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.

Pivmecillinam, the paradigm of an antibiotic with low resistance rates in Escherichia coli urine isolates despite high consumption.

OBJECTIVES: Pivmecillinam, the oral version of mecillinam, represents one of the major recommended and used antibiotics for empiric and targeted treatment of urinary tract infections in primary care in Denmark, Norway and Sweden. Mecillinam resistant mutants in Escherichia coli develop easily in vitro, but their fitness cost has been shown to be high. METHODS: We revisited the resistance and consumption data from the monitoring programmes in the three countries and compared pivmecillinam with ciprofloxacin from 2010 to 2020. RESULTS: Mecillinam resistance rates in Escherichia coli remained around 6% in Denmark and Norway relative to a constant consumption in Norway of 1.6-1.8 DID (defined daily doses per 1000 inhabitants per day), and even increasing in Denmark from 1.6 to 2.3 DID. In Sweden resistance was significantly lower at 4% related to the lower consumption of 0.5 DID. For ciprofloxacin, resistance rates fluctuated around 6%-12%, highest in Sweden with the highest consumption (0.8-0.6 DID) and lowest in Denmark (0.55-0.35 DID) and Norway (0.7-0.3 DID), although consumption declined significantly in all three countries. CONCLUSIONS: Pivmecillinam is an example of an antibiotic, which easily develops resistance in vitro, but apparently can be used broadly in primary care without increase in resistance rates.

SUsceptibility and Resistance to Fosfomycin and other antimicrobial agents among pathogens causing lower urinary tract infections: findings of the SURF study.

Urinary tract infections (UTIs) are prevalent worldwide, particularly among women. Their incidence increases with age, and treatment is increasingly challenging owing to antibiotic resistance and the lack of new agents. We investigated the susceptibility of current urinary isolates to fosfomycin and other antibiotics across Europe. This cross-sectional study collected consecutive urinary isolates from non-hospitalised women at 20 centres in Belgium, the UK, Italy, Spain and Russia. Bacteria were tested by disk diffusion with relevant antibiotics. As a quality control, a central laboratory re-tested, by agar dilution, (i) isolates found resistant to fosfomycin and (ii) every tenth isolate; all non-Russian sites were included. A total of 2848 isolates were analysed, principally Escherichia coli (2064; 72.5%), Klebsiella spp. (275; 9.7%) and Proteus spp. (103; 3.6%). For E. coli, agents active against >90% of isolates were nitrofurantoin (98.5%), fosfomycin (96.4%) and mecillinam (91.8%). Fosfomycin and nitrofurantoin remained active against >90% of cephalosporin-resistant E. coli. Among 143 E. coli recorded as susceptible locally by disk tests, 138 (96.5%) were confirmed susceptible by minimum inhibitory concentration (MIC) tests, however resistance was only confirmed in 29/58 (50.0%) of those reported resistant by local disk tests. Escherichia coli was found to be the most common uropathogen isolated and was highly susceptible to fosfomycin, nitrofurantoin and mecillinam, all used effectively for more than 30 years. Guidelines advocating fosfomycin for uncomplicated UTIs in women remain microbiologically valid.

Increased energy demand from anabolic-catabolic processes drives ß-lactam antibiotic lethality.

ß-Lactam antibiotics disrupt the assembly of peptidoglycan (PG) within the bacterial cell wall by inhibiting the enzymatic activity of penicillin-binding proteins (PBPs). It was recently shown that ß-lactam treatment initializes a futile cycle of PG synthesis and degradation, highlighting major gaps in our understanding of the lethal effects of PBP inhibition by ß-lactam antibiotics. Here, we assess the downstream metabolic consequences of treatment of Escherichia coli with the ß-lactam mecillinam and show that lethality from PBP2 inhibition is a specific consequence of toxic metabolic shifts induced by energy demand from multiple catabolic and anabolic processes, including accelerated protein synthesis downstream of PG futile cycling. Resource allocation into these processes is coincident with alterations in ATP synthesis and utilization, as well as a broadly dysregulated cellular redox environment. These results indicate that the disruption of normal anabolic-catabolic homeostasis by PBP inhibition is an essential factor for ß-lactam antibiotic lethality.

Chemical-genetic interrogation of RNA polymerase mutants reveals structure-function relationships and physiological tradeoffs.

