International Journal of Systematic and Evolutionary Microbiology (2010), 60, 801808

DOI 10.1099/ijs.0.011148-0

Brucella inopinata sp. nov., isolated from a breast implant infection

Holger C. Scholz,1 Karsten No ckler,2 Cornelia Go llner,2 Peter Bahn,2 Gilles Vergnaud,3,4 Herbert Tomaso,1 Sascha Al Dahouk,5 Peter Ka mpfer,6 Axel Cloeckaert,7 Marianne Maquart,7 Michel S. Zygmunt,7 Adrian M. Whatmore,8 Martin Pfeffer,1 Birgit Huber,9 Hans-Ju rgen Busse9 and Barun Kumar De10
Correspondence Holger C. Scholz [email protected]
1 2 3

Bundeswehr Institute of Microbiology, Neuherbergstrasse 11, D-80937 Munich, Germany Federal Institute for Risk Assessment, Diedersdorfer Weg 1, D-12277 Berlin, Germany DGA/D4S Mission pour la Recherche et lInnovation Scientifique, 7, rue des Mathurins, 92220 Bagneux, France

Paris-Sud 11, CNRS, UMR8621, Institut de Ge ne tique et Microbiologie, 91405 Orsay, Universite France RWTH Aachen University, Department of Internal Medicine III, Pauwelsstrae 30, D-52074 Aachen, Germany Institute for Applied Microbiology, Justus-Liebig-Universitat Giessen, IFZ, Heinrich-Buff-Ring 26-32, D-35392 Giessen, Germany Publique, IASP, F-37380 Nouzilly, France INRA, UR1282, Infectiologie Animale et Sante Veterinary Laboratories Agency, Woodham Lane, Addlestone KT15 3NB, UK

7 8 9

Institut fu rmedizinische Universita t, A-1210 Wien, r Bakteriologie, Mykologie und Hygiene, Veterina Austria Centers for Disease Control and Prevention, 1600 Clifton Rd, Atlanta, GA 30333, USA

10

A Gram-negative, non-motile, non-spore-forming coccoid bacterium (strain BO1T) was isolated recently from a breast implant infection of a 71-year-old female patient with clinical signs of brucellosis. Affiliation of strain BO1T to the genus Brucella was confirmed by means of polyamine pattern, polar lipid profile, fatty acid profile, quinone system, DNADNA hybridization studies and by insertion sequence 711 (IS711)-specific PCR. Strain BO1T harboured four to five copies of the Brucella-specific insertion element IS711, displaying a unique banding pattern, and exhibited a unique 16S rRNA gene sequence and also grouped separately in multilocus sequence typing analysis. Strain BO1T reacted with Brucella M-monospecific antiserum. Incomplete lysis was detected with bacteriophages Tb (Tbilisi), F1 and F25. Biochemical profiling revealed a high degree of enzymic activity and metabolic capabilities. In multilocus VNTR (variable-number tandem-repeat) analysis, strain BO1T showed a very distinctive profile and clustered with the other exotic Brucella strains, including strains isolated from marine mammals, and Brucella microti, Brucella suis biovar 5 and Brucella neotomae. Comparative omp2a and omp2b gene sequence analysis revealed the most divergent omp2 sequences identified to date for a Brucella strain. The recA gene sequence of strain BO1T differed in seven nucleotides from the Brucella recA consensus sequence. Using the Brucella species-specific multiplex PCR assay, strain BO1T displayed a unique banding pattern not observed in other Brucella species. From the phenotypic and molecular analysis it became evident that strain BO1T was clearly different from all other Brucella species, and therefore represents a novel species within the genus Brucella. Because of its unexpected isolation, the name Brucella inopinata with the type strain BO1T (5BCCN 09-01T5CPAM 6436T) is proposed.

