INTRODUCTION

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Streptococci that showed satellite growth around colonies of other microorganisms were first described by Frenkel and Hirsch in 1961 (6). They are the normal flora of the human mouth, pharynx, and intestinal and urogenital tracts and are often isolated from patients with so-called culture-negative bacterial endocardititis, bacteremia, and foci of various infectious diseases (2, 13, 16).

These organisms had formerly been known as nutritionally variant streptococci (NVS) and were supposed to be auxotrophic variants of viridans group streptococci (13). NVS require pyridoxal hydrochloride (vitamin B₆) analogs for growth and produce chromophore, pyrrolidonyl arylamidase, and bacteriolytic enzyme in common with some viridans group streptococci (2, 4, 7, 12, 16). However, NVS show considerable variations in their glycosidase and peptidase production profiles (1, 7) and the electrophoresis patterns of their penicillin-binding proteins (4). The species of clinical NVS isolates are often indicated as Gemella morbillorum (Gemella-like NVS) by the rapid identification systems (5, 7, 21). Four broad biotypes have been demonstrated so far in this unique group of cocci (4, 7).

The earlier DNA-DNA hybridization study divided fastidious NVS into two species, Streptococcus defectivus and Streptococcus adjacens (2). They were then transferred to the species Abiotrophia defectiva and Abiotrophia adiacens, respectively, on the basis of 16S rRNA gene sequence homology (8). A third, more fastidious species, Abiotrophia elegans, has recently been added to the genus Abiotrophia (14, 15). We have found that A. defectiva, A. adiacens, and A. elegans comprise 8, 84, and 8% of the NVS isolates from the human mouth, respectively (17). However, wider genetic heterogeneity among Abiotrophia spp. (18) and intraspecies variations in A. adiacens (3) have also been suggested.

The molecular genetic approaches such as restriction fragment length polymorphism (RFLP) analysis of PCR-amplified 16S rRNA genes and PCR assay of genomic DNA sequences have become applied to the specific identification and differentiation of Abiotrophia spp. (3, 11, 15). In the present study, we examined the genetic variations among 45 Abiotrophia strains including the type strains of A. defectiva, A. adiacens, and A. elegans and the oral isolates in our laboratory (7) and propose the presence of a fourth Abiotrophia species, Abiotrophia para-adiacens sp. nov. During the preparation of this article, a novel species, Abiotrophia balaenopterae sp. nov., isolated from a minke whale has been described (10); however, A. balaenopterae sp. nov. appears to be distinct from A. para-adiacens sp. nov. on the basis of some phenotypic properties that have been reported.

	MATERIALS AND METHODS

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Bacterial strains. Forty-five Abiotrophia strains were used (see Table 1). They included nine type or reference strains of A. defectiva (strains ATCC 49176^T, PE7, and NVS-47), A. adiacens (strains ATCC 49175^T, C50, L61, G40, and ATCC 27527), and A. elegans (strain DSM 11693^T), with all except one known to be isolates from patients with endocarditis or bacteremia (2, 4, 14, 20). Strain ATCC 27527 is a sputum isolate and is listed as Gemella morbillorum in the current American Type Culture Collection (ATCC) catalog but was confirmed to be an A. adiacens strain by the Rapid ID32 STREP system (Bio Mérieux SA, Marcy-l'Etoile, France) and other bacteriological and biochemical tests described previously (7) (see Results). The other 36 strains were from our laboratory and were isolates from the human mouth (7). The species of most of these strains have been determined and their phenotypes have been characterized, and some were phenotypically characterized and their species were provisionally determined to be A. defectiva, A. adiacens, or G. morbillorum (Gemella-like NVS) with the rapid identification system as described previously (7). The strains were biotyped with the emended typing system (17) and were serotyped (K. Kitada, T. Kanamoto, Y. Okada, and M. Inoue, submitted for publication). Strains of the other six genera and 23 species listed in Table 3 were used for comparisons; of these six species of the genera Streptococcus, Enterococcus, Dolosigranurum, and Aerococcus were bacteriolytic and 17 species of the genera Gemella, Streptococcus, Aerococcus, and Staphylococcus were nonbacteriolytic. Their Micrococcus luteus cell lysis activities were examined as described previously (7).

