PBP-3 Directed Therapy in VIM-producing Pseudomonas aeruginosa Creates Bacterial Transformers, Persisters in Disguise.

OBJECTIVES: The proliferation of metallo-beta-lactamase-producing Pseudomonas aeruginosa represents a significant public health threat. P. aeruginosa can undergo significant phenotypic changes that can drastically impair antibiotic efficacy. This study's objectives were (1) to quantify the time-course of killing of VIM-2-producing P. aeruginosa in response to aztreonam-based therapies (including avibactam for coverage of AmpC), and (2) to document the capacity of P. aeruginosa to undergo morphological transformations that facilitate persistence. METHODS: A well-characterized, clinical VIM-2-producing P. aeruginosa was studied in the Hollow Fiber Infection Model (HFIM) over 9 days (7 days of active antibiotic therapy, 2 days treatment withdrawal) at a 107.5 CFU/mL starting inoculum. HFIM treatment arms included: growth control, aztreonam, ceftazidime/avibactam, aztreonam/|ceftazidime/|avibactam, polymyxin B, and aztreonam/|ceftazidime/|avibactam/|polymyxin B. In addition, real-time imaging studies were conducted under static conditions to determine the time-course of the reversion of persister cells. RESULTS: A pronounced discrepancy was observed between OD620 and bacterial counts obtained from plating methods (hereafter referred to as 'OD-count discrepancy'). For aztreonam monotherapy, observed counts were 0 CFU/mL by 120 h. Despite this, there was a significant OD-count discrepancy as compared to the pre-treatment 0h. Between therapy withdrawal at 168h and 216h, all arms with suppressed counts had re-grown to the system carrying capacity. Real-time imaging of the P. aeruginosa filaments after drug removal showed rapid reversion from a long, filamentous phenotype to many individual rods within 2 h. CONCLUSION: Managing MBL-producing P. aeruginosa will require a multi-faceted approach, focused on maximizing killing and minimizing proliferation of resistant and persistent subpopulations, which will involve eliminating drug-induced phenotypic transformers.

Next generation antibiotic combinations to combat pan-drug resistant Klebsiella pneumoniae.

Antimicrobial resistance has emerged as one of the leading public health threats of the twenty-first century. Gram-negative pathogens have been a major contributor to the declining efficacy of antibiotics through both acquired resistance and tolerance. In this study, a pan-drug resistant (PDR), NDM-1 and CTX-M-15 co-producing isolate of K. pneumoniae, CDC Nevada, (Kp Nevada) was exposed to the clinical combination of aztreonam + ceftazidime/avibactam (ATM/CAZ/AVI) to overcome metallo-ß-lactamases. Unexpectedly, the ß-lactam combination resulted in long filamentous cell formation induced by PBP3 inhibition over 168 h in the hollow fiber infection model experiments with eventual reversion of the total population upon drug removal. However, the addition of imipenem to the two drug ß-lactam combination was highly synergistic with suppression of all drug resistant subpopulations over 5 days. Scanning electron microscopy and fluorescence microscopy for all imipenem combinations in time kill studies suggested a role for imipenem in suppression of long filamentous persisters, via the formation of metabolically active spheroplasts. To complement the imaging studies, salient transcriptomic changes were quantified using RT-PCR and novel cassette assay evaluated ß-lactam permeability. This showed significant upregulation of both spheroplast protein Y (SPY), a periplasmic chaperone protein that has been shown to be related to spheroplast formation, and penicillin binding proteins (PBP1, PBP2, PBP3) for all combinations involving imipenem. However, with aztreonam alone, pbp1, pbp3 and spy remained unchanged while pbp2 levels were downregulated by > 25%. Imipenem displayed 207-fold higher permeability as compared with aztreonam (mean permeability coefficient of 17,200 nm/s). Although the clinical combination of aztreonam/avibactam and ceftazidime has been proposed as an important treatment of MBL Gram-negatives, we report the first occurrence of long filamentous persister formation. To our knowledge, this is the first study that defines novel ß-lactam combinations involving imipenem via maximal suppression of filamentous persisters to combat PDR CDC Nevada K. pneumoniae.

Machine Learning-Led Optimization of Combination Therapy: Confronting the Public Health Threat of Extensively Drug Resistant Gram-Negative Bacteria.

