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.

Imipenem resistance associated with amino acid alterations of the OprD porin in Pseudomonas aeruginosa clinical isolates.

Globally, the spread of carbapenem-resistant strains has limited treatment options for multidrug-resistant (MDR) Pseudomonas aeruginosa infections. This study aimed to determine the role of point mutations as well as the expression level of the oprD gene in the emergence of imipenem-resistant P. aeruginosa strains isolated from patients referred to Ardabil hospitals. A total of 48 imipenem-resistant clinical isolates of P. aeruginosa collected between June 2019 and January 2022 were used in this study. Detection of the oprD gene and its amino acid alterations was performed using the polymerase chain reaction (PCR) and DNA sequencing techniques. The expression level of the oprD gene in imipenem-resistant strains was determined using the real-time quantitative reverse transcription PCR (RT-PCR) method. All imipenem-resistant P. aeruginosa strains were positive for the oprD gene based on the PCR results, and also five selected isolates indicated one or more amino acid alterations. Detected amino acid alterations in the OprD porin were Ala210Ile, Gln202Glu, Ala189Val, Ala186Pro, Leu170Phe, Leu127Val, Thr115Lys, and Ser103Thr. Based on the RT-PCR results, the oprD gene was downregulated in 79.1% of imipenem-resistant P. aeruginosa strains. However, 20.9% of strains showed overexpression of the oprD gene. Probably, resistance to imipenem in these strains is associated with the presence of carbapenemases, AmpC cephalosporinase, or efflux pumps. Owing to the high prevalence of imipenem-resistant P. aeruginosa strains due to various resistance mechanisms in Ardabil hospitals, the implementation of surveillance programs to reduce the spread of these resistant microorganisms along with rational selection and prescription of antibiotics is recommended.

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.

KPC-2 ß-lactamase enables carbapenem antibiotic resistance through fast deacylation of the covalent intermediate.

Serine active-site ß-lactamases hydrolyze ß-lactam antibiotics through the formation of a covalent acyl-enzyme intermediate followed by deacylation via an activated water molecule. Carbapenem antibiotics are poorly hydrolyzed by most ß-lactamases owing to slow hydrolysis of the acyl-enzyme intermediate. However, the emergence of the KPC-2 carbapenemase has resulted in widespread resistance to these drugs, suggesting it operates more efficiently. Here, we investigated the unusual features of KPC-2 that enable this resistance. We show that KPC-2 has a 20,000-fold increased deacylation rate compared with the common TEM-1 ß-lactamase. Furthermore, kinetic analysis of active site alanine mutants indicates that carbapenem hydrolysis is a concerted effort involving multiple residues. Substitution of Asn170 greatly decreases the deacylation rate, but this residue is conserved in both KPC-2 and non-carbapenemase ß-lactamases, suggesting it promotes carbapenem hydrolysis only in the context of KPC-2. X-ray structure determination of the N170A enzyme in complex with hydrolyzed imipenem suggests Asn170 may prevent the inactivation of the deacylating water by the 6α-hydroxyethyl substituent of carbapenems. In addition, the Thr235 residue, which interacts with the C3 carboxylate of carbapenems, also contributes strongly to the deacylation reaction. In contrast, mutation of the Arg220 and Thr237 residues decreases the acylation rate and, paradoxically, improves binding affinity for carbapenems. Thus, the role of these residues may be ground state destabilization of the enzyme-substrate complex or, alternatively, to ensure proper alignment of the substrate with key catalytic residues to facilitate acylation. These findings suggest modifications of the carbapenem scaffold to avoid hydrolysis by KPC-2 ß-lactamase.

Interplay between the Enamine and Imine Forms of the Hydrolyzed Imipenem in the Active Sites of Metallo-ß-lactamases and in Water Solution.

