Abstract
By DNA-DNA hybridization on microplates, we identified 1,230 strains of staphylococci from human clinical specimens and determined the distribution of species. The 10 Staphylococcus species isolated most often were S. epidermidis (31.3%), S. aureus (23.3%), S. haemolyticus (12.2%), S. caprae (10.7%), S. simulans (4.4%), S. hominis (4.0%), S. capitis (3.9%), S. saprophyticus (3.6%), S. warneri (2.2%), and S. lugdunensis (1.3%). From these results, we realized that S. caprae strains were widely distributed in human clinical specimens. The description in Bergey’s Manual of Systematic Bacteriology indicates that no strains of S. caprae produce acid from fructose and mannitol, but all our S. caprae strains produced acid from fructose and mannitol. Consequently, many strains of S. caprae isolated from human clinical specimens have been misidentified as S. haemolyticus or S. hominis by conventional biochemical tests. In this paper, we propose an emended description of S. caprae.
Recently, the coagulase-negative staphylococci (CNS) have been studied extensively because of their pathogenicity and involvement in some kinds of human and animal diseases (5, 20, 21, 23). Furthermore, the strains of several CNS species (i.e., Staphylococcus epidermidis, S. haemolyticus, S. warneri, S. hominis, and S. saprophyticus) were found to have high levels of resistance to various antibiotics (25, 30). The importance of being able to identify all CNS species routinely in clinical laboratories is increasing; however, the exact identification of CNS species is not easy, because the biochemical traits of the species are very similar and many clinical isolates show intermediate traits. Many clinical laboratories are currently using commercial identification kits to identify staphylococci; however, commercial kits do not include all Staphylococcus species, and their reliability for certain species is not sufficient (1, 8, 24).
We previously identified about 300 strains of staphylococci by quantitative hybridization (16) and noticed that many strains of S. haemolyticus, S. warneri, and S. hominis identified by biochemical tests or commercial kits were misidentified. Therefore, we decided to expand our study and proceeded to identify 1,230 human clinical strains isolated from various sources. Among the identified species, Staphylococcus caprae comprised an unexpectedly large number of strains (132 of 1,230). However, almost all these strains were misidentified as S. haemolyticus or S. hominis by the conventional identification method (biochemical tests). In this study, we report the distribution of Staphylococcus species among human clinical specimens and provide an emended description of S. caprae.
MATERIALS AND METHODS
Strains used.
We collected 1,230 human clinical strains from six hospitals in Japan. All strains had been identified at each hospital as gram-positive and catalase-positive cocci, so the strains were expected to be staphylococci. We collected only restrictive bacterial strains that grew well under aerobic conditions, and our distribution data did not include anaerobes such as S. aureus subsp. anaerobius and S. saccharolyticus. After receiving these strains, we checked each strain on a Trypticase soy agar (TSA; Difco) plate and reconfirmed the gram-positive staining and catalase reactivity (3% H2O2).
The type strains used for DNA-DNA hybridization were as follows: S. aureus subsp. aureus ATCC 12600, S. capitis subsp. capitis ATCC 9121, S. cohnii subsp. cohnii ATCC 29974, S. epidermidis ATCC 14990, S. haemolyticus ATCC 29970, S. hominis ATCC 27844, S. hyicus ATCC 11247, S. chromogenes NCTC 10530, S. saprophyticus ATCC 15305, S. sciuri ATCC 29062, S. simulans ATCC 27848, S. warneri ATCC 27836, S. xylosus ATCC 29971, S. caprae CCM 3573, S. auricularis ATCC 33753, S. gallinarum CCM 3572, S. carnosus DSM 20501, S. caseolyticus ATCC 13548, S. equorum DSM 20674, S. kloosii DSM 20676, S. arlettae DSM 20672, S. schleiferi subsp. schleiferi ATCC 43808, S. delphini DSM 20771, S. intermedius ATCC 29663, S. lugdunensis ATCC 43809, S. lentus ATCC 29070, Stomatococcus mucilaginosus CCM 2417, Kocuria kristinae (basonym, Micrococcus kristinae) ATCC 27570, Kocuria varians (basonym, Micrococcus varians) ATCC 15306, Micrococcus luteus JCM 1464, and Escherichia coli ATCC 11775 (as a negative control).
Microplate DNA-DNA hybridization.
