JOURNAL OF CLINICAL MICROBIOLOGY, Feb. 1994, p. 388-3920095-1137/94/$04.00+0Copyright X 1994, American Society for Microbiology

Clotting Activity in Staphylococcus schleiferi Subspecies fromHuman Patients

FRANOIS VANDENESCH,* CHRISTINE LEBEAU, MICHELE BES, GERARD LINA, BRUNO LINA,TIMOTHY GREENLAND, YVONNE BENITO, YVONNE BRUN, JEAN FLEURETTE,

AND JEROME ETIENNEDepartement de Recherche en Bactenriologie Medicale, Faculte de Medecine Alexis Carrel,

69372 Lyon Cedex 08, France

Received 6 July 1993/Returned for modification 28 September 1993/Accepted 4 November 1993

Staphylococcus schleiferi subsp. schleiferi is a coagulase-negative staphylococcus, usually present as acontaminant in human specimens. A near relative, S. schleiferi subsp. coagulans, possesses coagulase activitybut has not been reported from humans. We here describe three isolates of pseudocoagulase-positive S.schleiferi subsp. schleiferi and one isolate of S. schkiferi subsp. coagulans from human patients. Thepseudocoagulase from the S. schleiferi subsp. schleiferi isolates differs from S. aureus staphylocoagulase bybeing sensitive to a combination of protease inhibitors (aprotinin, N-ethylmaleimide, and heparin). Theseisolates 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. schkiferi subsp. coagulans produces proteinA, and S. schleiferi subsp. schleiferi expresses a clumping factor (fibrinogen affinity factor). Southern blothybridization with an S. aureus coa-specific probe revealed no sequence related to the coa gene in any of theS. schleiferi isolates, and their riboprobe profiles and biochemical characteristics were typical of S. schkeiferisubspecies, not of S. aureus. This study demonstrates that both subspecies of S. schleiferi can promote clottingof rabbit plasma in the standard tube test for coagulase.

Staphylococcal infections are frequent in humans andother mammals. Infections by Staphylococcus aureus canhave serious consequences, particularly in hospitalized pa-tients, and require aggressive antibiotic therapy, whichcarries the risk of adverse reactions. The therapeutic deci-sion relies on rapid laboratory identification of the infectiousagent, which usually involves evaluation of the production ofcoagulase, since S. aureus usually produces staphylocoagu-lase, whereas the less dangerous staphylococci are usuallycoagulase negative.

S. schleiferi subsp. schleiferi is a coagulase-negativestaphylococcus which was described in 1988 (7) and which,although usually benign, has been associated with humanconditions, such as infection of wounds, hip prostheses, orvascular devices, brain empyema, and bacteremia (6, 11,13). S. schleiferi subsp. schleiferi resembles S. aureus in thatboth species express a clumping factor (fibrinogen affinityfactor) and produce a heat-stable DNase but differ in thatonly S. aureus produces staphylocoagulase. In 1990, a new

subspecies, S. schleiferi subsp. coagulans, was obtainedfrom strains isolated from the external auditory meatus ofdogs suffering from external otitis (9). S. schleiferi subsp.coagulans is coagulase tube test positive, produces beta-hemolysin and a heat-stable nuclease but not a clumpingfactor, and has not previously been isolated from humans.

Staphylocoagulase reacts specifically with prothrombin,resulting in the formation of staphylothrombin, which hasspecific proteolytic activity and is resistant to natural throm-bin inhibitors, such as heparin (18). As S. aureus does notgrow in coagulase plasma, the tube test involves a delay of 2to 4 h, and more time is rarely needed (17).

* Corresponding author. Present address: Hopital Louis Pradel,BP Lyon Montchat, 69394 Lyon Cedex 03, France. Phone: (33) 7235 72 52. Fax: (33) 72 35 73 35.