Preparation of genomic DNA. Abiotrophia strains were normally grown anaerobically at 37°C for 18 h in Todd-Hewitt (TH) broth (Difco, Detroit, Mich.) supplemented with 0.001% pyridoxal hydrochloride. A. elegans DSM 11693^T was cultured in TH broth containing 5% horse serum (BioWhittaker, Walkersville, Md.) in place of pyridoxal. The other strains were grown in TH broth. The DNA was extracted from cells and was purified as described previously (19). Briefly, bacterial cells were collected by centrifugation (5,000 × g, 15 min), washed three times in saline, and lysed at 37°C for 1 h with egg white lysozyme (30 U/ml; Sigma Chemical Co., St. Louis, Mo.) and mutanolysin (60 U/ml; Sigma) in 0.1× standard saline citrate (SSC; 1× SSC is 0.15 M NaCl plus 0.015 M trisodium citrate). The sample was then incubated at 60°C for 30 min with 0.8% sodium dodecyl sulfate (SDS; Wako Pure Chemical Industries, Tokyo, Japan) in 1× SSC. The extracted DNA was deproteinized with phenol and with a chloroform-isoamyl alcohol (24:1 [vol/vol]) mixture, digested with RNase (0.1 mg/ml), and precipitated with 70% ethanol.

DNA-DNA hybridization. A DNA hybridization probe was labeled with [-³²P]dCTP (111 TBq/mmol; New England Nuclear Research Co., Boston, Mass.) with an oligolabeling kit (Ready-To-Go DNA Labeling Beads; Amersham Pharmacia Biotech, Uppsala, Sweden).

PCR of genomic DNA sequences. Nucleotide sequences of the 3.3- and 5.7-kb KpnI fragments of strain TKT1 (homology group 3) DNA were determined, and primers specific for the identification of strains of A. adiacens DNA homology group 3 (PR1072, 5'-GCGTGAATGCCATCTATCAG-3' and 5'-ATCCACCAGTCTAAGAACTG-3') and sequences common to strains of the genus Abiotrophia (PR257, 5'-TTATGGTCAGGTGGTAGGAG-3' and 5'-ACTTGTTGGTCTCGCAGTCA-3') were synthesized (Amersham Pharmacia Biothch, Tokyo, Japan). Premix Taq (Takara Shuzo Co., Shiga, Japan) containing Taq DNA polymerase and PCR buffer (25 µl), template DNA (0.5 µl), and 0.2 µM primers were added to 50 µl of the PCR mixture. PCR was performed for 30 cycles with a profile of 94°C for 1 min and 64°C for 1 min and for an additional extension at 64°C for 10 min with a thermal cycler (Zymoreactor II; ATTO). The PCR products were stained with 2.5% ethidium bromide in an agarose gel. The correct sizes of the amplicons are 1,072 bp for the A. adiacens homology group 3-specific PCR and 257 bp for the genus Abiotrophia-common PCR.

RFLP analysis of PCR-amplified 16S rRNA gene. Primers (5'-AGAGTTTGATCATGGCTCAG-3' and 5'-ACGGGCGGTGTGTAC-3') which correspond to the Escherichia coli 16S rRNA gene (positions 8 to 17 and 1405 to 1391, respectively; Amersham Pharmacia Biotech) were used to amplify the 16S rRNA genes of the test strains (9). PCR was carried out as described above, and the amplicon obtained was digested at 37°C for 40 min with 5 U each of the restriction enzymes, EcoRI, XbaI, and HindIII (Nippon Gene, Osaka, Japan). Profiles of the digests were analyzed in a 2.5% agarose gel after staining with ethidium bromide.

16S rRNA gene sequence. The PCR product was cloned into a pCR2.1 vector (TA cloning kit; Invitrogen Corporation, Carlsbad, Calif.) and was cut into two EcoRI fragments. Their single-stranded DNAs were obtained by subcloning into M13mp18 and were sequenced by using a Thermo Sequenase premixed cycle sequence kit (Amersham) with an automatic sequencer (SQ5500; Hitachi Ltd., Tokyo, Japan). The sequence of strain TKT1 was compared with those of the other related strains obtained from the data deposited in the DNA Data Bank of Japan (DDBJ; Mishima, Shizuoka, Japan) and was analyzed to construct a phylogenetic tree by using DDBJ Super Computer (VPP500; Fujitsu, Tokyo, Japan) and the program ClustalW (supplied by DDBJ).

Nucleotide sequence accession numbers. DNA sequence data for the 16S rRNA genes obtained from DDBJ had the indicated accession numbers: A. defectiva ATCC 49176^T, D50541; A. adiacens ATCC 49175^T, D50540; A. elegans DSM 11693^T, AF016390; G. morbillorum ATCC 27824^T, L14327; and E. coli, A14565. The sequence of strain TKT1 has been deposited in DDBJ under accession no. AB022027.

