Carbapenem-hydrolyzing Enzymes Research Articles

Genome-wide identification and oxacillinase OXA distribution characteristics of Acinetobacter spp. based on a global database.

To use genomic analysis to identify Acinetobacter spp. and to explore the distribution characteristics of ß-lactamase oxallicinases (blaOXA) among Acinetobacter species globally. Genomes of global Acinetobacter spp. were downloaded from GenBank using Aspera batch. After quality check using CheckM and QUAST software, the genomes were annotated using Prokka software to investigate the distribution of blaOXAs across Acinetobacter spp.; a phylogenetic tree was constructed to explore the evolutionary relationship among the blaOXA genes in Acinetobacter spp. Average-nucleotide identification (ANI) was performed to re-type the Acinetobacter spp. BLASTN comparison analysis was implemented to determine the sequence type (ST) of Acinetobacter baumannii strain. A total of 7,853 genomes were downloaded, of which only 6,639 were left for further analysis after quality check. Among them, 282 blaOXA variants were identified from the genomes of 5,893 Acinetobacter spp.; blaOXA-23 (n = 3,168, 53.8%) and blaOXA-66 (2,630, 44.6%) were the most frequent blaOXAs, accounting for 52.6% (3,489/6639), and the co-carriage of blaOXA-23 and blaOXA-66 was seen in 2223 (37.7%) strains. The 282 blaOXA variants were divided into 27 clusters according to the phylogenetic tree. The biggest clade was blaOXA-51-family carbapenem-hydrolyzing enzymes composed of 108 blaOXA variants. Overall, 4,923 A. baumannii were identified out of the 6,639 Acinetobacter spp. strains and 291 distinct STs were identified among the 4,904 blaOXA-carrying A. baumannii. The most prevalent ST was ST2 (n = 3,023, 61.6%) followed by ST1 (n = 228, 4.6%). OXA-like carbapenemases were the main blaOXA-type β-lactamase spread widely across Acinetobacter spp. Both blaOXA-23 and blaOXA-66 were the predominant blaOXAs, among all A. baumannii strains, with ST2 (belonging to CC2) being the main clone disseminated globally.

Efflux-Related Carbapenem Resistance in Acinetobacter baumannii Is Associated with Two-Component Regulatory Efflux Systems' Alteration and Insertion of ΔAbaR25-Type Island Fragment.

The efflux pumps, beside the class D carbapenem-hydrolysing enzymes (CHLDs), are being increasingly investigated as a mechanism of carbapenem resistance in Acinetobacter baumannii. This study investigates the contribution of efflux mechanism to carbapenem resistance in 61 acquired blaCHDL-genes-carrying A. baumannii clinical strains isolated in Warsaw, Poland. Studies were conducted using phenotypic (susceptibility testing to carbapenems ± efflux pump inhibitors (EPIs)) and molecular (determining expression levels of efflux operon with regulatory-gene and whole genome sequencing (WGS)) methods. EPIs reduced carbapenem resistance of 14/61 isolates. Upregulation (5-67-fold) of adeB was observed together with mutations in the sequences of AdeRS local and of BaeS global regulators in all 15 selected isolates. Long-read WGS of isolate no. AB96 revealed the presence of AbaR25 resistance island and its two disrupted elements: the first contained a duplicate ISAba1-blaOXA-23, and the second was located between adeR and adeA in the efflux operon. This insert was flanked by two copies of ISAba1, and one of them provides a strong promoter for adeABC, elevating the adeB expression levels. Our study for the first time reports the involvement of the insertion of the ΔAbaR25-type resistance island fragment with ISAba1 element upstream the efflux operon in the carbapenem resistance of A. baumannii.

421. In vitro and in vivo synergistic antimicrobial activities of the combinations of meropenem, colistin, tigecycline, and ceftolozane/tazobactam against carbapenem-resistant Klebsiella pneumoniae

