The mechanism of ceftazidime and cefiderocol hydrolysis by D179Y variants of KPC carbapenemases is similar and involves the formation of a long-lived covalent intermediate.

Klebsiella pneumoniae carbapenemase (KPC) variants have been described that confer resistance to both ceftazidime-avibactam and cefiderocol. Of these, KPC-33 and KPC-31 are D179Y-containing variants derived from KPC-2 and KPC-3, respectively. To better understand this atypical phenotype, the catalytic mechanism of ceftazidime and cefiderocol hydrolysis by KPC-33 and KPC-31 as well as the ancestral KPC-2 and KPC-3 enzymes was studied. Steady-state kinetics showed that the D179Y substitution in either KPC-2 or KPC-3 is associated with a large decrease in both kcat and KM such that kcat/KM values were largely unchanged for both ceftazidime and cefiderocol substrates. A decrease in both kcat and KM is consistent with a decreased and rate-limiting deacylation step. We explored this hypothesis by performing pre-steady-state kinetics and showed that the acylation step is rate-limiting for KPC-2 and KPC-3 for both ceftazidime and cefiderocol hydrolysis. In contrast, we observed a burst of acyl-enzyme formation followed by a slow steady-state rate for the D179Y variants of KPC-2 and KPC-3 with either ceftazidime or cefiderocol, indicating that deacylation of the covalent intermediate is the rate-limiting step for catalysis. Finally, we show that the low KM value for ceftazidime or cefiderocol hydrolysis of the D179Y variants is not an indication of tight binding affinity for the substrates but rather is a reflection of the deacylation reaction becoming rate-limiting. Thus, the hydrolysis mechanism of ceftazidime and cefiderocol by the D179Y variants is very similar and involves the formation of a long-lived covalent intermediate that is associated with resistance to the drugs.

The Structural Role of N170 in Substrate-Assisted Deacylation in KPC-2 ß-Lactamase.

The amino acid substitutions in Klebsiella pneumoniae carbapenemase 2 (KPC-2) that have arisen in the clinic are observed to lead to the development of resistance to ceftazidime-avibactam, a preferred treatment for KPC bearing Gram-negative bacteria. Specific substitutions in the omega loop (R164-D179) result in changes in the structure and function of the enzyme, leading to alterations in substrate specificity, decreased stability, and more recently observed, increased resistance to ceftazidime/avibactam. Using accelerated rare-event sampling well-tempered metadynamics simulations, we explored in detail the structural role of R164 and D179 variants that are described to confer ceftazidime/avibactam resistance. The buried conformation of D179 substitutions produce a pronounced structural disorder in the omega loop - more than R164 mutants, where the crystallographic omega loop structure remains mostly intact. Our findings also reveal that the conformation of N170 plays an underappreciated role impacting drug binding and restricting deacylation. The results further support the hypothesis that KPC-2 D179 variants employ substrate-assisted catalysis for ceftazidime hydrolysis, involving the ring amine of the aminothiazole group to promote deacylation and catalytic turnover. Moreover, the shift in the WT conformation of N170 contributes to reduced deacylation and an altered spectrum of enzymatic activity.

Glycopeptide Antibiotics Impair Neutrophil Effector Functions.

