Mechanisms of gelofusine protection in an in vitro model of polymyxin B-associated renal injury.

Polymyxins are a last-resort treatment option for multidrug-resistant gram-negative bacterial infections, but they are associated with nephrotoxicity. Gelofusine was previously shown to reduce polymyxin-associated kidney injury in an animal model. However, the mechanism(s) of renal protection has not been fully elucidated. Here, we report the use of a cell culture model to provide insights into the mechanisms of renal protection. Murine epithelial proximal tubular cells were exposed to polymyxin B. Cell viability, lactate dehydrogenase (LDH) release, polymyxin B uptake, mitochondrial superoxide production, nuclear morphology, and apoptosis activation were evaluated with or without concomitant gelofusine. A megalin knockout cell line was used as an uptake inhibition control. Methionine was included in selected experiments as an antioxidant control. A polymyxin B concentration-dependent reduction in cell viability was observed. Increased viability was observed in megalin knockout cells following comparable polymyxin B exposures. Compared with polymyxin B exposure alone, concomitant gelofusine significantly increased cell viability as well as reduced LDH release, polymyxin B uptake, mitochondrial superoxide, and apoptosis. Gelofusine and methionine were more effective at reducing renal cell injury in combination than either agent alone. In conclusion, the mechanisms of renal protection by gelofusine involve decreasing cellular drug uptake, reducing subsequent oxidative stress and apoptosis activation. These findings would be valuable for translational research into clinical strategies to attenuate drug-associated acute kidney injury.NEW & NOTEWORTHY Gelofusine is a gelatinous saline solution with the potential to attenuate polymyxin-associated nephrotoxicity. We demonstrated that the mechanisms of gelofusine renal protection involve reducing polymyxin B uptake by proximal tubule cells, limiting subsequent oxidative stress and apoptosis activation. In addition, gelofusine was more effective at reducing cellular injury than a known antioxidant control, methionine, and a megalin knockout cell line, indicating that gelofusine likely has additional pharmacological properties besides only megalin inhibition.

The emergence of cefiderocol resistance in Pseudomonas aeruginosa from a heteroresistant isolate during prolonged therapy.

Cefiderocol is a siderophore cephalosporin designed to target multi-drug-resistant Gram-negative bacteria. Previously, the emergence of cefiderocol non-susceptibility has been associated with mutations in the chromosomal cephalosporinase (PDC) along with mutations in the PirA and PiuA/D TonB-dependent receptor pathways. Here, we report a clinical case of cefiderocol-resistant P. aeruginosa that emerged in a patient during treatment. This resistance was associated with mutations not previously reported, suggesting potential novel pathways to cefiderocol resistance.

Zileuton ameliorates aminoglycoside and polymyxin-associated acute kidney injury in an animal model.

OBJECTIVES: Aminoglycosides and polymyxins are antibiotics with in vitro activity against MDR Gram-negative bacteria. However, their clinical use is hindered by dose-limiting nephrotoxicity. The objective of this project was to determine if zileuton can reduce nephrotoxicity associated with amikacin and polymyxin B in a rat model of acute kidney injury. METHODS: Sprague Dawley rats (n = 10, both genders) were administered either amikacin (300 mg/kg) or polymyxin B (20 mg/kg) daily for 10 days. Zileuton (4 and 10 mg/kg) was delivered intraperitoneally 15 min before antibiotic administration. Blood samples were collected at baseline and daily to determine serum creatinine concentration. Nephrotoxicity was defined as a ≥2× elevation of baseline serum creatinine. Time-to-event analysis and log rank test were used to compare the onset of nephrotoxicity in different cohorts. Histopathological analysis was also conducted to characterize the extent of kidney injury. RESULTS: Animals receiving amikacin or polymyxin B alone had nephrotoxicity rates of 90% and 100%, respectively. The overall rate was reduced to 30% in animals receiving adjuvant zileuton. The onset of nephrotoxicity associated with amikacin and polymyxin B was also significantly delayed by zileuton at 4 and 10 mg/kg, respectively. Histopathology confirmed reduced kidney injury in animals receiving amikacin concomitant with zileuton. CONCLUSIONS: Our pilot data suggest that zileuton has the potential to attenuate nephrotoxicity associated with last-line antibiotics. This would allow these antibiotics to treat MDR Gram-negative bacterial infections optimally without dose-limiting constraints. Further studies are warranted to optimize drug delivery and dosing in humans.

