Newly defined in vitro quality control ranges for oritavancin broth microdilution testing and impact of variation in testing parameters

A 9-laboratory M23-A2 quality control (QC) study was performed to evaluate reproducibility of oritavancin MICs against reference strains of Staphylococcus aureus, Enterococcus faecalis, and Streptococcus pneumoniae using broth microdilution assays in the presence of polysorbate 80. Polysorbate 80 has previously been shown to be required for accurate measurement of oritavancin broth microdilution MICs. Greater than 95% of replicate results (n = 270/organism) fell within the following QC ranges (in micrograms per milliliter): S. aureus ATCC 29213, 0.015 to 0.12; E. faecalis ATCC 29212, 0.008 to 0.03; and S. pneumoniae ATCC 49619, 0.001 to 0.004. Oritavancin MIC QC ranges were, thus, narrow and reproducible. Parameters affecting testing results in the presence of polysorbate 80 were also evaluated. Oritavancin MICs were equivalent to or within 1 doubling dilution of those obtained under standard Clinical and Laboratory Standards Institute testing conditions, regardless of incubation time (18, 24, or 48 h), Ca(2+) concentration, pH, or frozen panel storage time (up to 6 months).     

Susceptibility testing of fastidious organisms in the API microdilution panel

It has become increasingly important to perform routine susceptibility tests on certain anaerobic bacteria, Haemophilus influenzae and Streptococcus pneumoniae, as a result of the decreasing predictability of their antimicrobial susceptibility patterns. The antimicrobial susceptibilities of 49 anaerobic isolates, 25 H. influenzae isolates, and 25 S. pneumoniae isolates were determined concurrently by API Uniscept MIC trays and a conventional broth microdilution method using Wilkins-Chalgren broth, 5% Fildes in Schaedler broth, or 5% lysed horse blood in Mueller-Hinton broth, respectively. Analysis of 490 anaerobic organism-antibiotic combinations, 144 H. influenzae-antibiotic combinations, and 125 S. pneumoniae-antibiotic combinations showed that 98.9%, 100%, and 99.2%, respectively, of the API results were within +/- 1 log2 dilution of the reference system. The API Uniscept MIC panel would be acceptable for use as a routine susceptibility system for anaerobic organisms in a clinical microbiology laboratory. To eliminate trailing endpoints, however, further studies need to be performed to evaluate additional broth media for the susceptibility testing of H. influenzae and S. pneumoniae in the API panels.     

Use of a surfactant (polysorbate 80) to improve MIC susceptibility testing results for polymyxin B and colistin

Accurate determination of in vitro activity for polymyxin class agents has consistently been a problem due to their physical–chemical characteristics that can be influenced by the constituents of reference and/or standardized susceptibility testing methods. We evaluated the impact of using polysorbate 80 (P-80), a surfactant, in reference broth microdilution (BMD) methods when testing polymyxin B and colistin against 247 clinical strains of Enterobacteriaceae (124 strains), Acinetobacter spp. (60 strains), and Pseudomonas aeruginosa (63 strains). All testing was performed in frozen-form BMD panels with and without 0.002% P-80. MIC results for both polymyxins were generally 4- to 8-fold lower when P-80 was added to the testing broth compared to Mueller-Hinton broth without the surfactant. Decreases were greatest in organisms having MIC values at ≤2 μg/mL and among Acinetobacter spp. Polymyxins should be tested with P-80 to more accurately assess the potencies of these agents necessary to treat multidrug-resistant Gram-negative bacilli.     

Activity of moxifloxacin and twelve other antimicrobial agents against 216 clinical isolates of Streptococcus pneumoniae

