Methicillin-Resistant Staphylococci

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1 JOURNAL OF CLINICAL MICROBIOLOGY, Aug. 1980, p /80/ /06$02.00/0 Vol. 12, No. 2 Classification and Characteristics of Coagulase-Negative, Methicillin-Resistant Staphylococci BRIAN J. WILKINSON,' SARZ MAXWELL, AND SUSAN M. SCHAUS Department of Biological Sciences, Illinois State University, Normal, Illinois Sixty-five clinical isolates of coagulase-negative, methicillin-resistant staphylococci have been classified as Staphylococcus epidermidis (63.0%), "phosphatasenegative S. epidermidis" (12.3%), coagulase-negative Staphylococcus aureus (6.2%), Staphylococcus hemolyticus (6.2%), Staphylococcus hominis (3.1%), and Staphylococcus warneri (1.5%). Five of the organisms (7.7%) could not be classified with certainty as currently recognized species. Novobiocin resistance was encountered in eight of the strains, but these were not classified as the accepted novobiocin-resistant staphylococcal species. Some differences in antibiotic resistance patterns to those typical of methicillin-resistant S. aureus were noted in that, although 29 strains were resistant to methicillin, penicillin, sulfamethizole, streptomycin, and tetracycline, the remainder of the strains were sensitive to streptomycin or tetracycline or both. In a majority of the strains (42 of 65), methicillin susceptibility testing by the disk method at 30 or at 37 C in the presence of NaCI did not appear to enhance resistance expression. Most of the strains produced fl-lactamase (EC ), but none of the 21 strains tested produced enterotoxin B. There is an increasing awareness of the abilities of coagulase-negative staphylococci to cause disease. Coagulase-negative staphylococci have been recognized as the most frequent cause of prosthetic valve endocarditis, neurosurgical shunt infection, and infection of prosthetic orthopedic devices (25). In general, coagulase-negative staphylococci are reported as "Staphylococcus epidermidis." However, coagulase-negative staphylococci are a heterogeneous group of organisms, and several new species have been proposed by Kloos and Schleifer and their colleagues over the last few years (10). Two recent studies have found that a considerable number of infections were caused by the new coagulasenegative species in addition to the well-recognized staphylococcal species S. epidermidis and Staphylococcus saprophyticus (17, 18). Since many staphylococci are penicillin resistant through production of fl-lactamase (EC 3.5:2.6), methicillin and other,b-lactamase-resistant penicillins are important therapeutic agents for treatment of staphylococcal infections. However, a significant percentage of S. epidermidis is methicillin resistant (MR) (21); Siebert et al. (25) reported 41% of recent isolates to be MR. The incidence of methicillin resistance in strains recovered from infected prosthetic valves and cerebrospinal fluid shunts is 63 to 70% (1, 7). Although methicillin resistance has been extensively investigated in coagulasepositive staphylococci (Staphylococcus aureus), and several characteristics of such organisms are known (12, 13), the exact mechanism of resistance to the drug remains unknown (27). Limited information exists on the properties of coagulase-negative, MR staphylococci. The purpose of the present study was threefold: (i) to determine what species were present in a collection of clinically isolated, coagulasenegative, MR staphylococci; (ii) to determine the antibiotic resistance characteristics of these organisms to see whether they were similar to or different from MR S. aureus; and (iii) to characterize some coagulase-negative, MR organisms for future studies of the methicillin resistance mechanism in such strains. MATERIALS AND METHODS Orgamisms. The organisms were clinical isolates from a variety of sources collected in hospitals in Boston, Minneapolis, Saint