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H. C. Scholz and others

The genus Brucella was constituted in 1920 by Meyer & Shaw (1920) with Brucella melitensis, the causative agent of Malta fever, as the type species and Brucella abortus causing abortion in cattle as the second species. Within 48 years four additional Brucella species, namely Brucella suis (Huddleson, 1929), Brucella ovis (Buddle, 1956), Brucella neotomae (Stoenner & Lackman, 1957) and Brucella canis (Carmichael & Bruner, 1968) were described. It took nearly another 40 years before two additional species, Brucella pinnipedialis and Brucella ceti, originating from marine mammals were published (Foster et al., 2007). The most recently published description of a Brucella species is that for Brucella microti, isolated initially from common voles in the Czech Republic (Hubalek et al., 2007; Scholz et al., 2008a) and recently also from red foxes in Lower Austria (Vulpes vulpes) and directly from soil (Scholz et al., 2009, 2008c). Before the development of molecular techniques, differentiation of the various Brucella species and their biovars was solely based on phenotypic traits, i.e. CO2 requirement, H2S production, dye sensitivity, metabolic profiles, lysis by Brucella-specific bacteriophages and agglutination with monospecific antisera. Another important criterion for the differentiation of the various Brucella species was the natural host preference, which is why species names were given in accordance with their preferred host. Today it is known that all Brucella species are highly related at the genetic level, exhibiting identical 16S rRNA (Gee et al., 2004) and recA gene sequences (Scholz et al., 2008b) as well as highly similar genomes in terms of sequence identity and gene synteny (Del Vecchio et al., 2002; Paulsen et al., 2002; Halling et al., 2005). In the majority of housekeeping genes only single nucleotide polymorphisms are present among the various Brucella species (Whatmore et al., 2007). Indeed, applying the gold standard for species delineation, DNADNA hybridization (Wayne et al., 1987), all recognized Brucella species belong to a single species, as the DNADNA relatedness among all species is above 70 % (Verger et al., 1985). Consequently, Verger et al. (1985) suggested Brucella as a monospecific genus with B. melitensis as the sole true species and the other species to be recognized as biovars. This perception was later confirmed by using data from comparative whole genome
Abbreviations: MLST, multilocus sequence typing; MLVA, multilocus VNTR (variable-number tandem-repeat) analysis; RTD, routine test dilution. The GenBank/EMBL/DDBJ accession numbers for the omp2a, omp2b, omp25, omp31 and recA gene sequences of strain BO1T are FM177715, FM177716, FM177717, FM177718 and FM177719, respectively. Figures showing lysis of strain BO1T on Brucella agar by bacteriophage Tb (Tbilisi), a two-dimensional thin-layer chromatogram of total polar lipids of strain BO1T, IS711 element fingerprinting by using Southern blotting and a condensed dendrogram of clustered MLVA-16 genotypes obtained with more than 470 Brucella isolates corresponding to 324 different genotypes are available as supplementary material with the online version of this paper.

sequencing analysis of B. melitensis (Del Vecchio et al., 2002), B. suis (Paulsen et al., 2002), and more recently of Brucella abortus strain 9-941 (Halling et al., 2005) and B. abortus vaccine strain S19 (Crasta et al., 2008). However, in 2003, the Subcommittee on the Taxonomy of Brucella agreed unanimously on a return to pre-1986 Brucella taxonomy and reapproval of the six Brucella nomenspecies and their biovars (Meeting in Pamplona, n Spain in 2003), published in 2006 by Osterman & Moriyo (2006). Following the currently used Brucella taxonomy we describe a novel Brucella species, Brucella inopinata sp. nov., with the type strain BO1T, isolated from breast implant wound fluid and blood of a 71-year-old female patient with clinical symptoms consistent with brucellosis (De et al., 2008). B. inopinata sp. nov. is the first Brucella species that exhibits a unique 16S rRNA gene sequence and lower sequence similarities in various housekeeping genes and genes encoding outer-membrane proteins when compared with all other recognized Brucella species and hence represents the most unique species within this genus. Phenotypic analysis of strain BO1T Phenotypic analysis of strain BO1T, i.e. growth behaviour on different media at various temperatures, H2S production, CO2 requirement, agglutination with monospecific Aand M-antisera, cellular fatty acid analysis, lysis with bacteriophage Tb and growth in the presence of dyes (thionin and basic fuchsin), have been described previously (De et al., 2008) and are given in the species description. The antimicrobial susceptibility pattern of strain BO1T was determined using the CLSI interpretive criteria for Brucella spp. (De et al., 2008). Results are given in the species description. Additional phenotypic characterization of strain BO1T carried out in this study comprised other growth characteristics, standard biochemical characterization by using API 20E, API 20NE and API ZYM rieux), extended biochemical profiling using the (bioMe MICRONAUT (MERLIN Diagnostika GmbH) system, electron microscopy, and lysis with additional Brucella-specific bacteriophages, F1 and F25, as described for B. microti (Scholz et al., 2008a). The growth characteristics of strain BO1T resembled those of B. microti, characterized by very fast growth at 37 uC on various standard media, such as sheep blood agar and standard nutrient agar (both from Oxoid). On Brucella agar (Merck), growth became visible within 6 h of incubation at 37 uC with or without supplementary CO2 (data not shown). Single colonies of 12 mm were formed within 1224 h. As determined by using transmission electron microscopy (negative staining with 1 % uranyl acetate) with a JEM-1010 electron microscope at a magnification of 40 000, cells were non-flagellated and arranged individually or in irregular clusters, exhibiting a
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Brucella inopinata sp. nov. from a breast implant infection

