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Dose-range studies for polymyxin B (PMB) regimens of 0.75 to 12 mg/kg given every 12h (Q12h) were evaluated for bacterial killing and resistance prevention against an AmpC-overexpressing Pseudomonas aeruginosa (PA) and a blaKPC-3-harboring Klebsiella pneumoniae (KP) in 10-day in-vitro hollow fiber models. An exposure-response was observed. But all regimens failed due to regrowth. Lower-dose regimens amplified isolates that expressed transient, lower-level adaptive resistance to PMB (MICs: ≤ 4 mg/L). Higher PMB dosages amplified isolates that expressed this resistance mechanism, a higher-MIC "moderately stable" adaptive resistance, and a higher-MIC stable resistance to PMB. Failure of the highest dose regimens was solely due to subpopulations that expressed the two higher-level resistances. Total and bioactive PMB concentrations in broth declined below targeted PK profiles within hours of treatment initiation and prior to bacterial regrowth. With treatment failure, the total PMB measured in bacteria were substantially higher than in broth. But the bioactive PMB in broth and bacteria were low to non-detectable. Together these findings suggest a sequence of events for treatment failure of the clinical regimen. First, PMB concentrations in broth are diluted as PMB binds to bacteria, resulting in total and bioactive PMB in broth that are lower than targeted. Bacterial regrowth and treatment failure follow, with emergence of subpopulations that express transient lower-level adaptive resistance to PMB and possibly higher-level adaptive and stable resistances. Higher-dose PMB regimens can prevent the emergence of transient lower-level adaptive resistance but they do not prevent treatment failure due to isolates that express higher-level resistance mechanisms.

The repurposed agent moxifloxacin has become an important addition to the physician’s armamentarium for the therapy of Mycobacterium tuberculosis. When a drug is administered, we need to have metrics for success. As for most antimicrobial chemotherapy, we contend that for Mycobacterium tuberculosis therapy, these metrics should be a decline in the susceptible bacterial burden and the suppression of amplification of less-susceptible populations. To achieve optimal outcomes relative to these metrics, a dose and schedule of administration need to be chosen. For large populations of patients, there are true between-patient differences in important pharmacokinetic parameters. These distributions of parameter values may have an impact on these metrics, depending on what measure of drug exposure drives the metrics. To optimize dose and schedule choice of moxifloxacin, we performed a dose fractionation experiment in the hollow fiber infection model. We examined 12-, 24-, and 48-h dosing intervals with doses of 200, 400, and 800 mg for each interval, respectively. Within each interval, we had an arm where half-lives of 12, 8, and 4 h were simulated. We attempted to keep the average concentration (C(avg)) or area under the concentration-time curve (AUC) constant across arms. We found that susceptible bacterial load decline was linked to C(avg), as we had indicated previously. Resistance suppression, a nonmonotonic function, had minimum concentration (C(min)) as the linked index. The 48-h interval with the 4-h half-life had the largest less-susceptible population. Balancing bacterial kill, resistance suppression, toxicity (linked to peak concentration [C(peak)]), and adherence, we conclude that the dose of 400 mg daily is optimal for moxifloxacin.

The Mycobacterium tuberculosis drug discovery effort has generated a substantial number of new/repurposed drugs for therapy for this pathogen. The arrival of these drugs is welcome, but another layer of difficulty has emerged. Single agent therapy is insufficient for patients with late-stage tuberculosis because of resistance emergence. To achieve our therapeutic ends, it is requisite to identify optimal combination regimens. These regimens go through a lengthy and expensive evaluative process. If we have a modest group of 6 to 8 new or repurposed agents, this translates into 15 to 28 possible 2-drug combinations. There is neither time nor resources to give an extensive evaluation for all combinations. We sought a screening procedure that would identify combinations that had a high likelihood of achieving good bacterial burden decline. We examined pretomanid, moxifloxacin, linezolid, and bedaquiline in log-phase growth, acid-phase growth, and nonreplicative persister (NRP) phase in the Greco interaction model. We employed the interaction term α and the calculated bacterial burden decline as metrics to rank different regimens in different metabolic states. No relationship was found between α and bacterial kill. We chose bacterial kill as the prime metric. The combination of pretomanid plus moxifloxacin emerged as the clear frontrunner, as the largest bacterial declines were seen in log phase and acid phase with this regimen and it was second best in NRP phase. Bedaquiline also produced good kill. This screening process may identify optimal combinations that can be further evaluated in both the hollow-fiber infection model and in animal models of Mycobacterium tuberculosis infection.

