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.

Distinguishing Inducible and Non-Inducible Resistance to Colistin in Pseudomonas aeruginosa by Quantitative and Systems Pharmacology Modeling at Low and Standard Inocula.

Colistin is a polymyxin and peptide antibiotic that can yield rapid bacterial killing, but also leads to resistance emergence. We aimed to develop a novel experimental and Quantitative and Systems Pharmacology approach to distinguish between inducible and non-inducible resistance. Viable count profiles for the total and less susceptible populations of Pseudomonas aeruginosa ATCC 27853 from static and dynamic in vitro infection models were simultaneously modeled. We studied low and normal initial inocula to distinguish between inducible and non-inducible resistance. A novel cutoff filter approach allowed us to describe the eradication and inter-conversion of bacterial populations. At all inocula, 4.84 mg/L of colistin (sulfate) yielded ≥4 log10 killing, followed by >4 log10 regrowth. A pre-existing, less susceptible population was present at standard but not at low inocula. Formation of a non-pre-existing, less susceptible population was most pronounced at intermediate colistin (sulfate) concentrations (0.9 to 5 mg/L). Both less susceptible populations inter-converted with the susceptible population. Simultaneously modeling of the total and less susceptible populations at low and standard inocula enabled us to identify the de novo formation of an inducible, less susceptible population. Inducible resistance at intermediate colistin concentrations highlights the importance of rapidly achieving efficacious polymyxin concentrations by front-loaded dosage regimens.

Penicillin-Binding Protein 5/6 Acting as a Decoy Target in Pseudomonas aeruginosa Identified by Whole-Cell Receptor Binding and Quantitative Systems Pharmacology.

The ß-lactam antibiotics have been successfully used for decades to combat susceptible Pseudomonas aeruginosa, which has a notoriously difficult to penetrate outer membrane (OM). However, there is a dearth of data on target site penetration and covalent binding of penicillin-binding proteins (PBP) for ß-lactams and ß-lactamase inhibitors in intact bacteria. We aimed to determine the time course of PBP binding in intact and lysed cells and estimate the target site penetration and PBP access for 15 compounds in P. aeruginosa PAO1. All ß-lactams (at 2 × MIC) considerably bound PBPs 1 to 4 in lysed bacteria. However, PBP binding in intact bacteria was substantially attenuated for slow but not for rapid penetrating ß-lactams. Imipenem yielded 1.5 ± 0.11 log10 killing at 1h compared to <0.5 log10 killing for all other drugs. Relative to imipenem, the rate of net influx and PBP access was ~ 2-fold slower for doripenem and meropenem, 7.6-fold for avibactam, 14-fold for ceftazidime, 45-fold for cefepime, 50-fold for sulbactam, 72-fold for ertapenem, ~ 249-fold for piperacillin and aztreonam, 358-fold for tazobactam, ~547-fold for carbenicillin and ticarcillin, and 1,019-fold for cefoxitin. At 2 × MIC, the extent of PBP5/6 binding was highly correlated (r2 = 0.96) with the rate of net influx and PBP access, suggesting that PBP5/6 acted as a decoy target that should be avoided by slowly penetrating, future ß-lactams. This first comprehensive assessment of the time course of PBP binding in intact and lysed P. aeruginosa explained why only imipenem killed rapidly. The developed novel covalent binding assay in intact bacteria accounts for all expressed resistance mechanisms.

Penicillin-Binding Protein Occupancy Dataset for 18 ß-Lactams and 4 ß-Lactamase Inhibitors in Neisseria gonorrhoeae.