Some staphylococci can give false-positive reactions inthe coagulase tube test through their production of proteases(pseudocoagulases) capable of digesting prothrombin and/orplasminogen and thereby promoting clotting in the absenceof staphylocoagulase. This pseudocoagulase activity, but notstaphylocoagulase activity, is inhibited by the addition of a

combination of anticoagulants and protease inhibitors to therabbit plasma substrate (19). Chomarat and Flandrois (4)achieved satisfactory inhibition of nonspecific clotting in thepresence of EDTA, aprotinin, N-ethylmaleimide, and hepa-rin.We report on three coagulase tube test-positive isolates of

S. schleiferi subsp. schleiferi from two different patients.These isolates also expressed a clumping factor (fibrinogenaffinity factor) and a heat-stable DNase and could have beenmisidentified as S. aureus. Clotting activity was inhibited bya combination of protease inhibitors and anticoagulants,indicating the presence of a pseudocoagulase. Another co-agulase-positive isolate from another patient was identifiedas S. schleiferi subsp. coagulans; its coagulase activityremained in the presence of inhibitors.None of the four isolates and neither of two S. schleiferi

subsp. coagulans reference strains contained DNA thatcould hybridize to an S. aureus coagulase gene (coa)-specificprobe at a high stringency. The identity of the S. schleiferiisolates was confirmed by their biochemical profiles (6, 7)and by ribotyping (8).

MATERIALS AND METHODS

Bacterial isolates. Isolates N920213 and N920235 were

isolated in September 1992 from patient 1, who was operatedon for a subdural hematoma following cranial trauma at thePierre Wertheimer Hospital of Lyon, France (Table 1).These isolates were cultivated from an intracranial drain andan indwelling intravenous catheter 6 and 9 days after admis-

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CLOTrING ACTIVITY IN STAPHYLOCOCCUS SCHLEIFERI 389

TABLE 1. Origin and characteristics of isolates and strains

Isolate or strain Origin and/or characteristics Reference orsource

S. aureus 8325-4 NCTC 8325 cured of prophages; reference strain for the 14, 15coagulase gene

S. schleiferi subsp. schleiferi N920213 Patient 1 (indwelling catheter) This studyS. schleiferi subsp. schleiferi N920235 Patient 1 (intracranial drain) This studyS. schleifen subsp. schleifen N920246 Patient 2 (sternal wound) This studyS. schleiferi subsp. coagulans N920247 Patient 3 (finger wound) This studyS. schleifen subsp. schleifen ATCC 43808 Reference strain for this subspecies; HindIII digest ribotype A 7, 8S. schleifen subsp. coagulans GA211 (JCM 7470) Reference strain for this subspecies 9S. schleiferi subsp. coagulans GA64 Other strain of this subspecies 9S. schleiferi subsp. schleiferi N860173 HindIII digest ribotype B 8S. schleifen subsp. schleiferi N860346 HindIII digest ribotype C 8S. schleiferi subsp. schleifen N880223 HindIII digest ribotype D 8S. schleiferi subsp. schleifen N890081 HindIII digest ribotype E 8S. schleiferi subsp. schleiferi H89291012 HindIII digest ribotype F 8

sion to the hospital, respectively. Isolate N920246 wascultivated from a serous effusion of a sternal wound samplein October 1992 from patient 2, who had undergone acoronary artery bypass 4 weeks earlier at the Louis PradelHospital of Lyon, France. None of these isolates wereconsidered to be clinically significant, and no antistaphylo-coccal antibiotic was prescribed. The recovery of bothpatients proceeded normally, with no subsequent infection.Isolate N920247 was recovered from suppurating materialdrained from a whitlow occurring 7 days after surgery for abroken finger in patient 3, seen at the Edouard HerriotHospital of Lyon, France. A group G Streptococcus sp. wasalso isolated from the same sample, and the patient wastreated with amoxicillin plus clavulanic acid for 10 days.

Reference and control strains were S. schleiferi subsp.schleiferi ATCC 43808 (7), S. schleiferi subsp. coagulansGA211 (JCM 7470) and GA64 (9), and S. aureus 83254, fromwhich the coagulase gene has been cloned and sequenced(15) (Table 1). A series of S. schleiferi subsp. schleiferiisolates were included for ribotyping; together with ATCC43808, these isolates represented the six characteristic ri-botypes of this subspecies (8) (Table 1).

