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.

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.

Combating Multidrug-Resistant Bacteria by Integrating a Novel Target Site Penetration and Receptor Binding Assay Platform Into Translational Modeling.

Multidrug-resistant bacteria are causing a serious global health crisis. A dramatic decline in antibiotic discovery and development investment by pharmaceutical industry over the last decades has slowed the adoption of new technologies. It is imperative that we create new mechanistic insights based on latest technologies, and use translational strategies to optimize patient therapy. Although drug development has relied on minimal inhibitory concentration testing and established in vitro and mouse infection models, the limited understanding of outer membrane permeability in Gram-negative bacteria presents major challenges. Our team has developed a platform using the latest technologies to characterize target site penetration and receptor binding in intact bacteria that inform translational modeling and guide new discovery. Enhanced assays can quantify the outer membrane permeability of ß-lactam antibiotics and ß-lactamase inhibitors using multiplex liquid chromatography tandem mass spectrometry. While ß-lactam antibiotics are known to bind to multiple different penicillin-binding proteins (PBPs), their binding profiles are almost always studied in lysed bacteria. Novel assays for PBP binding in the periplasm of intact bacteria were developed and proteins identified via proteomics. To characterize bacterial morphology changes in response to PBP binding, high-throughput flow cytometry and time-lapse confocal microscopy with fluorescent probes provide unprecedented mechanistic insights. Moreover, novel assays to quantify cytosolic receptor binding and intracellular drug concentrations inform target site occupancy. These mechanistic data are integrated by quantitative and systems pharmacology modeling to maximize bacterial killing and minimize resistance in in vitro and mouse infection models. This translational approach holds promise to identify antibiotic combination dosing strategies for patients with serious infections.

First Penicillin-Binding Protein Occupancy Patterns for 15 ß-Lactams and ß-Lactamase Inhibitors in Mycobacterium abscessus.

Mycobacterium abscessus causes serious infections that often require over 18 months of antibiotic combination therapy. There is no standard regimen for the treatment of M. abscessus infections, and the multitude of combinations that have been used clinically have had low success rates and high rates of toxicities. With ß-lactam antibiotics being safe, double ß-lactam and ß-lactam/ß-lactamase inhibitor combinations are of interest for improving the treatment of M. abscessus infections and minimizing toxicity. However, a mechanistic approach for building these combinations is lacking since little is known about which penicillin-binding protein (PBP) target receptors are inactivated by different ß-lactams in M. abscessus We determined the preferred PBP targets of 13 ß-lactams and 2 ß-lactamase inhibitors in two M. abscessus strains and identified PBP sequences by proteomics. The Bocillin FL binding assay was used to determine the ß-lactam concentrations that half-maximally inhibited Bocillin binding (50% inhibitory concentrations [IC50s]). Principal component analysis identified four clusters of PBP occupancy patterns. Carbapenems inactivated all PBPs at low concentrations (0.016 to 0.5 mg/liter) (cluster 1). Cephalosporins (cluster 2) inactivated PonA2, PonA1, and PbpA at low (0.031 to 1 mg/liter) (ceftriaxone and cefotaxime) or intermediate (0.35 to 16 mg/liter) (ceftazidime and cefoxitin) concentrations. Sulbactam, aztreonam, carumonam, mecillinam, and avibactam (cluster 3) inactivated the same PBPs as cephalosporins but required higher concentrations. Other penicillins (cluster 4) specifically targeted PbpA at 2 to 16 mg/liter. Carbapenems, ceftriaxone, and cefotaxime were the most promising ß-lactams since they inactivated most or all PBPs at clinically relevant concentrations. These first PBP occupancy patterns in M. abscessus provide a mechanistic foundation for selecting and optimizing safe and effective combination therapies with ß-lactams.

Biochemical and Molecular Characterization of Five Bacillus Isolates Displaying Remarkable Carboxymethyl Cellulase Activities.

Cellulases have many useful applications in industry and biotechnology. So, identification of new bacterial strains expressing cellulases with better properties is desired. Five soil bacterial strains screened for high carboxymethyl cellulase (CMCase) activities were characterized and identified by 16S rRNA analysis as Bacillus amyloliquefaciens (FAY088), B. velezensis (FAY0103), B. tequilensis (FAY0117), B. subtilis (FAY0136), and B. subtilis (FAY0182). Their CMCase activities were 1.49, 1.26, 1.21, 1.21, and 1.24 U/ml, respectively. The maximum CMCase production was attained by growth at 35 °C, pH 6, and 180 rpm for 5 days. Residual activities of CMCases from FAY088 and FAY0117 were 88% or more after growth at 40 °C, which is same as FAY0182 CMCase at 40 and 45 °C. Additionally, FAY0182 retained 73% residual activity at 50 °C. FAY088 and FAY0182 retained more than 85% at pH 7 and 8. Conversely, residual activities from FAY0103 and FAY0136 declined a lot by increasing growth temperature beyond 40 °C and pH beyond 7. The maximum CMCase stability in all isolates was observed at pH 7, 3-h incubation, and 40 °C except for FAY0103 CMCase showed optimum temperature at 30 °C. More than 70% CMCase stability was retained in case of FAY088 at 50 °C, FAY0117 at 50-70 °C, and FAY0136 at 50-60 °C. FAY088 CMCase seemed to be the lest sensitive to temperature variation as it displayed residual activities 67, 72, 78, 84, 77, 74, and 72% at pH 3, 4, 5, 6, 8, 9, and 10, respectively. Finally, the five CMCase-producing isolates are recommended further enzyme applications in biotechnology and industry.