Comparative performances of six agglutination kits assessed by using typical and atypical strains of Staphylococcus aureus.

Six commercial agglutination tests designed for the identification of Staphylococcus aureus were compared by using a strain collection which included 512 staphylococci representing 33 species (318 isolates of Staphylococcus aureus [including 144 oxacillin resistant], 46 S. epidermidis isolates, 15 S. haemolyticus isolates, 12 S. saprophyticus isolates, 29 S. schleiferi isolates, 30 S. lugdunensis isolates, and 62 other coagulase-negative staphylococci). This group also included a proportion of strains with unusual phenotypes (e.g., 19 coagulase-negative S. aureus isolates, 26 clumping factor-negative S. aureus isolates, and 4 S. aureus isolates each with a double deficiency). The overall sensitivity for identification of typical and atypical S. aureus was high with the Staphaurex Plus test (Murex Biotech) (99.7%), the Pastorex Staph Plus test (Sanofi Diagnostics Pasteur) (99.7%), and the Slidex Staph Plus test (bioMérieux) (100%). The overall rate of specificity was affected by the unusual inclusion in this study of a high proportion of non-S. aureus species, such as S. lugdunensis and S. schleiferi, which express a clumping factor and therefore produce a positive result with the agglutination tests.     

Biotyping coagulase-negative staphylococci.

The biochemical profiles obtained with Staph-Ident (Analytab Products, Plainview, N.Y.) panels were combined with the results of adherence and synergistic hemolysis tests to define biotypes among 1,064 clinical isolates representing eight species of coagulase-negative staphylococci. The 672 isolates of Staphylococcus epidermidis were aligned in 69 of 144 potential biotypes in our scheme because of 18 different biochemical profiles and the eight physiologic subtypes. Isolates of most other species were in fewer biotypes because of more uniform adherence and synergistic hemolysis data, as well as fewer biochemical profiles. Since adherence and synergistic hemolysis may prove to be related to virulence and pathogenicity, biotyping with these test results would help evaluate the reliability of adherence and synergistic hemolysis as possible indices of the clinical significance of some of these organisms. When the antimicrobial susceptibility and plasmid profiles obtained on two clusters of S. epidermidis isolates were compared with the biotyping results, one cluster was not further differentiated by plasmid profiles, but was by antimicrobial profiles; the other cluster with only two biotypes was further divided into five distinct types by plasmid profiles but was not separated at all by antimicrobial profiles.     

Identification of coagulase-negative staphylococci other than Staphylococcus epidermidis by automated ribotyping

As routine identification of coagulase-negative staphylococci is problematic, the performance of automated ribotyping was evaluated for identification of coagulase-negative staphylococci other than Staphylococcus epidermidis. In total, 177 isolates were tested, comprising 149 isolates from blood samples, 15 isolates that were not identified by internal transcribed spacer (ITS)-PCR in a previous study, and 13 reference strains. The identification results were compared with those obtained by the API 20 Staph system, with standard phenotypic and molecular methods as reference. Most (n = 166; 93.8%) isolates were identified correctly by automated ribotyping. For 61 isolates, API 20 Staph and ribotyping were in agreement, but for 105 isolates, ribotyping provided correct identification and API 20 Staph did not. Four isolates not identified by automated ribotyping were recognised correctly by API 20 Staph. The remaining seven isolates could not be identified by either of the two methods. Automated ribotyping was able to distinguish Staphylococcus capitis reliably from Staphylococcus caprae. The results demonstrate the value of automated ribotyping for identification of coagulase-negative Staphylococcus (CoNS) isolates from human sources and may help to clarify the clinical relevance of CoNS species. In addition, automated ribotyping was able to detect polymorphisms that may be useful for epidemiological purposes within S. capitis, Staphylococcus hominis, Staphylococcus haemolyticus, Staphylococcus simulans, S. caprae, Staphylococcus warneri, Staphylococcus lugdunensis, Staphylococcus schleiferi, Staphylococcus sciuri, Staphylococcus pasteuri and Staphylococcus xylosus.     