Developing optimized regimens for combination antibiotic therapy is challenging and often performed empirically over many clinical studies. Novel implementation of a hybrid machine-learning pharmacokinetic/pharmacodynamic/toxicodynamic (ML-PK/PD/TD) approach optimizes combination therapy using human PK/TD data along with in vitro PD data. This study utilized human population PK (PopPK) of aztreonam, ceftazidime/avibactam, and polymyxin B along with in vitro PDs from the Hollow Fiber Infection Model (HFIM) to derive optimal multi-drug regimens de novo through implementation of a genetic algorithm (GA). The mechanism-based PD model was constructed based on 7-day HFIM experiments across 4 clinical, extensively drug resistant Klebsiella pneumoniae isolates. GA-led optimization was performed using 13 different fitness functions to compare the effects of different efficacy (60%, 70%, 80%, or 90% of simulated subjects achieving bacterial counts of 102 CFU/mL) and toxicity (66% of simulated subjects having a target polymyxin B area under the concentration-time curve [AUC] of 100 mg·h/L and aztreonam AUC of 1,332 mg·h/L) on the optimized regimen. All regimens, except those most heavily weighted for toxicity prevention, were able to achieve the target efficacy threshold (102 CFU/mL). Overall, GA-based regimen optimization using preclinical data from animal-sparing in vitro studies and human PopPK produced clinically relevant dosage regimens similar to those developed empirically over many years for all three antibiotics. Taken together, these data provide significant insight into new therapeutic approaches incorporating ML to regimen design and treatment of resistant bacterial infections.

Mechanistic Insights to Combating NDM- and CTX-M-Coproducing Klebsiella pneumoniae by Targeting Cell Wall Synthesis and Outer Membrane Integrity.

Metallo-ß-lactamase (MBL)-producing Gram-negative bacteria cause infections associated with high rates of morbidity and mortality. Currently, a leading regimen to treat infections caused by MBL-producing bacteria is aztreonam combined with ceftazidime-avibactam. The purpose of the present study was to evaluate and rationally optimize the combination of aztreonam and ceftazidime-avibactam with and without polymyxin B against a clinical Klebsiella pneumoniae isolate producing NDM-1 and CTX-M by use of the hollow fiber infection model (HFIM). A novel de-escalation approach to polymyxin B dosing was also explored, whereby a standard 0-h loading dose was followed by maintenance doses that were 50% of the typical clinical regimen. In the HFIM, the addition of polymyxin B to aztreonam plus ceftazidime-avibactam significantly improved bacterial killing, leading to eradication, including for the novel de-escalation dosing strategy. Serial samples from the growth control and monotherapies were explored in a Galleria mellonella virulence model to assess virulence changes. Weibull regression showed that low-level ceftazidime resistance and treatment with monotherapy resulted in increased G. mellonella mortality (P < 0.05). A neutropenic rabbit pneumonia model demonstrated that aztreonam plus ceftazidime-avibactam with or without polymyxin B resulted in similar bacterial killing, and these combination therapies were statistically significantly better than monotherapies (P < 0.05). However, only the polymyxin B-containing combination therapy produced a statistically significant decrease in lung weights (P < 0.05), indicating a decreased inflammatory process. Altogether, adding polymyxin B to the combination of aztreonam plus ceftazidime-avibactam for NDM- and CTX-M-producing K. pneumoniae improved bacterial killing effects, reduced lung inflammation, suppressed resistance amplification, and limited virulence changes.

Determining the optimal dosing of a novel combination regimen of ceftazidime/avibactam with aztreonam against NDM-1-producing Enterobacteriaceae using a hollow-fibre infection model.

