Molecular Basis of Class A β-Lactamase Inhibition by Relebactam

ABSTRACT β-Lactamase production is the major β-lactam resistance mechanism in Gram-negative bacteria. β-Lactamase inhibitors (BLIs) efficacious against serine β-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 β-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 β-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–β-lactam combinations.

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Structural Origins of Oxacillinase Specificity in Class D β-Lactamases

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01483-13 ◽

2013 ◽

Vol 58 (1) ◽

pp. 333-341 ◽

Cited By ~ 21

Author(s):

Cynthia M. June ◽

Beth C. Vallier ◽

Robert A. Bonomo ◽

David A. Leonard ◽

Rachel A. Powers

Keyword(s):

Resistance Mechanism ◽

Multidrug Resistant ◽

Health Concern ◽

Structural Basis ◽

Side Chain ◽

Gram Negative Bacteria ◽

Substrate Selectivity ◽

Major Health ◽

Content Type ◽

Class D

ABSTRACTSince the discovery and use of penicillin, the increase of antibiotic resistance among bacterial pathogens has become a major health concern. The most prevalent resistance mechanism in Gram-negative bacteria is due to β-lactamase expression. Class D β-lactamases are of particular importance due to their presence in multidrug-resistantAcinetobacter baumanniiandPseudomonas aeruginosa. The class D enzymes were initially characterized by their ability to efficiently hydrolyze isoxazolyl-type β-lactams like oxacillin. Due to this substrate preference, these enzymes are traditionally referred to as oxacillinases or OXAs. However, this class is comprised of subfamilies characterized by diverse activities that include oxacillinase, carbapenemase, or cephalosporinase substrate specificity. OXA-1 represents one subtype of class D enzyme that efficiently hydrolyzes oxacillin, and OXA-24/40 represents another with weak oxacillinase, but increased carbapenemase, activity. To examine the structural basis for the substrate selectivity differences between OXA-1 and OXA-24/40, the X-ray crystal structures of deacylation-deficient mutants of these enzymes (Lys70Asp for OXA-1; Lys84Asp for OXA-24) in complexes with oxacillin were determined to 1.4 Å and 2.4 Å, respectively. In the OXA-24/40/oxacillin structure, the hydrophobic R1 side chain of oxacillin disrupts the bridge between Tyr112 and Met223 present in the apo OXA-24/40 structure, causing the main chain of the Met223-containing loop to adopt a completely different conformation. In contrast, in the OXA-1/oxacillin structure, a hydrophobic pocket consisting of Trp102, Met99, Phe217, Leu161, and Leu255 nicely complements oxacillin's nonpolar R1 side chain. Comparison of the OXA-1/oxacillin and OXA-24/40/oxacillin complexes provides novel insight on how substrate selectivity is achieved among subtypes of class D β-lactamases. By elucidating important active site interactions, these findings can also inform the design of novel antibiotics and inhibitors.

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Inactivation of Polymyxin by Hydrolytic Mechanism

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02378-18 ◽

2019 ◽

Vol 63 (6) ◽

Cited By ~ 1

Author(s):

Jianhua Yin ◽

Gang Wang ◽

Dan Cheng ◽

Jianv Fu ◽

Juanping Qiu ◽

...

Keyword(s):

Multidrug Resistant ◽

Gram Negative Bacteria ◽

Peptide Antibiotics ◽

Peptide Bonds ◽

Gram Negative ◽

Content Type ◽

Gram Positive Bacteria ◽

Polymyxin E ◽

Cyclic Heptapeptide ◽

Hydrolytic Mechanism

ABSTRACTPolymyxins are nonribosomal peptide antibiotics used as the last-resort drug for treatment of multidrug-resistant Gram-negative bacteria. However, strains that are resistant to polymyxins have emerged in many countries. Although several mechanisms for polymyxin resistance have been well described, there is little knowledge on the hydrolytic mechanism of polymyxin. Here, we identified a polymyxin-inactivating enzyme fromBacillus licheniformisstrain DC-1 which was produced and secreted into the medium during entry into stationary phase. After purification, sequencing, and heterologous expression, we found that the alkaline protease Apr is responsible for inactivation of polymyxins. Analysis of inactivation products demonstrated that Apr cleaves polymyxin E at two peptide bonds: one is between the tripeptide side chain and the cyclic heptapeptide ring, the other betweenl-Thr andl-α-γ-diaminobutyric acid (l-Dab) within the cyclic heptapeptide ring. Apr is highly conserved among several genera of Gram-positive bacteria, includingBacillusandPaenibacillus. It is noteworthy that two peptidases S8 from Gram-negative bacteria shared high levels of sequence identity with Apr. Our results indicate that polymyxin resistance may result from inactivation of antibiotics by hydrolysis.