Staphylococcus lugdunensis: clinical spectrum, antibiotic susceptibility, and phenotypic and genotypic patterns of 39 isolates

Staphylococcus lugdunensis is a member of the coagulase-negative staphylococci with the potential to cause clinically significant infections. The spectrum of infections was investigated in 39 isolates of S. lugdunensis from 38 patients. Most (73%) infections were located below the waist, while those above the waist were mainly (5/7) breast abscesses. Most isolates were susceptible to the antibiotics tested, although 15.4% were beta-lactamase-positive and could be identified by the disk-diffusion method for penicillin G. There was very good concordance between the disk-diffusion method and the Etest method for oxacillin resistance. Pulsed-field gel electrophoresis (PFGE) showed that 56% of the isolates belonged to one SmaI pulsotype, while phenotypic analysis by the Phene Plate system identified three main phenotypic groups. Although the S. lugdunensis isolates analysed were obtained from different patients, treated in different wards and hospitals during a 4-year period, there was a low degree of diversity, both genotypically and phenotypically. For this reason, PFGE is not suitable for the analysis of an outbreak situation, and the homogeneity observed may indicate that S. lugdunensis is a genetically conserved species of coagulase-negative Staphylococcus.     

Identification of Staphylococcus species and subspecies by the chaperonin 60 gene identification method and reverse checkerboard hybridization.

A previous study (S. H. Goh et al., J. Clin. Microbiol. 34:818-823, 1996) demonstrated that a 600-bp region of the chaperonin 60 (Cpn60) genes from various bacterial isolates could be amplified by PCR with a pair of degenerate primers and that the products could be used as species-specific probes for Staphylococcus aureus, S. epidermidis, S. haemolyticus, S. lugdunensis, S. saprophyticus, and S. schleiferi. To further validate the utility of bacterial Cpn60 genes as universal targets for bacterial identification (ID), reverse checkerboard chemiluminescent hybridization experiments were performed with DNA probes from 34 different Staphylococcus species and subspecies. With the exception of probes from the Cpn60 genes of S. intermedius and S. delphini, which cross hybridized, all were species specific. Two subspecies of both S. capitis and S. cohnii were differentiated from one another, while DNAs from the two S. schleiferi subspecies cross hybridized. When 40 known Staphylococcus isolates were tested in a blind experiment by the Cpn60 gene method, 36 strains, representing six species and one subspecies (S. sciuri, S. caseolyticus, S. hominis, S. warneri, S. hyicus, S. haemolyticus, and S. capitis subsp. ureolyticus), were correctly identified. DNA from the four remaining isolates, known to be S. hyicus bovine strains, failed to hybridize to DNA from the S. hyicus target strain or any other Staphylococcus species. However, DNAs from these S. hyicus isolates did cross hybridize with each other. New DNA sequence data and evidence from previous studies suggest some genetic divergence between the two groups of S. hyicus isolates. Our results demonstrate that this Cpn60 gene-based ID method has the potential to be a basic method for bacterial ID. Studies are in progress to further validate the utility of this Cpn60 gene system for ID of Staphylococcus and other genera, including those of slow-growing microorganisms.     

Evaluation of Staf-Sistem 18-R for identification of staphylococcal clinical isolates to the species level.

The accuracy and efficiency of Staf-Sistem 18-R (Liofilchem s.r.l., Roseto degli Abruzzi, Teramo, Italy) were compared with those of conventional biochemical methods to identify 523 strains belonging to 16 different human Staphylococcus species. Overall, 491 strains (93.9%) were correctly identified (percentage of identification, > or = 90.0), with 28 (5.4%) requiring supplementary tests for complete identification. For 14 isolates (2.8%), the strains did not correspond to any key in the codebook and could not be identified by the manufacturer's computer service. Only 18 isolates (3.4%) were misidentified. The system is simple to use, is easy to handle, gives highly reproducible results, and is inexpensive. With the inclusion of more discriminating tests and adjustment in supplementary code numbers for some species, such as Staphylococcus lugdunensis and Staphylococcus schleiferi, Staf-Sistem 18-R is a suitable alternative for identification of human coagulase-positive and coagulase-negative Staphylococcus species in microbiological laboratories.     