mean diameter of 0.5 mm and a length of 1 mm (Fig. 1a and b). Strain BO1T agglutinated weakly with monospecific anti-M serum up to a dilution of 1 : 40 but not with anti-A serum. Incomplete lysis was observed with bacteriophages Tb, F1 and F25 at 1046routine test dilution (1046RTD) but not at any other dilutions (Supplementary Fig. S1, in IJSEM Online). This finding is in contradiction to previous results (De et al., 2008), in which no lysis could be detected with phage Tb. This can be explained by the rapid growth of strain BO1T. In order to detect lysis, the standard protocol had to be modified in terms of bacteria quantities and incubation periods. Briefly, Brucella agar plates were flooded with 5 ml Brucella bouillon including 5 ml of a 1011 cell suspension of BO1T ml21. Medium in excess was removed immediately and the plates were dried at 37 uC for 2 h before the various phage dilutions were applied. The incubation time of the phage dilutions was 10 s. Plates were incubated at 37 uC and inspected for phage lysis after 12 h. As described previously for B. microti (Scholz et al., 2008a), strain BO1T exhibited outstanding metabolic capabilities in comparison with recognized Brucella species, sharing a series of reactions with Ochrobactrum anthropi LMG 3331T and Ochrobactrum intermedium LMG 3301T. The results of the differential biochemical reactions of strain BO1T in

comparison with other Brucella species and their biovars as well as with Ochrobactrum species are summarized in Table 1. Hence, B. inopinata sp. nov. is the second fast-growing Brucella species with a highly active metabolism. Using the API 20E and API 20NE test systems, strain BO1T was misidentified as Ochrobactrum anthropi with identification scores of 98 and 99 %, respectively. Detailed results of API 20E, API 20NE, and API ZYM test systems are given in the species description. Quinones and polar lipids were extracted and analysed as described by Tindall (1990a, b) and Altenburger et al. (1996). The quinone system consisted of the major compound ubiquinone Q-10 (99 %) and ubiquinone Q-11 (1 %). The polar lipid profile consisted of five major compounds: phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, phosphatidylcholine and one unknown aminolipid (Supplementary Fig. S2, in IJSEM Online). The unknown aminolipid exhibited the same chromatographic behaviour as the unknown aminolipid AL1, which was also reported to be present in the type strains of B. microti, B. melitensis and B. abortus (Scholz et al., 2008a). In addition, moderate amounts of phosphatidylmonomethylethanolamine and minor to trace amounts of an unknown aminophospholipid and an unknown phospholipid corresponding to APL2 and PL, respectively, which were detected in B. microti, as well as four unknown polar lipids were detected. One of these unknown polar lipids showed the same chromatographic behaviour as the lipid L4 present in B. microti but not in other Brucella species (Scholz et al., 2008a). Unknown aminolipid AL2 found in B. microti (Scholz et al., 2008a) as a major compound was not detectable. Hence, the polar lipid profile clearly distinguished strain BO1T from B. microti as well as from B. melitensis and B. abortus based on the presence/absence of unknown aminolipid AL2 and unknown polar lipid L4. The polyamine pattern contained the major compounds spermidine (45 %) and putrescine (43 %) and small amounts of 1,3-diaminopropane (7 %), sym-homospermidine (1 %) and spermine (1 %). This polyamine pattern is in good agreement with those of other Brucella species (Scholz et al., 2008a). The whole-cell cellular fatty acid profile of strain BO1T was similar to those of B. abortus, B. melitensis, B. neotomae, B. ovis and B. suis, characterized by the presence of major amounts (546 %) of C19 : 0 cyclo 11-12, C18 : 1v7c, C18 : 0 and C16 : 0 and smaller amounts (14 %) of Br-C19 : 1. The antimicrobial susceptibility pattern of strain BO1T was comparable with profiles of other Brucella species described by Jevitt et al. (2005), with the exception of resistance to various cephalosporins (cefoxitin, cephotiam and cefuroxim). Details are given in the species description.

