Abstract
A total of 28 staphylococcal isolates from human clinical specimens belonging to the Staphylococcus sciuri group were identified and characterized. The API Staph and ID32 STAPH correctly identified S. sciuri and S. lentus but not S. vitulinus strains. Identification to the subspecies level was possible only by a PCR-based method.
Members of the Staphylococcus sciuri group are widespread in nature, and they can be isolated from a variety of farm animals, pets, and wild animals, as well as from various food products of animal origin (6, 9, 12, 14, 22, 26). This group is made up of Staphylococcus sciuri subsp. carnaticus, Staphylococcus sciuri subsp. rodentium, Staphylococcus sciuri subsp. sciuri, Staphylococcus lentus, and Staphylococcus vitulinus (12, 26). Staphylococcus pulvereri was a member of the S. sciuri group until recently, when it was shown that S. pulvereri is only a synonym of S. vitulinus (originally S. vitulus) (15, 23). Although they are principally associated with animals, members of the S. sciuri group may colonize humans, and it has been estimated that they may constitute 0.79 to 4.3% of the total number of coagulase-negative staphylococci isolated from clinical samples (8, 20). However, they have been associated with serious infections such as endocarditis (10), peritonitis (25), septic shock (11), urinary tract infection (20), endophthalmitis (1), pelvic inflammatory disease (21), and, most frequently, wound infections (16, 19). The aim of this study was to compare phenotypic (conventional, API Staph, ID32 Staph) and genotypic (PCR) methods for identification of isolates of the S. sciuri group.
A total of 28 isolates belonging to the S. sciuri group, recovered from 1998 to 2003 from clinical samples at the Institute of Microbiology, School of Medicine, Belgrade, Serbia, and Regional Hospital Příbram, Příbram, Czech Republic, were analyzed (Table 1). Half of them were isolated from urine samples. Some of these strains have been reported previously (18-21) but not investigated for the characteristics presented in this study. All the isolates were previously identified by conventional methods (5, 12, 18, 26) as S. sciuri (23 strains), S. lentus (3 strains), or S. vitulinus (2 strains).
TABLE 1.
Isolation and molecular identification of 28 clinical isolates of members of the S. sciuri group
| Strain no. | Yr of isolation | Specimena | Clinical significanceb | Identification by PCR amplification of the 16S-23S rRNA intergenic spacer region | Band obtained with species-specific primer for S. sciuri |
|---|---|---|---|---|---|
| 2 | 1998 | Tip of CVC | 0 | S. sciuri subsp. sciuri | Yes |
| 3 | 1998 | Skin around CVC | 0 | S. sciuri subsp. sciuri | Yes |
| 4 | 1998 | Tip of CVC | 0 | S. sciuri subsp. rodentium | Yes |
| 6 | 2001 | Vagina | 0 | S. sciuri subsp. rodentium | Yes |
| 7 | 2001 | Vagina | 0 | S. sciuri subsp. rodentium | Yes |
| 8 | 2001 | Wound | + | S. sciuri subsp. rodentium | Yes |
| 9 | 2000 | Urine | 0 | S. sciuri subsp. rodentium | Yes |
| 10 | 2000 | Urine | 0 | S. sciuri subsp. sciuri | Yes |
| 11 | 2000 | Urine | 0 | S. sciuri subsp. rodentium | Yes |
| 12 | 2001 | Urine | 0 | S. sciuri subsp. sciuri | Yes |
| 13 | 2000 | Tip of CVC | 0 | S. sciuri subsp. rodentium | Yes |
| 14 | 2000 | Cavum Douglasi | + | S. sciuri subsp. sciuri | Yes |
| 15 | 2001 | Urine | 0 | S. sciuri subsp. rodentium | Yes |
| 16 | 2001 | Urine | 0 | S. sciuri subsp. sciuri | Yes |
| 17 | 2001 | Urine | 0 | S. lentus | Yes |
| 18 | 2001 | Tip of CVC | Probably 0 | S. sciuri subsp. rodentium | Yes |
| 19 | 2001 | Urine | 0 | S. sciuri subsp. sciuri | Yes |
| 20 | 2001 | Vagina | 0 | S. sciuri subsp. rodentium | Yes |
| 23 | 2002 | Urine | 0 | S. sciuri subsp. sciuri | Yes |
| 177 | 2002 | Urine | 0 | S. sciuri subsp. rodentium | Yes |
| 179 | 2002 | Wound | + | S. sciuri subsp. rodentium | Yes |
| 201 | 2002 | Wound | Probably + | S. sciuri subsp. rodentium | Yes |
| 292 | 2003 | Wound | Probably + | S. sciuri subsp. rodentium | Yes |
| 293 | 2001 | Urine | 0 | S. lentus | Very weak |
| 294 | 2001 | Urine | 0 | S. vitulinus | Yes |
| 295 | 2002 | Urine | 0 | S. vitulinus | Very weak |
| 296 | 2002 | Urine | + | S. lentus | Very weak |
| 297 | 2003 | Cervix | 0 | S. sciuri subsp. rodentium | Yes |
CVC, central venous catheter.