BACKGROUND: MBL-producing strains of Enterobacteriaceae are a major public health concern. We sought to define optimal combination regimens of ceftazidime/avibactam with aztreonam in a hollow-fibre infection model (HFIM) of MBL-producing strains of Escherichia coli and Klebsiella pneumoniae. METHODS: E. coli ARLG-1013 (blaNDM-1, blaCTX-M, blaCMY, blaTEM) and K. pneumoniae ARLG-1002 (blaNDM-1, blaCTXM-15, blaDHA, blaSHV, blaTEM) were studied in the HFIM using simulated human dosing regimens of ceftazidime/avibactam and aztreonam. Experiments were designed to evaluate the effect of staggered versus simultaneous administration, infusion duration and aztreonam daily dose (6 g/day versus 8 g/day) on bacterial killing and resistance suppression. Prospective validation experiments for the most active combination regimens were performed in triplicate to ensure reproducibility. RESULTS: Staggered administration of the combination (ceftazidime/avibactam followed by aztreonam) was found to be inferior to simultaneous administration. Longer infusion durations (2 h and continuous infusion) also resulted in enhanced bacterial killing relative to 30 min infusions. The rate of killing was more pronounced with 8 g/day versus 6 g/day aztreonam combination regimens for both tested strains. In the prospective validation experiments, ceftazidime/avibactam with aztreonam dosed every 8 and 6 h, respectively (ceftazidime/avibactam 2/0.5 g every 8 h + aztreonam 2 g every 6 h), or ceftazidime/avibactam with aztreonam as continuous infusions resulted in maximal bacterial killing and resistance suppression over 7 days. CONCLUSIONS: Simultaneous administration of aztreonam 8 g/day given as a continuous or 2 h infusion with ceftazidime/avibactam resulted in complete bacterial eradication and resistance suppression. Further study of this combination is needed with additional MBL-producing Gram-negative pathogens. The safety of this double ß-lactam strategy also warrants further study in Phase 1 clinical trials.

Azithromycin Pharmacodynamics against Persistent Haemophilus influenzae in Chronic Obstructive Pulmonary Disease.

The pharmacodynamic profile of azithromycin against persistent strains of nontypeable Haemophilus influenzae (NTHi) from chronic obstructive pulmonary disease (COPD) patients was characterized. Azithromycin displayed differential concentration-dependent activities (R2 ≥ 0.988); the pharmacodynamic response was attenuated when we compared the "first" and "last" strains of NTHi that persisted in the airways of the same patient for 819 days (the 50% effective concentration [EC50] increased more than 50 times [0.0821 mg/liter versus 4.23 mg/liter]). In the hollow-fiber infection model, NTHi viability was maintained throughout simulated azithromycin (Zithromax) Z-Pak regimens over 10 days.

Polymyxin Combinations Combat Escherichia coli Harboring mcr-1 and blaNDM-5: Preparation for a Postantibiotic Era.

The rapid increase of carbapenem resistance in Gram-negative bacteria has resurrected the importance of the polymyxin antibiotics. The recent discovery of plasmid-mediated polymyxin resistance (mcr-1) in carbapenem-resistant Enterobacteriaceae serves as an important indicator that the golden era of antibiotics is under serious threat. We assessed the bacterial killing of 15 different FDA-approved antibiotics alone and in combination with polymyxin B in time-killing experiments against Escherichia coli MCR1_NJ, the first reported isolate in the United States to coharbor mcr-1 and a New Delhi metallo-ß-lactamase gene (blaNDM-5). The most promising regimens were advanced to the hollow-fiber infection model (HFIM), where human pharmacokinetics for polymyxin B, aztreonam, and amikacin were simulated over 240 h. Exposure to polymyxin B monotherapy was accompanied by MCR1_NJ regrowth but not resistance amplification (polymyxin B MIC from 0 to 240 h [MIC0h to MIC240h] of 4 mg/liter), whereas amikacin monotherapy caused regrowth and simultaneous resistance amplification (amikacin MIC0h of 4 mg/liter versus MIC240h of >64 mg/liter). No MCR1_NJ colonies were observed for any of the aztreonam-containing regimens after 72 h. However, HFIM cartridges for both aztreonam monotherapy and the polymyxin B-plus-aztreonam regimen were remarkably turbid, and the presence of long, filamentous MCR1_NJ cells was evident in scanning electron microscopy, suggestive of a nonreplicating persister (NRP) phenotype. In contrast, the 3-drug combination of polymyxin B, aztreonam, and amikacin provided complete eradication (>8-log10 CFU/ml reduction) with suppression of resistance and prevention of NRP formation. This is the first comprehensive pharmacokinetic/pharmacodynamic study to evaluate triple-drug combinations for polymyxin- and carbapenem-resistant E. coli coproducing MCR-1 and NDM-5 and will aid in the preparation for a so-called "postantibiotic" era.IMPORTANCE A global health crisis may be on the horizon, as the golden era of antibiotics is under serious threat. We recently reported the first case in the United States of a highly resistant, Escherichia coli so-called "superbug" (MCR1_NJ), coharboring two of the most worrying antibiotic resistance genes, encoding mobile colistin resistance (mcr-1) and a New Delhi metallo-ß-lactamase (blaNDM-5). Worryingly, the medical community is vulnerable to this emerging bacterial threat because optimal treatment strategies are undefined. Here, we report the activity of an optimized combination using simulated human doses of commercially available antibiotics against MCR1_NJ. A unique triple combination involving a cocktail of polymyxin B, aztreonam, and amikacin eradicated the MCR-1- and NDM-5-producing E. coli Each antimicrobial agent administered as monotherapy or in double combinations failed to eradicate MCR1_NJ at a high inoculum. To our knowledge, this is the first study to propose 3-drug therapeutic solutions against superbugs coharboring mcr-1 and blaNDM, seeking to prepare clinicians for future occurrences of these pathogens.