Fig. 1. Transmission electron micrographs (magnification of 40 000) showing two inactivated non-flagellated B. inopinata BO1T cells (a) and a cluster of B. inopinata BO1T cells (b). Bars, 0.2 mm (a) and 1 mm (b). http://ijs.sgmjournals.org

Molecular analyses of strain BO1T Previous molecular analyses of strain BO1T comprised 16S rRNA gene sequencing, DNADNA hybridization experi803

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Table 1. Differential physiological reactions of B. inopinata sp. nov. BO1T in comparison with other Brucella and Ochrobactrum species
Taxa: 1, B. microti CCM 4915T; 2, B. inopinata sp. nov. BO1T; 3, B. abortus strains: 544T (5NCTC 10093T) (bv. 1), 86/8/59 (5NCTC 10501) (bv. 2), Tulya (5NCTC 10502) (bv. 3), 292 (5NCTC 10503) (bv. 4), B3196 (5NCTC 10504) (bv. 5), 870 (5NCTC 10505) (bv. 6), 63175 (5NCTC 10506) (bv. 7), C68 (5NCTC 10507) (bv. 9); 4, B. melitensis strains: 16MT (5NCTC 10094T) (bv. 1), 63/9 (5NCTC 10508) (bv. 2), Ether (5NCTC 10509) (bv. 3); 5, B . suis strains: 1330T (5NCTC 10316T) (bv. 1), Thomsen (5NCTC 10510) (bv. 2), 686 (5NCTC 10511) (bv. 3), reference 40 (bv. 4), reference 513 (bv. 5); 6, B. ovis 63/290T (5NCTC 10512T); 7, B. canis RM6/66T (5NCTC 10854T); 8, B. neotomae 5K33T (5NCTC 10084T); 9, B. pinnipedialis NCTC 12890T; 10, B. ceti NCTC 12891T; 11, O. anthropi LMG 3331T; 12, O. intermedium LMG 3301T. oNP, o-Nitrophenyl; pNP, p-nitrophenyl; QO2N, mean of 10 repetitions per reaction. +, Positive (QO2N values .70); 2, negative (QO2N values ,50); V, variable (QO2N values .50 and ,70). Compound
L-Glutamine L-Histidine D-Histidine L-Citrulline DAPI Sarcosine hydrochloride