INTRODUCTION: Neutrophilic granulocytes represent the first line of defense against microorganisms. Granulocytes phagocytose microorganisms and specifically synthesize oxygen radicals against them, which eventually kills the invaders. METHODS: Neutrophilic granulocytes were isolated from peripheral blood of healthy volunteer donors. Putative interference of new-generation antibiotics with neutrophil function was tested using a collection of granulocyte-stimulating agents and Amplex™ Red-based plate assay and flow cytometry-based respiratory burst assays. In addition, phagocytosis of E. coli, IL-8 production, bactericidal activity, and CD62L expression of granulocytes were evaluated. RESULTS: Of note, we found that the two glycopeptide antibiotics dalbavancin and teicoplanin inhibited ROS production upon granulocyte activation via different signaling pathways in a dose-dependent manner. Dalbavancin also blocked the PMA-induced shedding of CD62L. In contrast, the oxazolidinone antibiotics tedizolid and linezolid had no effect on neutrophil function, while the combination of ceftazidime/avibactam dose dependently inhibited the fMLP/Cytochalasin B-induced granulocyte burst in a dose-dependent manner. Additionally, we showed that dalbavancin and teicoplanin as well as sulfametrole/trimethoprim and ceftazidime/avibactam inhibited baseline and PMA-induced IL-8 production by neutrophilic granulocytes. Moreover, dalbavancin impaired the bactericidal activity of neutrophilic granulocytes. CONCLUSION: We here identified hitherto unknown inhibitory effects of several classes of antibiotics on the effector functions of neutrophilic granulocytes.

Asp179 in the class A ß-lactamase from Mycobacterium tuberculosis is a conserved yet not essential residue due to epistasis.

Conserved residues are often considered essential for function, and substitutions in such residues are expected to have a negative influence on the properties of a protein. However, mutations in a few highly conserved residues of the ß-lactamase from Mycobacterium tuberculosis, BlaC, were shown to have no or only limited negative effect on the enzyme. One such mutant, D179N, even conveyed increased ceftazidime resistance upon bacterial cells, while displaying good activity against penicillins. The crystal structures of BlaC D179N in resting state and in complex with sulbactam reveal subtle structural changes in the Ω-loop as compared to the structure of wild-type BlaC. Introducing this mutation in four other ß-lactamases, CTX-M-14, KPC-2, NMC-A and TEM-1, resulted in decreased antibiotic resistance for penicillins and meropenem. The results demonstrate that the Asp in position 179 is generally essential for class A ß-lactamases but not for BlaC, which can be explained by the importance of the interaction with the side chain of Arg164 that is absent in BlaC. It is concluded that Asp179 though conserved is not essential in BlaC, as a consequence of epistasis.

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.

Zinc oxide nanoparticles impact the expression of the genes involved in toxin-antitoxin systems in multidrug-resistant Acinetobacter baumannii.

The aim of this study was to investigate the effect of zinc oxide nanoparticles (ZnO-NPs) on the expression of genes involved in toxin-antitoxin (TA) systems in multidrug-resistant (MDR) Acinetobacter baumannii. Seventy clinical isolates of A. baumannii were collected from variuos clinical samples. Antimicrobial susceptibility test was determined by disk diffusion. Type II TA system-related genes including GNAT, XRE-like, hipA, hipB, hicA, hicB were screened using polymerase chain reaction (PCR). ZnO-NPs prepared and characterized by field emission scanning electron microscopy and X-ray diffraction. MIC of ZnO-NPs of A. baumannii isolates was performed using the microdilution method. The expression of type II TA systems-related genes were assessed with and without exposure to ZnO-NPs using real-time PCR. The highest rate of resistance and sensitivity was observed against cefepime (77.14%), and ampicillin/sulbactam (42.85%), respectively. All A. baumannii isolates were considered as MDR. In this study, three TA loci were identified for A. baumannii including GNAT/XRE-like, HicA/HicB, and HipA/HipB and their prevalence was 100%, 42%, and 27.1%, respectively. There was no significant relationship between the prevalence of these systems and the origin of A. baumannii. Our data showed significant correlations between the presence of HicA/HicB system and resistance to ceftazidime, meropenem, imipenem, and cefepime (p < 0.05), and the presence of HipA/HipB system and resistance to ceftazidime, meropenem, imipenem, and cefepime (p < 0.05). In presence of ZnO-NPs, the expression of all studied genes decreased. GNAT and hicB showed the highest and lowest expression changes by 2.4 folds (p < 0.001) and 1.3 folds (p < 0.05), respectively. This study demonstrates the promising potential of nanoparticles to impact the expression of the genes involved in TA Systems. So, the application of ZnO-NPs may be helpful to design target-based strategies towards MDRs pathogens for empowered clinical applications by microbiologists and nanotechnologists.