BACKGROUND: An increased incidence of macrolide resistance in penicillin-resistant Streptococcus pneumoniae has been described. METHODS: With this in mind, 216 S. pneumoniae isolates were evaluated for their in vitro susceptibility to a new fluoroquinolone, moxifloxacin, which was compared with penicillin, amoxicillin, cefuroxime, cefotaxime, ceftriaxone, erythromycin, clarithromycin, ciprofloxacin, sparfloxacin, ofloxacin, vancomycin and teicoplanin. A broth microdilution assay was performed in cation- adjusted Mueller-Hinton broth with 5% (v/v) lysed horse blood according to NCCLS guidelines. RESULTS: Erythromycin resistance was observed in all the 22 penicillin-resistant S. pneumoniae (10.1%). All the penicillin- susceptible S. pneumoniae were susceptible to cephalosporins, whereas all the penicillin-resistant ones showed resistance to cefuroxime and only intermediate susceptibility to cefotaxime and ceftriaxone. The 216 tested strains were inhibited by sparfloxacin and moxifloxacin at concentrations of 0.12-0.5 mg/l and 0.06-0.25 mg/l, respectively, regardlesss of whether the strain was penicillin and/or erythromycin resistant. Seven penicillin-resistant strains displayed resistance to ofloxacin. All isolates were susceptible to vancomycin; teicoplanin MIC values ranged from 0.03 to 0.12 mg/l. The excellent in vitro activity of moxifloxacin against S. pneumoniae was not affected by penicillin and/or macrolides. CONCLUSION: Moxifloxacin appears to be a promising choice for the treatment of pneumococcal infections, including situations where therapeutic choices are limited due to penicillin and macrolide resistance.     

Increased rate of isolation of penicillin-resistant Streptococcus pneumoniae in a children's hospital and in vitro susceptibilities to antibiotics of potential therapeutic use.

The isolation of Streptococcus pneumoniae with both high and intermediate resistance to penicillin has increased in our institution since 1989 to an average of 12.1% of all isolates. We determined the susceptibilities of 95 isolates (34 susceptible to penicillin, 42 intermediate in resistance to penicillin, and 19 resistant to penicillin) to 16 antimicrobial agents of potential use in the treatment of disease caused by S. pneumoniae. Susceptibility to penicillin was determined by broth macrodilution with Mueller-Hinton broth supplemented with 5% lysed horse blood. Isolates were classified as highly resistant when the MIC was greater than or equal to 2.0 micrograms/ml, intermediate in resistance when the MIC was between 0.1 and 1.0 microgram/ml, and susceptible when the MIC was less than 0.1 microgram/ml. Fifteen of 19 isolates found to be highly resistant to penicillin were recovered from the middle ear of children. None of the isolates recovered from cerebrospinal fluid was highly resistant to penicillin. Fifteen of these isolates highly resistant to penicillin were found to be serogroup 6. Susceptibilities to other antibiotics were determined by the agar dilution method with Mueller-Hinton agar containing 5% lysed horse blood and an inoculum of 10(4) CFU per spot delivered by a replicator device. The MIC for 90% of isolates increased with increasing penicillin resistance for all antibiotics tested, except chloramphenicol, ciprofloxacin, rifampin, and vancomycin. Regardless of the classification of penicillin resistance, all isolates were classified as susceptible to cefotaxime, cefpirome, cefpodoxime, clarithromycin, imipenem, rifampin, and vancomycin on the basis of National Committee for Clinical Laboratory Standards interpretive guidelines. Interpretation of susceptibilities on the basis of currently available guidelines is difficult in that susceptibility guidelines applicable specifically to S. pneumoniae are not available.     

Use of In Vitro Vancomycin Testing Results To Predict Susceptibility to Oritavancin,a New Long-Acting Lipoglycopeptide