Clinical isolates of Staphylococcus lugdunensis and S. schleiferi: bacteriological characteristics and susceptibility to antimicrobial agents

The bacteriological characteristics and susceptibility to antimicrobial agents of 108 clinical isolates of Staphylococcus lugdunensis and Staphylococcus schleiferi are described. Fifty out of 108 isolates were considered to be responsible for 16 documented infections, including some severe infections (endocarditis, bacteraemia, osteitis). A number of bacteriological characteristics enabled the identification of these species in the clinical microbiology laboratory: the absence of coagulase and protein A, and the presence of a fibrinogen affinity factor and thermonuclease along with other biochemical characteristics (ornithine and arginine decarboxylases, carbohydrate acidification, novobiocin susceptibility) differentiated these new species from other staphylococci; however, they did not possess virulence markers such as toxins or haemagglutinin, but were haemolytic. In this series, almost all isolates were susceptible to 22 antibiotics and 4 antiseptics representative of the main groups of antimicrobial agents. More information is needed on the ecology and epidemiology of these new opportunistic pathogens.     

Clotting activity in Staphylococcus schleiferi subspecies from human patients.

Staphylococcus schleiferi subsp. schleiferi is a coagulase-negative staphylococcus, usually present as a contaminant in human specimens. A near relative, S. schleiferi subsp. coagulans, possesses coagulase activity but has not been reported from humans. We here describe three isolates of pseudocoagulase-positive S. schleiferi subsp. schleiferi and one isolate of S. schleiferi subsp. coagulans from human patients. The pseudocoagulase from the S. schleiferi subsp. schleiferi isolates differs from S. aureus staphylocoagulase by being sensitive to a combination of protease inhibitors (aprotinin, N-ethylmaleimide, and heparin). These isolates could all easily be confused with S. aureus in a typical clinical laboratory, since they all possess a heat-stable DNase and promote clotting formation. Moreover, S. schleiferi subsp. coagulans produces protein A, and S. schleiferi subsp. schleiferi expresses a clumping factor (fibrinogen affinity factor). Southern blot hybridization with an S. aureus coa-specific probe revealed no sequence related to the coa gene in any of the S. schleiferi isolates, and their riboprobe profiles and biochemical characteristics were typical of S. schleiferi subspecies, not of S. aureus. This study demonstrates that both subspecies of S. schleiferi can promote clotting of rabbit plasma in the standard tube test for coagulase.     

The production of fatty acid modifying enzyme (FAME) and lipase by various staphylococcal species.

Eighty-six strains encompassing 11 species of coagulase-negative staphylococci were examined for the production of fatty acid modifying enzyme (FAME) and lipase. Staphylococcus schleiferi and S. saprophyticus most closely resembled S. aureus in that 80% of the strains produced both enzymes. In contrast, no strains of S. lugdunensis and S. haemolyticus tested produced these enzymes. S. simulans was unusual in that eight of 10 strains produced FAME, but only one produced lipase. Among the other species the proportion of strains producing both enzymes ranged from 10 to 60%. Generally there was a strong correlation between FAME and lipase production.     

Characterization of unrelated strains of Staphylococcus schleiferi by using ribosomal DNA fingerprinting, DNA restriction patterns, and plasmid profiles.

The molecular characteristics of 31 unrelated strains of Staphylococcus schleiferi isolated from 13 hospitals between 1973 and 1991 were determined by ribosomal DNA fingerprinting by using a digoxigenin-labeled DNA probe, genomic DNA restriction patterns, and plasmid profiles. Only six strains harbored one or two plasmids. DNA restriction analysis, which was carried out with five endonucleases (EcoRI, HindIII, PstI, PvuII, and ClaI), did not allow us to discriminate between isolates. Ribotyping with HindIII, ClaI, or EcoRI enzymes generated six, seven, and nine distinct patterns, respectively. With the combination ClaI-EcoRI, 13 ribotypes were obtained among the 31 strains, suggesting a relative heterogeneity within the species. Moreover, all strains shared two or three common bands, according to the endonuclease used, which were relatively specific for S. schleiferi in comparison with the ribosomal banding patterns described for other coagulase-negative staphylococci. These results illustrate that ribotyping can be used for the epidemiological investigation of S. schleiferi isolates and possibly for taxonomic analysis in this species.     

