Inhibition Of Penicillin-binding Proteins Research Articles

2163. Prevalence and geographic distribution of mutations in TonB-dependent receptors in the Prospective Observational Pseudomonas aeruginosa (PA) Study

Abstract Background Cefiderocol (FDC) is a last line treatment for carbapenem-resistant Pseudomonas aeruginosa (CR-PA). FDC retains activity against CR-PA by inhibition of penicillin-binding proteins facilitated by active uptake of drug via TonB-dependent receptors (TBDR). Prior, we have shown an association between mutations in TBDR genes and loss of FDC susceptibility, particularly in association with changes at the PA-derived cephalosporinase (PDC/AmpC) active site. Here, we outline geographic variation and prevalence of TBDR mutations in the Prospective Observational Pseudomonas (POP) study.Figure 1.SNP frequency in the pirR, pirS, pirA, and piuA/piuD genes across geographic location.Figure 2.Genotypic variation of PirRS and PiuA/D genes in the 972 P. aeruginosa isolates of the POP cohort. Black versus gray differentiates presence of either PiuA versus PiuD homologous proteins. Color legend corresponds to presence of TBDR gene variants/SNPs per gene pirR, pirS, pirA, piuA/piuD, and ampC. Methods The genomes of 972 CR-PA isolates collected across 10 countries in North America, South America, Asia, and Oceania were screened for single nucleotide polymorphisms (SNP) resulting in missense, insertion/deletion (indel), frameshift, and early stop codon mutations in the TBDR genes pirR, pirS, pirA, piuA/piuD, and ampC using PAO1 as a reference. SNPs were defined as variant mutations occurring in ≤10% of isolates. A core genome maximum likelihood phylogenetic tree was used to assess distribution and prevalence of genotypic variants. Neighbor-joining trees of the variants in each gene were used to analyze clusters across lineages. Results Across all regions except Oceania, SNPs were most frequently seen in pirS and pirA (Fig 1). Gene variants comprised from 8% up to 50% of each geographic population. On phylogenetic mapping, isolates within the same lineage tended to have clustering of shared mutations across the TBDR genes pirS, pirR, and pirA, and the majority of piuA/piuD mutations clustered within two distinct lineages (Fig 2). Major mutations, including indel, frameshift, and early stop codon mutations occurred in 16 isolates (1.6%), predominantly in isolates from the United States (n=14). Variations in PDC were frequently observed, with 57 different variants across 35.8% of the population. Seven of the PDC variants (n=10 isolates) have been associated with decreased susceptibility to ceftolozane/tazobactam, but these variants did not co-occur with major TBDR mutations. Conclusion TBDR mutations occur in PA isolates worldwide. Mutations previously associated with reduced susceptibility to FDC occurred most frequently in isolates from the United States. Disclosures Michael J. Satlin, MD, AbbVie: IDMC member|Biomerieux: Grant/Research Support|Merck: Grant/Research Support|Shionogi: Advisor/Consultant|SNIPRBiome: Grant/Research Support William R. Miller, M.D., Merck: Grant/Research Support|UpToDate: Honoraria

Combined Structural Analysis and Molecular Dynamics Reveal Penicillin-Binding Protein Inhibition Mode with β-Lactones.

β-Lactam antibiotics comprise one of the most widely used therapeutic classes to combat bacterial infections. This general scaffold has long been known to inhibit bacterial cell wall biosynthesis by inactivating penicillin-binding proteins (PBPs); however, bacterial resistance to β-lactams is now widespread, and new strategies are urgently needed to target PBPs and other proteins involved in bacterial cell wall formation. A key requirement in the identification of strategies to overcome resistance is a deeper understanding of the roles of the PBPs and their associated proteins during cell growth and division, such as can be obtained with the use of selective chemical probes. Probe development has typically depended upon known PBP inhibitors, which have historically been thought to require a negatively charged moiety that mimics the C-terminus of the PBP natural peptidoglycan substrate, d-Ala-d-Ala. However, we have identified a new class of β-lactone-containing molecules that interact with PBPs, often in an isoform-specific manner, and do not incorporate this C-terminal mimetic. Here, we report a series of structural biology experiments and molecular dynamics simulations that we utilized to evaluate specific binding modes of this novel PBP inhibitor class. In this work, we obtained <2 Å resolution X-ray structures of four β-lactone probes bound to PBP1b from Streptococcus pneumoniae. Despite their diverging recognition modes beyond the site of covalent modification, these four probes all efficiently labeled PBP1b, as well as other PBPs from S. pneumoniae. From these structures, we analyzed protein-ligand interactions and characterized the β-lactone-bound active sites using in silico mutagenesis and molecular dynamics. Our approach has clarified the dynamic interaction profile in this series of ligands, expanding the understanding of PBP inhibitor binding.