0, no clinical significance; +, clinically significant.
Staphylocoagulase (free coagulase) activity was determined with rabbit plasma (Torlak, Belgrade, Serbia) by using the tube method (5). Oxidase activity was determined with oxidase diagnostic tablets (Rosco, Taastrup, Denmark). Novobiocin susceptibility was determined on Mueller-Hinton agar (Oxoid Limited, Basingstoke, Hampshire, United Kingdom) with a disk containing 5 μg of novobiocin (Bioanalyse, Ankara, Turkey). Strains were considered to be resistant to novobiocin if the zone of inhibition was ≤16 mm. Commercial identification kits, namely, API Staph and ID32 STAPH (bioMérieux, Marcy-l'Etoile, France), were used according to the manufacturer's instructions. All the strains were coagulase negative and oxidase positive. In addition, the disk diffusion method with the 5-μg novobiocin disk confirmed that all strains were resistant to novobiocin. However, only three S. sciuri strains showed resistance to novobiocin by use of the ID32 STAPH kit. The problem with determination of resistance to novobiocin by ID32 STAPH was also noted by Chesneau et al. (2). Moreover, the identification system in the instruction manual (identification table, version 2.0) indicated that only 26% of S. lentus isolates and 43% of S. sciuri isolates could exhibit resistance to novobiocin by use of the ID32 STAPH kit. Identification of isolates based on the conventional and commercial methods (API Staph and ID32 Staph) agreed in the identification of 26 out of 28 strains. The commercial methods agreed in the identification of all the S. sciuri and S. lentus strains, although some discrepancies between results obtained by API Staph versus ID32 STAPH were noted (Table 2). However, two isolates identified as S. vitulinus by the conventional method were identified as Staphylococcus capitis and S. sciuri by API Staph and as S. capitis by ID32 Staph. This discrepancy could be attributed to the fact that S. vitulinus is not included in the database of these tests. Misidentification of the members of the S. sciuri group by commercial identification systems has been reported previously (13, 17). Differentiation of S. sciuri from S. vitulinus is possible on the basis of utilization of mannose, l-arabinose, maltose, and 2-naphthyl phosphate (alkaline phosphatase) as substrates: S. sciuri utilizes some or all of these while S. vitulinus is unable to utilize any of them (5, 12, 26). However, we noted that production of acid from mannose by S. vitulinus could vary depending on the identification system (Table 2). Identification of the S. sciuri strains to the subspecies level was not possible on the basis of phenotypic characteristics, since S. sciuri isolates of different subspecies showed similar biochemical profiles.
TABLE 2.