Growth of Acinetobacter baumannii impacted by iron chelation.

Acinetobacter baumannii (AB) has become multidrug-resistant (MDR) in recent years, and, currently, there are limited effective treatment options. Nutrient metals (e.g. iron) are essential to the metabolic functions of AB. This study examined the impact of iron chelation on the growth of AB in vitro and in vivo. Susceptible and MDR-AB bloodstream isolates (n = 9) were recovered from different patients between 2011 and 2018. Clonal diversity was ascertained by Fourier-transform infrared spectroscopy. In vitro bacterial densities were measured over 20 h to determine growth profiles. Variable amounts of a chelating agent [deferiprone (DFP)] were added to create a concentration gradient. Galleria mellonella larvae were inoculated with an isolate, with and without DFP. Quantitative culture was used to ascertain the bacterial burden of aggregate larvae immediately and 4 h post-infection. Increasing concentrations of DFP caused a transient and concentration-dependent hindrance to in vitro growth, compared to the no-treatment group. In vivo bacterial burden immediately post-infection in both groups was comparable. After 4 h, the burden was much higher in the control group comparatively (8.7 and 6.7 log CFU g-1). These results support that micro-nutrient limitation has the potential of being a novel approach for treating high-risk infections due to MDR-AB.

Case Commentary: Novel Therapy for Multidrug-Resistant Acinetobacter baumannii Infection.

Acinetobacter baumannii is a pathogenic bacterium commonly associated with multidrug resistance. In this issue of Antimicrobial Agents and Chemotherapy, a challenging case of ventilator-associated pneumonia is presented in which bacteriophage therapy was used as a last resort treatment in combination with systemic antibiotics. The data are promising, and several key areas are highlighted for future research.

Optimal ceftazidime/avibactam dosing exposure against KPC-producing Klebsiella pneumoniae.

OBJECTIVES: Infections due to carbapenem-resistant Enterobacterales are considered urgent public health threats and often treated with a ß-lactam/ß-lactamase inhibitor combination. However, clinical treatment failure and resistance emergence have been attributed to inadequate dosing. We used a novel framework to provide insights of optimal dosing exposure of ceftazidime/avibactam. METHODS: Seven clinical isolates of Klebsiella pneumoniae producing different KPC variants were examined. Ceftazidime susceptibility (MIC) was determined by broth dilution using escalating concentrations of avibactam. The observed MICs were characterized as response to avibactam concentrations using an inhibitory sigmoid Emax model. Using the best-fit parameter values, %fT>MICi was estimated for various dosing regimens of ceftazidime/avibactam. A hollow-fibre infection model (HFIM) was subsequently used to ascertain the effectiveness of selected regimens over 120 h. The drug exposure threshold associated with bacterial suppression was identified by recursive partitioning. RESULTS: In all scenarios, ceftazidime MIC reductions were well characterized with increasing avibactam concentrations. In HFIM, bacterial regrowth over time correlated with emergence of resistance. Overall, suppression of bacterial regrowth was associated with %fT>MICi ≥ 76.1% (100% versus 18.2%; P < 0.001). Using our framework, the optimal drug exposure could be achieved with ceftazidime/avibactam 2.5 g every 12 h in 5 out of 7 isolates. Furthermore, ceftazidime/avibactam 2.5 g every 8 h can suppress an isolate deemed resistant based on conventional susceptibility testing method. CONCLUSIONS: An optimal drug exposure to suppress KPC-producing bacteria was identified. The novel framework is informative and may be used to guide optimal dosing of other ß-lactam/ß-lactamase inhibitor combinations. Further in vivo investigations are warranted.

Discerning in vitro pharmacodynamics from OD measurements: A model-based approach.