Oritavancin is a recently approved lipoglycopeptide antimicrobial agent with activity against Gram-positive pathogens. Its extended serum elimination half-life and concentration-dependent killing enable single-dose treatment of acute bacterial skin and skin structure infections. At the time of regulatory approval, new agents, including oritavancin, are not offered in the most widely used susceptibility testing devices and therefore may require application of surrogate testing using a related antimicrobial to infer susceptibility. To evaluate vancomycin as a predictive susceptibility marker for oritavancin, 26,993 recent Gram-positive organisms from U.S. and European hospitals were tested using reference MIC methods. Organisms included Staphylococcus aureus, coagulase-negative staphylococci (CoNS), beta-hemolytic streptococci (BHS), viridans group streptococci (VGS), and enterococci (ENT). These five major pathogen groups were analyzed by comparing results with FDA-approved susceptible breakpoints for both drugs, as well as those suggested by epidemiological cutoff values and supported by pharmacokinetic/pharmacodynamic analyses. Vancomycin susceptibility was highly accurate (98.1 to 100.0%) as a surrogate for oritavancin susceptibility among the indicated pathogen species. Furthermore, direct MIC comparisons showed high oritavancin potencies, with vancomycin/oritavancin MIC₉₀ results of 1/0.06, 2/0.06, 0.5/0.12,1/0.06, and >16/0.06 μg/ml for S. aureus, CoNS, BHS, VGS, and ENT, respectively. In conclusion, vancomycin demonstrated acceptable accuracy as a surrogate marker for predicting oritavancin susceptibility when tested against the indicated pathogens. In contrast, 93.3% of vancomycin-nonsusceptible enterococci had oritavancin MIC values of ≤0.12 μg/ml, indicating a poor predictive value of vancomycin for oritavancin resistance against these organisms. Until commercial oritavancin susceptibility testing devices are readily available, isolates that when tested show vancomycin susceptibility can be inferred to be susceptible to oritavancin by using FDA-approved breakpoints.     

Susceptibility of pseudomonads to Melaleuca alternifolia (tea tree) oil and components

OBJECTIVES: Thirty isolates of Pseudomonas aeruginosa, 15 isolates of Pseudomonas putida and 11 isolates of Pseudomonas fluorescens were tested for susceptibility to tea tree oil (TTO), the essential oil of Melaleuca alternifolia, and the components terpinen-4-ol, alpha-terpineol, cineole, gamma-terpinene and rho-cymene. METHODS: MICs were determined by broth microdilution in Mueller-Hinton medium supplemented with 0.002% (v/v) Tween 80. RESULTS: The MIC90 of TTO for all isolates tested was 4% (v/v) or less. Susceptibility to components tested varied between species. CONCLUSIONS: Pseudomonas spp. are susceptible to TTO and some of its components although they are less susceptible than many other bacteria tested previously.     

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.     

Comparative Evaluation of Colistin Susceptibility Testing Methods among Carbapenem-Nonsusceptible Klebsiella pneumoniae and Acinetobacter baumannii Clinical Isolates

We compared six colistin susceptibility testing (ST) methods on 61 carbapenem-nonsusceptible Klebsiella pneumoniae (n = 41) and Acinetobacter baumannii (n = 20) clinical isolates with provisionally elevated colistin MICs by routine ST. Colistin MICs were determined by broth microdilution (BMD), BMD with 0.002% polysorbate 80 (P80) (BMD-P80), agar dilution (AD), Etest, Vitek2, and MIC test strip (MTS). BMD was used as the reference method for comparison. The EUCAST-recommended susceptible and resistant breakpoints of ≤2 and >2 μg/ml, respectively, were applied for both K. pneumoniae and A. baumannii. The proportions of colistin-resistant strains were 95.1, 77, 96.7, 57.4, 65.6, and 98.4% by BMD, BMD-P80, AD, Etest, MTS, and Vitek2, respectively. The Etest and MTS methods produced excessive rates of very major errors (VMEs) (39.3 and 31.1%, respectively), while BMD-P80 produced 18% VMEs, AD produced 3.3% VMEs, and Vitek2 produced no VMEs. Major errors (MEs) were rather limited by all tested methods. These data show that gradient diffusion methods may lead to inappropriate colistin therapy. Clinical laboratories should consider the use of automated systems, such as Vitek2, or dilution methods for colistin ST.     

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.     

Dalbavancin activity against selected populations of antimicrobial-resistant Gram-positive pathogens