Paul, and Winnipeg, Canada, and were kindly provided by Michel Laverdiere. The organisms were maintained frozen in 15% (vol/ vol) glycerol at -80 C. Control organisms included S. aureus H, MR S. aureus DU4916, MR S. aureus Meuse, MR S. aureus Col, S. epidermidis ATCC 14990, S. saprophyticus ATCC 15305, and Micrococcus luteus (4, 27). Tests used in classification. Inocula for various tests were taken from overnight cultures (5 ml) of organisms in tryptic soy broth (Difco Laboratories, Detroit, Mich.) incubated without shaking at 37 C, with the exception of the phosphatase test, in which 161

2 162 WILKINSON, MAXWELL, AND SCHAUS the inocula were taken from P agar (9). The organisms were examined in two different series. In series I, organisms were first grown in liquid culture from frozen glycerol cultures and were then maintained on slants of brain heart infusion agar (BBL Microbiology Systems, Cockeysville, Md.). This practice was discontinued when it was discovered that several strains lost antibiotic resistance and some strains became coagulase positive (see below). Consequently, all antibiotic resistances reported are derived from organisms taken from frozen glycerol cultures and grown in tryptic soy broth overnight. The organisms in series Il were all cultured from frozen glycerol cultures. The tests used in classification were those recommended by Kloos and Schleifer (9) and were performed at 37 C. Colony morphology was observed on P agar (9). Lysozyme and lysostaphin susceptibilities were determined by the method of Kloos et al. (11). Coagulase production was determined by tube assay using rabbit plasma (Difco) according to the manufacturer's instructions. Anaerobic growth in a thioglycolate medium, hemolysis of bovine blood agar, and aerobic acid production from carbohydrates were determined by the method of Kloos and Schleifer (9). Phosphatase activity was determined by the method of Pennock and Huddy (19), and nitrate reductase was determined after aerobic growth for 2 days in Difco nitrate broth by using the alternate coupling reagent described by Miller and Neville (15). The aerobic production of acid from glycerol in the presence of erythromycin was used to aid in separating staphylococci from micrococci as described by Schleifer and Kloos (23). Heat-stable deoxyribonuclease activity was assayed by the method of Barry et al. (2).,f-Lactamase production was determined by the Gots test as modified by Haight and Finland (5). Antibiotic susceptibilities. Susceptibilities were determined according to the method of Bauer et al. (3); zone diameter was measured after 24 and 48 h of incubation at 30 or 37 C. The antibiotics (BBL) used and their disk potencies were as follows: methicillin (5 lig), novobiocin (30,ug), penicillin G (10 U), tetracycline (30,ig), streptomycin (10,g), and sulfamethizole (1 mg). In addition, methicillin susceptibility was determined on plates supplemented with 5% (wt/vol) NaCl. The zone diameters were interpreted as sensitive or resistant according to the chart supplied by the manufacturer. RESULTS AND DISCUSSION Preliminary examination of strains. The test strains were classified in the family Micrococcaceae, being nonmotile, gram-positive, catalase-positive cocci, approximately 1 ym in diameter, which divide in uneven planes to form irregular clusters. Of 73 strains initially examined, 65 were classified as staphylococci on the basis of lysostaphin susceptibility and lysozyme resistance and their ability to produce acid aerobically from glycerol in the presence of erythromycin (9, 22). The remaining eight strains did not meet the criteria of the above reactions to justify their classification as staphylococci. J. CLIN. MICROBIOL. Classification of strains. The phenotypic characteristics of the strains and their identification at the species level are given in Table 1. In this table, the reactions of the