Bacterial characterization. Isolates were identified by abiochemical gallery (ID32 Staph; bioMerieux, Marcyl'Etoile, France). Fibrinogen affinity factor (clumping factor)was evaluated by the Staphyslide agglutination test (bio-Merieux) protein A was evaluated by an immunodiffusiontest (Jackson Immunoresearch Laboratories, Baltimore,Md.), heat-stable DNase was evaluated by a diagnostic kit(Sanofi Diagnostics Pasteur, Mames-la-Coquette, France),and DNA-rRNA hybridization was evaluated by a DNAprobe specific for S. aureus rRNA (Accuprobe system;Gen-Probe, Biotechnie, Rungis, France).The coagulase tube test was performed with rabbit coag-

ulase plasma containing EDTA (Difco Laboratories, Detroit,Mich.) in the presence or absence of clotting inhibitors.Overnight cultures of staphylococci on sheep blood agarplates (bioMerieux) were inoculated into brain heart infusionbroth (Difco) and incubated for 3 h at 37°C with vigorousagitation. Samples of these exponential-phase cultures (100,ul) were added to 400 ,ul of rehydrated Bacto CoagulasePlasma EDTA (Difco) alone or supplemented with 2 mMN-ethylmaleimide (Aldrich-Chemie, Steinheim, Germany),100 mM aprotinin (Choay Laboratories, Gentilly, France),and 20 IU of heparin per ml (Dakota Pharm Laboratories,Creteil, France). Clotting was evaluated hourly for the first 6h and then after 18 h of incubation at 37°C.

Total DNA extraction and Southern blotting. Whole-cellDNA was isolated from staphylococci according to a stan-dard procedure (16). HindIII restriction digests were pre-pared following the supplier's instructions (BoehringerMannheim, Meylan, France). DNAs were separated byagarose gel electrophoresis, vacuum transferred to posi-tively charged nylon membranes (Boehringer), and cross-linked with UV light.DNA probes. The coagulase gene (coa)-specific probe was

an 871-bp EcoRI-HindIII fragment internal to the coa geneisolated from pCOA5 (15). Plasmid pKK3535, a pBR322derivative containing the rnB ribosomal operon of Esche-nichia coli, encoding 5S, 16S, and 23S RNAs and tRNAGlU(3), was used as the probe specific for genes coding for rRNAas described previously (8). The probes were labelled withdigoxigenin-11-dUTP by random priming according to themanufacturer's instructions (Boehringer).

Hybridization. Hybridization was performed under strin-gent conditions and detected with the DIG kit (Boehringer)according to the manufacturer's instructions. Chemilumines-cence was generated by hydrolysis of the Lumigen PPDsubstrate [4-methoxy-4-(3-phosphatephenyl)spiro(1,2-diox-etane-3,2'-adamantane)] by alkaline phosphatase conjugatedto antidigoxigenin antibodies (Boehringer). Filters were thenexposed to X-ray films (Appligene, Illkirch, France) for 15min.Data analysis. The degree of similarity between ribotype

patterns was measured by determining Dice's coefficients ofsimilarity (5).

RESULTS

The phenotypic characteristics permitting the identifica-tion of the three isolates as S. schleiferi subsp. schleiferi andthe fourth as S. schleifeni subsp. coagulans are listed inTable 2. The two subspecies differ in that S. schleifeni subsp.coagulans possesses protein A (a characteristic not men-tioned by Igimi and colleagues [9]) and produces urease butlacks clumping factor. Both subspecies differ from S. aureusin their inability to ferment maltose, lactose, mannitol,sucrose, or turanose. Confusion between the S. schleiferisubspecies with clotting activity and S. aureus is conceiv-able, since they all possess heat-stable DNases. Further-more, S. schleiferi subsp. coagulans possesses protein Aand urease activity, while S. schleiferi subsp. schleiferiexpresses clumping factor (Table 2). The standard coagulasetube test gave positive results with all four isolates from the

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390 VANDENESCH ET AL.

