Reliability of high-content disks and modified broth dilution tests for detecting staphylococcal resistance to the penicillinase-resistant penicillins.

In vitro susceptibility tests were performed with 271 isolates of Staphylococcus species (204 Staphylococcus aureus), including 110 strains resistant to the penicillinase-resistant penicillins. Disks containing 5 or 10 micrograms of methicillin, 1 or 4 micrograms of oxacillin, and 1 or 4 micrograms of nafcillin were evaluated. After a full 24 h of incubation at 35 degrees C, tests with 1-microgram oxacillin disks provided optimal results. Use of the more potent oxacillin, nafcillin, or methicillin disks only increased the number of false-susceptible test results. For broth microdilution tests, 2% NaCl should be added to cation-supplemented Mueller-Hinton broth, and MICs should be recorded after a full 24 h at 35 degrees C. Microdilution tests with oxacillin in broth with 2% NaCl were more reliable than similar tests with methicillin.     

Screening method for rapid detection of methicillin-resistant (heteroresistant) Staphylococcus aureus.

A broth screening method was developed to detect methicillin-resistant (heteroresistant) strains of Staphylococcus aureus from primary plates. Lyophilized vials containing cation-supplemented Mueller-Hinton broth, 2% NaCl, and 10 micrograms of methicillin and 6 micrograms of oxacillin per ml were hydrated and tested with 129 methicillin-resistant and 35 methicillin-susceptible strains of S. aureus. With the addition of a tetrazolium indicator after 5 h of incubation, 96.9% of resistant strains were detected. No false-positive results occurred.     

Reliability of the microdilution technic for detection of methicillin-resistant strains of staphylococcus aureus.

To determine whether the microdilution technic for antimicrobic susceptibility testing could detect the hetero-resistance of "methicillin"-resistant Staphylococcus aureus, 100 stock cultures (54 methicillin-susceptible and 46 methicillin-resistant) were tested against methicillin, oxacillin, nafcillin and cephalothin. Each drug was diluted in Mueller-Hinton broth and in Mueller-Hinton broth with 5% NaCl. Minimum inhibitory concentration (MIC) determinations were made after 20-24 hours at 35 C and again after 48 hours of incubation. With one exception, methicillin resistance was detected in Mueller-Hinton broth when the trays were reincubated and read after 48 hours. Earlier detection of methicillin resistance was possible when the penicillinase-stable penicillins were diluted in broth containing 5% NaCl. Further studies suggest that the addition of NaCl to the broth medium might improve the stabilities of methicillin, oxacillin, and nafcillin during storage of the frozen microdilution trays. Either nafcillin or oxacillin would appear to be the best representative of the penicillinase-stable penicillins. Standardized disk-diffusion tests with cephalothin disks indicated that most methicillin-resistant strains are susceptible to cephalothin. However, there is reason to believe that such in-vitro test results might be inappropriate. With microdilution tests, most methicillin-resistant isolates were more resistant to cephalothin than were the methicillin-susceptible strains, but the MIC's for both types of staphylococci were generally within the range of concentrations that can be obtained during therapy. The addition of NaCl to the microdilution tests with cephalothin did not alter the test results significantly.     

Detection of methicillin-resistant Staphylococcus epidermidis.

To determine whether methods suggested for detecting methicillin-resistant Staphylococcus aureus apply equally to methicillin-resistant Staphylococcus epidermidis, 135 S. epidermidis isolates were tested by the Vitek AMS gram-positive susceptibility card (Vitek Systems, Inc., Hazelwood, Mo.) and by modifications of agar screen, disk diffusion, and microdilution methods. Modifications included 24- versus 48-h incubation, unsupplemented versus 2% NaCl-supplemented broth, and standard versus direct inoculum. At 24 h, the highest number of resistant strains, 59, was detected by oxacillin (1 microgram) disk diffusion. At 48 h, three additional strains were judged resistant. With one exception, results for oxacillin disk diffusion and agar screen were equivalent at 24 and 48 h. Vitek detected 50 resistant strains. Significantly fewer resistant strains were detected at 24 h by methicillin disk diffusion (5 micrograms) and methicillin microdilution with 2% NaCl. For oxacillin microdilution, neither 2% NaCl supplementation nor the method of inoculum preparation significantly affected the results. Oxacillin microdilution with cation- rather than non-cation-supplemented broth detected significantly fewer (n = 33) resistant strains at 24 h; 51 were resistant at 48 h. To detect methicillin-resistant S. epidermidis, a direct inoculum with either 24-h oxacillin disk diffusion and reincubation of intermediate strains for an additional 24 h or 24-h oxacillin agar screen and reincubation of strains with no growth for a total of 48 h is recommended.     