1 + + + + + + + + + + + + + 2 + + + +
V

2 + + + + + + + + + + + + + 2 + + + + + + + + + + + + + + + + 2 + + +

3 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2

4 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2

5 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2
V

6 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2

7 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2
V

8 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2

9 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2

10 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2

11 + + + + + + + + + 2 2 +
V

12 + + + +

Substrate class Amino Amino Amino Amino acid acid acid acid

hydrochloride

Fumaric acid Succinic acid a-Ketoglutaric acid Itaconic acid Mesaconic acid Glutaric acid Arginine p-nitroanilide L-Pyroglutamic acid b-naphthylamide pNP N-Acetyl-b-D-galactosaminide (pH 7.5) oNP N-Acetyl-a-D-galactosaminide (pH 7.5) oNP N-Acetyl-b-D-galactosaminide (pH 7.5) pNP N-Acetyl-b-D-glucosaminide (pH 7.5) pNP N-Acetyl-1-thio-b-D-glucosaminide (pH 7.5) 4-Nitrophenyl-a-D-maltoheptaoside-4,6,O-ethylidene (pH 7.5) pNP N-Acetyl-b-D-glucosamide (pH 5.5) pNP N-a-L-Arabinopyranoside (pH 7.5) oNP N-b-D-Xylopyranoside (pH 7.5) pNP N-1-thio-b-D-Galactopyranoside (pH 7.5) 2-Methoxy-4-(2-nitrovinyl)-phenyl b-Dgalactopyranoside pNP b-L-Fucopyranoside (pH 7.5) pNP b-D-Thiofucopyranoside (pH 7.5) pNP b-D-Maltoside (pH 7.5) pNP b-D-Lactopyranoside (pH 7.5) pNP b-D-Glucuronide (pH 5.5) Aesculin hydrolysis VogesProskauer reaction

+
V

+ Amino acid + Amino acid derivative + Org. acid + Org. acid + Org. acid 2 Org. acid 2 Org. acid + Org. acid + Amino peptidase + Amino peptidase + + + + + + 2 + + + + + + + + 2 + Esterase Esterase Esterase Esterase Esterase Esterase Esterase Glucosidase Glucosidase Glucosidase Glucosidase

+ + + + + + + + +
V

+ + + + + + + + + + + + + + + + + + +

+ 2 +

2 2 2

2 2 2

+ oNP b-D-Galactopyranoside-6-phosphate pNP Phosphate-di(2-amino-2-ethyl-1,3-propanediol) + (pH 5.5)

Glucosidase Glucosidase Glucosidase Glucosidase Glucosidase Classical reaction + Classical reaction + Phosphatase + Phosphatase

ments, multilocus sequence typing (MLST) analysis and IS711-specific PCR (De et al., 2008). DNADNA reassociation of .70 % between strain BO1T and the type strains of B. melitensis (80 %) and B. suis (78 %) as well as the
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presence of the Brucella-specific IS711 element clearly demonstrated that strain BO1T is a member of the genus Brucella. However, the data from 16S rRNA gene sequencing and MLST analysis also revealed that strain BO1T was
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markedly different from all recognized Brucella species and their biovars. For example, strain BO1T is characterized by a unique 16S rRNA gene sequence differing in five nucleotides when compared with the 16S rRNA gene consensus sequence of Brucella species and also exhibits a significantly lower level of sequence similarity in various housekeeping genes (De et al., 2008). In this study, we have additionally sequenced the recA, omp2a/b, omp25 and omp31 genes as described for B. microti (Scholz et al., 2008a). As shown previously, all recognized Brucella species, including B. microti, are identical in their recA gene sequences (Scholz et al., 2008b). However, the partial recA gene sequence of strain BO1T (GenBank no. FM177719) differed in seven of 631 nucleotides (98.9 % identity). These findings again emphasized the unique position of strain BO1T within Brucella. As in MLST analysis, the various omp gene sequences of strain BO1T also

exhibited lower sequence similarities. The omp31 gene (723 bp) was only 96 % identical to the respective sequences of other Brucella species. The omp25 gene (642 bp) sequence of strain BO1T was most closely related to omp25 of B. suis ATCC 23445 (GenBank no. CP000911) with a similarity of 99 %. In the phylogenetic analysis using the neighbourjoining method, the omp2a and omp2b gene sequences of BO1T were most closely related to omp2a/b of Brucella strain 83-210 (83/13), an isolate with uncertain affiliation (Fig. 2), and differed considerably (9798 % sequence similarity) from omp gene sequences of other Brucella species. In addition to sequencing of the above-mentioned genes, strain BO1T was characterized by using AMOS PCR (Bricker & Halling, 1994) and species-specific multiplex PCR (Garcia-Yoldi et al., 2006). In multiplex PCR analysis a unique pattern of six PCR fragments (150 bp, 272 bp, 450 bp, 587 bp, 794 bp and 1682 bp), not observed in

