- METHODS ella (Tatlockia) maceachernii (colonies are blue) and the other species of Legionella (colonies are green) (Vickers et Isolation procedure. Water samples (1 l) were collected from al., 1987). three different locations in France: a thermal spa in the city Direct immunofluorescence assays. The eight Legionella of Gre! oux-les-Bains, a potash mine near Mulhouse and a isolates were identified by performing direct immuno- hospital in Montbe! liard. These samples were concentrated fluorescence assays (DFA) using 60 hyperimmune rabbit by continuous centrifugation and\or filtration to a 10 ml antisera prepared as described previously (Cherry et al., final volume and either left untreated or treated by acid- 1978). Antisera were not available for four species: Legion- ification according to previously described procedures ella genomospecies 1, Legionella lansingensis, Legionella et al (Bornstein ., 1989). The resulting samples were cultured shakespearei and Legionella waltersii. Conversely, all of the on: BCYE agar plates supplemented with 0n1% 2-oxo- 65 serogroup reference strains were tested by DFA using glutarate (BCYEα); BCYEα supplemented with glycine T −" −" −" antisera prepared against strain Gre! oux 11 D13 and against (3 g l ), vancomycin (5 mg l ) and colistin (5 mg l ) (GVC strain Montbe! liard A1T. medium); and BAB agar which is a BCYE medium " supplemented with colistin (16 mg l− ), vancomycin Slide agglutination test. Both type strains were tested at " " (0n5mgl− ) and cefalothin (4n23 mg l− ). Controls were done the Centers for Disease Control (Atlanta, GA, USA) with Table 1. Biochemical characteristics of the isolates ................................................................................................................................................................................................................................................................................................................. , Weak; j, weakly positive; , variable. Strain Oxidase β-Lactamase