C. Hesje

MICs, MPCs and PK/PDs: a match (sometimes) made in hosts

There is little doubt that we have a global pandemic of antimicrobial-resistant microorganisms. Despite regional variations in resistance rates for various bacteria/drug combinations, the overall impact of this trend has impacted on individual patients and the economics of managing infections, as well as our approach to the empirical use of antimicrobial compounds for both inand outpatient management. Indeed, various treatment guidelines from expert working groups recommend different treatment options based on the likelihood of resistant pathogens. Currently, antibiotics are approved based on the demonstration of noninferiority of a new drug when compared with a standard antibiotic agent already approved for a specific indication. However, such trials may fail to take into account various microbiological or pharmacological parameters that could be used to determine optimal versus suboptimal dosing. As such, these parameters may not necessarily affect clinical outcome but may have a huge impact on the selection of drug-resistant pathogens.

Low Correlation between MIC and Mutant Prevention Concentration

Soon after the term mutant prevention concentration (MPC) was coined to define the MIC of the least susceptible mutant subpopulation of a microbial culture (2), we noticed that MPCs and MICs correlated poorly (r2 = 0.39) for a set of closely related fluoroquinolones when determined with Mycobacterium smegmatis (9). Subsequently, isolated examples were described in which correlation was low for a variety of fluoroquinolones with strains of Escherichia coli, Salmonella enterica, and Staphylococcus aureus (5, 8, 10), and a set of 20 clinical isolates of E. coli showed a low correlation (r2 = 0.58) for ciprofloxacin (6). To determine whether a low correlation between MICs and MPCs is likely to be a general phenomenon, we calculated the correlation coefficients for several quinolones with five bacterial species and for three macrolides with Streptococcus pneumoniae using data from published and unpublished studies of clinical isolates. As shown in Table ​Table1,1, r2,determined by linear regression, was below 0.5 for fluoroquinolones with E. coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, S. aureus, and S. pneumoniae (an exception was levofloxacin with K. pneumoniae [r2 = 0.7]). Values of r2 were slightly above 0.5 for three macrolides with S. pneumoniae (Table ​(Table11). TABLE 1. Relationship between MICs and MPCs Low correlations between MICs and MPCs with clinical isolates are likely to require a complex explanation. These isolates probably contain mutant subpopulations that vary considerably in relative abundance and drug susceptibility, which will contribute to a wide variation in MICs when the mutants are abundant enough to be scored. The isolates may also contain many different multistep mutants (1) which may or may not represent the least susceptible subpopulations that determine MPCs. Added complexity derives from some resistance mutations having a much larger effect on MPCs than on MICs (4). Indeed, isolates with the same MIC were found to have values of MPC that ranged over 5 twofold dilutions. A consequence of a low correlation between MICs and MPCs is that MPCs cannot be estimated accurately from MICs on an individual patient basis. Thus, using antimutant strategies for individual patients will require measurement of the MPC. Likewise, empirical estimates of antimutant activity that are keyed to MIC-based pharmacokinetic-pharmacodynamic indices, such as area under the concentration-time curve at 24 h/MIC, will tend to exhibit more patient-to-patient variability than indices using MPCs.

Benzalkonium chloride enhances antibacterial activity of gatifloxacin and reduces its propensity to select for fluoroquinolone-resistant strains.

