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.     

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.     

Staphylococcus aureus and Coagulase-Negative Staphylococci from Blood Stream Infections: Frequency of Occurrence,Antimicrobial Susceptibility,and Molecular (mecA) Characterization of Oxacillin Resistance in the SCOPE Program

Staphylococci are major causes of nosocomial blood stream infection. The recently completed SCOPE Surveillance Program found that coagulase-negative staphylococci (CoNS) and Staphylococcus aureus were the first and second most common etiologic agents, respectively, causing nosocomial blood stream infection in the USA. The frequency of oxacillin resistance was 68% among 1553 strains of CoNS and 26% among 787 strains of S. aureus in this study. Extended susceptibility profiles were generated for a subset of 150 S. aureus and 300 CoNS against 16 antimicrobial agents. Oxacillin-susceptible strains of both CoNS and S. aureus were uniformly susceptible to β-lactam agents with the exception of ampicillin and penicillin. Oxacillin-susceptible S. aureus were also highly susceptible to the fluoroquinolones, aminoglycosides, and trimethoprim/sulfamethoxazole. The oxacillin-susceptible CoNS were less susceptible to these agents, and only glycopeptides were reliably active against oxacillin-resistant strains. PCR detection of the mecA gene was used to scrutinize current NCCLS interpretive breakpoint MICs for determining susceptibility or resistance to oxacillin. We found complete concordance between the presence or absence of mecA and the NCCLS oxacillin interpretive breakpoint categories for S. aureus. In contrast, the NCCLS breakpoints for oxacillin significantly underestimate the degree of true oxacillin resistance among CoNS. Using the presence of mecA as the reference standard, we detected 15.7% false susceptibility to oxacillin using a MIC susceptible breakpoint concentration of ≤2 μg/mL. Lowering the oxacillin MIC breakpoint to ≤0.25 μg/mL for CoNS would greatly improve the accuracy of the MIC test performance. We found that both the current oxacillin disk test and the 30-μg ceftizoxime disk test functioned quite well in predicting those strains of CoNS that contain mecA. These studies have demonstrated both a high level of antimicrobial resistance among nosocomial blood stream isolates of staphylococci as well as significant problems with the current NCCLS breakpoints for oxacillin when testing CoNS.     

Vitek GPS card susceptibility testing accuracy using direct inoculation from Bactec 9240 blood culture bottles

The emergent need for antimicrobial susceptibility testing (AST) data for the therapy of bacteremic patients has led to the development of rapid methods and local procedure modification of some commercial AST products such as the direct inoculation from blood culture systems. We compared the Vitek GPS card results using direct and standardized inoculation with a reference broth microdilution method for 112 consecutive staphylococcal bloodstream infections (seven drugs). Among the 28 Staphylococcus aureus strains, 0%–3.6% total error/drug was observed with both Vitek inoculation procedures. However, the only oxacillin-resistant strain was not detected (100% true very-major error). For 84 coagulase-negative staphylococci (CNS), the direct inoculation procedure had an 11.9% very-major error rate for oxacillin, ampicillin-sulbactam, and cephalothin, plus 4.8% very-major error rate for ciprofloxacin and trimethoprim-sulfamethoxazole (total error rate 4.8%–16.7% for five of seven drugs compared). The Vitek direct inoculation procedure routinely missed 20.4% of oxacillin-resistant CNS strains. The use of Vitek direct inoculation procedures for staphylococcal bloodstream infection isolates (from Bactec 9240 cultures) produced serious false-susceptible results; this procedure should be avoided in favor of routine package insert-recommended Vitek procedures or other reference-quality overnight incubation susceptibility tests.     