Antimicrobial resistance in Neisseria gonorrhoeae is threatening the treatment and control of gonorrhea globally, and new treatment options are imperative. Utilizing our dynamic in vitro hollow fiber infection model (HFIM), we examined the pharmacodynamics of the first-in-class spiropyrimidinetrione (DNA gyrase B inhibitors), zoliflodacin, against the N. gonorrhoeae reference strains World Health Organization F (susceptible to all relevant antimicrobials) and WHO X (extensively drug resistant, including resistance to ceftriaxone) over 7 days. Dose-range experiments with both strains, simulating zoliflodacin single oral dose regimens of 0.5–8 g, and dose-fractionation experiments with WHO X, simulating zoliflodacin oral dose therapy with 1–4 g administered as q12 h and q8 h for 24 h, were performed. A kill-rate constant that reflected a rapid bacterial kill during the first 6.5 h for both strains and all zoliflodacin doses was identified. In the dose-range experiments, the zoliflodacin 2–8 g single-dose treatments successfully eradicated both WHO strains, and resistance to zoliflodacin was not observed. However, zoliflodacin as a single 0.5 g dose failed to eradicate both WHO strains, and a 1 g single dose failed to eradicate WHO X in one of two experiments. The zoliflodacin 1 g/day regimen also failed to eradicate WHO X when administered as two and three divided doses given at q12 h and q8 h in the dose-fractionation studies, respectively. All failed regimens selected for zoliflodacin-resistant mutants. In conclusion, these data demonstrate that zoliflodacin should be administered at >2 g as a single oral dose to provide effective killing and resistance suppression of N. gonorrhoeae. Future studies providing pharmacokinetic data for zoliflodacin (and other gonorrhea therapeutic antimicrobials) in urogenital and extragenital infection sites, particularly in the pharynx, and evaluation of gonococcal strains with different gyrB mutations would be important.

Poor penetration through the outer membrane (OM) of Gram-negative bacteria is a major barrier of antibiotic development. While β-lactam antibiotics are commonly used against Klebsiella pneumoniae and Enterobacter cloacae, there are limited data on OM permeability especially in K. pneumoniae. Here, we developed a novel cassette assay, which can simultaneously quantify the OM permeability to five β-lactams in carbapenem-resistant K. pneumoniae and E. cloacae. Both clinical isolates harbored a blaKPC-2 and several other β-lactamases. The OM permeability of each antibiotic was studied separately (“discrete assay”) and simultaneously (“cassette assay”) by determining the degradation of extracellular β-lactam concentrations via multiplex liquid chromatography-tandem mass spectrometry analyses. Our K. pneumoniae isolate was polymyxin resistant, whereas the E. cloacae was polymyxin susceptible. Imipenem penetrated the OM at least 7-fold faster than meropenem for both isolates. Imipenem penetrated E. cloacae at least 258-fold faster and K. pneumoniae 150-fold faster compared to aztreonam, cefepime, and ceftazidime. For our β-lactams, OM permeability was substantially higher in the E. cloacae compared to the K. pneumoniae isolate (except for aztreonam). This correlated with a higher OmpC porin production in E. cloacae, as determined by proteomics. The cassette and discrete assays showed comparable results, suggesting limited or no competition during influx through OM porins. This cassette assay allowed us, for the first time, to efficiently quantify the OM permeability of multiple β-lactams in carbapenem-resistant K. pneumoniae and E. cloacae. Characterizing the OM permeability presents a critical contribution to combating the antimicrobial resistance crisis and enables us to rationally optimize the use of β-lactam antibiotics. IMPORTANCE Antimicrobial resistance is causing a global human health crisis and is affecting all antibiotic classes. While β-lactams have been commonly used against susceptible isolates of Klebsiella pneumoniae and Enterobacter cloacae, carbapenem-resistant isolates are spreading worldwide and pose substantial clinical challenges. Rapid penetration of β-lactams leads to high drug concentrations at their periplasmic target sites, allowing β-lactams to more completely inactivate their target receptors. Despite this, there are limited tangible data on the permeability of β-lactams through the outer membranes of many Gram-negative pathogens. This study presents a novel, cassette assay, which can simultaneously characterize the permeability of five β-lactams in multidrug-resistant clinical isolates. We show that carbapenems, and especially imipenem, penetrate the outer membrane of K. pneumoniae and E. cloacae substantially faster than noncarbapenem β-lactams. The ability to efficiently characterize the outer membrane permeability is critical to optimize the use of β-lactams and combat carbapenem-resistant isolates.