A whole-genome assay identifies four principal gene functions that confer tolerance of meropenem stress upon Escherichia coli

We report here the identification of four gene functions of principal importance for the tolerance of meropenem stress in Escherichia coli: cell division, cell envelope synthesis and maintenance, ATP metabolism, and transcription regulation. The primary mechanism of β-lactam antibiotics such as meropenem is inhibition of penicillin binding proteins, thus interfering with peptidoglycan crosslinking, weakening the cell envelope, and promoting cell lysis. However, recent systems biology approaches have revealed numerous downstream effects that are triggered by cell envelope damage and involve diverse cell processes. Subpopulations of persister cells can also arise, which can survive elevated concentrations of meropenem despite the absence of a specific resistance factor. We used Transposon-Directed Insertion Sequencing with inducible gene expression to simultaneously assay the effects of upregulation, downregulation, and disruption of every gene in a model E. coli strain on survival of exposure to four concentrations of meropenem. Automated Gene Functional Classification and manual categorization highlighted the importance at all meropenem concentrations of genes involved in peptidoglycan remodeling during cell division, suggesting that cell division is the primary function affected by meropenem. Genes involved in cell envelope synthesis and maintenance, ATP metabolism, and transcriptional regulation were generally important at higher meropenem concentrations, suggesting that these three functions are therefore secondary or downstream targets. Our analysis revealed the importance of multiple two-component signal transduction mechanisms, suggesting an as-yet unexplored coordinated transcriptional response to meropenem stress. The inclusion of an inducible, transposon-encoded promoter allowed sensitive detection of genes involved in proton transport, ATP production and tRNA synthesis, for which modulation of expression affects survival in the presence of meropenem: a finding that would not be possible with other technologies. We were also able to suggest new targets for future antibiotic development or for synergistic effects between gene or protein inhibitors and existing antibiotics. Overall, in a single massively parallel assay we were able to recapitulate many of the findings from decades of research into β-lactam antibiotics, add to the list of genes known to be important for meropenem tolerance, and categorize the four principal gene functions involved.

Intrinsic Antibacterial Activity of Xeruborbactam In Vitro: Assessing Spectrum and Mode of Action.

ABSTRACTXeruborbactam (formerly QPX7728) is a cyclic boronate inhibitor of numerous serine and metallo-beta-lactamases. At concentrations generally higher than those required for beta-lactamase inhibition, xeruborbactam has direct antibacterial activity against some Gram-negative bacteria, with MIC50/MIC90 values of 16/32 μg/mL and 16/64 μg/mL against carbapenem-resistant Enterobacterales and carbapenem-resistant Acinetobacter baumannii, respectively (the MIC50/MIC90 values against Pseudomonas aeruginosa are >64 μg/mL). In Klebsiella pneumoniae, inactivation of OmpK36 alone or in combination with OmpK35 resulted in 2- to 4-fold increases in the xeruborbactam MIC. In A. baumannii and P. aeruginosa, AdeIJK and MexAB-OprM, respectively, affected xeruborbactam’s antibacterial potency (the MICs were 4- to 16-fold higher in efflux-proficient strains). In Escherichia coli and K. pneumoniae, the 50% inhibitory concentrations (IC50s) of xeruborbactam’s binding to penicillin-binding proteins (PBPs) PBP1a/PBP1b, PBP2, and PBP3 were in the 40 to 70 μM range; in A. baumannii, xeruborbactam bound to PBP1a, PBP2, and PBP3 with IC50s of 1.4 μM, 23 μM, and 140 μM, respectively. Treating K. pneumoniae and P. aeruginosa with xeruborbactam at 1× and 2× MIC resulted in changes of cellular morphology similar to those observed with meropenem; the morphological changes observed after treatment of A. baumannii were consistent with inhibition of multiple PBPs but were unique to xeruborbactam compared to the results for control beta-lactams. No single-step xeruborbactam resistance mutants were obtained after selection at 4× MIC of xeruborbactam using wild-type strains of E. coli, K. pneumoniae, and A. baumannii; mutations selected at 2× MIC in K. pneumoniae did not affect antibiotic potentiation by xeruborbactam through beta-lactamase inhibition. Consistent with inhibition of PBPs, xeruborbactam enhanced the potencies of beta-lactam antibiotics even against strains that lacked beta-lactamase. In a large panel of KPC-producing clinical isolates, the MIC90 values of meropenem tested with xeruborbactam (8 μg/mL) were at least 4-fold lower than those in combination with vaborbactam at 64 μg/mL, the concentration of vaborbactam that is associated with complete inhibition of KPC. The additional enhancement of the potency of beta-lactam antibiotics beyond beta-lactamase inhibition may contribute to the potentiation of beta-lactam antibiotics by xeruborbactam.

