Improved characterization of aminoglycoside penetration into human lung epithelial lining fluid via population pharmacokinetics.

Aminoglycosides are important treatment options for serious lung infections, but modeling analyses to quantify their human lung epithelial lining fluid (ELF) penetration are lacking. We estimated the extent and rate of penetration for five aminoglycosides via population pharmacokinetics from eight published studies. The area under the curve in ELF vs plasma ranged from 50% to 100% and equilibration half-lives from 0.61 to 5.80 h, indicating extensive system hysteresis. Aminoglycoside ELF peak concentrations were blunted, but overall exposures were moderately high.

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.

Aminoglycoside-resistance gene signatures are predictive of aminoglycoside MICs for carbapenem-resistant Klebsiella pneumoniae.

BACKGROUND: Aminoglycoside-containing regimens may be an effective treatment option for infections caused by carbapenem-resistant Klebsiella pneumoniae (CR-Kp), but aminoglycoside-resistance genes are common in these strains. The relationship between the aminoglycoside-resistance genes and aminoglycoside MICs remains poorly defined. OBJECTIVES: To identify genotypic signatures capable of predicting aminoglycoside MICs for CR-Kp. METHODS: Clinical CR-Kp isolates (n = 158) underwent WGS to detect aminoglycoside-resistance genes. MICs of amikacin, gentamicin, plazomicin and tobramycin were determined by broth microdilution (BMD). Principal component analysis was used to initially separate isolates based on genotype. Multiple linear regression was then used to generate models that predict aminoglycoside MICs based on the aminoglycoside-resistance genes. Last, the performance of the predictive models was tested against a validation cohort of 29 CR-Kp isolates. RESULTS: Among the original 158 CR-Kp isolates, 91.77% (145/158) had at least one clinically relevant aminoglycoside-resistance gene. As a group, 99.37%, 84.81%, 82.28% and 10.76% of the CR-Kp isolates were susceptible to plazomicin, amikacin, gentamicin and tobramycin, respectively. The first two principal components explained 72.23% of the total variance in aminoglycoside MICs and separated isolates into four groups with aac(6')-Ib, aac(6')-Ib', aac(6')-Ib+aac(6')-Ib' or no clinically relevant aminoglycoside-resistance genes. Regression models predicted aminoglycoside MICs with adjusted R2 values of 56%-99%. Within the validation cohort, the categorical agreement when comparing the observed BMD MICs with the predicated MICs was 96.55%, 89.66%, 86.21% and 82.76% for plazomicin, gentamicin, amikacin and tobramycin, respectively. CONCLUSIONS: Susceptibility to each aminoglycoside varies in CR-Kp. Detection of aminoglycoside-resistance genes may be useful to predict aminoglycoside MICs for CR-Kp.

Generating Genotype-Specific Aminoglycoside Combinations with Ceftazidime/Avibactam for KPC-Producing Klebsiella pneumoniae.

Antibiotic combinations, including ceftazidime/avibactam (CAZ/AVI), are frequently employed to combat KPC-producing Klebsiella pneumoniae (KPC-Kp), though such combinations have not been rationally optimized. Clinical KPC-Kp isolates with common genes encoding aminoglycoside-modifying enzymes (AMEs), aac(6')-Ib' or aac(6')-Ib, were used in static time-kill assays (n = 4 isolates) and the hollow-fiber infection model (HFIM; n = 2 isolates) to evaluate the activity of gentamicin, amikacin, and CAZ/AVI alone and in combinations. A short course, one-time aminoglycoside dose was also evaluated. Gentamicin plus CAZ/AVI was then tested in a mouse pneumonia model. Synergy with CAZ/AVI was more common with amikacin for aac(6')-Ib'-containing KPC-Kp but more common with gentamicin for aac(6')-Ib-containing isolates in time-kill assays. In the HFIM, although the isolates were aminoglycoside-susceptible at baseline, aminoglycoside monotherapies displayed variable initial killing, followed by regrowth and resistance emergence. CAZ/AVI combined with amikacin or gentamicin resulted in undetectable counts 50 h sooner than CAZ/AVI monotherapy against KPC-Kp with aac(6')-Ib'. CAZ/AVI monotherapy failed to eradicate KPC-Kp with aac(6')-Ib and a combination with gentamicin led to undetectable counts 70 h sooner than with amikacin. A one-time aminoglycoside dose with CAZ/AVI provided similar killing to aminoglycosides dosed for 7 days. In the mouse pneumonia model (n = 1 isolate), gentamicin and CAZ/AVI achieved a 6.0-log10 CFU/lung reduction at 24 h, which was significantly greater than either monotherapy (P < 0.005). Aminoglycosides in combination with CAZ/AVI were promising for KPC-Kp infections; this was true even for a one-time aminoglycoside dose. Selecting aminoglycosides based on AME genes or susceptibilities can improve the pharmacodynamic activity of the combination.

