Carbapenem-hydrolyzing Class D Β-lactamase Research Articles

Co-production of metallo-β-lactamase and OXA-type β-lactamases in carbapenem-resistant Acinetobacter baumannii clinical isolates in North East India.

Carbapenem-resistant Acinetobacter baumannii poses a significant threat to public health globally, especially due to its ability to produce multiple carbapenemases, leading to treatment challenges. This study aimed to investigate the antibiotic resistance pattern of carbapenem-resistant A. baumannii isolates collected from different clinical settings in North East India, focusing on their genotypic and phenotypic resistance profiles. A total of 172 multidrug-resistant A. baumannii isolates were collected and subjected to antibiotic susceptibility test using the Kirby-Bauer disk diffusion method. Various phenotypic tests were performed to detect extended-spectrum β-lactamase (ESBL), metallo-β-lactamase (MBL), class C AmpC β-lactamase (AmpC), and carbapenem hydrolyzing class D β-lactamase (CHDL) production among the isolates. Overexpression of carbapenemase and cephalosporinase genes was detected among the isolates through both phenotypic and genotypic investigation. The antibiotic resistance profile of the isolates revealed that all were multidrug-resistant; 25% were extensively drug-resistant, 9.30% were pan-drug-resistant, whereas 91.27% were resistant to carbapenems. In the genotypic investigation, 80.81% of isolates were reported harbouring at least one metallo-β-lactamase encoding gene, with blaNDM being the most prevalent at 70.34%, followed by blaIMP at 51.16% of isolates. Regarding class D carbapenemases, blaOXA-51 and blaOXA-23 genes were detected in all the tested isolates, while blaOXA-24, blaOXA-48, and blaOXA-58 were found in 15.11%, 6.97%, and 1.74% isolates respectively. Further analysis showed that 31.97% of isolates co-harboured ESBL, MBL, AmpC, and CHDL genes, while 31.39% of isolates co-harboured ESBL, MBL, and CHDL genes with or without ISAba1 leading to extensively drug-resistant or pan drug-resistant phenotypes. This study highlights the complex genetic profile and antimicrobial-resistant pattern of the isolates circulating in North East India, emphasizing the urgent need for effective infection control measures and the development of alternative treatment strategies to combat these challenging pathogens.

Epidemiological and genetic characteristics of clinical carbapenem-resistant Acinetobacter baumannii strains collected countrywide from hospital intensive care units (ICUs) in China

ABSTRACT Acinetobacter baumannii is one of the key Gram-negative pathogens that can cause serious nosocomial infections. In China, a large proportion of clinical A. baumannii strains are multidrug resistant, among which strains resistant to carbapenems are particularly worrisome, as infections caused by such strains may limit the choice of existing antibiotics. We conducted a nationwide and genome-based surveillance on the prevalence and antibiotic susceptibility profile of carbapenem-resistant A. baumannii (CRAB) strains collected from intensive care units (ICUs) in hospitals in different provinces and investigated the routes of transmission and mechanism of resistance by whole-genome sequencing and phylogenetic analysis. We found that CRAB strains were prevalent in 71.4% (55/77) of the ICUs surveyed. Clonal spread of CRAB was found in 37.6% (29/77) of ICUs and a total of 22 different clones were identified. Most clones were transmissible within one ICU, but up to six clones could be detected in at least three hospitals. In addition, carbapenem-hydrolysing class D β-lactamases (CHDL) were found to be mainly responsible for carbapenem-resistance in A. baumannii and the ST2 global-clone is the predominant type of CRAB in China. Importantly, we found that CRAB isolates currently exhibited an extremely low rate of resistance to colistin (0.4%) and tigecycline (2.5%), but a high rate of resistance to ceftazidime–avibactam (70.2%). Findings in this work shall facilitate development of appropriate antimicrobial regimens for treatment of CRAB infections. Further surveillance and research on the evolutionary and epidemiological features of clinical CRAB strains are necessary.

Genomic analysis of CTX-M-115 and OXA-23/-72 co-producing Acinetobacter baumannii, and their potential to spread resistance genes by natural transformation.

