Comparison of agar dilution, microdilution, Etest and disc diffusion to test the activity of trovafloxacin against Pseudomonas aeruginosa, methicillin-resistant Staphylococcus aureus and Streptococcus pneumoniae.

To investigate the ability of four in-vitro methods to test trovafloxacin activity, this study evaluated susceptibility of 101 isolates of each of Pseudomonas aeruginosa, methicillin-resistant Staphylococcus aureus and Streptococcus pneumoniae to trovafloxacin by agar dilution, microdilution, Etest and disc diffusion methodologies. MIC50 and MIC90 values were very similar for all three species with all four methods. For S. aureus and P. aeruginosa, good correlation was obtained between breakpoints of > or =17 mm, 14-16 mm and < or =13 mm with agar and microdilution MICs. For both species, Etests yielded susceptibility rates lower than the other three methods. For pneumococci, excellent correlation was obtained with all four methods.     

Oxacillin susceptibility testing of Staphylococcus saprophyticus using disk diffusion, agar dilution, broth microdilution, and the Vitek GPS-105 card

Eighty-three mecA negative isolates of S. saprophyticus had oxacillin zone diameters ≤ 15 mm or MICs ranging from ≤ 0.25–1.0 μg/ml when tested by either agar dilution, broth microdilution, or the Vitek GPS-105 card. Greater than 90% of these isolates would be considered resistant using NCCLS M7-A5, M100-S10 criteria. These results suggest that the current NCCLS MIC and zone diameter breakpoints for oxacillin resistance in coagulase-negative Staphylococci are not appropriate for S. saprophyticus as they do not correlate with the presence of the mecA gene.     

Determination of methicillin-resistance in Staphylococcus aureus by agar dilution and disc diffusion methods

Four-hundred and seventy-six isolates of Staphylococcus aureus from patients in Hong Kong were tested for methicillin-resistance by agar dilution and disc diffusion methods, using heavy inocula. With Mueller-Hinton agar incubated at 30 degrees C for 24 h, 216 (MRSA) isolates were resistant to 8 mg/l of methicillin and grew up to the edge of a 10 micrograms methicillin disc, and 260 strains were susceptible to greater than or equal to 4 mg/l methicillin and produced inhibition zones of at least 20 mm diameter. Incubation for 48 h, the addition of sodium chloride, or the use of a 5 micrograms disc had little effect on these results, but a significant number of MRSA strains produced inhibition zones when disc diffusion tests were incubated at 35 degrees C, and many sensitive strains showed scanty growth on salt-containing agar at high methicillin concentrations in agar dilution tests. Iso-Sensitest agar did not appear to support the growth of the minority resistant populations of MRSA unless supplemented with menadione and thiamine, and even with supplemented Iso-Sensitest medium a few presumptively resistant strains appeared methicillin-sensitive in both disc diffusion and agar dilution tests.     

Single- and multistep resistance selection studies on the activity of retapamulin compared to other agents against Staphylococcus aureus and Streptococcus pyogenes

Retapamulin had the lowest rate of spontaneous mutations by single-step passaging and the lowest parent and selected mutant MICs by multistep passaging among all drugs tested for all Staphylococcus aureus strains and three Streptococcus pyogenes strains which yielded resistant clones. Retapamulin has a low potential for resistance selection in S. pyogenes, with a slow and gradual propensity for resistance development in S. aureus.     

Novel Bacteriophage Lysin with Broad Lytic Activity Protects against Mixed Infection by Streptococcus pyogenes and Methicillin-Resistant Staphylococcus aureus

Methicillin-resistant Staphylococcus aureus (MRSA) and Streptococcus pyogenes (group A streptococcus [GrAS]) cause serious and sometimes fatal human diseases. They are among the many Gram-positive pathogens for which resistance to leading antibiotics has emerged. As a result, alternative therapies need to be developed to combat these pathogens. We have identified a novel bacteriophage lysin (PlySs2), derived from a Streptococcus suis phage, with broad lytic activity against MRSA, vancomycin-intermediate S. aureus (VISA), Streptococcus suis, Listeria, Staphylococcus simulans, Staphylococcus epidermidis, Streptococcus equi, Streptococcus agalactiae (group B streptococcus [GBS]), S. pyogenes, Streptococcus sanguinis, group G streptococci (GGS), group E streptococci (GES), and Streptococcus pneumoniae. PlySs2 has an N-terminal cysteine-histidine aminopeptidase (CHAP) catalytic domain and a C-terminal SH3b binding domain. It is stable at 50°C for 30 min, 37°C for >24 h, 4°C for 15 days, and −80°C for >7 months; it maintained full activity after 10 freeze-thaw cycles. PlySs2 at 128 μg/ml in vitro reduced MRSA and S. pyogenes growth by 5 logs and 3 logs within 1 h, respectively, and exhibited a MIC of 16 μg/ml for MRSA. A single, 2-mg dose of PlySs2 protected 92% (22/24) of the mice in a bacteremia model of mixed MRSA and S. pyogenes infection. Serially increasing exposure of MRSA and S. pyogenes to PlySs2 or mupirocin resulted in no observed resistance to PlySs2 and resistance to mupirocin. To date, no other lysin has shown such notable broad lytic activity, stability, and efficacy against multiple, leading, human bacterial pathogens; as such, PlySs2 has all the characteristics to be an effective therapeutic.     

