Carbapenem-hydrolyzing Class Research Articles

Bacterial Phosphorylation Suppresses Carbapenemase Activity of the Class-D β-Lactamase OXA-24/40 from Acinetobacter baumannii.

The resistance of Gram-negative bacteria to β-lactam antibiotics is mostly due to deactivation of the antibiotics by bacterial enzymes, β-lactamases. Disclosing the factors regulating β-lactamase activity is vital for developing therapies to combat multidrug-resistant pathogens, such as Acinetobacter baumannii. Recent A. baumannii studies have revealed post-translational phosphorylation of serine β-lactamases at the active site serine. However, the functional consequences of such phosphorylation are unclear. We have taken the first steps to define these consequences through studies of OXA-24/40, a carbapenem-hydrolyzing class D β-lactamase in A. baumannii. We generated OXA-24/40 phosphorylated at its active site serine, S81, and explored its effects via NMR and MS. Phosphorylation inhibits carbapenemase activity by altering the active site conformation and impeding the carboxylation of an active site lysine, a requirement for class D β-lactamase activity. The inhibition varies with the carbapenem side chain properties. Phosphorylation-induced chemical shift perturbations extend beyond the active site, suggesting allosteric effects. Our findings offer the first atomic-level insights into the functional consequences of serine phosphorylation of class D β-lactamases.

Insertion Sequences within Oxacillinases Genes as Molecular Determinants of Acinetobacter baumannii Resistance to Carbapenems-A Pilot Study.

Carbapenem-resistant Acinetobacter baumannii is one of the major problems among hospitalized patients. The presence of multiple virulence factors results in bacteria persistence in the hospital environment. It facilitates bacterial transmission between patients, causing various types of infections, mostly ventilator-associated pneumonia and wound and bloodstream infections. A. baumannii has a variable number of resistance mechanisms, but the most commonly produced are carbapenem-hydrolyzing class D β-lactamases (CHDLs). In our study, the presence of blaOXA-23, blaOXA-40 and blaOXA-51 genes was investigated among 88 clinical isolates of A. baumannii, including 53 (60.2%) strains resistant to both carbapenems (meropenem and imipenem) and 35 (39.8%) strains susceptible to at least meropenem. Among these bacteria, all the isolates carried the blaOXA-51 gene. The blaOXA-23 and blaOXA-40 genes were detected in two (5.7%) and three (8.6%) strains, respectively. Among the OXA-23 carbapenemase-producing A. baumannii strains (n = 55), insertion sequences (ISAba1) were detected upstream of the blaOXA-23 gene in fifty-two (94.5%) carbapenem-resistant and two (3.6%) meropenem-susceptible isolates. A. baumannii clinical strains from Poland have a similar antimicrobial resistance profile as those worldwide, with the presence of ISAba1 among blaOXA-23-positive isolates also being quite common. Carbapenem resistance among A. baumannii strains is associated with the presence of CHDLs, especially when insertion sequences are present.

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.

