Phenotypic detection of mec A-positive staphylococcal blood stream isolates: High accuracy of simple disk diffusion tests

Detection of oxacillin-resistance in staphylococci by phenotypic methods remains problematic. Although standardized susceptibility test methods are adequate for Staphylococcus aureus, many are less satisfactory for the coagulase-negative staphylococci (CNS). We have studied 108 consecutive blood culture isolates of staphylococci. The mec A gene was detected by PCR in one S. aureus and 55 CNS isolates. Susceptibility testing was performed as follows: oxacillin (1-μg), ceftizoxime (30-μg), and cephalothin (30-μg) by disk diffusion; oxacillin, ceftizoxime, cephalothin, methicillin, ampicillin, ampicillin/sulbactam, penicillin, cefazolin, imipenem, and meropenem by the broth microdilution method. In addition, isolates were tested by the oxacillin agar screen plate method. The single oxacillin-resistant S. aureus strain was detected by all oxacillin susceptibility test methods and by the ceftizoxime disk and MIC methods. Two oxacillin-susceptible S. aureus were intermediate (minor error) by ceftizoxime broth microdilution (MIC, 16 μg/mL). The most sensitive, simple phenotypic methods for detection of oxacillin-resistant CNS (mec A positive) were as follows: oxacillin disk diffusion at 98%, oxacillin screen plate at 91%, oxacillin broth microdilution at 87%, ceftizoxime disk diffusion at 100%, ceftizoxime broth microdilution at 87%, and methicillin broth microdilution at 83%. These results indicate that oxacillin and ceftizoxime disk diffusion tests are the most accurate phenotypic methods in routine clinical use for detection of oxacillin-resistant CNS. Oxacillin broth microdilution MIC testing (2% NaCl supplement) would perform more satisfactorily (100% sensitivity) with an adjusted interpretive breakpoint at ⩽0.5 μg/mL, in contrast to the lower accuracy of the “so-called” reference agar screen test.     

Evaluation of the Mastalex latex agglutination test for methicillin resistance in Staphylococcus aureus grown on different screening media

The Mastalex MRSA latex agglutination method was evaluated with 52 methicillin-resistant and 27 methicillin-susceptible strains of Staphylococcus aureus grown on various media. All tests were correct with colonies grown on blood agar with or without oxacillin 2 mg/L. Tests on colonies grown on mannitol salt agar were less reliable, although addition of oxacillin 2 mg/L improved performance. One of the 26 MRSA which grew on Baird-Parker medium with 8 mg/L ciprofloxacin gave a false-negative result. Agglutination was faster when strains were grown on media with oxacillin. The method would be particularly useful for urgent confirmation of resistance.     

Evaluation of antibacterial sensitivity testing methods for methicillin-resistant Staphylococcus aureus in a dermatology outpatient population

Over a period of one year, 1986-1987, 116 strains of Staphylococcus aureus were isolated from patients attending two outpatient dermatology clinics in Houston, Texas. The purpose of this study was to evaluate the adequacy of routine antibiotic sensitivity testing methods for detecting methicillin-resistant Staphylococcus aureus (MRSA). The Kirby-Bauer disk diffusion method was compared with a commercially available screening medium containing 6 micrograms/ml of oxacillin and 4% NaCl. The minimal inhibitory concentration (MIC) of methicillin, oxacillin, and oxacillin with 4% NaCl to S aureus using the agar dilution method was also determined. Approximately 90% of S aureus strains produced beta-lactamase and were resistant to penicillin and ampicillin. By disk diffusion, no strains were resistant to methicillin, though diameters of zones of inhibition were between 10 and 14 mm in seven strains. All strains proved to be sensitive to methicillin by MIC determinations and on the oxacillin-NaCl screening medium. The MIC of methicillin was 2.5 micrograms/ml for the majority of strains of S aureus, between 0.16 and 0.31 microgram/ml for oxacillin, and 0.08 to 0.16 microgram for oxacillin with 4% NaCl. We concluded that the incidence of MRSA in an outpatient dermatology population is low, and a combination of disk diffusion and oxacillin-NaCl screening is adequate for testing sensitivity.     

