Comprehensive stability analysis of 13 ß-lactams and ß-lactamase inhibitors in in vitro media, and novel supplement dosing strategy to mitigate thermal drug degradation.

Non-clinical antibiotic development relies on in vitro susceptibility and infection model studies. Validating the achievement of the targeted drug concentrations is essential to avoid under-estimation of drug effects and over-estimation of resistance emergence. While certain ß-lactams (e.g., imipenem) and ß-lactamase inhibitors (BLIs; clavulanic acid) are believed to be relatively unstable, limited tangible data on their stability in commonly used in vitro media are known. We aimed to determine the thermal stability of 10 ß-lactams and 3 BLIs via LC-MS/MS in cation-adjusted Mueller Hinton broth at 25 and 36°C as well as agar at 4 and 37°C, and in water at -20, 4, and 25°C. Supplement dosing algorithms were developed to achieve broth concentrations close to their target over 24 h. During incubation in broth (pH 7.25)/agar, degradation half-lives were 16.9/21.8 h for imipenem, 20.7/31.6 h for biapenem, 29.0 h for clavulanic acid (studied in broth only), 23.1/71.6 h for cefsulodin, 40.6/57.9 h for doripenem, 46.5/64.6 h for meropenem, 50.8/97.7 h for cefepime, 61.5/99.5 h for piperacillin, and >120 h for all other compounds. Broth stability decreased at higher pH. All drugs were ≥90% stable for 72 h in agar at 4°C. Degradation half-lives in water at 25°C were >200 h for all drugs except imipenem (14.7 h, at 1,000 mg/L) and doripenem (59.5 h). One imipenem supplement dose allowed concentrations to stay within ±31% of their target concentration. This study provides comprehensive stability data on ß-lactams and BLIs in relevant in vitro media using LC-MS/MS. Future studies are warranted applying these data to antimicrobial susceptibility testing and assessing the impact of ß-lactamase-related degradation.

Successful cure of daptomycin-non-susceptible, vancomycin-intermediate Staphylococcus aureus prosthetic aortic valve endocarditis directed by synergistic in vitro time-kill study.

Infectious complications following surgical valve replacements are extremely difficult to treat, often requiring prolonged antimicrobials therapy with or without surgery. Vancomycin-intermediate Staphylococcus aureus is an infrequent pathogen, with an estimated prevalence of less than 0.3%, but presents even greater challenges. We report a case of successful cure of daptomycin-non-susceptible and vancomycin-intermediate Staphylococcus aureus prosthetic valve endocarditis using an eight-week course of combination antimicrobial therapy. Using time-kill study, the combination of daptomycin plus ceftaroline and rifampin resulted in a greater than 4 log reduction of bacterial growth at 24 hours. This antimicrobial combination was used for a total of eight weeks with a successful outcome.

Native valve Proteus mirabilis endocarditis: successful treatment of a rare entity formulated by in vitro synergy antibiotic testing.

Infective endocarditis caused by Proteus mirabilis is a rare and poorly reported disease, with no well-defined effective antibiotic regimen. Here, we present a case of P. mirabilis aortic valve endocarditis. We reviewed prior cases and treatment regimens, and devised effective treatment, which was guided by in vitro sensitivity and synergy testing on the pathogen. Our patient survived without complications or the need for a surgical intervention.

Framework for optimisation of the clinical use of colistin and polymyxin B: the Prato polymyxin consensus.

In the face of diminishing therapeutic options for the treatment of infections caused by multidrug-resistant, Gram-negative bacteria, clinicians are increasingly using colistin and polymyxin B. These antibiotics became available clinically in the 1950s, when understanding of antimicrobial pharmacology and regulatory requirements for approval of drugs was substantially less than today. At the 1st International Conference on Polymyxins in Prato, Italy, 2013, participants discussed a set of key objectives that were developed to explore the factors affecting the safe and effective use of polymyxins, identify the gaps in knowledge, and set priorities for future research. Participants identified several factors that affect the optimum use of polymyxins, including: confusion caused by several different conventions used to describe doses of colistin; an absence of appropriate pharmacopoeial standards for polymyxins; outdated and diverse product information; and uncertainties about susceptibility testing and breakpoints. High-priority areas for research included: better definition of the effectiveness of polymyxin-based combination therapy compared with monotherapy via well designed, randomised controlled trials; examination of the relative merits of colistin versus polymyxin B for various types of infection; investigation of pharmacokinetics in special patient populations; and definition of the role of nebulised polymyxins alone or in combination with intravenous polymyxins for the treatment of pneumonia. The key areas identified provide a roadmap for action regarding the continued use of polymyxins, and are intended to help with the effective and safe use of these important, last-line antibiotics.