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.     

Antimicrobial susceptibility of Moraxella bovis determined by agar disk diffusion and broth microdilution.

The antimicrobial susceptibility of 84 isolates of Moraxella bovis was evaluated by the standard agar disk diffusion and broth microdilution procedures. All isolates were resistant to cloxacillin by disk diffusion, with 97% of isolates having a minimal inhibitory concentration of greater than or equal to 2 micrograms/ml. Of the hemolytic isolates, 68% were resistant to streptomycin. A high frequency of susceptibility was recorded for all other antimicrobial agents tested. Quantitative data supported the use of sulfonamides, but not tylosin, for parenteral infectious bovine keratoconjunctivitis therapy.     

Correlation between E-test,disk diffusion,and microdilution methods for antifungal susceptibility testing of fluconazole and voriconazole

The activities of fluconazole and voriconazole against isolates of Candida spp. (n = 400) were tested by the E-test, disk diffusion, and the National Committee for Clinical Laboratory Standards (NCCLS) M27-A2 broth microdilution-based reference methods. More than 96% of isolates found to be susceptible to fluconazole by the reference method were identified as susceptible by the agar-based methods. Lesser degrees of correlation with the reference method were seen for isolates identified as resistant by the agar-based methods. Interpretive categories are not available for voriconazole, but results qualitatively similar to those for fluconazole were seen. The agar-based E-test and disk diffusion methods are reliable alternatives to the NCCLS M27-A2 reference microdilution method for isolates that test susceptible to fluconazole.     

Oxacillin susceptibility testing of Staphylococcus saprophyticus using disk diffusion, agar dilution, broth microdilution, and the Vitek GPS-105 card

Eighty-three mecA negative isolates of S. saprophyticus had oxacillin zone diameters ≤ 15 mm or MICs ranging from ≤ 0.25–1.0 μg/ml when tested by either agar dilution, broth microdilution, or the Vitek GPS-105 card. Greater than 90% of these isolates would be considered resistant using NCCLS M7-A5, M100-S10 criteria. These results suggest that the current NCCLS MIC and zone diameter breakpoints for oxacillin resistance in coagulase-negative Staphylococci are not appropriate for S. saprophyticus as they do not correlate with the presence of the mecA gene.     

Activities of beta-lactams against Acinetobacter genospecies as determined by agar dilution and E-test MIC methods.

The agar dilution MIC method was used to test activities of ticarcillin, ticarcillin-clavulanate, amoxicillin, amoxicillin-clavulanate, ampicillin, ampicillin-sulbactam, piperacillin, piperacillin-tazobactam, inhibitors alone, ceftazidime, and imipenem against 237 Acinetobacter genospecies. A total of 93.2% of strains were beta-lactamase positive by the chromogenic cephalosporin method. Overall, ampicillin-sulbactam was the most active combination against all strains (MIC at which 50% of the isolates are inhibited [MIC50] and MIC90, 4.0 and 32.0 microg/ml; 86.9% susceptible at < or = 16 microg/ml), followed by ticarcillin-clavulanate (16.0 and 128.0 microg/ml; 85.7% susceptible at < or = 64 microg/ml), piperacillin-tazobactam (16.0 and 128.0 microg/ml; 84.8% susceptible at < or = 64 microg/ml), and amoxicillin-clavulanate (16.0 and 64.0 microg/ml; 54.4% susceptible at < or =16 microg/ml). Ceftazidime and imipenem yielded MIC50s and MIC90s of 8.0 and 64.0 microg/ml (ceftazidime) and 0.5 and 1.0 microg/ml (imipenem), respectively; 71.3% of strains were susceptible to ceftazidime at < or = 16 microg/ml, and 99.2% were susceptible to imipenem at < or = 8 microg/ml. Sulbactam was the most active beta-lactamase inhibitor alone (MIC50 and MIC90, 2.0 and 16.0 microg/ml); clavulanate and tazobactam were less active (16.0 and 32.0 microg/ml for both compounds). Enhancement of beta-lactams by beta-lactamase inhibitors was not always seen in beta-lactamase-positive strains, and activity of combinations such as ampicillin-sulbactam was due to the inhibitor alone. Acinetobacter baumannii was the most resistant genospecies. By contrast, Acinetobacter haemolyticus, Acinetobacter calcoaceticus, Acinetobacter johnsonii, Acinetobacter junii, Acinetobacter radioresistens, and other non-Acinetobacter baumannii strains were more susceptible to all compounds tested. E-test MICs were within 1 dilution of agar dilution MICs in 38.4 to 89.6% of cases and within 2 dilutions in 61.6 to 98.6% of cases.     