Detection of Methicillin Resistance in Coagulase-Negative Staphylococci Initially Reported as Methicillin Susceptible Using Automated Methods

Reliable detection of methicillin resistance in coagulase-negative staphylococci (CNS) is required for appropriate therapy of serious infections from these pathogens. To determine the most accurate method of measuring methicillin resistance in CNS initially reported as methicillin susceptible by automated methods, we compared mecA detection by polymerase chain reaction (PCR) with phenotypic methods. One hundred eighty-eight blood culture isolates of CNS that were initially reported as susceptible to methicillin using commercial methods (Vitek or MicroScan) were tested by agar dilution, disk diffusion, oxacillin salt agar screen plate, and a multiplex PCR assay using primer sets for mecA and 16S rRNA. Sixteen isolates (8.5%) previously reported as methicillin susceptible by automated methods contained the mecA gene. MICs of these isolates ranged from 0.5 μg/mL to ≥128 μg/mL. Ten of these isolates had MICs equal to or below the NCCLS breakpoint of 2 μg/mL. Six of the 10 isolates (4 with MICs of 0.5 μg/mL and 2 with MICs of 2 μg/mL) did not grow on any of the oxacillin screen plates after 48 h of incubation at 30°C or 35°C. All six isolates were induced to grow in the presence of oxacillin at 128 μg/mL by serial passaging on plates containing increasing concentrations of antibiotic. Retesting with MicroScan and Vitek detected methicillin resistance in 7 and 10 isolates, respectively. Disk diffusion testing with incubation for 48 h proved to be the next best method after PCR for detection of methicillin resistance (15 of 16 isolates). Commercial automated methods and some methods recommended by National Committee for Clinical Laboratory Standards may not detect methicillin resistance in CNS that carry the mecA gene and have MICs just below breakpoint.     

In vitro susceptibilities of four species of coagulase-negative staphylococci.

The in vitro susceptibilities of 260 strains of coagulase-negative staphylococci to penicillin G, oxacillin, nafcillin, methicillin, cephalothin, and seven non-beta-lactam antimicrobial agents were determined and compared with the susceptibilities of 54 strains of Staphylococcus aureus with known patterns of susceptibility. Penicillin G susceptibility for S. aureus, Staphylococcus epidermidis, Staphylococcus haemolyticus, and Staphylococcus hominis was readily determined by using beta-lactamase tests with induced cells and with a standardized microdilution test. MIC criteria for susceptibility used for S. aureus were applicable to the coagulase-negative species. Percentages of organisms susceptible were as follows: S. epidermidis, 7%; S. haemolyticus, 5%; and S. hominis, 47%. Oxacillin susceptibility for these four species was readily determined by using a modification of the microdilution test. MIC criteria for susceptibility used for S. aureus were applicable to S. haemolyticus and S. hominis, but alternate criteria were necessary for S. epidermidis. Percentages of organisms susceptible were as follows: S. epidermidis, 29%; S. haemolyticus, 36%; and S. hominis, 97%. Staphylococcus saprophyticus differed from the other staphylococcal species; all strains were beta-lactamase negative and were penicillin susceptible but had higher penicillin G MICs than did susceptible strains of the other species. There was total cross resistance among the penicillinase-resistant penicillins and cephalothin for the coagulase-negative staphylococci as well as for S. aureus; oxacillin MICs were more reliable than MICs of the other drugs or a standardized disk diffusion test for distinguishing resistant from susceptible strains. Vancomycin, rifampin, and ciprofloxacin were consistently active against all staphylococci. Erythromycin, clindamycin, gentamicin, and trimethoprim-sulfamethoxazole were more active against oxacillin-susceptible staphylococci than against oxacillin-resistant staphylococci.     

Routine use of the Gen-Probe MTD2 amplification test for detection of Mycobacterium tuberculosis in clinical specimens in a large public health mycobacteriology laboratory

The antimicrobial susceptibility of 239 coagulase-negative staphylococci (CNS) isolates consecutively collected from blood culture in patients admitted in a 600-bed teaching hospital was evaluated. The isolates were identified to the species level by conventional methods and the MicroScan Positive Combo Panel type 6 system, and their susceptibility to vancomycin, teicoplanin, and oxacillin were tested by agar dilution, disk diffusion, and MicroScan-WalkAway system. The species distribution was as follows: Staphylococcus epidermidis 120 (50.2%), S. hominis 29 (12.1%), S. haemolyticus 24 (10.0%), S. cohnii 14 (5.9%), and isolates from other CNS species 52 (21.8%). The percentage of resistance to oxacillin was 74.5% by agar dilution. The highest percentages of oxacillin resistance were found among S. haemolyticus (95.8%) and S. epidermidis (80.8%). Teicoplanin resistance (MIC > or = 32 micrograms/mL) was detected in five S. haemolyticus isolates, whereas intermediate resistance (MIC = 16 micrograms/mL) was detected in nine strains. These isolates with reduced susceptibility to teicoplanin were resistant to oxacillin, but remained susceptible to vancomycin (MIC < or = 4 micrograms/mL). Two isolates, one S. haemolyticus and one S. epidermidis, showed a vancomycin MIC of 8 micrograms/mL, and both MicroScan and disk diffusion methods classified these isolates as susceptible. Our results showed that glycopeptide resistance is emerging among CNS isolates in our institution and the disk diffusion method may not detect isolates with decreased susceptibility to these antimicrobial agents.     