Predictors of agr dysfunction in methicillin-resistant Staphylococcus aureus (MRSA) isolates among patients with MRSA bloodstream infections.

Despite emerging evidence that dysfunction in the accessory gene regulator (agr) locus is associated with deleterious outcomes among patients treated with vancomycin for methicillin-resistant Staphylococcus aureus (MRSA) infections, factors predictive of agr dysfunction have not been evaluated. This study describes the epidemiology of agr dysfunction, identifies predictors of agr dysfunction in MRSA isolates among those with MRSA bloodstream infections, and describes the relationship between agr dysfunction and other microbiologic phenotypes. A cross-sectional study of patients with MRSA bloodstream infections at two institutions in upstate New York was performed. Clinical data on demographics, comorbidities, disease severity, hospitalization history, and antibiotic history were collected. Microbiologic phenotypes, including agr dysfunction, MIC values by broth microdilution (BMD) and Etest, and vancomycin heteroresistance (hVISA) were tested. Multivariable analyses were performed to identify factors predictive of agr dysfunction. Among 200 patients with an MRSA bloodstream infection, the proportion of strains with agr dysfunction was 31.5%. The distribution of MICs determined by both BMD and Etest were equivalent across agr groups, and there was no association between agr dysfunction and the presence of hVISA. Severity of illness, comorbidities, and hospitalization history were comparable between agr groups. In the multivariate analysis, prior antibiotic exposure was the only factor of variables studied found to be predictive of agr dysfunction. This relationship was predominantly driven by prior beta-lactam and fluoroquinolone administration in the bivariate analysis. Identifying these institution-specific risk factors can be used to develop a process to assess the risk of agr dysfunction and guide empirical antibiotic therapy decisions.

Short-course gentamicin in combination with daptomycin or vancomycin against Staphylococcus aureus in an in vitro pharmacodynamic model with simulated endocardial vegetations.

The ability to maximize bactericidal activity while minimizing toxicity is a therapeutic goal in the treatment of infective endocarditis. We evaluated the impact of administering short-course regimens of gentamicin in combination with daptomycin or vancomycin against one methicillin-susceptible (MSSA 1199) and one methicillin-resistant (MRSA 494) Staphylococcus aureus isolate using an in vitro pharmacodynamic model with simulated endocardial vegetations over 96 h. Human therapeutic dosing regimens for daptomycin (6 and 8 mg/kg of body weight), vancomycin, and gentamicin were simulated. Short-course combination regimens involving gentamicin were administered either as a single 5-mg/kg dose or three 1-mg/kg doses for only the first 24 h and compared to the regimens administered for the full 96-h duration. For all experiments, physiologic conditions of albumin, calcium, and pH were simulated. Both regimens of daptomycin achieved 99.9% kill by 32 h and maintained bactericidal activity against both isolates, which was significantly different from vancomycin, which displayed bacteriostatic activity (P < 0.05). The effects of all short-course regimens of gentamicin were equal to those of the full-duration regimens in combination with daptomycin. Adding three doses of gentamicin (1 mg/kg) to daptomycin resulted in enhancement and bactericidal activity at 24 h against both MRSA and MSSA. The addition of a single dose of gentamicin (5 mg/kg) enhanced or improved the activity of daptomycin and resulted in early bactericidal activity at 4 h against both isolates. The addition of three doses of gentamicin (1 mg/kg) did not improve the activity of vancomycin. However, the addition of a single 5-mg/kg dose of gentamicin to vancomycin resulted in early enhancement at 4 h and 99.9% kill at 32 h for MRSA. These results suggest that a single high dose of gentamicin in combination with daptomycin or vancomycin may be of utility to maximize synergistic and bactericidal activity and minimize toxicity. Further investigation is warranted.