DNAs of the type strains were prepared by the standard procedure of Marmur (22) with minor modifications (9). DNAs from the clinical strains were extracted by a small-scale DNA extraction method described previously (10) and labeled with photobiotin (Vector Laboratories, Inc., Burlingame, Calif.). Microplate quantitative DNA-DNA hybridization was performed by the previously described method (10, 11, 16). Briefly, the purified DNA (100 μg/ml) of each type strain was heat denatured and then diluted to 10 μg/ml with ice-cold phosphate-buffered saline (pH 7.4) containing 0.1 M MgCl2. The diluted DNA solution was distributed in a microplate (Maxsorp; Nunc, Roskilde, Denmark) at 100 μl/well, and the plate was incubated at 30°C for 12 h. The solution was discarded, and the plate was dried at 60°C. The plate was prehybridized for 30 min and then hybridized (in a solution of 2× SSC [1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate]–50% formamide) with biotinylated DNA at 45°C for 2 h. The plate was washed three times with 1× SSC. Then 100 μl of streptavidin–beta-d-galactosidase (diluted 1:1,000 in phosphate-buffered saline containing 0.5% bovine serum albumin) (GIBCO BRL) was added to each well. The plate was incubated at 37°C for 30 min and washed three times with 1× SSC. Substrate (100 μl, 100 μg/ml; 4-methylumbelliferyl-beta-galactopyranoside; Wako Pure Chemical, Ltd., Osaka, Japan) was added to each well and the fluorescence intensity was measured by Titertek Fluoroskan II (Labosystems, Osaka, Japan).
When a labeled DNA of each type strain was hybridized with the DNAs from all other type strains, the hybridization signal was less than 40% of the homologous hybridization signal (data shown previously) (16). From these results, we concluded that our method is quite applicable to the identification of members of the genera Staphylococcus and Stomatococcus.
Phenotypic tests and conventional identification.
We selected 22 biochemical tests including 13 key characteristics selected by Kloos and Schleifer (17). The following characteristics were determined: cell morphology in Gram-stained smears; catalase test (3% H2O2); free coagulase test (rabbit plasma; Eiken Co., Tokyo, Japan); hemolysis test on TSA plate supplemented with 5% defibrinated bovine blood; anaerobic growth and glucose fermentation test (GF medium; Nissui Pharmaceutical Co., Tokyo, Japan); nitrate reduction test (nitrate broth; Difco); acid production tests from fructose, xylose, arabinose, ribose, maltose, lactose, sucrose, trehalose, mannitol, xylitol, cellobiose, and mannose on purple agar base (Difco), with a final concentration of 1% for each substrate; and susceptibility tests to novobiocin (5 μg/disk; Difco), bacitracin (0.04 U/disk; BBL), oxolonic phosphate (10 μg/disk; Sigma), and lysostaphin (4.5 U/disk; Sigma) on Mueller-Hinton agar (Difco). We identified species from the reaction patterns in accordance with the method previously reported (17) and Bergey’s Manual of Systematic Bacteriology (18).
Commercial kit.
When necessary, we tested other biochemical traits such as arginine dihydrolase, urease, beta-glucosidase, beta-glucuronidase, and beta-galactosidase activity by using STAPHYOGRAM (Wako Pure Chemical, Ltd.) according to the recommended procedure.
RESULTS
Distribution of staphylococci among human clinical specimens.
The distribution of Staphylococcus species among 1,230 strains by the DNA-DNA hybridization test is presented in Table 1. The 10 species isolated the most often were S. epidermidis (385 strains [31.3%]), S. aureus (286 strains [23.3%]), S. haemolyticus (150 strains [12.2%]), S. caprae (132 strains [10.7%]), S. simulans (54 strains [4.4%]), S. hominis (49 strains [4.0%]), S. capitis (48 strains [3.9%]), S. saprophyticus (44 strains [3.6%]), S. warneri (27 strains [2.2%]), and S. lugdunensis (16 strains [1.3%]).
TABLE 1.