Abstract Background The aim of this study was to evaluate the in vitro and in vivo activities of various antimicrobial combinations against carbapenem-resistant Klebsiella pneumoniae (CRKP). Methods The in vitro activity of six two-drug combinations against CRKP isolates collected from the rectal swab samples of patients with intestinal CRKP colonization was evaluated using the checkerboard method and time-kill assay to identify potential synergistic and bactericidal two-drug combinations against CRKP isolates. The in vivo efficacy of the combinations of colistin, meropenem and ceftolozane/tazobactam (C/T) as therapeutic options, was assessed in a mouse model of intraperitoneal CRKP sepsis. Antimicrobial susceptibility and genotypes of carbapenem-resistance were also analyzed. Results A total of 5 clinical isolates of CRKP were collected from nonduplicate patients with CRKP infection. Of 5 CRKP isolates, 4 carried the blaKPC-2-type carbapenem-hydrolyzing enzymes except 1 blaNDM-1-type. Antimicrobial susceptibility rates were tigecycline 80%, colistin 40%, and C/T 0%, respectively. The checkerboard assay showed that in vitro synergistic activity against CRKP isolates was 90% for the meropenem-tigecycline combinations and 20% for colistin-C/T combinations. None of the various combinations of the six antibiotics showed a synergistic effect in the time-kill assay. However, the meropenem-colistin (100%) and colistin-C/T (100%) combinations showed bactericidal effects in the time-kill assay (using an antibiotic concentration of 1× MIC) (Table). The survival rates of the mice was 80% (4/5) at 48 h in the treated group using low dose combinations of colistin (10 mg/kg) and C/T (25 mg/kg), unlike both antibiotics, which were 60% in a monotherapy with the same dose (Figure). Comparison of checkerboard assay and time-kill assay Comparison of checkerboard assay and time-kill assay associated with the bactericidal activity of two-drug combinations (1 × MIC) against each carbapenem-resistant Klebsiella pneumoniae isolate In vivo survival tests for antimicrobial combinations Comparison of the survival rates of the mice in the treated group using antimicrobial combinations Conclusion In conclusion, the present in vivo study demonstrated that the low-dose combination of colistin and C/T may be a promising alternative to nephrotoxic high-dose colistin alone for treating CRKP infections. However, it was difficult to predict this possibility in in vitro tests, and even there is great discordance of antimicrobial synergistic activities between the checkerboard microdilution and time-kill assays. Disclosures All Authors: No reported disclosures.

Resistance and virulence features of hypermucoviscous Klebsiella pneumoniae from bloodstream infections: Results of a nationwide Italian surveillance study.

Among Enterobacterales, Klebsiella pneumoniae (Kp) is one of the major opportunistic pathogens causing hospital-acquired infections. The most problematic phenomenon linked to Kp is related to the dissemination of multi-drug resistant (MDR) clones producing carbapenem-hydrolyzing enzymes, representing a clinical and public health threat at a global scale. Over the past decades, high-risk MDR clones (e.g., ST512, ST307, ST101 producing blaKPC–type carbepenemases) have become endemic in several countries, including Italy. Concurrently, the spread of highly virulent Kp lineages (e.g., ST23, ST86) able to cause severe, community-acquired, pyogenic infections with metastatic dissemination in immunocompetent subjects has started to be documented. These clones, designated as hypervirulent Kp (hvKp), produce an extensive array of virulence factors and are highly virulent in previously validated animal models. While the prevalence and distribution of MDR Kp has been previously assessed at local and national level knowledge about dissemination of hvKp remains scarce. In this work, we studied the phenotypic and genotypic features of hypermucoviscous (HMV, as possible marker of increased virulence) Kp isolates from bloodstream infections (BSI), obtained in 2016–17 from 43 Italian Laboratories. Antimicrobial susceptibility testing, whole genome sequencing and the use of two animal models (G. mellonella and murine) were employed to characterize collected isolates. Over 1502 BSI recorded in the study period, a total of 19 Kp were selected for further investigation based on their HMV phenotype. Results showed that hvKp isolates (ST5, ST8, ST11, ST25) are circulating in Italy, although with a low prevalence and in absence of a clonal expansion; convergence of virulence (yersiniabactin and/or salmochelin, aerobactin, regulators of mucoid phenotype) and antimicrobial-resistance (extended-spectrum beta-lactamases) features was observed in some cases. Conventional MDR Kp clones (ST307, ST512) may exhibit an HMV phenotype, but with a low virulence potential in the animal models. To the best of our knowledge, this work represents the first systematic survey on HMV and hvKp in Italy, employing a functional characterization of collected isolates. Future surveillance programs are warranted to monitor the threatening convergence of virulence and resistance among MDR Kp and the spread of hvKp.