Beta-lactam susceptibility of coagulase-negative staphylococci causing catheter sepsis in pediatric patients

We evaluated standard oxacillin and methicillin disk diffusion (DD) and broth microdilution (MD)-MIC tests with and without 2% NaCl for detecting heteroresistance among 47 blood isolates of coagulase-negative staphylococci (CNS) causing catheter sepsis in pediatric patients. The 24-hr oxacillin DD test detected the greatest number (40) of apparent hetero-resistant isolates, but methicillin DD and oxacillin MD-MIC with 2% NaCl performed equally as well (38 and 37 resistant isolates, respectively). An additional 24-hr incubation did not significantly increase the number of apparent heteroresistant isolates detected by these methods. Discrepant results with the various test methods occurred most commonly among Staphylococcus epidermidis isolates with MD-MIC values near the breakpoint concentrations for interpretation of susceptible and resistant strains. For detection of heteroresistance among the CNS, we encourage use of standard oxacillin DD and MD-MIC tests but would suggest that isolates with MIC values ranging from 1-2 micrograms/ml be interpreted cautiously until clinical studies demonstrate the efficacy of treating patients with infections caused by such strains.     

纸片法头孢西丁敏感的青霉素耐药葡萄球菌分析

目的 评估不同检测方法 对纸片法头孢西丁敏感,苯唑西林耐药葡萄球菌耐药性状的检测能力,并对非mecA基因介导苯唑西林耐药的匍萄球菌进行药敏谱分析.方法 收集2007年1月至2009年5月间复旦大学附属华山医院就诊患者呼吸道、尿、分泌物和无菌体液标本中分离得到的255株金黄色葡萄球菌,采用苯唑西林纸片法、苯唑西林MIC法、头孢西丁纸片法、头孢西丁MIC法枪测金黄色葡萄球菌对苯唑西林的敏感性:用苯唑西林MIC法和头孢西丁纸片法检测75株凝固酶阴性萄萄球菌对苯唑西林的敏感性:将所有葡萄球菌进行mecA基因检测,结合试验结果 分析葡萄球菌苯唑西林耐药原因,并对非mecA基因介导的苯唑西林耐药葡萄球菌用MIC法进行抗菌药敏谱分析.结果 255株纸片法头孢西丁敏感、青霉素耐药的金黄色葡萄球菌苯唑西林纸片法检测出6株中介,4株耐药;头孢西丁纸片法、头孢西丁MIC法、苯唑西林MIC法全敏感、mecA基因检测全阴性.75株纸片法头孢西丁敏感、青霉素耐药的凝固酶阴性葡萄球菌,苯唑西林MIC法59株敏感,16株耐药;mecA基因阴性为71株,阳性为4株.12株非mecA基因介导苯唑西林耐药的葡萄球菌庆大霉素敏感为10株,克林霉素8株、环丙沙星11株、红霉素6株、甲氧苄啶/磺胺甲噁唑11株,头孢菌素类、替考拉宁、万古霉素、哌拉西林/他唑巴坦、四环素12株.结论 金黄色葡萄球菌用头孢西丁检测mecA基因介导的苯唑西林耐药,具有很好的可靠性.凝固酶阴性葡萄球菌最好同时使用头孢西丁纸片法和苯唑西林MIC法检测mecA基因介导的苯唑西林耐药,以提高检出率.非mecA基因介导的苯唑西林耐药,临床可依据实际药物敏感实验结果 选择性使用β内酰胺酶稳定的青霉素、β内酰胺酶抑制剂复合药、头孢类和碳青霉烯类药物治疗.     