Distribution of staphylococcal species among 1,230 human clinical strains identified genetically by DNA-DNA hybridization method and comparison of results with those of conventional biochemical tests
| Genetic identification
|
Conventional tests
|
||
|---|---|---|---|
| Species | No. of strains (%) | No. of strains correctly identified (%) | Most common misidentification (%) |
| S. epidermidis | 385 (31.3) | 344 (89) | S. haemolyticus (6), S. hominis (4) |
| S. aureus | 286 (23.3) | 266 (93) | S. haemolyticus (5), S. epidermidis (2), S. capitis (1) |
| S. haemolyticus | 150 (12.2) | 121 (81) | S. epidermidis (13), S. warneri (3), undetermined (2) |
| S. caprae | 132 (10.7) | 0 (0) | S. haemolyticus (48), S. hominis (45), S. warneri (6) |
| S. simulans | 54 (4.4) | 43 (80) | S. haemolyticus (11), undetermined (7), S. epidermidis (2) |
| S. hominis | 49 (4.0) | 30 (61) | S. epidermidis (24), S. haemolyticus (10) |
| S. capitis | 48 (3.9) | 37a (77) | S. hominis (11), S. haemolyticus (6), S. epidermidis (5) |
| S. saprophyticus | 44 (3.6) | 41 (93) | S. haemolyticus (5), S. warneri (2) |
| S. warneri | 27 (2.2) | 2 (7) | S. haemolyticus (85), S. hominis (7) |
| S. lugdunensis | 16 (1.3) | 15 (94) | S. haemolyticus (6) |
| S. sciuri | 6 (0.5) | 5 (83) | S. xylosus (17) |
| S. cohnii | 3 (0.2) | 3 (100) | |
| S. gallinarum | 2 (0.2) | 2 (100) | |
| S. auricularis | 2 (0.2) | 2 (100) | |
| S. delphini | 1 (0.1) | 0 (0) | S. aureus (100) |
| S. xylosus | 1 (0.1) | 1 (100) | |
| Stomatococcus mucilaginosus | 7 (0.6) | 0 (0) | S. epidermidis (43), S. haemolyticus (14), S. hominis (14) |
| Staphylococcus strainsb | 17 (1.4) | S. xylosus (24), S. haemolyticus (18), S. epidermidis (12) | |
| Total | 1,230 (100) | 912 (74) | |
Twenty-four strains were identified as S. capitis subsp. ureolyticus.
These strains did not hybridize to any of the type strains used (see Materials and Methods).
By the genetic method (DNA-DNA hybridization), two strains were identified as S. gallinarum, and one strain was identified as S. delphini. Seven strains were identified as Stomatococcus mucilaginosus. Seventeen strains could not be identified because they did not hybridize to any of the type strains we used.
Comparison of identification results by the genetic method and conventional biochemical tests.
The results of conventional biochemical tests are also presented in Table 1. Of S. epidermidis strains identified genetically (DNA-DNA hybridization), 89% were correctly identified by the conventional tests. Similarly, more than 80% of S. aureus, S. haemolyticus, S. simulans, S. saprophyticus, and S. lugdunensis strains were correctly identified by the conventional tests. In contrast, no strain of S. caprae (0%) and two strains of S. warneri (7%) were correctly identified by the conventional tests (Table 1). In total, 912 of 1,230 strains (74.1%) were identified correctly by the conventional tests.
Of the 286 S. aureus strains, 20 strains (7.0%) had coagulase-negative reactions, and these atypical strains were mainly misidentified as S. haemolyticus or S. epidermidis by the conventional biochemical tests. The DNase test and beta-glucosidase test are helpful biochemical traits to correctly identify coagulase-negative S. aureus (data not shown). Similarly, 38 strains (9.9%) of S. epidermidis had atypical reactions; namely, 23, 5, and 10 strains had positive reactions for trehalose acidification, mannitol acidification, and both acidification tests, respectively. These atypical strains could not be identified as S. epidermidis by the conventional tests.
Biochemical traits of S. caprae.
To clarify the reason for the misidentification of S. caprae, we compared each biochemical trait with the description in the original study and Bergey’s Manual of Systematic Bacteriology (Table 2) (7, 18). In our 60 clinical strains, a positive reaction for >90% of strains was obtained for the following tests: nitrate reduction, arginine dihydrolase, urease activity, and acid production from fructose, maltose, lactose, trehalose, and mannitol under aerobic conditions. In contrast, a negative or positive reaction for <10% of strains was obtained for the following tests: beta-glucosidase, beta-glucuronidase, beta-galactosidase, and acid production from xylose, arabinose, ribose, xylitol, and cellobiose under aerobic conditions. Thirty-two strains (53%) showed hemolysis on bovine blood agar. These data were different from previous descriptions of acid production from fructose, maltose, sucrose, and mannitol under aerobic conditions.
TABLE 2.