The lack of effective first-line antibiotic treatments against Neisseria gonorrhoeae, and the worldwide dissemination of resistant strains, are the main drivers of a worsening global health crisis. ß-lactam antibiotics have been the backbone of therapeutic armamentarium against gonococci. However, we are lacking critical insights to design rationally optimized therapies. In the present work, we generated the first PBP-binding data set on 18 currently available and clinically relevant ß-lactams and 4 ß-lactamase inhibitors in two N. gonorrhoeae ATCC type collection strains, 19424 and 49226 (PBP2 type XXII and A39T change in mtrR). PBP binding (IC50) was determined via the Bocillin FL binding assay in isolated membrane preparations. Three clusters of differential PBP IC50s were identified and were mostly consistent across both strains, but with quantitative differences. Carbapenems were coselective for PBP2 and PBP3 (0.01 to 0.03 mg/L). Third- and fourth-generation cephalosporins cefixime, cefotaxime, ceftazidime, cefepime, and ceftriaxone showed the lowest IC50 values for PBP2 (0.01 mg/L), whereas cefoxitin, ceftaroline, and ceftolozane required higher concentrations (0.04 to >2 mg/L). Aztreonam was selective for PBP2 in both strains (0.03 to 0.07 mg/L); amdinocillin bound this PBP at higher concentrations (1.33 to 2.94 mg/L). Penicillins specifically targeted PBP2 in strain ATCC 19424 (0.02 to 0.19 mg/L) and showed limited inhibition in strain ATCC 49226 (0.01 to >2 mg/L). Preferential PBP2 binding was observed by ß-lactam-based ß-lactamase inhibitors sulbactam and tazobactam (1.07 to 6.02 mg/L); meanwhile, diazabicyclooctane inhibitors relebactam and avibactam were selective for PBP3 (1.27 to 5.40 mg/L). This data set will set the bar for future studies that will help the rational use and translational development of antibiotics against multidrug-resistant (MDR) N. gonorrhoeae. IMPORTANCE The manuscript represents the first N. gonorrhoeae PBP-binding data set for 22 chemically different drugs in two type strains with different genetic background. We have identified three clusters of drugs according to their PBP binding IC50s and highlighted the binding differences across the two strains studied. With the currently available genomic information and the PBP-binding data, we have been able to correlate the target attainment differences and the mutations that affect the drug uptake with the MIC changes. The results of the current work will allow us to develop molecular tools of great practical use for the study and the design of new rationally designed therapies capable of combating the growing MDR gonococci threat.

PBP Target Profiling by ß-Lactam and ß-Lactamase Inhibitors in Intact Pseudomonas aeruginosa: Effects of the Intrinsic and Acquired Resistance Determinants on the Periplasmic Drug Availability.

The lack of effective treatment options against Pseudomonas aeruginosa is one of the main contributors to the silent pandemic. Many antibiotics are ineffective against resistant isolates due to poor target site penetration, efflux, or ß-lactamase hydrolysis. Critical insights to design optimized antimicrobial therapies and support translational drug development are needed. In the present work, we analyzed the periplasmic drug uptake and binding to PBPs of 11 structurally different ß-lactams and 4 ß-lactamase inhibitors (BLIs) in P. aeruginosa PAO1. The contribution of the most prevalent ß-lactam resistance mechanisms to MIC and periplasmic target attainment was also assessed. Bacterial cultures (6.5 log10 CFU/mL) were exposed to 1/2× PAO1 MIC of each antibiotic for 30 min. Unbound PBPs were labeled with Bocillin FL and analyzed using a FluorImager. Imipenem extensively inactivated all targets. Cephalosporins preferentially targeted PBP1a and PBP3. Aztreonam and amdinocillin bound exclusively to PBP3 and to PBP2 and PBP4, respectively. Penicillins bound preferentially to PBP1a, PBP1b, and PBP3. BLIs displayed poor PBP occupancy. Inactivation of oprD elicited a notable reduction of imipenem target attainment, and it was to a lesser extent in the other carbapenems. Improved PBP occupancy was observed for the main targets of the widely used antipseudomonal penicillins, cephalosporins, meropenem, aztreonam, and amdinocillin upon oprM inactivation, in line with MIC changes. AmpC constitutive hyperexpression caused a substantial PBP occupancy reduction for the penicillins, cephalosporins, and aztreonam. Data obtained in this work will support the rational design of optimized ß-lactam-based combination therapies against resistant P. aeruginosa infections. IMPORTANCE The growing problem of antibiotic resistance in Gram-negative pathogens is linked to three key aspects, (i) the progressive worldwide epidemic spread of multidrug-resistant (MDR), extensively drug-resistant (XDR), and pandrug-resistant (PDR) Gram-negative strains, (ii) a decrease in the number of effective new antibiotics against multiresistant isolates, and (iii) the lack of mechanistically informed combinations and dosing strategies. Our combined efforts should focus not only on the development of new antimicrobial agents but the adequate administration of these in combination with other agents currently available in the clinic. Our work determined the effectiveness of these compounds in the clinically relevant bacteria Pseudomonas aeruginosa at the molecular level, assessing the net influx rate and their ability to access their targets and achieve bacterial killing without generating resistance. The data generated in this work will be helpful for translational drug development.

Comparative analysis of in vitro dynamics and mechanisms of ceftolozane/tazobactam and imipenem/relebactam resistance development in Pseudomonas aeruginosa XDR high-risk clones.