New recommendations for disk diffusion antimicrobial susceptibility tests for methicillin-resistant (heteroresistant) staphylococci

The agar disk diffusion susceptibility test was reevaluated for its ability to discriminate between susceptible and resistant Staphylococcus aureus (128 strains) and coagulase-negative staphylococci (19 strains) when tested with methicillin, oxacillin, and nafcillin. The results show that the current recommendations for disk potencies and interpretive zone diameters do not fit well with MIC correlates that we now recommend. Based on data from this study, we suggest that these parameters of the test be changed. For methicillin, we recommend a 10-micrograms disk with breakpoints of less than or equal to 11 mm (greater than or equal to 16 micrograms/ml) to indicate resistance and greater than or equal to 15 mm (less than or equal to 4 micrograms/ml) to indicate susceptibility. For oxacillin and nafcillin, we recommend 4-micrograms disks with breakpoints of less than or equal to 12 mm (greater than or equal to 8 micrograms/ml) to indicate resistance and greater than or equal to 16 mm (less than or equal to 2 micrograms/ml) to indicate susceptibility. MIC breakpoints were from a broth microdilution system which used a medium containing salt. If one of these three penicillins were to be selected for routine tests, we would recommend oxacillin, based on our data, but we recognize that this may depend upon the population of staphylococci within a particular hospital.     

Evaluation of MicroScan MIC panels for detection of oxacillin-resistant staphylococci.

Clinical isolates of staphylococci (420 Staphylococcus aureus isolates and 248 coagulase-negative staphylococci) were tested by both MicroScan MIC panels (MicroScan, West Sacramento, Calif.) and an oxacillin agar screen (Mueller-Hinton agar [Difco Laboratories, Detroit, Mich.] containing 6 micrograms of oxacillin per ml and 4% NaCl) to evaluate the ability of MicroScan to detect oxacillin-resistant strains. MicroScan panels and oxacillin agar screen plates were incubated at 35 degrees C for 24 h and at 30 degrees C for an additional 24 h. Endpoints were recorded at 24 and 48 h. By MicroScan, 23 (5.5%) and 30 (7%) S. aureus isolates and 161 (65%) and 162 (65%) coagulase-negative staphylococci were oxacillin resistant at 24 and 48 h, respectively. At both 24 and 48 h, 23 (5.5%) S. aureus isolates and 162 (65%) coagulase-negative staphylococci were resistant by the oxacillin agar screen. Five strains for which the oxacillin MIC was 2 or 4 micrograms/ml and eight strains resistant to oxacillin only at 48 h were further evaluated by broth macrodilution testing for oxacillin with and without clavulanic acid, by oxacillin and amoxicillin-clavulanic acid disk diffusion, and by oxacillin agar screen comparing Mueller-Hinton agars purchased from Difco and BBL Microbiology Systems, Cockeysville, Md. By this additional testing, all 10 S. aureus isolates and 1 of 3 coagulase-negative staphylococci examined produced increased amounts of beta-lactamase. One coagulase-negative staphylococcus appeared to be truly intermediately oxacillin susceptible. There was no significant difference in the rate of detection of oxacillin resistance between MicroScan and the agar screen. MicroScan panels should be incubated for 24 h only, because prolonged incubation caused strains producing excessive amounts of beta-lactamase to appear to be falsely oxacillin resistant.     

Evaluation of laboratory tests for detection of methicillin-resistant Staphylococcus aureus and Staphylococcus epidermidis.