The multi-subunit bacterial RNA polymerase (RNAP) and its associated regulators carry out transcription and integrate myriad regulatory signals. Numerous studies have interrogated RNAP mechanism, and RNAP mutations drive Escherichia coli adaptation to many health- and industry-relevant environments, yet a paucity of systematic analyses hampers our understanding of the fitness trade-offs from altering RNAP function. Here, we conduct a chemical-genetic analysis of a library of RNAP mutants. We discover phenotypes for non-essential insertions, show that clustering mutant phenotypes increases their predictive power for drawing functional inferences, and demonstrate that some RNA polymerase mutants both decrease average cell length and prevent killing by cell-wall targeting antibiotics. Our findings demonstrate that RNAP chemical-genetic interactions provide a general platform for interrogating structure-function relationships in vivo and for identifying physiological trade-offs of mutations, including those relevant for disease and biotechnology. This strategy should have broad utility for illuminating the role of other important protein complexes.

CombiANT: Antibiotic interaction testing made easy.

Antibiotic combination therapies are important for the efficient treatment of many types of infections, including those caused by antibiotic-resistant pathogens. Combination treatment strategies are typically used under the assumption that synergies are conserved across species and strains, even though recent results show that the combined treatment effect is determined by specific drug-strain interactions that can vary extensively and unpredictably, both between and within bacterial species. To address this problem, we present a new method in which antibiotic synergy is rapidly quantified on a case-by-case basis, allowing for improved combination therapy. The novel CombiANT methodology consists of a 3D-printed agar plate insert that produces defined diffusion landscapes of 3 antibiotics, permitting synergy quantification between all 3 antibiotic pairs with a single test. Automated image analysis yields fractional inhibitory concentration indices (FICis) with high accuracy and precision. A technical validation with 3 major pathogens, Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus, showed equivalent performance to checkerboard methodology, with the advantage of strongly reduced assay complexity and costs for CombiANT. A synergy screening of 10 antibiotic combinations for 12 E. coli urinary tract infection (UTI) clinical isolates illustrates the need for refined combination treatment strategies. For example, combinations of trimethoprim (TMP) + nitrofurantoin (NIT) and TMP + mecillinam (MEC) showed synergy, but only for certain individual isolates, whereas MEC + NIT combinations showed antagonistic interactions across all tested strains. These data suggest that the CombiANT methodology could allow personalized clinical synergy testing and large-scale screening. We anticipate that CombiANT will greatly facilitate clinical and basic research of antibiotic synergy.

Stability of ß-lactam antibiotics in bacterial growth media.

Laboratory assays such as MIC tests assume that antibiotic molecules are stable in the chosen growth medium-but rapid degradation has been observed for antibiotics including ß-lactams under some conditions in aqueous solution. Degradation rates in bacterial growth medium are less well known. Here, we develop a 'delay time bioassay' that provides a simple way to estimate antibiotic stability in bacterial growth media, using only a plate reader and without the need to measure the antibiotic concentration directly. We use the bioassay to measure degradation half-lives of the ß-lactam antibiotics mecillinam, aztreonam and cefotaxime in widely-used bacterial growth media based on MOPS and Luria-Bertani (LB) broth. We find that mecillinam degradation can occur rapidly, with a half-life as short as 2 hours in MOPS medium at 37°C and pH 7.4, and 4-5 hours in LB, but that adjusting the pH and temperature can increase its stability to a half-life around 6 hours without excessively perturbing growth. Aztreonam and cefotaxime were found to have half-lives longer than 6 hours in MOPS medium at 37°C and pH 7.4, but still shorter than the timescale of a typical minimum inhibitory concentration (MIC) assay. Taken together, our results suggest that care is needed in interpreting MIC tests and other laboratory growth assays for ß-lactam antibiotics, since there may be significant degradation of the antibiotic during the assay.

Efficacy of mecillinam against clinical multidrug-resistant Escherichia coli in a murine urinary tract infection model.

Pivmecillinam, a pro-drug of mecillinam, has been used extensively in Scandinavia for the treatment of acute lower urinary tract infections (UTIs) caused by Enterobacterales. It is still an attractive first-line drug for the empirical treatment of UTIs owing to the low prevalence of resistance as well as its favourable impact on the intestinal microbiota as a pro-drug and good in vitro efficacy against extended-spectrum ß-lactamase (ESBL)- and plasmid-mediated AmpC ß-lactamase-producing Escherichia coli. However, optimal dosing of pivmecillinam as well as its in vivo efficacy against UTIs caused by multidrug-resistant (MDR) broad-spectrum ß-lactamase-producing E. coli has not been thoroughly studied. In this study, the efficacy of two mimicked human dosing regimens of pivmecillinam (200 mg and 400 mg three times daily) against clinical E. coli strains, including isolates producing ESBLs (CTX-M-14 and CTX-M-15), plasmid-mediated AmpCs (CMY-4 and CMY-6) and carbapenemases (NDM-1 and VIM-29), in a murine UTI model was compared. Both dosing regimens reduced the number of CFU/mL in urine for all strains, including mecillinam-resistant strains. Combining the effect for all six strains showed no significant differences in effect between doses for all three fluids/organs, but for each dose there was a highly significant effect in urine, kidney and bladder compared with vehicle-treated mice. Overall, this highlights the need for further studies to elucidate the role of mecillinam in the treatment of infections caused by MDR E. coli producing broad-spectrum ß-lactamases, including specific carbapenemases.