Pharmacodynamics of dose-escalated 'front-loading' polymyxin B regimens against polymyxin-resistant mcr-1-harbouring Escherichia coli.

Objectives: Gram-negative bacteria harbouring the mcr-1 plasmid are resistant to the 'last-line' polymyxins and have been reported worldwide. Our objective was to define the impact of increasing the initial polymyxin B dose intensity against an mcr-1 -harbouring strain to delineate the impact of plasmid-mediated polymyxin resistance on the dynamics of bacterial killing and resistance. Methods: A hollow fibre infection model (HFIM) was used to simulate polymyxin B regimens against an mcr-1 -harbouring Escherichia coli (MIC 8 mg/L) over 10 days. Four escalating polymyxin B 'front-loading' regimens (3.33, 6.66, 13.3 or 26.6 mg/kg for one dose followed by 1.43 mg/kg every 12 h starting 12 h later) simulating human pharmacokinetics were utilized in the HFIM. A mechanism-based, mathematical model was developed using S-ADAPT to characterize bacterial killing. Results: The 3.33 mg/kg 'front-loading' regimen resulted in regrowth mirroring the growth control. The 6.66, 13.3 and 26.6 mg/kg 'front-loading' regimens resulted in maximal bacterial reductions of 1.91, 3.79 and 6.14 log 10 cfu/mL, respectively. Irrespective of the early polymyxin B exposure (24 h AUC), population analysis profiles showed similar growth of polymyxin B-resistant subpopulations. The HFIM data were well described by the mechanism-based model integrating three subpopulations (susceptible, intermediate and resistant). Compared with the susceptible subpopulation of mcr-1 -harbouring E. coli , the resistant subpopulation had an approximately 10-fold lower rate of killing due to polymyxin B treatment. Conclusions: Manipulating initial dose intensity of polymyxin B was not able to overcome plasmid-mediated resistance due to mcr-1 in E. coli . This reinforces the need to develop new combinatorial strategies to combat these highly resistant Gram-negative bacteria.

New Polymyxin B Dosing Strategies To Fortify Old Allies in the War against KPC-2-Producing Klebsiella pneumoniae.

Pharmacodynamics of a polymyxin B, meropenem, and rifampin triple combination were examined against Klebsiella pneumoniae carbapenemase-producing Klebsiella pneumoniae (KPC-Kp) ST258. In time-kill experiments against three KPC-Kp isolates, triple combination generated 8.14, 8.19, and 8.29 log10 CFU/ml reductions within 24 h. In the hollow-fiber infection model, the triple combination caused maximal killing of 5.16 log10 CFU/ml at 78 h and the time required for regrowth was more than doubled versus the 2-drug combinations. Remarkably, combinations with a high single-dose polymyxin B burst plus rifampin preserved KPC-Kp polymyxin susceptibility (MIC240 h = 0.5 mg/liter) versus the same combination with traditionally dosed polymyxin B, where resistance was amplified (MIC240 h = 32 mg/liter).

Influence of rhlR and lasR on Polymyxin Pharmacodynamics in Pseudomonas aeruginosa and Implications for Quorum Sensing Inhibition with Azithromycin.

The impact of quorum sensing on polymyxin and azithromycin pharmacodynamics was assessed in Pseudomonas aeruginosa PAO1 and an isogenic rhlR/lasR double knockout. For polymyxin B, greater killing against the rhlR/lasR knockout than against PAO1 was observed at 108 CFU/ml (polymyxin B half-maximal effective concentration [EC50], 5.61 versus 12.5 mg/liter, respectively; P < 0.005). Polymyxin B combined with azithromycin (256 mg/liter) was synergistic against each strain, significantly reducing the respective polymyxin B EC50 compared to those with monotherapy (P < 0.005), and is a promising strategy by which to combat P. aeruginosa.

