INTRODUCTION

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Nutritionally variant streptococci (NVS) were originally described by Frenkel and Hirsch in 1961 (7) as a new type of streptococci exhibiting satellitism around colonies of other bacteria. Isolates have been recovered from blood, abscesses, oral ulcers, and urethral samples (15). Because of both difficulties in culturing these organisms and the variety of appearances that they present on primary detection, such strains have caused major diagnostic difficulties. By DNA-DNA hybridization studies, Bouvet et al. in 1989 (3) demonstrated that NVS isolates could be divided into two groups, Streptococcus defectivus and Streptococcus adiacens; these two new species showed low DNA relatedness to reference strains of other Streptococcus species. In 1995, a new genus, Abiotrophia, was created, and the two species were transferred hereto as Abiotrophia defectiva and Abiotrophia adiacens (9). "Abiotrophia" means life nutrition deficiency and refers to the species' requirements for supplemented media for growth. Since then, three new species have been added, Abiotrophia elegans (human endocarditis patients) (13), Abiotrophia balaenopterae (isolated from a minke whale) (10), and most recently Abiotrophia para-adiacens (human endocarditis patients) (8), which has been proposed for some strains similar to A. adiacens. Phylogenetically, the genus Abiotrophia consists of two distinct lines, A. defectiva and a similar group consisting of A. adiacens, A. balaenopterae, and A. elegans (3, 4). Therefore, Collins and Lawson (4) recently have proposed that these last three species be reclassified in a new genus, Granulicatella.

Many strains of NVS have been received at the Centers for Disease Control and Prevention Streptococcus Laboratory over the years for confirmation of identification and species determination. In many cases, the species of the strains could not be determined because the phenotypic characteristics did not correlate with the species descriptions published in the literature. With the change of the genus identification and the addition of new species for this group of bacteria, we found it of interest to test a sample of the strains identified as NVS in the Centers for Disease Control and Prevention collection with more up-to-date procedures.

	MATERIALS AND METHODS

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Strains. One hundred one NVS strains were retrieved from 70°C storage. These strains were received from 37 different state health department laboratories.

Clinical data. Information given on the report submitted together with the isolate was recorded.

Phenotypic characteristics. Strains were grown on Danish blood agar (DBA) (5 and 10%; a peptone [5.0 g/1,050 ml] agar [10.0 g/1,050 ml] supplemented with cysteine HCl [0.05 g/1,050 ml]) and yeast extract (3.0 g/1,050 ml), and 5% sheep blood agar (SBA) (Trypticase soy agar with 5% sheep blood). The cultures were phenotypically characterized and compared with the type strains of G. adiacens (ATCC 49175^T), A. defectiva (ATCC 49176^T), and G. elegans (DSM 11693^T). Strains were examined for the following characteristics: Gram stain reaction; growth (satellitism and dependence on pyridoxal HCl); production of pyrrolidonyl aminopeptidase (PYR), leucine aminopeptidase (LAP), -galactosidase, -galactosidase, -glucuronidase, alkaline phosphatase, and -fucosidase (Rosco Diagnostic Tablets; A/S Rosco, Taastrup, Denmark); acetoin production (6); deamination of arginine (according to the method of Möller [6]); hippurate hydrolysis (tested in broth supplemented with 1% sodium hippurate [6] and with Rosco Diagnostic Tablets); and acid production from inulin, lactose, maltose, raffinose, sucrose, trehalose, amygdalin, salicin, pullulan, and tagatose (Difco phenol red broth base supplemented with 1% carbohydrate; tubes were observed for 14 days). Standard inoculum was 1 drop of an 18-h-old Todd-Hewitt broth culture supplemented with 5 drops of pyridoxal HCl (10 mg/liter) (repeated just before inoculation of tests) when testing for acid production from carbohydrates and 3 drops when testing for enzyme production.

	RESULTS

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Phenotypic characteristics. Many cultures were submitted with the presumptive identification of unknown bacterial species that grow on only chocolate agar and with pleomorphic Gram stain characteristics, much like the characteristics described by Bottone et al. (2). Isolates exhibited Gram stain variability and pleomorphism with forms ranging from bacilli with spore-like swellings to cocci arranged predominantly in pairs and chains when Gram stain preparations were made from agar plates. Addition of pyridoxal HCl (10 mg/liter; 5 drops in 7 ml of Todd-Hewitt broth or 5 drops in 5 ml of fluid thioglycolate broth) or growth on 10% DBA showed that isolates were arranged predominantly in pairs and chains. By recognition, isolates were alpha-hemolytic and showed satellitism around colonies of Staphylococcus epidermidis (except for a few strains, depending on inoculum size), and addition of pyridoxal HCl (streaking the plate with a swab immersed in pyridoxal HCl [10 mg/liter]) was required for and/or enhanced growth on blood agar plates. Isolates could be maintained on 10% DBA. For growth on 5 and 10% DBA and 5% SBA, the largest colonies were found on 5% SBA, followed by 10% DBA, on which colony sizes were approximately twice what was seen on 5% DBA; on 10% DBA, colonies were grayish-white and approximately 1 to 3 mm in diameter after 48 h. Approximately 10% of strains showed dimorphism, with small and large colony variants which by reculturing showed the same dimorphism and had identical phenotypic reactions. No growth was seen for any isolate in 6.5% NaCl.