Evaluation of cefiderocol activity for Gram-negative bacteria and performance of cefiderocol disk diffusion testing using multiple brands of Mueller Hinton Agar

Abstract Cefidericol is a cephalosporin-siderophore antibiotic for the treatment of multidrug resistant Gram-negative bacteria. Similar to other cephalosporin antibiotics, the lethal mechanism of action is due to inhibition of penicillin binding proteins leading to lysis of the bacteria. However, unlike previously developed antibiotics, the siderophore portion of cefidericol is able to bind iron and then be actively transported into the periplasmic space. To ascertain the feasibility of cefidericol antibiotic susceptibility testing in the Barnes-Jewish Clinical Microbiology Laboratory, we collected a cohort of multidrug Enterobacteriacae (5 Enterobacter cloace, 8 Escherichia coli, 12 Klebsiella pneumoniae), Pseudomonas aeruginosa (n=23), Stenotrophomonas maltophila (n=24), and Acinetobacter baumannii (n=25). We evaluated activity of cefidericol on these strains, and the performance of disk diffusion using three different brands of Mueller-Hinton Agar (BD, Hardy, and Remel). The reference method for comparison was an FDA-cleared broth microdilution panel containing cefidericol (ThermoFisher Scientific). Using CLSI breakpoints, we found that disk diffusion with BD agar had 96% categorical agreement for Enterobacterales, 100% for P. aeruginosa, 92% for A. baumannii, 96% for S. maltophila. We found that Hardy had 96% categorical agreement for Enterobacterales, 92% for P. aeruginosa, 92% for A. baumannii, 96% for S. maltophila. Finally, we found that Hardy had 96% categorical agreement for Enterobacterales, 92% for P. aeruginosa, 92% for A. baumannii, 96% for S. maltophila. Minor errors on any media never exceed 4% and there were no very major errors. Resistance to cefidericol within our cohort of selected antibiotic resistant bacteria was rare, one E. coli isolate and two P. aeruginosa isolates had minimal inhibitory concentrations (MICs) &amp;gt; 32 μg/mL. The highest MICs for one isolate of A. baumannii and one isolate S. maltophila was 8 μg/mL and 4 μg/mL, respectively, both of which were intermediate. There was no difference in the distribution of zone disk diffusion diameter for A. baumannii or Enterobacterales. However, there was a significant difference in the distribution of zone disk diffusion diameters for P. aeruginosa and S. maltophila on BD vs Hardy agar. The median for P. aeruginosa on BD is 25 mm while it is 29 mm on Hardy. The trend for S. maltophila is the opposite as the median for BD was 31.5 mm and 28.5 mm for Hardy. Use of FDA vs CLSI vs EUCAST breakpoints significantly changes outcome of susceptibility testing for broth microdilution and disk diffusion. As one example for broth microdilution of A. baumannii, we had one isolate intermediate using CLSI breakpoints, 4 resistant using EUCAST breakpoints, and 4 resistant and 3 intermediate isolates using FDA breakpoints. Our work demonstrates that cefedericol testing can be performed in a routine format, with certain organismal differences on Mueller-Hinton agar, and that different interpretative criteria significantly change outcomes.

Conjugation to Enterobactin and Salmochelin S4 Enhances the Antimicrobial Activity and Selectivity of β-Lactam Antibiotics against Nontyphoidal Salmonella.