Few studies evaluating susceptibility testing of methicillin-resistant staphylococci have included isolates of Staphylococcus epidermidis, a known pathogen in many types of serious infections. We tested 175 S. epidermidis and 95 Staphylococcus aureus isolates to determine the most sensitive procedures for detecting methicillin-resistant staphylococci. Reference procedures included agar dilution with methicillin and 4% NaCl in the agar and broth microdilution with methicillin and 2% NaCl in cation-supplemented Mueller-Hinton broth. After 24 h of incubation, the results from both methods correlated well and were within 1 log2 dilution for all isolates tested. Only one-half of all resistant isolates (92 of 183) were detected at 18 h by using the standard disk diffusion technique with 5-micrograms methicillin disks, and even fewer were detected with 10-micrograms methicillin disks and newly recommended zone-size criteria. However, the standard disk diffusion method with 4% NaCl in the agar increased the sensitivity and specificity for identification of the proper phenotype to greater than 92%. The spread plate and new spot techniques, both using agar with 4% NaCl, were also sensitive methods. Of 47 S. epidermidis isolates tested against oxacillin, 6 (13%) were oxacillin susceptible but methicillin resistant. Two automated systems, the Automicrobic system (Vitek Systems) and MicroScan (American MicroScan), as well as two broth screening systems available from Remel and Austin Biological Laboratories, failed to detect several resistant isolates, depending on the species.     

Variation in the abilities of automated, commercial, and reference methods to detect methicillin-resistant (heteroresistant) Staphylococcus aureus.

The abilities of commercial MIC, automated, and reference methods for in vitro detection of methicillin-resistant Staphylococcus aureus were determined on 49 strains from eight hospitals. Micro-Media, MicroScan, Sensititre, Sceptor, API Uniscept KB, Abbott MS-2, Vitek AMS, Autobac MTS, NCCLS disk diffusion, and broth microdilution antimicrobial susceptibility testing procedures were evaluated. All testing was performed by using manufacturers' or reference procedures, and results were determined at no later than 24 h of incubation at 35 degrees C. With NCCLS disk diffusion, all strains were resistant to oxacillin (1 microgram), 47 (96%) were resistant to methicillin (4 micrograms), and 48 (98%) were resistant to nafcillin (1 microgram). The percentages of strains resistant to methicillin (greater than 8 micrograms/ml) were 98% with API Uniscept KB, 86% with Sceptor, MicroScan, and Autobac MTS, 84% with Sensititre, 71% with Micro-Media, and 70% with NCCLS MIC. Abbott MS-2 detected 86% of strains resistant to methicillin (greater than 5 micrograms/ml). With oxacillin (greater than 2 micrograms/ml), 90% were detected with Vitek AMS and 70% were detected with NCCLS MIC. With nafcillin (greater than 2 micrograms), 82% were resistant with Micro-Media, 57% were resistant by NCCLS MIC, and 50% (three of six) were resistant by MicroScan. Two strains from one hospital and one strain from another gave susceptible results with all automated and commercial methods. All strains from three centers were detected by all methods. Variability also occurred among the systems with cephalothin, clindamycin, gentamicin, chloramphenicol, and trimethoprim sulfamethoxazole.     

The role of beta-lactamase in staphylococcal resistance to penicillinase-resistant penicillins and cephalosporins.

We showed that most Staphylococcus aureus strains that have borderline or intermediate susceptibility to the penicillinase-resistant penicillins (PRPs) react this way because of the activity of their beta-lactamase on these antimicrobial agents. These strains produced large amounts of staphylococcal beta-lactamase that rapidly hydrolyzed penicillin and partially hydrolyzed the PRPs. Susceptibility to hydrolysis was penicillin greater than oxacillin greater than cephalothin greater than methicillin. The borderline results and the hydrolysis could be prevented by the beta-lactamase inhibitors clavulanic acid and sulbactam. For intrinsically methicillin-resistant (heteroresistant) S. aureus, the inhibitors reduced the penicillin MICs, but the strains remained resistant to all the beta-lactam antimicrobial agents, including penicillin. We conclude that the borderline in vitro susceptibility or resistance to PRPs in most of these S. aureus strains is mediated by beta-lactamase and they are not heteroresistant or intrinsically resistant. We do not know whether this in vitro resistance is expressed clinically.     

Detection of intrinsically resistant (heteroresistant) Staphylococcus aureus with the Sceptor and AutoMicrobic systems.