Prevalence of Efflux Pump and Porin-Related Antimicrobial Resistance in Clinical Klebsiella pneumoniae in Baghdad, Iraq.

Klebsiella pneumoniae is an opportunistic bacterium that causes many infections, including septicemia, pneumonia, urinary tract infection, and liver abscesses. There are many mechanisms for antibiotic resistance and K. pneumonia is considered a multidrug-resistant pathogen. This study aimed to find the correlation between the susceptibility of K. pneumonia to certain antibiotics with the porin-related resistance and pumps mechanisms. In total, two genes that are responsible for porin formation were considered in the current study OmpK-35gene and OmpK-36 gene, in addition to other four genes (CfiaS, CfiaL, MFS, and MdtK genes) related to an efflux pump mechanism of antibiotic resistance. The bacterial resistance was investigated towards five cephalosporins (Cefazolin, Cefoxitin, Ceftazidime, Ceftriaxone, and Cefepime) and two carbapenems (imipenem and ertapenem). Clinical samples, including blood, swabs, and urine, consisting of 20 specimens for each group, were collected from patients who attended three hospitals in Baghdad. The VITEK-2 system and genetic tests (polymerase chain reaction and sequencing) of bacterial isolates were applied to confirm the diagnosis of K. pneumoniae and detect the antibiotic sensitivity profile. The results showed that 51 (85%) and 15 (25%) of the total 60 isolates had positive results for OmpK-35 and Omp-K36 genes, respectively. The MFS and MdtK genes were observed (70-88.3%) in cephalosporin-resistant isolates of K. pneumoniae. There were no significant variations of bacterial resistance genes of antibiotics within the specimen groups. It was concluded that the bacterial resistance of the selected antibiotics was elevated markedly with the loss of the OmpK-36 gene with a high expression of MFS and MdtK genes and a slight minimal occurrence in the new generation of carbapenems. The best antimicrobial agent was ertapenem with a percentage of 0% of resistance in all bacterial isolates.

Relevance of preclinical rodent pharmacokinetics in the selection of a companion antibiotic for combining with beta-lactamase inhibitor.

Recent approvals of beta-lactamase inhibitor (BLI) drug in combination with cephalosporins/penems have provided the right impetus for novel BLIs. One important research question, hitherto not addressed, is pertaining to the relevance of preclinical pharmacokinetics for pairing the antibiotic with existing/novel BLI.Two BLI combination drugs: (a) approved (i.e. ceftazidime/avibactam); (b) clinical development (i.e. cefepime/zidebactam) were explored to provide insights to address the research question.Individual intravenous dosing of ceftazidime, avibactam, cefepime and zidebactam was done at 1 mg/kg by intravenous route in Balb/c mice and Wistar rats. Serial blood samples were collected and analysed by LC-MS/MS method.Examination of the ratios of pharmacokinetic parameters (CL, VSS and T1/2) for individual drugs in combinations (for instance, CL (ceftazidime)/CL (avibactam); CL (cefepime)/CL (zidebactam)) suggested that the pharmacokinetic data gathered in rats were generally within 0.5- to 2-fold; but mouse data revealed larger disparity for VSS (0.11- to 8.25-fold) or CL (0.49- to 4.03-fold).The observed ratio for CL/VSS observed in rats agreed with corresponding human ratios for the pairwise comparison of the individual drugs in the combinations.Retrospectively, current pharmacokinetic findings suggest rat pharmacokinetic data may aid the combination of BLI with an appropriate antibiotic.

Molecular Basis of Class A ß-Lactamase Inhibition by Relebactam.