The pathogen Salmonella enterica is a leading cause of infection worldwide. Nontyphoidal Salmonella (NTS) serovars typically cause inflammatory diarrhea in healthy individuals, and can cause bacteremia in immunocompromised patients, children, and the elderly. Management of NTS infection poses a challenge because antibiotic treatment prolongs fecal shedding of the pathogen and is thus not recommended for most patients. In recent years, the emergence of antibiotic resistance in NTS has also become a major issue. Thus, new therapeutic strategies to target NTS are needed. Here, we evaluated whether six siderophore-β-lactam conjugates based on enterobactin (Ent) and salmochelin S4 (digulcosylated Ent, DGE) provide antimicrobial activity against the two highly prevalent NTS serovars Typhimurium and Enteritidis by targeting the siderophore receptors FepA and/or IroN. The conjugates showed 10- to 1000-fold lower minimum inhibitory concentrations against both serovars Typhimurium and Enteritidis compared to the parent antibiotics under iron limitation and were recognized and transported by FepA and/or IroN. NTS treated with the Ent/DGE-β-lactam conjugates exhibited aberrant cellular morphologies suggesting inhibition of penicillin-binding proteins, and the conjugates selectively killed NTS in coculture with Staphylococcus aureus. Lastly, the DGE-based conjugates proved to be effective at inhibiting growth of NTS in the presence of the Ent-sequestering protein lipocalin-2. This work describes the successful use of siderophore-antibiotic conjugates against NTS and highlights the opportunity for narrowing the activity spectrum of antibiotics by using Ent and DGE to target enteric bacterial pathogens.

Enterobactin- and salmochelin-β-lactam conjugates induce cell morphologies consistent with inhibition of penicillin-binding proteins in uropathogenic Escherichia coli CFT073.

The design and synthesis of narrow-spectrum antibiotics that target a specific bacterial strain, species, or group of species is a promising strategy for treating bacterial infections when the causative agent is known. In this work, we report the synthesis and evaluation of four new siderophore-β-lactam conjugates where the broad-spectrum β-lactam antibiotics cephalexin (Lex) and meropenem (Mem) are covalently attached to either enterobactin (Ent) or diglucosylated Ent (DGE) via a stable polyethylene glycol (PEG3) linker. These siderophore-β-lactam conjugates showed enhanced minimum inhibitory concentrations against Escherichia coli compared to the parent antibiotics. Uptake studies with uropathogenic E. coli CFT073 demonstrated that the DGE-β-lactams target the pathogen-associated catecholate siderophore receptor IroN. A comparative analysis of siderophore-β-lactams harboring ampicillin (Amp), Lex and Mem indicated that the DGE-Mem conjugate is advantageous because it targets IroN and exhibits low minimum inhibitory concentrations, fast time-kill kinetics, and enhanced stability to serine β-lactamases. Phase-contrast and fluorescence imaging of E. coli treated with the siderophore-β-lactam conjugates revealed cellular morphologies consistent with the inhibition of penicillin-binding proteins PBP3 (Ent/DGE-Amp/Lex) and PBP2 (Ent/DGE-Mem). Overall, this work illuminates the uptake and cell-killing activity of Ent- and DGE-β-lactam conjugates against E. coli and supports that native siderophore scaffolds provide the opportunity for narrowing the activity spectrum of antibiotics in clinical use and targeting pathogenicity.

Synthesis, In-Vitro and In-Silico Evaluation of Silver Nanoparticles with Root Extract of Withania somnifera for Antibacterial Activity via Binding of Penicillin-Binding Protein-4.

Metal Nanoparticles (NPs) have been widely used for various applications in biomedical sciences, including in drug delivery, and as therapeutic agents, but limited owing to their toxicity towards the healthy tissue. This warrants an alternative method, which can achieve the desired activity with much reduced or no toxicity. Being a biological product, Withania somnifera (W. somnifera) is environment friendly, besides being less toxic as compared to metal-based NPs. However, the exact mechanism of action of W. somnifera for its antibacterial activities has not been studied so far. To develop "silver nanoparticles with root extract of W. somnifera (AgNPs-REWS)" for antimicrobial and anticancer activities. Furthermore, the analysis of their mechanism of action will be studied. Using the in-silico approach, the molecular docking study was performed to evaluate the possible antibacterial mechanism of W. somnifera phytochemicals such as Anaferine, Somniferine, Stigmasterol, Withaferin A, Withanolide- A, G, M, and Withanone by the inhibition of Penicillin- Binding Protein 4 (PBP4). Next, we utilized a bottom-up approach for the green synthesis of AgNPs- REWS, performed an in-detail phytochemical analysis, confirmed the AgNPs-REWS by SEM, UVvisible spectroscopy, XRD, FT-IR, and HPLC. Eventually, we examined their antibacterial activity. The result of molecular docking suggests that WS phytochemicals (Somniferine, Withaferin A, Withanolide A, Withanolide G, Withanolide M, and Withanone) possess the higher binding affinity toward the active site of PBP4 as compared to the Ampicillin (-6.39 kcal/mol) reference molecule. These phytochemicals predicted as potent inhibitors of PBP4. Next, as a proof-of-concept, AgNPs- REWS showed significant antibacterial effect as compared to crude, and control; against Xanthomonas and Ralstonia species. The in-silico and molecular docking analysis showed that active constituents of W. somnifera such as Somniferine, Withaferin A, Withanolide A, Withanolide G, Withanolide M, and Withanone possess inhibition potential for PBP4 and are responsible for the anti-bacterial property of W. somnifera extract. This study also establishes that AgNPs via the green synthesis with REWS showed enhanced antibacterial activity towards pathogenic bacteria.