In vitro pharmacodynamic evaluation of ceftolozane/tazobactam against ß-lactamase-producing Escherichia coli in a hollow-fibre infection model.

The proliferation of multidrug-resistant Gram-negative pathogens has been exacerbated by a lack of novel agents in current development by pharmaceutical companies. Ceftolozane/tazobactam was recently approved by the FDA for the treatment of complicated intra-abdominal infections and complicated urinary tract infections. In the present study, the activity of ceftolozane/tazobactam against four isogenic Escherichia coli strains was investigated in a hollow-fibre infection model simulating various clinical dosing regimens. The four investigational E. coli strains included #2805 (no ß-lactamase), #2890 (AmpC ß-lactamase), #2842 (CMY-10 ß-lactamase) and #2807 (CTX-M-15 ß-lactamase). Each strain was exposed to regimens simulating 1 g ceftolozane, 2 g ceftolozane, 1 g ceftolozane/0.5 g tazobactam, and 2 g ceftolozane/1 g tazobactam utilising a starting inoculum of ca. 106 CFU/mL. Whereas 1 g of ceftolozane eradicated strains #2805 and #2842 without subsequent regrowth, 1 g ceftolozane/0.5 g tazobactam was required to eradicate strain #2890. For strain #2890, ceftolozane monotherapy led to bacterial growth on plates impregnated with 20 mg/L ceftolozane by 24 h, whilst combination treatment with tazobactam completely suppressed the development of ceftolozane resistance. In contrast, none of the regimens, including 2 g ceftolozane/1 g tazobactam, were able to entirely suppress bacterial growth in strain #2807, with bacterial counts exceeding 108 CFU/mL by 48 h and ceftolozane-resistant populations being amplified after 24 h. Thus, the combination of ceftolozane and tazobactam achieved bactericidal activity followed by sustained killing over 10 days for three of four isogenic E. coli strains. Ceftolozane/tazobactam is a promising new agent to counter multidrug-resistant Gram-negative bacteria.

High-intensity meropenem combinations with polymyxin B: new strategies to overcome carbapenem resistance in Acinetobacter baumannii.

OBJECTIVES: The pharmacodynamics of polymyxin/carbapenem combinations against carbapenem-resistant Acinetobacter baumannii (CRAB) are largely unknown. Our objective was to determine whether intensified meropenem regimens in combination with polymyxin B enhance killing and resistance suppression of CRAB. METHODS: Time-kill experiments for meropenem and polymyxin B combinations were conducted against three polymyxin B-susceptible (MIC of polymyxin B = 0.5 mg/L) CRAB strains with varying meropenem MICs (ATCC 19606, N16870 and 03-149-1; MIC of meropenem = 4, 16 and 64 mg/L, respectively) at 108 cfu/mL. A hollow-fibre infection model was then used to simulate humanized regimens of polymyxin B and meropenem (2, 4, 6 and 8 g prolonged infusions every 8 h) versus N16870 at 108 cfu/mL over 14 days. New mathematical mechanism-based models were developed using S-ADAPT. RESULTS: Time-kill experiments were well described by the mathematical mechanism-based models, with the presence of polymyxin B drastically decreasing the meropenem concentration needed for half-maximal activity against meropenem-resistant populations from 438 to 82.1 (ATCC 19606), 158 to 93.6 (N16870) and 433 to 76.0 mg/L (03-149-1). The maximum killing effect of combination treatment was similar among all three strains despite divergent meropenem MIC values (Emax = 2.13, 2.08 and 2.15; MIC of meropenem = 4, 16 and 64 mg/L, respectively). Escalating the dose of meropenem in hollow-fibre combination regimens from 2 g every 8 h to 8 g every 8 h resulted in killing that progressed from a >2.5 log10 cfu/mL reduction with regrowth by 72 h (2 g every 8 h) to complete eradication by 336 h (8 g every 8 h). CONCLUSION: Intensified meropenem dosing in combination with polymyxin B may offer a unique strategy to kill CRAB irrespective of the meropenem MIC.

Polymyxin B in combination with doripenem against heteroresistant Acinetobacter baumannii: pharmacodynamics of new dosing strategies.