species according to Kloos and Schleifer (9) are shown for those strains that are classified as recognized species. The number of strains giving positive reactions in each test is shown in the table. The organisms were classified as S. epidermidis (63.0%), "phosphatase-negative S. epidermidis" (12.3%), coagulase-negative S. aureus (6.2%) Staphylococcus hemolyticus (6.2%), Staphylococcus hominis (3.1%), and Staphylococcus warneri (1.5%). The remaining five strains (7.7%) were designated Staphylococcus simulans?. Thus, the large majority of the coagulase-negative, MR strains were S. epidermidis. S. epidermidis. The strains classified as S. epidermidis showed close correlation with the characteristics described by Kloos and Schleifer (9). Of 41 strains, 16 showed some hemolysis of bovine blood; 12 showed weak acid production from melezitose, which is somewhat higher than the incidence noted by Kloos and Schleifer (9); 17 showed some acid production from ribose; and 1 strain (1-41) produced acid from trehalose. A low incidence of trehalose-positive S. epidermidis has been reported (10). Six strains (1-47, II-1, II-9, II-43, II-44, and II-47) were found to be novobiocin resistant. In the scheme of Kloos and Schleifer, novobiocin resistance is a taxonomic characteristic of S. saprophyticus, Staphylococcus cohnii, and Staphylococcus xylosus. Upon comparison of the test reactions of our strains with those quoted for the above species, it was concluded that the strains could not be classified as any species but S. epidermidis. Kloos and Schleifer (9) point out that additional staphylococcal species may acquire novobiocin resistance with exposure to this antimicrobial agent in the environment. Phosphatase-negative S. epidermidis. Eight of the strains showed the closest correlation to S. epidermidis, with the exception that they did not show phosphatase activity. Strains with similar characteristics have recently been noted by Oeding and Digranes (18) and Varaldo and Satta (26) and have been described by Kloos et al. (10). Coagulase-negative S. aureus. With the exception of coagulase production, four of the strains showed extremely close correlation to S. aureus, according to Kloos and Schleifer (9). When the inocula of strains I-4, I-52, I-53, and I- 59 were taken from slants of brain heart infusion agar, on which they had been maintained for about 6 months with monthly transfers, positive coagulase reactions were noted. However, when

3 VOL. 12, 1980 TABLE 1. METHICILLIN-RESISTANT STAPHYLOCOCCAL SPECIES 163 Characteristics of the strains and their identification as staphylococcal species Test reaction/no. of strains giving positive results' Phospha- Characteristic S. tase-nega- Coagulase- S he. dts d ep-srm 4>tieS. ne.tv. S.molyti- S. hominis S. warneri S. (41)b tive S. negatives. cus (4' (2)f 1 lans? simu- (5)" idermidis aureus (4)d CU(4(2 1) ias'(h (8Y Colony diameter (5 -/2 0 +/2 (+)/3 -/0 -/0 (+)/5 days 2 5 mm) Hemolysis (bovine) -, ±/16 3 (+)/2 (+)/4 (->/1 -, ±/0 ±, -/0 Nitrate reduction (+, ±)/39 8 +/4 (+)/4 (+, ±)/2 (-)/1 +/5 Phosphatase +/41 0 +/4 (-)/1 (-)/0 (-)/0 (±)/0 Acid (aerobically) from:,8-d-(-)-fructose +/41 8 +/4 +,-/4 +/2 +/1 +/5 D-(+)-Galactose (+)/41 8 +/4 v/3 v/l (-)/1 -/4 D-(+)-Mannose (+)/35 8 (+)/4 -/0 -/1 -/0 (+)/0 D-(+)-Xylose -/0 0 -/0 -/0 -/0 -/0 -/0 D-(+)-Arabinose -/0 0 -/0 -/0 -/0 -/0 -/0 D-(-)-Ribose -/17 2 +/4 (-)/2 -/1 +, -/1 v/5 Maltose +/40 8 +/4 +/4 +/2 +, ±/1 -,/5 a-lactose (+)/41 7 (+)/4 -, +/3 v/l (-)/1 +/4 Sucrose +/41 8 +/4 +/4 +/2 +/1 +/5 D-(+)-Trehalose -/1 0 +/4 +/2 +/O +/1 (+)/5 D-(+)-Turanose v/21 3 (+)/4 v/o (+)/1 (-)/1 -/3 D-(+)-Melezitose (-)/12 0 -/4 -/0 v/i -/0 -/O D-(+)-Mannitol -/0 0 +/4 +,-/2 -/0 +, -/1 (+)/5 Xylitol -/0 O -/0 -/0 -/O -/O -/0 'athe reactions designated by the symbols are those defining the various species according to Kloos and Schleifer (9). Numbers within parentheses in the column