Capability of Enterococcus faecalis to shield Gram-negative pathogens from aminoglycoside exposure.

BACKGROUND: Enterococcus faecalis commonly produce aminoglycoside-modifying enzymes (AMEs) and are implicated in polymicrobial infections. OBJECTIVES: To determine if AME-producing E. faecalis is capable of protecting Enterobacteriaceae and Pseudomonas aeruginosa from gentamicin exposure. METHODS: Two Klebsiella pneumoniae isolates, two Escherichia coli isolates, and two Pseudomonas aeruginosa isolates were investigated in monoculture time-kill experiments, and each Gram-negative organism was also evaluated during co-culture with either AME-producing or AME-deficient E. faecalis. A pharmacokinetic/pharmacodynamics analysis that utilized Log Ratio Areas and a Hill-type mathematical model was used to determine if the maximal killing or potency of gentamicin against the Gram-negative organisms was altered by the presence of the E. faecalis. RESULTS: The maximal killing and potency of gentamicin was the same during monoculture and co-culture experiments for both K. pneumoniae isolates and one E. coli isolate (P > 0.05). In contrast, the maximal killing of gentamicin was attenuated against one E. coli isolate and both P. aeruginosa isolates during co-culture with E. faecalis (P < 0.05). The potency of gentamicin was variable against the three aforementioned isolates. Against the E. coli isolate, the potency of gentamicin was significantly reduced by the presence of either E. faecalis isolate (EC50 95% CI = 4.23-4.43 mg/L monoculture versus 3.86-4.19 mg/L and 3.55-3.96 mg/L during co-culture with AME-producing and AME-deficient E. faecalis, respectively). The potency of gentamicin increased or decreased for P. aeruginosa depending on which E. faecalis isolate was investigated. CONCLUSIONS: The AME-producing E. faecalis did not provide a consistent protective effect from aminoglycosides for the Gram-negative pathogens.

Optimizing aminoglycoside selection for KPC-producing Klebsiella pneumoniae with the aminoglycoside-modifying enzyme (AME) gene aac(6')-Ib.

OBJECTIVES: KPC-producing Klebsiella pneumoniae (KPC-Kp) isolates commonly co-harbour the aminoglycoside-modifying enzyme (AME) gene aac(6')-Ib, which encodes an AME that can confer resistance to some of the commercially available aminoglycosides. We sought to determine the influence of AAC(6')-Ib in KPC-Kp on the pharmacodynamic activity of aminoglycosides. METHODS: Six KPC-Kp clinical isolates, three with and three without aac(6')-Ib, were analysed. Using these isolates, the bacterial killing of amikacin, gentamicin and tobramycin was assessed in static time-kill experiments. The pharmacodynamic activity of the aminoglycosides was then assessed in a dynamic one-compartment infection model over 72 h using simulated human pharmacokinetics of once-daily dosing with amikacin (15 mg/kg), gentamicin (5 mg/kg) and tobramycin (5 mg/kg). RESULTS: At clinically relevant aminoglycoside concentrations in time-kill experiments and the dynamic one-compartment model, gentamicin was more active than amikacin or tobramycin against the isolates harbouring aac(6')-Ib. Amikacin, gentamicin and tobramycin all showed progressively reduced bacterial killing with exposure to repeated doses against most isolates in the dynamic one-compartment model. MIC values were generally not a good predictor of gentamicin pharmacodynamic activity against KPC-Kp, but were more reliable for amikacin and tobramycin. CONCLUSIONS: Gentamicin may be preferred over amikacin or tobramycin for treatment of KPC-Kp infections. However, gentamicin MICs are not a consistent predictor of its pharmacodynamic activity and unexpected treatment failures are possible.