Survey of antimicrobial activity of four commonly used third generation cephalosporins tested against recent bacterial isolates from ten American medical centers,and assessment of disk diffusion test performance

Over 2,000 clinical isolates from ten American medical centers were tested for susceptibility to cefotaxine, ceftriaxone, ceftizoxime, and ceftazidime by both broth microdilution and disk diffusion methods. Typically resistant (e.g. enterococci) and highly susceptible (e.g. streptococci) isolates whowed no change in susceptibility patterns compared to previous surveys. Pseudomonas aeruginosa and Stenotrophomonas maltophilia exhibited significant decreases in susceptibility to these cephalosporins. Among Escherichia coli and Klebsiella spp isolates, two to three percent were resistant to one of the four drugs whereas they were very susceptible to the other three. Of the four antibiotics, the cefotaxime disk diffusion test results correlated best with the microdilution results. With the other three drugs the disk diffusion test yielded 1 to 9% more susceptible test results and 1 to 20% fewer resistant test results than broth microdilution when testing gram negative bacilli. The clinical significance of such discrepancies is not known, but the impact on antibiotic susceptibility surveys and antibiogram comparisons could be significant.     

Evaluation of a Commercial Microdilution System for Quantitative Susceptibility Testing of Aminoglycosides Against Multidrug-Resistant, Gram-Negative Bacilli

Susceptibility of clinical isolates of Pseudomonas aeruginosa (29 isolates), Klebsiella species (54 isolates), Escherichia coli (28 isolates), Serratia marcescens (28 isolates), and Enterobacter species (29 isolates) to gentamicin, tobramycin, and amikacin was determined by the following three methods: commercial broth microdilution trays, standard agar dilution, and disk diffusion susceptibility. A total of 504 tests were performed by each method, and overall susceptibility or resistance determined by the broth microdilution method agreed with that determined by the agar dilution method in 92.7% of the tests, whereas results from the disk diffusion method agreed with those from the agar dilution method in 91.9% of the tests. The broth microdilution and disk diffusion methods agreed with each other 88.7% of the time. The broth microdilution system results varied from the agar dilution method results by more than one dilution in 121 of 504 determinations (24%); however, this altered susceptibility determinations in only 7.3% of the assays. E. coli isolates were found to be quantitatively more resistant to the aminoglycosides with the broth microdilution method than with the agar dilution method. In contrast, the broth microdilution method demonstrated P. aeruginosa to be quantitatively more susceptible to the aminoglycosides than when the results were obtained by the agar dilution method. The Micro-Media Systems method is economical, reliable, rapid, and simple to perform and yields quantitative minimum inhibitory concentrations.     

Antimicrobial Activity of Ceftaroline Tested against Staphylococci with Reduced Susceptibility to Linezolid,Daptomycin, or Vancomycin from U.S. Hospitals, 2008 to 2011

Vancomycin, linezolid, and daptomycin are very active against staphylococci, but isolates with decreased susceptibility to these antimicrobial agents are isolated sporadically. A total of 19,350 Staphylococcus aureus isolates (51% methicillin resistant [MRSA]) and 3,270 coagulase-negative staphylococci (CoNS) were collected consecutively from 82 U.S. medical centers from January 2008 to December 2011 and tested for susceptibility against ceftaroline and comparator agents by the reference broth microdilution method. Among S. aureus strains, 14 isolates (0.07%) exhibited decreased susceptibility to linezolid (MIC, ≥8 μg/ml), 18 (0.09%) to daptomycin (MIC, ≥2 μg/ml), and 369 (1.9%) to vancomycin (MIC, ≥2 μg/ml; 368 isolates at 2 μg/ml and 1 at 4 μg/ml). Fifty-one (1.6%) CoNS were linezolid resistant (MIC, ≥8 μg/ml), and four (0.12%) were daptomycin nonsusceptible (MIC, ≥2 μg/ml). Ceftaroline was very active against S. aureus overall (MIC_50/90, 0.5/1 μg/ml; 98.5% susceptible), including MRSA (MIC_50/90, 0.5/1 μg/ml; 97.2% susceptible). All daptomycin-nonsusceptible and 85.7% of linezolid-resistant S. aureus isolates were susceptible to ceftaroline. Against S. aureus isolates with a vancomycin MIC of ≥2 μg/ml, 91.9, 96.2, and 98.9% were susceptible to ceftaroline, daptomycin, and linezolid, respectively. CoNS strains were susceptible to ceftaroline (MIC_50/90, 0.25/0.5 μg/ml; 99.1% inhibited at ≤1 μg/ml), including methicillin-resistant (MIC_50/90, 0.25/0.5 μg/ml), linezolid-resistant (MIC_50/90, 0.5/0.5 μg/ml), and daptomycin-nonsusceptible (4 isolates; MIC range, 0.03 to 0.12 μg/ml) strains. In conclusion, ceftaroline demonstrated potent in vitro activity against staphylococci with reduced susceptibility to linezolid, daptomycin, or vancomycin, and it may represent a valuable treatment option for infections caused by these multidrug-resistant staphylococci.     