Clinical data. Clinical data for 97 patients are given in Table 2. The lowest median age of patients was seen for patients from whom A. defectiva was isolated, which could be attributed to the presence of more patients with congenital heart disease in this group. Blood cultures were by far the most common source, though three isolates from eye ulcers were also included. Infective endocarditis was reported in 58% of patients for whom clinical diagnosis was reported, while in 26% of patients septicemia or bacteremia was given as the clinical diagnosis.

	DISCUSSION

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Microscopic appearance and results of Gram staining may vary considerably among NVS strains; in particular, the extremely pleomorphic appearance of NVS strains may be confusing (2, 15). Addition of pyridoxal HCl causes most isolates to convert to Streptococcus-like cellular arrangements and gram positivity. For the recently described species G. elegans, however, addition of cysteine HCl has this effect, but addition of pyridoxal HCl does not (13). The compositions of primary isolation media have changed considerably over time and are still being improved, influencing the description of growth characteristics. Furthermore, it seems that some isolates appear to adapt to growth without addition of pyridoxal or cysteine. Satellitism around S. epidermidis is an important phenotypic characteristic, as strains belonging to the miscellaneous group of Streptococcus-like Gemella strains (6) characteristically have not exhibited this feature. Some isolates; however, have adapted to growth well enough not to require satellitism or excessive inoculum, and perhaps carryover of primary isolation medium containing pyridoxal may be enough enrichment for satellitism not to be required.

Kanamoto et al. (8) recently investigated 45 Abiotrophia strains (including the type strains of A. defectiva, G. adiacens, and G. elegans) from endocarditis patients for DNA homology, PCR of genomic DNA sequences, 16S rRNA gene PCR-restriction fragment length polymorphism analysis, 16S rRNA gene sequence homology, and phenotypic characteristics. The endocarditis isolates could be divided into four genetic groups representing the three type strains and a new group closely related to G. adiacens. These investigators proposed that this new group be named A. para-adiacens sp. nov. The 45 endocarditis isolates were identified as 9 strains of A. defectiva, 15 strains of G. adiacens, 13 strains of A. para-adiacens, and 8 strains of G. elegans. Phenotypic characteristics useful in differentiating between species within the genera Abiotrophia and Granulicatella are production of - and -galactosidase; production of -glucuronidase; hippurate hydrolysis; arginine dihydrolase activity; and acid production from trehalose, sucrose, pullulan, and tagatose (1, 10; this study). The most often reported results for these reactions, including results from the present study, are given in Table 3. For the type strains, phenotypic reactions resembled results obtained for the majority of the clinical strains. However, the type strain of G. adiacens produced -galactosidase, in contrast to 51 of 55 clinical strains. Some of the phenotypic characteristics are, however, not 100% positive or negative (Table 1).

Twenty-three of the A. defectiva strains, 31 of the G. adiacens strains, and the 3 G. elegans strains examined in this study have been examined by partial 16S rRNA sequence analysis; species identifications based on phenotypic reactions and sequencing were in accordance for all strains (M. D. Collins, unpublished data). Molecular methods will in the future have a more prominent place as a diagnostic tool. Primer sets for detection and identification by PCR have been described and tested by Roggenkamp et al. (14). They examined 4 G. elegans, 8 G. adiacens, and 3 A. defectiva strains in addition to 57 non-NVS strains and succeeded in separating NVS strains from non-NVS strains and correctly identifying the NVS strains. Phylogenetically, the reclassified Abiotrophia species exhibit a close phylogenetic relationship, with 16S rRNA sequence divergences within 3% and a sequence divergence of approximately 7% (4). Granulicatella and Abiotrophia species are related to members of the more recently described genera Facklamia, Ignavigranum, Globicatella, Eremococcus, Dolosicoccus, and Aerococcus, which also may be isolated in human infections and with whom they share various amounts of phenotypic identity (4). Cellular arrangement in Gram stains, tests for production of LAP and PYR, and susceptibility to vancomycin are important characteristics when differentiating among this group of bacteria (6). It is worth emphasizing that often the production of LAP and PYR is weak for Abiotrophia and Granulicatella strains. It is therefore very important to use sufficient inocula even though the strains are often difficult to cultivate.

Granulicatella and Abiotrophia species are part of the normal oral flora and the human urogenital and intestinal tracts (15). Among 91 isolates from the human mouth, a proportion of isolates of A. defectiva, G. adiacens, and G. elegans of about 1:11:1 was found (16). Approximately 5% of infective endocarditis cases are caused by NVS strains (15). A number of studies referring to endocarditis caused by NVS have appeared in the literature. Of those, only a few include cases in children and/or cases with prosthetic valve endocarditis. In this study, five patients were less than 15 years old, and in two patients, prosthetic valves were present. Besides endocarditis, NVS have been implicated in a variety of other infections anatomically related to their natural habitats (15). Notably, among the isolates received at the Centers for Disease Control and Prevention three isolates were from patients with corneal ulcers (n = 2) and keratitis (n = 1). Recently, two cases of vitreous infections by A. defectiva and G. adiacens following cataract extraction for two elderly patients were described in the literature (11). In addition, NVS strains have been isolated from horses with corneal ulcers (5) and from humans with infectious crystalline keratopathy (12). In a review of 30 cases of infective endocarditis caused by NVS isolates (17), it was noted that the clinical course often was more severe than that in cases caused by enterococci or viridans streptococci. Infections were difficult to treat, with a relapse rate of 41% despite treatment with appropriate antibiotics.