	RESULTS

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DNA homology. By using the probe DNAs prepared from two strains each of NVS biotypes 1 to 4 (emended) (7, 17), the 45 strains tested were divided into four homology groups, although DNAs from a few homology group 3 and 4 strains gave low percent homologies (less than 50%) with all eight probe DNA preparations used (Tables 1 and 2). Homology group 1 strains were clearly distinguished from strains of the other groups. Homology group 2 and 3 strains were also separated, although their DNA homology levels were not so distantly related to each other; e.g., the mean ± standard deviation homologies were 75.7% ± 18.2% and 50.1% ± 16.9%, respectively, when the strains were probed with ATCC 49175 DNA. Homology group 4 strains constituted an independent group. In this group, DNA of A. elegans DSM 11693^T showed a low degree of homology (23.3%) when its DNA was hybridized with the HHC5 probe DNA, but the HHC5 DNA had a degree of high homology (70.7%) with the DSM 11693 probe DNA. Mean homology values for these two probe DNAs for the homology group 4 strains were very similar (63.6 versus 61.3%; Table 2).

PCR of genomic DNA sequences. The genetic variations among the Abiotrophia strains described above, particularly the differentiation of homology group 3 strains from the other strains, were confirmed by PCR-based assays.

16S rRNA gene PCR-RFLP analysis. Genetic heterogeneities were examined further by use of the 16S rRNA gene PCR-RFLP profiles of the DNA homology groups. All strains of A. adiacens homology group 2 (strains ATCC 49175^T, L61, C1-3, and C2-2) and group 3 (strains TKT1, TKT2, C4-1, HKT1-1, and ATCC 27527) tested gave four fragments of 0.21, 0.34, 0.40, and 0.46 kb. In contrast, strains of A. elegans homology group 4 (strains HHC5, YTM1, and DSM 11693^T) as well as strains of A. defectiva homology group 1 (strains ATCC 49176^T, PE7, and C8-3) had three fragments of 0.34, 0.40, and 0.68 kb. Thus, the four homology groups were not satisfactorily discriminated from each other by the PCR-RFLP analysis in this study, but the 16S rRNA gene of G. morbillorum ATCC 27824^T gave a unique profile with three fragments of 0.26, 0.48, and 0.68 kb.

16S rRNA gene sequence homology. The TKT1 16S rRNA gene sequence was 1,407 bp in length and included the forward and reverse primers in the 5' and 3' directions, respectively. A multiple alignment analysis showed that the sequence homologies between TKT1 and the type strains of A. adiacens, A. elegans, A. defectiva, and G. morbillorum were 99, 97, 92, and 85%, respectively. As depicted in an unrooted phylogenetic tree (Fig. 1), DNA homology group 3 strain TKT1 was most closely related to A. adiacens ATCC 49175^T (homology group 2) and was considerably more distantly related to the other Abiotrophia species (homology groups 4 and 1), and all the Abiotrophia type strains were remote in terms of their phylogenetic distance from G. morbillorum ATCC 27824^T.

View larger version (12K): [in this window] [in a new window]   FIG. 1.   The 16S rRNA gene sequence-based phylogenetic distance between strain TKT1 (DNA homology group 3) and A. defectiva (homology group 1), A. adiacens (homology group 2), and A. elegans (homology group 4).

Phenotypic characteristics of Abiotrophia genotypes. As described above, the genus Abiotrophia was differentiated into four genetic categories (designated genotypes 1 to 4). The physiological and serological properties of strains of these genotypes are summarized from the data reported previously (7, 17; Kitada et al.; submitted) (Table 4).

	DISCUSSION

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The Abiotrophia spp. are known to be pyridoxal dependent and to produce chromophore, pyrrolidonyl arylamidase, and bacteriolytic enzyme (2, 4, 7, 12, 16), but it has been known that the genus Abiotrophia represents a heterogeneous group of bacteria (1, 4, 15, 18) and three species and four biotypes of this unique genus have long been known (4, 7, 17). Very recently, during the preparation of this article, a new species has been added to the genus Abiotrophia (10).

The present studies demonstrated that the genus Abiotrophia is divided into four genetic groups. DNA-DNA hybridization analysis (Table 1) and analysis of the PCR product profiles of the genomic DNA and 16S rRNA gene sequences (Fig. 1; Table 3) differentiated the genotype 3 strains from the other strains and separated most strains of A. adiacens identified phenotypically (7) into genotypes 2 and 3, although the two genotypes appear to be rather closely related to each other. These results collectively indicate that along with A. defectiva (genotype 1), A. adiacens (genotype 2), and A. elegans (genotype 4), an additional Abiotrophia species (genotype 3) exists, and that it is most closely related to but distinct from A. adiacens (genotype 2). We propose here that Abiotrophia genotype 3 be called Abiotrophia para-adiacens sp. nov.