Case of Staphylococcus schleiferi Endocarditis and a Simple Scheme To Identify Clumping Factor-Positive Staphylococci

Staphylococcus schleiferi is a coagulase-negative staphylococcus infrequently reported as a human pathogen. We report a case of prosthetic valve endocarditis attributed to this organism, contrast it to another Staphylococcus species that gives similar clumping factor results (S. lugdunensis), and propose a simple, effective identification scheme for identification of clumping factor-positive staphylococci.     

Occurrence of Staphylococcus lugdunensis in consecutive clinical cultures and relationship of isolation to infection.

Consecutive record review over a 63-month period revealed 229 Staphylococcus lugdunensis isolates, or 10.1% of the staphylococcal species that were not Staphylococcus aureus or Staphylococcus epidermidis. A total of 155 S. lugdunensis specimens were isolated from sites over the entire bodies of the 143 patients studied. The most common clinical diagnoses were skin and skin structure infections (55.4%) and blood and vascular catheter infections (17.4%). For 40% of the reviewed specimens, S. lugdunensis was the sole agent isolated, and for 60% of specimens, S. lugdunensis was isolated as part of mixed flora. In only 15.4% of clinically reviewed specimens was S. lugdunensis clearly a culture contaminant or colonizing organism. The pattern of human infection identified in this study emphasizes the predominance of skin and soft tissue S. lugdunensis infections over deep serious infections such as endocarditis, peritonitis, infected hip prosthesis, and osteomyelitis and vascular-associated infections. S. lugdunensis should be included along with S. epidermidis, Staphylococcus haemolyticus, and Staphylococcus saprophyticus as a coagulase-negative species of Staphylococcus pathogenic for humans.     

Use of genotypic identification by sodA sequencing in a prospective study to examine the distribution of coagulase-negative Staphylococcus species among strains recovered during septic orthopedic surgery and evaluate their significance

A total of 212 coagulase-negative Staphylococcus strains recovered prospectively during 119 surgeries for proven or suspected bone and joint infection (BJI) were identified by sodA sequencing. These strains were identified as 151 Staphylococcus epidermidis isolates, 15 S. warneri isolates, 14 S. capitis isolates, 9 S. hominis isolates, 6 S. lugdunensis isolates, 5 S. haemolyticus isolates, 4 S. caprae isolates, 4 S. pasteuri isolates, 3 S. simulans isolates, and 1 S. cohnii isolate. Only S. epidermidis, S. lugdunensis, S. capitis, and S. caprae were found to be infecting organisms and were involved, respectively, in 35 (81.4%), 3 (7.0%), 3 (7.0%), and 2 (4.6%) cases of BJI.     

Gas-liquid chromatography of cellular fatty acids for identification of staphylococci.

A commercially available, computer-assisted microbial identification system (MIS) employs gas-liquid chromatographic analyses of bacterial fatty acids. The MIS was used to identify 470 isolates of Staphylococcus species. The accuracy of the MIS was compared with the accuracies of conventional methods. There was a complete agreement between the MIS and conventional methods in the identification of 413 (87.8%) strains. For 36 of 45 misidentified strains, the correct identification was listed by the MIS as a choice but not as the first choice. Twelve strains could not be matched. All strains of Staphylococcus cohnii, S. epidermidis, S. intermedius, S. lugdunensis, S. schleiferi, S. sciuri, S. simulans, and S. xylosus were correctly identified. Two species, S. hominis and S. saprophyticus, accounted for 52.6% (30 of 57) of the misidentifications. Seventy-eight organisms were retested. Identification of 73 organisms remained unchanged, and for five organisms, the second choice became first and vice versa. The overall performance of the MIS is acceptable, and the system can be used as an alternate identification method for staphylococci.     

