Killing of Streptococcus pneumoniae by azithromycin, clarithromycin, erythromycin, telithromycin and gemifloxacin using drug minimum inhibitory concentrations and mutant prevention concentrations.

Streptococcus pneumoniae continues to be a significant respiratory pathogen, and increasing antimicrobial resistance compromises the use of ß-lactam and macrolide antibiotics. Bacterial eradication impacts clinical outcome, and bacterial loads at the site of infection may fluctuate. Killing of two macrolide- and quinolone-susceptible clinical S. pneumoniae isolates by azithromycin, clarithromycin, erythromycin, telithromycin and gemifloxacin against varying bacterial densities was determined using the measured minimum inhibitory concentration (MIC) and mutant prevention concentration (MPC). For kill experiments, 10(6)-10(9) CFU/mL were exposed to the drug and were sampled at 0, 0.5, 1, 2, 3, 4, 6, 12 and 24 h following drug exposure. The log(10) reduction and percent reduction (kill) of viable cells was recorded. MICs and MPCs (mg/L) for azithromycin, clarithromycin, erythromycin, telithromycin and gemifloxacin were 0.063-0.125/0.5-1, 0.031-0.063/0.25-0.5, 0.063/0.25-0.5, 0.008/0.016 and 0.031/0.25, respectively. Killing 10(6)-10(9) CFU/mL of bacteria by the drug MIC yielded incomplete killing, however log10 reductions occurred by 12 h and 24 h for all drugs. Exposure of 10(6)-10(9) CFU/mL to MPC drug concentrations resulted in the following log(10) reduction by 6h of drug exposure: azithromycin, 1.3-3.9; clarithromycin, 1.9-5.8; erythromycin, 0.8-4.7; telithromycin, 0.3-1.7; and gemifloxacin, 1.8-4.2. Bacterial loads at the site of infection may range from 10(6) to 10(9), and kill experiments utilising a higher bacterial inoculum provided a more accurate measure of antibiotic performance in high biomass situations. Killing was slower with telithromycin. Kill was greater and fastest with MPC versus MIC drug concentrations.

Mutant prevention concentration of tigecycline for clinical isolates of Streptococcus pneumoniae and Staphylococcus aureus.

BACKGROUND: The mutant prevention concentration (MPC) reflects the antimicrobial susceptibility of the resistant mutant subpopulations present in large bacterial populations. In principle, combining the MPC with pharmacokinetic measurements can guide treatment to restrict the enrichment of resistant subpopulations, just as the MIC is used with pharmacokinetics to restrict the growth of bulk, susceptible populations. Little is known about the MPC of tigecycline, one of the more recently approved antimicrobials. Tigecycline is particularly interesting because it shows good activity against Gram-positive pathogens. METHODS: MPCs were determined using tigecycline-containing agar plates for clinical isolates of Streptococcus pneumoniae (n=47), MRSA (n=50) and MSSA (n=50). RESULTS: Trypticase soy agar containing sheep red blood cells, commonly used for the growth of S. pneumoniae, gave tigecycline MPC90 values that were two orders of magnitude higher than expected. The addition of agar to Todd-Hewitt broth (solidified Todd-Hewitt broth) allowed the high-density growth of S. pneumoniae in the absence of red blood cells and lowered the MPC90 of tigecycline by 100-fold to 0.5 mg/L. The addition of red blood cells to solidified Todd-Hewitt broth raised the MPC90 by 100-fold. Thus, red blood cells reduce the efficacy of tigecycline against S. pneumoniae. The growth of Staphylococcus aureus was not sensitive to red blood cells; values of MPC90 were 2 and 4 mg/L for MSSA and MRSA, respectively. CONCLUSIONS: Values of MPC constitute a concentration threshold for restricting the emergence of tigecycline resistance that can now be used in animal studies to determine pharmacodynamic thresholds. The off-label treatment of S. pneumoniae blood infections with tigecycline may require caution due to blood-cell-mediated interference with the antimicrobial.

Comparative minimum inhibitory and mutant prevention drug concentrations of enrofloxacin, ceftiofur, florfenicol, tilmicosin and tulathromycin against bovine clinical isolates of Mannheimia haemolytica.