Dual \u03b2-lactam combination therapy for multi-drug resistant Pseudomonas aeruginosa infection: enhanced efficacy in vivo and comparison with monotherapies of penicillin-binding protein inhibition

The aim of the study was to determine the efficacy of dual β-lactam combination treatments derived from eight approved drugs against Galleria mellonella larvae infected with MDR strains of P. aeruginosa. Carbapenem-resistant P. aeruginosa NCTC 13437 and an unrelated clinical isolate were used to infect G. mellonella larvae and the efficacy of twenty-eight dual β-lactam combination therapies were compared to their constituent monotherapies. For the most potent combinations identified, penicillin-binding protein (PBP) inhibition profiles were measured and compared with each constituent antibiotic. Five of the dual β-lactam combinations resulted in greater than 70% survival of infected G. mellonella. Two combinations showed potent, enhanced efficacy versus both strains − ceftazidime + meropenem and aztreonam + meropenem. Comparison of PBP inhibition profiles revealed that the enhanced efficacy of these two dual β-lactam combinations could not be explained by more potent inhibition of PBPs or inhibition of a broader range of PBPs. A possible contribution to the enhanced efficacy of the combinations could be stimulation of innate immunity via increased haemocyte numbers compared to their constituent monotherapies. Combinations of β-lactam antibiotics show promise in overcoming MDR P. aeruginosa and are worthy of additional study and development.

The Novel β-Lactam Enhancer Zidebactam Augments the In Vivo Pharmacodynamic Activity of Cefepime in a Neutropenic Mouse Lung Acinetobacter baumannii Infection Model.

WCK 5222 is a combination of cefepime and the high-affinity PBP2-binding β-lactam enhancer zidebactam. The cefepime-zidebactam combination is active against multidrug-resistant Gram-negative bacteria, including carbapenemase-expressing Acinetobacter baumannii The mechanism of action of the combination involves concurrent multiple penicillin binding protein inhibition, leading to the enhanced bactericidal action of cefepime. The aim of the present study was to assess the impact of the zidebactam-mediated enhanced in vitro bactericidal action in modulating the percentage of the time that the free drug concentration remains above the MIC (percent fT>MIC) for cefepime required for the in vivo killing of A. baumannii Cefepime and cefepime-zidebactam MICs were comparable and ranged from 2 to 16 mg/liter for the A. baumannii strains (n = 5) employed in the study. Time-kill studies revealed the improved killing of these strains by the cefepime-zidebactam combination compared to that by the constituents alone. Employing a neutropenic mouse lung infection model, exposure-response analyses for all the A. baumannii strains showed that the cefepime fT>MIC required for 1-log10 kill was 38.9%. In the presence of a noneffective dose of zidebactam, the cefepime fT>MIC requirement dropped significantly to 15.5%, but it still rendered a 1-log10 kill effect. Thus, zidebactam mediated the improvement in cefepime's bactericidal effect observed in time-kill studies, manifested in vivo through the lowering of cefepime's pharmacodynamic requirement. This is a first-ever study demonstrating a β-lactam enhancer role of zidebactam that helps augment the in vivo activity of cefepime by reducing the magnitude of its pharmacodynamically relevant exposures against A. baumannii.

Optimization of a β-Lactam Scaffold for Antibacterial Activity via the Inhibition of Bacterial Type I Signal Peptidase.