Antimicrobial susceptibility of Gram-positive bacteria isolated from US medical centers: results of the Daptomycin Surveillance Program (2007–2008)

Gram-positive bacterial strains (12?443) consecutively collected during 2007 to 2008 in hospitals located in the United States were tested by reference broth microdilution methods against daptomycin and comparison agents. Methicillin (oxacillin) resistance rates were 55.9% and 74.0% for Staphylococcus aureus and coagulase-negative staphylococci, respectively, and the vancomycin resistance rate among Enterococcus faecalis and Enterococcus faecium were 5.4% and 75.4%, respectively. Daptomycin was very active against all Gram-positive species with MIC₉₀ values of 0.5, 0.25, 0.5, and 2 μg/mL for staphylococci, β-hemolytic streptococci, viridans group streptococci, and enterococci, respectively. Overall, 99.9% of S. aureus, 100.0% of E. faecalis, and 99.5% of E. faecium were susceptible to daptomycin. In addition, daptomycin MIC distributions for S. aureus and enterococci from 2007 to 2008 were very similar to those from 2002 to 2003. In summary, high rates of methicillin-resistant staphylococci and vancomycin-resistant enterococci were observed in US hospitals, but daptomycin remains active against these clinically important Gram-positive organisms with no evidence of potency loss since its approval for clinical use in late 2003.     

In vitro susceptibility testing and quality control parameters for sarafloxacin (A-56620): a fluoroquinolone used for treatment and control of colibacillosis in poultry

Sarafloxacin (formerly A-56620) is a fluoroquinolone recently introduced into veterinary practice for the therapy of colibacillosis and other indicated infections. Sarafloxacin was noted to be very active and comparable to ciprofloxacin and enrofloxacin for inhibiting 823 strains from a wide variety of species at ≤1 or ≤2 μg/mL. In vitro susceptibility tests for sarafloxacin using National Committee for Clinical Laboratory Standards (NCCLS) methods were also studied and interpretive criteria for Escherichia coli–associated colibacillosis isolates were proposed: susceptible at ≤0.06 μg/mL (≥25 mm) and resistant at ≥0.25 μg/mL (≤21 mm). Sarafloxacin agar dilution MIC results were approximately one log₂ dilution higher than broth microdilution endpoints. The interpretive criteria demonstrated a low error rate of 5.9%, with a very major rate of only 0.5% (correlation coefficient between methods = 0.94). Quality control trials in six laboratories established MIC and zone diameter (5-μg disk) limits for the NCCLS recommended strains: for the broth microdilution test, E. coli ATCC 25922 = 0.008 to 0.03 μg/mL, Pseudomonas aeruginosa ATCC 27853 = 0.12 to 1 μg/mL, Enterococcus faecalis ATCC 29212 = 0.5 to 2 μg/mL, and Staphylococcus aureus ATCC 29213 = 0.06 to 0.25 μg/mL; and for the disk diffusion test, E. coli ATCC 25922 = 30 to 36 mm, S. aureus ATCC 25923 = 25 to 30 mm, and P. aeruginosa ATCC 27853 = 23 to 29 mm. These criteria for in vitro tests with sarafloxacin should enable the longitudinal monitoring of its activity against the indicated pathogens and allow detection of emerging resistant populations that may necessitate altered dosing regimens.     