头孢西丁纸片扩散法检测耐甲氧西林金黄色葡萄球菌

目的　评价头孢西丁纸片扩散法检测耐甲氧西林金黄色葡萄球菌 (MRSA)在临床的应用价值。方法　用头孢西丁纸片扩散法检测临床分离的 94株金黄色葡萄球菌 ,并与苯唑西林纸片扩散法、琼脂稀释法及mecA基因检测进行比较。结果　mecA基因阳性的 77株金黄色葡萄球菌 ,头孢西丁纸片扩散法均显示耐药。结论　头孢西丁纸片扩散法与mecA基因检测高度一致 ,是筛选和确认MRSA的一种可靠的试验方法。     

Evaluation of different methods for detecting methicillin (oxacillin) resistance in Staphylococcus aureus

OBJECTIVES: To evaluate the performance of oxacillin, cefazolin, cefoxitin, cefotaxime and imipenem discs; Etest for oxacillin; microdilution; agar screening plates with 2 and 6 mg/L of oxacillin; and PBP2' agglutination for detection of methicillin-resistant Staphylococcus aureus (MRSA). METHODS: A total of 102 clinical S. aureus isolates, including 51 MRSA isolates, tested by PCR for the presence or absence of the mecA gene (gold standard method), isolated from different patients and at different times, were tested with: oxacillin (1 microg), cefazolin, cefoxitin, cefotaxime and imipenem (all 30 microg) discs; Etest for oxacillin; microdilution with oxacillin; agar screening tests (ORSAB medium) with 2 mg/L or 6 mg/L of oxacillin; and PBP2' agglutination with two different kits for detection of MRSA strains. RESULTS: The cefoxitin disc, ORSAB medium and PBP2' detection all showed 100% sensitivity. The cefoxitin, cefazolin and imipenem discs, Etest for oxacillin, microdilution and agar screening method with 6 mg/L at 24 h showed the highest specificity (100%), although variable degrees of sensitivity. The cefoxitin disc, which showed negative and positive predictive values of 100% and 98%, respectively was the best method for detecting MRSA isolates. CONCLUSIONS: In the absence of availability of molecular biology techniques, the cefoxitin disc was the best predictor of methicillin resistance in S. aureus from among the techniques tested.     

Comparison of a polymerase chain reaction assay and a conventional microbiologic method for detection of methicillin-resistant Staphylococcus aureus.

The presence or absence of a methicillin resistance gene in 58 clinical isolates of Staphylococcus aureus was examined by the polymerase chain reaction (PCR) and Southern blot analyses. The results were analyzed in relation to those of the MIC assay of methicillin and oxacillin. PCR assay results were identical to those of Southern blot analysis of genomic DNA digested with HindIII (positive, 28 strains; negative, 30 strains). Among the 28 PCR-positive strains, 6 strains showed methicillin susceptibility by the conventional susceptibility test (MICs, less than or equal to 8 micrograms/ml). Culturing of the six strains with ceftizoxime led to an increase in the phenotypic level of resistance to methicillin and oxacillin, indicating that these strains should be classified as methicillin-resistant S. aureus (MRSA). The PCR assay was found to be a sensitive and reliable procedure for the rapid diagnosis of MRSA infection, even in cases in which the conventional MIC assay failed to detect MRSA.     