TABLE 2. Biochemical characteristics of the strains

Reaction of:

Characteristic S. schleiferi S. schleiferi S. schleiferi S. schleiferi S. schleiferi ssubsp. schleiferi subsp. schleiferi subsp. schleifeni subsp. coagulans subsp. coagulans S.ChlJifl 5 aureusN920213 N920235 N920246 N920247 (patient GA211 (JCM ATCC 43808 8325-4(patient 1) (patient 1) (patient 2) 3) 7470) ATCC 43808

Protein A - - - + + - +Heat-stable DNase + + + + + + +Clumping factor + + + - - + +Tube coagulase (Difco + + Wa + + _ +EDTA)

Resistance to:Novobiocin sb S S S S S SNitrofurantoin S S S S S S S

Nitrate reduction + + + + + + +Urease - - - + + - +Arginine dihydrolase + + + + + + +Ornithine decarboxylaseAlkaline phosphatase + + + + + + +Acetoin production + + + + + + +Acid production from:

Glucose + + + + + + +Mannose + + + + + + +Maltose - - - - - - +Lactose - - - - - - +Mannitol - - - - - - +Raffinose - - - - - -

Sucrose - - - + - - +Turanose - - - - - - +Arabinose - - - - - -

Ribose - - - - - -

Cellobiose - - - - - - -

a w, weak reaction after 6 h, but positive clotting was detected after 18 h (see Table 3).b S, susceptible.

three patients described here, generally with a time course HindIII restriction fragment of 7 kb from S. aureus 8325-4similar to that observed with the reference S. aureus or S. was recognized (data not shown). No DNA hybridizing at aschleiferi subsp. coagulans strains (Table 3). The addition of high stringency to this probe was observed either in theprotease inhibitors and anticoagulants to the plasma sub- pseudocoagulase-positive S. schleiferi subsp. schleiferi iso-strate somewhat delayed the reaction by S. aureus 8325-4, S. lates or in S. schleiferi subsp. coagulans.schleiferi subsp. coagulans GA211, and isolate N920247 but The ribotypes of our four isolates and of the two typeabrogated all the reactivity of the S. schleiferi subsp. schlei- strains of S. schleiferi subsp. coagulans were compared withfeni isolates and, unexpectedly, that of S. schleifen subsp. those already described for S. schleifen (Fig. 1). All strainscoagulans GA64, suggesting that its clotting activity was due displayed the three HindIII bands in the size range of 1.6 toto pseudocoagulase. 2 kb that are present in all S. schleifen strains (8) but are also

Southern blot analysis of genomic DNA was performed on found in S. intennedius (10). Isolates N920213 and N920235these strains with a coagulase gene-specific probe. A single (both from patient 1) and N920246 (from patient 2) showed

TABLE 3. Clotting of rabbit plasma by staphylococcal cultures with and without inhibitors of proteolytic enzymes

Reaction of the following strain with the indicated substrate:

S. schleiferi S. schleiferi S. schleiferi S. schleiferi Se. S. schleiferi S.

Time S. aureus subsp. subsp. subsp. subsp. scbsp subsp. schleifeni(h)e S83A25 schleifeni schleiferi schleiferi schleiferi susp. coagulans subsp.(h)o 8325-4 ATCC N920213 N920235 N920246 coagulans GA211 coagulans43808 (patient 1) (patient 1) (patient 2) (patient 3) (JCM 7470) GA64

pa Pib P Pi P PI P PI P Pi P Pi P Pi P PI

2 + - - - + - + _ _ _ _ _ + - wc _4 + w - - + - + - - - + - + w + -6 + + - - + - + - w - + - + w + -

18 + + -+ + + + + + + +

a p, Difco rabbit plasma containing EDTA.b PI, Difco rabbit plasma containing EDTA, 2 mM N-ethylmaleimide, 100 mM aprotinin, and 20 IU of heparin per ml.c w, weak clotting.