Deactivation of the ß-lactam antibiotics in the active sites of the ß-lactamases is among the main mechanisms of bacterial antibiotic resistance. As drugs of last resort, carbapenems are efficiently hydrolyzed by metallo-ß-lactamases, presenting a serious threat to human health. Our study reveals mechanistic aspects of the imipenem hydrolysis by bizinc metallo-ß-lactamases, NDM-1 and L1, belonging to the B1 and the B3 subclasses, respectively. The results of QM(PBE0-D3/6-31G**)/MM simulations show that the enamine product with the protonated nitrogen atom is formed as the major product in NDM-1 and as the only product in the L1 active site. In NDM-1, there is also another reaction pathway that leads to the formation of the (S)-enantiomer of the imine form of the hydrolyzed imipenem; this process occurs with the higher energy barriers. The absence of the second pathway in L1 is due to the different amino acid composition of the active site loop. In L1, the hydrophobic Pro226 residue is located above the pyrroline ring of imipenem that blocks protonation of the carbon atom. Electron density analysis is performed at the stationary points to compare reaction pathways in L1 and NDM-1. Tautomerization from the enamine to the imine form likely happens in solution after the dissociation of the hydrolyzed imipenem from the active site of the enzyme. Classical molecular dynamics simulations of the hydrolyzed imipenem in solution, both with the neutral enamine and the negatively charged N-C2-C3 fragment, demonstrate a huge diversity of conformations. The vast majority of conformations blocks the C3-atom from the side required for the (S)-imine formation upon tautomerization. Thus, according to our calculations, formation of the (R)-imine is more likely. QM(PBE0-D3/6-31G**)/MM molecular dynamics simulations of the hydrolyzed imipenem with the negatively charged N-C2-C3 fragment followed by the Laplacian bond order analysis demonstrate that the N═C2-C3- resonance structure is the most pronounced that facilitates formation of the imine form. The proposed mechanism of the enzymatic enamine formation and its subsequent tautomerization to the imine form in solution is in agreement with the recent spectroscopic and NMR studies.

Metallo-beta-lactamase CphA evolving into more efficient hydrolases through gene mutation is a novel pathway for the resistance of super bacteria.

The evolution of metallo-beta-lactamase CphA in discontinuous gradient concentration of imipenem was investigated in this work. The results suggested that single-base mutations K218R, K249T, K249M, Q253H, and a frameshift mutation M1 were observed. Compared with wild type, the minimum inhibitory concentration (MICs) of K249T, K249M, and M1 increased by at least 128 times and that of K218R increased by 64 times. And the catalytic efficiency increased by 312% and 653%, respectively. It is speculated from the details of the structural changes revealed by molecular dynamics simulations that the carbon skeleton migration caused by the outward motion of the loop 3 in the mutant may have significantly increased the cavity volume of the binding pocket, which is more conducive to the entry and expulsion of imipenem and its hydrolytic product. And the conformational change of the TDRAGGN (71-77) is located at the bottom of the binding pocket from order α-helix to disorder random coil enabled the binding pocket to be more conducive to accommodate and hold the imipenem respectively. All these indicated that during the repeated drug resistance, the wild-type achieved gene mutations and conformational change and evolved to the mutant enzymes with a more delicate structure and stronger hydrolysis ability. KEY POINTS: • The mutation and evolution of CphA under the selective pressure of imipenem. • The CphA evolved to the mutants with stronger hydrolysis capacity. • A novel pathway for the resistance of super bacteria.

Quantitative Determination of Unbound Piperacillin and Imipenem in Biological Material from Critically Ill Using Thin-Film Microextraction-Liquid Chromatography-Mass Spectrometry.

ß-Lactam antibiotics are most commonly used in the critically ill, but their effective dosing is challenging and may result in sub-therapeutic concentrations that can lead to therapy failure and even promote antimicrobial resistance. In this study, we present the analytical tool enabling specific and sensitive determination of the sole biologically active fraction of piperacillin and imipenem in biological material from the critically ill. Thin-film microextraction sampling technique, followed by rapid liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis, was optimized and validated for the quantitative determination of antibiotics in blood and bronchoalveolar lavage (BAL) specimens collected from intensive care unit (ICU) patients suffering from ventilation-associated pneumonia (n = 18 and n = 9, respectively). The method was optimized and proved to meet the criteria of US Food and Drug Administration (FDA) guidelines for bioanalytical method validation. Highly selective, sensitive, accurate and precise analysis by means of thin-film microextraction-LC-MS/MS, which is not affected by matrix-related factors, was successfully applied in clinical settings, revealing poor penetration of piperacillin and imipenem from blood into BAL fluid (reflecting the site of bacterial infection), nonlinearity in antibiotic binding to plasma-proteins and drug-specific dependence on creatinine clearance. This work demonstrates that only a small fraction of biologically active antibiotics reach the site of infection, providing clinicians with a high-throughput analytical tool for future studies on personalized therapeutic drug monitoring when tailoring the dosing strategy to an individual patient.