In June 2017, the National Institute of Allergy and Infectious Diseases, part of the National Institutes of Health, organized a workshop entitled "Pharmacokinetics-Pharmacodynamics (PK/PD) for Development of Therapeutics against Bacterial Pathogens." The aims were to discuss details of various PK/PD models and identify sound practices for deriving and utilizing PK/PD relationships to design optimal dosage regimens for patients. Workshop participants encompassed individuals from academia, industry, and government, including the United States Food and Drug Administration. This and the accompanying review on clinical PK/PD summarize the workshop discussions and recommendations. Nonclinical PK/PD models play a critical role in designing human dosage regimens and are essential tools for drug development. These include in vitro and in vivo efficacy models that provide valuable and complementary information for dose selection and translation from the laboratory to human. It is crucial that studies be designed, conducted, and interpreted appropriately. For antibacterial PK/PD, extensive published data and expertise are available. These have been leveraged to develop recommendations, identify common pitfalls, and describe the applications, strengths, and limitations of various nonclinical infection models and translational approaches. Despite these robust tools and published guidance, characterizing nonclinical PK/PD relationships may not be straightforward, especially for a new drug or new class. Antimicrobial PK/PD is an evolving discipline that needs to adapt to future research and development needs. Open communication between academia, pharmaceutical industry, government, and regulatory bodies is essential to share perspectives and collectively solve future challenges.

In 2017, the WHO identified Acinetobacter baumannii as the top priority for the development of new antibiotics. Despite the need for new antibiotics, there remains a lack of well validated preclinical tools for A. baumannii. Here, we characterize and validate a mouse model for A. baumannii translational research. Antibiotic sensitivity for meropenem, amikacin, and polymyxin b was determined by the broth microdilution MIC assay. LD100 inoculums, in both blood and lung infection models, were determined in male and female C3HeB/FeJ mice that were challenged with various A. baumannii clinical isolates. Blood (blood infection model) or blood and lung tissue (lung infection model) were collected from infected mice at 2 and 18 hours and the bacterial burden was determined by quantitative culture. Blood chemistry was analyzed using the iStat system. Cytokines (IL-1s, TNF, IL-6, and IL-10) were measured in the blood and lung homogenate by ELISA assay. Lung sections (H&E stains) were scored by a pathologist. In the blood infection model, the cytokines and physiological data indicate that mice become moribund due to sepsis (low blood pH, falling bicarbonate, and a rising base deficit), whereas mice become moribund due to respiratory failure (low blood pH, rising bicarbonate, and a falling base deficit) in the oral aspiration pneumonia model. We also characterized the timing of changes in various clinical and biomarker endpoints, which can serve as a basis for future interventional studies. Susceptibility was generally similar across gender and infection route. However, we did observe that female mice were approximately 2-fold more sensitive to LAC-4 ColR in the blood infection model. We also observed that female mice were more than 10-fold more resistant to VA-AB41 in the oral aspiration pneumonia model. These results establish parameters to follow in order to assess efficacy of novel preventative and therapeutic approaches for these infections.