Unraveling the Gentamicin Drug Product Complexity Reveals Variation in Microbiological Activities and Nephrotoxicity.

The gentamicin drug product is a complex mixture of numerous components, many of which have not individually undergone safety and efficacy assessments. This is in contrast to the majority of medicines that require rigorous characterizations of trace impurities and are dosed as single components. In gentamicin, four components, known as gentamicin congeners C1, C1a, C2, and C2a, comprise the majority of the mixture. A liquid chromatography-mass spectroscopy analysis revealed that the relative abundances of each gentamicin congener in commercial formulations can vary up to 1.9-fold depending on the commercial source of the gentamicin. To determine if the gentamicin used for antibiotic susceptibility testing (AST) would be predictive of the microbiological activity of the product used to dose patients, the relative abundances of the four congeners contained on commercial AST disks were measured. It was found that the congener abundances on the commercial AST disks varied up to 4.1-fold. After purification of the four gentamicin congeners, similar potencies against bacterial strains lacking aminoglycoside-modifying enzymes (AMEs) were observed. However, the potency of the congeners against strains harboring a common AME differed up to 128-fold. Nephrotoxicity of the individual gentamicin congeners also differed significantly in cell-based and repeat-dose rat nephrotoxicity studies. Variations in the composition of commercial gentamicin products combined with toxicity differences between gentamicin congeners suggest that some gentamicin formulations may be more nephrotoxic. Our results also raise the concern that gentamicin susceptibility test results may not be predictive of patient outcomes and could lead to unexpected clinical treatment failures.

Inoculum effect of ß-lactam antibiotics.

The phenomenon of attenuated antibacterial activity at inocula above those utilized for susceptibility testing is referred to as the inoculum effect. Although the inoculum effect has been reported for several decades, it is currently debatable whether the inoculum effect is clinically significant. The aim of the present review was to consolidate currently available evidence to summarize which ß-lactam drug classes demonstrate an inoculum effect against specific bacterial pathogens. Review of the literature showed that the majority of studies that evaluated the inoculum effect of ß-lactams were in vitro investigations of Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Haemophilus influenzae and Staphylococcus aureus. Across all five pathogens, cephalosporins consistently displayed observable inoculum effects in vitro, whereas carbapenems were less susceptible to an inoculum effect. A handful of animal studies were available that validated that the in vitro inoculum effect translates into attenuated pharmacodynamics of ß-lactams in vivo. Only a few clinical investigations were available and suggested that an in vitro inoculum effect of cefazolin against MSSA may correspond to an increased likeliness of adverse clinical outcomes in patients receiving cefazolin for bacteraemia. The presence of ß-lactamase enzymes was the primary mechanism responsible for an inoculum effect, but the observation of an inoculum effect in multiple pathogens lacking ß-lactamase enzymes indicates that there are likely multiple mechanisms that may result in an inoculum effect. Further clinical studies are needed to better define whether interventions made in the clinic in response to organisms displaying an in vitro inoculum effect will optimize clinical outcomes.

Rational Combinations of Polymyxins with Other Antibiotics.