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CLOTTING ACIIVITY IN STAPHYLOCOCCUS SCHLEIFERI 391

S.schleiferisubsp. coagulaons

Iar-

,^-^ 7

r-

[ -t7eNq 'I

4..1-. ..fr

r ~ ~~~~~~~~~~~~4:

_ IftION_ _

_*, - ... II. -'_'i ""'

a.

,,O= "

a.0

FIG. 1. rRNA restriction patterns of strains of S. aureus, S.schleifeni subsp. coagulans, and S. schleiferi subsp. schleiferi withHindIII.

patterns identical to those of ATCC 43808 (ribotype A) andisolate N860346 (ribotype C), respectively. On the contrary,our isolate of S. schleifen subsp. coagulans (N920247;patient 3) as well as the two type strains (GA211 and GA64)displayed patterns that were not encountered previously inS. schleiferi. The degree of similarity between these patternsshowed that the S. schleifen subsp. coagulans patterns werenot closer to each other (56 to 67% similarity) than they wereto those of S. schleiferi subsp. schleiferi (44 to 67% similar-ity) (Table 4). Additionally, the degree of similarity betweenthe different patterns of S. schleiferi subsp. schleiferi can be

as low as 44% (Table 4), suggesting that ribotyping cannotdifferentiate between strains at the subspecies level.

DISCUSSION

S. aureus, unlike most other staphylococci, is a majorhuman pathogen, and its isolation from a patient is usually asignal for aggressive antibiotic therapy, with its attendantrisks. In routine laboratory practice, the production ofcoagulase is frequently used as a criterion to distinguish S.aureus from other staphylococci, since other coagulase-positive staphylococcal species, such as S. hyicus, S. inter-medius, or S. schleiferi subsp. coagulans, have not until nowbeen found in humans (9, 12). Other S. aureus characteris-tics, such as the presence of a fibrinogen affinity factor or aheat-stable DNase, are useful for completing the identifica-tion of S. aureus but may be detected in S. lugdunensis or S.schleiferi subsp. schleiferi, both of which are responsible forhuman infections (6, 11, 13). The two subspecies of S.schleiferi (schleifen and coagulans) both express a heat-stable DNase, the former produces a fibrinogen affinityfactor, and the later produces a coagulase (9) and protein A(this study).We report four non-S. aureus human staphylococcal iso-

lates giving positive results in the standard tube test forcoagulase. Three of the isolates were identified as S. schlei-feri subsp. schleiferi, and their presence was not clinicallysignificant; the two patients resolved their infection withoutantibiotic therapy. The fourth isolate was identified as S.schleiferi subsp. coagulans, which had not previously beenfound in humans. The isolate appeared pathogenic in asso-ciation with a group G streptococcal isolate. Both isolateswere acquired in circumstances suggesting nosocomialtransmission, and the patient received antibiotic treatment.The staphylocoagulase produced by S. aureus may be

differentiated from the proteases giving pseudocoagulaseactivity by the addition of anticoagulants and proteaseinhibitors to the plasma substrate in the coagulase tube test.The EDTA present in the standard tube test is not sufficientto inhibit certain proteases capable of pseudocoagulaseactivity, as reported for two of the three types of proteases

TABLE 4. Dice's coefficients of similarity between ribotype patterns of S. schleifen strains

% Similarity among:

Strain S. aureu S. schleiferi subsp. S. schleiferi subsp. schleifenS.aureus coagulans8325-4

N920247 GA211 GA64 Ribotype A' Ribotype Bb Ribotype Cc Ribotype Db Ribotype Eb Ribotype Fb

S. aureus 8325-4 100

S. schleiferi subsp. coagulansN920247 (patient 3) 21 100GA211 0 67 100GA64 0 56 67 100

S. schleiferi subsp. schleiferiRibotype Aa 0 56 56 56 100Ribotype B" 11 67 56 56 56 100Ribotype cc 0 67 67 56 89 67 100Ribotype D" 11 67 56 56 67 89 78 100Ribotype Eb 0 44 44 56 78 56 67 56 100Ribotype F" 0 56 56 56 78 44 67 44 78 100a Corresponds to ATCC 43808 and isolates N920213 and N920235 (both from patient 1).bSee Table 1.c Corresponds to isolates N860346 and N920246 (the latter from patient 2).