Antimicrobial susceptibility of flavobacteria as determined by agar dilution and disk diffusion methods.

A total of 106 clinical isolates of flavobacteria, including 41 isolates of Flavobacterium meningosepticum, 59 of Flavobacterium indologenes, and 6 of Flavobacterium odoratum were collected from January 1992 to December 1995 from patients in Taiwan. The in vitro activities of antimicrobial agents were determined concomitantly by the standard agar dilution and disk diffusion methods. More than 90% of the flavobacterial isolates were resistant to cephalothin, cefotaxime, ceftriaxone, moxalactam, aztreonam, imipenem, aminoglycosides, erythromycin, and glycopeptides. The majority of F. meningosepticum isolates were susceptible to piperacillin and to minocycline but resistant to ceftazidime, with MICs at which 90% of the isolates are inhibited being 8, 4, and > 128 microg/ml, respectively. Approximately half of the F. indologenes isolates were susceptible to piperacillin, cefoperazone, ceftazidime, and minocycline, with MICs at which 50% of the isolates are inhibited being 4, 16, 8, and 4 microg/ml, respectively. The majority of F. odoratum isolates were resistant to all the antimicrobial agents tested except minocycline, to which five of six isolates were susceptible. With least-squares regression analysis and error rate-bounded analysis methods, the following resistant and susceptible zone diameter breakpoints were established: < or = 12 and > or = 17 mm, respectively, for piperacillin against F. meningosepticum and F. indologenes; < or = 13 and > or = 18 mm, respectively, for ceftazidime against F. meningosepticum and F. indologenes, < or = 17 and > or = 21 mm, respectively, for ofloxacin against F. indologenes; < or = 16 and > or = 20 mm, respectively, for ciprofloxacin against F. meningosepticum. Valid breakpoints for the disk diffusion method could not be established for cefoperazone and ofloxacin against F. meningosepticum and for minocycline against F. meningosepticum and F. indologenes due to a poor correlation coefficient for the regression line or for cefoperazone and ciprofloxacin against F. indologenes due to the presence of remarkable error rates.     

Comparative activity of garenoxacin and other agents by susceptibility and time-kill testing against Staphylococcus aureus, Streptococcus pyogenes and respiratory pathogens

OBJECTIVES: Garenoxacin is a novel des-F(6)quinolone that has shown excellent antimicrobial activity against a wide range of clinically important microorganisms. In this study, its activity was examined, in comparison with that of other antimicrobial agents, by susceptibility and time-kill testing against Staphylococcus aureus, Streptococcus pyogenes and respiratory pathogens. METHODS: Overall, 200 bacterial strains were tested. The antimicrobial activity of garenoxacin was compared with that of ciprofloxacin, levofloxacin, moxifloxacin, amoxicillin, co-amoxiclav, cefuroxime, cefotaxime, ceftriaxone, imipenem, erythromycin and clarithromycin. In addition, the bactericidal activity of garenoxacin, moxifloxacin, levofloxacin and ciprofloxacin was evaluated by time-kill analysis against four strains each of staphylococci [two methicillin-susceptible (MSSA) and two methicillin-resistant (MRSA)], pneumococci (two penicillin-susceptible and two penicillin-resistant) and Streptococcus pyogenes (two erythromycin-susceptible and two erythromycin-resistant). Antibiotics were tested at concentrations 1-8 x MIC. RESULTS: MIC90 values of garenoxacin for the MSSA and MRSA strains were 0.03 and 2 mg/L, respectively. Among all the quinolones tested, garenoxacin yielded the lowest MIC values against all pneumococci (MIC90 0.12 mg/L) irrespective of macrolide resistance; the rank order of activity was garenoxacin> moxifloxacin>levofloxacin>ciprofloxacin. Excellent activity was shown also against Haemophilus influenzae (MIC90 or= 3 log10 decrease in viable counts (cfu/mL) within 3 h at 4 x MIC, whereas a moderate, slower killing rate was observed versus streptococci. CONCLUSIONS: This investigational des-F(6)quinolone represents a promising alternative for the treatment of respiratory tract infections.     