Combinations of antimicrobial agents are often used in the management of infectious diseases. Antimicrobial agents used as part of combination therapy are often selected empirically. As regrowth and the emergence of polymyxin (either colistin or polymyxin B) resistance has been observed with polymyxin monotherapy, polymyxin combination therapy has been suggested as a possible means by which to increase antimicrobial activity and reduce the development of resistance. This chapter provides an overview of preclinical and clinical investigations of CMS/colistin and polymyxin B combination therapy. In vitro data and animal model data suggests a potential clinical benefit with many drug combinations containing clinically achievable concentrations of polymyxins, even when resistance to one or more of the drugs in combination is present and including antibiotics normally inactive against Gram-negative organisms. The growing body of data on the emergence of polymyxin resistance with monotherapy lends theoretical support to a benefit with combination therapy. Benefits include enhanced bacterial killing and a suppression of polymyxin resistant subpopulations. However, the complexity of the critically ill patient population, and high rates of treatment failure and death irrespective of infection-related outcome make demonstrating a potential benefit for polymyxin combinations extremely challenging. Polymyxin combination therapy in the clinic remains a heavily debated and controversial topic. When combinations are selected, optimizing the dosage regimens for the polymyxin and the combinatorial agent is critical to ensure that the benefits outweigh the risk of the development of toxicity. Importantly, patient characteristics, pharmacokinetics, the site of infection, pathogen and resistance mechanism must be taken into account to define optimal and rational polymyxin combination regimens in the clinic.

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.

High-Dose Ampicillin-Sulbactam Combinations Combat Polymyxin-Resistant Acinetobacter baumannii in a Hollow-Fiber Infection Model.

Acinetobacter baumannii is emerging with resistance to polymyxins. In 24-h time-kill experiments, high-dose ampicillin-sulbactam in combination with meropenem and polymyxin B achieved additivity or synergy against 108 CFU/ml of two clinical A. baumannii isolates resistant to all three drugs (maximum reductions, 1.6 and 3.1 logs). In a 14-day hollow-fiber infection model, high-dose ampicillin-sulbactam (8/4 g every 8 h, respectively) in combination with meropenem (2 g every 8 h) and polymyxin B (1.43 mg/kg of body weight every 12 h with loading dose) resulted in rapid (96 h) eradication of A. baumannii.

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).

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.

Pharmacodynamics of colistin and fosfomycin: a 'treasure trove' combination combats KPC-producing Klebsiella pneumoniae.

Objectives: KPC-producing Klebsiella pneumoniae are an emerging public health problem around the globe. We defined the combinatorial pharmacodynamics and ability to suppress resistance of two 'old' antibiotics, fosfomycin and colistin, in time-kill experiments and hollow-fibre infection models (HFIM). Methods: Two KPC-2-producing K. pneumoniae isolates were used: one susceptible to both colistin and fosfomycin (KPC 9A: MIC colistin 0.25 mg/L and MIC fosfomycin ≤8 mg/L) and the other resistant to colistin and susceptible to fosfomycin (KPC 5A: MIC colistin 64 mg/L and MIC fosfomycin 32 mg/L). Time-kill experiments assessed an array of colistin and fosfomycin concentrations against both isolates. Colistin and fosfomycin pharmacokinetics from critically ill patients were simulated in the HFIM to define the pharmacodynamic activity of humanized regimens over 5 days against KPC 9A. Results: In time-kill experiments, synergy was demonstrated for all colistin/fosfomycin combinations containing >8 mg/L fosfomycin against the double-susceptible KPC strain, 9A. Synergy versus KPC strain 5A was only achieved at the highest concentrations of colistin (4 mg/L) and fosfomycin (512 mg/L) at 48 h. In the HFIM, colistin or fosfomycin monotherapies resulted in rapid proliferation of resistant subpopulations; KPC 9A regrew by 24 h. In contrast to the monotherapies, the colistin/fosfomycin combination resulted in a rapid 6.15 log 10 cfu/mL reduction of KPC 9A by 6 h and complete suppression of resistant subpopulations until 120 h. Conclusions: Colistin and fosfomycin may represent an important treatment option for KPC-producing K. pneumoniae otherwise resistant to traditional antibiotics.

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.

Sequential Evolution of Vancomycin-Intermediate Resistance Alters Virulence in Staphylococcus aureus: Pharmacokinetic/Pharmacodynamic Targets for Vancomycin Exposure.