Biochemical traits of S. caprae, S. haemolyticus, and S. hominisa
| Test or characteristic |
S. caprae data from this study
|
Data from Bergey’s manual
|
|||
|---|---|---|---|---|---|
| CCM 3573T | Human isolates (%)b | S. caprae | S. haemolyticus | S. hominis | |
| Hemolysis on bovine blood agar | + | d (53) | (+) | (+) | −w |
| Nitrate reduction | + | + (90) | + | d | d |
| Production of acid from: | |||||
| Fructose | + | + (100) | − | d | + |
| Xylose | − | − (0) | − | − | − |
| Arabinose | − | − (0) | − | − | − |
| Ribose | − | − (0) | − | d | − |
| Maltose | − | + (100) | d | + | + |
| Lactose | + | + (95) | + | d | d |
| Sucrose | − | d (27) | − | + | (+) |
| Trehalose | + | + (90) | + | + | d |
| Xylitol | − | − (0) | − | − | − |
| Mannitol | + | + (95) | d[−]c | d | − |
| Cellobiose | − | − (0) | − | − | − |
| Beta-glucosidase | − | − (0) | − | d | − |
| Beta-glucuronidase | − | − (0) | − | d | − |
| Beta-galactosidase | − | − (0) | − | − | − |
| Arginine dihydrolase | + | + (100) | + | + | d |
| Urease | + | + (90) | + | − | + |
+, more than 90% strains positive; −, less than 10% strains positive; d, 11 to 89% strains positive; (+), delayed reaction; −w, negative or weak reaction.
Data collected from 60 human clinical strains.
DISCUSSION
We first identified the species of all our isolates by conventional biochemical tests. However, after collecting data on all biochemical traits, it was very difficult to determine or estimate the species name because characteristic traits of each species were not clear and many of our isolates showed intermediate traits. Finally, we decided to identify all our clinical isolates by the DNA-DNA hybridization method. With the publication of the “Report of the Ad Hoc Committee on Reconciliation of Approaches to Bacterial Systematics” (28), the species are clearly defined and can be identified exactly by the DNA-DNA hybridization method.
Our results on the distribution of Staphylococcus species in human clinical specimens are similar to those reviewed by Kloos and Bannerman (20), except for S. caprae and S. lugdunensis.
Of our clinical strains, 132 strains hybridized most strongly with the type strain DNA of S. caprae and reacted very weakly (similarity values of <40%) with DNAs from other type strains. From these data, we conclude that these strains truly belonged to the species S. caprae. S. caprae was originally isolated from goat milk in 1983 (7) and has been primarily associated with animal specimens. Only a few researchers have reported S. caprae strains isolated from human specimens (3, 13, 15, 26), but from our data, it is clear that there are many S. caprae strains in human clinical specimens. We have not received the detailed report of the isolate site of S. caprae strains, but some strains were isolated from blood, skin abscess, and urine samples.
S. lugdunensis was originally isolated from human clinical specimens (12), and it was reported that this species accounted for 10% of all Staphylococcus species in human clinical specimens if S. aureus and S. epidermidis strains are excluded (14). We also expected that 5 to 10% of our isolates would be S. lugdunensis, but we found only 1.3%. We cannot identify the reason for this very different percentage, but from our data, the population of S. lugdunensis in human clinical specimens seems to be not very significant in Japan.
S. gallinarum and S. delphini were originally isolated from poultry (7) and dolphins (27), respectively, and this is the first report in which these species were isolated from human clinical specimens. We did not obtain the report of clinical symptoms and patient personal history from each hospital, so we could not judge whether these patients had been in contact with poultry or dolphins and whether these species were human pathogens or were only contaminants or colonizing flora.
In 1991, new subspecies of S. capitis and S. cohnii were identified: S. capitis subsp. ureolyticus (2) and S. cohnii subsp. urealyticum (19), respectively. We used only DNA from the type strain of each species for immobilization on our hybridization plate, so we could not identify each subspecies genetically. However, 24 strains (50%) of S. capitis showed positive reaction of both urease activity and acid production from maltose, which coincides with the description of S. capitis subsp. ureolyticus, while no strains of S. cohnii (we used only three strains) showed positive reaction of urease activity, which fits the description of S. cohnii subsp. urealyticum.
Of our 1,230 clinical strains, no strains were identified as Kocuria kristinae (basonym, Micrococcus kristinae), K. varians (basonym, M. varians), and M. luteus, while 7 strains were genetically identified as Stomatococcus mucilaginosus. The description of Stomatococcus mucilaginosus indicates that its catalase activity is weak or negative and that it may be misidentified as Streptococcus spp. (4). However, it is clear from our study that some clinical strains of Stomatococcus mucilaginosus were misidentified as staphylococci because these strains showed a catalase-positive reaction almost identical to those of ordinary staphylococci strains.