OBJECTIVES: To analyse the dynamics and mechanisms of stepwise resistance development to ceftolozane/tazobactam and imipenem/relebactam in XDR Pseudomonas aeruginosa clinical strains. METHODS: XDR clinical isolates belonging to ST111 (main resistance mechanisms: oprD-, dacB-, CARB-2), ST175 (oprD-, ampR-G154R) and ST235 (oprD-, OXA-2) high-risk clones were incubated for 24 h in Müeller-Hinton Broth with 0.125-64 mg/L of ceftolozane + tazobactam 4 mg/L or imipenem + relebactam 4 mg/L. Tubes from the highest antibiotic concentration showing growth were reinoculated into fresh medium containing concentrations up to 64 mg/L for 7 consecutive days. Two colonies per strain from each of the triplicate experiments were characterized by determining the susceptibility profiles, whole genome sequencing (WGS), and in vitro fitness through competitive growth assays. RESULTS: Resistance development occurred more slowly and reached a lower level for imipenem/relebactam than for ceftolozane/tazobactam in all tested XDR strains. Moreover, resistance development to imipenem/relebactam remained low even for ST175 isolates that had developed ceftolozane/tazobactam resistance during therapy. Lineages evolved in the presence of ceftolozane/tazobactam showed high-level resistance, imipenem/relebactam hypersusceptibility and low fitness cost, whereas lineages evolved in the presence of imipenem/relebactam showed moderate (borderline) resistance, no cross-resistance to ceftolozane/tazobactam and high fitness cost. WGS evidenced that ceftolozane/tazobactam resistance was mainly caused by mutations in the catalytic centres of intrinsic (AmpC) or acquired (OXA) ß-lactamases, whereas lineages evolved in imipenem/relebactam frequently showed structural mutations in MexB or in ParS, along with some strain-specific mutations. CONCLUSIONS: Imipenem/relebactam could be a useful alternative for the treatment of XDR P. aeruginosa infections, potentially reducing resistance development during therapy.

In vitro evolution of cefepime/zidebactam (WCK 5222) resistance in Pseudomonas aeruginosa: dynamics, mechanisms, fitness trade-off and impact on in vivo efficacy.

OBJECTIVES: To study the dynamics, mechanisms and fitness cost of resistance selection to cefepime, zidebactam and cefepime/zidebactam in Pseudomonas aeruginosa. METHODS: WT P. aeruginosa PAO1 and its ΔmutS derivative (PAOMS) were exposed to stepwise increasing concentrations of cefepime, zidebactam and cefepime/zidebactam. Selected mutants were characterized for change in susceptibility profiles, acquired mutations, fitness, virulence and in vivo susceptibility to cefepime/zidebactam. Mutations were identified through WGS. In vitro fitness was assessed by measuring growth in minimal medium and human serum-supplemented Mueller-Hinton broth. Virulence was determined in Caenorhabditis elegans and neutropenic mice lung infection models. In vivo susceptibility to a human-simulated regimen (HSR) of cefepime/zidebactam was studied in neutropenic mice lung infection. RESULTS: Resistance development was lower for the cefepime/zidebactam combination than for the individual components and high-level resistance was only achieved for PAOMS. Cefepime resistance development was associated with mutations leading to the hyperexpression of AmpC or MexXY-OprM, combined with PBP3 mutations and/or large chromosomal deletions involving galU. Zidebactam resistance was mainly associated with mutations in PBP2. On the other hand, resistance to cefepime/zidebactam required multiple mutations in genes encoding MexAB-OprM and its regulators, as well as PBP2 and PBP3. Cumulatively, these mutations inflicted significant fitness cost and cefepime/zidebactam-resistant mutants (MIC = 16-64 mg/L) remained susceptible in vivo to the HSR. CONCLUSIONS: Development of cefepime/zidebactam resistance in P. aeruginosa required multiple simultaneous mutations that were associated with a significant impairment of fitness and virulence.

Molecular Basis of AmpC ß-Lactamase Induction by Avibactam in Pseudomonas aeruginosa: PBP Occupancy, Live Cell Binding Dynamics and Impact on Resistant Clinical Isolates Harboring PDC-X Variants.