To characterize Acinetobacter baumannii strains co-producing the ESBL CTX-M-115 and carbapenem-hydrolysing class D β-lactamases (CHDLs), and to assess the potential diffusion of their resistance genes by horizontal transfer. Nineteen CTX-M-115/CHDL-positive A. baumannii were collected between 2015 and 2019 from patients hospitalized in France. Their whole-genome sequences were determined on Illumina and Oxford Nanopore platforms and were compared through core-genome MLST (cgMLST) and SNP analyses. Transferability of resistance genes was investigated by natural transformation assays. Eighteen strains were found to harbour CHDL OXA-72, and another one CHDL OXA-23, in addition to CTX-M-115, narrow-spectrum β-lactamases and aminoglycoside resistance determinants including ArmA. cgMLST typing, as well as Oxford Scheme ST and K locus typing, confirmed that 17 out of the 18 CTX-M-115/OXA-72 isolates belonged to new subclades within clonal complex 78 (CC78). The chromosomal region carrying the blaCTX-M-115 gene appeared to vary greatly both in gene content and in length (from 20 to 79 kb) among the strains, likely because of IS26-mediated DNA rearrangements. The blaOXA-72 gene was localized on closely related plasmids showing structural variations that occurred between pdif sites. Transfer of all the β-lactamase genes, as well as aminoglycoside resistance determinants to a drug-susceptible A. baumannii recipient, was easily obtained in vitro by natural transformation. This work highlights the propensity of CC78 isolates to collect multiple antibiotic resistance genes, to rearrange and to pass them to other A. baumannii strains via natural transformation. This process, along with mobile genetic elements, likely contributes to the considerable genomic plasticity of clinical strains, and to the diversity of molecular mechanisms sustaining their multidrug resistance.

Arginine Modulates Carbapenem Deactivation by OXA-24/40 in Acinetobacter baumannii

The resistance of Gram-negative bacteria to β-lactam antibiotics stems mainly from β-lactamase proteins that hydrolytically deactivate the β-lactams. Of particular concern are the β-lactamases that can deactivate a class of β-lactams known as carbapenems. Carbapenems are among the few anti-infectives that can treat multi-drug resistant bacterial infections. Revealing the mechanisms of their deactivation by β-lactamases is a necessary step for preserving their therapeutic value. Here, we present NMR investigations of OXA-24/40, a carbapenem-hydrolyzing Class D β-lactamase (CHDL) expressed in the gram-negative pathogen, Acinetobacter baumannii. Using rapid data acquisition methods, we were able to study the “real-time” deactivation of the carbapenem known as doripenem by OXA-24/40. Our results indicate that OXA-24/40 has two deactivation mechanisms: canonical hydrolytic cleavage, and a distinct mechanism that produces a β-lactone product that has weak affinity for the OXA-24/40 active site. The mechanisms issue from distinct active site environments poised either for hydrolysis or β-lactone formation. Mutagenesis reveals that R261, a conserved active site arginine, stabilizes the active site environment enabling β-lactone formation. Our results have implications not only for OXA-24/40, but the larger family of CHDLs now challenging clinical settings on a global scale.

Genomic Analysis of Carbapenem-Resistant Acinetobacter baumannii Isolates Belonging to Major Endemic Clones in South America.