ß-Lactamase production is the major ß-lactam resistance mechanism in Gram-negative bacteria. ß-Lactamase inhibitors (BLIs) efficacious against serine ß-lactamase (SBL) producers, especially strains carrying the widely disseminated class A enzymes, are required. Relebactam, a diazabicyclooctane (DBO) BLI, is in phase 3 clinical trials in combination with imipenem for the treatment of infections by multidrug-resistant Enterobacteriaceae We show that relebactam inhibits five clinically important class A SBLs (despite their differing spectra of activity), representing both chromosomal and plasmid-borne enzymes, i.e., the extended-spectrum ß-lactamases L2 (inhibition constant 3 µM) and CTX-M-15 (21 µM) and the carbapenemases KPC-2, -3, and -4 (1 to 5 µM). Against purified class A SBLs, relebactam is an inferior inhibitor compared with the clinically approved DBO avibactam (9- to 120-fold differences in half maximal inhibitory concentration [IC50]). MIC assays indicate relebactam potentiates ß-lactam (imipenem) activity against KPC-producing Klebsiella pneumoniae, with similar potency to avibactam (with ceftazidime). Relebactam is less effective than avibactam in combination with aztreonam against Stenotrophomonas maltophilia K279a. X-ray crystal structures of relebactam bound to CTX-M-15, L2, KPC-2, KPC-3, and KPC-4 reveal its C2-linked piperidine ring can sterically clash with Asn104 (CTX-M-15) or His/Trp105 (L2 and KPCs), rationalizing its poorer inhibition activity than that of avibactam, which has a smaller C2 carboxyamide group. Mass spectrometry and crystallographic data show slow, pH-dependent relebactam desulfation by KPC-2, -3, and -4. This comprehensive comparison of relebactam binding across five clinically important class A SBLs will inform the design of future DBOs, with the aim of improving clinical efficacy of BLI-ß-lactam combinations.

Antibacterial Countermeasures via Metal-Organic Framework-Supported Sustained Therapeutic Release.

Long-term antimicrobial therapies are necessary to treat infections caused by virulent intracellular pathogens, including biothreat agents. Current treatment plans include injectable therapeutics given multiple times daily over a period for up to 8 weeks. Here, we present a metal-organic framework (MOF), zeolitic imidazolate framework-8 (ZIF-8), as a robust platform to support the sustained release of ceftazidime, an important antimicrobial agent for many critical bacterial infections. Detailed material characterization confirms the successful encapsulation of ceftazidime within the ZIF-8 matrix, indicating sustained drug release for up to a week. The antibacterial properties of ceftazidime@ZIF-8 particles were confirmed against Escherichia coli, chosen here as a representative of Gram-negative bacteria infection model in a proof-of-concept study. Further, we showed that this material system is compatible with macrophage and lung epithelial cell lines, relevant targets for antibacterial therapy for pulmonary and intracellular infections. A promising methodology to enhance the treatment of intracellular infections is to deliver the antibiotic cargo intracellularly. Importantly, this is the first study to unequivocally demonstrate direct MOF particle internalization using confocal microscopy via 3D reconstructions of z-stacks, taking advantage of the intrinsic emission properties of ZIF-8. This is an important development as it circumvents the need to use any staining dyes and addresses current methodology limitations concerning false impression of cargo uptake in the event of the carrier particle breakdown within biological media.

The Reaction Mechanism of Metallo-ß-Lactamases Is Tuned by the Conformation of an Active-Site Mobile Loop.

Carbapenems are "last resort" ß-lactam antibiotics used to treat serious and life-threatening health care-associated infections caused by multidrug-resistant Gram-negative bacteria. Unfortunately, the worldwide spread of genes coding for carbapenemases among these bacteria is threatening these life-saving drugs. Metallo-ß-lactamases (MßLs) are the largest family of carbapenemases. These are Zn(II)-dependent hydrolases that are active against almost all ß-lactam antibiotics. Their catalytic mechanism and the features driving substrate specificity have been matter of intense debate. The active sites of MßLs are flanked by two loops, one of which, loop L3, was shown to adopt different conformations upon substrate or inhibitor binding, and thus are expected to play a role in substrate recognition. However, the sequence heterogeneity observed in this loop in different MßLs has limited the generalizations about its role. Here, we report the engineering of different loops within the scaffold of the clinically relevant carbapenemase NDM-1. We found that the loop sequence dictates its conformation in the unbound form of the enzyme, eliciting different degrees of active-site exposure. However, these structural changes have a minor impact on the substrate profile. Instead, we report that the loop conformation determines the protonation rate of key reaction intermediates accumulated during the hydrolysis of different ß-lactams in all MßLs. This study demonstrates the existence of a direct link between the conformation of this loop and the mechanistic features of the enzyme, bringing to light an unexplored function of active-site loops on MßLs.