Time-kill experiments can discern the pharmacodynamics of infectious bacteria exposed to antibiotics in vitro, and thus help guide the design of effective therapies for challenging clinical infections. This task is resource-limited, therefore typically bypassed in favor of empirical shortcuts. The resource limitation could be addressed by continuously assessing the size of a bacterial population under antibiotic exposure using optical density measurements. However, such measurements count both live and dead cells and are therefore unsuitable for declining populations of live cells. To fill this void, we develop here a model-based method that infers the count of live cells in a bacterial population exposed to antibiotics from continuous optical-density measurements of both live and dead cells combined. The method makes no assumptions about the underlying mechanisms that confer resistance and is widely applicable. Use of the method is demonstrated by an experimental study on Acinetobacter baumannii exposed to levofloxacin.

New ß-Lactam-ß-Lactamase Inhibitor Combinations.

The limited armamentarium against drug-resistant Gram-negative bacilli has led to the development of several novel ß-lactam-ß-lactamase inhibitor combinations (BLBLIs). In this review, we summarize their spectrum of in vitro activities, mechanisms of resistance, and pharmacokinetic-pharmacodynamic (PK-PD) characteristics. A summary of available clinical data is provided per drug. Four approved BLBLIs are discussed in detail. All are options for treating multidrug-resistant (MDR) Enterobacterales and Pseudomonas aeruginosa Ceftazidime-avibactam is a potential drug for treating Enterobacterales producing extended-spectrum ß-lactamase (ESBL), Klebsiella pneumoniae carbapenemase (KPC), AmpC, and some class D ß-lactamases (OXA-48) in addition to carbapenem-resistant Pseudomonas aeruginosa Ceftolozane-tazobactam is a treatment option mainly for carbapenem-resistant P. aeruginosa (non-carbapenemase producing), with some activity against ESBL-producing Enterobacterales Meropenem-vaborbactam has emerged as treatment option for Enterobacterales producing ESBL, KPC, or AmpC, with similar activity as meropenem against P. aeruginosa Imipenem-relebactam has documented activity against Enterobacterales producing ESBL, KPC, and AmpC, with the combination having some additional activity against P. aeruginosa relative to imipenem. None of these drugs present in vitro activity against Enterobacterales or P. aeruginosa producing metallo-ß-lactamase (MBL) or against carbapenemase-producing Acinetobacter baumannii Clinical data regarding the use of these drugs to treat MDR bacteria are limited and rely mostly on nonrandomized studies. An overview on eight BLBLIs in development is also provided. These drugs provide various levels of in vitro coverage of carbapenem-resistant Enterobacterales, with several drugs presenting in vitro activity against MBLs (cefepime-zidebactam, aztreonam-avibactam, meropenem-nacubactam, and cefepime-taniborbactam). Among these drugs, some also present in vitro activity against carbapenem-resistant P. aeruginosa (cefepime-zidebactam and cefepime-taniborbactam) and A. baumannii (cefepime-zidebactam and sulbactam-durlobactam).

Optimizing pharmacokinetics/pharmacodynamics of ß-lactam/ß-lactamase inhibitor combinations against high inocula of ESBL-producing bacteria.

OBJECTIVES: Reduced in vitro ß-lactam activity against a dense bacterial population is well recognized. It is commonly attributed to the presence of ß-lactamase(s) and it is unknown whether the inoculum effect could be diminished by a ß-lactamase inhibitor. We evaluated different ß-lactam/ß-lactamase inhibitor combinations in suppressing a high inoculum of ESBL-producing bacteria. METHODS: Three clinical isolates expressing representative ESBLs (CTX-M-15 and SHV-12) were examined. The impact of escalating ß-lactamase inhibitor (tazobactam or avibactam) concentrations on ß-lactam (piperacillin or ceftazidime) MIC reduction was characterized by an inhibitory sigmoid Emax model. The effect of various dosing regimens of ß-lactam/ß-lactamase inhibitor combinations was predicted using %T>MICi and selected exposures were experimentally validated in a hollow-fibre infection model over 120 h. The threshold exposure to suppress bacterial regrowth was identified using recursive partitioning. RESULTS: A concentration-dependent reduction in ß-lactam MIC was observed (r2 ≥0.93). Regrowth could be suppressed in all six experiments using %T>MICi ≥73.6%, but only one out of six experiments below the threshold (P = 0.015). The exposures to suppress regrowth might be attained using the clinical dose of avibactam, but a much higher dose than the standard dose would be needed for tazobactam. CONCLUSIONS: A dense population of ESBL-producing bacteria could be suppressed by an optimized dosing regimen of selected ß-lactam/ß-lactamase inhibitor combinations. The reversibility of enzyme inhibition could play an important role in diminishing the inoculum effect. In vivo investigations to validate these findings are warranted.