Ventilator-associated bacterial pneumonia (VABP) is a difficult therapeutic problem. Considerable controversy exists regarding the optimal chemotherapy for this entity. The recent guidelines of the Infectious Diseases Society of America and the American Thoracic Society recommend a 7-day therapeutic course for VABP based on the balance of no negative impact on all-cause mortality, less resistance emergence, and fewer antibiotic treatment days, counterbalanced with a higher relapse rate for patients whose pathogen is a nonfermenter. The bacterial burden causing an infection has a substantial impact on treatment outcome and resistance selection. We describe the baseline bronchoalveolar lavage (BAL) fluid burden of organisms in suspected VABP patients screened for inclusion in a clinical trial. We measured the urea concentrations in plasma and BAL fluid to provide an index of the dilution of the bacterial and drug concentrations in the lung epithelial lining fluid introduced by the BAL procedure. We were then able to calculate the true bacterial burden as the diluted colony count times the dilution factor. The median dilution factor was 28.7, with the interquartile range (IQR) being 11.9 to 53.2. Median dilution factor-corrected colony counts were 6.18 log

Ventilator-associated bacterial pneumonia (VABP) is a difficult therapeutic problem. Considerable controversy exists regarding the optimal chemotherapy for this entity. The recent guidelines of the Infectious Diseases Society of America and the American Thoracic Society recommend a 7-day therapeutic course for VABP based on the balance of no negative impact on all-cause mortality, less resistance emergence, and fewer antibiotic treatment days, counterbalanced with a higher relapse rate for patients whose pathogen is a nonfermenter. The bacterial burden causing an infection has a substantial impact on treatment outcome and resistance selection. We describe the baseline bronchoalveolar lavage (BAL) fluid burden of organisms in suspected VABP patients screened for inclusion in a clinical trial. We measured the urea concentrations in plasma and BAL fluid to provide an index of the dilution of the bacterial and drug concentrations in the lung epithelial lining fluid introduced by the BAL procedure. We were then able to calculate the true bacterial burden as the diluted colony count times the dilution factor. The median dilution factor was 28.7, with the interquartile range (IQR) being 11.9 to 53.2. Median dilution factor-corrected colony counts were 6.18 log 10 (CFU/ml) [IQR, 5.43 to 6.46 log 10 (CFU/ml)]. In a subset of patients, repeat BAL on day 5 showed a good stability of the dilution factor. We previously showed that large bacterial burdens reduce or stop bacterial killing by granulocytes. (This study has been registered at ClinicalTrials.gov under registration no. NCT01570192.)

A recent report found that generic parenteral vancomycin products may not have in vivo efficacies equivalent to those of the innovator in a neutropenic murine thigh infection model despite having similar in vitro microbiological activities and murine serum pharmacokinetics. We compared the in vitro and in vivo activities of six of the parenteral vancomycin products available in the United States. The in vitro assessments for the potencies of the vancomycin products included MIC/minimal bactericidal concentration (MBC) determinations, quantifying the impact of human and murine serum on the MIC values, and time-kill studies. Also, the potencies of the vancomycin products were quantified with a biological assay, and the human and mouse serum protein binding rates for the vancomycin products were measured. The in vivo studies included dose-ranging experiments with the 6 vancomycin products for three isolates of Staphylococcus aureus in a neutropenic mouse thigh infection model. The pharmacokinetics of the vancomycin products were assessed in infected mice by population pharmacokinetic modeling. No differences were seen across the vancomycin products with regard to any in vitro evaluation. Inhibitory sigmoid maximal bacterial kill ( E max ) modeling of the relationship between vancomycin dosage and the killing of the bacteria in mice in vivo yielded similar E max and EC 50 (drug exposure driving one-half E max ) values for bacterial killing. Further, there were no differences in the pharmacokinetic clearances of the 6 vancomycin products from infected mice. There were no important pharmacodynamic differences in the in vitro or in vivo activities among the six vancomycin products evaluated.