Modified procedures for the Sceptor Gram-Positive MIC Panel and the Vitek AutoMicrobic System GPS-M Card were evaluated for their ability to detect methicillin-resistant (heteroresistant) Staphylococcus aureus. A total of 398 clinical isolates (including 222 methicillin-resistant S. aureus) obtained from 10 hospitals were tested. Both systems had 2% NaCl in the oxacillin wells. Sceptor MIC panels were inoculated with an organism suspension prepared from an 18- to 24-h blood agar plate and were inoculated for a full 24 h at 35 degrees C before MICs were read. All methicillin-resistant S. aureus isolates were detected as resistant to oxacillin at greater than or equal to 8 micrograms/ml by the Sceptor method and at greater than 2 micrograms/ml by the Vitek method. All 176 oxacillin-susceptible, methicillin-susceptible S. aureus isolates were correctly distinguished from methicillin-resistant S. aureus isolates by Sceptor. However, with the Vitek system 29 methicillin-susceptible S. aureus isolates tested as falsely resistant to oxacillin and four isolates tested as falsely resistant to vancomycin. The modified testing procedure with the Sceptor system can be used reliably for accurate susceptibility testing of methicillin-resistant and methicillin-susceptible S. aureus. The Vitek GPS-M card does not accurately discriminate between methicillin-resistant and methicillin-susceptible S. aureus with an oxacillin breakpoint of greater than 2 micrograms/ml.     

Correlation between genotype and phenotypic categorization of staphylococci based on methicillin susceptibility and resistance

Positive correlation between methicillin and oxacillin susceptibility test results and the detection of the mecA gene was observed for Staphylococcus aureus, S. epidermidis, and S. haemolyticus as well as among mecA(+) strains of other species of coagulase-negative staphylococci (CNS). However, at least 50% of the mecA-negative strains of these other species of CNS were falsely classified as methicillin and oxacillin resistant.     

Choice of the NaCl concentration for optimizing the detection of methicillin resistance in Staphylococcus using the gel diffusion method

The detection of oxacillin resistance is evaluated by the disk diffusion method in a collection of 374 Staphylococcus sp strains. The disk diffusion assay is performed with 5-microgram oxacillin disk and a 10(8) CFU/mL inoculum on Mueller-Hinton agar plates supplemented with 2 or 5% NaCl and incubated at 37 degrees C for 24 h. Strains are considered resistant in accordance to the French recommendations (any growth around the disk is observed). The detection of mecA gene is performed by PCR almost for resistant and discordant strains. Results are concordant for 246 of 256 Staphylococcus aureus strains (182 susceptible and 64 resistant strains) and for 105 of 118 S. epidermidis isolates tested (37 susceptible and 68 resistant strains). Six mecA-negative strains (3 S. aureus and 3 S. epidermidis) give false resistant results on agar with 2 and 5% NaCl. Seventeen isolates are discordant on 5% NaCl: 7 mecA-negative S. aureus strains are susceptible on 2% NaCl agar but resistant at 5% (3% false-positive results), 10 mecA-positive S. epidermidis strains are resistant on 2% NaCl agar but susceptible at 5% NaCl (5% false-negative results). The detection of meticillin resistance is improved on agar supplemented with 2% NaCl.     

Comparison of different techniques for the detection of heterogeneous resistance to methicillin in Staphylococcus aureus]

The methods for detection of methicillin resistant S. aureus (MRSA) can fail to detect resistance because phenotypic expression is often heterogeneous (40% of strains). Seventy four strains of S. aureus [4 methicillin susceptible strains, 10 homogeneous MRSA (Ro) and 60 heterogeneous MRSA (Rh)] were isolated from different french hospitals in Paris. These strains were tested by different methods: oxacillin screen plate with 6 micrograms/ml oxacillin and 4% NaCl, agar diffusion method with 5 micrograms oxacillin disk tested either at 30 degrees C on Mueller-Hinton medium or at 37 degrees C on Mueller-Hinton plus 5% NaCl, BBL Crystal MRSA ID system tested with two inocula (0.5 and 1 McFarland equivalent bacterial suspension) at 37 degrees C for 4 h and 5 h. Dot-blot hybridization was performed under stringent condition with the mecA probe. The accuracy of the different methods for the detection of methicillin resistance is equivalent, except for the BBL crystal system with a 0.5 McFarland inoculum wich detects the resistance with an accuracy of 86% for Ro strains and 69% for Rh strains. In other respects, there was a close correlation with the detection of the phenotypic resistance and the presence of mecA gene. So this study demonstrates that these various methods can be used for the detection of methicillin resistant S. aureus. For a rapid detection (below 5 h) the BBL crystal system with a 1 McFarland inoculum can be used; the agar diffusion method remains a good method provided some conditions (inoculum, incubation temperature, addition of salt, incubation period); the oxicillin screnn plate is a very attractive method for it is easy and reliable.     