PURPOSE To assess the impact of benzalkonium chloride (BAK) on the minimum inhibitory concentration (MIC) and mutant prevention concentration (MPC) of gatifloxacin against Gram-positive pathogens in comparison to gatifloxacin and moxifloxacin alone, moxifloxacin plus BAK, and/or levofloxacin. METHODS The MIC was measured following incubation of 10(5) colony-forming units (CFU)/mL of coagulase-negative staphylococci (CNS; n = 20), methicillin-susceptible Staphylococcus aureus (MSSA; n = 20), and methicillin-resistant S. aureus (MRSA; n = 20) with gatifloxacin, levofloxacin, or moxifloxacin. When present, BAK was added from 3.125 microg/mL to 6.25 microg/mL. The MPC was measured following incubation of 10(10) CFU/mL of MRSA (n = 9) and a commercially available MSSA strain with gatifloxacin or moxifloxacin in the absence and presence of BAK at concentrations from 7 microg/mL to 10 microg/mL. RESULTS CNS was more susceptible to gatifloxacin (MIC(90) = 2 microg/mL) than levofloxacin (MIC(90) = 8 microg/mL) or moxifloxacin (MIC(90) = 4 microg/mL). MSSA was more susceptible to moxifloxacin (MIC(90) = 1 microg/mL) than gatifloxacin (MIC(90) = 4 microg/mL) or levofloxacin (MIC(90) = 4 microg/mL). MRSA were resistant to gatifloxacin, levofloxacin, and moxifloxacin. In the presence of BAK, however, the MIC(90) of gatifloxacin and moxifloxacin against CNS, MSSA, and MRSA was < or =0.008 microg/mL. Gatifloxacin and moxifloxacin had similar MPCs against MRSA (> or =4 microg/mL). In the presence of BAK, the MPC of gatifloxacin and moxifloxacin against MRSA ranged from < or =0.004 microg/mL to 0.125 microg/mL. CONCLUSIONS BAK substantially lowered the MIC and MPC of gatifloxacin and moxifloxacin against Gram-positive staphylococci compared to gatifloxacin alone, moxifloxacin alone, and/or levofloxacin. These findings suggest that the presence of BAK in the ophthalmic formulation of gatifloxacin (Zymar) may serve to enhance the potency of gatifloxacin and decrease its propensity to select for fluoroquinolone-resistant S. aureus strains.

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 β-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.

Comparative efficacy of besifloxacin and other fluoroquinolones in a prophylaxis model of penicillin-resistant Streptococcus pneumoniae rabbit endophthalmitis.

PURPOSE To study the efficacy of topical administration of gatifloxacin (0.3%), moxifloxacin (0.5%) ophthalmic solutions, and besifloxacin (0.6%) ophthalmic suspension as prophylaxis and treatment of pneumococcal endophthalmitis. METHODS Four groups of New Zealand white rabbits were topically treated with gatifloxacin, moxifloxacin, besifloxacin, and phosphate-buffered saline (PBS) at the following time points: 60, 45, 30, and 15 min before infection, immediately after infection, and then 6, 12, 18, and 24 h postinfection. Penicillin-resistant Streptococcus pneumoniae (PRSP; 10(6) colony-forming unit [CFU] in 50 microL) was injected into the aqueous humor of each eye. The clinical severity of the eyes was assessed by 2 masked observers 24 h postinfection. Aqueous and vitreous samples were collected, diluted, and plated to determine recovered CFU. RESULTS Fluoroquinolone-treated eyes had significantly lower clinical scores and bacteria recovered from the aqueous humor than the PBS-treated eyes. There was no difference, however, among the fluoroquinolone-treated groups. In contrast, none of the fluoroquinolones reduced the number of bacteria recovered (CFU) from the vitreous humor. CONCLUSIONS Besifloxacin is as effective as moxifloxacin and gatifloxacin in a rabbit model for topical prophylaxis and treatment of PRSP-induced endophthalmitis.

Contribution of the R8 substituent to the in vitro antibacterial potency of besifloxacin and comparator ophthalmic fluoroquinolones

Introduction Previous work has shown that besifloxacin, an 8-chloro-fluoroquinolone, has more potent activity against gram-positive pathogens than moxifloxacin and gatifloxacin, which carry an 8-methoxy group. This study was conducted to determine the contribution of the R7 and R8 substituent to fluoroquinolone antibacterial activity. Materials and methods Besifloxacin, moxifloxacin, gatifloxacin, their R8 structural analogs, and ciprofloxacin were tested against representative isolates of various gram-positive and gram-negative species and previously characterized fluoroquinolone-resistant mutants of Staphylococcus aureus. Minimum inhibitory and minimum bactericidal concentrations were determined according to Clinical and Laboratory Standards Institute (CLSI) guidelines. Reserpine was used to determine the effect of efflux pumps on antibacterial activity. Results In general, exchanging the R8 residue in besifloxacin slightly reduced the molecule’s potency, while introducing an 8-chloro group in moxifloxacin increased its potency. A similar change in gatifloxacin had little to no effect. Substituting the R8 residues did not increase the susceptibility to the efflux pump inhibitor reserpine or result in a loss of bactericidal activity. In contrast, the positive control, ciprofloxacin, was shown to be a substrate for reserpine and lost bactericidal activity against some fluoroquinolone-resistant isolates of S. aureus. Conclusion The data presented here show that, depending on the R7 substituent, replacing an 8-methoxy group with an 8-chloro substituent can improve potency or can have little-to-no effect. These findings highlight the importance of the interplay between the R7 and R8 substituents in determining antibacterial potency.