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In Vitro Activity of Ceftazidime-Avibactam against Isolates from Respiratory and Blood Specimens from Patients with Nosocomial Pneumonia, Including Ventilator-Associated Pneumonia, in a Phase 3 Clinical Trial

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02356-19 ◽

2020 ◽

Vol 64 (5) ◽

Cited By ~ 2

Author(s):

Gregory G. Stone ◽

Patricia A. Bradford ◽

Margaret Tawadrous ◽

Dianna Taylor ◽

Mary Jane Cadatal ◽

...

Keyword(s):

Nosocomial Pneumonia ◽

Bacterial Pathogen ◽

Multidrug Resistant ◽

Ventilator Associated Pneumonia ◽

Phase 3 ◽

Gram Positive ◽

Gram Negative ◽

Content Type ◽

Highly Active

ABSTRACT Nosocomial pneumonia (NP), including ventilator-associated pneumonia (VAP), is increasingly associated with multidrug-resistant Gram-negative pathogens. This study describes the in vitro activity of ceftazidime-avibactam, ceftazidime, and relevant comparator agents against bacterial pathogens isolated from patients with NP, including VAP, enrolled in a ceftazidime-avibactam phase 3 trial. Gram-positive pathogens were included if coisolated with a Gram-negative pathogen. In vitro susceptibility was determined at a central laboratory using Clinical and Laboratory Standards Institute broth microdilution methods. Of 817 randomized patients, 457 (55.9%) had ≥1 Gram-negative bacterial pathogen(s) isolated at baseline, and 149 (18.2%) had ≥1 Gram-positive pathogen(s) coisolated. The most common isolated pathogens were Klebsiella pneumoniae (18.8%), Pseudomonas aeruginosa (15.8%), and Staphylococcus aureus (11.5%). Ceftazidime-avibactam was highly active in vitro against 370 isolates of Enterobacteriaceae, with 98.6% susceptible (MIC90, 0.5 μg/ml) compared with 73.2% susceptible for ceftazidime (MIC90, >64 μg/ml). The percent susceptibility values for ceftazidime-avibactam and ceftazidime against 129 P. aeruginosa isolates were 88.4% and 72.9% (MIC90 values of 16 μg/ml and 64 μg/ml), respectively. Among ceftazidime-nonsusceptible Gram-negative isolates, ceftazidime-avibactam percent susceptibility values were 94.9% for 99 Enterobacteriaceae and 60.0% for 35 P. aeruginosa. MIC90 values for linezolid and vancomycin (permitted per protocol for Gram-positive coverage) were within their respective MIC susceptibility breakpoints against the Gram-positive pathogens isolated. This analysis demonstrates that ceftazidime-avibactam was active in vitro against the majority of Enterobacteriaceae and P. aeruginosa isolates from patients with NP, including VAP, in a phase 3 trial. (This study has been registered at ClinicalTrials.gov under identifier NCT01808092.)

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Heterogeneous and Flexible Transmission ofmcr-1in Hospital-AssociatedEscherichia coli

mBio ◽

10.1128/mbio.00943-18 ◽

2018 ◽

Vol 9 (4) ◽

Cited By ~ 23

Author(s):

Yingbo Shen ◽

Zuowei Wu ◽

Yang Wang ◽

Rong Zhang ◽

Hong-Wei Zhou ◽

...

Keyword(s):