Carbapenem resistance determinants in Klebsiella pneumoniae strains isolated from blood cultures-comparative analysis of molecular and phenotypic methods

Abstract Introduction: This study provides data on carbapenemases identified in carbapenem-resistant Klebsiella pneumoniae (CR-KP) isolated from blood-cultures by the multiplex molecular method. Material and method: Between October 2016 and September 2017, 47 non-duplicate Klebsiella pneumoniae (KP) were isolated from blood cultures, from hospitalized patients in the Regional Institute of Gastroenterology and Hepathology, Cluj-Napoca, Romania. Identification and antimicrobial susceptibility tests (AST) were performed by Vitek 2 Compact. The combination disks test (CDT) was used for phenotypic analysis and the LightCycler® Multiplex DNA assay was used to detect and identify the carbapenemases by the LightCycler®z 480 Instrument. The following targets were chosen: blaKPC, blaNDM, blaGES, blaIMP and blaOXA-48 genes and the Cobas® 4800 software variant 2.2.0 was used for the results interpretation. Results: Taking into consideration the meropenem minimum inhibitory concentration (MIC), 29 KP were susceptible and 18 were not-susceptible (MIC≥0.5 µg ml-1). In the CR-KP group, the CDT identified OXA-48 (10/18) and KPC (7/18) producers. One isolate showed a noninterpretable profile. The multiplex molecular analyses confirmed the carbapenemases production as: 9 CR-KP were KPC and OXA-48 co-producers, 8 were OXA-48 and one was KPC producing strains. In CR-KP group, we found a significant correlation between the CDT and RT-PCR tests results, concerning KPC (p = 0.671). Eight phenotypic results were confirmed by molecular Light-Cycler® Multiplex DNA assay. For CR-KP co-producers (KPC and OXA-48), the CDT could indicate only one carbapenem-hydrolyzing enzyme. Conclusion: This study highlights the CR-KP co-producers (OXA-48 and KPC). OXA-48-like is more frequently encountered in our area than other carbapenemases.

To Be or Not to Be an OXA-48 Carbapenemase.

Since the first description of OXA-48, more than forty variants have been recovered from Enterobacterales isolates. Whereas some OXA-48-related enzymes have been reported as conferring similar resistance patterns, namely, the hydrolysis of carbapenems and penicillins with very weak or almost no activity against expanded-spectrum cephalosporins, some have reduced carbapenem and temocillin hydrolysis, and others hydrolyze expanded-spectrum cephalosporins and carbapenems only marginally. With such drastic differences in the hydrolytic profile, especially of carbapenems, it becomes urgent to establish hydrolytic cutoffs in order to determine when an OXA-48-like enzyme may be considered as a carbapenemase or not. With this aim, the coefficient of activity for imipenem (kcat/Km) was determined for a total of 30 enzymes, including OXA-48, OXA-48-like natural variants, and OXA-48 synthetic mutants. In addition, six different methods for the detection of carbapenemase-producers were performed. The coefficients of activity for imipenem for all the different enzymes went from 550 mM−1·s−1 to 0.02 mM−1·s−1. In order to match the coefficient of activity results with the biochemical confirmatory tests, we suggest the value of 0.27 mM−1·s−1 as the cutoff above which an OXA-48 variant may be considered a carbapenem-hydrolyzing enzyme.

Genetic support of carbapenemases: a One Health systematic review and meta-analysis of current trends in Africa.

Antimicrobial resistance (AMR) is a public health threat globally. Carbapenems are β-lactam antibiotics used as last-resort agents for treating antibiotic-resistant infections. Mobile genetic elements (MGEs) play an important role in the dissemination and expression of antimicrobial resistance genes (ARGs), including the mobilization of ARGs within and between species. The presence of MGEs around carbapenem-hydrolyzing enzymes, called carbapenemases, in bacterial isolates in Africa is concerning. The association between MGEs and carbapenemases is described herein. Specific plasmid replicons, integrons, transposons, and insertion sequences were found flanking specific and different carbapenemases across the same and different clones and species isolated from humans, animals, and the environment. Notably, similar genetic contexts have been reported in non-African countries, supporting the importance of MGEs in driving the intra- and interclonal and species transmission of carbapenemases in Africa and globally. Technical and budgetary limitations remain challenges for epidemiological analysis of carbapenemases in Africa, as studies undertaken with whole-genome sequencing remained relatively few. Characterization of MGEs in antibiotic-resistant infections can deepen our understanding of carbapenemase epidemiology and facilitate the control of AMR in Africa. Investment in genomic epidemiology will facilitate faster clinical interventions and containment of outbreaks.

Crystallography and QM/MM Simulations Identify Preferential Binding of Hydrolyzed Carbapenem and Penem Antibiotics to the L1 Metallo-β-Lactamase in the Imine Form.