Exploring the Role of L10 Loop in New Delhi Metallo-ß-lactamase (NDM-1): Kinetic and Dynamic Studies.

Four NDM-1 mutants (L218T, L221T, L269H and L221T/Y229W) were generated in order to investigate the role of leucines positioned in L10 loop. A detailed kinetic analysis stated that these amino acid substitutions modified the hydrolytic profile of NDM-1 against some ß-lactams. Significant reduction of kcat values of L218T and L221T for carbapenems, cefazolin, cefoxitin and cefepime was observed. The stability of the NDM-1 and its mutants was explored by thermofluor assay in real-time PCR. The determination of TmB and TmD demonstrated that NDM-1 and L218T were the most stable enzymes. Molecular dynamic studies were performed to justify the differences observed in the kinetic behavior of the mutants. In particular, L218T fluctuated more than NDM-1 in L10, whereas L221T would seem to cause a drift between residues 75 and 125. L221T/Y229W double mutant exhibited a decrease in the flexibility with respect to L221T, explaining enzyme activity improvement towards some ß-lactams. Distances between Zn1-Zn2 and Zn1-OH- or Zn2-OH- remained unaffected in all systems analysed. Significant changes were found between Zn1/Zn2 and first sphere coordination residues.

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.

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.

Multiple substitutions lead to increased loop flexibility and expanded specificity in Acinetobacter baumannii carbapenemase OXA-239.

OXA-239 is a class D carbapenemase isolated from an Acinetobacter baumannii strain found in Mexico. This enzyme is a variant of OXA-23 with three amino acid substitutions in or near the active site. These substitutions cause OXA-239 to hydrolyze late-generation cephalosporins and the monobactam aztreonam with greater efficiency than OXA-23. OXA-239 activity against the carbapenems doripenem and imipenem is reduced ∼3-fold and 20-fold, respectively. Further analysis demonstrated that two of the substitutions (P225S and D222N) are largely responsible for the observed alteration of kinetic parameters, while the third (S109L) may serve to stabilize the protein. Structures of OXA-239 with cefotaxime, doripenem and imipenem bound as acyl-intermediates were determined. These structures reveal that OXA-239 has increased flexibility in a loop that contains P225S and D222N. When carbapenems are bound, the conformation of this loop is essentially identical with that observed previously for OXA-23, with a narrow active site that makes extensive contacts to the ligand. When cefotaxime is bound, the loop can adopt a different conformation that widens the active site to allow binding of that bulky drug. This alternate conformation is made possible by P225S and further stabilized by D222N. Taken together, these results suggest that the three substitutions were selected to expand the substrate specificity profile of OXA-23 to cephalosporins and monobactams. The loss of activity against imipenem, however, suggests that there may be limits to the plasticity of class D enzymes with regard to evolving active sites that can effectively bind multiple classes of ß-lactam drugs.

Genetic and Biochemical Characterization of OXA-519, a Novel OXA-48-Like ß-Lactamase.

A multidrug-resistant Klebsiella pneumoniae 1210 isolate with reduced carbapenem susceptibility revealed the presence of a novel plasmid-encoded blaOXA-48-like gene, named blaOXA-519 The 60.7-kb plasmid (pOXA-519) was similar to the IncL-OXA-48 prototypical plasmid except for a ca. 2-kb deletion due to an IS1R insertion. OXA-519 differed from OXA-48 by a Val120Leu substitution, which resulted in an overall reduced ß-lactam-hydrolysis profile, except those for ertapenem and meropenem, which were increased. Thus, detection of OXA-519 producers using biochemical tests that monitor imipenem hydrolysis will be difficult.

The reduction of l-cystine to l-cysteine in the supernatant of A549 cell culture causes imipenem inactivation.