OBJECTIVES: Polymyxin B is being increasingly utilized as a last resort against resistant Gram-negative bacteria. We examined the pharmacodynamics of novel dosing strategies for polymyxin B combinations to maximize efficacy and minimize the emergence of resistance and drug exposure against Acinetobacter baumannii. METHODS: The pharmacodynamics of polymyxin B together with doripenem were evaluated in time-kill experiments over 48 h against 108 cfu/mL of two polymyxin-heteroresistant A. baumannii isolates (ATCC 19606 and N16870). Pharmacokinetic/pharmacodynamic relationships were mathematically modelled using S-ADAPT. A hollow-fibre infection model (HFIM) was also used to simulate clinically relevant polymyxin B dosing strategies (traditional, augmented 'front-loaded' and 'burst' regimens), together with doripenem, against an initial inoculum of 109 cfu/mL of ATCC 19606. RESULTS: In static time-kill studies, polymyxin B concentrations >4 mg/L in combination with doripenem 25 mg/L resulted in rapid bactericidal activity against both strains with undetectable bacterial counts by 24 h. The mathematical model described the rapid, concentration-dependent killing as subpopulation and mechanistic synergy. In the HFIM, the traditional polymyxin B combination regimen was synergistic, with a >7.5 log10 reduction by 48 h. The polymyxin B 'front-loaded' combination resulted in more rapid and extensive initial killing (>8 log10) within 24 h, which was sustained over 10 days. With only 25% of the cumulative drug exposure, the polymyxin B 'burst' combination demonstrated antibacterial activity similar to traditional and 'front-loaded' combination strategies. The polymyxin B 'front-loaded' and 'burst' combination regimens suppressed the emergence of resistance. CONCLUSIONS: Early aggressive dosing regimens for polymyxin combinations demonstrate promise for treatment of heteroresistant A. baumannii infections.

Combinatorial pharmacodynamics of polymyxin B and tigecycline against heteroresistant Acinetobacter baumannii.

The prevalence of heteroresistant Acinetobacter baumannii is increasing. Infections due to these resistant pathogens pose a global treatment challenge. Here, the pharmacodynamic activities of polymyxin B (PMB) (2-20 mg/L) and tigecycline (0.15-4 mg/L) were evaluated as monotherapy and in combination using a 4 × 4 concentration array against two carbapenem-resistant and polymyxin-heteroresistant A. baumannii isolates. Time Kill Experiments was employed at starting inocula of 10(6) and 10(8) CFU/mL over 48 h. Clinically relevant combinations of PMB (2 mg/L) and tigecycline (0.90 mg/L) resulted in greater reductions in the bacterial population compared with polymyxin alone by 8 h (ATCC 19606, -6.38 vs. -3.43 log10 CFU/mL; FADDI AB115, -1.38 vs. 2.08 log10 CFU/mL). At 10× the clinically achievable concentration (PMB 20 mg/L in combination with tigecycline 0.90 mg/L), there was bactericidal activity against FADDI AB115 by 4 h that was sustained until 32 h, and against ATCC 19606 that was sustained for 48 h. These studies show that aggressive polymyxin-based dosing in combination with clinically achievable tigecycline concentrations results in early synergistic activity that is not sustained beyond 8 h, whereas combinations with higher tigecycline concentrations result in sustained bactericidal activity against both isolates at both inocula. These results indicate a need for optimised front-loaded polymyxin-based combination regimens that utilise high polymyxin doses at the onset of treatment to achieve good pharmacodynamic activity whilst minimising adverse events.

Defining the Active Fraction of Daptomycin against Methicillin-Resistant Staphylococcus aureus (MRSA) Using a Pharmacokinetic and Pharmacodynamic Approach.

Our objective was to study the pharmacodynamics of daptomycin in the presence of varying concentrations of human serum (HS) in vitro to quantify the fraction of daptomycin that is 'active'. Time kill experiments were performed with daptomycin (0 to 256 mg/L) against two MRSA strains at log-phase growth, in the presence of HS (0%, 10%, 30%, 50%, 70%) combined with Mueller-Hinton broth. Daptomycin ≥ 2 mg/L achieved 99.9% kill within 8 h at all HS concentrations; early killing activity was slightly attenuated at higher HS concentrations. After 1 h, bacterial reduction of USA300 upon exposure to daptomycin 4 mg/L ranged from -3.1 to -0.5 log10CFU/mL in the presence of 0% to 70% HS, respectively. Bactericidal activity was achieved against both strains at daptomycin ≥ 4 mg/L for all fractions of HS exposure. A mechanism-based mathematical model (MBM) was developed to estimate the active daptomycin fraction at each %HS, comprising 3 bacterial subpopulations differing in daptomycin susceptibility. Time-kill data were fit with this MBM with excellent precision (r2 >0.95). The active fraction of daptomycin was estimated to range from 34.6% to 25.2% at HS fractions of 10% to 70%, respectively. Despite the reported low unbound fraction of daptomycin, the impact of protein binding on the activity of daptomycin was modest. The active fraction approach can be utilized to design in vitro experiments and to optimize therapeutic regimens of daptomycin in humans.