heads indicate the number of strains tested. b Series I: 2, 9, 10, 11, 13, 17, 32A, 33, 34, 41, 47, 56, 57, 58A. Series II: 1, 2, 3, 5, 9, 11, 12, 13, 14, 17, 33, 34, 36, 43, 44, 46, 47, 53, 54, 56, 57W, 57Y, 58, 60, 61, 62, 70. C Series I: 43, 44, 45, 60. Series IL: 52, 75, 79, 80. d Series I: 4, 52, 53, 59. ' Series IL: 15, 63, 64, 65. f Series I: 36. Series IL: 39. ' Series II: 59. h Series IL: 55, 66, 72, 76, 78. the inocula were taken from frozen glycerol cultures, none of these strains gave a positive coagulase test, whether they were grown in tryptic soy broth or brain heart infusion broth. Strains I-4 and I-52, maintained as frozen glycerol cultures, did show heat-stable deoxyribonuclease activity; strain I-59 was negative, and strain I-53 was not tested. The elaboration of coagulase after maintenance on brain heart infusion agar slants and production of heat-stable deoxyribonuclease by some of the strains supports their identification as coagulase-negative S. aureus (4). Lotter and Genigeorgis (14) have reported the isolation of coagulase-positive variants from cultures of coagulase-negative, enterotoxigenic staphylococci transferred on brain heart infusion agar at least nine times. However, streak plates of strains I-4, I-52, I-53, and I-59 did not yield evidence of more than one colony type. S. hemolyticus. Good correlation between our findings and the characteristics described by Kloos and Schleifer was noted. One of the strains showed weak phosphatase activity. Also, one of the strains (II-15). was novobiocin resistant. S. hominis. The two strains correlated reasonably well with the characteristics of S. hominis described by Kloos and Schleifer, although neither of the strains produced acid from trehalose. S. warneri One of the strains showed closest correlation to S. warneri, although acid production from turanose, lactose, and galactose was noted, and the strain reduced nitrate. S. simulanst. Five of the strains showed closest similarity to S. simulans, although they also resembled S. aureus. Acid production from turanose and galactose and lack of production of phosphatase contraindicated their classification as S. simulans and S. aureus, respectively. One of the strains (II-55) was novobiocin resistant. The results of the classification of the strains support the usefulness of the Kloos and Schleifer

4 164 WILKINSON, MAXWELL, AND SCHAUS scheme for identification of coagulase-negative staphylococci, since most of the strains could be readily classified without many anomalous reactions. However, we did not find anaerobic growth in thioglycolate medium to be particularly useful in species identification. Our results did not correlate well with those of Kloos and Schleifer, and variations within species were noted. Similar conclusions regarding this test have recently been made by Namavar et al. (16). In a study of 522 coagulase-negative staphylococci from various infections, Nord et al. (17) found the following distribution of species: S. epidermidis, 43%; S. saprophyticus, 25%; S. hemolyticus, 22.8%; S. hominis, 8.8%; S. cohnii, 3.1%; Staphylococcus capitis, 2.3%; S. warneri, 2.3%; and S. simulans, 2.1%. The most striking difference between our study and theirs was that we did not encounter any S. saprophyticus strains. The significance of this is unclear, but it may be that methicillin resistance does not become readily established in certain staphylococcal species. Further species identification studies of coagulase-negative, MR species will be required to evaluate this possibility. Antibiotic resistance patterns. Lacey (12) has reported that typical MR S. aureus strains show a heterogeneity of methicillin resistance expression, are resistant to other penicillins and cephalosporins, sulfonamides, streptomycin, and tetracycline, and produce penicillinase and enterotoxin B. This has been cited as evidence for the single-clone