In the course of measuring the intracellular antibacterial activity of antibiotics using a human alveolar epithelial cell line A549, we discovered that the antimicrobial activity of several carbapenems (CPs) decreased in the supernatant of the cells cultured with fetal calf serum (FCS)-free RPMI1640 medium (RPMI). Further investigation revealed A549 culture supernatant inhibited the antibacterial activity of CPs but did not inactivate other types of antibiotics. CE-TOFMS and LC-TOFMS metabolomics analysis of the supernatant revealed the presence of l-cysteine (Cys), which is not an original component in RPMI. Cys is known to hydrolyze and inactivate CPs in a time- and concentration-dependent manner. In this study, the inactivating effects of A549 culture supernatant on the imipenem (IPM) were examined. Antimicrobial activity of 100 µg/mL IPM decreased to 25% with two-fold dilution of A549 supernatant incubated for 3 h. l-Cystine (CS), the Cys oxide, and an original component in RPMI did not inactivate IPM. However, the inactivating effects of A549 supernatant on IPM corresponds with the Cys concentration and depends on the CS content of the culture medium. Addition of FCS to the culture medium decreased the Cys concentration and reduced inactivation of IPM in a dose-dependent manner. Our data suggest that IPM were inactivated by Cys reduced from CS, and this CS-to-Cys conversion must be considered when evaluating the antimicrobial activity of CPs in cell culture. Further studies are needed to understand if the same inactivation occurs around the cells in the human body.

The Acinetobacter Outer Membrane Contains Multiple Specific Channels for Carbapenem ß-Lactams as Revealed by Kinetic Characterization Analyses of Imipenem Permeation into Acinetobacter baylyi Cells.

The number and type of outer membrane (OM) channels responsible for carbapenem uptake in Acinetobacter are still not well defined. Here, we addressed these questions by using Acinetobacter baylyi as a model species and a combination of methodologies aimed to characterize OM channels in their original membrane environment. Kinetic and competition analyses of imipenem (IPM) uptake by A. baylyi whole cells allowed us to identify different carbapenem-specific OM uptake sites. Comparative analyses of IPM uptake by A. baylyi wild-type (WT) cells and ΔcarO mutants lacking CarO indicated that this OM protein provided a carbapenem uptake site displaying saturable kinetics and common binding sites for basic amino acids compatible with a specific channel. The kinetic analysis uncovered another carbapenem-specific channel displaying a somewhat lower affinity for IPM than that of CarO and, in addition, common binding sites for basic amino acids as determined by competition studies. The use of A. baylyi gene deletion mutants lacking OM proteins proposed to function in carbapenem uptake in Acinetobacter baumannii indicated that CarO and OprD/OccAB1 mutants displayed low but consistent reductions in susceptibility to different carbapenems, including IPM, meropenem, and ertapenem. These two mutants also showed impaired growth on l-Arg but not on other carbon sources, further supporting a role of CarO and OprD/OccAB1 in basic amino acid and carbapenem uptake. A multiple-carbapenem-channel scenario may provide clues to our understanding of the contribution of OM channel loss or mutation to the carbapenem-resistant phenotype evolved by pathogenic members of the Acinetobacter genus.

Effect of ß-lactamase production and ß-lactam instability on MIC testing results for Mycobacterium abscessus.

OBJECTIVES: Limited treatment options available for Mycobacterium abscessus infections include the parenteral ß-lactam antibiotics cefoxitin and imipenem, which show moderate in vitro activity. Other ß-lactam antibiotics (except meropenem) have no considerable in vitro activity, due to their rapid hydrolysis by a broad-spectrum ß-lactamase (Bla_Mab). We here addressed the impact of ß-lactamase production and ß-lactam in vitro stability on M. abscessus MIC results and determined the epidemiological cut-off (ECOFF) values of cefoxitin, imipenem and meropenem. METHODS: By LC high-resolution MS (LC-HRMS), we assessed the in vitro stability of cefoxitin, imipenem and meropenem. M. abscessus ATCC 19977 strain and its isogenic blaMab deletion mutant were used for MIC testing. Based on MIC distributions for M. abscessus clinical strains, we determined ECOFFs of cefoxitin, imipenem and meropenem. RESULTS: A functional Bla_Mab increased MICs of penicillins, ceftriaxone and meropenem. LC-HRMS data showed significant degradation of cefoxitin, imipenem and meropenem during standard antibiotic susceptibility testing procedures. MIC, MIC50 and ECOFF values of cefoxitin, imipenem and meropenem are influenced by incubation time. CONCLUSIONS: The results of our study support administration of imipenem, meropenem and cefoxitin, for treatment of patients infected with M. abscessus. Our findings on in vitro instability of imipenem, meropenem and cefoxitin explain the problematic correlation between in vitro susceptibility and in vivo activity of these antibiotics and question the clinical utility of susceptibility testing of these chemotherapeutic agents.

Development of an algorithm for phenotypic screening of carbapenemase-producing Enterobacteriaceae in the routine laboratory.