Widespread bacterial resistance to carbapenem antibiotics is an increasing global health concern. Resistance has emerged due to carbapenem-hydrolyzing enzymes, including metallo-β-lactamases (MβLs), but despite their prevalence and clinical importance, MβL mechanisms are still not fully understood. Carbapenem hydrolysis by MβLs can yield alternative product tautomers with the potential to access different binding modes. Here, we show that a combined approach employing crystallography and quantum mechanics/molecular mechanics (QM/MM) simulations allow tautomer assignment in MβL:hydrolyzed antibiotic complexes. Molecular simulations also examine (meta)stable species of alternative protonation and tautomeric states, providing mechanistic insights into β-lactam hydrolysis. We report the crystal structure of the hydrolyzed carbapenem ertapenem bound to the L1 MβL from Stenotrophomonas maltophilia and model alternative tautomeric and protonation states of both hydrolyzed ertapenem and faropenem (a related penem antibiotic), which display different binding modes with L1. We show how the structures of both complexed β-lactams are best described as the (2S)-imine tautomer with the carboxylate formed after β-lactam ring cleavage deprotonated. Simulations show that enamine tautomer complexes are significantly less stable (e.g., showing partial loss of interactions with the L1 binuclear zinc center) and not consistent with experimental data. Strong interactions of Tyr32 and one zinc ion (Zn1) with ertapenem prevent a C6 group rotation, explaining the different binding modes of the two β-lactams. Our findings establish the relative stability of different hydrolyzed (carba)penem forms in the L1 active site and identify interactions important to stable complex formation, information that should assist inhibitor design for this important antibiotic resistance determinant.

On the Offensive: the Role of Outer Membrane Vesicles in the Successful Dissemination of New Delhi Metallo-β-lactamase (NDM-1).

ABSTRACTThe emergence and worldwide dissemination of carbapenemase-producing Gram-negative bacteria are a major public health threat. Metallo-β-lactamases (MBLs) represent the largest family of carbapenemases. Regrettably, these resistance determinants are spreading worldwide. Among them, the New Delhi metallo-β-lactamase (NDM-1) is experiencing the fastest and largest geographical spread. NDM-1 β-lactamase is anchored to the bacterial outer membrane, while most MBLs are soluble, periplasmic enzymes. This unique cellular localization favors the selective secretion of active NDM-1 into outer membrane vesicles (OMVs). Here, we advance the idea that NDM-containing vesicles serve as vehicles for the local dissemination of NDM-1. We show that OMVs with NDM-1 can protect a carbapenem-susceptible strain of Escherichia coli upon treatment with meropenem in a Galleria mellonella infection model. Survival curves of G. mellonella revealed that vesicle encapsulation enhances the action of NDM-1, prolonging and favoring bacterial protection against meropenem inside the larva hemolymph. We also demonstrate that E. coli cells expressing NDM-1 protect a susceptible Pseudomonas aeruginosa strain within the larvae in the presence of meropenem. By using E. coli variants engineered to secrete variable amounts of NDM-1, we demonstrate that the protective effect correlates with the amount of NDM-1 secreted into vesicles. We conclude that secretion of NDM-1 into OMVs contributes to the survival of otherwise susceptible nearby bacteria at infection sites. These results disclose that OMVs play a role in the establishment of bacterial communities, in addition to traditional horizontal gene transfer mechanisms.

Pathobiology of Multidrug Resistant Acinetobacter baumannii: An update

Acinetobacter baumannii is an opportunistic nosocomial pathogen responsible for mortality and mobidity asscoaited with hospital acquired infections worldwide. Antimicrobial resistance due to natural and acquired modes facilitates A. baumannii as one of the most significant nosocomial pathogens. Challenging most issue is the carbapenem resistance in A. baumannii in the healthcare settings worldwide, which is conferred by several mechanisms including: beta-lactamases, reduced permeability of the outer membrane, efflux pumps, and modification of penicillinbinding proteins. Among these, carbapenem-hydrolysing enzymes are the foremost mechanism that belongs to Ambler class D and B beta-lactamases. Several techniques are used to study the molecular epidemiology like WGS, PFGE, MLST, RFLP and RAPD. Among these, MLST is the gold standard, which are prevalently used to study the evolutionary geentics and clonal relatedness of A. baumannii clones. A. baumannii has been investigated in a number of outbreaks in clinical settings and spread of infections in hospital environments. To enhance our knowledge, in this review, we study the taxonomy, lab diagnosis, molecular techniques of identification, mechanisms of its antimicrobial resistance, molecular epidemiology and outbreaks, bacterial cell firness and use of animal models in multidrug resistant (MDR) A. baumannii.