Avibactam belongs to the new class of diazabicyclooctane ß-lactamase inhibitors. Its inhibitory spectrum includes class A, C and D enzymes, including P. aeruginosa AmpC. Nonetheless, recent reports have revealed strain-dependent avibactam AmpC induction. In the present work, we wanted to assess the mechanistic basis underlying AmpC induction and determine if derepressed PDC-X mutated enzymes from ceftazidime/avibactam-resistant clinical isolates were further inducible. We determined avibactam concentrations that half-maximally inhibited (IC50) bocillin FL binding. Inducer ß-lactams were also studied as comparators. Live cells' time-course penicillin-binding proteins (PBPs) occupancy of avibactam was studied. To assess the ampC induction capacity of avibactam and comparators, qRT-PCR was performed in wild-type PAO1, PBP4, triple PBP4, 5/6 and 7 knockout derivatives and two ceftazidime/avibactam-susceptible/resistant XDR clinical isolates belonging to the epidemic high-risk clone ST175. PBP4 inhibition was observed for avibactam and ß-lactam comparators. Induction capacity was consistently correlated with PBP4 binding affinity. Outer membrane permeability-limited PBP4 binding was observed in the live cells' assay. As expected, imipenem and cefoxitin showed strong induction in PAO1, especially for carbapenem; avibactam induction was conversely weaker. Overall, the inducer effect was less remarkable in ampC-derepressed mutants and nonetheless absent upon avibactam exposure in the clinical isolates harboring mutated AmpC variants and their parental strains.

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.

In vitro dynamics and mechanisms of resistance development to imipenem and imipenem/relebactam in Pseudomonas aeruginosa.

OBJECTIVES: We analysed the dynamics and mechanisms of resistance development to imipenem alone or combined with relebactam in Pseudomonas aeruginosa WT (PAO1) and mutator (PAOMS; ΔmutS) strains. METHODS: PAO1 or PAOMS strains were incubated for 24 h in Mueller-Hinton Broth with 0.125-64 mg/L of imipenem ± relebactam 4 mg/L. Tubes from the highest antibiotic concentration showing growth were reinoculated in fresh medium containing concentrations up to 64 mg/L of imipenem ± relebactam for 7 days. Two colonies per strain, replicate experiment and antibiotic from early (Day 1) and late (Day 7) cultures were characterized by determining the susceptibility profiles, WGS and determination of the expression of ampC and efflux-pump-coding genes. Virulence was studied in a Caenorhabditis elegans infection model. RESULTS: Relebactam reduced imipenem resistance development for both strains, although resistance emerged much faster for PAOMS. WGS indicated that imipenem resistance was associated with mutations in the porin OprD and regulators of ampC, while the mutations in imipenem/relebactam-resistant mutants were located in oprD and regulatoras of MexAB-OprM. High-level imipenem/relebactam resistance was only documented in the PAOMS strain and was associated with an additional specific (T680A) mutation located in the catalytic pocket of ponA (PBP1a) and with reduced virulence in the C. elegans model. CONCLUSIONS: Imipenem/relebactam could be a useful alternative for the treatment of MDR P. aeruginosa infections, potentially reducing resistance development during treatment. Moreover, this work deciphers the potential resistance mechanisms that may emerge upon the introduction of this novel combination into clinical practice.

Effective inhibition of PBPs by cefepime and zidebactam in the presence of VIM-1 drives potent bactericidal activity against MBL-expressing Pseudomonas aeruginosa.

OBJECTIVES: The combination of cefepime and the novel ß-lactam enhancer zidebactam (WCK 5222) is under development for the treatment of difficult-to-treat Gram-negative infections. Against MBL-producing pathogens, cefepime and zidebactam induce cell elongation and spheroplast formation, indicating PBP3 and PBP2 dysfunction, respectively, having a potent bactericidal effect as a combination. The objective of the present study was to determine the mechanistic basis of the bactericidal effect of cefepime/zidebactam on MBL-expressing pathogens. METHODS: Pseudomonal PBP-binding affinities of cefepime, zidebactam and imipenem were assessed at different timepoints and also in the presence of purified VIM-1 using a Bocillin FL competition assay. The antibacterial activity of cefepime/zidebactam against three VIM-expressing Pseudomonas aeruginosa isolates was assessed by time-kill and neutropenic mouse lung/thigh infection studies. RESULTS: Amidst cefepime-hydrolysing concentrations of VIM-1, substantial cefepime binding to target PBPs was observed. High-affinity binding of zidebactam to PBP2 remained unaltered in the presence of VIM-1; however, MBL addition significantly affected imipenem PBP2 binding. Furthermore, the rate of cefepime binding to the primary target PBP3 was found to be higher compared with the imipenem PBP2 binding rate. Finally, complementary PBP inhibition by cefepime/zidebactam resulted in enhanced bactericidal activity in time-kill and neutropenic mouse lung/thigh infection studies against VIM-6-, VIM-10- and VIM-11-expressing P. aeruginosa, thus revealing the mechanistic basis of ß-lactam enhancer action. CONCLUSIONS: For the first time ever (to the best of our knowledge), this study demonstrates that in the presence of VIM-1 MBL, ß-lactamase-labile cefepime and ß-lactamase-stable zidebactam produce effective inhibition of respective target PBPs. For cefepime, this seems to be a result of a faster rate of PBP binding, which helps it overcome ß-lactamase-mediated hydrolysis.