Species-level identification of staphylococcal isolates by real-time PCR and melt curve analysis

A real-time PCR assay was developed to identify common staphylococcal species. A single set of consensus primers was designed to amplify a portion of the 16S rRNA gene, and a pair of fluorescence resonance energy transfer probes was used to identify species based on the unique melt properties of the probes resulting from sequence variations in the amplicons from each species. Nine common staphylococcal strains (S. aureus, S. capitis, S. epidermidis, S. haemolyticus, S. hominis, S. lugdunensis, S. schleiferi, S. simulans, and S. warneri) were used for assay development. The species-specific melting profiles were validated by correctly identifying 36 of 37 coagulase-negative staphylococcal (CoNS) isolates identified by ribotyping. In a study of clinical isolates, the PCR/melt curve approach correctly identified 56/56 S. aureus isolates identified by coagulase/protein A latex agglutination. Fifty-four CoNS clinical isolates characterized using the API Staph assay were studied, with the PCR/melt curve approach yielding matching identifications for 32/54 (59%). The API Staph assay was unable to identify 18 CoNS isolates, and differing results were obtained for 4 isolates. Sequencing of the 22 discrepant or unidentified CoNS samples revealed that the PCR/melt curve results were correct for all but one isolate. Thus, PCR/melt curve analysis achieved a nearly 100% accuracy and performed better than biochemical testing. Performance of the PCR/melt curve approach requires less than 2 h after colony selection. This method thus provides a rapid and accurate approach to the identification of staphylococcal species in the clinical laboratory.     

Four-year prospective study of STAPH-IDENT system and conventional method for reference identification of Staphylococcus, Stomatococcus, and Micrococcus spp..

A 4-year prospective study compared the accuracy of the STAPH-IDENT system (bioMérieux Vitek, Inc., Hazelwood, Mo.) with that of the reference procedure of the Centers for Disease Control and Prevention for the identification of Staphylococcus species, Stomatococcus mucilaginosus, and Micrococcus species. The study compared the results from 1,106 cultures (500 eye cultures, 217 strains submitted for reference identification, and 389 known stock strains) representing 21 species of the family Micrococcaceae. The overall agreement of genus and species identifications was 81.1%. The percent agreement for the five most common clinical isolates was as follows: Staphylococcus epidermidis, 97.1% (517 isolates); Staphylococcus hominis, 82.5% (57 isolates); Staphylococcus aureus, 77.2% (162 isolates); Staphylococcus haemolyticus, 75.8% (61 isolates); and Staphylococcus warneri, 64.1% (39 isolates). The lowest percent agreement was with Staphylococcus cohnii (11.1%; (9 isolates). Of the 217 isolates sent to the Centers for Disease Control and Prevention for identification, 60.4% (131) were correctly identified by the STAPH-IDENT system. Of these, S. epidermidis accounted for 23.9%, S. aureus accounted for 15.6%, S. warneri accounted for 6.9%, Staphylococcus lugdunensis accounted for 6.5%, S. haemolyticus accounted for 5.5%, and S. hominis accounted for 4.1%. The STAPH-IDENT system did not perform adequately when dealing with commonly encountered organisms and is unsuitable for identifying uncommon isolates.     

Practical disk diffusion method for detection of inducible clindamycin resistance in Staphylococcus aureus and coagulase-negative staphylococci

Resistance to macrolides in staphylococci may be due to active efflux (encoded by msrA) or ribosomal target modification (macrolide-lincosamide-streptogramin B [MLSB] resistance; usually encoded by ermA or ermC). MLSB resistance is either constitutive or inducible following exposure to a macrolide. Induction tests utilize closely approximated erythromycin and clindamycin disks; the flattening of the clindamycin zone adjacent to the erythromycin disk indicates inducible MLSB resistance. The present study reassessed the reliability of placing erythromycin and clindamycin disks in adjacent positions (26 to 28 mm apart) in a standard disk dispenser, compared to distances of 15 or 20 mm. A group of 130 clinical isolates of Staphylococcus aureus and 100 isolates of erythromycin-resistant coagulase-negative staphylococci (CNS) were examined by disk approximation; all CNS isolates and a subset of S. aureus isolates were examined by PCR for ermA, ermC, and msrA. Of 114 erythromycin-resistant S. aureus isolates, 39 demonstrated constitutive resistance to clindamycin, while 33 showed inducible resistance by disk approximation at all three distances. Only one isolate failed to clearly demonstrate induction at 26 mm. Of 82 erythromycin-resistant CNS isolates that contained ermA or ermC, 57 demonstrated constitutive clindamycin resistance, and 25 demonstrated inducible resistance, at 20 and 26 mm. None of the 42 S. aureus isolates or 18 CNS isolates containing only msrA and none of the erythromycin-susceptible isolates yielded positive disk approximation tests. Simple placement of erythromycin and clindamycin disks at a distance achieved with a standard disk dispenser allowed detection of 97% of S. aureus strains and 100% of CNS strains with inducible MLSB resistance in this study.     