BlaOXA-48-like genome architecture among carbapenemase-producing Escherichia coli and Klebsiella pneumoniae in the Netherlands.

Carbapenem-hydrolysing enzymes belonging to the OXA-48-like group are encoded by bla OXA-48-like alleles and are abundant among Enterobacterales in the Netherlands. Therefore, the objective here was to investigate the characteristics, gene content and diversity of the bla OXA-48-like carrying plasmids and chromosomes of Escherichia coli and Klebsiella pneumoniae collected in the Dutch national surveillance from 2014 to 2019 in comparison with genome sequences from 29 countries. A combination of short-read genome sequencing with long-read sequencing enabled the reconstruction of 47 and 132 complete bla OXA-48-like plasmids for E. coli and K. pneumoniae , respectively. Seven distinct plasmid groups designated as pOXA-48-1 to pOXA-48-5, pOXA-181 and pOXA-232 were identified in the Netherlands which were similar to internationally reported plasmids obtained from countries from North and South America, Europe, Asia and Oceania. The seven plasmid groups varied in size, G+C content, presence of antibiotic resistance genes, replicon family and gene content. The pOXA-48-1 to pOXA-48-5 plasmids were variable, and the pOXA-181 and pOXA-232 plasmids were conserved. The pOXA-48-1, pOXA-48-2, pOXA-48-3 and pOXA-48-5 groups contained a putative conjugation system, but this was absent in the pOXA-48-4, pOXA-181 and pOXA-232 plasmid groups. pOXA-48 plasmids contained the PemI antitoxin, while the pOXA-181 and pOXA-232 plasmids did not. Furthermore, the pOXA-181 plasmids carried a virB2-virB3-virB9-virB10-virB11 type IV secretion system, while the pOXA-48 plasmids and pOXA-232 lacked this system. A group of non-related pOXA-48 plasmids from the Netherlands contained different resistance genes, non-IncL-type replicons or no replicons. Whole genome multilocus sequence typing revealed that the bla OXA-48-like plasmids were found in a wide variety of genetic backgrounds in contrast to chromosomally encoded bla OXA-48-like alleles. Chromosomally localized bla OXA-48 and bla OXA-244 alleles were located on genetic elements of variable sizes and comprised regions of pOXA-48 plasmids. The bla OXA-48-like genetic element was flanked by a direct repeat upstream of IS1R, and was found at multiple locations in the chromosomes of E. coli . Lastly, K. pneumoniae isolates carrying bla OXA-48 or bla OXA-232 were mostly resistant for meropenem, whereas E. coli bla OXA-48, bla OXA-181 and chromosomal bla OXA-48 or bla OXA-244 isolates were mostly sensitive. In conclusion, the overall bla OXA-48-like plasmid population in the Netherlands is conserved and similar to that reported for other countries, confirming global dissemination of bla OXA-48-like plasmids. Variations in size, presence of antibiotic resistance genes and gene content impacted pOXA-48, pOXA-181 and pOXA-232 plasmid architecture.

IS26-mediated amplification of blaOXA-1 and blaCTX-M-15 with concurrent outer membrane porin disruption associated with de novo carbapenem resistance in a recurrent bacteraemia cohort.

Approximately half of clinical carbapenem-resistant Enterobacterales (CRE) isolates lack carbapenem-hydrolysing enzymes and develop carbapenem resistance through alternative mechanisms. To elucidate development of carbapenem resistance mechanisms from clonal, recurrent ESBL-positive Enterobacterales (ESBL-E) bacteraemia isolates in a vulnerable patient population. This study investigated a cohort of ESBL-E bacteraemia cases in Houston, TX, USA. Oxford Nanopore Technologies long-read and Illumina short-read sequencing data were used for comparative genomic analysis. Serial passaging experiments were performed on a set of clinical ST131 Escherichia coli isolates to recapitulate in vivo observations. Quantitative PCR (qPCR) and qRT-PCR were used to determine copy number and transcript levels of β-lactamase genes, respectively. Non-carbapenemase-producing CRE (non-CP-CRE) clinical isolates emerged from an ESBL-E background through a concurrence of primarily IS26-mediated amplifications of blaOXA-1 and blaCTX-M-1 group genes coupled with porin inactivation. The discrete, modular translocatable units (TUs) that carried and amplified β-lactamase genes mobilized intracellularly from a chromosomal, IS26-bound transposon and inserted within porin genes, thereby increasing β-lactamase gene copy number and inactivating porins concurrently. The carbapenem resistance phenotype and TU-mediated β-lactamase gene amplification were recapitulated by passaging a clinical ESBL-E isolate in the presence of ertapenem. Clinical non-CP-CRE isolates had stable carbapenem resistance phenotypes in the absence of ertapenem exposure. These data demonstrate IS26-mediated mechanisms underlying β-lactamase gene amplification with concurrent outer membrane porin disruption driving emergence of clinical non-CP-CRE. Furthermore, these amplifications were stable in the absence of antimicrobial pressure. Long-read sequencing can be utilized to identify unique mobile genetic element mechanisms that drive antimicrobial resistance.