BACKGROUND: Carbapenemase-producing Enterobacteriaceae (CPE) are difficult to identify among carbapenem non-susceptible Enterobacteriaceae (NSE). We designed phenotypic strategies giving priority to high sensitivity for screening putative CPE before further testing. METHODS: Presence of carbapenemase-encoding genes in ertapenem NSE (MIC > 0.5 mg/l) consecutively isolated in 80 French laboratories between November 2011 and April 2012 was determined by the Check-MDR-CT103 array method. Using the Mueller-Hinton (MH) disk diffusion method, clinical diameter breakpoints of carbapenems other than ertapenem, piperazicillin+tazobactam, ticarcillin+clavulanate and cefepime as well as diameter cut-offs for these antibiotics and temocillin were evaluated alone or combined to determine their performances (sensitivity, specificity, positive and negative likelihood ratios) for identifying putative CPE among these ertapenem-NSE isolates. To increase the screening specificity, these antibiotics were also tested on cloxacillin-containing MH when carbapenem NSE isolates belonged to species producing chromosomal cephalosporinase (AmpC) but Escherichia coli. RESULTS: Out of the 349 ertapenem NSE, 52 (14.9%) were CPE, including 39 producing OXA-48 group carbapenemase, eight KPC and five MBL. A screening strategy based on the following diameter cut offs, ticarcillin+clavulanate <15 mm, temocillin <15 mm, meropenem or imipenem <22 mm, and cefepime <26 mm, showed 100% sensitivity and 68.1% specificity with the better likelihood ratios combination. The specificity increased when a diameter cut-off <32 mm for imipenem (76.1%) or meropenem (78.8%) further tested on cloxacillin-containing MH was added to the previous strategy for AmpC-producing isolates. CONCLUSION: The proposed strategies that allowed for increasing the likelihood of CPE among ertapenem-NSE isolates should be considered as a surrogate for carbapenemase production before further CPE confirmatory testing.

Role of Residues W228 and Y233 in the Structure and Activity of Metallo-ß-Lactamase GIM-1.

Metallo-ß-lactamases (MBLs) hydrolyze virtually all ß-lactam antibiotics, including penicillins, cephalosporins, and carbapenems. The worldwide emergence of antibiotic-resistant bacteria harboring MBLs poses an increasing clinical threat. The MBL German imipenemase-1 (GIM-1) possesses an active site that is narrower and more hydrophobic than the active sites of other MBLs. The GIM-1 active-site groove is shaped by the presence of the aromatic side chains of tryptophan at residue 228 and tyrosine at residue 233, positions where other MBLs harbor hydrophilic residues. To investigate the importance of these two residues, eight site-directed mutants of GIM-1, W228R/A/Y/S and Y233N/A/I/S, were generated and characterized using enzyme kinetics, thermostability assays, and determination of the MICs of representative ß-lactams. The structures of selected mutants were obtained by X-ray crystallography, and their interactions with ß-lactam substrates were modeled in silico. Steady-state kinetics revealed that both positions are important to GIM-1 activity but that the effects of individual mutations vary depending on the ß-lactam substrate. Activity against type 1 substrates bearing electron-donating C-3/C-4 substituents (cefoxitin, meropenem) could be enhanced by mutations at position 228, whereas hydrolysis of type 2 substrates (benzylpenicillin, ampicillin, ceftazidime, imipenem) with methyl or positively charged substituents was favored by mutations at position 233. The crystal structures showed that mutations at position 228 or the Y233A variant alters the conformation of GIM-1 loop L1 rather than that of loop L3, on which the mutations are located. Taken together, these data show that point mutations at both positions 228 and 233 can influence the catalytic properties and the structure of GIM-1.

Active-Site Plasticity Is Essential to Carbapenem Hydrolysis by OXA-58 Class D ß-Lactamase of Acinetobacter baumannii.