Escherichia Coli ◽

Genomic Analysis ◽

Multidrug Resistant ◽

Fluoroquinolone Resistance ◽

Gram Negative Bacteria ◽

Colistin Resistance ◽

Gram Negative ◽

Content Type ◽

E Coli ◽

Genes Encoding

ABSTRACTThe recent emergence of a transferable colistin resistance mechanism, MCR-1, has gained global attention because of its threat to clinical treatment of infections caused by multidrug-resistant Gram-negative bacteria. However, the possible transmission route ofmcr-1amongEnterobacteriaceaespecies in clinical settings is largely unknown. Here, we present a comprehensive genomic analysis ofEscherichia coliisolates collected in a hospital in Hangzhou, China. We found thatmcr-1-carrying isolates from clinical infections and feces of inpatients and healthy volunteers were genetically diverse and were not closely related phylogenetically, suggesting that clonal expansion is not involved in the spread ofmcr-1. Themcr-1gene was found on either chromosomes or plasmids, but in most of theE. coliisolates,mcr-1was carried on plasmids. The genetic context of the plasmids showed considerable diversity as evidenced by the different functional insertion sequence (IS) elements, toxin-antitoxin (TA) systems, heavy metal resistance determinants, and Rep proteins of broad-host-range plasmids. Additionally, the genomic analysis revealed nosocomial transmission ofmcr-1and the coexistence ofmcr-1with other genes encoding β-lactamases and fluoroquinolone resistance in theE. coliisolates. These findings indicate thatmcr-1is heterogeneously disseminated in both commensal and pathogenic strains ofE. coli, suggest the high flexibility of this gene in its association with diverse genetic backgrounds of the hosts, and provide new insights into the genome epidemiology ofmcr-1among hospital-associatedE. colistrains.IMPORTANCEColistin represents one of the very few available drugs for treating infections caused by extensively multidrug-resistant Gram-negative bacteria. The recently emergentmcr-1colistin resistance gene threatens the clinical utility of colistin and has gained global attention. Howmcr-1spreads in hospital settings remains unknown and was investigated by whole-genome sequencing ofmcr-1-carryingEscherichia coliin this study. The findings revealed extraordinary flexibility ofmcr-1in its spread among genetically diverseE. colihosts and plasmids, nosocomial transmission ofmcr-1-carryingE. coli, and the continuous emergence of novel Inc types of plasmids carryingmcr-1and newmcr-1variants. Additionally,mcr-1was found to be frequently associated with other genes encoding β-lactams and fluoroquinolone resistance. These findings provide important information on the transmission and epidemiology ofmcr-1and are of significant public health importance as the information is expected to facilitate the control of this significant antibiotic resistance threat.

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Clinical Experience of Colistin-Glycopeptide Combination in Critically Ill Patients Infected with Gram-Negative Bacteria

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00871-13 ◽

2013 ◽

Vol 58 (2) ◽

pp. 851-858 ◽

Cited By ~ 62

Author(s):

Nicola Petrosillo ◽

Maddalena Giannella ◽

Massimo Antonelli ◽

Mario Antonini ◽

Bruno Barsic ◽

...

Keyword(s):

Protective Factor ◽

Multidrug Resistant ◽

Gram Negative Bacteria ◽

Gram Negative ◽

Content Type ◽

Carbapenem Resistant ◽

Causative Agents ◽

Treatment Onset ◽

Cox Analysis

ABSTRACTA colistin-glycopeptide combination (CGC) has been shownin vitroto be synergistic against multidrug-resistant Gram-negative bacteria (MDR GNB), especiallyAcinetobacter baumannii, and to prevent further resistance. However, clinical data are lacking. We carried out a retrospective multicenter study of patients hospitalized in intensive care units (ICUs) who received colistin for GNB infection over a 1-year period, to assess the rates of nephrotoxicity and 30-day mortality after treatment onset among patients treated with and without CGC for ≥48 h. Of the 184 patients treated with colistin, GNB infection was documented for 166. The main causative agents were MDRA. baumannii(59.6%), MDRPseudomonas aeruginosa(18.7%), and carbapenem-resistantKlebsiella pneumoniae(14.5%); in 16.9% of patients, a Gram-positive bacterium (GPB) coinfection was documented. Overall, 68 patients (40.9%) received CGC. Comparison of patients treated with and without CGC showed significant differences for respiratory failure (39.7% versus 58.2%), ventilator-associated pneumonia (54.4% versus 71.4%), MDRA. baumanniiinfection (70.6% versus 52%), and GPB coinfection (41.2% versus 0%); there were no differences for nephrotoxicity (11.8% versus 13.3%) and 30-day mortality (33.8% versus 29.6%). Cox analysis performed on patients who survived for ≥5 days after treatment onset showed that the Charlson index (hazard ratio [HR], 1.26; 95% confidence interval [CI], 1.01 to 1.44;P= 0.001) and MDRA. baumanniiinfection (HR, 2.51; 95% CI, 1.23 to 5.12;P= 0.01) were independent predictors of 30-day mortality, whereas receiving CGC for ≥5 days was a protective factor (HR, 0.42; 95% CI, 0.19 to 0.93;P= 0.03). We found that CGC was not associated with higher nephrotoxicity and was a protective factor for mortality if administered for ≥5 days.