origin of MR S. aureus. The susceptibilities of the coagulase-negative, MR strains to the above antibiotics were determined for comparison with MR S. aureus. It was discovered that some of the strains in series 1, which had ail been maintained on brain heart infusion agar for several months, lost resistance to methicillin and other antibiotics. Consequently, the antibiotic resistance patterns reported are from results obtained with inocula taken from frozen glycerol cultures (Table 2). In addition to being resistant to methicillin, all the experimental strains were also resistant to penicillin and sulfamethizole. In addition to resistance to these three antibiotics, 29 of 65 strains were resistant to both streptomycin and tetracycline, 19 of 65 strains were resistant to streptomycin but not tetracycline, 7 of 65 strains were tetracycline resistant and streptomycin sensitive, and 10 of 65 were resistant to neither streptomycin nor tetracycline. The distribution of resistance patterns among the different species is also shown in Table 2; 15 of 41 S. epidermidis, 4 of 4 coagulase-negative S. aureus, 4 of 4 S. hemolyticus, and 4 of 5 S. simulans? strains were resistant to all five antibiotics. This was J. CLIN. MICROBIOL. the most common susceptibility pattern among the species, with the exception of the phosphatase-negative S. epidermidis, for which tetracycline sensitivity was encountered in seven of eight strains. Eight of the strains were novobiocin resistant, but were not classified as S. saprophyticus, S. cohnii, or S. xylosus, for which novobiocin resistance is used as a taxonomic characteristic. This observation raises the possibility that novobiocin resistance is fairly common in other staphylococcal species that are resistant to multiple antibiotics. With the exception of strain I-47 (S. epidermidis), all of the strains produced,8-lactamase. Of the organisms in series I, twenty-one were tested for enterotoxin B production by the method of Iandolo and Shafer (6), and none was found to produce it (J. J. Iandolo, personal communication). This included all four of the coagulase-negative S. aureus strains. Enterotoxin production is rare in coagulase-negative staphylococci (4), and Shafer and Iandolo (24) have recently concluded that the genetic determinant for toxin production is not physically linked to the methicillin resistance gene. Effect of incubation temperature and 5% NaCI on expression of methicillin resistance. It is a common characteristic of MR S. aureus that only one in 105 cells can form a colony on methicillin-containing agar at 37 C, but that virtually 100% of an inoculum can form a colony at 30 C, or at 37 C in the presence of 5% NaCI (20). This is referred to as heterogeneity of methicillin resistance expression. To evaluate this, the diameters of methicillin inhibition zones on plates incubated at 37 and 30 C and plates supplemented with 5% NaCI incubated at 37 C were compared (Table 3) (22). It can be seen that in the majority of cases (42 of 65) incubation at 30 C or inclusion of NaCI in the plate did not appear to enhance methicillin resistance expression. With these strains, growth was up to the disk under all three conditions, which indicates a high degree of resistance expression. This method, of course, represents a cruder estimate of resistance expression than colony counts on methicillin-containing agar. In 15 of 65 strains, an incubation temperature of 30 C or inclusion of 5% NaCI in the agar and incubation at 37 C led to smaller zones of inhibition than those of organisms incubated at 37 C in the absence of NaCI. This indicates that greater expression of resistance occurred under these conditions, as is typically found in MR S. aureus. In 9 of 65 strains, a surprising result was found. Incubation at 30 C or at 37 C in the presence of NaCI, or both, led to larger zones of inhibition than were noted at 37 C in the ab-