Vitamin E Increases Antimicrobial Sensitivity by Inhibiting Bacterial Lipocalin Antibiotic Binding.

Burkholderia cenocepacia is an opportunistic Gram-negative bacterium that causes serious respiratory infections in patients with cystic fibrosis. Recently, we discovered that B. cenocepacia produces the extracellular bacterial lipocalin protein BcnA upon exposure to sublethal concentrations of bactericidal antibiotics. BcnA captures a range of antibiotics outside bacterial cells, providing a global extracellular mechanism of antimicrobial resistance. In this study, we investigated water-soluble and liposoluble forms of vitamin E as inhibitors of antibiotic binding by BcnA. Our results demonstrate that in vitro, both vitamin E forms bind strongly to BcnA and contribute to reduce the MICs of norfloxacin (a fluoroquinolone) and ceftazidime (a ß-lactam), both of them used as model molecules representing two different chemical classes of antibiotics. Expression of BcnA was required for the adjuvant effect of vitamin E. These results were replicated in vivo using the Galleria mellonella larva infection model whereby vitamin E treatment, in combination with norfloxacin, significantly increased larva survival upon infection in a BcnA-dependent manner. Together, our data suggest that vitamin E can be used to increase killing by bactericidal antibiotics through interference with lipocalin binding.IMPORTANCE Bacteria exposed to stress mediated by sublethal antibiotic concentrations respond by adaptive mechanisms leading to an overall increase of antibiotic resistance. One of these mechanisms involves the release of bacterial proteins called lipocalins, which have the ability to sequester antibiotics in the extracellular space before they reach bacterial cells. We speculated that interfering with lipocalin-mediated antibiotic binding could enhance the efficacy of antibiotics to kill bacteria. In this work, we report that when combined with bactericidal antibiotics, vitamin E contributes to enhance bacterial killing both in vitro and in vivo. This adjuvant effect of vitamin E requires the presence of BcnA, a bacterial lipocalin produced by the cystic fibrosis pathogen Burkholderia cenocepacia Since most bacteria produce lipocalins like BcnA, we propose that our findings could be translated into making novel antibiotic adjuvants to potentiate bacterial killing by existing antibiotics.

Loss of activity of ceftazidime-avibactam due to MexAB-OprM efflux and overproduction of AmpC cephalosporinase in Pseudomonas aeruginosa isolated from patients suffering from cystic fibrosis.

In Pseudomonas aeruginosa (P. aeruginosa) collected from cystic fibrosis (CF) patients, 24% resistance to ceftazidime-avibactam in isolates negative for carbapenemases and extended-spectrum ß-lactamases (ESBLs) has previously been observed. The current study aimed to unravel the underlying mechanism(s). Using the laboratory strain PAO1 and derivatives thereof, with ampC expression induced by a sub-minimum inhibitory concentration (MIC) of imipenem, a higher MIC of ceftazidime-avibactam was found for those overexpressing MexAB-OprM (quantitative polymerase chain reaction (PCR) of mexA) and, to a lesser extent, MexEF-OprN (PCR of mexE), or without OprD expression (SDS-Page and Coomassie blue staining). This was ascribed to (i) an efflux of avibactam (efflux mutants) and (ii) a lack of avibactam penetration (OprD mutants), respectively. We then used 10 CF clinical isolates resistant to ceftazidime (MIC ≥ 128 mg/L) and with (i) variable basal levels of ampC overexpression, (ii) mutations in mexA or mexB inactivating to variable extent the MexAB-OprM transport capacity (assessed by extrusion of N-phenyl-1-naphthylamine [NPN]), and (iii) expression or not of mexE and of OprD porin. The reduction of ceftazidime MIC in the presence of avibactam was partially lost for isolates with large efflux activity of MexAB-OprM and/or increased ampC expression, but not significantly with mexE expression or lack of OprD (non-parametric and parametric tests). This identified MexAB-OprM as a main avibactam efflux transporter in P. aeruginosa that, together with ampC overexpression, reduced avibactam potency. Since about 30% of CF isolates show mutations in MexAB-OprM compromising efflux (Chalhoub, et al. Sci Reports 2017;7:40208), routine susceptibility testing of CF P. aeruginosa with ceftazidime-avibactam is warranted.