Background FOS (ZTI-01, fosfomycin for injection) is an epoxide antibiotic that covalently binds MurA, an earlier step in cell wall synthesis inhibition. FOS has demonstrated synergistic killing with other classes of agents, including carbapenems. PA, among other non-fermentors, are difficult to treat pathogens and combination therapy is important to ensure killing and suppress emergence of resistance. Here, we examine the combination of FOS + MER in the HFIM against PA. Methods The experimental design was fully factorial (3 FOS and 3 MER arms and all combinations). Simulated FOS doses were 4, 6 and 8 g 8 hourly (Q8). MER doses were 0.5, 1 and 2 g Q8. The experiment lasted 10 days. Concentrations from the central compartment were measured in all arms by LC/MS/MS. Total bacterial burden and resistant subpopulations for both drugs were measured. Resistance plates were infused with 3XBaseline MIC. Starting inoculum was 6.86 Logs. Results FOS/MER MIC’s were 64 mg/L (broth) and 0.5 mg/L. Mutational frequency to resistance were -5.27 (FOS) and -6.7 (MER). There were 15 drug-containing arms and a no-treatment control. All drug arms had concentration-time profiles accurately reproduced. All FOS alone arms had rapid resistance emergence. MER 0.5 gm alone had resistance emerge at Day1. FOS 4 g + MER 0.5 g allowed resistance to both agents as did FOS 6g + MER 0.5 g. FOS 6 g + MER 1 g allowed MER resistance, but not FOS. All other combination regimens fully suppressed all resistant mutants. The effect of combination therapy is shown in Figures 1–3. MIC’s for MER-resistant isolates were 2 mg/L early and up to 16 mg/L late (day4 and after). FOS-resistant isolates generally had MIC values between 128 and >1024 mg/L. Conclusion The combination of FOS + MER is promising for therapy of a wild-type PA. Doses of FOS of 6 and 8 g Q8 tended to suppress resistance emergence to either agent when combined with 1 or 2 g Q8 of MER. We intend on examining the impact of resistance mutations to MER (oprD downregulated and MexAB upregulated) with this combination to identify any potential therapeutic advantage of this combination regimen Disclosures G. L. Drusano, Zavante: Scientific Advisor, Consulting fee

It has been previously shown that mice subjected to an aerosol exposure to Yersinia pestis and treated with β-lactam antibiotics after a delay of 42 h died at an accelerated rate compared to controls. It was hypothesized that endotoxin release in antibiotic-treated mice accounted for the accelerated death rate in the mice exposed to aerosol Y. pestis . Imipenem, a β-lactam antibiotic, binds to penicillin binding protein 2 with the highest affinity and produces rounded cells. The binding of imipenem causes cells to lyse quickly and thereby to release less free endotoxin. Two imipenem regimens producing fractions of time that the concentration of free, unbound drug was above the MIC ( f T>MIC) of approximately 25% (6/24 h) and 40% (9.5/24 h) were evaluated. In the postexposure prophylaxis study, the 40% and 25% regimens produced 90% and 40% survivorship, respectively. In the 42-h treatment study, both regimens demonstrated a 40 to 50% survivorship at therapy cessation and some deaths thereafter, resulting in a 30% survivorship. As this was an improvement over the results with other β-lactams, a comparison of both endotoxin and cytokine levels in mice treated with imipenem and ceftazidime (a β-lactam previously demonstrated to accelerate death in mice during treatment) was performed and supported the original hypotheses; however, the levels observed in animals treated with ciprofloxacin (included as an unrelated antibiotic that is also bactericidal but should cause little lysis due to a different mode of action) were elevated and significantly (7-fold) higher than those with ceftazidime.

Coadministration of moxifloxacin and rifampin was evaluated in a murine model of Mycobacterium tuberculosis pulmonary infection to determine whether the finding of antagonism documented in a hollow-fiber infection model could be recapitulated in vivo . Colony counts were followed in a no-treatment control group, groups administered moxifloxacin or rifampin monotherapy, and a group administered a combination of the two agents. Following 18 days of once-daily oral administration to mice infected with M. tuberculosis , there was a reduction in the plasma exposure to rifampin that decreased further when rifampin was coadministered with moxifloxacin. Pharmacodynamic analysis demonstrated a mild antagonistic interaction between moxifloxacin and rifampin with respect to cell kill in the mouse model for tuberculosis (TB). No emergence of resistance was noted over 28 days of therapy, even with monotherapy. This was true even though one of the agents in the combination (moxifloxacin) induces error-prone replication. The previously noted antagonism with respect to cell kill shown in the hollow-fiber infection model was recapitulated in the murine TB lung model, although to a lesser extent.