Carbapenem-resistant Acinetobacter baumannii (CRAB) are emerging worldwide. In South America, clinical isolates presenting such a phenotype usually do not belong to the globally distributed international clone 2 (IC2). The majority of these isolates are also resistant to multiple other antimicrobials and are often designated extremely drug-resistant (XDR). The aim of this study was to characterize the resistance mechanisms presented by 18 carbapenem-resistant A. baumannii isolates from five different Brazilian hospitals. Species identification was determined by rpoB sequencing, and antimicrobial susceptibility was determined by broth microdilution. Isolates were submitted to whole genome sequencing using Illumina platform and genetic similarity was determined by PFGE, MLST, and cgMLST. Genome analysis was used to identify intrinsic and acquired resistance determinants, including mutations in the AdeRSABC efflux system and in outer membrane proteins (OMPs). All isolates were identified as A. baumannii and grouped into 4 pulsotypes by PFGE, which belonged to clonal complexes (CC) 15Pas/103Ox (n = 4) and 79Pas/113Ox (n = 14), corresponding to IC4 and IC5, respectively. High MIC values to carbapenems, broad-spectrum cephalosporins, amikacin, and ciprofloxacin were observed in all isolates, while MICs of ampicillin/sulbactam, gentamicin, and tigecycline varied among the isolates. Minocycline was the most active antimicrobial agent tested. Moreover, 12 isolates (66.7%) were considered resistant to polymyxins. Besides intrinsic OXA-51 and ADC variants, all isolates harbored an acquired carbapenem-hydrolyzing class D β-lactamase (CHDL) encoding gene, either blaOXA–23 or blaOXA–72. A diversity of aminoglycoside modifying enzymes and resistance determinants to other antimicrobial classes were found, as well as mutations in gyrA and parC. Non-synonymous mutations have also been identified in the AdeRSABC efflux system and in most OMPs, but they were considered natural polymorphisms. Moreover, resistance to polymyxins among isolates belonging to IC5 were associated to non-synonymous mutations in pmrB, but no known polymyxin resistance mechanism was identified in isolates belonging to IC4. In conclusion, A. baumannii clinical isolates belonging to South America’s major clones present a myriad of antimicrobial resistance determinants. Special attention should be paid to natural polymorphisms observed in each clonal lineage, especially regarding non-synonymous mutations in constitutive genes associated with distinct resistance phenotypes.

In vitro and in vivo activities of imipenem combined with BLI-489 against class D β-lactamase-producing Acinetobacter baumannii.

According to our preliminary study, BLI-489 has the potential to inhibit the hydrolysing activity of OXA-51-like β-lactamase produced by carbapenem-resistant Acinetobacter baumannii (CRAb). In the present study, the in vitro and in vivo activities of imipenem combined with BLI-489 against CRAb producing carbapenem-hydrolysing class D β-lactamases (CHDLs), namely OXA-23, OXA-24, OXA-51 and OXA-58, were determined. A chequerboard analysis of imipenem and BLI-489 was performed using 57 and 7 clinical CRAb isolates producing different CHDLs and MBLs, respectively. Four representative strains harbouring different CHDL genes were subjected to a time-kill assay to evaluate the synergistic effects. An in silico docking analysis was conducted to simulate the interactions between BLI-489 and the different families of CHDLs. The in vivo activities of this combination were assessed using a Caenorhabditis elegans survival assay and a mouse pneumonia model. Chequerboard analysis showed that imipenem and BLI-489 had a synergistic effect on 14.3, 92.9, 100, 16.7 and 100% of MBL-, OXA-23-, OXA-24-like-, OXA-51-like- and OXA-58-producing CRAb isolates, respectively. In the time-kill assay, imipenem and BLI-489 showed synergy against OXA-24-like-, OXA-51-like- and OXA-58-, but not OXA-23-producing CRAb isolates after 24 h. The in silico docking analysis showed that BLI-489 could bind to the active sites of OXA-24 and OXA-58 to confer strong inhibition activity. The combination of imipenem and BLI-489 exhibited synergistic effects for the rescue of CRAb-infected C. elegans and mice. Imipenem combined with BLI-489 has synergistic effects against CHDL-producing CRAb isolates.

Genomic and phenotypic characterisation of antimicrobial resistance in carbapenem-resistant Acinetobacter baumannii hyperendemic clones CC1, CC15, CC79 and CC25