Effect of Fluoroquinolones and Macrolides on Eradication and Resistance of Haemophilus influenzae in Chronic Obstructive Pulmonary Disease.

Little is known about the effect of antibiotics on eradication of carriage and development of resistance in Haemophilus influenzae in individuals with chronic obstructive pulmonary disease (COPD). Our goals were to assess antibiotic susceptibilities, prevalence of resistance genes, and development of resistance in H. influenzae and to evaluate the effect of macrolide and fluoroquinolone administration on H. influenzae eradication. Data were from a 15-year longitudinal study of COPD. Genome sequence data were used to determine genotype and identify resistance genes. MICs of antibiotics were determined by reference broth microdilution. Generalized linear mixed models were used to evaluate associations between antibiotic use and H. influenzae eradication. We examined 267 H. influenzae isolates from 77 individuals. All newly acquired H. influenzae isolates were susceptible to azithromycin. Five of 27 (19%) strains developed 4-fold increases in azithromycin MICs and reached or exceeded the susceptibility breakpoint (≤4 µg/ml) during exposure. H. influenzae isolates were uniformly susceptible to ciprofloxacin, levofloxacin, and moxifloxacin (MIC90s of 0.015, 0.015, and 0.06, respectively); there were no mutations in quinolone resistance-determining regions. Fluoroquinolone administration was associated with increased H. influenzae eradication compared to macrolides (odds ratio [OR], 16.67; 95% confidence interval [CI], 2.67 to 104.09). There was no difference in H. influenzae eradication when comparing macrolide administration to no antibiotic (OR, 1.89; 95% CI, 0.43 to 8.30). Fluoroquinolones are effective in eradicating H. influenzae in individuals with COPD. Macrolides are ineffective in eradicating H. influenzae, and their use in COPD patients may lead to decreased macrolide susceptibility and resistance.

Paradoxical Effect of Polymyxin B: High Drug Exposure Amplifies Resistance in Acinetobacter baumannii.

Administering polymyxin antibiotics in a traditional fashion may be ineffective against Gram-negative ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) pathogens. Here, we explored increasing the dose intensity of polymyxin B against two strains of Acinetobacter baumannii in the hollow-fiber infection model. The following dosage regimens were simulated for polymyxin B (t1/2 = 8 h): non-loading dose (1.43 mg/kg of body weight every 12 h [q12h]), loading dose (2.22 mg/kg q12h for 1 dose and then 1.43 mg/kg q12h), front-loading dose (3.33 mg/kg q12h for 1 dose followed by 1.43 mg/kg q12h), burst (5.53 mg/kg for 1 dose), and supraburst (18.4 mg/kg for 1 dose). Against both A. baumannii isolates, a rapid initial decline in the total population was observed within the first 6 h of polymyxin exposure, whereby greater polymyxin B exposure resulted in greater maximal killing of -1.25, -1.43, -2.84, -2.84, and -3.40 log10 CFU/ml within the first 6 h. Unexpectedly, we observed a paradoxical effect whereby higher polymyxin B exposures dramatically increased resistant subpopulations that grew on agar containing up to 10 mg/liter of polymyxin B over 336 h. High drug exposure also proliferated polymyxin-dependent growth. A cost-benefit pharmacokinetic/pharmacodynamic relationship between 24-h killing and 336-h resistance was explored. The intersecting point, where the benefit of bacterial killing was equal to the cost of resistance, was an fAUC0-24 (area under the concentration-time curve from 0 to 24 h for the free, unbound fraction of drug) of 38.5 mg · h/liter for polymyxin B. Increasing the dose intensity of polymyxin B resulted in amplification of resistance, highlighting the need to utilize polymyxins as part of a combination against high-bacterial-density A. baumannii infections.

Optimization of Polymyxin B in Combination with Doripenem To Combat Mutator Pseudomonas aeruginosa.