Staphylococcus lugdunensis infections: high frequency of inguinal area carriage

Following a change in surgical practice, we noted that the rate at which Staphylococcus lugdunensis was isolated from samples from the plastic surgery unit of our hospital increased considerably. We investigated the sources of these S. lugdunensis strains, and we found that in the case of drain colonization or surgical site infection, the strain was more likely to have come from the patient's skin bacteria when the pubic site had been shaved preoperatively. To test the hypothesis of pubic site colonization, we evaluated the prevalence of S. lugdunensis carriage among the cutaneous flora of the inguinal area. We found that 22% of 140 incoming patients carried S. lugdunensis in this area and that carriage at both inguinal folds was frequent (68% of carriers). A study of the genetic structure of the total population, including the clinical (n = 18) and the commensal (n = 53) strains, revealed that the diversity of the species was low and that the population was composed of two major groups that diverged at a distance of 35%. No particular characteristics made it possible to distinguish between clinical and commensal strains. Only isolates producing beta-lactamase were homogeneous; six of the eight beta-lactamase-positive strains displayed the same pulsed-field gel electrophoresis pattern.     

Rapid and economical method for species identification of clinically significant coagulase-negative staphylococci.

Four methods for the species identification of coagulase-negative staphylococci in the medical microbiology laboratory were compared with 444 consecutive isolates. The methods included (i) the reference method based on growth tests, (ii) API ID 32 Staph (bioMérieux), (iii) Staph-Zym (Rosco), and (iv) a rapid 4-h method developed in our laboratory (UZA method). The last method is based on the detection within 4 h of enzymatic activity of heavy bacterial suspensions in three substrate solutions (nongrowth tests). For 16.5% of the isolates some supplementary growth tests read after 24 h had to be added to the enzyme data for satisfactory identification. The reference method failed to identify four isolates. Of the 440 isolates identified by the reference method, API ID 32 Staph, Staph-Zym, and the UZA method correctly identified 419 (95.2%), 429 (97.5%), and 430 (97.7%) and misidentified 8 (1.8%), 4 (0.9%), and 1 (0.2%), respectively. Staphylococcus epidermidis, S. haemolyticus, S. lugdunensis, S. schleiferi, and S. capitis were identified with an accuracy of 98 to 100% by all the systems tested. S. capitis subsp. ureolyticus was not recognized by the API ID 32 system because the biochemical profiles for it are not yet included in the corresponding database. Whereas API ID 32 identified all 13 S. warneri isolates, both Staph-Zym and the UZA method missed 2 of these. S. hominis was identified with the least accuracy by the API ID 32 system (26 of 39 isolates), whereas the UZA and Staph-Zym methods identified 36 of the isolates belonging to this species.(ABSTRACT TRUNCATED AT 250 WORDS)     

Frequency of isolation of Staphylococcus lugdunensis in consecutive urine cultures and relationship to urinary tract infection

Recent reports associate Staphylococcus lugdunensis with severe infection in humans. The frequency of this microorganism in urine cultures is unknown. Five hundred isolates of coagulase-negative staphylococci (CoNS) were recovered from 4,652 consecutive urine specimens submitted for culture to the Mayo Clinic Microbiology Laboratory. Thirty-one (6%) of 500 isolates of CoNS were identified as S. lugdunensis. In no case was S. lugdunensis isolated in pure culture; 29 (94%) of 31 S. lugdunensis isolates were part of mixed nonpathogenic flora. Medical records were reviewed for 30 of the 31 patients from whom these 31 isolates were isolated. Twenty-one (70%) of the 30 evaluable patients were not treated with antibiotics; the remaining 9 (30%) of 30 patients were treated with antibiotics that may be effective against S. lugdunensis. S. lugdunensis may be an unrecognized yet infrequent cause of urinary tract infection.