Comparative evaluation of the MRS test. A 4 to 6 hour screening test for detecting oxacillin-resistant staphylococci

A total of 131 strains of S. aureus and 25 strains of unspeciated coagulase-negative staphylococci (CNS) initially tested by automated methods for susceptibility to oxacillin were concurrently retested using standardized disk diffusion, reference 2% NaCl-supplemented broth microdilution, oxacillin salt agar, and the MRS test (a commercially prepared broth screening method). Compared to the reference broth microdilution test results, the MRS test was 97% sensitive for S. aureus, 95% sensitive for CNS, and 100% specific for all staphylococci. Results were available in 4 hr for S. aureus and less than 6 hr for CNS. The oxacillin salt agar screen had sensitivities of 93 to 99% with a specificity of 100%. Although the disk diffusion method was the most sensitive method (100%), it was the least specific (83% for S. aureus and 80% for CNS). Differences in manufacturers' agar affected results with most discrepancies resulting in a false-resistant interpretation. Although inoculum standardization was important for accurate susceptibility test results, overinoculation alone could not account for the 30 isolates falsely-resistant to oxacillin by the Vitek AMS or Abbott MS-2. Contaminants or card-fill problems may have also have been responsible for some of the discrepancies. The MRS test was considered to be an acceptable alternative screen or a supplement to other methods for same-day testing for ORS.     

两种检测耐甲氧西林金黄色葡萄球菌的K-B法应用评价

目的评价两种分别使用MH琼脂(头孢西丁)和高盐琼脂(苯唑西林)纸片扩散法检测耐甲氧西林金黄色葡萄球菌的临床应用价值。方法用头孢西丁纸片在MH琼脂平板上进行纸片扩散法(K-B法)检测临床分离的89株耐甲氧西林金黄色葡萄球菌,并与苯唑西林纸片扩散法(K-B法)、mecA基因检测进行比较。结果在MH琼脂使用头孢西丁纸片检测MRSA优于高盐琼脂苯唑西林纸片扩散法,并与PCR检测mecA基因的方法高度一致。结论头孢西丁纸片扩散法是筛选和确认耐甲氧西林金黄色葡萄球菌(MRSA)一种简单、可靠的实验方法。     

The influence of salt concentration on the detection of methicillin resistance in coagulase-negative staphylococci.

The detection of methicillin resistance by the breakpoint method was examined using three different media containing varying quantities of added salt and 4 mg/L methicillin or 1 mg/L oxacillin. Three hundred clinical isolates of eight species of coagulase-negative staphylococci were tested. In 68 strains methicillin resistance was expressed only at certain salt concentrations and four distinct susceptibility phenotypes were observed. A correlation between the susceptibility phenotype and the species of the isolate was found. Testing on Columbia agar (CA) containing 4 mg/L methicillin with 0% and 4% added salt was required to detect resistance in all 68 strains. Resistance was detected less frequently using Balanced Sensitivity Test (BST) agar or Diagnostic Sensitivity Test (DST) agar containing methicillin or CA, BST or DST agar containing oxacillin. Increased production of beta-lactamase was shown to be an unlikely cause of MR in these strains. Disc sensitivity tests were performed on the 68 strains using five different media. Columbia agar gave optimum results as the other media gave enhanced zones of inhibition for some isolates. Further tests were performed on CA containing varying salt concentrations using both oxacillin and methicillin discs. A close relationship between the staphylococcal species, and the influence of increasing salt concentration on zone size was found. Discrepancies were noted between results obtained by breakpoint and the results obtained with methicillin discs particularly with Staphylococcus simulans and some Staphylococcus epidermidis strains. Results obtained with oxacillin discs more closely correlated with those obtained by breakpoint, but only when disc tests were performed on media with low and high salt content. To identify methicillin resistance in strains of CNS by disc tests, the use of Columbia agar with 0% and 5% added salt and oxacillin discs is recommended.     

Development of the novel chromogenic screening medium for methicillin-resistant Staphylococcus aureus

MRSA-chrom, a novel chromogenic screening agar medium for methicillin-resistant Staphylococcus aureus (MRSA), was developed. There were all MRSA strains recovered in 24 h as a specific blue-colored colony among 130 microbes including 42 MRSA strains. MRSA-chrom showed the highest detection ratio among 4 commercially available selective media using 50 clinical specimens.     