Disk diffusion interpretive criteria for fusidic acid susceptibility testing of Staphylococci by the National Committee for Clinical Laboratory Standards method

Fusidic acid is used in many countries for the treatment of multiresistant staphylococcal infection, especially multiresistant Staphylococcus aureus infection (MRSA). We collected consecutive fusidic acid-resistant isolates of staphylococci from the routine laboratory over several years, and compared these strains with fusidic acid-susceptible staphylococci to establish interpretive criteria for disk diffusion testing by National Committee for Clinical Laboratory Standards (NCCLS) methods. The minimum inhibitory concentrations (MICs) and zone diameters for strains of S. aureus (n = 102), including MRSA, S. saprophyticus (n = 20) and other coagulase-negative staphylococci (n = 115) were determined by NCCLS agar dilution and disk diffusion tests using a 2.5-μg disk of fusidic acid. MICs were bimodally distributed. No isolates had MICs of 0.5 or 1 μg/ml; thus, we chose these values to define strains of intermediate susceptibility. The error-rate-bounded method was used to determine interpretive zone diameters for disk testing. Interpretive zone diameter criteria were found to be: susceptible ⩾22 mm, intermediate 18–21 mm, and resistant ⩽17 mm. All S. saprophyticus were intrinsically resistant to fusidic acid (MIC ⩾2 μg/ml).     

Antistaphylococcal activity of amoxicillin and ticarcillin when combined with clavulanic acid. Evaluation of oxacillin-resistant and oxacillin-susceptible isolates

The beta-lactamase inhibitor, clavulanic acid, was combined with amoxicillin and with ticarcillin for in vitro studies with 586 staphylococci: 97 stock cultures of oxacillin-resistant strains recovered before 1982, and 489 blood or wound isolates collected from 40 separate medical centers during 1987-1988 (300 were oxacillin resistant). Over 92% of the staphylococci produced beta-lactamase enzymes and were thus resistant to both penicillins. However, with the addition of clavulanic acid, oxacillin-susceptible strains were rendered susceptible to low concentrations of amoxicillin and ticarcillin. Staphylococcus aureus strains with borderline or partial borderline resistance to penicillinase-resistant penicillins occurred infrequently (72 of 325 S. aureus isolates). Those strains were susceptible to both clavulanic acid combinations, because their methicillin resistance is thought to be due to an excess beta-lactamase production. Strains with chromosomally mediated intrinsic heteroresistance were relatively resistant to both drug combinations. Minimal inhibitory concentration (MIC) breakpoints that best separated those heteroresistant strains from oxacillin-susceptible isolates were as follows: amoxicillin/clavulanic acid, less than or equal to 2.0/1.0 micrograms/ml for susceptible; and ticarcillin/clavulanic acid, less than or equal to 4.0/2.0 micrograms/ml for susceptible. When the broth was supplemented by 2% NaCl, MICs for both drug combinations were increased by less than one doubling dilution. Although oxacillin and methicillin broth microdilution tests were more reliable when 2% NaCl was added, tests with the two drug combinations were only minimally improved by adding 2% NaCl to the broth medium.     

Antimicrobial susceptibility testing of Helicobacter pylori comparison of E-test,broth microdilution,and disk diffusion for ampicillin,clarithromycin, and metronidazole

The optimal method for the determination of the minimum inhibitory concentration (MIC) of antimicrobials against Helicobacter pylori has not been established. The epsilometer agar diffusion gradient test (E-Test; AB Biodisk, Solna, Sweden) was compared with broth microdilution, the reference method, and disk diffusion for the antimicrobial susceptibility testing of 122 clinical isolates of H. pylori to ampicillin, clarithromycin, and metronidazole. Isolates were considered to be resistant when the MIC value was >8 μg/ml for either ampicillin or metronidazole and >2 μg/ml for clarithromycin. For an individual isolate, the MICs for ampicillin and clarithromycin determined by broth microdilution and the E-test were highly reproducible, with replicate results being within ±1 log₂ dilution. The correlation between the MICs determined by E-test and broth microdilution was excellent for both ampicillin and clarithromycin (90.1% and 88.5% were within ±log₂ dilution, and 98.3% and 96.7% of the values were within ±2 log₂ dilution, respectively). In no instance did the interpretation of “sensitive” or “resistant” differ. Conversely, only 70.5% of the E-test results for metronidazole were within ±1 log₂ dilution of the broth microdilution results. In addition, 15 (12.3%) of the H. pylori isolates interpreted as resistant by the E-test were sensitive by the broth microdilution method. All discrepancies occurred when the E-test MIC values fell between 8 and 32 μg/ml. The results of the ampicillin and clarithromycin disk diffusion assay correlated 100% with the results of the broth microdilution. However, these data suggest that when the E-test MIC results for metronidazale yield values between 8 and 32 μg/ml, the MIC should be reevaluated by another method.     