Mannheimia haemolytica is the most prevalent cause of bovine respiratory disease (BRD) and this disease accounts for 75% of morbidity, 50-70% of feedlot deaths and is estimated to cost up to $1 billion dollars annually in the USA. Antimicrobial therapy is essential for reducing morbidity, mortality and impacting on the financial burden of this disease. Due to the concern of increasing antimicrobial resistance, investigation of antibacterial agents for their potential for selecting for resistance is of paramount importance. A novel in vitro measurement called the mutant prevention concentration (MPC) defines the antimicrobial drug concentration necessary to block the growth of the least susceptible cells present in high density (≥10(7) colony forming units/ml) bacterial populations such as those seen in acute infection. We compared the minimum inhibitory concentration (MIC) and MPC values for 5 antimicrobial agents (ceftiofur, enrofloxacin, florfenicol, tilmicosin, tulathromycin) against 285 M. haemolytica clinical isolates. The MIC(90)/MPC(90) values for each agent respectively were as follows: 0.016/2, 0.125/1, 2/≥16, 8/≥32, 2/8. Dosing to achieve MPC concentrations (where possible) may serve to reduce the selection of bacterial subpopulations with reduced antimicrobial susceptibility. The rank order of potency based on MIC(90) values was ceftiofur > enrofloxacin > florfenicol = tulathromycin > tilmicosin. The rank order of potency based on MPC(90) values was enrofloxacin > ceftiofur > tulathromycin > florfenicol ≥ tilmicosin.

Evaluation of the effect of bacterial efflux pumps on the antibacterial activity of the novel fluoroquinolone besifloxacin.

This study examined the susceptibility of a variety of wild-type strains and efflux pump mutants to besifloxacin and the comparator agents sparfloxacin, ciprofloxacin, norfloxacin, moxifloxacin, tetracycline, and ethidium bromide. Organisms tested included Staphylococcus aureus (mepA or norA), Streptococcus pneumoniae (pmrA, patB), Escherichia coli (acrAB::Tn903, tolC::Tn10), Haemophilus influenzae (acrAB) and Pseudomonas aeruginosa (mepAB-oprM, oprM::ΩHg(r) rpsL). The minimal inhibitory concentrations (MIC) of besifloxacin and comparators were also measured in the presence of the efflux pump inhibitors reserpine, carbonyl cyanide mchlorophenyl- hydrazone, or sodium orthovanadate. Overall, very few meaningful changes (>2-fold) in besifloxacin MIC values resulted from the presence of efflux pump mutations or efflux pump inhibitors. In summary, the novel fluoroquinolone besifloxacin is no exception to the observation that newer fluoroquinolones are generally less affected by efflux pump-mediated resistance than older fluoroquinolones.

Antimicrobial efficacy of gatifloxacin and moxifloxacin with and without benzalkonium chloride compared with ciprofloxacin and levofloxacin against methicillin-resistant Staphylococcus aureus.

We compared the antimicrobial activity of gatifloxacin and moxifloxacin with and without benzalkonium chloride (BAK) against clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA). Minimum inhibitory concentrations (MICs) against clinical isolates of MRSA were evaluated. Approximately 10(5 )CFU/ml of methicillinresistant S. aureus was added to Mueller-Hinton broth containing two-fold concentration increments of drug. For the evaluation of gatifloxacin with BAK, 50 microg/ml of BAK were added to the first well of the plate with gatifloxacin or moxifloxacin and then serially diluted. The combination of gatifloxacin or moxifloxacin with BAK was more active than either fluoroquinolone without BAK. The MICs ranged from

Application of two methods to determine killing of Streptococcus pneumoniae by various fluoroquinolones.

Minimum inhibitory concentration (MIC) testing measures the lowest drug concentration that prevents microbial growth using an inoculum of 10(5) colony forming units/ml (cfu/ml) whereas the mutant prevention concentration (MPC) (inoculum approximately 10(10) cells) defines the antimicrobial drug concentration threshold that would require an organism to possess two simultaneous mutations for continued growth in the presence of the drug. The rates at which multidrug-resistant Streptococcus pneumoniae [MDRSP] were killed by the respiratory fluoroquinolones, gatifloxacin, gemfloxacin, levofloxacin and moxifloxacin, were compared based on the MIC and MPC drug concentrations and at inocula ranging from 10(6)-10(9) cfu/ml. The MIC drug concentration failed to eradicate all viable cells whereas the MPC drug concentration resulted in 99.9% to 100% cellular reduction following 12-24 hours of drug exposure. MPC values against S. pneumoniae were different for each fluoroquinolone. The MPC drug concentration prevents the selection of multidrug-resistant or fluoroquinolone-resistant S. pneumoniae. The value of dosing of antimicrobial agents based on MPC thresholds results in a rapid reduction in viable cells--even at higher inocula which are more reflective of organism burden in pneumonia. The rapid reduction in viable cells observed at MPC drug concentrations may not only have an impact on preventing the selection of resistant mutants but may also help explain the rapid symptom resolution seen with new fluoroquinolones since these agents lead to little or low release of cell contents which are known to drive the inflammatory response.