Reevaluation of the ability of the standardized disk diffusion test to detect methicillin-resistant strains of Staphylococcus aureus.

To reevaluate the ability of the disk diffusion method to detect methicillin-resistant Staphylococcus aureus, 73 such isolates from 13 cities were tested for antimicrobial susceptibility with the standardized disk diffusion test. Duplicate plates were incubated at 30 and 35 degrees C and read after 18, 24, and 48 h. After incubation at 35 degrees C for 24 h, 97% of isolates appeared resistant to methicillin, and 99% appeared resistant to oxacillin. A significantly smaller proportion of isolates appeared resistant to cephalothin (P less than 0.001) and cefamandole (P less than 0.001). Isolates from some cities had no zones of inhibition around methicillin and oxacillin disks, whereas those from other cities had measurable zones of inhibition, with light growth inside the zones. Patterns of growth around cephalothin and cefamandole disks also varied among isolates from different cities. Incubation at 30 degrees C for 24 h did not result in better detection of methicillin or oxacillin resistance. All study isolates appeared resistant to methicillin and oxacillin after 48 h of incubation at 35 degrees C. The results suggest that methicillin-resistant S. aureus strains from many areas will be detected if standardized disk diffusion tests are incubated at 35 degrees C for 24 h.     

Identification of methicillin-resistant strains of staphylococci by polymerase chain reaction.

A simple and reliable method using a polymerase chain reaction (PCR) was devised to identify methicillin-resistant staphylococci. By using lysates of the strain to be tested as templates and 22-mer oligonucleotides as primers, a 533-bp region of mecA, the structural gene of a low-affinity penicillin-binding protein (PBP 2'), was amplified by PCR and detected by agarose gel electrophoresis. Results obtained by this method were compared with those obtained by broth microdilution MIC determination for 210 and 100 clinical isolates of Staphylococcus aureus and coagulase-negative staphylococci, respectively. Of 99 mecA-negative S. aureus isolates, 100% of the strains were methicillin susceptible and 98% of the strains were oxacillin susceptible. Three strains (3%) of 111 mecA-positive S. aureus isolates exhibited almost the same susceptibility to beta-lactams as the mecA-negative ones and did not produce detectable amounts of PBP 2' despite the presence of the mecA gene. One of them yielded typically methicillin-resistant variants at a low frequency with concomitant recovery of PBP 2' production. The mecA gene was also found in coagulase-negative Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus sciuri, Staphylococcus saprophyticus, and Staphylococcus caprae and conferred resistance on most of the bacteria.     

Two percent sodium chloride is required for susceptibility testing of staphylococci with oxacillin when using agar-based dilution methods.

The need to add NaCl to agar media to ensure accuracy of results when testing staphylococci with oxacillin was investigated. The results of four antimicrobial susceptibility testing methods (agar and broth dilution, E test, and disk diffusion) in which the growth medium contained 0, 2, 4, or 5% NaCl were compared with the results of a hybridization assay using a mec gene probe. We tested 223 strains of staphylococci, 128 of which were mec gene positive. A total of 7 of the 128 positive strains were coagulase-negative staphylococci with 24-h oxacillin MICs of < or = 2 micrograms/ml. Ninety-five isolates were mec gene negative, including seven strains of Staphylococcus aureus with oxacillin MICs of > or = 4 micrograms/ml. The oxacillin MICs for mec gene-positive, oxacillin-resistant strains of staphylococci increased two- to fourfold with the addition of NaCl to the test medium, while the MICs for mec gene-negative strains did not change in the presence of added salt. Very major error rates for the agar dilution and E test methods in the absence of salt ranged from 18.2 to 20.2%. Major error rates for mec gene-negative S. aureus isolates were > 17% for all test methods when 4 or 5% NaCl was added to the test medium. The addition of 2% NaCl to Mueller-Hinton agar for testing of oxacillin resulted in very major error rates of < 1% for the agar dilution and E test methods although the major error rates for the two methods with added NaCl were 8.5 and 6.9%, respectively. The disk diffusion test did not perform well in this study, showing essential error rates of > or = 18.3%. We recommended the addition of 2% NaC1 to Mueller-Hinton agar when testing staphylococci with oxacillin by either the agar dilution or E test method. NaC1 should not be added for the disk diffusion test.     