Getting Drugs through Small Pores: Exploiting the Porins Pathway in Pseudomonas aeruginosa.

Understanding molecular properties of outer membrane channels of Gram-negative bacteria is of fundamental significance as they are the entry point of polar antibiotics into bacteria. Outer membrane proteomics revealed OccK8 (OprE) to be among the five most expressed substrate specific channels of the clinically important Pseudomonas aeruginosa. The high-resolution X-ray structure and electrophysiology highlighted a very narrow pore. However, experimental in vitro methods showed the transport of natural amino acids and antibiotics, among them ceftazidime. We used molecular dynamics simulations to reveal the importance of the physicochemical properties of ceftazidime in modulating the translocation through OccK8, proposing a structure-function relationship. As in general porins, the internal electric field favors the translocation of polar molecules by gainful energy compensation in the central constriction region. Importantly, the comparatively narrow OccK8 pore can undergo a substrate-induced expansion to accommodate relatively large-sized substrates.

Defining Substrate Specificity in the CTX-M Family: the Role of Asp240 in Ceftazidime Hydrolysis.

The natural diversification of CTX-M ß-lactamases led to the emergence of Asp240Gly variants in the clinic that confer reduced susceptibility to ceftazidime (CAZ). In this study, we compared the impact of this substitution on CAZ and ceftazidime-avibactam (CZA) MICs against isogenic Escherichia coli strains with different porin deficiencies. Our results show a noticeable increase in CAZ resistance in clones expressing Asp240Gly-harboring CTX-M when combined with OmpF porin deficiency. Kinetic analysis revealed that the kcat/Km for CAZ was 5- to 15-fold higher for all Asp240Gly variants but remained 200- to 725-fold lower than that for cefotaxime (CTX). In vitro selection of CAZ-resistant clones yielded nonsusceptible CTX-M producers (MIC of >16 µg/ml) only after overnight incubation; the addition of avibactam (AVI) decreased MICs to a susceptible range against these variants. In contrast, the use of CZA as a selective agent did not yield resistant clones. AVI inactivated both CTX-M-12 and CTX-M-96, with an apparent inhibition constant comparable to that of SHV-2 and 1,000-fold greater than that of PER-2 and CMY-2, and k2/K for CTX-M-12 was 24- and 35-fold higher than that for CTX-M-96 and CTX-M-15, respectively. Molecular modeling suggests that AVI interacts similarly with CTX-M-96 and CTX-M-15. We conclude that the impact of Asp240Gly in resistance may arise when other mechanisms are also present (i.e., OmpF deficiency). Additionally, CAZ selection could favor the emergence of CAZ-resistant subpopulations. These results define the role of Asp240 and the impact of the -Gly substitution and allow us to hypothesize that the use of CZA is an effective preventive strategy to delay the development of resistance in this family of extended-spectrum ß-lactamases.

Klebsiella pneumoniae Carbapenemase-2 (KPC-2), Substitutions at Ambler Position Asp179, and Resistance to Ceftazidime-Avibactam: Unique Antibiotic-Resistant Phenotypes Emerge from ß-Lactamase Protein Engineering.