Novel Cassette Assay To Quantify the Outer Membrane Permeability of Five ß-Lactams Simultaneously in Carbapenem-Resistant Klebsiella pneumoniae and Enterobacter cloacae.

Poor penetration through the outer membrane (OM) of Gram-negative bacteria is a major barrier of antibiotic development. While ß-lactam antibiotics are commonly used against Klebsiella pneumoniae and Enterobacter cloacae, there are limited data on OM permeability especially in K. pneumoniae Here, we developed a novel cassette assay, which can simultaneously quantify the OM permeability to five ß-lactams in carbapenem-resistant K. pneumoniae and E. cloacae Both clinical isolates harbored a blaKPC-2 and several other ß-lactamases. The OM permeability of each antibiotic was studied separately ("discrete assay") and simultaneously ("cassette assay") by determining the degradation of extracellular ß-lactam concentrations via multiplex liquid chromatography-tandem mass spectrometry analyses. Our K. pneumoniae isolate was polymyxin resistant, whereas the E. cloacae was polymyxin susceptible. Imipenem penetrated the OM at least 7-fold faster than meropenem for both isolates. Imipenem penetrated E. cloacae at least 258-fold faster and K. pneumoniae 150-fold faster compared to aztreonam, cefepime, and ceftazidime. For our ß-lactams, OM permeability was substantially higher in the E. cloacae compared to the K. pneumoniae isolate (except for aztreonam). This correlated with a higher OmpC porin production in E. cloacae, as determined by proteomics. The cassette and discrete assays showed comparable results, suggesting limited or no competition during influx through OM porins. This cassette assay allowed us, for the first time, to efficiently quantify the OM permeability of multiple ß-lactams in carbapenem-resistant K. pneumoniae and E. cloacae Characterizing the OM permeability presents a critical contribution to combating the antimicrobial resistance crisis and enables us to rationally optimize the use of ß-lactam antibiotics.IMPORTANCE Antimicrobial resistance is causing a global human health crisis and is affecting all antibiotic classes. While ß-lactams have been commonly used against susceptible isolates of Klebsiella pneumoniae and Enterobacter cloacae, carbapenem-resistant isolates are spreading worldwide and pose substantial clinical challenges. Rapid penetration of ß-lactams leads to high drug concentrations at their periplasmic target sites, allowing ß-lactams to more completely inactivate their target receptors. Despite this, there are limited tangible data on the permeability of ß-lactams through the outer membranes of many Gram-negative pathogens. This study presents a novel, cassette assay, which can simultaneously characterize the permeability of five ß-lactams in multidrug-resistant clinical isolates. We show that carbapenems, and especially imipenem, penetrate the outer membrane of K. pneumoniae and E. cloacae substantially faster than noncarbapenem ß-lactams. The ability to efficiently characterize the outer membrane permeability is critical to optimize the use of ß-lactams and combat carbapenem-resistant isolates.

Regulation of AmpC-Driven ß-Lactam Resistance in Pseudomonas aeruginosa: Different Pathways, Different Signaling.