Biochemical and phenotypic characteristics of 28 members of the S. sciuri group
| Characteristic | No. of strains of the following speciesa testing positiveb for the indicated characteristic:
|
|||
|---|---|---|---|---|
| S. sciuri subsp. sciuri (n = 8) | S. sciuri subsp. rodentium (n = 15) | S. lentus (n = 3) | S. vitulinus (n = 2) | |
| Tube coagulase | 0 | 0 | 0 | 0 |
| Oxidase | 8 | 15 | 3 | 2 |
| Novobiocin resistance (5 μg) | 8 | 15 | 3 | 2 |
| Urease | 0 | 0 | 0 | 0 |
| Arginine dihydrolase | 0 | 0 | 0 | 0 |
| Ornithine decarboxylase | 0 | 0 | 0 | 0 |
| Esculin | 8 | 15 | 3 | 0 |
| d-Glucose | 8 | 15 | 3 | 2 |
| d-fructose | 8 | 15 | 3 | 2 |
| d-Mannose | 8 | 15 | 3 | 0 (2) |
| d-Maltose | 8 | 15 | 3 | 0 |
| d-Lactose | 4 (2w) | 9, 1w (9, 2w) | 3 | 0 |
| d-Trehalose | 8 | 15 | 3 | 0 |
| d-Mannitol | 8 | 15 | 3 | 1 |
| d-Raffinose | 0 | 0 | 3 | 0 |
| Nitrate reduction | 8 | 15 | 3 | 2 |
| Voges-Proskauer | 0 | 0 | 0 | 0 (1) |
| β-Galactosidase | 0 | 0 | 0 | 0 |
| Arginine arylamidase | 0 | 0 | 0 | 0 |
| Alkaline phosphatase | 8 | 15 | 1 | 0 |
| Pyrrolidonyl arylamidase | 0 | 0 | 0 | 0 |
| d-Saccharose | 8 | 15 | 3 | 2 |
| N-Acetyl glucosamine | 5, 3w (6, 2w) | 10, 5w (13, 1w) | 2, 1w (3) | 0 |
| d-Turanose | 8 | 11, 4w | 1, 2w | 0 |
| l-Arabinose | 4 | 11 | 2, 1w | 0 |
| β-Glucuronidase | 5 | 4 | 0 | 0 |
| d-Ribose | 8 | 15 | 3 | 0 |
| d-Cellobiose | 8 | 14, 1w | 3 | 0 |
| Xylitol | 0 | 0 | 0 | 0 |
| d-Melibiose | 0 | 0 | 3 | 0 |
| Xylose | 0 | 1 | 3 | 2 |
| α-Methyl-β-glucoside | 0 | 0 | 1 | 0 |
The number of strains identified by PCR as belonging to each species is given after the species name.
By API Staph and ID32 STAPH. Where discrepancies between results by these two tests were noted (d-mannose, d-lactose, Voges-Proskauer, and N- acetylglucosamine), results obtained by API Staph are given in parentheses w, weak reaction.
Generally, molecular approaches proposed for the identification of staphylococci can be divided into those based on the detection of species-specific sequences and those based on the detection of sequence variations in ubiquitous elements such as rRNA and tRNA operons or chaperonin-encoding genes (3). To the best of our knowledge, species-specific primers have been published only for S. sciuri (of all the staphylococci in the S. sciuri group) (7). All the strains were tested by PCR using species-specific primers for S. sciuri based on previously published primers and methods (7). Bright bands indicating a positive reaction were obtained for all 23 S. sciuri strains (Table 1) and agreed with the identification by API Staph and ID32 STAPH. However, the fact that strong or weak hybridization signals were obtained for all S. lentus and S. vitulinus strains suggests that this set of primers may not be reliable in the identification of S. sciuri. PCR amplification of the 16S-23S rRNA intergenic spacer region was performed in accordance with the protocols previously described by Couto et al. (4) and Shittu et al. (16). This PCR method enabled identification of all isolates to the species or subspecies level. Out of the 28 isolates, 8 strains were identified as S. sciuri subsp. sciuri, 15 were identified as S. sciuri subsp. rodentium, 3 were identified as S. lentus, and 2 were identified as S. vitulinus by the 16S-23S rRNA PCR method.
In conclusion, our study showed that the members of the S. sciuri group can be identified and differentiated from other staphylococci from human clinical samples on the basis of the oxidase test. Only the recently described Staphylococcus fleurettii is also novobiocin resistant, coagulase negative, and oxidase positive as well (24). It should be noted that this bacterium has not been isolated from clinical samples of humans. However, certain problems could arise in the identification of these bacteria to the species level by use of API Staph and ID32 STAPH, since S. vitulinus is not included in the database for these tests.
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