Detection of oxacillin-resistance in Staphylococcus aureus by MicroScan MIC panels in comparison to four other methods

Two hundred fifty-two isolates of Staphylococcus aureus were tested for oxacillin susceptibility by MicroScan Gram positive overnight and rapid MIC panels. Results were compared with nonautomated methods including disk diffusion, MRSA Crystal ID, and Etests using MRSA Screen Agar as reference. One hundred sixty-nine isolates (67.1%) were oxacillin-susceptible and 83 (32.9%) were resistant. All methods agreed for 234 (92.9%) isolates. Very major error rates were 1.2% for disk diffusion, 3.6% for Etest, and 0 for all other methods. Major error rates were 5.3% for MicroScan overnight panels, 3% for rapid panels, 2.4% for disk diffusion, 1.2% for Etest, and 0.6% for MRSA Crystal ID. Nine oxacillin-susceptible isolates with borderline MICs and discrepant results for 1 or more methods were tested for the mec A gene and all were negative. Each was susceptible to β lactam/β lactamase inhibitor combinations, suggesting that false resistance may have been due to excessive β lactamase production. Oxacillin-resistant S. aureus with borderline MICs determined by MicroScan should be confirmed by an alternate method. The most practical and cost-effective means among those we tested is the MRSA Screen Agar.     

Evaluation of a Disk Diffusion Method for Determining Susceptibility of Mycobacterium avium Complex to Clarithromycin

We evaluated an agar disk diffusion method for determining the susceptibility of Mycobacterium avium complex to clarithromycin. Isolates were inoculated onto the surface of a Middlebrook 7H11 plate, followed by the application of a 15-μg clarithromycin disk. Zone sizes were read after 5–7 days of incubation. Zone sizes had a bimodal distribution; 40 isolates (10%) had no zone of inhibition, whereas the zone sizes for the remaining isolates ranged from 11 to 60 mm. Most isolates (37/40) having no zone of inhibition came from patients who had been treated previously with clarithromycin. Fifty-one isolates were also tested for clarithromycin susceptibility using a microdilution broth method. Defining susceptibility as a zone size of >10 mm, disk diffusion test results agreed with the results by the microdilution broth method for 50 of 51 (98%) isolates tested by both methods. Agar disk diffusion is a promising method for the determination of clarithromycin susceptibility testing for M. avium complex.     

Evaluation of selection media for the detection of borderline MRSA

Recently, hospital-associated as well as community-acquired methicillin-resistant Staphylococcus aureus (MRSA) strains showing a low level of resistance to oxacillin have emerged worldwide, and as a result, a highly sensitive method to detect MRSA has become more important. To prevent MRSA being overlooked, some selection agar media have recently been developed. We evaluated six commercially available selection agar media in regard to the detection of 35 borderline MRSA (BOMRSA) strains which were mecA-positive but showed low resistance to oxacillin. The MIC values of oxacillin differed between the broth dilution method and the agar dilution method, and 11 of the 35 BOMRSA strains were judged as sensitive by the broth dilution method and 14 of the 35 strains were judged as sensitive by the agar dilution method. Thirty-two of the 35 strains were also judged as sensitive by an oxacillin disk diffusion test. Moreover, there was no consistent pattern of resistance to the tested beta-lactams among the BOMRSA strains. Some commercially available selection media designed for the detection of MRSA contain a beta-lactam antibiotic; oxacillin, ceftizoxime, or cefoxitin, and we evaluated media containing each of these agents. The detection sensitivities of cefoxitin-based agar media, such as CHROMagar MRSA and MRSA ID, for BOMRSA were 100% at 24-h culture. On the other hand, the media containing oxacillin or ceftizoxime gave lower results at 24 h, suggesting that, possibly, BOMRSA strains may not to be able to grow on these media. These results suggest that cefoxitin-based agar media should be recommended for the first-round screening of BOMRSA.