Fig. 2. Phylogenetic tree reconstructed with omp2a (1104 nt) and omp2b (1089 nt) sequences using CLUSTREE neighbourjoining analysis (Thompson et al., 1994) and Kimura distances. The significance of each branch is indicated by a bootstrap value calculated for 1000 subsets. The tree was rooted by outgrouping sequence omp2a of O. anthropi LMG 3331T. Bar, 0.1 divergent residues per site. http://ijs.sgmjournals.org 805

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other Brucella species, was generated. AMOS PCR was negative using all primer combinations (data not shown). Finally, strain BO1T was typed by using IS711 fingerprinting using the Southern blotting technique and by multilocus VNTR (variable-number tandem-repeat) analysis (MLVA) using eight mini- and eight micro-satellite VNTR markers as described recently for B. microti (Scholz et al., 2008a). Of the Brucella-specific insertion sequence IS711, four to five copies, displaying a unique banding pattern, were detected (Supplementary Fig. S3, in IJSEM Online). The MLVA typing pattern obtained was significantly different from those for previously analysed strains ` che et al., 2006; Al Dahouk et al., 2007). A new (Le Fle intermediate size allele was observed at locus Bruce42 (coded 2.5). No amplification product could be obtained at four loci, bruce07, bruce18, bruce21 and bruce30 (Le ` che et al., 2006), an exceptional behaviour not observed Fle previously. All these loci are associated with an interspersed repeat element (containing one or two octameric tandem repeats) present in about 20 locations in B. melitensis 16MT strain as reported previously by Bricker et al. (2003). This suggests that these four loci are the result of recent spreading events of this presumably mobile element within the Brucella genus, after the occurrence of the last common ancestor to B. inopinata sp. nov. and to the other Brucellae. Alternatively, these four loci might have been lost or rearranged along the inopinata lineage as a result of their peculiar structure. MLVA cluster analysis using the categorical distance coefficient and the unweighted pairgroup method with arithmetic averages (UPGMA) was carried out as described previously (Scholz et al., 2008a). In these analyses, strain BO1T clustered together with the B. microti, B. suis biovar 5 and B. neotomae strains and three representative marine mammal strains (Supplementary Fig. S4, in IJSEM Online) in a very loosely connected group with long branches. The inopinata branch is in proportion markedly shorter than the relatively very long branch observed by using MLST (De et al., 2008). This might be because of the inherent homoplasy at MLVA loci. Furthermore, relatively long evolutionary distances will be underestimated by MLVA as 16 markers were used in this MLVA assay, and the distance is the categorical coefficient; an MLVA distance between two strains cannot be more than 16. Another possibility is that VNTR markers do not evolve as fast in these presumably environmental species, in comparison with the highly pathogenic and host-adapted variants (the more classical Brucellae), which are under different stress conditions. However, as yet nothing is known about the natural reservoir or the host-range of this novel Brucella species. From the phenotypic and molecular data it was obvious that strain BO1T belonged to the genus Brucella but also differed from all recognized species and their biovars. We therefore conclude that BO1T represents a novel species of the genus Brucella, for which the name Brucella inopinata sp. nov. is proposed.