Dissemination of carbapenem-resistant Acinetobacter baumannii (CRAB) is mainly driven by the spread of clonal lineages. High frequencies of CRAB are reported in South America, and clonal complexes CC1, CC15, CC79 and CC25 are predominant. A total of 79 non-redundant CRAB recovered from 26 Brazilian hospitals were selected for antimicrobial susceptibility testing by microdilution and whole-genome sequencing (WGS). Multilocus sequence typing (MLST), acquired antimicrobial resistance genes and phylogeny based on high-quality SNPs were extracted from WGS data. XDR (86.1%), MDR (12.7%) and one PDR isolate from CC15 (1.3%) were identified. Colistin resistance was more frequent in CC25 isolates (P < 0.01). Prevalence of CC79 (n=22; 27.8%) CC1 (n=21; 26.6%), CC15 (n=21; 26.6%) and CC25 (n=12; 15.2%) was observed. Regarding carbapenem-hydrolysing class D β-lactamases (CHDLs), blaOXA-23 was frequently detected in CC1, CC15 and CC25 isolates, whereas blaOXA-72 was the most frequent CHDL in CC79 isolates [n=12/22 (54.5%); P < 0.01]. High-quality SNP analysis correlated well with sequence type and revealed that CRAB clones are highly conversed and present some clone-specific resistance determinants. This study provides essential information to understand the antimicrobial resistance patterns of CRAB in Brazilian hospitals, where hyperendemic XDR-CRAB clones are disseminated. Phenotypic and genomic analysis of CRAB recovered from Brazilian hospitals revealed the predominance of XDR phenotype in the majority of international clonal complex CC79, CC1, CC15 and CC25. Dissemination of specific CRAB lineages in Brazil is suggested to be driven by their resistance determinants under antimicrobial selective pressure.

Prevalence of Carbapenem Resistant Acinetobacter baumannii (CRAB) in some Egyptian Hospitals: Evaluation of the Use of blaOXA-51-like Gene as Species Specific Marker for CRAB

CARBAPENEM resistant Acinetobacter baumannii (CRAB) now leads the WHO’s critical priority list of nosocomial pathogens worldwide. Carbapenem Hydrolysing Class D β-lactamases (CHDL) -“OXA-type β-lactamases”- is of great concern. In this study, bacterial isolates recovered from different clinical samples collected from three different hospitals in Cairo from October 2014 to May 2015. The isolates were identified using Vitek 2 and partial 16S rRNA gene sequencing. Acinetobacter baumannii isolates represented 16.1% of the total Gram negative isolates. Susptibility test revealed that 88.9% were CRAB, 65% of imipenem resistant isolates and 57.5% of meropenem resistant isolates had chromosomal mediated resistance. Forty CRAB isolates were identified and screened for blaOXA-23-like, blaOXA-24-like, blaOXA-51-like and blaOXA-58-like genes.The most prevalent gene was blaOXA-51-like which was detected in 95% of the isolates followed by blaOXA-23-like and blaOXA-24-like in 90% of the isolates. However, blaOXA-58-like was not detected in any of the isolates. The absence of blaOXA-51-like in 5% of CRAB isolates proving the inconvenience of using blaOXA-51-like as a species specific region for identification of CRAB by PCR. In addition, all isolates harbored more than one OXA gene, 75% were blaOXA-23/OXA-24/OXA-51 carriers while 25% harbored two OXA genes. This study revealed the emergence of blaOXA-24-like gene in CRAB for the first time in Egyptian hospitals.

Site-Specific Recombination at XerC/D Sites Mediates the Formation and Resolution of Plasmid Co-integrates Carrying a blaOXA-58- and TnaphA6-Resistance Module in Acinetobacter baumannii.