Dalbavancin, a dimethylaminepropyl amide derivative of the lipoglycopeptide A40926, was tested against 375 antimicrobial-resistant Gram-positive pathogens collected worldwide during 2001-2003. The isolates were tested by reference and Clinical Laboratory Standards Institute broth microdilution susceptibility methods, and dalbavancin was compared with over 20 other antimicrobials. Vancomycin resistance determinants among enterococci were identified using PCR primer sets for vanA and vanB. Dalbavancin was generally more potent than vancomycin or teicoplanin. Dalbavancin was highly active against penicillin- and ceftriaxone-resistant Streptococcus pneumoniae strains (MIC(90), < or = 0.016 microg/mL). Dalbavancin was also very active against teicoplanin-nonsusceptible coagulase-negative staphylococci (CoNS; MIC range, 0.03-0.25 microg/mL), but dalbavancin MIC results were slightly elevated compared with wild type strains. Dalbavancin inhibited vanB enterococci (MIC range, 0.03-0.12 microg/mL) and was active against other resistant, non-vanA enterococcal species. However, vanA enterococcal strains were not as susceptible to dalbavancin (MIC50, 16 microg/mL). In summary, dalbavancin was very active against a wide spectrum of resistant Gram-positive isolates and demonstrated greater potency than vancomycin or teicoplanin.     

Comparative evaluation of a new fluorescent carboxyfluorescein diacetate-modified microdilution method for antifungal susceptibility testing of Candida albicans isolates

This report presents a fluorescent carboxyfluorescein diacetate (CFDA)-modified microdilution method used for the susceptibility testing of Candida albicans to amphotericin B, fluconazole, ketoconazole, itraconazole, voriconazole, and flucytosine. Four different broth microdilution susceptibility testing methods were simultaneously evaluated at 24 and 48 h. The MICs determined using the CFDA-modified method (MIC(cfda)) were compared to those obtained by the standard broth microdilution method (MIC(visual)) and a procedure employing the indicator Alamar blue (MIC(alamar)). The reference MIC was determined visually as recommended by the NCCLS M27-A protocol, and then quantified spectrophotometrically following agitation (MIC(spec)). The CFDA-modified microdilution method was demonstrated to effectively determine the MICs for all the antifungal drugs tested at both 24 and 48 h. The results from both the MIC(spec) and MIC(cfda) methods yielded >80% agreement within +/-1 dilution and >90% agreement within +/-2 dilutions at 24 h in comparison to the reference MIC(visual) method, respectively. The trailing growth phenomenon that occurs with azole antifungal drugs and many strains of C. albicans did not inhibit the effectiveness of the MIC(spec) and MIC(cfda) methods. The MIC(spec) and MIC(cfda) methods shared 92.8% agreement within +/-1 dilution at 24 h and 87.6% agreement within +/-1 dilution at 48 h.     

Broth microdilution and E-test for determining fluoroquinolone activity against Streptococcus pneumoniae

OBJECTIVE: To compare broth microdilution and E-test minimum inhibitory concentrations (MICs) of 4 fluoroquinolones against Streptococcus pneumoniae and to determine the effect of these in vitro MIC methods on the calculation of AUC00-24/MIC ratios. METHODS: Levofloxacin, gatifloxacin, moxifloxacin, and gemifloxacin MICs were determined by broth microdilution (incubated in air) and E-test (incubated in CO2) for 100 clinical isolates of S. pneumoniae. MIC50, MIC90, and geometric mean MIC were calculated. Steady-state serum concentration-time profiles were simulated for once-daily, oral dosing of levofloxacin 500 mg, gatifloxacin 400 mg, moxifloxacin 400 mg, and gemifloxacin 320 mg. After correcting for protein binding, AUC0-24 of unbound drug was calculated for each regimen, and AUC0-24/MIC ratios were calculated using MIC data from both in vitro methods. Differences in MICs between methods were determined for each agent using the paired t-test (after logarithmic transformation of MICs) and the Wilcoxon signed-rank test. Differences in AUC0-24/MIC ratios were also determined using the paired t-test and the Wilcoxon signed-rank test. The level of significance for all analyses was p < 0.05. RESULTS: Broth microdilution and E-test MICs were within +/- 1 log2 dilution for 94%, 93%, 61%, and 35% of the isolates for levofloxacin, gatifloxacin, moxifloxacin, and gemifloxacin, respectively. Broth microdilution MICs were significantly lower than E-test MICs for all 4 agents (p < 0.001). However, a categorical change in susceptibility was seen for only 1 isolate with gatifloxacin and moxifloxacin (intermediate by broth microdilution, resistant by E-test). AUC0-24/MIC ratios were significantly higher for each regimen when MICs were determined by broth microdilution compared with E-test (p < 0.001). CONCLUSIONS: There is a significant difference in the activity of the newer fluoroquinolones against S. pneumoniae when MICs are determined by broth microdilution and E-test. When evaluating fluoroquinolone activity and pharmacodynamics against this organism, clinicians must be aware that MIC testing methodology may have a significant impact on the results.     