Identification of beta-lactamase-negative, ampicillin-resistant strains of Haemophilus influenzae with four methods and eight media

A challenge set of 143 non-beta-lactamase-producing strains of Haemophilus influenzae was tested for ampicillin susceptibility on two broth media and six agar media, using broth microdilution, agar dilution, disk diffusion, and E-test procedures. When beta-lactamase-negative, ampicillin-resistant (BLNAR) strains were defined as those for which the ampicillin MIC was > or = 4.0 microg/ml, 5 to 44% of our selected strains were BLNAR depending on the medium and/or test method used. If nonsusceptible strains for which ampicillin MICs were intermediate were included in the BLNAR category, 32 to 50% of our isolates would be considered BLNAR. These data emphasize the need for a standardized testing procedure and a universal definition of BLNAR strains before the clinical relevance of such strains can be evaluated. NCCLS dilution tests with haemophilus test medium broth or agar are preferred for testing ampicillin against H. influenzae.     

Rapid MRSA detection by a latex kit.

Methicillin resistant strains of Staphylococcus aureus (MRSA) are implicated in serious infections and nosocomial outbreaks, and show resistance to a wide range of antibiotics, thus limiting the treatment options. Therefore, rapid detection is clinically crucial for both treatment and infection control measures. This study assessed the performance of a rapid latex agglutination kit marketed to detect MRSA clinical isolates (MRSA-Screen test Denka Seiken Co Ltd, Tokyo, Japan) based on detecting a specific penicillin binding protein 2a (PBP2a) in comparison to the NCCLS oxacillin salt agar screen plate, the 1 microg oxacillin disk diffusion test, and the oxacillin MIC by E-test. Testing was carried out on 133 isolates consisting of 99 MRSA and 34 methicillin sensitive strains of S. aureus (MSSA). Concordant results were observed between the latex kit and all the other tests for the 99 MRSA isolates. Only 1 of the 34 MSSA isolates gave a positive agglutination reaction in the latex kit. The kit sensitivity and specificity were determined to be 100% and 97%, respectively. This reliable performance indicates that the MRSA-Screen latex test is very useful test for the rapid detection of MRSA isolates in the clinical microbiology laboratory.     

Susceptibility testing of clinical isolates of Methylobacterium species.

Methylobacterium species represent a relatively new genus which is being increasingly isolated from cases of opportunistic infections. This study reports on 3 reference strains and 15 clinical isolates of Methylobacterium species. Susceptibility tests were performed by the agar dilution and commercial broth microdilution methods at both 30 and 35 degrees C. Readings were made at 24, 48, and 72 h. Incubation conditions of 48 h and 30 degrees C were found to be optimum. Both the agar dilution and broth microdilution methods gave equivalent results. Drugs tested and their MICs for 90% of isolates (in micrograms per milliliter) were as follows: amikacin, less than or equal to 1; gentamicin, 1; ciprofloxacin, 1; trimethoprim-sulfamethoxazole, 2/38; ceftriaxone, 16; and ceftizoxime, 16. The majority of our isolates were resistant to six other beta-lactam drugs tested. Nine of the 15 Methylobacterium isolates were beta-lactamase positive.     

Comparison of in vitro antibiograms of Bacteroides fragilis group isolates: differences in resistance rates in two institutions because of differences in susceptibility testing methodology.

With 120 clinical isolates of the Bacteroides fragilis group, a comparison of rates of resistance to selected antimicrobial agents by using two susceptibility tests was performed in two medical institutions. The broth microdilution method produced MICs significantly lower than those determined by the agar dilution method. With ceftizoxime and cefoxitin, 88 and 18%, respectively, of the MICs were greater than or equal to 2 twofold dilutions apart. These differences in MIC results produced major interpretive discrepancies for ceftizoxime and cefoxitin, whereas no significant differences in resistance rates were noted for clindamycin and metronidazole.     