The hyperproduction of the chromosomal AmpC ß-lactamase is the main mechanism driving ß-lactam resistance in Pseudomonas aeruginosa, one of the leading opportunistic pathogens causing nosocomial acute and chronic infections in patients with underlying respiratory diseases. In the current scenario of the shortage of effective antipseudomonal drugs, understanding the molecular mechanisms mediating AmpC hyperproduction in order to develop new therapeutics against this fearsome pathogen is of great importance. It has been accepted for decades that certain cell wall-derived soluble fragments (muropeptides) modulate AmpC production by complexing with the transcriptional regulator AmpR and acquiring different conformations that activate/repress ampC expression. However, these peptidoglycan-derived signals have never been characterized in the highly prevalent P. aeruginosa stable AmpC hyperproducer mutants. Here, we demonstrate that the previously described fragments enabling the transient ampC hyperexpression during cefoxitin induction (1,6-anhydro-N-acetylmuramyl-pentapeptides) also underlie the dacB (penicillin binding protein 4 [PBP4]) mutation-driven stable hyperproduction but differ from the 1,6-anhydro-N-acetylmuramyl-tripeptides notably overaccumulated in the ampD knockout mutant. In addition, a simultaneous greater accumulation of both activators appears linked to higher levels of AmpC hyperproduction, although our results suggest a much stronger AmpC-activating potency for the 1,6-anhydro-N-acetylmuramyl-pentapeptide. Collectively, our results propose a model of AmpC control where the activator fragments, with qualitative and quantitative particularities depending on the pathways and levels of ß-lactamase production, dominate over the repressor (UDP-N-acetylmuramyl-pentapeptide). This study represents a major step in understanding the foundations of AmpC-dependent ß-lactam resistance in P. aeruginosa, potentially useful to open new therapeutic conceptions intended to interfere with the abovementioned cell wall-derived signaling.IMPORTANCE The extensive use of ß-lactam antibiotics and the bacterial adaptive capacity have led to the apparently unstoppable increase of antimicrobial resistance, one of the current major global health challenges. In the leading nosocomial pathogen Pseudomonas aeruginosa, the mutation-driven AmpC ß-lactamase hyperproduction stands out as the main resistance mechanism, but the molecular cues enabling this system have remained elusive until now. Here, we provide for the first time direct and quantitative information about the soluble cell wall-derived fragments accounting for the different levels and pathways of AmpC hyperproduction. Based on these results, we propose a hierarchical model of signals which ultimately govern ampC hyperexpression and resistance.

Comparable Efficacy and Better Safety of Double ß-Lactam Combination Therapy versus ß­Lactam plus Aminoglycoside in Gram-Negative Bacteria in Randomized, Controlled Trials.

There is a great need for efficacious therapies against Gram-negative bacteria. Double ß-lactam combination(s) (DBL) are relatively safe, and preclinical data are promising; however, their clinical role has not been well defined. We conducted a metaanalysis of the clinical and microbiological efficacy of DBL compared to ß-lactam plus aminoglycoside combinations (BLAG). PubMed, Embase, ISI Web of Knowledge, and Cochrane Controlled Trials Register database were searched through July 2018. We included randomized controlled clinical trials that compared DBL with BLAG combinations. Clinical response was used as the primary outcome and microbiological response in Gram-negative bacteria as the secondary outcome; sensitivity analyses were performed for Pseudomonas aeruginosa, Klebsiella spp., and Escherichia coli Heterogeneity and risk of bias were assessed. Safety results were classified by systems and organs. Thirteen studies evaluated 2,771 cases for clinical response and 665 cases for microbiological response in various Gram-negative species. DBL achieved slightly, but not significantly, better clinical response (risk ratio, 1.05; 95% confidence interval [CI], 0.99 to 1.11) and microbiological response in Gram-negatives (risk ratio, 1.11; 95% CI, 0.99 to 1.25) compared with BLAG. Sensitivity analyses by pathogen showed the same trend. No significant heterogeneity across studies was found. DBL was significantly safer than BLAG regarding renal toxicity (6.6% versus 8.8%, P = 0.0338) and ototoxicity (0.7 versus 3.1%, P = 0.0137). Other adverse events were largely comparable. Overall, empirically designed DBL showed comparable clinical and microbiological responses across different Gram-negative species, and were significantly safer than BLAG. Therefore, DBL should be rationally optimized via the latest translational approaches, leveraging mechanistic insights and newer ß-lactams for future evaluation in clinical trials.

In Vitro and In Vivo Activities of ß-Lactams in Combination with the Novel ß-Lactam Enhancers Zidebactam and WCK 5153 against Multidrug-Resistant Metallo-ß-Lactamase-Producing Klebsiella pneumoniae.