Reproducibility assessment of tigecycline MIC results by broth microdilution methods using commercially prepared dry-form panels

We assessed the reproducibility of tigecycline and piperacillin/tazobactam MIC results tested by broth microdilution methods on commercially prepared dry-format panels. Fifteen bacterial isolates were tested 3 times daily for 3 days for a total of 9 replicate results per strain. The within-day and between-days reproducibility was 100% for tigecycline at +/-1 log(2) dilution step and >98% for piperacillin/tazobactam at +/-1 log(2) dilution step. Identical MIC results were observed for 84.4% and 78.7% of tests for same-day replicates for tigecycline and piperacillin/tazobactam, respectively. These results demonstrate the excellent reproducibility of in vitro susceptibility tests for both tigecycline and piperacillin/tazobactam using a commercially prepared dry-form broth microdilution MIC panel.     

Correlation of various in vitro testing methods with clinical outcomes in patients with Bacteroides fragilis group infections treated with cefoxitin: a retrospective analysis.

There is limited information regarding the correlation of anaerobic susceptibility testing and outcome in the treatment of Bacteroides fragilis infections. We retrospectively analyzed the clinical outcomes of B. fragilis infections in patients treated with cefoxitin; the analysis was blinded for susceptibility results. Isolates of B. fragilis were tested by multiple agar dilution methods, disk elution, and commercial broth microdilution methods. Of 19 patients analyzed, 11 were cured and 8 were treatment failures. No significant differences existed between the groups with respect to age, sex distribution, weight, APACHE II score, dose of cefoxitin, or bacteremia. Failure was associated with a longer cefoxitin dosing interval (P = 0.019), a longer duration of hospitalization (P = 0.038), and decreased duration of cefoxitin treatment (P = 0.05). Four agar dilution systems (brucella plus blood, Wilkins-Chalgren, Wilkins-Chalgren plus blood, brain heart infusion plus blood) and two broth systems (Wilkins-Chalgren microdilution and a commercial system [Micromedia; Beckman, Carlsbad, Calif.]) all demonstrated lower geometric mean MICs for isolates from the group of patients that could be cured. Only the commercial broth microdilution medium (Micromedia) demonstrated a significantly reduced geometric mean MIC (P = 0.056). By using a logistic regression analysis, the shorter cefoxitin dosing interval (P = 0.0004) and the lower geometric mean MIC (P = 0.0088) in the commercial broth microdilution system were shown to be independent predictors of treatment success. These data suggest that the time that the concentration of cefoxitin is over the MIC for B. fragilis may be an important predictor of treatment success.     

National surveillance of species distribution in blood isolates of Candida species in Japan and their susceptibility to six antifungal agents including voriconazole and micafungin

OBJECTIVES: The aim of this study was to evaluate species distribution and antifungal susceptibility of Candida blood isolates in Japan. METHODS: In a 1 year surveillance programme, 535 Candida blood isolates were collected. Identification of species was followed by examination with the broth microdilution method, as described in NCCLS M27-A2, of antifungal susceptibility to six agents, including voriconazole and micafungin, with readings after 24 and 48 h of incubation. RESULTS: The overall species distribution was: 41% Candida albicans, 23% Candida parapsilosis, 18% Candida glabrata, 12% Candida tropicalis and 2% Candida krusei. The concentrations of fluconazole necessary to inhibit 90% of the isolates (MIC(90)) at 24/48 h were 0.25/1 mg/L for C. albicans, 0.5/2 mg/L for C. parapsilosis, 4/32 mg/L for C. glabrata and 4/>128 mg/L for C. tropicalis. Percentages of fluconazole resistance were 1.8% for C. albicans, 0.8% for C. parapsilosis, 5.2% for C. glabrata and 3.2% for C. tropicalis, taking the tendency of trailing growth of C. tropicalis into account. MIC(90) of voriconazole was 0.5 mg/L, although 35% of isolates less susceptible (>/=16 mg/L) to fluconazole showed resistance (>/=2 mg/L). Micafungin was very active against all species (MIC(90), 0.03 mg/L) except for C. parapsilosis (MIC(90), 2 mg/L). CONCLUSIONS: These data suggest that, in Japan, the species distribution of Candida bloodstream infections and the fluconazole resistance rate are similar to those reported previously in North America and Europe. Voriconazole and micafungin appear to have strong in vitro activity against Candida blood isolates, although continuing surveillance and further clinical research are needed.     