Evaluation of a new 3-h hybridization method for detecting the mecA gene in Staphylococcus aureus and comparison with existing genotypic and phenotypic susceptibility testing methods.

A new 3-h hybridization assay for detection of the staphylococcal mecA gene and the Staphylococcus aureus nuclease gene was evaluated by comparing the assay with existing genotypic and phenotypic methods. A total of 275 S. aureus strains were tested, including 257 epidemiologically unrelated strains (135 mecA-positive and 122 mecA-negative; collection I), and 18 strains with known borderline resistance to methicillin (collection II). Complete agreement was obtained for both collections when comparing the new assay with genotypic methods. We further evaluated a range of phenotypic susceptibility methods recommended in Europe and/or USA using the presence of the mecA gene as the defining standard. For collection I a high degree of agreement was found for both Etests (256 strains) and the oxacillin screen plate test (255 strains); the degree of agreement was lower for agar dilution methicillin (250 strains) and oxacillin 1 microg discs (239 strains). For the borderline strains a high degree of agreement was only obtained by the oxacillin screen plate test (17 of 18 strains). The other tests were less accurate, in the following order: agar dilution methicillin, Etest methicillin, Etest oxacillin and oxacillin discs with disagreement for four, five, nine and 13 strains, respectively. In conclusion, the new hybridization assay is a rapid and exact method for detecting the mecA gene and the S. aureus nuclease gene. This study confirms that phenotypic tests for methicillin resistance in S. aureus strains creates both false-susceptible and false-resistant results, especially for borderline resistant strains.     

Detection and expression of methicillin/oxacillin resistance in multidrug-resistant and non-multidrug-resistant Staphylococcus aureus in Central Sydney,Australia

Ninety clinical Staphylococcus aureus isolates from separate patients were examined phenotypically and genotypically for susceptibility to methicillin/oxacillin. Thirty were methicillin/oxacillin susceptible and 60 were methicillin and oxacillin resistant (MRSA). The 60 MRSA isolates examined were subdivided into two groups according to their antibiotic profiles and comprised 30 non-multidrug-resistant (NMDR) isolates, resistant to less than two non-beta-lactam antibiotics, and 30 multidrug-resistant (MDR) isolates, resistant to three or more non-beta-lactam antibiotics. Phenotypic and genotypic analysis of methicillin/oxacillin showed that despite use of the guidelines published by the NCCLS for the testing of S. aureus susceptibility to methicillin/oxacillin, MIC values of some NMDR MRSA isolates fell below the NCCLS-recommended breakpoints. Etest strips failed to detect two NMDR MRSA isolates tested with oxacillin and four tested with methicillin. Lowering the NCCLS-recommended oxacillin screen agar concentration from 6 to 2 mg/L and temperature of incubation to 30 degrees C, improved the specificity and sensitivity of NMDR MRSA detection from 87% to 100%. On PFGE analysis these NMDR MRSA strains were genotypically different. Genotypic tests, such as multiplex PCR for the mecA/nuc genes and DNA hybridization for the mecA gene, or phenotypic monoclonal antibody-based tests to detect penicillin-binding protein 2a (PBP2a) offer advantages for problematic isolates in detecting or confirming low-level phenotypic heterogeneous mecA expression of oxacillin and methicillin resistance in NMDR MRSA.     