The development of new antiviral compounds active against hepatitis C virus (HCV) has surged in recent years. In order for these new compounds to be efficacious in humans, optimal dosage regimens for each compound must be elucidated. We have developed a novel in vitro pharmacokinetic/pharmacodynamic system, the BelloCell system, to identify optimal dosage regimens for anti-HCV compounds. In these experiments, genotype 1b HCV replicon-bearing cells (2209-23 cells) were inoculated onto carrier flakes in BelloCell bottles and treated with MK-4519, a serine protease inhibitor. Our dose-ranging studies illustrated that MK-4519 inhibited replicon replication in a dose-dependent manner, yielding a 50% effective concentration (EC 50 ) of 1.8 nM. Dose-fractionation studies showed that shorter dosing intervals resulted in greater replicon suppression, indicating that the time that the concentration is greater than the EC 50 is the pharmacodynamic parameter for MK-4519 linked with inhibition of replicon replication. Mutations associated with resistance to serine protease inhibitors were detected in replicons harvested from all treatment arms. These data suggest that MK-4519 is highly active against genotype 1b HCV, but monotherapy is not sufficient to prevent the amplification of resistant replicons. In summary, our findings show that the BelloCell system is a useful and clinically relevant tool for predicting optimal dosage regimens for anti-HCV compounds.

Some trials administered antituberculosis agents for 5 of 7 days (5/7-day regimen) to optimize adherence. Since moxifloxacin has a longer half-life than rifampin, rifampin concentrations are 2 log10 CFU/ml above the low-exposure 7/7-day arm. Pharmacokinetic mismatching of drugs in the therapy of tuberculosis may result in emergence of resistance when a drug holiday is imposed during which there is functional monotherapy and where the remaining agent induces error-prone replication. This is particularly true for the portion of the population where the clearance is higher (1 SD above the mean)., IMPORTANCE Directly observed therapy is a cornerstone of treatment of Mycobacterium tuberculosis. Patients are often given a drug holiday to facilitate the direct observation of therapy. With rifampin and moxifloxacin, there is a discordance between the half-lives of these agents (1.9 versus 6.5 h when employed in combination). In addition, moxifloxacin induces error-prone replication in Mycobacterium tuberculosis. In this experiment, we demonstrate that the drug holiday (5 of 7 days of therapy [5/7-day regimen]) allows the emergence of resistance to moxifloxacin, which was not seen with 7/7-day therapy. If drug holidays are used, it is imperative to better match pharmacokinetics to minimize the risk of emergence of resistance.

Pseudomonas aeruginosa pneumonia remains a most-difficult-to-treat nosocomial bacterial infection. We used mathematical modeling to identify drug exposure targets for meropenem in the epithelial lining fluid (ELF) of mice with Pseudomonas pneumonia driving substantial [2 to 3 log 10 (CFU/g)] killing and which suppressed resistant subpopulation amplification. We bridged to humans to estimate the frequency with which the largest licensed meropenem dose would achieve these exposure targets. Cell kills of 2 and 3 log 10 (CFU/g) and resistant subpopulation suppression were mediated by achieving time > MIC in ELF of 32%, 50%, and 50%. Substantial variability in meropenem's ability to penetrate into ELF of both mice and humans was observed. Penetration variability and high exposure targets combined to prevent even the largest licensed meropenem dose from achieving the targets at an acceptable frequency. Even a highly potent agent such as meropenem does not adequately suppress resistant subpopulation amplification as single-agent therapy administered at maximal dose and optimal schedule. Combination chemotherapy is likely required in humans if we are to minimize resistance emergence in Pseudomonas aeruginosa pneumonia. This combination needs evaluation both in the murine pneumonia model and in humans.