Primer aislamiento de enterobacteria productora de carbapenemasa tipo-NDM en un hospital público de Rosario, Argentina

Introducción: La emergencia de enterobacterias productoras de carbapenemasas en el ámbito hospitalario representa un verdadero problema de salud pública mundial. Las carbapenemasas son enzimas que producen resistencia a los antibióticos carbapenémicos, teniendo un directo impacto en la disponibilidad de alternativas terapéuticas. En Argentina, a partir de 2013 han emergido carbapenemasas tipo-NDM (Nueva Delhi Metalo-β-lactamasa, MβL), que constituyen una resistencia emergente a nivel global.
 Objetivo: Reportar el primer aislamiento clínico de enterobacteria portadora de NDM en nuestra institución.
 Materiales y métodos: El aislamiento estudiado fue recuperado de una muestra ósea de un paciente adulto. La identificación bacteriana y los ensayos de susceptibilidad antibiótica se realizaron mediante metodología manual y sistema automatizado Vitek 2C (Biomérieux). La detección y caracterización de carbapenemasas se efectuó por ensayos fenotípicos y moleculares.
 Resultados: Los ensayos revelaron que el aislamiento, tipificado como Citrobacter freundii, es productor de carbapenemasa tipo NDM. Resultó sensible a aztreonam, colistina y fosfomicina. No se detectó fenotípicamente la presencia de beta lactamasas de espectro extendido.
 Discusión: Se reporta el primer aislamiento de enterobacteria productor de MβL tipo-NDM en nuestro nosocomio, siendo multirresistente, con escasas alternativas terapéuticas. Dado que la presencia de este tipo de aislamiento es considerado de alto riesgo, se requiere un monitoreo activo de este mecanismo de resistencia y la instauración de medidas de control adecuadas para hacer frente a la amenaza que suponen.

Characterization of the First OXA-10 Natural Variant with Increased Carbapenemase Activity.

While carbapenem resistance in Gram-negative bacteria is mainly due to the production of efficient carbapenemases, β-lactamases with a narrower spectrum may also contribute to resistance when combined with additional mechanisms. OXA-10-type class D β-lactamases, previously shown to be weak carbapenemases, could represent such a case. In this study, two novel OXA-10 variants were identified as the sole carbapenem-hydrolyzing enzymes in meropenem-resistant enterobacteria isolated from hospital wastewater and found by next-generation sequencing to express additional β-lactam resistance mechanisms. The new variants, OXA-655 and OXA-656, were carried by two related IncQ1 broad-host-range plasmids. Compared to the sequence of OXA-10, they both harbored a Thr26Met substitution, with OXA-655 also bearing a leucine instead of a valine in position 117 of the SAV catalytic motif. Susceptibility profiling of laboratory strains replicating the natural blaOXA plasmids and of recombinant clones expressing OXA-10 and the novel variants in an isogenic background indicated that OXA-655 is a more efficient carbapenemase. The carbapenemase activity of OXA-655 is due to the Val117Leu substitution, as shown by steady-state kinetic experiments, where the kcat of meropenem hydrolysis was increased 4-fold. In contrast, OXA-655 had no activity toward oxyimino-β-lactams, while its catalytic efficiency against oxacillin was significantly reduced. Moreover, the Val117Leu variant was more efficient against temocillin and cefoxitin. Molecular dynamics indicated that Val117Leu affects the position 117-Leu155 interaction, leading to structural shifts in the active site that may alter carbapenem alignment. The evolutionary potential of OXA-10 enzymes toward carbapenem hydrolysis combined with their spread by promiscuous plasmids indicates that they may pose a future clinical threat.

Prevalence of VIM- and GIM-producing Acinetobacter baumannii from patients with severe urinary tract infection.