头孢西丁纸片扩散法检测耐甲氧西林葡萄球菌

目的以PCR法检测葡萄球菌的mecA基因(mecA基因法)为标准，评价头孢西丁纸片扩散法、苯唑西林纸片扩散法和苯唑西林盐平板法检测葡萄球菌中耐甲氧西林葡萄球菌的灵敏度和特异性。方法PCR扩增葡萄球菌的特异性mecA基因片段，头孢西丁纸片扩散法、苯唑西林纸片扩散法和苯唑西林盐平板法检测葡萄球菌中耐甲氧西林葡萄球菌，药敏试验方法按标准K—B(Kirby-Bauer)法进行。结果在190株临床分离的葡萄球菌中金黄色葡萄球菌(金葡菌)138株，表皮葡萄球菌30株，溶血葡萄球菌22株，经。PCR法检测mecA基因，金葡菌中耐甲氧西林金葡菌(MRSA)和凝固酶阴性葡萄球菌中耐甲氧西林凝固酶阴性葡萄球菌(MRCNS)的发生率分别为81.2％(112／138)、96．1％(50／52)。头孢西丁纸片扩散法检测金葡菌中MRSA和CNS中MRCNS的发生率分别为81.2％(112／138)、94．2％(49／52)，与mecA基因法结果相比较，头孢西丁纸片扩散法检测葡萄球菌中MRS的灵敏度和特异度分别为99．4％(161／162)、100．0％(28／28)，两者符合率为99．5％(189／190)。2种方法所获得的结果经统计学处理，两者差异无显著性。结论头孢西丁纸片扩散法检测耐甲氧西林葡萄球菌具有很高的灵敏度和特异度，适合在临床微生物实验室中进行推广。     

Evaluation of alternative disk diffusion methods for detecting mecA-mediated oxacillin resistance in an international collection of staphylococci: validation report from the SENTRY Antimicrobial Surveillance Program

To validate the current National Committee for Clinical Laboratory Standards recommendations of the cefoxitin disk as a preferred surrogate marker to detect oxacillin resistance in staphylococcal isolates, 304 staphylococcal isolates originating from 49 sites in 16 countries in the SENTRY Antimicrobial Surveillance Program (2003) were tested. Two hundred three Staphylococcus aureus and 101 coagulase-negative staphylococci (CoNS), of which >95% were bloodstream isolates, were evaluated by comparing the results of the National Committee for Clinical Laboratory Standards broth microdilution method for oxacillin with those of the disk diffusion test using oxacillin, cefoxitin and ceftizoxime disks. Discrepancies were resolved using the PBP2a latex agglutination test. For S. aureus, the cefoxitin disk performed without interpretive error followed by the ceftizoxime disk (1% major and 0.5% minor errors; > or =20 mm = susceptible); use of the oxacillin disk test had the highest error rates with 4.4% major and 1.5% minor errors, whereas the oxacillin minimal inhibitory concentration (MIC) test was 99.0% accurate. For CoNS, the oxacillin disk test had the highest error rate with 4.0% major errors, followed by the cefoxitin (3.0% major error rate) and the ceftizoxime (1% very major and 1% minor error: > or =20 mm = susceptible) disk tests. The oxacillin MIC test was also 99.0% accurate for CoNS testing. Modification of the ceftizoxime disk diffusion breakpoints for CoNS resulted in complete intermethod categorical agreement. The overall accuracy of the four tests was as follows: modified ceftizoxime disk (99.3%) > oxacillin MIC = cefoxitin disk (99.0%) > current ceftizoxime disk (98.4%) > oxacillin disk (94.7%). In conclusion, these results confirm the superior performance characteristics of cefoxitin and ceftizoxime disk tests as surrogate markers to detect oxacillin resistance; by using an international collection of clinically significant staphylococcal isolates, we also demonstrate its wide global application.     