β-Lactam antibiotics, one of the most important class of human therapeutics, act via the inhibition of penicillin-binding proteins (PBPs). The unparalleled success in their development has inspired efforts to develop them as inhibitors of other targets. Bacterial type I signal peptidase is evolutionarily related to the PBPs, but the stereochemistry of its substrates and its catalytic mechanism suggest that β-lactams with the 5S stereochemistry, as opposed to the 5R stereochemistry of the traditional β-lactams, would be required for inhibition. We report the synthesis and evaluation of a variety of 5S penem derivatives and identify several with promising activity against both a Gram-positive and a Gram-negative bacterial pathogen. To our knowledge these are the first 5S β-lactams to possess significant antibacterial activity and the first β-lactams imparted with antibacterial activity via optimization of the inhibition of a target other than a PBP. Along with the privileged status of their scaffold and the promise of bacterial signal peptidase I (SPase) as a target, this activity makes these compounds promising leads for development as novel therapeutics.

Mechanisms of proton relay and product release by Class A β-lactamase at ultrahigh resolution.

Structural data are available in the PDB database under the accession numbers 5TOP, 5TOY, and 5VLE.

Structural basis of metallo-β-lactamase, serine-β-lactamase and penicillin-binding protein inhibition by cyclic boronates.

β-Lactamases enable resistance to almost all β-lactam antibiotics. Pioneering work revealed that acyclic boronic acids can act as ‘transition state analogue' inhibitors of nucleophilic serine enzymes, including serine-β-lactamases. Here we report biochemical and biophysical analyses revealing that cyclic boronates potently inhibit both nucleophilic serine and zinc-dependent β-lactamases by a mechanism involving mimicking of the common tetrahedral intermediate. Cyclic boronates also potently inhibit the non-essential penicillin-binding protein PBP 5 by the same mechanism of action. The results open the way for development of dual action inhibitors effective against both serine- and metallo-β-lactamases, and which could also have antimicrobial activity through inhibition of PBPs.

Allosteric site-mediated active site inhibition of PBP2a using Quercetin 3-O-rutinoside and its combination

Recent crystallographic study revealed the involvement of allosteric site in active site inhibition of penicillin binding protein (PBP2a), where one molecule of Ceftaroline (Cef) binds to the allosteric site of PBP2a and paved way for the other molecule (Cef) to bind at the active site. Though Cef has the potency to inhibit the PBP2a, its adverse side effects are of major concern. Previous studies have reported the antibacterial property of Quercetin derivatives, a group of natural compounds. Hence, the present study aims to evaluate the effect of Quercetin 3-o-rutinoside (Rut) in allosteric site-mediated active site inhibition of PBP2a. The molecular docking studies between allosteric site and ligands (Rut, Que, and Cef) revealed a better binding efficiency (G-score) of Rut (−7.790318) and Cef (−6.194946) with respect to Que (−5.079284). Molecular dynamic (MD) simulation studies showed significant changes at the active site in the presence of ligands (Rut and Cef) at allosteric site. Four different combinations of Rut and Cef were docked and their G-scores ranged between −6.320 and −8.623. MD studies revealed the stability of the key residue (Ser403) with Rut being at both sites, compared to other complexes. Morphological analysis through electron microscopy confirmed that combination of Rut and Cefixime was able to disturb the bacterial cell membrane in a similar fashion to that of Rut and Cefixime alone. The results of this study indicate that the affinity of Rut at both sites were equally good, with further validations Rut could be considered as an alternative for inhibiting MRSA growth.

Experimental and computational approaches of a novel methyl (2E)-2-{[N-(2-formylphenyl)(4-methylbenzene)sulfonamido]methyl}-3-(4-chlorophenyl)prop-2-enoate: A potential antimicrobial agent and an inhibition of penicillin-binding protein

The title compound methyl(2E)-2-{[N-(2-formylphenyl) (4-methylbenzene)sulfonamido]methyl}-3-(4-chlorophenyl) prop-2-enoate (MFMSC) has been synthesized and single crystals were grown by slow evaporation solution growth technique at room temperature. Structural and vibrational spectroscopic studies were carried out by using single crystal X-ray diffraction, FT-IR and NMR spectral analysis together with DFT method using Gaussian'03 software. The detailed interpretation of the vibrational spectra has been carried out by VEDA program. NBO analysis, Mulliken charge analysis, HOMO-LUMO, MEP, Global chemical reactivity descriptors and thermodynamic properties have been analyzed. The hyperpolarisability calculation reveals the present material has a reasonably good propensity for nonlinear optical activity. The obtained antimicrobial activity results indicate that the compound shows good to moderate activity against all tested bacterial and fungal pathogens. A computational study was also carried out to predict the drug-likeness and ADMET properties of the title compound. Due to the different potential biological activity of the title compound, molecular docking study is also reported and the compound might exhibit inhibitory activity against penicillin-binding protein PBP-2X.