Seventeen strains could not be identified by our microplate DNA-DNA hybridization method because they did not hybridize strongly to any of the type strains used. We did not use the DNA from newly described species, such as S. pasteuri (6), S. pulvereri (31), and S. vitulus (29). However, from the biochemical traits, these unidentified strains may not belong to these new species (data not shown), and the strains may be candidates for other new species.
We could identify only 7% (2 of 27 strains) of S. warneri correctly by conventional biochemical tests. From the description in Bergey’s manual (18), most strains of this species show a negative reaction to a nitrate reduction test. However, 15 (56%) S. warneri strains identified by DNA-DNA hybridization showed a positive reaction to this test, and 13 (48%) of S. warneri strains showed strong hemolysis on bovine blood agar. For these reasons, we could not correctly identify the species by conventional tests. A positive reaction rate should be considered insufficient data when researchers attempt to identify S. warneri by conventional tests.
Biochemical traits and conventional identification of S. caprae.
We compared biochemical traits from our 60 strains of S. caprae with the description in Bergey’s manual (18) and the original study describing S. caprae (7). Our data were different from these descriptions of acid production under aerobic conditions from maltose and sucrose and especially from fructose and mannitol (Table 2). In the description in Bergey’s manual and in the original study, all strains of S. caprae (10 strains isolated from goat milk) did not produce acid from fructose and mannitol, but 100 and 95% of our strains produced acid from fructose and mannitol, respectively. Therefore, by using the previous descriptions, these strains were misidentified as S. haemolyticus when they produced acid from fructose and mannitol and showed hemolysis (63 of 132 [48%] of genetically identified S. caprae strains), and they were misidentified as S. hominis when they produced acid from fructose and mannitol (some strains were negative) and did not exhibit hemolysis (60 of 132 strains [45%]) (Table 2). We believe that the misidentification was mainly due to the result for acid production from fructose and mannitol.
From our correction of biochemical traits of S. caprae human isolates shown in Table 2, urease test and acid production from mannitol test are useful biochemical traits to differentiate S. caprae from S. haemolyticus or S. hominis, respectively, and S. caprae strains can now be correctly identified by biochemical tests.
There is little possibility for geographic variation for biochemical traits of S. caprae human isolates. Our described biochemical traits (isolates from Japan) are almost identical to those in the report of Vandenesch et al. (26), who used S. caprae isolated from humans in France and United Kingdom. Therefore, we believed that our described traits of S. caprae (Table 2) is useful not only for Japan but also for other countries.
We have only one strain of S. caprae from animal (CCM 3573T, isolated from goat milk) which also produced acid from fructose and mannitol on purple agar base (Table 2). We could not find other animal-derived S. caprae strains (we tested about 150 isolates) and thus could not compare the biochemical traits of S. caprae isolated from humans and animals in Japan. However, Vandenesch et al. (26) did report the biochemical traits of S. caprae from human and goat isolates. In their report, all human and goat isolates produced acid from fructose, but 33% (5 of 15 strains) and 55% (8 of 15 strains) of goat isolates did not produce acid from mannitol and maltose, respectively, while all human isolates showed positive reactions for both tests. Moreover, these two different derivatives were clearly differentiated genetically by SmaI digestion of chromosomal DNA (13) or by ribotyping with an EcoRI enzyme and a 16S-23S rRNA probe (26). These observations and our DNA-DNA hybridization results suggest that the S. caprae species contains two different evolutionary groups.
Emended description of S. caprae (Devriese, Poutrel, Kilpper-Bälz, and Schleifer 1983).
The following description is based on our study (60 human isolates) and on a previously published study (15 goat isolates) (26).
A positive reaction for >90% of isolates was obtained for the following biochemical reaction: nitrate reduction, arginine dihydrolase, urease activity, and acid production from fructose, lactose, and trehalose under aerobic conditions. More than 90% of human isolates also produced acid from mannitol and maltose; however, in the goat isolates, these rates were less than 70% (67 and 47%, respectively). A negative or positive reaction for <10% of isolates was obtained for the following tests: beta-glucosidase, beta-glucuronidase, beta-galactosidase, and acid production from xylose, arabinose, ribose, xylitol, and cellobiose under aerobic conditions. Sixteen strains (27%) produced acid from sucrose under aerobic conditions. Thirty-two strains (53%) showed hemolysis on bovine blood agar. Strains were originally isolated from animals but have also been isolated from human clinical specimens, such as blood, skin abscess, and urine samples.
Biochemical traits useful for distinguishing S. caprae and S. haemolyticus or S. hominis are shown in Table 2.
ACKNOWLEDGMENTS
This work was partially supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan, grant (B)-09557024.
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