Comparison of BSAC agar dilution and NCCLS broth microdilution MIC methods for in vitro susceptibility testing of Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis: the BSAC Respiratory Resistance Surveillance Programme

OBJECTIVE: The aim of this study was to establish the degree of comparability between the NCCLS broth microdilution and BSAC agar dilution MIC methods of antimicrobial susceptibility testing. METHODS: Six hundred and sixty-one clinical isolates of Streptococcus pneumoniae, 936 Haemophilus influenzae and 421 Moraxella catarrhalis, collected in the winter of 1999-2000 by 20 laboratories in the UK and Eire from patients with presumed community-acquired lower respiratory infections, were tested by the two methods. MIC agreement was defined as excellent (good) if results were within +/- 1 doubling dilution for > or =90% (> or =80%) of isolates and within +/- 2 doubling dilutions for > or =95%. Isolates were categorized as susceptible, intermediate or resistant using the breakpoints appropriate to the testing method. RESULTS: MIC agreement was good or excellent in 27 of 36 organism-agent combinations. Agreement was less for M. catarrhalis than for other species, and lower in all three species for cefaclor and trimethoprim than for other antimicrobials. Discrepancies in categorization occurred only occasionally, and were generally explained by differences in breakpoints rather than in measured MICs. One exception was S. pneumoniae with penicillin. Despite excellent MIC agreement and identical breakpoints, 9% of these had minor discrepancies, mainly because 7% of isolates were found intermediate by the BSAC method but resistant by the NCCLS method. CONCLUSION: There is generally very good agreement between the MICs obtained by the BSAC agar dilution and NCCLS broth microdilution methods in this population of isolates, comparable to the level of agreement achieved between different laboratories using a single method. Breakpoint differences contribute to most of the discrepancies in susceptibility categorization.     

Comparison of a spectrophotometric microdilution method with RPMI-2% glucose with the National Committee for Clinical Laboratory Standards reference macrodilution method M27-P for in vitro susceptibility testing of amphotericin B, flucytosine, and fluconazole against Candida albicans.

The National Committee for Clinical Laboratory Standards has proposed a reference broth macrodilution method for in vitro antifungal susceptibility testing of yeasts (the M27-P method). This method is cumbersome and time-consuming and includes MIC endpoint determination by visual and subjective inspection of growth inhibition after 48 h of incubation. An alternative microdilution procedure was compared with the M27-P method for determination of the amphotericin B, flucytosine, and fluconazole susceptibilities of 8 American Type Culture Collection strains (6 of them were quality control or reference strains) and 50 clinical isolates of candida albicans. This microdilution method uses as culture medium RPMI 1640 supplemented with 18 g of glucose per liter (RPMI-2% glucose). Preparation of drugs, basal medium, and inocula was done by following the recommendations of the National Committee for Clinical Laboratory Standards. The MIC endpoint was calculated objectively from the turbidimetric data read at 24 h. Increased growth of C. albicans in RPMI-2% glucose and its spectrophotometric reading allowed for the rapid (24 h) and objective calculation of MIC endpoints compared with previous microdilution methods with standard RPMI 1640. Nevertheless, good agreement was shown between the M27-P method and this microdilution test. The MICs obtained for the quality control or reference strains by the microdilution method were in the ranges published for those strains. For clinical isolates, the percentages of agreement were 100% for amphotericin B and fluconazole and 98.1% for flucytosine. These data suggest that this microdilution method may serve as a less subjective and more rapid alternative to the M27-P method for antifungal susceptibility testing of yeasts.     