Proteochemometric model for predicting the inhibition of penicillin-binding proteins.

Neisseria gonorrhoeae infection threatens to become an untreatable sexually transmitted disease in the near future owing to the increasing emergence of N. gonorrhoeae strains with reduced susceptibility and resistance to the extended-spectrum cephalosporins (ESCs), i.e. ceftriaxone and cefixime, which are the last remaining option for first-line treatment of gonorrhea. Alteration of the penA gene, encoding penicillin-binding protein 2 (PBP2), is the main mechanism conferring penicillin resistance including reduced susceptibility and resistance to ESCs. To predict and investigate putative amino acid mutations causing β-lactam resistance particularly for ESCs, we applied proteochemometric modeling to generalize N. gonorrhoeae susceptibility data for predicting the interaction of PBP2 with therapeutic β-lactam antibiotics. This was afforded by correlating publicly available data on antimicrobial susceptibility of wild-type and mutant N. gonorrhoeae strains for penicillin-G, cefixime and ceftriaxone with 50 PBP2 protein sequence data using partial least-squares projections to latent structures. The generated model revealed excellent predictability (R2=0.91, Q2=0.77, QExt2=0.78). Moreover, our model identified amino acid mutations in PBP2 with the highest impact on antimicrobial susceptibility and provided information on physicochemical properties of amino acid mutations affecting antimicrobial susceptibility. Our model thus provided insight into the physicochemical basis for resistance development in PBP2 suggesting its use for predicting and monitoring novel PBP2 mutations that may emerge in the future.

Ceftolozane/Tazobactam: A Novel Cephalosporin/β-Lactamase Inhibitor Combination with Activity Against Multidrug-Resistant Gram-Negative Bacilli

Ceftolozane is a novel cephalosporin currently being developed with the β-lactamase inhibitor tazobactam for the treatment of complicated urinary tract infections (cUTIs), complicated intra-abdominal infections (cIAIs), and ventilator-associated bacterial pneumonia (VABP). The chemical structure of ceftolozane is similar to that of ceftazidime, with the exception of a modified side-chain at the 3-position of the cephem nucleus, which confers potent antipseudomonal activity. As a β-lactam, its mechanism of action is the inhibition of penicillin-binding proteins (PBPs). Ceftolozane displays increased activity against Gram-negative bacilli, including those that harbor classical β-lactamases (e.g., TEM-1 and SHV-1), but, similar to other oxyimino-cephalosporins such as ceftazidime and ceftriaxone, it is compromised by extended-spectrum β-lactamases (ESBLs) and carbapenemases. The addition of tazobactam extends the activity of ceftolozane to include most ESBL producers as well as some anaerobic species. Ceftolozane is distinguished from other cephalosporins by its potent activity versus Pseudomonas aeruginosa, including various drug-resistant phenotypes such as carbapenem, piperacillin/tazobactam, and ceftazidime-resistant isolates, as well as those strains that are multidrug-resistant (MDR). Its antipseudomonal activity is attributed to its ability to evade the multitude of resistance mechanisms employed by P.aeruginosa, including efflux pumps, reduced uptake through porins and modification of PBPs. Ceftolozane demonstrates linear pharmacokinetics unaffected by the coadministration of tazobactam; specifically, it follows a two-compartmental model with linear elimination. Following single doses, ranging from 250 to 2,000mg, over a 1-h intravenous infusion, ceftolozane displays a mean plasma half-life of 2.3h (range 1.9-2.6h), a steady-state volume of distribution that ranges from 13.1 to 17.6L, and a mean clearance of 102.4mL/min. It demonstrates low plasma protein binding (20%), is primarily eliminated via urinary excretion (≥92%), and may require dose adjustments in patients with a creatinine clearance <50mL/min. Time-kill experiments and animal infection models have demonstrated that the pharmacokinetic-pharmacodynamic index that is best correlated with ceftolozane's in vivo efficacy is the percentage of time in which free plasma drug concentrations exceed the minimum inhibitory concentration of a given pathogen (%fT >MIC), as expected of β-lactams. Two phaseII clinical trials have been conducted to evaluate ceftolozane±tazobactam in the settings of cUTIs and cIAIs. One trial compared ceftolozane 1,000mg every 8h (q8h) versus ceftazidime 1,000mg q8h in the treatment of cUTI, including pyelonephritis, and demonstrated similar microbiologic and clinical outcomes, as well as a similar incidence of adverse effects after 7-10days of treatment, respectively. A second trial has been conducted comparing ceftolozane/tazobactam 1,000/500mg and metronidazole 500mg q8h versus meropenem 1,000mg q8h in the treatment of cIAI. A number of phaseI and phaseII studies have reported ceftolozane to possess a good safety and tolerability profile, one that is consistent with that of other cephalosporins. In conclusion, ceftolozane is a new cephalosporin with activity versus MDR organisms including P.aeruginosa. Tazobactam allows the broadening of the spectrum of ceftolozane versus β-lactamase-producing Gram-negative bacilli including ESBLs. Potential roles for ceftolozane/tazobactam include empiric therapy where infection by a resistant Gram-negative organism (e.g., ESBL) is suspected, or as part of combination therapy (e.g., with metronidazole) where a polymicrobial infection is suspected. In addition, ceftolozane/tazobactam may represent alternative therapy to the third-generation cephalosporins after treatment failure or for documented infections due to Gram-negative bacilli producing ESBLs. Finally, the increased activity of ceftolozane/tazobactam versus P.aeruginosa, including MDR strains, may lead to the treatment of suspected and documented P.aeruginosa infections with this agent. Currently, ceftolozane/tazobactam is being evaluated in three phaseIII trials for the treatment of cUTI, cIAI, and VABP.