S.schleiferi suhsp. schleiferi

N 00 .- y-. NC N

00 00 -. es. N '? 'i. N 00

Z. N N ff. N N CI. -' C C C C C C Na - N N .C . N 00 O. .a . 00 00 . 00 00 00. . Z .¶ Z Z .

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392 VANDENESCH ET AL.

present in S. aureus (1, 2). The activity present in our S.

schleiferi subsp. schleiferi isolates was of the pseudocoagu-lase type and was abrogated in the presence of inhibitors.Unfortunately, the presence of inhibitors significantly pro-

longs the reaction time to positivity for some genuine coag-

ulase-positive strains (Table 3 and reference 19), an undesir-able situation in a clinical context.

Isolate N920247, like the reference strain S. schleiferisubsp. coagulans GA211 but not strain GA64, appears toproduce an exoproduct similar to the S. aureus coagulase,capable of clotting plasma in the presence of a combinationof inhibitors, although more slowly. As mentioned earlier, S.schleiferi subsp. coagulans GA64 appeared to producepseudocoagulase activity, demonstrating that both subspe-cies of S. schleiferi can produce pseudocoagulase. Thus, thedistinction between the two subspecies cannot be basedsolely upon the results of the standard tube test for coagu-

lase.Southern blot hybridization with an S. aureus coa-specific

probe showed no sequence related to the coa gene in any ofthe S. schleiferi isolates tested, suggesting significant diver-gence between the two species in their coagulase determi-nant.

Ribotyping did not accurately separate the two subspeciesinto two distinct groups of related ribotypes. Indeed, if allthe ribotypes observed clearly belong to the S. schleiferigroup, as assessed by the presence of a number of commonbands (8), the ribotypes of the S. schleiferi subsp. schleiferistrains were not more related to each other (44 to 89%similarity) than they were to the ribotypes of the three S.schleiferi subsp. coagulans strains (44 to 67% similarity)(Table 4). This result may reflect the failure of ribotyping insubspecies delineation. Alternatively, this result may reflecta higher relatedness between these subspecies than was

previously expected. This hypothesis is supported by theobservation of Igimi et al. (9) that the level of DNA-DNArelatedness between S. schleiferi subsp. schleiferi and S.schleiferi subsp. coagulans can be as high (73%) as thelowest level of relatedness between different strains of S.schleiferi subsp. coagulans (72%).

Finally, the incidence of isolation of non-S. aureus staph-ylococci expressing coagulase activity, a fibrinogen affinityfactor, and a heat-stable DNase could be underestimated,since these strains are probably sometimes misidentified as

S. aureus, leading to overprescription of antibiotics. Thenonacidification of maltose, mannitol, and lactose by S.schleiferi subsp. schleiferi or S. schleiferi subsp. coagulanscould help in recognizing these strains in routine laboratorypractice.

ACKNOWLEDGMENTS

This work was supported by a grant from the Conseil RegionalRh6ne-Alpes.We thank V. Delorme and C. Mouren for technical assistance.

REFERENCES1. Arvidson, S. 1973. Hydrolysis of casein by three extracellular

proteolytic enzymes from Staphylococcus aureus. Acta Pathol.Microbiol. Scand. Sect. B 81:538-544.

2. Bjorklind, A., and H. Jornvall. 1974. Substrate specificity ofthree different extracellular proteolytic enzymes from Staphy-lococcus aureus. Biochim. Biophys. Acta 370:524-529.

3. Brosius, J., A. Ulirich, M. A. Raker, A. Gray, T. J. Dull, R. R.Gutell, and H. F. Notell. 1991. Construction and fine mapping ofrecombinant plasmid containing rrnB ribosomal RNA operon.Plasmid 6:112-118.