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.     

Effect of Elevated Atmospheric Pressure on Penicillin Binding by Staphylococcus aureus and Streptococcus pyogenes

A gas pressure of 68 atm, elicited by helium-oxygen gas mixtures, reduced the susceptibility to penicillin of Staphylococcus aureus but not of Streptococcus pyogenes. The elevated pressure also caused a reduction in the binding of (14)C-penicillin to S. aureus, but not to S. pyogenes. When these studies were extended to glycine incorporation, it was shown that, even without penicillin, pressurization reduced glycine incorporation into the cell wall of S. aureus. Incorporation into other cellular components was not altered by pressurization. Cells grown in a pressurized environment were slightly more susceptible than those grown at 1 atm to rapid change in osmotic pressure. In the presence of penicillin, glycine incorporation into the cell wall was reduced to the same low level at 68 atm and at 1 atm. These results suggest that pressurization renders S. aureus less susceptible to penicillin because it reduces the enzymatic activity of the binding component on the cell, a penicillin-sensitive transpeptidase.     

In vitro activities of retapamulin and 16 other antimicrobial agents against recently obtained Streptococcus pyogenes isolates

Retapamulin in vitro activity against 400 Streptococcus pyogenes clinical isolates obtained from skin, pharynx, ear fluid, and blood samples recovered from 2007 to 2009 was studied. The isolates belonged to 26 different emm types, including isolates nonsusceptible to erythromycin (n=187), tetracycline (n=99), ciprofloxacin (n=59), and bacitracin (n=43). Results were compared to the activities of 16 other antibiotics for topical and systemic use. Retapamulin MICs ranged from ≤0.015 to 0.12 μg/ml, showing the highest intrinsic activity among the topical antimicrobial drugs studied.     

In vitro development of resistance to six quinolones in Streptococcus pneumoniae, Streptococcus pyogenes, and Staphylococcus aureus

Streptococcus pneumoniae, Streptococcus pyogenes, and Staphylococcus aureus isolates were exposed to subinhibitory MICs of ciprofloxacin, sparfloxacin, gatifloxacin, moxifloxacin, clinafloxacin, and gemifloxacin during a 10-day period. Subculturing led to resistance development, regardless of the initial potencies of the quinolones. None of the quinolones was associated with a significantly slower rate of resistance development.     

In vitro activity of metronidazole against Helicobacter pylori as determined by agar dilution and agar diffusion.

Metronidazole activity against 25 clinical isolates of Helicobacter pylori was evaluated by agar dilution, epsilometer (E-test; AB Biodisk, Solna, Sweden), and disk diffusion methods after 3 and 5 days of incubation in a microaerophilic atmosphere. Agar dilution, performed in duplicate, provided reproducible results with MICs for 50% of the isolates of less than or equal to 0.12 microgram/ml after 3 and 5 days of incubation and MICs for 90% of the isolates of 2 and 4 micrograms/ml after 3 and 5 days of incubation, respectively. Reproducibility of MICs was slightly better after 5 days than after 3 days of incubation. MICs obtained with the E-test were higher, with 76 and 68% of isolates inhibited by less than or equal to 16 micrograms of metronidazole per ml after 3 and 5 days, respectively, in contrast with corresponding values of 92 and 88% for agar dilution. Zone diameters obtained with the commercially available 80-micrograms metronidazole elution disk were too large (greater than or equal to 41 mm) to allow discrimination between susceptible and resistant isolates, although resistant subpopulations were detected by the appearance of inner colonies in four isolates. In conclusion, the E-test was easy to perform and interpret, and it appeared to be more likely than agar dilution to detect metronidazole resistance in vitro in H. pylori.     

Oxacillin killing curve patterns of Staphylococcus aureus isolates by agar dilution plate count method.

The bactericidal dynamics of oxacillin against four Staphylococcus aureus isolates with known 24-h "persister" percentages were studied by using the agar dilution plate count method. Isolates were selected to provide a representative spectrum whose individual 24-h trough intrinsic persister percentages ranged from greater than 1 to less than 0.01%. Resultant agar dilution plate count method killing curve patterns were found to be reproducible and served to characterize each isolate. The paradoxical effect was observed for each isolate, with paradoxical peaks tending to develop and diminish sequentially during the course of oxacillin action. The observed strain-dependent dynamics of oxacillin killing underscore the artifactual nature of the so-called tolerance phenomenon and negate the usefulness of MBCs and MBC/MIC ratios for the characterization of S. aureus isolates.