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Pseudomonas aeruginosa Contact-Dependent Growth Inhibition Plays Dual Role in Host-Pathogen Interactions

mSphere ◽

10.1128/msphere.00336-17 ◽

2017 ◽

Vol 2 (6) ◽

Cited By ~ 19

Author(s):

Jeffrey A. Melvin ◽

Jordan R. Gaston ◽

Shawn N. Phillips ◽

Michael J. Springer ◽

Christopher W. Marshall ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Acute Infection ◽

Multidrug Resistant ◽

Gram Negative Bacteria ◽

Microbial Pathogenesis ◽

Host Pathogen Interactions ◽

Gram Negative ◽

Content Type ◽

Disease Outcomes ◽

Host Pathogen

ABSTRACT How bacteria compete and communicate with each other is an increasingly recognized aspect of microbial pathogenesis with a major impact on disease outcomes. Gram-negative bacteria have recently been shown to employ a contact-dependent toxin-antitoxin system to achieve both competition and regulation of their physiology. Here, we show that this system is vital for virulence in acute infection as well as for establishment of chronic infection in the multidrug-resistant pathogen Pseudomonas aeruginosa. Greater understanding of the mechanisms underlying bacterial virulence and infection is important for the development of effective therapeutics in the era of increasing antimicrobial resistance. Microorganisms exist in a diverse ecosystem and have evolved many different mechanisms for sensing and influencing the polymicrobial environment around them, utilizing both diffusible and contact-dependent signals. Contact-dependent growth inhibition (CDI) is one such communication system employed by Gram-negative bacteria. In addition to CDI mediation of growth inhibition, recent studies have demonstrated CDI-mediated control of communal behaviors such as biofilm formation. We postulated that CDI may therefore play an active role in host-pathogen interactions, allowing invading strains to establish themselves at polymicrobial mucosal interfaces through competitive interactions while simultaneously facilitating pathogenic capabilities via CDI-mediated signaling. Here, we show that Pseudomonas aeruginosa produces two CDI systems capable of mediating competition under conditions of growth on a surface or in liquid. Furthermore, we demonstrated a novel role for these systems in contributing to virulence in acute infection models, likely via posttranscriptional regulation of beneficial behaviors. While we did not observe any role for the P. aeruginosa CDI systems in biofilm biogenesis, we did identify for the first time robust CDI-mediated competition during interaction with a mammalian host using a model of chronic respiratory tract infection, as well as evidence that CDI expression is maintained in chronic lung infections. These findings reveal a previously unappreciated role for CDI in host-pathogen interactions and emphasize their importance during infection. IMPORTANCE How bacteria compete and communicate with each other is an increasingly recognized aspect of microbial pathogenesis with a major impact on disease outcomes. Gram-negative bacteria have recently been shown to employ a contact-dependent toxin-antitoxin system to achieve both competition and regulation of their physiology. Here, we show that this system is vital for virulence in acute infection as well as for establishment of chronic infection in the multidrug-resistant pathogen Pseudomonas aeruginosa. Greater understanding of the mechanisms underlying bacterial virulence and infection is important for the development of effective therapeutics in the era of increasing antimicrobial resistance.

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Prominence of an O75 Clonal Group (Clonal Complex 14) among Non-ST131 Fluoroquinolone-Resistant Escherichia coli Causing Extraintestinal Infections in Humans and Dogs in Australia

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.06120-11 ◽

2012 ◽

Vol 56 (7) ◽

pp. 3898-3904 ◽

Cited By ~ 32

Author(s):

Joanne L. Platell ◽

Darren J. Trott ◽

James R. Johnson ◽

Peter Heisig ◽

Anke Heisig ◽

...