4. Chomarat, M., and J. P. Flandrois. 1984. Frequency of pseudo-coagulase production in Staphylococcus aureus. Zentralbl. Bak-teriol. 258:441-448.

5. Dice, L. R. 1945. Measures of the amount of ecological associ-ation between species. Ecology 26:297-302.

6. Fleurette, J., M. Bes, Y. Brun, J. Freney, F. Forey, M. Coulet,M. E. Reverdy, and J. Etienne. 1989. Clinical isolates of Staph-ylococcus lugdunensis and S. schleiferi: bacteriological charac-teristics and susceptibility to antimicrobial agents. Res. Micro-biol. 140:107-118.

7. Freney, J., Y. Brun, M. Bes, H. Meugnier, F. Grimont, P. A. D.Grimont, C. Nervi, and J. Fleurette. 1988. Staphylococcuslugdunensis sp. nov. and Staphylococcus schleiferi sp. nov.,two species from human clinical specimens. Int. J. Syst. Bac-teriol. 38:168-172.

8. Grattard, F., J. Etienne, B. Pozzetto, F. Tardy, 0. G. Gaudin,and J. Fleurette. 1993. Characterization of unrelated strains ofStaphylococcus schleiferi by using ribosomal DNA fingerprint-ing, DNA restriction patterns, and plasmid profiles. J. Clin.Microbiol. 31:812-818.

9. Igimi, S., E. Takahashi, and T. Mitsuoka. 1990. Staphylococcusschleiferi subsp. coagulans subsp. nov., isolated from theexternal auditory meatus of dogs with external ear otitis. Int. J.Syst. Bacteriol. 40:409-411.

10. Izard, N. C., H. Hachier, M. Grehn, and F. H. Kayser. 1992.Ribotyping of coagulase-negative staphylococci with specialemphasis on intraspecific typing of Staphylococcus epidernidis.J. Clin. Microbiol. 30:817-823.

11. Jean-Pierre, H., H. Darbas, A. Jean-Roussenq, and G. Boyer.1989. Pathogenicity in two cases of Staphylococcus schleiferi, arecently described species. J. Clin. Microbiol. 27:2110-2111.

12. Kloos, W. E., and D. W. Lambe, Jr. 1991. Staphylococcus, p.222-237. In A. Balows, W. J. Hausler, Jr., K. L. Herrmann,H. D. Isenberg, and H. J. Shadomy (ed.), Manual of clinicalmicrobiology, 5th ed. American Society for Microbiology,Washington, D.C.

13. Latorre, M., P. M. Rojo, M. J. Unzaga, and R. Cisterna. 1993.Staphylococcus schleifen: a new opportunistic pathogen. Clin.Infect. Dis. 16:589-590.

14. Novick, R. 1967. Properties of a cryptic high-frequency trans-ducing phage in Staphylococcus aureus. Virology 33:155-166.

15. Phonimdaeng, P., M. O'Reilly, P. O'Toole, and T. J. Foster.1988. Molecular cloning and expression of the coagulase gene ofStaphylococcus aureus 8325.4. J. Gen. Microbiol. 134:75-83.

16. Renaud, F., J. Etienne, A. Bertrand, Y. Brun, T. B. Greenland,J. Freney, and J. Fleurette. 1991. Molecular epidemiology ofStaphylococcus haemolyticus strains isolated in an Albanianhospital. J. Clin. Microbiol. 29:1493-1497.

17. Selepak, S. T., and F. G. Witebsky. 1985. Inoculum size andlot-to-lot variation as significant variables in the tube coagulasetest for Staphylococcus aureus. J. Clin. Microbiol. 22:835-837.

18. Soulier, J. P., 0. Prou, and L. Halle. 1970. Further studies onthrombin coagulase. Thromb. Diath. Haemorrh. 23:37-49.

19. Wegrynowicz, Z., P. B. Heczko, J. Jeljaszewicz, M. Neugebauer,and G. Pulverer. 1979. Pseudocoagulase activity of staphylo-cocci. J. Clin. Microbiol. 9:15-19.

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