Moxifloxacin is under development for expanded use against Mycobacterium tuberculosis. Rifampin is a mainstay of therapy. We examined the interaction of moxifloxacin plus rifampin for log-phase and nonreplicating persister (NRP) organisms. For this evaluation, we employed our hollow-fiber infection model, in which organisms are exposed to clinically relevant drug concentration-time profiles and the impact on bacterial cell kill and resistant subpopulation amplification is determined. In log phase, resistance emergence was observed in all monotherapy regimens and in no combination therapy regimen. No difference was seen in time to a 3-log reduction in the bacterial burden; there was a significant difference in time to resistance emergence (P = 0.0006). In the NRP experiment, no resistance emergence was seen. There was a significant difference between the monotherapy and combination therapy regimens in time to a 3-log reduction in the bacterial burden (P = 0.042). The combination is efficacious for suppressing resistant organisms but is antagonistic for cell kill., IMPORTANCE M. tuberculosis infects one-third of the world’s population. Multiresistant organisms have become more frequent, threatening our ability to provide adequate chemotherapy. Moxifloxacin has been seen as an important new agent with the potential to supplant isoniazid or add to the rifampin/isoniazid combination. M. tuberculosis also exists in different physiological states, including the nonreplicating persister phenotype. We examined the moxifloxacin/rifampin combination in a new in vitro system to allow judgment of how moxifloxacin would interact with rifampin and allow its performance in clinical trials to be placed into perspective. Importantly, the combination suppressed resistance emergence, but at the price of slightly slowing bacterial cell kill. This new combination is a welcome addition to the physician’s armamentarium.

Bacillus anthracis is complex because of its spore form. The spore is invulnerable to antibiotic action. It also has an impact on the emergence of resistance. We employed the hollow-fiber infection model to study the impacts of different doses and schedules of moxifloxacin on the total-organism population, the spore population, and the subpopulations of vegetative- and spore-phase organisms that were resistant to moxifloxacin. We then generated a mathematical model of the impact of moxifloxacin, administered by continuous infusion or once daily, on vegetative- and spore-phase organisms. The ratio of the rate constant for vegetative-phase cells going to spore phase ( K vs ) to the rate constant for spore-phase cells going to vegetative phase ( K sv ) determines the rate of organism clearance. The continuous-infusion drug profile is more easily sensed as a threat; the K vs / K sv ratio increases at lower drug exposures (possibly related to quorum sensing). This movement to spore phase protects the organism but makes the emergence of resistance less likely. Suppression of resistance requires a higher level of drug exposure with once-daily administration than with a continuous infusion, a difference that is related to vegetative-to-spore (and back) transitioning. Spore biology has a major impact on drug therapy and resistance suppression. These findings explain why all drugs of different classes have approximately the same rate of organism clearance for Bacillus anthracis .

Rifampin is a cornerstone of modern antituberculosis therapy. However, rifampin's half-life of 3 h is believed to limit its utility for intermittent therapy, so new congeners with long half-lives are being developed. Using an in vitro pharmacokinetic-pharmacodynamic model of tuberculosis, we examined the relationships between rifampin exposure, microbial killing of log-phase-growth Mycobacterium tuberculosis , and suppression of resistance. Rifampin's microbial killing was linked to the area under the concentration-time curve-to-MIC ratio. The suppression of resistance was associated with the free peak concentration ( C max )-to-MIC ratio and not the duration that the rifampin concentration was above MIC. Rifampin prevented resistance to itself at a free C max /MIC ratio of ≥175. The postantibiotic effect duration was ≥5.2 days and was most closely related to the C max /MIC ratio ( r 2 = 0.96). To explain rifampin's concentration-dependent effect, we examined the kinetics of rifampin entry into M. tuberculosis . Rifampin achieved concentration-dependent intracellular steady-state concentrations within 15 min. Our results suggest that doses of rifampin higher than those currently employed would optimize the effect of rifampin, if patients could tolerate them. Another major implication is that in the design of new rifampin congeners for intermittent therapy, the important properties may include (i) the efficient entry of the rifamycin into M. tuberculosis , (ii) the achievement of a free C max /MIC of >175 that can be tolerated by patients, and (iii) a long postantibiotic effect duration.