1932. Molecular Epidemiology of NDM-producing Acinetobacter baumannii in the US—October 2013—March 2022

Abstract Background In the United States, most carbapenem-resistant Acinetobacter baumannii (CRAB) produce carbapenem-hydrolyzing class D β-lactamases (OXA); other carbapenemase classes have historically been uncommon. We describe the epidemiology and molecular characteristics of New Delhi Metallo-β-lactamase (NDM)-producing CRAB reported to CDC since October 2013.Figure 1:A core genome multilocus sequence typing (cgMLST) scheme phylogenetic tree of 208 the New Delhi Metallo-β-lactamase (NDM), sequenced by US public health laboratories and with publicly available sequences, with dates of collection between November 1, 2018 – March 31, 2022. Four regional clusters were identified in Mountain, Mid-Atlantic, Midwest and West regions. Isolates within each cluster were closely related to each other. Methods We included a patient’s first NDM-CRAB isolate reported to CDC through reference antimicrobial susceptibility testing, outbreak response, or Antimicrobial Resistance Laboratory Network, with specimen collection dates during 10/1/2013–3/31/2022. Two or more NDM-CRAB linked in space and time were classified as cluster-associated; other NDM-CRAB as sporadic. We described patient demographics and clinical characteristics and analyzed whole genome sequence (WGS) data for relatedness using both traditional multilocus sequence typing (MLST) with the Oxford scheme (STOX) and core genome (cg) MLST. We compared publicly available sequences of US NDM-CRAB to NDM-CRAB from non-US locations. Results CDC received reports of 263 NDM-CRAB from unique patients in 21 states. Patients had a median age of 65 years (IQR: 55-74) (Table 1). NDM-CRAB were from clinical cultures of respiratory (60), wound (44), urine (32), blood (11), and other sites (11) and from rectal or axilla/groin cultures (96). Eleven of 103 patients with information available were hospitalized outside the US ≤12 months prior to NDM-CRAB specimen collection, 82% were sporadic cases and 19% were a part of clusters. Overall, 191 were from cluster-associated cases in four distinct geographic regions that reported multifacility outbreaks; all cluster-associated cases were from cultures collected after June 15, 2019. WGS data were available for 208 (79%) isolates. The four regional outbreaks identified in the epidemiologic investigation were different STOX (Fig. 1); three (ST218, ST281, and ST1697) were closely related to NDM-CRAB isolates from outside the US. Conclusion NDM-CRAB reported to CDC appears to reflect domestic acquisition rather than importation. ST differences across the regional outbreaks indicate they arose from unique introductions rather than inter-regional spread or dissemination of a successful clone. Disclosures All Authors: No reported disclosures

Clonal relatedness of carbapenem-resistant Acinetobacter baumannii: high prevalence of ST136pas in a burn center

BackgroundCarbapenem-resistant Acinetobacter baumannii (CRAB) is a global health crisis. This study aimed to determine the clonal relatedness of antibiotic-resistant A. baumannii isolates in hospitalized patients who suffered from burn wound infection.MethodsOne hundred and six A. baumannii isolates from 562 patients with burn wound infections, were identified and examined for antimicrobial susceptibility. Detection and characterization of carbapenem-hydrolyzing class D OXA-type beta-lactamases (CHDLs) were performed by PCR assays. The clonal relatedness of A. baumannii isolates was determined by multilocus sequence typing (MLST) according to the Pasteur scheme, dual-sequence typing of blaOXA−51-like and ampC genes, and RAPD-PCR method.ResultsAll isolates were carbapenem-resistant while susceptible to colistin, minocycline, doxycycline, and ampicillin-sulbactam. The intrinsic blaOXA−51-like was detected in all isolates, and blaOXA−23-like was identified in 92.5% of isolates. However, blaOXA−143-like and blaOXA−58-like genes were not detected among isolates. Four distinct blaOXA−51-like alleles were determined as follows: blaOXA−317 (67.0%), blaOXA−90 (9.4%), blaOXA−69 (17.0%), and blaOXA−64 (6.6%) and four ampC (blaADC) allele types including ampC-25 (6.6%), ampC-39 (9.4%), ampC-1 (17.0%), and blaADC−88 (67.0%) were identified. MLST (Pasteur scheme) analysis revealed four ST types including ST136 (singleton), ST1 (CC1), ST25 (CC25), and ST78 (singleton) in 71, 18, 7, and 10 of A. baumannii strains, respectively. Five RAPD clusters including A (1.9%), B (26.4%), C (57.5%), D (7.5%), and E (1.9%) were characterized and 5 (4.7%) strains were found to be singletons.ConclusionThe present study demonstrated that there was a high prevalence of blaOXA−23-like producing CRAB in the clinical setting. The majority of isolates belonged to ST136 (singleton). However, blaOXA−23-like producing multi-drug resistant international clones including ST1, and emerging lineages (e.g. ST25 and ST78) were also identified. Interestingly, in this study ST2 was not detected.

Phenotypic and Molecular Characteristics of Carbapenem-Resistant Acinetobacter baumannii Isolates from Bulgarian Intensive Care Unit Patients.