Mutational change of CTX-M-15 to CTX-M-127 resulting in mecillinam resistant Escherichia coli during pivmecillinam treatment of a patient.

Pivmecillinam (amdinocillin pivoxil) is the recommended first-choice antibiotic used to treat urinary tract infections (UTIs) in Denmark. The frequency of mutation to mecillinam (MEC) resistance is described as high in vitro; however, treatment of UTI has a good clinical response and prevalence of mecillinam resistance in Escherichia coli remains low despite many years of use. We describe occurrence of in vivo mecillinam resistance in a clinical isolate of ESBL-producing E. coli following pivmecillinam treatment. The identified phenotypic differences in the mecillinam resistant isolate compared with the original mecillinam susceptible isolate were a full-length LPS with O-antigen (O25), mecillinam resistance and a lower MIC for ceftazidime. Regarding genotype, the resistant isolate differed with a mutation in blaCTX-M-15 to blaCTX-M-127 , loss of a part of a plasmid and a genomic island, respectively, and insertion of a transposase in wbbL, causing the rough phenotype. The observed mecillinam resistance is expected to be caused by the mutation in blaCTX-M-15 with additional contribute from the serotype shift. We continue to recommend the use of pivmecillinam as first-line treatment for UTI.

Spheroplast-Mediated Carbapenem Tolerance in Gram-Negative Pathogens.

Antibiotic tolerance, the ability to temporarily sustain viability in the presence of bactericidal antibiotics, constitutes an understudied and yet potentially widespread cause of antibiotic treatment failure. We have previously shown that the Gram-negative pathogen Vibrio cholerae can tolerate exposure to the typically bactericidal ß-lactam antibiotics by assuming a spherical morphotype devoid of detectable cell wall material. However, it is unclear how widespread ß-lactam tolerance is. Here, we tested a panel of clinically significant Gram-negative pathogens for their response to the potent, broad-spectrum carbapenem antibiotic meropenem. We show that clinical isolates of Enterobacter cloacae, Klebsiella aerogenes, and Klebsiella pneumoniae, but not Escherichia coli, exhibited moderate to high levels of tolerance of meropenem, both in laboratory growth medium and in human serum. Importantly, tolerance was mediated by cell wall-deficient spheroplasts, which readily recovered wild-type morphology and growth upon removal of antibiotic. Our results suggest that carbapenem tolerance is prevalent in clinically significant bacterial species, and we suggest that this could contribute to treatment failure associated with these organisms.

Upregulation of PBP1B and LpoB in cysB Mutants Confers Mecillinam (Amdinocillin) Resistance in Escherichia coli.

Mecillinam (amdinocillin) is a ß-lactam antibiotic that inhibits the essential penicillin-binding protein 2 (PBP2). In clinical isolates of Escherichia coli from urinary tract infections, inactivation of the cysB gene (which encodes the main regulator of cysteine biosynthesis, CysB) is the major cause of resistance. How a nonfunctional CysB protein confers resistance is unknown, however, and in this study we wanted to examine the mechanism of resistance. Results show that cysB mutations cause a gene regulatory response that changes the expression of ∼450 genes. Among the proteins that show increased levels are the PBP1B, LpoB, and FtsZ proteins, which are known to be involved in peptidoglycan biosynthesis. Artificial overexpression of either PBP1B or LpoB in a wild-type E. coli strain conferred mecillinam resistance; conversely, inactivation of either the mrcB gene (which encodes PBP1B) or the lpoB gene (which encodes the PBP1B activator LpoB) made cysB mutants susceptible. These results show that expression of the proteins PBP1B and LpoB is both necessary and sufficient to confer mecillinam resistance. The addition of reducing agents to a cysB mutant converted it to full susceptibility, with associated downregulation of PBP1B, LpoB, and FtsZ. We propose a model in which cysB mutants confer mecillinam resistance by inducing a response that causes upregulation of the PBP1B and LpoB proteins. The higher levels of these two proteins can then rescue cells with mecillinam-inhibited PBP2. Our results also show how resistance can be modulated by external conditions such as reducing agents.