Synergy of daptomycin with oxacillin and other beta-lactams against methicillin-resistant Staphylococcus aureus

We previously observed marked synergy between daptomycin and both rifampin and ampicillin against vancomycin-resistant enterococci (VRE). Because the synergy between daptomycin and ampicillin was observed for 100% of VRE strains with high-level ampicillin resistance (ampicillin MIC of > or =128 microg/ml), we looked for synergy between daptomycin and other beta-lactams against 18 strains of methicillin-resistant Staphylococcus aureus (MRSA) by employing a time-kill method using Mueller-Hinton broth supplemented to 50 mg of Ca2+/liter. All strains were resistant to oxacillin (16 of 18 strains were resistant at drug concentrations of > or =256 microg/ml), and all strains were susceptible to daptomycin (the MIC at which 90% of the tested isolates were inhibited was 1 microg/ml). Daptomycin was tested at concentrations of 2, 1, 0.5, 0.25, 0.125, and 0.0625 microg/ml alone or in combination with oxacillin at a fixed concentration of 32 microg/ml. Synergy was found for all 18 strains with daptomycin at one-half the MIC in combination with 32 microg of oxacillin/ml, and synergy was found for 11 of 18 strains (61%) with daptomycin at one-fourth the MIC or less in combination with oxacillin. At 24 h, the daptomycin-oxacillin combination with daptomycin at one-half the MIC showed bactericidal activity against all 18 strains, and the combination with one-fourth the daptomycin MIC showed bactericidal activity against 9 of 18 strains. We also used a novel screening method to look for synergy between daptomycin and other beta-lactams. In this approach, daptomycin was incorporated into Ca(2+)-supplemented Mueller-Hinton agar at subinhibitory concentrations, and synergy was screened by comparing test antibiotic Kirby-Bauer disks on agar with and without daptomycin. By this method, daptomycin with ampicillin-sulbactam, ticarcillin-clavulanate, or piperacillin-tazobactam showed synergy comparable to or greater than daptomycin with oxacillin. For seven of the eight strains tested, time-kill studies confirmed synergy between daptomycin and ampicillin-sulbactam with ampicillin in the range of 2 to 8 microg/ml. The combination of daptomycin and beta-lactams may be useful for the treatment of MRSA infection, but further studies are needed to elucidate the mechanisms and to determine the in vivo efficacy of the combination.     

The prevalence of staphylococcal resistance to penicillinase-resistant penicillins. A retrospective and prospective national surveillance trial of isolates from 40 medical centers

In a retrospective survey of resistance to penicillinase-resistant penicillins (PRPs) in 152,076 clinical staphylococcal strains isolated in 40 United States Hospitals in 1985 and 1986, rates of resistance to oxacillin were found to be 11 and 13%, respectively, among Staphylococcus aureus isolates. The rates were approximately four times higher among coagulase-negative staphylococcal strains. In a prospective study of 1,408 wound or bacteremia isolates from the participant hospitals, oxacillin and methicillin agar screening, disk diffusion, and broth microdilution testing were conducted at a single reference laboratory. These tests yielded PRP resistance rates of 15% among S. aureus, 75% among S. epidermidis, and 48% among other coagulase-negative strains. No major changes in the distribution of resistance rates among hospitals or by hospital type were observed. Dilution susceptibility testing of several antimicrobial agents against PRP-resistant isolates and species-matched susceptible isolates from the same hospital showed that teicoplanin and vancomycin were the most active drugs (100% of S. aureus isolates were susceptible). Teicoplanin and vancomycin disk diffusion testing of all PRP-resistant staphylococcal strains also showed that these isolates were susceptible to the glycopeptides. However, agar dilution screening and broth microdilution tests revealed that several coagulase-negative strains, predominantly S. haemolyticus, had teicoplanin MICs greater than or equal to 8 micrograms/ml. S. haemolyticus isolates represented a very small number of the total stains tested. Teicoplanin and vancomycin were also the most active drugs when tested against older (1962-82) clinical PRP-resistant S. aureus strains from the reference laboratory collection. The methods found to be superior in detecting PRP-resistant strains were the oxacillin 6 micrograms/ml agar screening test in 4% NaCl-supplemented Mueller-Hinton agar and the 1 microgram oxacillin disk test. By reference laboratory standards, participant laboratories were incorrect in only 2.3% of species identifications and 4.5% of oxacillin-susceptibility determinations, indicating acceptable contemporary agreement and accuracy.