Carbapenem-resistant Acinetobacter baumannii (CRAB) is designated as an urgent public health threat, both due to its remarkable multidrug resistance and propensity for clonal spread. This study aimed to explore the phenotypic and molecular characteristics of antimicrobial resistance in CRAB isolates (n = 73) from intensive care unit (ICU) patients in two university hospitals in Bulgaria (2018-2019). The methodology included antimicrobial susceptibility testing, PCR, whole-genome sequencing (WGS), and phylogenomic analysis. The resistance rates were as follows: imipenem, 100%; meropenem, 100%; amikacin, 98.6%; gentamicin, 89%; tobramycin, 86.3%; levofloxacin, 100%; trimethoprim-sulfamethoxazole, 75.3%; tigecycline, 86.3%; colistin, 0%; and ampicillin-sulbactam, 13.7%. All isolates harbored blaOXA-51-like genes. The frequencies of distribution of other antimicrobial resistance genes (ARGs) were: blaOXA-23-like, 98.6%; blaOXA-24/40-like, 2.7%; armA, 86.3%; and sul1, 75.3%. The WGS of selected extensively drug-resistant A. baumannii (XDR-AB) isolates (n = 3) revealed the presence of OXA-23 and OXA-66 carbapenem-hydrolyzing class D β-lactamases in all isolates, and OXA-72 carbapenemase in one of them. Various insertion sequencies, such as ISAba24, ISAba31, ISAba125, ISVsa3, IS17, and IS6100, were also detected, providing increased ability for horizontal transfer of ARGs. The isolates belonged to the widespread high-risk sequence types ST2 (n = 2) and ST636 (n = 1) (Pasteur scheme). Our results show the presence of XDR-AB isolates, carrying a variety of ARGs, in Bulgarian ICU settings, which highlights the crucial need for nationwide surveillance, especially in the conditions of extensive antibiotic usage during COVID-19.

Detection of blaOXA-145, blaOXA-224, blaOXA-539, and blaOXA-675 Genes and Carbapenem-Hydrolyzing Class D β-Lactamases (CHDLs) in Clinical Isolates of Pseudomonas aeruginosa Collected from West of Iran, Hamadan.

Carbapenem-hydrolyzing class D β-lactamases (CHDLs) are on the rise and are a major public health problem worldwide. Pseudomonas aeruginosa is resistant to carbapenem; currently, the most effective treatment option is being increasingly reported. This study aimed to identify blaOXA-145, blaOXA-224, blaOXA-539, and blaOXA-675 genes in CHDL strains. Samples were collected from clinical specimens admitted to the hospital. Antibiotic susceptibility was determined using the disk diffusion methods. CHDL strains were detected using a phenotypic method (disk diffusion). The PCR method was used to identify blaOXA-145, blaOXA-224, blaOXA-539, and blaOXA-675 genes. Piperacillin-resistant strains (n = 9, 17.4%) had the lowest frequency, and cefoxitin-resistant strains (n = 100, 91.7%) had the highest distribution in P. aeruginosa isolates. Also, 29.35%, 12.8%, and 8.2% were multidrug-resistant, extensively drug-resistant, and pan drug-resistant, respectively. MBL-producing P. aeruginosa and KPC-producing P. aeruginosa were detected, respectively, in 47.7% and 37.6% of isolates. Biofilm formation was observed in 63.3% of P. aeruginosa isolates. The frequency of OXA genes was as follows: blaOXA-145 gene in 30 isolates (27.5%), blaOXA-224 in 24 isolates (22.0%), blaOXA-539 in 22 isolates (20.1%), and blaOXA-675 in 13 isolates (11.9%). However, 19 (17.4%) isolates carry all blaOXA-145, blaOXA-224, blaOXA-539, and blaOXA-675 genes. The antimicrobial resistance and OXA genes among biofilm former strains were significantly higher than those of nonbiofilm former strains (p < 0.05). The emergence of carbapenem-resistant isolates of P. aeruginosa has posed serious threats to the community because they exhibit multiple drug resistance, thus limiting the therapeutic options for clinicians.

Molecular Characterization of Carbapenem-Resistant Acinetobacter baumannii Isolated from Intensive Care Unit Patients in Jordanian Hospitals.