High Proportion of Nontypeable Streptococcus pneumoniae Isolates among Sporadic, Nonoutbreak Cases of Bacterial Conjunctivitis

Purpose: Outbreaks of bacterial conjunctivitis have been linked to nontypeable strains of Streptococcus pneumoniae that lack a capsule, a key virulence factor for invasive infections. In contrast, isolates from sporadic, nonoutbreak cases of conjunctivitis were thought to be similar to invasive or nasopharyngeal isolates with respect to their capsular serotype and antibiotic resistance profile. This hypothesis was tested for 302 strains isolated during three prospective, multicenter clinical studies of bacterial conjunctivitis. Materials and methods: S. pneumoniae capsular serotypes were determined by agglutination assay and confirmed by the Statens Serum Institute. The presence of the cpsAB capsule genes was determined by polymerase chain reaction (PCR). Minimum inhibitory concentrations were measured for 17 antibacterial drugs by the broth microdilution method. Results: Only 25 (8.3%) isolates reacted with the capsule-specific antisera and only one (0.3%) of these serotypes was covered by the capsule-specific PCV7 vaccine. The remaining 277 (91.7%) isolates were nontypeable, suggesting that they did not produce a capsule. PCR analysis indicated the loss of the capsule operon in 24/25 randomly selected nontypeable strains. Resistance rates were highest for azithromycin, trimethoprim, and tetracycline, while no resistance was detected for the fluoroquinolones, linezolid, and vancomycin. Antibiotic resistance rates were generally lower than those reported for invasive isolates, although some highly resistant or multidrug-resistant isolates were identified. Conclusions: The prevalence of nontypeable strains of S. pneumoniae was higher than expected, while the number of isolates responsive to the PCV7 vaccine was surprisingly low. These results highlight the need for new vaccines that can target all S. pneumoniae strains regardless of the presence or nature of a capsule. In addition, resistance to azithromycin, erythromycin, tetracycline, and trimethoprim was greater than 10%, which may be relevant when selecting empiric treatments for ocular surface infections. Trial registration: ClinicalTrials.gov identifier: NCT00622908. Trial registration: ClinicalTrials.gov identifier: NCT00347932. Trial registration: ClinicalTrials.gov identifier: NCT00348348.

Bactericidal effects of various concentrations of enrofloxacin, florfenicol, tilmicosin phosphate, and tulathromycin on clinical isolates of Mannheimia haemolytica

OBJECTIVE To determine bactericidal effects of enrofloxacin, florfenicol, tilmicosin, and tulathromycin on clinical isolates of Mannheimia haemolytica at various bacterial densities and drug concentrations. SAMPLE 4 unique isolates of M haemolytica recovered from clinically infected cattle. PROCEDURES Minimum inhibitory concentration (MIC) and mutant prevention concentration (MPC) were determined for each drug and isolate. Mannheimia haemolytica suspensions (10(6) to 10(9) CFUs/mL) were exposed to the determined MIC and MPC and preestablished maximum serum and tissue concentrations of each drug. Log10 reduction in viable cells (percentage of cells killed) was measured at various points. RESULTS Bacterial killing at the MIC was slow and incomplete. After 2 hours of isolate exposure to the MPC and maximum serum and tissue concentrations of the tested drugs, 91% to almost 100% cell killing was achieved with enrofloxacin, compared with 8% growth to 93% cell killing with florfenicol, 199% growth to 63% cell killing with tilmicosin, and 128% growth to 43% cell killing with tulathromycin over the range of inoculum tested. For all drugs, killing of viable organisms was evident at all bacterial densities tested; however, killing was more substantial at the MPC and maximum serum and tissue drug concentrations than at the MIC and increased with duration of drug exposure. Rank order of drugs by killing potency was enrofloxacin, florfenicol, tilmicosin, and tulathromycin. CONCLUSIONS AND CLINICAL RELEVANCE Findings suggested that antimicrobial doses that equaled or exceeded the MPC provided rapid killing of M haemolytica by the tested drugs, decreasing opportunities for antimicrobial-resistant subpopulations of bacteria to develop during drug exposure.