Keyword(s):

Escherichia Coli ◽

Clonal Complex ◽

Resistance Mechanism ◽

Multidrug Resistant ◽

Phylogenetic Group ◽

Mechanism Analysis ◽

Content Type ◽

Clonal Group ◽

E Coli ◽

Animal Medicine

ABSTRACTFluoroquinolone (FQ)-resistant extraintestinal pathogenicEscherichia coli(FQrExPEC) strains from phylogenetic group B2 are undergoing epidemic spread. Isolates belonging to phylogenetic group B2 are generally more virulent than otherE. coliisolates; therefore, resistance to FQs among group B2 isolates is concerning. Although clonal expansion of sequence type 131 (ST131) is a major factor, the contribution of additional clonal groups has not been quantified. Group B2 FQrExPEC isolates from humans (n= 250) and dogs (n= 12) in Australia were screened for ST131, a recently recognized and rapidly emerging multidrug-resistant and virulent clonal group that is important in both human and companion animal medicine. Non-ST131 isolates underwent virulence genotyping, PCR-based O typing, partial multilocus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE), and FQ resistance mechanism analysis. Of 49 non-ST131 isolates (45 human, 4 canine), 49% (24 human, 2 canine) represented O-type O75 and exhibited conserved virulence genotypes (F10papAallele,iha,fimH,sat,vat,fyuA,iutA,kpsMII,usp,ompT,malX, K1/K5 capsule) and MLST allele profiles corresponding with clonal complex CC14. Two clusters, each containing canine and human isolates, were identified by PFGE (differentiated by K1 and K5 capsules). Australian FQrO75 isolates exhibited commonality with an historical FQ-susceptible O75 urosepsis isolate (also CC14). The isolation from humans and dogs of highly similar FQrderivatives of the classic O75:K1/K5 (CC14) ExPEC lineage suggests recent acquisition of FQ resistance and potential cross-host-species transfer. This lineage should be targeted with ST131 in future epidemiological investigations of FQrExPEC.

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Species Diversity of Environmental GIM-1-Producing Bacteria Collected during a Long-Term Outbreak

Applied and Environmental Microbiology ◽

10.1128/aem.00424-16 ◽

2016 ◽

Vol 82 (12) ◽

pp. 3605-3610 ◽

Cited By ~ 13

Author(s):

Andreas F. Wendel ◽

Sofija Ressina ◽

Susanne Kolbe-Busch ◽

Klaus Pfeffer ◽

Colin R. MacKenzie

Keyword(s):

Resistance Genes ◽

Multidrug Resistant ◽

Hospital Environment ◽

Resistant Bacteria ◽

Environmental Sampling ◽

Gram Negative Bacteria ◽

Multidrug Resistant Bacteria ◽

Gram Negative ◽

Content Type

ABSTRACTReports of outbreaks concerning carbapenemase-producing Gram-negative bacteria in which the main source of transmission is the hospital environment are increasing. This study describes the results of environmental sampling in a protracted polyspecies metallo-beta-lactamase GIM-1 outbreak driven by plasmids and bacterial clones ofEnterobacter cloacaeandPseudomonas aeruginosain a tertiary care center. Environmental sampling targeting wet locations (especially sinks) was carried out on a surgical intensive care unit and on a medical ward on several occasions in 2012 and 2013. We were able to demonstrate 43blaGIM-1-carrying bacteria (mainly nonfermenters but alsoEnterobacteriaceae) that were either related or unrelated to clinical strains in 30 sinks and one hair washbasin. GIM-1 was found in 12 different species, some of which are described here as carriers of GIM-1. Forty out of 43 bacteria displayed resistance to carbapenems and, in addition, to various non-beta-lactam antibiotics. Colistin resistance was observed in twoE. cloacaeisolates with MICs above 256 mg/liter. TheblaGIM-1gene was harbored in 12 different class 1 integrons, some without the typical 3′ end. TheblaGIM-1gene was localized on plasmids in five isolates.In vitroplasmid transfer by conjugation was successful in one isolate. The environment, with putatively multispecies biofilms, seems to be an important biological niche for multidrug-resistant bacteria and resistance genes. Biofilms may serve as a “melting pot” for horizontal gene transfer, for dissemination into new species, and as a reservoir to propagate future hospital outbreaks.IMPORTANCEIn Gram-negative bacteria, resistance to the clinically relevant broad-spectrum carbapenem antibiotics is a major public health concern. Major reservoirs for these resistant organisms are not only the gastrointestinal tracts of animals and humans but also the (hospital) environment. Due to the difficulty in eradicating biofilm formation in the latter, a sustained dissemination of multidrug-resistant bacteria from the environment can occur. In addition, horizontal transfer of resistance genes on mobile genetic elements within biofilms adds to the total “resistance gene pool” in the environment. To gain insight into the transmission pathways of a rare and locally restricted carbapenemases resistance gene (blaGIM-1), we analyzed the genetic background of theblaGIM-1gene in environmental bacteria during a long-term polyspecies outbreak in a German hospital.