Zidebactam and WCK 5153 are novel bicyclo-acyl hydrazide (BCH) agents that have previously been shown to act as ß-lactam enhancer (BLE) antibiotics in Pseudomonas aeruginosa and Acinetobacter baumannii The objectives of this work were to identify the molecular targets of these BCHs in Klebsiella pneumoniae and to investigate their potential BLE activity for cefepime and aztreonam against metallo-ß-lactamase (MBL)-producing strains in vitro and in vivo Penicillin binding protein (PBP) binding profiles were determined by Bocillin FL assay, and 50% inhibitory concentrations (IC50s) were determined using ImageQuant TL software. MICs and kill kinetics for zidebactam, WCK 5153, and cefepime or aztreonam, alone and in combination, were determined against clinical K. pneumoniae isolates producing MBLs VIM-1 or NDM-1 (plus ESBLs and class C ß-lactamases) to assess the in vitro enhancer effect of BCH compounds in conjunction with ß-lactams. Additionally, murine systemic and thigh infection studies were conducted to evaluate BLE effects in vivo Zidebactam and WCK 5153 showed specific, high PBP2 affinity in K. pneumoniae The MICs of BLEs were >64 µg/ml for all MBL-producing strains. Time-kill studies showed that a combination of these BLEs with either cefepime or aztreonam provided 1 to >3 log10 kill against MBL-producing K. pneumoniae strains. Furthermore, the bactericidal synergy observed for these BLE-ß-lactam combinations translated well into in vivo efficacy even in the absence of MBL inhibition by BLEs, a characteristic feature of the ß-lactam enhancer mechanism of action. Zidebactam and WCK 5153 are potent PBP2 inhibitors and display in vitro and in vivo BLE effects against multidrug-resistant (MDR) K. pneumoniae clinical isolates producing MBLs.

Four Decades of ß-Lactam Antibiotic Pharmacokinetics in Cystic Fibrosis.

The pharmacokinetics (PK) of ß-lactam antibiotics in cystic fibrosis (CF) patients has been compared with that in healthy volunteers for over four decades; however, no quantitative models exist that explain the PK differences between CF patients and healthy volunteers in older and newer studies. Our aims were to critically evaluate these studies and explain the PK differences between CF patients and healthy volunteers. We reviewed all 16 studies that compared the PK of ß-lactams between CF patients and healthy volunteers within the same study. Analysis of covariance (ANCOVA) models were developed. In four early studies that compared adolescent, lean CF patients with adult healthy volunteers, clearance (CL) in CF divided by that in healthy volunteers was 1.72 ± 0.90 (average ± standard deviation); in four additional studies comparing age-matched (primarily adult) CF patients with healthy volunteers, this ratio was 1.46 ± 0.16. The CL ratio was 1.15 ± 0.11 in all eight studies that compared CF patients and healthy volunteers who were matched in age, body size and body composition, or that employed allometric scaling by lean body mass (LBM). Volume of distribution was similar between subject groups after scaling by body size. For highly protein-bound ß-lactams, the unbound fraction was up to 2.07-fold higher in older studies that compared presumably sicker CF patients with healthy volunteers. These protein-binding differences explained over half of the variance for the CL ratio (p < 0.0001, ANCOVA). Body size, body composition and lower protein binding in presumably sicker CF patients explained the PK alterations in this population. Dosing CF patients according to LBM seems suitable to achieve antibiotic target exposures.

First population pharmacokinetic analysis showing increased quinolone metabolite formation and clearance in patients with cystic fibrosis compared to healthy volunteers.

Understanding the pharmacokinetics in patients with cystic fibrosis (CF) is important for dosing. For antibiotics with extensive metabolism, however, a comparison of metabolite formation and elimination between patients with CF and healthy volunteers has never been performed via population modeling. We aimed to compare the population pharmacokinetics of fleroxacin and its N­oxide and demethyl metabolites between patients with CF and healthy volunteers. Our analysis included eleven adult patients with CF and twelve healthy volunteers who received 800 mg fleroxacin as a single oral dose followed by five doses every 24 h from a previously published study. All plasma concentrations and amounts in urine for fleroxacin and its metabolites were simultaneously modelled. The estimates below accounted for differences in body size and body composition via allometric scaling by lean body mass. Oral absorption was slower in patients with CF than in healthy volunteers. For fleroxacin, the population mean in patients with CF divided by that in healthy volunteers was 1.12 for renal clearance, 1.01 for linear nonrenal clearance, 0.83 for saturable exsorption clearance into intestine, and 0.81 for volume of distribution. The formation clearances of N­oxide fleroxacin and N­demethylfleroxacin were 0.520 L/h and 0.496 L/h in patients with CF; these formation clearances were 0.378 L/h and 0.353 L/h in healthy volunteers. Renal clearance in patients with CF divided by that in healthy volunteers was 1.53 for N­oxide fleroxacin and 1.70 for N­demethyl fleroxacin. Allometric scaling by lean body mass best explained the variability. While fleroxacin pharmacokinetics was comparable, both formation and elimination clearances of its two metabolites were substantially larger in patients with CF compared to those in healthy volunteers.