Flow cytometry antifungal susceptibility testing of pathogenic yeasts other than Candida albicans and comparison with the NCCLS broth microdilution test

Candida species other than Candida albicans frequently cause nosocomial infections in immunocompromised patients. Some of these pathogens have either variable susceptibility patterns or intrinsic resistance against common azoles. The availability of a rapid and reproducible susceptibility-testing method is likely to help in the selection of an appropriate regimen for therapy. A flow cytometry (FC) method was used in the present study for susceptibility testing of Candida glabrata, Candida guilliermondii, Candida krusei, Candida lusitaniae, Candida parapsilosis, Candida tropicalis, and Cryptococcus neoformans based on accumulation of the DNA binding dye propidium iodide (PI). The results were compared with MIC results obtained for amphotericin B and fluconazole using the NCCLS broth microdilution method (M27-A). For FC, the yeast inoculum was prepared spectrophotometrically, the drugs were diluted in either RPMI 1640 or yeast nitrogen base containing 1% dextrose, and yeast samples and drug dilutions were incubated with amphotericin B and fluconazole, respectively, for 4 to 6 h. Sodium deoxycholate and PI were added at the end of incubation, and fluorescence was measured with a FACScan flow cytometer (Becton Dickinson). The lowest drug concentration that showed a 50% increase in mean channel fluorescence compared to that of the growth control was designated the MIC. All tests were repeated once. The MICs obtained by FC for all yeast isolates except C. lusitaniae were in very good agreement (within 1 dilution) of the results of the NCCLS broth microdilution method. Paired t test values were not statistically significant (P = 0.377 for amphotericin B; P = 0.383 for fluconazole). Exceptionally, C. lusitaniae isolates showed higher MICs (2 dilutions or more) than in the corresponding NCCLS broth microdilution method for amphotericin B. Overall, FC antifungal susceptibility testing provided rapid, reproducible results that were statistically comparable to those obtained with the NCCLS method.     

Anti-streptococcal activity of SB-265805 (LB20304), a novel fluoronaphthyridone, compared with five other compounds, including quality control guidelines

SB-265805 (formerly LB20304) is a novel C-7 pyrrolidine-substituted naphthyridone that has a broad spectrum of activity, especially against Gram-positive cocci. SB-265805 activity was compared with ciprofloxacin, grepafloxacin, moxifloxacin, sparfloxacin, and penicillin against 599 Streptococcus spp. isolated recently from more than 30 medical centers in North and South America. These included 70 isolates with decreased susceptibility to recently released fluoroquinolones (levofloxacin MIC, > or = 4 micrograms/mL). All strains were tested by reference microdilution methods in lysed horse blood-supplemented Mueller-Hinton broth. Sixteen percent of 148 beta-haemolytic streptococci (strains of gr. B and C) were not susceptible to penicillin, whereas 38% and 42% of viridans group streptococci and Streptococcus pneumoniae were resistant to penicillin, respectively. SB-265805 potency against 301 pneumococci (MIC90, 0.06 microgram/mL) was fourfold more active than moxifloxacin and was > or = eightfold more potent than other quinolones. Against beta-haemolytic streptococci, SB-265805 and moxifloxacin were the most active (MIC90, 0.06 and 0.25 microgram/mL, respectively), whereas sparfloxacin, grepafloxacin, and ciprofloxacin (MIC90, 0.5-1 microgram/mL) were less potent. SB-265805 MICs versus viridans group streptococci (MIC90, 0.12 microgram/mL) were fourfold lower than sparfloxacin or grepafloxacin, and twofold more active than moxifloxacin. A nine-laboratory quality control (QC) protocol conforming to NCCLS M23-T3 guidelines demonstrated a modal SB-265805 MIC of 0.016 microgram/mL for S. pneumoniae ATCC 49619 (proposed QC range, 0.008 to 0.03 microgram/mL). The SB-265805 disk (5-microgram) QC range was 28-34 mm (97.3% of qualifying results). In general, SB-265805 in vitro activity against Streptococcus species was superior to sparfloxacin, grepafloxacin, and moxifloxacin and markedly greater than ciprofloxacin. This degree of antimicrobial potency warrants further investigation of this newer drug for its potential human clinical application against streptococcal infections.