5 VOL. 12, 1980 METHICILLIN-RESISTANT STAPHYLOCOCCAL SPECIES 165 TABLE 2. Antibiotic resistance patterns of coagulase-negative, MR staphylococcia Resistance pattemb No. of strains with pattern/total no. of strains Phospha- Meth MTAll Pen Sul Strep Tet srains Coagu- S. epidermi- tasenegalasenegadis tive S. tive S. epider- aureus S. hemolyticus S. hommis S. warneri S. simuplans? midis R R R R R 29/65 15/41 1/8 4/4 4/4 0/2 1/1 4/5 R R R S R 7/65 5/41 0/8 0/4 0/4 2/2 0/1 0/5 R R R R S 19/65 13/41 5/8 0/4 0/4 0/2 0/1 1/5 R R R S S 10/65 8/41 2/8 0/4 0/4 0/2 0/1 0/5 a Antibiotic susceptibilities are reported after 24 h of incubation at 37 C. In some cases, incubation at 300C or for 48 h at 37 C was necessary to observe methicillin resistance. b Meth, methicillin; pen, penicillin; sul, sulfamethizole; strep, streptomycin; tet, tetracycline; R, resistance; S, sensitivity. TABLE 3. Influence of incubation temperature and NaCI on the expression of methicillin resistance of coagulase-negative, MR staphylococci0 No. of strains with given zone of inhibition/total no. of strains Zone of inhibition compared Phospha- Coaguto that at 37 C Alstreams S. epidermi- tase-nega- lase-nega- S. hemoly- Sḣmis w S. simudis tive S. epi- tive S. au- ticus arnert lans? dermidis reus Same at 300C and 37 C 41/65 26/41 1/8 4/4 3/4 1/2 1/1 5/5 plus 5% NaCI Decreased at 30 C and 15/65 11/41 4/8 0/4 0/4 0/2 0/1 0/5 37 C plus 5% NaCI Increased at 30 C and/ 9/65 4/41 3/8 0/4 1/4 1/2 0/1 0/5 or 37 C plus 5% NaCI a Organisms were grown overnight at 37 C and then swabbed onto three plates, one of which was supplemented with 5% NaCI. After applying the methicillin disk, the plate supplemented with NaCI and another one were incubated at 37 C; the remaining plate was incubated at 30 C. The zones of inhibition were measured after 24 and 48 h. Reported results are after 48 h of incubation. sence of NaCI. In these strains, average zone diameters were: 37 C, 7.8 mm; 30 C, 17.9 mm; 37 C plus NaCI, 15.3 mm (disk diameter, 7 mm). Thus, some differences were noted in the antibiotic resistance patterns of the strains as compared with MR S. aureus in that several were not resistant to streptomycin or tetracycline or both. Most of the strains produced fl-lactamase. However, none of the strains tested produced enterotoxin B. Also, in contrast to MR S. aureus, a majority of the strains did not show a large heterogeneity in methicillin resistance expression. It has been proposed that MR S. aureus strains isolated in England and Switzerland have derived from a single strain (8, 12, 13). However, Schaefler et al. (22) have considered the possibility that certain MR S. aureus strains have a different origin. The origin of coagulase-negative, MR staphylococcal species is uncertain. It will be interesting to see whether methicillin resistance is encountered in all the newly recognized, coagulase-negative staphylococcal species. The existence of coagulase-negative strains showing differences from MR S. aureus and different phenotypical characteristics within the strains may indicate more than one evolutionary pedigree. ACKNOWLEDGMENTS This study was supported in part by an Illinois State University Faculty Research Grant and a Grant-in-Aid from the Illinois Heart Association. We are grateful to J. A. Sagebiel for drawing bovine blood. We thank J. J. Iandolo for enterotozin determinations. LITERATURE CITED 1. Archer, G. L Antimicrobial susceptibility and selection of resistance among Staphylococcus epidermidis isolates recovered from patients with infections of indwelling foreign devices. Antimicrob. Agents Chemother. 14: Barry, A. L., R V. F. Lachica, and F. W. Atkinson Identification of Staphylococcus aureus by simultaneous use of tube coagulase and thermonuclease tests. Apple. Microbiol. 25: Bauer, A. W., W. M. Kirby, J. C. Sherris, and M.

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