SIMULTANEOUS IN VITRO SIMULATION OF MULTIPLE ANTIMICROBIAL AGENTS WITH DIFFERENT ELIMINATION HALF-LIVES IN A PRE-CLINICAL INFECTION MODEL.

Combination therapy for treatment of multi-drug resistant bacterial infections is becoming common. In vitro testing of drug combinations under realistic pharmacokinetic conditions is needed before a corresponding combination is eventually put into clinical use. The current standard for design of such in vitro simulations for drugs with different half-lives is heuristic and limited to two drugs. To address that void, we develop a rigorous design method suitable for an arbitrary number of N drugs with different half-lives. The method developed offers substantial flexibility and produces novel designs even for two drugs. Explicit design equations are rigorously developed and are suitable for immediate use by experimenters. These equations were used in experimental verification using a combination of three antibiotics with distinctly different half-lives. In addition to antibiotics, the method is applicable to any anti-infective or anti-cancer drugs with distinct elimination pharmacokinetics.

Optimizing Pharmacokinetics-Pharmacodynamics of Antimicrobial Management in Patients with Sepsis: A Review.

Critically ill patients with sepsis or septic shock are at an increased risk of death. Early and aggressive interventions are essential for improving clinical outcomes. There are a number of therapeutic and practical challenges in the management of antimicrobials in patients with sepsis. These include the timely selection and administration of appropriate antimicrobials, significant physiological alterations that can influence antimicrobial pharmacokinetics, and significant interpatient variability of antimicrobial concentrations using standard dosing approaches. Understanding the impact of these factors on the probability of attaining pharmacokinetic-pharmacodynamic target goals is essential to guide optimal therapy. Using rapid diagnostic technology could facilitate timely selection of antimicrobials, and therapeutic drug monitoring would provide a more individualized dosing approach. Using an interdisciplinary sepsis team would also be beneficial in coordinating efforts to overcome the challenges encountered during this critical period to ensure optimal care.

Characterization of Amikacin Drug Exposure and Nephrotoxicity in an Animal Model.

Despite excellent in vitro activity, aminoglycosides are used conservatively to treat multidrug-resistant bacterial infections due to their associated nephrotoxicity. Aminoglycosides are known to accumulate in the kidneys, but the quantitative relationship between drug exposures and nephrotoxicity is not well established. To bridge the knowledge gap, the objective of this study was to develop an animal model with clinically relevant conditions to mimic human disease progression. Single-dose pharmacokinetics were studied in Sprague-Dawley rats dosed either with 100 or 500 mg/kg of body weight of amikacin subcutaneously. Serial blood samples were collected, and serum amikacin concentrations were measured using liquid chromatography tandem mass spectrometry. Rats were also dosed with amikacin once daily for up to 10 days; blood samples were taken at baseline and daily to detect nephrotoxicity (defined as doubling of serum creatinine from baseline). Kidneys from both studies were harvested from selected rats, and amikacin concentrations in renal tissues were measured. A dose-dependent increase in systemic area under the curve (AUC) was observed, which ranged from approximately 1/3 (AUC of 53 mg·h/liter) to 3 times (AUC of 650 mg·h/liter) the expected exposure resulting from standard dosing in humans. Nephrotoxicity was significantly higher in rats given 500 mg/kg (100% versus 30%, P = 0.003). Kaplan-Meier analysis also showed a significant difference in nephrotoxicity onset between the two groups (P = 0.001). Finally, analysis of the renal tissues showed that the accumulation of amikacin could be associated with nephrotoxicity. These results are consistent with clinical observations, which support using this model in the future to investigate an intervention(s) that can be used clinically to alleviate nephrotoxicity.