A. balaenopterae sp. nov., which was very recently described, was recovered from the lung of a minke whale and has also been shown to be closely related to but distinct from A. adiacens and A. elegans (10). The novel Abiotrophia species ferments pullulan but not sucrose and produces arginine dihydrolase and thus appears to be distinguished from A. para-adiacens which was segregated from A. adiacens (Table 4). The genetic relation between these two new species should be determined in further studies.

The A. para-adiacens (genotype 3) strains constituted a large cluster almost equal in size to the A. adiacens (genotype 2) cluster (Table 1). Both of these species, however, were mixture of biotype 2 and 3 (emended) strains (Tables 1 and 4) and were rather difficult to differentiate by the tests in the rapid identification system. However, the confidence level in the identification of the strain as A. adiacens by the Rapid ID32 STREP system was "excellent" to "good" for most of the genotype 2 strains (80.0%), whereas it was "low" to "id not valid" for most genotype 3 strains (76.9%) (7). In addition, so far tagatose fermentation appears to be a putative key property for the differentiation of these two species: generally, the A. adiacens strains produced -glucosidase and/or fermented tagatose, while the A. para-adiacens strains produced -glucosidase and/or did not ferment tagatose. Moreover, most strains of the former species were serotype II or III, but those of the latter species were serotype IV, V, or VI (Table 4). In fact, all except a few serotype II or III strains maintained in our laboratory (39 strains) ferment tagatose (89.7%), whereas only a few tagatose-positive but serotype IV, V, or VI strains (9.4%) are found in A. para-adiacens (Kitada et al., submitted).

In contrast, the remaining two Abiotrophia species were phenotypically distinct. A. elegans, which consisted of emended biotype 4 strains (17) of A. adiacens or Gemella-like NVS (7), was clearly separated from the other Abiotrophia species in that it produced arginine dihydrolase. A. defectiva corresponded strictly to NVS biotype 1 (7) and was also distinct from the others by the production of - and -galactosidases and the fermentation of trehalose (4, 7) (Table 4).

Not many A. elegans strains have so far been isolated (14, 15). They occupy ca. 10% or less of the clinical Abiotrophia isolates in our laboratory (17). A. elegans is reported to be more fastidious than the other Abiotrophia spp. and to show unique nutritional requirements: it does not use pyridoxal hydrochloride but uses L-cysteine in TH broth as a growth factor (14). In fact, unlike the other genotype 4 strains, the DSM 11693^T substrain that we maintain cannot grow in TH broth and other complex media supplemented with vitamin B₆ instead of horse serum (preliminary results). However, the substrain and the genotype 4 oral isolates resembled each other genetically, biochemically, and serologically (Tables 1, 3, and 4), thus forming a homologous cluster, A. elegans. Strains of the other Abiotrophia spp. did not hydrolyze arginine but A. elegans strains did hydrolyze arginine (17) (Table 4). The existence of an atypical A. adiacens strain which produces arginine dihydrolase has been reported (18), and A. elegans has been known to be genetically most closely related to A. adiacens (14, 17).

Strain ATCC 27527, known as G. morbillorum, was identified as A. adiacens by the rapid identification system (Table 1), showed phenotypic characteristics typical of those of Abiotrophia spp. (Table 4), and, together with some Gemella-like NVS and A. adiacens strains (7), belonged to genotype 3 and was not related to G. morbillorum ATCC 27824^T (Tables 1 and 3). Thus, strain ATCC 27527 is certainly a member of A. para-adiacens genotype 3. The genera Abiotrophia and Gemella are shown to be located far apart in genetic distance, as estimated by comparison of the 16S rRNA gene sequences (8, 17), but Abiotrophia strains are occasionally identified as G. morbillorum by the rapid identification tests (7) (Table 1) and are misidentified, as in the case of strain ATCC 27527 in the present study.

The PCR-based molecular approaches such as RFLP analysis of universal 16S rRNA PCR products have been attempted for the identification and differentiation of Abiotrophia spp. (8, 11, 15). Primer PR1072 was specific for the separation of A. para-adiacens from A. adiacens as well as A. defectiva and A. elegans. The PCR assay with primer PR1072 was negative for the nonbacteriolytic and bacteriolytic strains of the other six genera and 23 species tested, including 13 species of oral viridans group streptococci and 10 species including a bona fide G. morbillorum strain (Table 3). Thus, the PCR assay with PR1072 would facilitate the specific and sensitive identification of A. para-adiacens.