Members of the genus Acinetobacter possess distinct plasmid types which provide effective platforms for the acquisition, evolution, and dissemination of antimicrobial resistance structures. Many plasmid-borne resistance structures are bordered by short DNA sequences providing potential recognition sites for the host XerC and XerD site-specific tyrosine recombinases (XerC/D-like sites). However, whether these sites are active in recombination and how they assist the mobilization of associated resistance structures is still poorly understood. Here we characterized the plasmids carried by Acinetobacter baumannii Ab242, a multidrug-resistant clinical strain belonging to the ST104 (Oxford scheme) which produces an OXA-58 carbapenem-hydrolyzing class-D β-lactamase (CHDL). Plasmid sequencing and characterization of replication, stability, and adaptive modules revealed the presence in Ab242 of three novel plasmids lacking self-transferability functions which were designated pAb242_9, pAb242_12, and pAb242_25, respectively. Among them, only pAb242_25 was found to carry an adaptive module encompassing an ISAba825-blaOXA-58 arrangement accompanied by a TnaphA6 transposon, the whole structure conferring simultaneous resistance to carbapenems and aminoglycosides. Ab242 plasmids harbor several XerC/D-like sites, with most sites found in pAb242_25 located in the vicinity or within the adaptive module described above. Electrotransformation of susceptible A. nosocomialis cells with Ab242 plasmids followed by imipenem selection indicated that the transforming plasmid form was a co-integrate resulting from the fusion of pAb242_25 and pAb242_12. Further characterization by cloning and sequencing studies indicated that a XerC/D site in pAb242_25 and another in pAb242_12 provided the active sister pair for the inter-molecular site-specific recombination reaction mediating the fusion of these two plasmids. Moreover, the resulting co-integrate was found also to undergo intra-molecular resolution at the new pair of XerC/D sites generated during fusion thus regenerating the original pAb242_25 and pAb242_12 plasmids. These observations provide the first evidence indicating that XerC/D-like sites in A. baumannii plasmids can provide active pairs for site-specific recombination mediating inter-molecular fusions and intra-molecular resolutions. The overall results shed light on the evolutionary dynamics of A. baumannii plasmids and the underlying mechanisms of dissemination of genetic structures responsible for carbapenem and other antibiotics resistance among the Acinetobacter clinical population.

Dissemination and Characteristics of a Novel Plasmid-Encoded Carbapenem-Hydrolyzing Class D β-Lactamase, OXA-436, Found in Isolates from Four Patients at Six Different Hospitals in Denmark.

The diversity of OXA-48-like carbapenemases is continually expanding. In this study, we describe the dissemination and characteristics of a novel carbapenem-hydrolyzing class D β-lactamase (CHDL) named OXA-436. In total, six OXA-436-producing Enterobacteriaceae isolates, including Enterobacter asburiae (n = 3), Citrobacter freundii (n = 2), and Klebsiella pneumoniae (n = 1), were identified in four patients in the period between September 2013 and April 2015. All three species of OXA-436-producing Enterobacteriaceae were found in one patient. The amino acid sequence of OXA-436 showed 90.4 to 92.8% identity to the amino acid sequences of other acquired OXA-48-like variants. Expression of OXA-436 in Escherichia coli and kinetic analysis of purified OXA-436 revealed an activity profile similar to that of OXA-48 and OXA-181, with activity against penicillins, including temocillin; limited or no activity against extended-spectrum cephalosporins; and activity against carbapenems. The blaOXA-436 gene was located on a conjugative ∼314-kb IncHI2/IncHI2A plasmid belonging to plasmid multilocus sequence typing sequence type 1 in a region surrounded by chromosomal genes previously identified to be adjacent to blaOXA genes in Shewanella spp. In conclusion, OXA-436 is a novel CHDL with functional properties similar to those of OXA-48-like CHDLs. The described geographical spread among different Enterobacteriaceae and the plasmid location of blaOXA-436 illustrate its potential for further dissemination.

Expanded Substrate Activity of OXA-24/40 in Carbapenem-Resistant Acinetobacter baumannii Involves Enhanced Binding Loop Flexibility

Gram-negative bacteria resist β-lactam antibiotics primarily by deploying β-lactamase proteins that hydrolytically destroy the antibiotics. In clinical settings, these bacteria are producing variant β-lactamases with "gain-of-activity" mutations that inactivate a broader range of β-lactams. Learning how these mutations broaden substrate activity is important for coping with β-lactam resistance. Here, we investigate a gain of activity mutation in OXA-24/40, a carbapenem-hydrolyzing class D β-lactamase (CHDL) in Acinetobacter baumannii. OXA-24/40 was originally active against penicillin and carbapenem classes of β-lactams, but a clinical variant of OXA-24/40, the single-site substitution mutant P227S, has emerged with expanded activity that now includes advanced cephalosporins and the monobactam aztreonam. Using solution-state nuclear magnetic resonance (NMR) spectroscopy, we have compared the site-specific backbone dynamics of wild-type OXA-24/40 and the P227S variant. P227S changes local backbone flexibility in segments that are important for both binding and hydrolysis of carbapenem and cephalosporin substrates. Our results suggest that mutation-induced changes in sequence-specific dynamics can expand substrate activity and thus highlight the role of protein conformational dynamics in antibiotic resistance. To the best of our knowledge, this is the first NMR study of CHDL conformational dynamics and its impact on the expansion of β-lactam antibiotic resistance.