Carbapenem-hydrolyzing class D ß-lactamases (CHDLs) are a subgroup of class D ß-lactamases, which are enzymes that hydrolyze ß-lactams. They have attracted interest due to the emergence of multidrug-resistant Acinetobacter baumannii, which is not responsive to treatment with carbapenems, the usual antibiotics of choice for this bacterium. Unlike other class D ß-lactamases, these enzymes efficiently hydrolyze carbapenem antibiotics. To explore the structural requirements for the catalysis of carbapenems by these enzymes, we determined the crystal structure of the OXA-58 CHDL of A. baumannii following acylation of its active-site serine by a 6α-hydroxymethyl penicillin derivative that is a structural mimetic for a carbapenem. In addition, several point mutation variants of the active site of OXA-58, as identified by the crystal structure analysis, were characterized kinetically. These combined studies confirm the mechanistic relevance of a hydrophobic bridge formed over the active site. This structural feature is suggested to stabilize the hydrolysis-productive acyl-enzyme species formed from the carbapenem substrates of this enzyme. Furthermore, our structural studies provide strong evidence that the hydroxyethyl group of carbapenems samples different orientations in the active sites of CHDLs, and the optimum orientation for catalysis depends on the topology of the active site allowing proper closure of the active site. We propose that CHDLs use the plasticity of the active site to drive the mechanism of carbapenem hydrolysis toward efficiency.

Escherichia coli Overexpressing a Baeyer-Villiger Monooxygenase from Acinetobacter radioresistens Becomes Resistant to Imipenem.

Antimicrobial resistance is a global issue currently resulting in the deaths of hundreds of thousands of people a year worldwide. Data present in the literature illustrate the emergence of many bacterial species that display resistance to known antibiotics; Acinetobacter spp. are a good example of this. We report here that Acinetobacter radioresistens has a Baeyer-Villiger monooxygenase (Ar-BVMO) with 100% amino acid sequence identity to the ethionamide monooxygenase of multidrug-resistant (MDR) Acinetobacter baumannii. Both enzymes are only distantly phylogenetically related to other canonical bacterial BVMO proteins. Ar-BVMO not only is capable of oxidizing two anticancer drugs metabolized by human FMO3, danusertib and tozasertib, but also can oxidize other synthetic drugs, such as imipenem. The latter is a member of the carbapenems, a clinically important antibiotic family used in the treatment of MDR bacterial infections. Susceptibility tests performed by the Kirby-Bauer disk diffusion method demonstrate that imipenem-sensitive Escherichia coli BL21 cells overexpressing Ar-BVMO become resistant to this antibiotic. An agar disk diffusion assay proved that when imipenem reacts with Ar-BVMO, it loses its antibiotic property. Moreover, an NADPH consumption assay with the purified Ar-BVMO demonstrates that this antibiotic is indeed a substrate, and its product is identified by liquid chromatography-mass spectrometry to be a Baeyer-Villiger (BV) oxidation product of the carbonyl moiety of the ß-lactam ring. This is the first report of an antibiotic-inactivating BVMO enzyme that, while mediating its usual BV oxidation, also operates by an unprecedented mechanism of carbapenem resistance.

Interspecies scaling of excretory amounts using allometry - retrospective analysis with rifapentine, aztreonam, carumonam, pefloxacin, miloxacin, trovafloxacin, doripenem, imipenem, cefozopran, ceftazidime, linezolid for urinary excretion and rifapentine, cabotegravir, and dolutegravir for fecal excretion.

1. Interspecies allometry scaling for prediction of human excretory amounts in urine or feces was performed for numerous antibacterials. Antibacterials used for urinary scaling were: rifapentine, pefloxacin, trovafloxacin (Gr1/low; <10%); miloxacin, linezolid, PNU-142300 (Gr2/medium; 10-40%); aztreonam, carumonam, cefozopran, doripenem, imipenem, and ceftazidime (Gr3/high; >50%). Rifapentine, cabotegravir, and dolutegravir was used for fecal scaling (high; >50%). 2. The employment of allometry equation: Y = aW(b) enabled scaling of urine/fecal amounts from animal species. Corresponding predicted amounts were converted into % recovery by considering the respective human dose. Comparison of predicted/observed values enabled fold difference and error calculations (mean absolute error [MAE] and root mean square error [RMSE]). Comparisons were made for urinary/fecal data; and qualitative assessment was made amongst Gr1/Gr2/Gr3 for urine. 3. Average correlation coefficient for the allometry scaling was >0.995. Excretory amount predictions were largely within 0.75- to 1.5-fold differences. Average MAE and RMSE were within ±22% and 23%, respectively. Although robust predictions were achieved for higher urinary/fecal excretion (>50%), interspecies scaling was applicable for low/medium excretory drugs. 4. Based on the data, interspecies scaling of urine or fecal excretory amounts may be potentially used as a tool to understand the significance of either urinary or fecal routes of elimination in humans in early development.