Acinetobacter baumannii is a common cause of healthcare-associated infections (HAI) worldwide, mostly occurring in intensive care units (ICUs). Extended-spectrum beta lactamases (ESBL)-positive A. baumannii strains have emerged as highly resistant to most currently used antimicrobial agents, including carbapenems. The most common mechanism for carbapenem resistance in this species is β-lactamase-mediated resistance. Carbapenem-hydrolyzing class D oxacillinases are widespread among multidrug-resistant (MDR) A. baumannii strains. The present study was conducted to determine the presence and distribution of blaOXA genes among multidrug-resistant A. baumannii isolated from ICU patients and genes encoding insertion sequence (IS-1) in these isolates. Additionally, the plasmid DNA profiles of these isolates were determined. A total of 120 clinical isolates of A. baumannii from various ICU clinical specimens of four main Jordanian hospitals were collected. Bacterial isolate identification was confirmed by biochemical testing and antibiotic sensitivity was then assessed. PCR amplification and automated sequencing were carried out to detect the presence of blaOXA-51, blaOXA-23, blaOXA-24, and blaOXA-58 genes, and ISAba1 insertion sequence. Out of the 120 A. baumannii isolates, 95% of the isolates were resistant to three or more classes of the antibiotics tested and were identified as MDR. The most frequent resistance of the isolates was against piperacillin (96.7%), cephalosporins (97.5%), and β-lactam/β-lactamase inhibitor combinations antibiotics (95.8%). There were 24 (20%) ESBL-producing isolates. A co-existence of blaOXA-51 gene and ISAba1 in all the 24 ESBL-producing isolates was determined. In addition, in the 24 ESBL-producing isolates, 21 (87.5%) carried blaOXA-51 and blaOXA-23 genes, 1 (4.2%) carried blaOXA-51 and blaOXA-24, but all were negative for the blaOXA-58 gene. Plasmid DNA profile A and profile B were the most common (29%) in ESBL-positive MDR A. baumannii isolates while plasmid DNA profile A was the most common in the ESBL-negative isolates. In conclusion, there was an increase in prevalence of MDR-A. baumannii in ICU wards in Jordanian hospitals, especially those having an ESBL phenotype. Thus, identification of ESBL genes is necessary for the surveillance of their transmission in hospitals.

Conformational flexibility in carbapenem hydrolysis drives substrate specificity of the class D carbapenemase OXA-24/40

The evolution of multidrug resistance in Acinetobacter spp. increases the risk of our best antibiotics losing their efficacy. From a clinical perspective, the carbapenem-hydrolyzing class D β-lactamase subfamily present in Acinetobacter spp. is particularly concerning because of its ability to confer resistance to carbapenems. The kinetic profiles of class D β-lactamases exhibit variability in carbapenem hydrolysis, suggesting functional differences. To better understand the structure–function relationship between the carbapenem-hydrolyzing class D β-lactamase OXA-24/40 found in Acinetobacter baumannii and carbapenem substrates, we analyzed steady-state kinetics with the carbapenem antibiotics meropenem and ertapenem and determined the structures of complexes of OXA-24/40 bound to imipenem, meropenem, doripenem, and ertapenem, as well as the expanded-spectrum cephalosporin cefotaxime, using X-ray crystallography. We show that OXA-24/40 exhibits a preference for ertapenem compared with meropenem, imipenem, and doripenem, with an increase in catalytic efficiency of up to fourfold. We suggest that superposition of the nine OXA-24/40 complexes will better inform future inhibitor design efforts by providing insight into the complicated and varying ways in which carbapenems are selected and bound by class D β-lactamases.