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Exploring the Landscape of Diazabicyclooctane (DBO) Inhibition: Avibactam Inactivation of PER-2 β-Lactamase

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02476-16 ◽

2017 ◽

Vol 61 (6) ◽

Cited By ~ 5

Author(s):

Melina Ruggiero ◽

Krisztina M. Papp-Wallace ◽

Magdalena A. Taracila ◽

Maria F. Mojica ◽

Christopher R. Bethel ◽

...

Keyword(s):

Active Site ◽

Therapeutic Option ◽

Structural Features ◽

Atomic Mass ◽

Stable Complex ◽

Water Molecules ◽

Gram Negative Bacteria ◽

Biophysical Properties ◽

Content Type ◽

Class A

ABSTRACT PER β-lactamases are an emerging family of extended-spectrum β-lactamases (ESBL) found in Gram-negative bacteria. PER β-lactamases are unique among class A enzymes as they possess an inverted omega (Ω) loop and extended B3 β-strand. These singular structural features are hypothesized to contribute to their hydrolytic profile against oxyimino-cephalosporins (e.g., cefotaxime and ceftazidime). Here, we tested the ability of avibactam (AVI), a novel non-β-lactam β-lactamase inhibitor to inactivate PER-2. Interestingly, the PER-2 inhibition constants (i.e., k 2/K = 2 × 103 ± 0.1 × 103 M−1 s−1, where k 2 is the rate constant for acylation (carbamylation) and K is the equilibrium constant) that were obtained when AVI was tested were reminiscent of values observed testing the inhibition by AVI of class C and D β-lactamases (i.e., k 2/K range of ≈103 M−1 s−1) and not class A β-lactamases (i.e., k 2/K range, 104 to 105 M−1 s−1). Once AVI was bound, a stable complex with PER-2 was observed via mass spectrometry (e.g., 31,389 ± 3 atomic mass units [amu] → 31,604 ± 3 amu for 24 h). Molecular modeling of PER-2 with AVI showed that the carbonyl of AVI was located in the oxyanion hole of the β-lactamase and that the sulfate of AVI formed interactions with the β-lactam carboxylate binding site of the PER-2 β-lactamase (R220 and T237). However, hydrophobic patches near the PER-2 active site (by Ser70 and B3-B4 β-strands) were observed and may affect the binding of necessary catalytic water molecules, thus slowing acylation (k 2/K) of AVI onto PER-2. Similar electrostatics and hydrophobicity of the active site were also observed between OXA-48 and PER-2, while CTX-M-15 was more hydrophilic. To demonstrate the ability of AVI to overcome the enhanced cephalosporinase activity of PER-2 β-lactamase, we tested different β-lactam–AVI combinations. By lowering MICs to ≤2 mg/liter, the ceftaroline-AVI combination could represent a favorable therapeutic option against Enterobacteriaceae expressing bla PER-2. Our studies define the inactivation of the PER-2 ESBL by AVI and suggest that the biophysical properties of the active site contribute to determining the efficiency of inactivation.

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Pathogenicity Genomic Island-Associated CrpP-Like Fluoroquinolone-Modifying Enzymes among Pseudomonas aeruginosa Clinical Isolates in Europe

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00489-20 ◽

2020 ◽

Vol 64 (7) ◽

Cited By ~ 1

Author(s):

José Manuel Ortiz de la Rosa ◽

Patrice Nordmann ◽

Laurent Poirel

Keyword(s):

Pseudomonas Aeruginosa ◽

Genomic Island ◽

Multidrug Resistant ◽

Resistance Mechanisms ◽

Genomic Islands ◽

Clinical Isolates ◽

Gram Negative Bacteria ◽

Gram Negative ◽

Content Type ◽

Genes Encoding

ABSTRACT Many transferable quinolone resistance mechanisms have been identified in Gram-negative bacteria. The plasmid-encoded 65-amino-acid-long ciprofloxacin-modifying enzyme CrpP was recently identified in Pseudomonas aeruginosa isolates. We analyzed a collection of 100 clonally unrelated and multidrug-resistant P. aeruginosa clinical isolates, among which 46 were positive for crpP-like genes, encoding five CrpP variants conferring variable levels of reduced susceptibility to fluoroquinolones. These crpP-like genes were chromosomally located as part of pathogenicity genomic islands.

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