The emergence of Klebsiella pneumoniae carbapenemases (KPCs), ß-lactamases that inactivate "last-line" antibiotics such as imipenem, represents a major challenge to contemporary antibiotic therapies. The combination of ceftazidime (CAZ) and avibactam (AVI), a potent ß-lactamase inhibitor, represents an attempt to overcome this formidable threat and to restore the efficacy of the antibiotic against Gram-negative bacteria bearing KPCs. CAZ-AVI-resistant clinical strains expressing KPC variants with substitutions in the Ω-loop are emerging. We engineered 19 KPC-2 variants bearing targeted mutations at amino acid residue Ambler position 179 in Escherichia coli and identified a unique antibiotic resistance phenotype. We focus particularly on the CAZ-AVI resistance of the clinically relevant Asp179Asn variant. Although this variant demonstrated less hydrolytic activity, we demonstrated that there was a prolonged period during which an acyl-enzyme intermediate was present. Using mass spectrometry and transient kinetic analysis, we demonstrated that Asp179Asn "traps" ß-lactams, preferentially binding ß-lactams longer than AVI owing to a decreased rate of deacylation. Molecular dynamics simulations predict that (i) the Asp179Asn variant confers more flexibility to the Ω-loop and expands the active site significantly; (ii) the catalytic nucleophile, S70, is shifted more than 1.5 Å and rotated more than 90°, altering the hydrogen bond networks; and (iii) E166 is displaced by 2 Å when complexed with ceftazidime. These analyses explain the increased hydrolytic profile of KPC-2 and suggest that the Asp179Asn substitution results in an alternative complex mechanism leading to CAZ-AVI resistance. The future design of novel ß-lactams and ß-lactamase inhibitors must consider the mechanistic basis of resistance of this and other threatening carbapenemases.IMPORTANCE Antibiotic resistance is emerging at unprecedented rates and threatens to reach crisis levels. One key mechanism of resistance is the breakdown of ß-lactam antibiotics by ß-lactamase enzymes. KPC-2 is a ß-lactamase that inactivates carbapenems and ß-lactamase inhibitors (e.g., clavulanate) and is prevalent around the world, including in the United States. Resistance to the new antibiotic ceftazidime-avibactam, which was designed to overcome KPC resistance, had already emerged within a year. Using protein engineering, we uncovered a mechanism by which resistance to this new drug emerges, which could arm scientists with the ability to forestall such resistance to future drugs.

Early postnatal gentamicin and ceftazidime treatment in normal and food restricted neonatal wistar rats: Implications for kidney development.

BACKGROUND: Up to two-thirds of premature born neonates are treated for infections with aminoglycosides such as gentamicin. Although acute toxicities are well described, there is uncertainty on developmental changes after treatment of premature born neonates. We studied the effect of gentamicin and ceftazidime on kidney development in the rat. Additionally, we evaluated the modulating effect of extrauterine growth restriction. METHODS: On postnatal day (PND) 2, Wistar rats were cross-fostered into normal sized litters (12 pups) or large litters (20 pups) to create normal food (NF) or food restricted (FR) litters to simulate growth restriction and dosed daily intraperitoneally with placebo, 4 mg/kg of gentamicin or 50 mg/kg ceftazidime until PND 8. Gentamicin pharmacokinetics were studied in a separate group of animals. Kidneys were weighed. Renal expression of 18 developmental genes was evaluated by quantitative PCR on PND 8. On PND 35, glomerular number was assessed by stereology and glomerular generations were counted. RESULTS: Food restricted litters showed 22% less body weight compared with controls by day 35 (p < 0.001), 1.4- to 1.5-fold down regulation of Renin, Oat1, and Agtr1a (p < 0.05) expression and a 12% reduction in glomerular numbers (mean 30841 vs. 35187, p < 0.001), whereas glomerular generation count was unaffected. Gentamicin pharmacokinetic parameters were found to be in a human clinical range (mean maximum concentration in plasma of 4.88 mg/L and mean area under the plasma-concentration time curve up to the last measured concentration after 4 hr of 10.71 mg.h/L for sexes combined) and all endpoints were unaffected. Ceftazidime reduced Renin expression by 1.7-fold (p < 0.01). CONCLUSION: Our experiments showed that gentamicin at clinical levels did not disturb kidney development, ceftazidime can affect Renin expression, and extrauterine growth restriction impairs kidney development, but did not modulate potential drug toxicity. Birth Defects Research 109:1228-1235, 2017. © 2017 Wiley Periodicals, Inc.