In vitro activity of SecA inhibitors in combination with carbapenems against carbapenem-hydrolysing class D β-lactamase-producing Acinetobacter baumannii.

According to our previous study, OXA-58 translocates to the periplasm via the Sec pathway in carbapenem-resistant Acinetobacter baumannii (CRAb). In the present study, carbapenem-hydrolysing class D β-lactamases (CHDLs) belonging to the OXA-23, OXA-40 and OXA-51 families were examined to determine whether they are also Sec-dependent. Additionally, the effects of SecA inhibitors combined with carbapenems against CHDL-producing CRAb were examined. Cell fractionation and western blot analyses were performed to detect periplasmic His-tagged CHDLs. A chequerboard analysis with pairwise combinations of carbapenems (imipenem or meropenem) and SecA inhibitors (rose bengal, sodium azide or erythrosin B) was performed using six clinical CRAb isolates harbouring different CHDL genes. The fractional inhibitory concentration (FIC) index was determined. The combination with the lowest FIC index was subjected to a time-kill analysis to examine synergistic effects. In an in silico analysis, the CHDLs OXA-23, OXA-40 and OXA-51 were preferentially translocated via the Sec system. The SecA inhibitor rose bengal decreased periplasmic translocation of His-tagged OXA-23 and OXA-83 (belonging to the OXA-51 family), but not OXA-72 (belonging to the OXA-40 family) from ATCC 15151 transformants. Imipenem or meropenem with rose bengal showed synergistic effects (FIC index, ≤0.5) for six and four clinical isolates, respectively. Imipenem or meropenem with sodium azide showed no interactions (FIC index, 0.5-4) against all clinical isolates. Imipenem and rose bengal had the lowest FIC index and showed synergy at 24 h in the time-kill assay. Combinations of SecA inhibitors and carbapenems have synergistic effects against CHDL-producing CRAb.

Detection of SPM-1-Producing Pseudomonas aeruginosa and Class D β-Lactamase-Producing Acinetobacter baumannii Isolates by Use of Liquid Chromatography-Mass Spectrometry and Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry

This study evaluates the accuracy of liquid chromatography-mass spectrometry (LC-MS) and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF) for detecting carbapenem hydrolytic activity among SPM-1-, GIM-1-, and GES-5-producing Pseudomonas aeruginosa isolates and OXA-143-, IMP-10-, and OXA-58-producing Acinetobacter baumannii isolates. Class A and B carbapenemase activities were rapidly detected by MALDI-TOF in a 2-h assay. However, an extended incubation time was necessary for detection of carbapenem-hydrolyzing class D β-lactamase (CHDL) activity in Acinetobacter spp.

Evidence of Diversity among Epidemiologically Related Carbapenemase-Producing Acinetobacter baumannii Strains Belonging to International Clonal Lineage II