Local Tissue Response to Subcutaneous Administration of Ceftriaxone in an Animal Model.

Subcutaneous administration is a novel way to deliver antibiotics for an infection, but intolerability has been reported. Evaluating the local tolerability of subcutaneously administered antibiotics is not standardized. The goal of this study was to develop an animal model to assess the subcutaneous administration of ceftriaxone. Sprague-Dawley rats were given daily subcutaneous injections for 12 days. The back of each animal was divided into 4 quadrants, with injections rotating each day among the quadrants. Ceftriaxone (1,000 mg/kg of body weight daily) was given in different concentrations and durations. Normal saline and potassium chloride solutions (2 meq/2 ml) were used as negative and positive controls, respectively. After the treatment course, skin samples were biopsied, and the local inflammatory response was assessed histologically using a semiquantitative scoring system. The histopathology scores were compared using a Kruskal-Wallis test. Injections with potassium chloride resulted in full-thickness skin necrosis with subcutaneous atrophy that was not seen in the saline-injected animals; inflammation of the muscular panniculus was observed, with various degrees of myocyte injury. Serosanguinous cavity formation in the subcutaneous compartment was observed when ceftriaxone (125 mg/ml) was given as a bolus injection, but the extent of the local tissue response was remarkably reduced when the same ceftriaxone dose was given at a lower concentration (25 mg/ml) over 120 min (P = 0.63, compared to saline controls). At a low concentration, ceftriaxone infusion was found to be well tolerated in this animal tissue necrosis model. If validated, the model could be an instrumental platform to evaluate different pharmaceutical formulations for subcutaneous delivery.

Optimal Piperacillin-Tazobactam Dosing Strategies against Extended-Spectrum-ß-Lactamase-Producing Enterobacteriaceae.

Piperacillin-tazobactam has been proposed as an alternative to carbapenems for the treatment of infections caused by extended-spectrum-ß-lactamase (ESBL)-producing Enterobacteriaceae However, limited understanding of optimal dosing strategies for this combination may curtail its utility. In this study, we correlated various exposures of piperacillin-tazobactam to efficacy, using a modified pharmacokinetic/pharmacodynamic index. Using a clinical Klebsiella pneumoniae isolate expressing CTX-M-15, piperacillin MIC values were determined with increasing tazobactam concentrations and fitted to a sigmoid inhibitory maximum effect (Emax) model. A hollow-fiber infection model (HFIM) was used to evaluate the efficacy of escalating tazobactam dosing with a fixed piperacillin exposure. Simulated drug concentrations from the HFIM were incorporated in the Emax model to determine the percentage of free time above instantaneous MIC (%fT>MICi) associated with each experimental exposure. The target %fT>MICi associated with growth suppression was prospectively validated using an SHV-12-producing isolate of Escherichia coli and 2 other CTX-M-15-producing K. pneumoniae isolates. Based on our reference isolate, piperacillin-tazobactam exposures of %fT>MICi of ≥55.1% were associated with growth suppression. Despite underlying differences, these findings were consistent with prospective observations in 3 other clinical isolates. Our modeling approach can be applied relatively easily in the clinical setting, and it appeared to be robust in predicting the effectiveness of various piperacillin-tazobactam exposures. This modified pharmacokinetic/pharmacodynamic index could be used to characterize response to other ß-lactam/ß-lactamase inhibitor combinations.

Generating Robust and Informative Nonclinical In Vitro and In Vivo Bacterial Infection Model Efficacy Data To Support Translation to Humans.

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.

Toxicity in Patients.

Polymyxin toxicity remains a significant concern that limits the clinical utility of this class of antibacterials for patient care. The most notable adverse event is the dose- and treatment-limiting nephrotoxicity that occurs in roughly 30-60% of patients receiving a systemic polymyxin. This chapter focuses on this adverse event with a detailed assessment of the incidence of, and risk factors for, polymyxin-associated nephrotoxicity. In particular, the text focuses on the impact of dose, serum concentrations, and polymyxin selection on nephrotoxicity. Additionally, less common, but clinically important adverse events are discussed.

Transcriptional profiles of pulmonary innate immune responses to isogenic antibiotic-susceptible and multidrug-resistant Pseudomonas aeruginosa.