The preferential use of older antimicrobial agents is, in general, sound public health policy and is meant to maintain susceptibility to newer agents. In the case of fluoroquinolones, however, this strategy is flawed and may actually hasten the spread of Streptococcus pneumoniae strains resistant to newer members of the class. In a mouse thigh infection model, we were unable to isolate clones of pneumococci resistant to the newer fluoroquinolone levofloxacin at 2 × or 4 × the baseline MIC. An initial exposure in vivo to the older agent, ciprofloxacin, allowed straightforward selection of clones resistant to levofloxacin in a subsequent experiment. The original ciprofloxacin exposure generated clones without changes in the parC / E and gyrA / B quinolone target sites almost exclusively but did allow overexpression of a reserpine-responsive pump. While this caused only minimal change in the levofloxacin MIC (0.6 mg/liter to 0.8 mg/liter), it allowed a major change in the mutational frequency to resistance for levofloxacin (8.5 to approximately 1/10 4.5 ), which allowed levofloxacin-resistant clones to be isolated in a subsequent in vivo experiment. The reason underlying ciprofloxacin's propensity to select for pump-overexpressed clones is likely related to its hydrophilicity. To preserve the susceptibility of Streptococcus pneumoniae to newer members of the class of quinolones, use of ciprofloxacin for community-acquired respiratory infections should be minimized.

Pharmacokinetic and pharmacodynamic studies were conducted in a murine model of systemic candidiasis to determine the pharmacodynamic parameter linked with caspofungin efficacy. Additional studies defined the importance of persistent tissue drug concentrations to treatment outcome. The pharmacokinetics of caspofungin were determined in the serum and kidneys of infected mice over 96 h. Population pharmacokinetic analysis demonstrated a serum terminal half-life ( t 1/2 ) for caspofungin of 20.2 h when only serum concentrations were considered, but the terminal t 1/2 increased to 59.2 h when serum and kidney concentration-time data were comodeled. In dose-range studies, the dose-response effect was well described by an inhibitory sigmoid curve for the exposure-effect killing caused by the drug ( r 2 > 0.96; P ≪ 0.001). In dose-fractionation studies, fungal counts in kidneys were not statistically different for total doses given as one, two, or four equally divided doses over 96 h, indicating that the area under the concentration-time curve/MIC is the pharmacodynamic parameter that predicts caspofungin efficacy in our infection model. In a separate study, mice infected with Candida albicans 24 h after serum concentrations of caspofungin fell below the MIC for the fungal isolate had significant reductions in fungal densities in their kidneys compared with the growth of fungi in the kidneys of untreated controls ( P = 0.005). This in vivo biological assay demonstrates that therapeutic concentrations of caspofungin persist at the site of infection in kidney tissue well after serum concentrations fall below the MIC, underscoring the primacy of caspofungin levels in tissues on determining treatment outcome.

In this study we defined the pharmacodynamic parameter that optimizes outcome in deep-seated Candida albicans infections treated with fluconazole. Using a murine model of systemic candidiasis, we conducted single-dose dose-ranging studies with fluconazole to determine the dosage of this drug that resulted in a 50% reduction in fungal densities (50% effective dose [ED 50 ]) in kidneys versus the fungal densities in the kidneys of untreated controls. We found that the ED 50 of fluconazole given intraperitoneally was 4.56 mg/kg of body weight/day (95% confidence interval, 3.60 to 5.53 mg/kg/day), and the dose-response relationship was best described by an inhibitory sigmoid maximal effect ( E max ) curve. To define the pharmacodynamics of fluconazole, we gave dosages lower than, approximating, and higher than the ED 50 of fluconazole (range, 3.5 to 5.5 mg/kg/day, equivalent to the ED 16 to the ED 75 ) to various groups of infected animals using three dose-fractionation schedules. For each total dose of fluconazole examined, the dose-fractionation schedules optimized the ratio of the area under the concentration-time curve (AUC) to the MIC (the AUC/MIC ratio), the ratio of the maximum concentration of drug in serum ( C max ) to the MIC, and the time that the drug remained above the MIC for the infecting C. albicans isolate. Similar reductions in fungal densities in kidneys were seen between groups that received the same total dose of fluconazole in one, two, or four equally divided doses. Thus, dose-fractionation studies demonstrated that the pharmacodynamic parameter of fluconazole that best predicted outcome was the AUC/MIC ratio.