Molecular Detection of VEB and OXA-23 Resistance Gene in Pseudomonas Aeruginosa Isolates at Dr. Wahidin Sudirohusodo Hospital Makassar

Pseudomonas aeruginosa is a common gram-negative pathogen in nosocomial infections in immune-compromised patients. It exhibits high rates of intrinsic resistance to many classes of antibiotics, especially beta-lactam antibiotics. Production of extended-spectrum beta-lactamase (ESBL) and genes belonging to the carbapenem-hydrolyzing class D subgroup β-lactamases (CHDL) are a problem for increasing antibiotic resistance worldwide. This study aimed to identify P. aeruginosa containing the VEB and OXA-23 genes. Eighty-five clinical isolates of P. aeruginosa from various clinical samples were identified and tested for antimicrobial susceptibility using VITEK 2 compact. VEB and OXA-23 genes were detected using the Polymerase Chain Reaction (PCR) method. The PCR results revealed that 13 (15.3%) of P. aeruginosa isolates were positive OXA-23 gene, but no isolate positive for the VEB gene in P. aeruginosa isolates. The study results demonstrated the spread of the OXA-23 gene in P. aeruginosa isolates at Dr. Wahidin Sudirohusodo Hospital Makassar.

Specific Amino Acid Substitutions in OXA-51-Type β-Lactamase Enhance Catalytic Activity to a Level Comparable to Carbapenemase OXA-23 and OXA-24/40.

The chromosomal blaOXA-51-type gene encodes carbapenem-hydrolyzing class D β-lactamases (CHDLs), specific variants shown to mediate carbapenem resistance in the Gram-negative bacterial pathogen Acinetobacter baumannii. This study aims to characterize the effect of key amino acid substitutions in OXA-51 variants of carbapenem-hydrolyzing class D β-lactamases (CHDLs) on substrate catalysis. Mutational and structural analyses indicated that each of the L167V, W222G, or I129L substitutions contributed to an increase in catalytic activity. The I129L mutation exhibited the most substantial effect. The combination of W222G and I129L substitutions exhibited an extremely strong catalytic enhancement effect in OXA-66, resulting in higher activity than OXA-23 and OXA-24/40 against carbapenems. These findings suggested that specific arrangement of residues in these three important positions in the intrinsic OXA-51 type of enzyme can generate variants that are even more active than known CHDLs. Likewise, mutation leading to the W222M change also causes a significant increase in the catalytic activity of OXA-51. blaOXA-51 gene in A. baumannii may likely continue to evolve, generating mutant genes that encode carbapenemase with extremely strong catalytic activity.

C6 Hydroxymethyl-Substituted Carbapenem MA-1-206 Inhibits the Major Acinetobacter baumannii Carbapenemase OXA-23 by Impeding Deacylation.

ABSTRACTAcinetobacter baumannii has become a major nosocomial pathogen, as it is often multidrug-resistant, which results in infections characterized by high mortality rates. The bacterium achieves high levels of resistance to β-lactam antibiotics by producing β-lactamases, enzymes which destroy these valuable agents. Historically, the carbapenem family of β-lactam antibiotics have been the drugs of choice for treating A. baumannii infections. However, their effectiveness has been significantly diminished due to the pathogen’s production of carbapenem-hydrolyzing class D β-lactamases (CHDLs); thus, new antibiotics and inhibitors of these enzymes are urgently needed. Here, we describe a new carbapenem antibiotic, MA-1-206, in which the canonical C6 hydroxyethyl group has been replaced with hydroxymethyl. The antimicrobial susceptibility studies presented here demonstrated that this compound is more potent than meropenem and imipenem against A. baumannii producing OXA-23, the most prevalent CHDL of this pathogen, and also against strains producing the CHDL OXA-24/40 and the class B metallo-β-lactamase VIM-2. Our kinetic and mass spectrometry studies revealed that this drug is a reversible inhibitor of OXA-23, where inhibition takes place through a branched pathway. X-ray crystallographic studies, molecular docking, and molecular dynamics simulations of the OXA-23-MA-1-206 complex show that the C6 hydroxymethyl group forms a hydrogen bond with the carboxylated catalytic lysine of OXA-23, effectively preventing deacylation. These results provide a promising strategy for designing a new generation of CHDL-resistant carbapenems to restore their efficacy against deadly A. baumannii infections.

Carbapenem Resistance in Acinetobacter nosocomialis and Acinetobacter junii Conferred by Acquisition of blaOXA-24/40 and Genetic Characterization of the Transmission Mechanism between Acinetobacter Genomic Species.