Early Screening of oxacillin-resistant Staphylococcus aureus and Staphylococcus epidermidis from blood culture

The timely detection of blood-borne pathogens is one of the most important functions of the microbiology laboratory. Recently, methicillin-resistant staphylococci have become the most important pathogens seen by the laboratory. The purpose of this study was to evaluate Staphy agar, a novel screening medium, for the detection methicillin-resistant Staphylococcus aureus, S. epidermidis, or other coagulase-negative staphylococci (CNS) from positive blood cultures showing Gram-positive cocci in clusters. Eighty-six blood cultures that yielded Gram-positive cocci in clusters were included in this study. The organisms were finally identified by the Vitek system, and oxacillin resistance was confirmed by polymerase chain reaction (PCR)-based mecA gene detection. The identification and oxacillin resistance of all S. aureus strains showed complete agreement with the Vitek and PCR results. The presumptive detection of S. epidermidis and other CNS were consistent with the Vitek system in 94.7%, and the screening of oxacillin resistance was consistent with the result of PCR in 92.1% of 38 strains. The Staphy agar method is reliable and rapid for differentiating Gram-positive cocci in clusters in blood and for determining their methicillin resistance.     

Salt-supplemented medium for testing methicillin-resistant staphylococci with newer beta-lactams.

The addition of 2% NaCl to cation-supplemented Mueller-Hinton broth (CSMHB) was evaluated for microdilution testing of the susceptibility of staphylococci to five cephalosporins, imipenem, amoxicillin-clavulanate, and ticarcillin-clavulanate. With Staphylococcus aureus, NaCl improved the recognition of methicillin (oxacillin) resistance to cefamandole, imipenem, or ticarcillin-clavulanate. Resistance to amoxicillin-clavulanate was readily determined, irrespective of the presence of added salt. The addition of 2% NaCl to CSMHB did not significantly improve detection of resistance to any of the beta-lactams among coagulase-negative staphylococci. Since the addition of NaCl did not have significant adverse effects on tests with coagulase-negative staphylococci, the routine addition of 2% NaCl to oxacillin or methicillin tests with staphylococci may be justifiable on the basis of convenience or standardization. However, addition of NaCl to susceptibility tests of other beta-lactams does not consistently improve recognition of resistance among staphylococci and thus cannot be recommended for routine use.     

Survey of Staphylococcus isolates among hospital personnel, environment and their antibiogram with special emphasis on methicillin resistance

The objective of this study was to find the prevalence of Staphylococcus spp. carriage among hospital personnel and hospital environment and their antibiogram with special emphasis on methicillin resistance. A total of 205 samples from hospital personnel and environment were collected from casualty, oncology and multidisciplinary cardiac unit ward of Kasturba Medical College Hospital, Manipal. Samples were collected using sterile cotton wool swabs and inoculated into brain heart infusion broth. Subcultures were done onto blood agar and MacConkey's agar. Isolates were identified by standard methods up to species level. Antimicrobial susceptibility test was performed according to standardized disc diffusion Kirby-Bauer method. Each of the isolates was screened for methicillin resistance using oxacillin disc on Mueller Hinton agar plate followed by MIC for methicillin and cefoxitin susceptibility test by disc diffusion method. Sixty five out of 205 strains (31.7%) were Staphylococcus spp. and all of them were coagulase negative. Most of the strains belonged to S.epidermidis 49.23% (32/65) followed by S. saprophyticus 26.15% (17/65). Maximum isolates of S.epidermidis were from anterior nares 28.12% (9/32 strains of S.epidermidis). Highest number of methicillin resistant coagulase negative strains (3/9, 33.33%) were isolated from stethoscope of multidisciplinary cardiac unit ward followed by carriers in the anterior nares (2/9, 22.22%). Methicillin resistant coagulase negative staphylococci are prevalent in anterior nares of hospital personnel and in the hospital environment thereby providing a definite source for hospital acquired infection. All isolates were sensitive to vancomycin, ciprofloxacin and amikacin.