Carbapenem-resistant Acinetobacter baumannii strains belonging to international clonal lineage II (ICL-II) have become predominant in intensive care units (ICUs) throughout Italy. Between 2005 and 2009, the carbapenem-hydrolyzing class D β-lactamase (CHDL) bla(OXA-23) gene became more prevalent than bla(OXA-58) among epidemic ICL-II strains showing extensive genetic similarity. These findings posed the question of whether CHDL gene replacement occurred in the homogeneous ICL-II population or a new OXA-23 clone(s) emerged and spread in ICUs. In this study, the changes in the ICL-II A. baumannii population and CHDL gene carriage were investigated in 30 genetically related isolates collected during the bla(OXA-58)-to-bla(OXA-23) transition period. Pulsotyping, randomly amplified polymorphic DNA (RAPD) analysis, and multilocus sequence typing (MLST) results were combined with multilocus variable-number tandem-repeat (VNTR) analysis (MLVA-8), siderotyping, and plasmid profiling to improve genotype-based discrimination between isolates. Pulsotyping, RAPD analysis, and MLST clustered isolates into a single type. MLVA-8 identified 19 types that clustered into three complexes. All OXA-23-producing isolates formed a single complex, while OXA-58 producers were split into two complexes. Southern blot analysis of the physical localization and genetic context of the CHDL genes showed that bla(OXA-58) was invariably located on plasmids, while bla(OXA-23) was present within Tn2006 on the chromosome or both the chromosome and plasmids. These data indicate that the apparently homogeneous population of CHDL-producing ICL-II strains was composed of several independent strains and that, between 2005 and 2009, distinct OXA-23 producers displaced the preexisting OXA-58 producers. Thus, MLVA-8 appears to be a suitable tool not only for investigating A. baumannii population structure but also for high-resolution epidemiological typing.

Understanding the dynamics of imipenem-resistant Acinetobacter baumannii lineages within Portugal

A recent collection of 213 imipenem-resistant Acinetobacter baumannii (IRAB) clinical isolates was characterized for the presence of acquired carbapenem-hydrolysing class D β-lactamases (CHDLs) and clonality. A population structure analysis of IRAB was also conducted, with five molecular typing methods. Three main clusters, each one associated with a specific CHDL, were observed with multilocus sequence typing. Overall, our results suggest a switch in the dominant clone, with sequence type (ST) 92, carrying blaOXA-23 (63.4%), replacing the closely related ST98, carrying blaOXA-24/40 (22%). In addition, ST103, an independent lineage, was associated with blaOXA-58-carrying isolates (14.6%).

High Prevalence of Carbapenem-Resistant Acinetobacter baumannii Carrying the bla OXA-143 Gene in Brazilian Hospitals