The virulence of an isogenic pair of Pseudomonas aeruginosa strains was studied under similar experimental conditions in two animal infection models. The time to death was significantly longer for the multidrug resistant (MDR) than the wild-type strain. The transcriptional profiles of 84 innate immune response genes in the lungs of immune competent Balb/C mice were further compared. Significantly weaker expression of genes involved in production of soluble pattern recognition receptor and complement were observed in animals infected with the MDR strain. Altered patterns of innate immune system activation may explain the attenuated virulence in MDR bacteria.

Pharmacokinetics and Pharmacodynamics of Minocycline against Acinetobacter baumannii in a Neutropenic Murine Pneumonia Model.

Multidrug-resistant (MDR) Acinetobacter baumannii is increasingly more prevalent in nosocomial infections. Although in vitro susceptibility of A. baumannii to minocycline is promising, the in vivo efficacy of minocycline has not been well established. In this study, the in vivo activity of minocycline was evaluated in a neutropenic murine pneumonia model. Specifically, we investigated the relationship between minocycline exposure and bactericidal activity using five A. baumannii isolates with a broad range of susceptibility (MIC ranged from 0.25 mg/liter to 16 mg/liter). The pharmacokinetics of minocycline (single dose of 25 mg/kg of body weight, 50 mg/kg, 100 mg/kg, and a humanized regimen, given intraperitoneally) in serum and epithelial lining fluid (ELF) were characterized. Dose linearity was observed for doses up to 50 mg/kg and pulmonary penetration ratios (area under the concentration-time curve in ELF from 0 to 24 h [AUCELF,0-24]/area under the concentration time curve in serum from 0 to 24 h [AUCserum,0-24]) ranged from 2.5 to 2.8. Pharmacokinetic-pharmacodynamics (PK-PD) index values in ELF for various dose regimens against different A. baumannii isolates were calculated. The maximum efficacy at 24 h was approximately 1.5-log-unit reduction of pulmonary bacterial burdens from baseline. The AUC/MIC ratio was the PK-PD index most closely correlating to the bacterial burden (r2 = 0.81). The required AUCELF,0-24/MIC for maintaining stasis and achieving 1-log-unit reduction were 140 and 410, respectively. These findings could guide the treatment of infections caused by A. baumannii using minocycline in the future. Additional studies to examine resistance development during therapy are warranted.

Efficacy of Ceftaroline against Methicillin-Susceptible Staphylococcus aureus Exhibiting the Cefazolin High-Inoculum Effect in a Rat Model of Endocarditis.

Certain Staphylococcus aureus strains exhibit an inoculum effect (InE) with cefazolin (CFZ) that has been associated with therapeutic failures in high-inoculum infections. We assessed the in vitro activities of ceftaroline (CPT), CFZ, and nafcillin (NAF) against 17 type A ß-lactamase (ßla)-producing, methicillin-susceptible S. aureus (MSSA) strains, including the previously reported TX0117, which exhibits the CFZ InE, and its ßla-cured derivative, TX0117c. Additionally, we determined the pharmacokinetics of CPT in rats after single intramuscular doses of 20 and 40 mg/kg of body weight and evaluated the activities of CPT (40 mg/kg every 8 h [q8h]), CFZ, and NAF against TX0117 and TX0117c in a rat model of infective endocarditis. No InE was observed for CPT or NAF, whereas a marked InE was detected for CFZ (MIC, 8 to ≥128 µg/ml). CPT and NAF treatment against TX0117 resulted in mean bacterial counts of 2.3 and 2.1 log10 CFU/g in vegetations, respectively, compared to a mean of 5.9 log10 CFU/g in the CFZ-treated group (CPT and NAF versus CFZ, P = 0.001; CPT versus NAF, P = 0.9830). Both CFZ and CPT were efficacious against the ßla-cured derivative, TX0117c, compared to time zero (t0) (P = <0.0001 and 0.0015, respectively). Our data reiterate the in vivo consequences of the CFZ InE and show that CPT is not affected by this phenomenon. CPT might be considered for high-inoculum infections caused by MSSA exhibiting the CFZ InE.