In vitro activity of mecillinam and nitroxoline against Neisseria gonorrhoeae - re-purposing old antibiotics in the multi-drug resistance era.

In 2018, the European Centre for Disease Prevention and Control reported the first cases of extensively drug-resistant Neisseria gonorrhoeae infections in Europe. Seeking new options for antimicrobial therapy we investigated the susceptibility of N. gonorrhoeae to nitroxoline (NIT) and mecillinam (MCM), both of which are currently only indicated to treat uncomplicated urinary tract infections. Clinical N. gonorrhoeae isolates with non-susceptibility to penicillin from two German medical centres were included (n =27). Most isolates were also non-susceptible to a range of other anti-gonococcal antimicrobials (cefotaxime, ciprofloxacin, azithromycin, tetracycline). All isolates were further characterized by multi-locus sequence typing. MICs of penicillin and cefotaxime were determined by agar gradient diffusion. Production of penicillinase was tested by cefinase disk test. Susceptibility of MCM was investigated by agar dilution, NIT by agar dilution and disk diffusion. Penicillin MICs ranged from 0.125 to 64 mg l-1 and MICs of cefotaxime ranged from < 0.016 to 1 mg l-1 . Five isolates were penicillinase-producers. MICs of MCM ranged from 16 to > 128 mg l-1 whereas MICs of NIT ranged from 0.125 to 2 mg l-1 . NIT disk diffusion (median zone diameter 32 mm) correlated well with results from agar dilution. We demonstrated excellent in vitro activity of NIT against clinical N. gonorrhoeae isolates with non-susceptibility to standard anti-gonococcal antibiotics. MCM activity was unsatisfactory. Correlation of agar dilution and disk diffusion in NIT susceptibility testing is an important aspect with potential clinical implications.

Results from a Prospective In Vitro Study on the Mecillinam (Amdinocillin) Susceptibility of Enterobacterales.

The activity of mecillinam (amdinocillin) was assessed in Enterobacterales (n = 420) isolated from urine samples between 2016 and 2017. Mecillinam susceptibilities were 97.4% in Escherichia coli isolates (294/302), 89.7% in Klebsiella spp. isolates (52/58), and 93.3% in Proteus mirabilis isolates (28/30). Among extended-spectrum ß-lactamase (ESBL) producers, 95.2% (99/104) were mecillinam susceptible, including two OXA-48-producing Klebsiella pneumoniae isolates. In Enterobacter spp. and Citrobacter spp., MICs were low (MIC50 = 0.5 mg/liter). In conclusion, the activity of mecillinam was high in Enterobacterales, even among multidrug-resistant isolates.

Collateral sensitivity constrains resistance evolution of the CTX-M-15 ß-lactamase.

Antibiotic resistance is a major challenge to global public health. Discovery of new antibiotics is slow and to ensure proper treatment of bacterial infections new strategies are needed. One way to curb the development of antibiotic resistance is to design drug combinations where the development of resistance against one drug leads to collateral sensitivity to the other drug. Here we study collateral sensitivity patterns of the globally distributed extended-spectrum ß-lactamase CTX-M-15, and find three non-synonymous mutations with increased resistance against mecillinam or piperacillin-tazobactam that simultaneously confer full susceptibility to several cephalosporin drugs. We show in vitro and in mice that a combination of mecillinam and cefotaxime eliminates both wild-type and resistant CTX-M-15. Our results indicate that mecillinam and cefotaxime in combination constrain resistance evolution of CTX-M-15, and illustrate how drug combinations can be rationally designed to limit the resistance evolution of horizontally transferred genes by exploiting collateral sensitivity patterns.

Pathway-Directed Screen for Inhibitors of the Bacterial Cell Elongation Machinery.