A recent article by Higgins et al. reported the identification of a novel carbapenem-hydrolyzing class D β-lactamase (CHDL), defined as OXA-143, in a carbapenem-resistant Acinetobacter baumannii strain isolated in Brazil in 2004 (5). This enzyme, which has 88% identity with OXA-40, is the first representative of a novel subgroup of CHDLs whose prevalence remains to be determined (5). In an attempt to carry out this task, we have conducted a surveillance study of carbapenem-resistant A. baumannii isolates in order to determine the antimicrobial susceptibility patterns and prevalence of blaOXA-type carbapenemase genes in medical centers in both the southeastern and southern regions of Brazil. We report here a high prevalence of carbapenem-resistant A. baumannii carrying the blaOXA-143 and blaOXA-23 genes, along with the first isolation of strains carrying the blaOXA-72 (OXA-72 is a single-amino-acid variant of OXA-40) and blaOXA-58 genes in Brazilian hospitals. From 2004 to 2008, 36 carbapenem-resistant A. baumannii isolates recovered from different patients hospitalized in eight medical centers were screened for the presence of genes encoding metallo-β-lactamases (MβLs) and OXA-type β-lactamases (6, 15). Flanking sequences of blaOXA-type genes were characterized by PCR using primers targeting ISAba-1 or -3 (12). PCR products were confirmed by sequencing, and the genetic diversity of blaOXA-positive strains was determined by enterobacterial repetitive intergenic consensus (ERIC)-PCR analysis (11). All isolates were found to be resistant to imipenem, meropenem, ceftazidime, aztreonam, piperacillin-tazobactam, and ciprofloxacin. Tobramycin (61.1%), ampicillin-sulbactam (61.1%), gentamicin (47.2%), amikacin (27.8%), and cefepime (11.1%) susceptibility rates were determined. MβL-encoding genes were not identified, whereas a high prevalence of blaOXA genes was noticed among carbapenem-resistant isolates (Table ​(Table1).1). In this regard, 21 strains (58.3%) carried the blaOXA-143 gene, 15 strains (41.7%) carried the ISAba-1/blaOXA-23 gene array, one strain carried the ISAba-3/blaOXA-58/ISAba-3 gene array, and two isolates carried the blaOXA-72 gene (GenBank accession no. {type:entrez-nucleotide,attrs:{text:FJ628170,term_id:223585696,term_text:FJ628170}}FJ628170, {type:entrez-nucleotide,attrs:{text:FJ492877,term_id:219563065,term_text:FJ492877}}FJ492877, {type:entrez-nucleotide,attrs:{text:FJ969387,term_id:238866561,term_text:FJ969387}}FJ969387, and {type:entrez-nucleotide-range,attrs:{text:HM804278 to HM804281,start_term:HM804278,end_term:HM804281,start_term_id:308737170,end_term_id:308737176}}HM804278 to HM804281). Although the presence of the blaOXA-51 and ISAba-1 genes was confirmed in all of the isolates, no colinearity of the two genes (i.e., ISAba-1 adjacent to the blaOXA-23 gene) was observed. Isolates harboring the blaOXA-143, blaOXA-72, and blaOXA-58 genes were restricted to hospitals located in Sao Paulo (the largest and most populous metropolitan area in southeastern Brazil), while blaOXA-23-harboring A. baumannii isolates were obtained from hospitals in Sao Paulo and Parana (in southern Brazil), confirming previous reports of widespread dissemination of OXA-23-producing A. baumannii in this region (2, 9). Finally, ERIC-PCR typing revealed genetic diversity among blaOXA-143- and blaOXA-23-positive A. baumannii isolates. TABLE 1. Characteristics of carbapenem-resistant A. baumannii isolates carrying blaOXA-type genes encoding class D carbapenemasesa In summary, the blaOXA-143 gene was commonly identified among the carbapenem-resistant A. baumannii isolates surveyed in this study. Also, we report the first isolation of blaOXA-72- and blaOXA-58-carrying strains in Brazilian hospitals. OXA-143 is a new subgroup of CHDL identified in isolates of A. baumannii from Brazil (5). We would only bring to mind that the blaOXA-72 gene was first reported from an A. baumannii strain in Thailand in 2004 (GenBank accession no. {type:entrez-nucleotide,attrs:{text:AY739646,term_id:52699554,term_text:AY739646}}AY739646), and so far it has been restricted to Acinetobacter sp. isolates from Asian and Mediterranean countries (1, 4, 7, 8, 14).

First Isolation of the bla OXA-23 Carbapenemase Gene from an Environmental Acinetobacter baumannii Isolate

Acinetobacter baumannii is frequently associated with nosocomial infections, and its increasing resistance to carbapenems may significantly reduce the choice of effective antibiotics ([2][1]). Since the first description of a carbapenem-hydrolyzing class D β-lactamase (CHDL), ARI-1 (renamed OXA-23

OXA-143, a Novel Carbapenem-Hydrolyzing Class D β-Lactamase in Acinetobacter baumannii

A carbapenem-resistant Acinetobacter baumannii strain was isolated in Brazil in 2004 in which no known carbapenemase gene was detected by PCR. Cloning experiments, followed by expression in Escherichia coli, gave an E. coli recombinant strain expressing a novel carbapenem-hydrolyzing class D beta-lactamase (CHDL). OXA-143 showed 88% amino acid sequence identity with OXA-40, 63% identity with OXA-23, and 52% identity with OXA-58. It hydrolyzed penicillins, oxacillin, meropenem, and imipenem but not expanded-spectrum cephalosporins. The bla(OXA-143) gene was located on a ca. 30-kb plasmid. After transformation into reference strain A. baumannii ATCC 19606, it conferred resistance to carbapenems. Analysis of the genetic environment of bla(OXA-143) revealed that it was associated with neither insertion sequences nor integron structures. However, it was bracketed by similar replicase-encoding genes at both ends, suggesting acquisition through a homologous recombination process. This study identified a novel class D beta-lactamase involved in carbapenem resistance in A. baumannii. This enzyme is the first member of a novel subgroup of CHDLs whose prevalence remains to be determined.