Staphylococcus aureus possesses exceptional virulence and a remarkable ability to adapt in the face of antibiotic therapy. We examined the in vitro evolution of S. aureus in response to escalating vancomycin exposure by evaluating bacterial killing and the progression of resistance. A hollow-fiber infection model was utilized to simulate human doses of vancomycin increasing from 0.5 to 4 g every 12 h (q12h) versus a high inoculum (10(8) CFU/ml) of methicillin-resistant S. aureus (MRSA) USA300 and USA400. Host-pathogen interactions using Galleria mellonella and accessory gene regulator (agr) expression were studied in serially obtained isolates. In both USA300 and USA400 MRSA isolates, vancomycin exposure up to 2 g q12h resulted in persistence and regrowth, whereas 4 g administered q12h achieved sustained killing against both strains. As vancomycin exposure increased from 0.5 to 2 g q12h, the bacterial population shifted toward vancomycin-intermediate resistance, and collateral increases in the MICs of daptomycin and televancin were observed over 10 days. Guideline-recommended exposure of a ratio of the area under the concentration-time curve for the free, unbound fraction of the drug to the MIC (fAUC/MIC ratio) of 200 displayed a 0.344-log bacterial reduction in area, whereas fAUC/MICs of 371 and 554 were needed to achieve 1.00- and 2.00-log reductions in area, respectively. The stepwise increase in resistance paralleled a decrease in G. mellonella mortality (P = 0.021) and a gradual decline of RNAIII expression over 10 days. Currently recommended doses of vancomycin resulted in amplification of resistance and collateral damage to other antibiotics. Decreases in agr expression and virulence during therapy may be an adaptive mechanism of S. aureus persistence.

Evolution of Staphylococcus aureus under vancomycin selective pressure: the role of the small-colony variant phenotype.

Staphylococcus aureus small-colony variants (SCVs) often persist despite antibiotic therapy. Against a 10(8)-CFU/ml methicillin-resistant S. aureus (MRSA) (strain COL) population of which 0%, 1%, 10%, 50%, or 100% was an isogenic hemB knockout (Ia48) subpopulation displaying the SCV phenotype, vancomycin achieved maximal reductions of 4.99, 5.39, 4.50, 3.28, and 1.66 log10 CFU/ml over 48 h. Vancomycin at ≥16 mg/liter shifted a population from 50% SCV cells at 0 h to 100% SCV cells at 48 h, which was well characterized by a Hill-type model (R2>0.90).

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.

Assessment of antibiotic release and antibacterial efficacy from pendant glutathione hydrogels using ex vivo porcine skin.

Acute bacterial skin and skin structure infections (ABSSSIs) confer a substantial burden on the healthcare system. Local antibiotic delivery systems can provide controlled drug release directly to the site of infection to maximize efficacy and minimize systemic toxicity. The purpose of this study was to examine the antibacterial activity of antibiotic-loaded glutathione-conjugated poly(ethylene glycol) hydrogels (GSH-PEG) against ABSSSIs utilizing an ex vivo porcine dermal explant model. Vancomycin- or meropenem-loaded GSH-PEG hydrogels at 3 different dose levels were loaded over 1 h. Drug release was monitored in vitro under submerged conditions, by the Franz cell diffusion method, and ex vivo utilizing a porcine dermis model. Antibacterial activity was assessed ex vivo on porcine dermis explants inoculated with Staphylococcus aureus or Pseudomonas aeruginosa isolates treated with vancomycin- or meropenem-loaded GSH-PEG hydrogels, respectively. Histological assessment of the explants was conducted to evaluate tissue integrity and viability in the context of the experimental conditions. A dose-dependent release was observed from vancomycin and meropenem hydrogels, with in vitro Franz cell diffusion data closely representing ex vivo vancomycin release, but not high dose meropenem release. High dose vancomycin-loaded hydrogels resulted in a >3 log10 clearance against all S. aureus isolates at 48 h. High dose meropenem-loaded hydrogels achieved 6.5, 4, and 2 log10 reductions in CFU/ml against susceptible, intermediate, and resistant P. aeruginosa isolates, respectively. Our findings demonstrate the potential application of GSH-PEG hydrogels for flexible, local antibiotic delivery against bacterial skin infections.