Development of spontaneous mutations in Pseudomonas aeruginosa has been associated with antibiotic failure, leading to high rates of morbidity and mortality. Our objective was to evaluate the pharmacodynamics of polymyxin B combinations against rapidly evolving P. aeruginosa mutator strains and to characterize the time course of bacterial killing and resistance via mechanism-based mathematical models. Polymyxin B or doripenem alone and in combination were evaluated against six P. aeruginosa strains: wild-type PAO1, mismatch repair (MMR)-deficient (mutS and mutL) strains, and 7,8-dihydro-8-oxo-deoxyguanosine system (GO) base excision repair (BER)-deficient (mutM, mutT, and mutY) strains over 48 h. Pharmacodynamic modeling was performed using S-ADAPT and facilitated by SADAPT-TRAN. Mutator strains displayed higher mutation frequencies than the wild type (>600-fold). Exposure to monotherapy was followed by regrowth, even at high polymyxin B concentrations of up to 16 mg/liter. Polymyxin B and doripenem combinations displayed enhanced killing activity against all strains where complete eradication was achieved for polymyxin B concentrations of >4 mg/liter and doripenem concentrations of 8 mg/liter. Modeling suggested that the proportion of preexisting polymyxin B-resistant subpopulations influenced the pharmacodynamic profiles for each strain uniquely (fraction of resistance values are -8.81 log10 for the wild type, -4.71 for the mutS mutant, and -7.40 log10 for the mutM mutant). Our findings provide insight into the optimization of polymyxin B and doripenem combinations against P. aeruginosa mutator strains.

Combinatorial Pharmacodynamics of Ceftolozane-Tazobactam against Genotypically Defined ß-Lactamase-Producing Escherichia coli: Insights into the Pharmacokinetics/Pharmacodynamics of ß-Lactam-ß-Lactamase Inhibitor Combinations.

Despite a dearth of new agents currently being developed to combat multidrug-resistant Gram-negative pathogens, the combination of ceftolozane and tazobactam was recently approved by the Food and Drug Administration to treat complicated intra-abdominal and urinary tract infections. To characterize the activity of the combination product, time-kill studies were conducted against 4 strains ofEscherichia colithat differed in the type of ß-lactamase they expressed. The four investigational strains included 2805 (no ß-lactamase), 2890 (AmpC ß-lactamase), 2842 (CMY-10 ß-lactamase), and 2807 (CTX-M-15 ß-lactamase), with MICs to ceftolozane of 0.25, 4, 8, and >128 mg/liter with no tazobactam, and MICs of 0.25, 1, 4, and 8 mg/liter with 4 mg/liter tazobactam, respectively. All four strains were exposed to a 6 by 5 array of ceftolozane (0, 1, 4, 16, 64, and 256 mg/liter) and tazobactam (0, 1, 4, 16, and 64 mg/liter) over 48 h using starting inocula of 10(6)and 10(8)CFU/ml. While ceftolozane-tazobactam achieved bactericidal activity against all 4 strains, the concentrations of ceftolozane and tazobactam required for a ≥3-log reduction varied between the two starting inocula and the 4 strains. At both inocula, the Hill plots (R(2)> 0.882) of ceftolozane revealed significantly higher 50% effective concentrations (EC50s) at tazobactam concentrations of ≤4 mg/liter than those at concentrations of ≥16 mg/liter (P< 0.01). Moreover, the EC50s at 10(8)CFU/ml were 2.81 to 66.5 times greater than the EC50s at 10(6)CFU/ml (median, 10.7-fold increase;P= 0.002). These promising results indicate that ceftolozane-tazobactam achieves bactericidal activity against a wide range of ß-lactamase-producingE. colistrains.

Emergence of polymyxin B resistance influences pathogenicity in Pseudomonas aeruginosa mutators.

The interplay between polymyxin B pharmacodynamics and pathogenicity was examined in Pseudomonas aeruginosa PAO1 and isogenic DNA repair-deficient mutators (mutM and mutS strains). Against mutS mutators, polymyxin B initial killing was concentration dependent, with >99.9% bacterial reduction at 2 h followed by regrowth and resistance. The pre- versus postexposed strains were inoculated real time into Galleria mellonella waxworms, resulting in increased median survival times from 20 h to 23 h (P < 0.001). Emergence of resistance in mutS P. aeruginosa resulted in attenuation of virulence.