Probing the interaction of cephalosporin antibiotic-ceftazidime with human serum albumin: A biophysical investigation.

The fate of drug administered to a living organism depends on drug's pharmacokinetics as well as pharmacological behavior. Serum albumins (proteins in blood plasma of human) act as a carrier molecule to deliver the drug at specific site. In the present study, we have explored the mechanism of interaction between cephalosporin antibiotic-ceftazidime (CFD) and human serum albumin (HSA) by spectroscopic and molecular docking studies. Quenching of HSA fluorescence by CFD inferred that it binds to HSA through static quenching mechanism; with binding affinity in order of 104M-1. Fluorescence resonance energy transfer (FRET) results shows that donor and acceptor molecule are at 2.08nm apart and also reflects the high probability of energy transfer between HSA and CFD. Change in secondary structure as well as microenvironment around both tryptophan and tyrosine residue, were monitored by Circular Dichroism (CD) and Synchronous fluorescence spectroscopy respectively; confirms that CFD increases the alpha helical secondary structure as well as altered the environment around tryptophan and tyrosine. The specific binding site of CFD on HSA was determined by site-specific markers and molecular docking methods. CFD preferably bind to subdomain IIIA (Sudlow site II) on HSA.

The Drug-Resistant Variant P167S Expands the Substrate Profile of CTX-M ß-Lactamases for Oxyimino-Cephalosporin Antibiotics by Enlarging the Active Site upon Acylation.

CTX-M ß-lactamases provide resistance against the ß-lactam antibiotic, cefotaxime, but not a related antibiotic, ceftazidime. ß-Lactamases that carry the P167S substitution, however, provide ceftazidime resistance. In this study, CTX-M-14 was used as a model to study the structural changes caused by the P167S mutation that accelerate ceftazidime turnover. X-ray crystallography was used to determine the structures of the P167S apoenzyme along with the structures of the S70G/P167S, E166A/P167S, and E166A mutant enzymes complexed with ceftazidime as well as the E166A/P167S apoenzyme. The S70G and E166A mutations allow capture of the enzyme-substrate complex and the acylated form of ceftazidime, respectively. The results showed a large conformational change in the Ω-loop of the ceftazidime acyl-enzyme complex of the P167S mutant but not in the enzyme-substrate complex, suggesting the change occurs upon acylation. The change results in a larger active site that prevents steric clash between the aminothiazole ring of ceftazidime and the Asn170 residue in the Ω-loop, allowing accommodation of ceftazidime for hydrolysis. In addition, the conformational change was not observed in the E166A/P167S apoenzyme, suggesting the presence of acylated ceftazidime influences the conformational change. Finally, the E166A acyl-enzyme structure with ceftazidime did not exhibit the altered conformation, indicating the P167S substitution is required for the change. Taken together, the results reveal that the P167S substitution and the presence of acylated ceftazidime both drive the structure toward a conformational change in the Ω-loop and that in CTX-M P167S enzymes found in drug-resistant bacteria this will lead to an increased level of ceftazidime hydrolysis.

Ceftazidime-avibactam: novel antimicrobial combination for the treatment of complicated urinary tract infections.

Ceftazidime-avibactam is a combination of a third-generation cephalosporin and a novel non-beta-lactam beta-lactamase inhibitor. This combination was recently recommended for the treatment of complicated urinary tract infections, including acute pyelonephritis, in adults with limited or no alternative treatment options. The current review is aimed to determine activity, efficacy and safety of ceftazidime-avibactam in the treatment of patients with complicated urinary tract infections.