ABSTRACTCarbapenem resistance is increasing among Gram-negative bacteria, including the genus Acinetobacter. This study aimed to characterize, for the first time, the development of carbapenem resistance in clinical isolates of Acinetobacter junii and Acinetobacter nosocomialis conferred by the acquisition of a plasmid-borne blaOXA-24/40 gene and also to characterize the dissemination of this gene between species of Acinetobacter. Carbapenem-resistant A. nosocomialis HUAV-AN66 and A. junii HUAV-AJ77 strains were isolated in the Arnau de Vilanova Hospital (Spain). The genomes were sequenced, and in silico analysis were performed to characterize the genetic environment and the OXA-24/40 transmission mechanism. Antibiotic MICs were determined, and horizontal transfer assays were conducted to evaluate interspecies transmission of OXA-24/40. Carbapenems MICs obtained were ≥64 mg/L for HUAV-AN66 and HUAV-AJ77. Genome analysis revealed the presence in both strains of a new plasmid, designated pHUAV/OXA-24/40, harboring the carbapenem-resistance gene blaOXA-24/40 and flanked by sequences XerC/XerD. pHUAV/OXA-24/40 was successfully transferred from A. nosocomialis and A. junii to a carbapenem-susceptible A. baumannii strain, thus conferring carbapenem resistance. A second plasmid (pHUAV/AMG-R) was identified in both clinical isolates for the successful horizontal transfer of pHUAV/OXA-24/40. blaOXA-24/40-carrying plasmids of the GR12 group and showing high identity with pHUAV/OXA-24/40 were identified in at least 8 Acinetobacter species. In conclusion the carbapenemase OXA-24/40 is described for the first time in A. nosocomialis and A. junii. In both isolates the blaOXA-24/40 gene was located in the GR12 pHUAV/OXA-24/40 plasmid. GR12 plasmids are implicated in the dissemination and spread of carbapenem resistance among Acinetobacter species.IMPORTANCE Acinetobacter baumannii is one of the most relevant pathogens in terms of antibiotic resistance. The main resistance mechanisms are the carbapenem-hydrolyzing class D β-lactamases (CHDLs), especially OXA-23 and OXA-24/40. In addition to A. baumannii, there are other species within the genus Acinetobacter, which in general exhibit much lower resistance rates. In this work we characterize for the first time two clinical isolates of Acinetobacter nosocomialis and Acinetobacter junii, isolated in the same hospital, carrying the carbapenemase OXA-24/40 and displaying high resistance rates to carbapenems. By means of bioinformatics analysis we have also been able to characterize the mechanism by which this carbapenemase is horizontally transferred interspecies of Acinetobacter spp. The dissemination of carbapenemase OXA-24/40 between non-baumannii Acinetobacter species is concerning since it prevents the use of most β-lactam antibiotics in the fight against these resistant isolates.

Genetic Environment Surrounding blaOXA-55-like in Clinical Isolates of Shewanella algae Clade and Enhanced Expression of blaOXA-55-like in a Carbapenem-Resistant Isolate.