First Penicillin-Binding Protein Occupancy Patterns of ß-Lactams and ß-Lactamase Inhibitors in Klebsiella pneumoniae.

Penicillin-binding proteins (PBPs) are the high-affinity target sites of all ß-lactam antibiotics in bacteria. It is well known that each ß-lactam covalently binds to and thereby inactivates different PBPs with various affinities. Despite ß-lactams serving as the cornerstone of our therapeutic armamentarium against Klebsiella pneumoniae, PBP binding data are missing for this pathogen. We aimed to generate the first PBP binding data on 13 chemically diverse and clinically relevant ß-lactams and ß-lactamase inhibitors in K. pneumoniae PBP binding was determined using isolated membrane fractions from K. pneumoniae strains ATCC 43816 and ATCC 13883. Binding reactions were conducted using ß-lactam concentrations from 0.0075 to 256 mg/liter (or 128 mg/liter). After ß-lactam exposure, unbound PBPs were labeled by Bocillin FL. Binding affinities (50% inhibitory concentrations [IC50]) were reported as the ß-lactam concentrations that half-maximally inhibited Bocillin FL binding. PBP occupancy patterns by ß-lactams were consistent across both strains. Carbapenems bound to all PBPs, with PBP2 and PBP4 as the highest-affinity targets (IC50, <0.0075 mg/liter). Preferential PBP2 binding was observed by mecillinam (amdinocillin; IC50, <0.0075 mg/liter) and avibactam (IC50, 2 mg/liter). Aztreonam showed high affinity for PBP3 (IC50, 0.06 to 0.12 mg/liter). Ceftazidime bound PBP3 at low concentrations (IC50, 0.06 to 0.25 mg/liter) and PBP1a/b at higher concentrations (4 mg/liter), whereas cefepime bound PBPs 1 to 4 at more even concentrations (IC50, 0.015 to 2 mg/liter). These PBP binding data on a comprehensive set of 13 clinically relevant ß-lactams and ß-lactamase inhibitors in K. pneumoniae enable, for the first time, the rational design and optimization of double ß-lactam and ß-lactam-ß-lactamase inhibitor combinations.

Potent ß-Lactam Enhancer Activity of Zidebactam and WCK 5153 against Acinetobacter baumannii, Including Carbapenemase-Producing Clinical Isolates.

Multidrug-resistant Acinetobacter baumannii has rapidly spread worldwide, resulting in a serious threat to hospitalized patients. Zidebactam and WCK 5153 are novel non-ß-lactam bicyclo-acyl hydrazide ß-lactam enhancer antibiotics being developed to target multidrug-resistant A. baumannii The objectives of this work were to determine the 50% inhibitory concentrations (IC50s) for penicillin-binding proteins (PBP), the OXA-23 inhibition profiles, and the antimicrobial activities of zidebactam and WCK 5153, alone and in combination with ß-lactams, against multidrug-resistant A. baumannii MICs and time-kill kinetics were determined for an A. baumannii clinical strain producing the carbapenemase OXA-23 and belonging to the widespread European clone II of sequence type 2 (ST2). Inhibition of the purified OXA-23 enzyme by zidebactam, WCK 5153, and comparators was assessed. All of the compounds tested displayed apparent Ki values of >100 µM, indicating poor OXA-23 ß-lactamase inhibition. The IC50s of zidebactam, WCK 5153, cefepime, ceftazidime, meropenem, and sulbactam (range of concentrations tested, 0.02 to 2 µg/ml) for PBP were also determined. Zidebactam and WCK 5153 demonstrated specific high-affinity binding to PBP2 of A. baumannii (0.01 µg/ml for both of the compounds). The MICs of zidebactam and WCK 5153 were >1,024 µg/ml for wild-type and multidrug-resistant Acinetobacter strains. Importantly, combinations of cefepime with 8 µg/ml of zidebactam or WCK 5153 and sulbactam with 8 µg/ml of zidebactam or WCK 5153 led to 4- and 8-fold reductions of the MICs, respectively, and showed enhanced killing. Notably, several of the combinations resulted in full bacterial eradication at 24 h. We conclude that zidebactam and WCK 5153 are PBP2 inhibitors that show a potent ß-lactam enhancer effect against A. baumannii, including a multidrug-resistant OXA-23-producing ST2 international clone.