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Description of Brucella inopinata sp. nov. Brucella inopinata (in.o.pi.na9ta. L. fem. adj. inopinata unexpected). Aerobic, non-fermentative, non-motile, non-spore-forming, Gram-negative coccobacilli or short rods, 0.50.7 mm in diameter and 0.61.2 mm in length arranged individually or in irregular clusters. Non-fastidious, fast growth (visible within 6 h) occurs on Brucella agar, trypticase soy agar (Becton Dickinson), sheep blood agar and standard nutrient agar at 2542 uC. Positive for growth on MacConkey agar (Becton Dickinson). Smooth, opaque colonies are formed within 18 h, with a diameter of approximately 12 mm. Good growth does not require CO2, supplementary serum or blood. No haemolysis is observed. Growth occurs in the presence of thionin at concentrations of 1/25 000, 1/50 000 and 1/100 000, and with basic fuchsin at concentrations of 1/50 000 and 1/100 000. Incomplete lysis occurs with bacteriophages Tb, F1 and F25 at 1046RTD, but not at other dilutions. Agglutinates with monospecific anti-M serum up to a dilution of 1 : 40. Oxidase- and catalase-positive. Fast urease reaction (,5 min). Growth occurs in nutrient broth with or without 6 % NaCl. Nitrate and nitrite are reduced (with gas formation from nitrate). Production of H2S and VogesProskauer reaction are positive. Negative for hydrolysis of aesculin, gelatin liquefaction, production of indole, citrate utilization, growth on cetrimide and SalmonellaShigella (SS) agar. Acid production is observed in Kings oxidationfermentation base from D-glucose and D-xylose, whereas no acid production is observed in Kings oxidationfermentation base from mannitol, lactose, sucrose and maltose. Positive (API ZYM) for acid phosphatase, alkaline phosphatase, trypsin, leucine arylamidase and naphthol-AS-BI-phosphohydrolase. Negative (API ZYM) for esterase, esterase lipase, lipase, valine arylamidase, cystine arylamidase, a-chymotrypsin, a- and b-galactosidase, b-glucuronidase, a- and b-glucosidase, N-acetyl-b-glucosaminidase, a-mannosidase and a-fucosidase. Positive (API 20NE) for D-glucose, maltose, L-arabinose, D-mannose, N-acetylglucosamine and adipic acid. Negative for D-mannitol, citric acid, gluconate, capric acid, malic acid and phenylacetic acid. In API 20E, positive for fermentation of L-arabinose. Additional differential physiological reactions (MICRONAUT) of strain BO1T in comparison with other Brucella species and biovars and O. anthropi LMG 3331T are given in Table 1. Susceptible to the following antimicrobial agents: doxycycline (0.12 mg ml21), tetracycline (0.25 mg ml21), streptomycin (2 mg ml21), gentamicin (1 mg ml21) and trimethoprimsulfamethoxazole (,0.5 and 9.5 mg ml21). Major whole-cell cellular fatty acids (546 %) are C19 : 0 cyclo 11-12, C18 : 1v7c, C18 : 0 and C16 : 0, and smaller amounts (14 %) of Br-C19 : 1. Quinone system is composed of the major compound ubiquinone Q-10 and minor amounts of Q-11. The polar lipid profile contains the major compounds phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, phosphatidylcholine and one unknown
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aminolipid, moderate amounts of phosphatidylmonomethylethanolamine and minor to trace amounts of an unknown aminophospholipid, an unknown phospholipid and two unknown polar lipids. The polyamine pattern consists of the major compounds spermidine and putrescine and small amounts of 1.3-diaminopropane, symhomospermidine and spermine. The type strain, BO1 (5BCCN 09-01 5CPAM 6436 ), was isolated in 2005 from a breast implant infection of a 71-year-old female patient in the USA.
T T T

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confirmatory identification of Brucella isolates. J Clin Microbiol 42, 36493654.

Halling, S. M., Peterson-Burch, B. D., Bricker, B. J., Zuerner, R. L., Qing, Z., Li, L. L., Kapur, V., Alt, D. P. & Olsen, S. C. (2005).

Acknowledgements
We are grateful to Csilla Lodri, Stefan Schatz, Robert Schneider, and Angelika Draeger for excellent technical assistance. The development of genotyping methods for the precise strain identification of dangerous pathogens as part of microbial forensics is supported by le gation Ge ne rale pour lArmement). MRIS and the French DGA (De BIM are members of the European Biodefense Laboratory Network (EBLN) supported by the European Defence Agency (EDA). We ` che for the MLVA typing and Mark S Koylass for thank Philippe Le Fle MLST analysis of strain BO1T.

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