Carbapenems are administered as the final drug of choice for treating complicated nosocomial infections caused by multidrug-resistant Acinetobacter baumannii strains. It is currently a worldwide issue that metallo-β-lactamases (MBLs) as carbapenem-hydrolyzing enzymes are one of the major drug resistance mechanisms. This investigation is thus aimed to assess the prevalence and characterize the MBL-producing strains of A. baumannii both by phenotypic assays and by genotypic characterization. A total of 73 isolates of A. baumannii were phenotypically and genotypically characterized from patients (N = 1,000) with severe urinary tract infection. Tested strains were subjected to double disc synergy testing (DDST) by Kirby-Bauer disc diffusion method with imipenem (IMP) and IMP/EDTA combination discs. Plasmid DNA was molecularly screened for MBL-encoding blaIMP, blaVIM, blaGIM, and blaNDM genes by PCR for the genetic relatedness of the MBL genes with carbapenem resistance. Carbapenem resistance profile showed 100%, 45%, and 49% non-susceptibility against imipenem, doripenem, and meropenem, respectively. Altogether 42.46% (n = 31) of the isolates showed MBL production upon double disc phenotypic test with IMP and IMP/EDTA discs. The blaVIM and blaGIM were detected in 34.24% (n = 25) and 16.43% (n = 12) of the isolates, respectively, while the co-occurrence of blaVIM and blaGIM was 2.73% among the isolates. DDST-positive isolates showed 21.19% and 9.58% strains positive for blaVIM and blaGIM, respectively, whereas 1.36% of the strains for both genes. None of the strains yielded blaIMP and blaNDM genes. The findings of this study showed prevalence of carbapenem resistance among A. baumannii from urine samples and the frequency of blaVIM and blaGIM.

Diversity of Carbapenemase-Producing Escherichia coli Isolates in France in 2012-2013.

With the dissemination of carbapenemase-producing Enterobacteriaceae (CPE) strains worldwide, carbapenem-hydrolyzing enzymes are increasingly reported among isolates of Escherichia coli, the first hospital and community-acquired opportunistic pathogen. Here, we have performed an epidemiological survey of carbapenemase-producing E. coli (CP-Ec) isolates received at the French National Reference Centre (F-NRC) in 2012 and 2013. Antimicrobial susceptibilities for last-resort antibiotics and antimicrobial compounds commonly used to treat urinary tract infections were determined by broth microdilution. Clonal relationship was assessed using repetitive sequence-based PCR (rep-PCR) and multilocus sequence typing (MLST). From this collection of 140 carbapenemase-producing E. coli isolates, 74% produced an OXA-48-like carbapenemase and 21% produced an NDM carbapenemase. A link with a foreign country was suspected for 37% of infected/colonized patients. Most of the isolates were from screening (56%) and from urine samples (26%). Colistin, fosfomycin, and nitrofurantoin possessed the most consistent activity, with 100%, 95%, and 96% isolates susceptible, respectively. A wide diversity of carbapenemase-producing E. coli isolates has been found (50 different sequence types [STs]). The most prevalent clones were (i) E. coli sequence type 38 (ST38) producing OXA-48 (n = 21), a clone linked to Turkey and North African countries, (ii) E. coli ST-90 producing OXA-204 (n = 9), which was responsible for an outbreak related to a contaminated duodenoscope, and (iii) E. coli ST-410 producing OXA-181 (n = 5), which was recovered from patients of different geographical origins. These specific clones might be considered high-risk clones for the dissemination of carbapenemases in E. coli The wide diversity of STs, combined with the increasing number of CP-Ec isolates received by the F-NRC, suggests a likely dissemination of CP-Ec isolates in the community.

Global survey of Klebsiella pneumoniae major porins from ertapenem non-susceptible isolates lacking carbapenemases.

To understand the diversity of porin disruption in Klebsiella pneumoniae, the major outer membrane protein (OMP) porins, OmpK35 and OmpK36, were examined in a set of isolates that did not harbour traditional carbapenem-hydrolysing enzymes, but nevertheless tested non-susceptible to ertapenem. A world-wide collection of Klebsiella pneumoniae isolates that were part of the Study for Monitoring Antimicrobial Resistance Trends (SMART) surveillance project over the years 2008-2014 were characterised with regard to their β-lactamase gene carriage and potential permeability defects. Four hundred and eighty-seven isolates that did not carry carbapenemase genes, but were non-susceptible to ertapenem, were investigated by sequence analysis of the genes encoding OmpK35 and OmpK36. Isolates without obvious genetic lesions in either major porin gene were further examined by outer membrane protein SDS-PAGE. The majority of isolates, 83.0 % (404/487), exhibited clear genetic disruption in either or both of the ompK35 and ompK36 genes. Among the proportion of the collection with the highest ertapenem MIC value (>4 mg l-1), 60.5 % (115/190) showed mutation in both porin genes. Isolates without obvious genetic mutations were examined by SDS-PAGE, and 90.4 % (75/83) were found to lack or show altered expression of at least one of the major OMPs when compared to an ertapenem sensitive control strain. This study illustrates that porin deficiency in Klebsiella pneumoniae is a widespread phenomenon, and in combination with ESBLs and/or AmpC enzymes, likely accounts for the elevated ertapenem MICs observed in this study.