New antibiotics are needed to combat the growing problem of resistant bacterial infections. An attractive avenue toward the discovery of such next-generation therapies is to identify novel inhibitors of clinically validated targets, like cell wall biogenesis. We have therefore developed a pathway-directed whole-cell screen for small molecules that block the activity of the Rod system of Escherichia coli This conserved multiprotein complex is required for cell elongation and the morphogenesis of rod-shaped bacteria. It is composed of cell wall synthases and membrane proteins of unknown function that are organized by filaments of the actin-like MreB protein. Our screen takes advantage of the conditional essentiality of the Rod system and the ability of the beta-lactam mecillinam (also known as amdinocillin) to cause a toxic malfunctioning of the machinery. Rod system inhibitors can therefore be identified as molecules that promote growth in the presence of mecillinam under conditions permissive for the growth of Rod- cells. A screen of ∼690,000 compounds identified 1,300 compounds that were active against E. coli Pathway-directed screening of a majority of this subset of compounds for Rod inhibitors successfully identified eight analogs of the MreB antagonist A22. Further characterization of the A22 analogs identified showed that their antibiotic activity under conditions where the Rod system is essential was strongly correlated with their ability to suppress mecillinam toxicity. This result combined with those from additional biological studies reinforce the notion that A22-like molecules are relatively specific for MreB and suggest that the lipoprotein transport factor LolA is unlikely to be a physiologically relevant target as previously proposed.

Synergistic and bactericidal activities of mecillinam, amoxicillin and clavulanic acid combinations against extended-spectrum ß-lactamase (ESBL)-producing Escherichia coli in 24-h time-kill experiments.

This study aimed to evaluate the potential synergistic and bactericidal effects of mecillinam in combination with amoxicillin and clavulanic acid against extended-spectrum ß-lactamase (ESBL)-producing Escherichia coli. Eight clinical E. coli isolates with varying susceptibility to mecillinam [minimum inhibitory concentrations (MICs) of 0.125 mg/L to >256 mg/L] and high-level resistance to amoxicillin (MICs > 256 mg/L) were used. Whole-genome sequencing was performed to determine the presence of ß-lactamase genes and mutations in the cysB gene. The activities of single drugs and the combinations of two or three drugs were tested in 24-h time-kill experiments. Population analysis was performed for two strains before and after experiments. Only one strain had a mutation in the cysB gene resulting in an amino acid substitution. With the two-drug combinations, initial killing was observed both with mecillinam and amoxicillin when combined with clavulanic acid. Synergy was observed with mecillinam and clavulanic acid against one strain and with amoxicillin and clavulanic acid against three strains. However, following significant re-growth, a bactericidal effect was found only with amoxicillin and clavulanic acid against two strains. Pre-existing subpopulations with elevated mecillinam MICs were detected before experiments and were selected with mecillinam alone or in two-drug combinations. In contrast, the three-drug combination showed enhanced activity with synergy against six strains, a bactericidal effect against all eight strains, and suppression of resistance during 24-h antibiotic exposure. This combination may be of clinical interest in the treatment of urinary tract infections caused by ESBL-producing E. coli.

Conserved collateral antibiotic susceptibility networks in diverse clinical strains of Escherichia coli.

There is urgent need to develop novel treatment strategies to reduce antimicrobial resistance. Collateral sensitivity (CS), where resistance to one antimicrobial increases susceptibility to other drugs, might enable selection against resistance during treatment. However, the success of this approach would depend on the conservation of CS networks across genetically diverse bacterial strains. Here, we examine CS conservation across diverse Escherichia coli strains isolated from urinary tract infections. We determine collateral susceptibilities of mutants resistant to relevant antimicrobials against 16 antibiotics. Multivariate statistical analyses show that resistance mechanisms, in particular efflux-related mutations, as well as the relative fitness of resistant strains, are principal contributors to collateral responses. Moreover, collateral responses shift the mutant selection window, suggesting that CS-informed therapies may affect evolutionary trajectories of antimicrobial resistance. Our data allow optimism for CS-informed therapy and further suggest that rapid detection of resistance mechanisms is important to accurately predict collateral responses.

MreB polymers and curvature localization are enhanced by RodZ and predict E. coli's cylindrical uniformity.

The actin-like protein MreB has been proposed to coordinate the synthesis of the cell wall to determine cell shape in bacteria. MreB is preferentially localized to areas of the cell with specific curved geometries, avoiding the cell poles. It remains unclear whether MreB's curvature preference is regulated by additional factors, and which specific features of MreB promote specific features of rod shape growth. Here, we show that the transmembrane protein RodZ modulates MreB curvature preference and polymer number in E. coli, properties which are regulated independently. An unbiased machine learning analysis shows that MreB polymer number, the total length of MreB polymers, and MreB curvature preference are key correlates of cylindrical uniformity, the variability in radius within a single cell. Changes in the values of these parameters are highly predictive of the resulting changes in cell shape (r2 = 0.93). Our data thus suggest RodZ promotes the assembly of geometrically-localized MreB polymers that lead to the growth of uniform cylinders.

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.