Structure guided development of potent reversibly binding penicillin binding protein inhibitors.

Following from the evaluation of different types of electrophiles, combined modeling and crystallographic analyses are used to generate potent boronic acid based inhibitors of a penicillin binding protein. The results suggest that a structurally informed approach to penicillin binding protein inhibition will be useful for the development of both improved reversibly binding inhibitors, including boronic acids, and acylating inhibitors, such as β-lactams.

ホスホマイシンの前投与がテイコプラニンの効果を増強したと考えられた重症メチシリン耐性黄色ブドウ球菌（ＭＲＳＡ）の一症例

Teicoplanin is a glycopeptide antibiotic that is highly effective against methicillin-resistant Staphylococcus aureus (MRSA).However,it is recommended that a loading procedure is used to enable teicoplanin to rapidly reach optimal plasma trough levels.Fosfomycin,a cell wall synthesis-inhibiting antibiotic,prevents biofilm formation through the inhibition of penicillin-binding protein 2’in MRSA.We report a case in which teicoplanin and fosfomycin had a marked synergistic effect in a patient with a serious MRSA infection,in whom C-reactive protein levels stayed high despite teicoplanin having reached an optimal plasma concentration.The combination of fosfomycin and teicoplaninachievedobviousimprovementsininflammatorymarkersandMRSAwas eradicated from the blood and sputum.

The Rcs Phosphorelay Is a Cell Envelope Stress Response Activated by Peptidoglycan Stress and Contributes to Intrinsic Antibiotic Resistance

Gram-negative bacteria possess stress responses to maintain the integrity of the cell envelope. Stress sensors monitor outer membrane permeability, envelope protein folding, and energization of the inner membrane. The systems used by gram-negative bacteria to sense and combat stress resulting from disruption of the peptidoglycan layer are not well characterized. The peptidoglycan layer is a single molecule that completely surrounds the cell and ensures its structural integrity. During cell growth, new peptidoglycan subunits are incorporated into the peptidoglycan layer by a series of enzymes called the penicillin-binding proteins (PBPs). To explore how gram-negative bacteria respond to peptidoglycan stress, global gene expression analysis was used to identify Escherichia coli stress responses activated following inhibition of specific PBPs by the beta-lactam antibiotics amdinocillin (mecillinam) and cefsulodin. Inhibition of PBPs with different roles in peptidoglycan synthesis has different consequences for cell morphology and viability, suggesting that not all perturbations to the peptidoglycan layer generate equivalent stresses. We demonstrate that inhibition of different PBPs resulted in both shared and unique stress responses. The regulation of capsular synthesis (Rcs) phosphorelay was activated by inhibition of all PBPs tested. Furthermore, we show that activation of the Rcs phosphorelay increased survival in the presence of these antibiotics, independently of capsule synthesis. Both activation of the phosphorelay and survival required signal transduction via the outer membrane lipoprotein RcsF and the response regulator RcsB. We propose that the Rcs pathway responds to peptidoglycan damage and contributes to the intrinsic resistance of E. coli to beta-lactam antibiotics.