Multiple Antibiotic Resistance, Antibiogram and Phenotypic Detection of Metallo-Beta-Lactamase (MBL) from Escherichia coli of Poultry Origin

Background: Bacteria produce antibiotic-degrading enzymes such as carbapenemases. Carbapenemases are a consortium of carbapenem-hydrolyzing enzymes such as metallo-β-lactamase (MBL) that gives Gram-negative bacteria the exceptional ability to degrade and render the carbapenems inefficacious. Aim: This study evaluated the antibiogram, multiple antibiotic resistance and occurrence of MBL-producing E. coli from cloacal swabs of poultry birds in a local poultry farm in Abakaliki, Nigeria. Materials and methods: A total of 40 cloacal swab samples from the cloacal region of poultry birds were bacteriologically analyzed for the isolation of E. coli. E. coli isolates were identified using standard microbiology techniques and the antibiogram of the isolates was determined using the disk diffusion technique. The multidrug resistance nature of the E. coli isolates was determined using multiple antibiotics resistance index (MARI) protocol while MBL production was phenotypically confirmed using the inhibition based assay. Results: A total of 29 (72.5%) E. coli isolates was recovered from the 40 cloacal swab samples. The E. coli isolates were highly resistant to imipenem (31%), meropenem (58.6%), ertapenem (75.9%), cefotaxime (55.2%), ciprofloxacin (89.7%), cefoxitin (93.1%). and ceftazidime (69.0%). MBL production was phenotypically detected in 3 (10.3%) E. coli isolates out of the 29 isolates of E. coli recovered in this study. The resistant E. coli isolates were multiply resistant to antibiotics in the class of fluoroquinolones, cephalosporins, aminoglycosides and carbapenems; and they had a multiple antibiotic resistance of 0.4 on average. Conclusion: This presumptive study has shown that E. coli isolates of poultry origin produce MBL. The emergence and spread of drug resistant bacteria in the community can be contained if we use antibiotics rationally and find alternative measures for promoting animal growth without the use of antimicrobial agents.

β-Lactamases: A Focus on Current Challenges.

β-Lactamases, the enzymes that hydrolyze β-lactam antibiotics, remain the greatest threat to the usage of these agents. In this review, the mechanism of hydrolysis is discussed for both those enzymes that use serine at the active site and those that require divalent zinc ions for hydrolysis. The β-lactamases now include >2000 unique, naturally occurring amino acid sequences. Some of the clinically most important of these are the class A penicillinases, the extended-spectrum β-lactamases (ESBLs), the AmpC cephalosporinases, and the carbapenem-hydrolyzing enzymes in both the serine and metalloenzyme groups. Because of the versatility of these enzymes to evolve as new β-lactams are used therapeutically, new approaches to antimicrobial therapy may be required.

From "An Enzyme Able to Destroy Penicillin" to Carbapenemases: 70 Years of Beta-lactamase Misbehaviour.

As early as 1940, Abraham and Chain described "an enzyme able to destroy penicillin". In the late 1940's, penicillin-resistant strains of Staphylococcus aureus were found to be a clinical problem. They produced a penicillinase that could hydrolyze the amide bond in the β-lactam ring. Later, an enzyme mediated by an R-factor was isolated from Enterobacteriaceae. Methicillin and cephalosporins, both very poor substrates of the S. aureus enzyme, were found to be sensitive to this new enzyme. Third generation cephalosporins appeared to solve the problem, but further enzymes were selected that exhibited extended spectra and could for instance hydrolyze cefotaxime and/or ceftazidime. The discovery of carbapenems constituted a major advance for our antimicrobial arsenal: they inactivated most of the essential penicillin binding proteins effectively and escaped the activity of nearly all known -β lactamases. However, the metallo-β-lactamases, which had not been recognised as a major danger before 1990, were found to act as effective carbapenemases and started to spread in a worrying way. Moreover, carbapenem-hydrolyzing enzymes were found in each of the 3 classes of active-site serine β-lactamases.