ABSTRACTAlthough Shewanella spp. are most frequently isolated from marine environments; more rarely, they have been implicated in human infections. Shewanella spp. are also recognized as the origin of genes for carbapenem-hydrolyzing class D β-lactamases. Due to the spread globally among Enterobacterales in recent years, risk assessments of both clinical and environmental Shewanella strains are urgently needed. In this study, we analyzed the whole-genome sequences of 10 clinical isolates and 13 environmental isolates of Shewanella spp. and compared them with those of Shewanella species strains registered in public databases. In addition, the levels of blaOXA-55-like transcription and β-lactamase activity of a carbapenem-resistant Shewanella algae isolate were compared with those of carbapenem-susceptible S. algae clade isolates. All clinical isolates were genetically identified as S. algae clade (S. algae, Shewanella chilikensis, and Shewanella carassii), whereas all but one of the environmental isolates were identified as various Shewanella spp. outside the S. algae clade. Although all isolates of the S. algae clade commonly possessed an approximately 12,500-bp genetic region harboring blaOXA-55-like, genetic structures outside this region were different among species. Among S. algae clade isolates, only one showed carbapenem resistance, and this isolate showed a high level of blaOXA-55-like transcription and β-lactamase activity. Although this study documented the importance of the S. algae clade in human infections and the relationship between enhanced production of OXA-55-like and resistance to carbapenems in S. algae, further studies are needed to elucidate the generalizability of these findings.IMPORTANCE Shewanella spp., which are known to carry chromosomally located blaOXA genes, have mainly been isolated from marine environments; however, they can also cause infections in humans. In this study, we compared the molecular characteristics of clinical isolates of Shewanella spp. with those originating from environmental sources. All 10 clinical isolates were genetically identified as members of the Shewanella algae clade (S. algae, S. chilikensis, and S. carassii); however, all but one of the 13 environmental isolates were identified as Shewanella species members outside the S. algae clade. Although all the S. algae clade isolates possessed an approximately 12,500-bp genetic region harboring blaOXA-55-like, only one isolate showed carbapenem resistance. The carbapenem-resistant isolate showed a high level of blaOXA-55-like transcription and β-lactamase activity compared with the carbapenem-susceptible isolates. To confirm the clinical significance and antimicrobial resistance mechanisms of the S. algae clade members, analysis involving more clinical isolates should be performed in the future.

OXA-900, a Novel OXA Sub-Family Carbapenemase Identified in Citrobacter freundii, Evades Detection by Commercial Molecular Diagnostics Tests.

Using whole-genome sequencing and cloning of the target gene, we identified blaOXA-900 carbapenemase, a novel blaOXA belonging to a distant and distinct sub-family of blaOXA-48-like. The plasmid-mediated gene was identified in a C. freundii isolate with elevated carbapenem MICs that evaded detection by commercial DNA-based methods. The novel gene, an OXA-48 family carbapenem-hydrolyzing class D β-lactamase, OXA-900, likely originates from marine environmental Shewanella. Since this plasmid-mediated gene has entered a member of the Enterobacterales and evades detection by commonly used tests, it may gain wide dissemination among Enterobacterales.

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.

Diversity of Oxacillinases and Sequence Types in Carbapenem-Resistant Acinetobacter baumannii from Austria.

Carbapenem-resistant Acinetobacter baumannii is a significant health problem worldwide. A multicenter study on A. baumannii was performed to investigate the molecular epidemiology and genetic background of carbapenem resistance of A. baumannii isolates collected from 2014–2017 in Austria. In total, 117 non-repetitive Acinetobacter spp. assigned to A. baumannii (n = 114) and A. pittii (n = 3) were collected from four centers in Austria. The isolates were uniformly resistant to piperacillin/tazobactam, ceftazidime, and carbapenems, and resistance to imipenem and meropenem was 97.4% and 98.2%, respectively. The most prominent OXA-types were OXA-58-like (46.5%) and OXA-23-like (41.2%), followed by OXA-24-like (10.5%), with notable regional differences. Carbapenem-hydrolyzing class D carbapenemases (CHDLs) were the only carbapenemases found in A. baumannii isolates in Austria since no metallo-β-lactamases (MBLs) nor KPC or GES carbapenemases were detected in any of the isolates. One-third of the isolates harbored multiple CHDLs. The population structure of A. baumannii isolates from Austria was found to be very diverse, while a total of twenty-three different sequence types (STs) were identified. The most frequent was ST195 found in 15.8%, followed by ST218 and ST231 equally found in 11.4% of isolates. Two new ST types, ST2025 and ST2026, were detected. In one A. pittii isolate, blaOXA-143-like was detected for the first time in Austria.

Activity of Imipenem, Meropenem, Cefepime, and Sulbactam in Combination with the β-Lactamase Inhibitor LN-1-255 against Acinetobacter spp.

Treatment of infections caused by Acinetobacter spp., particularly A. baumannii, is a major clinical problem due to its high rates of antibiotic resistance. New strategies must be developed; therefore, restoration of β-lactam efficacy through the use of β-lactamase inhibitors is paramount. Activities of the antibiotics imipenem, meropenem, cefepime, and sulbactam in combination with the penicillin-sulfone inhibitor LN-1-255 were tested by microdilution against 148 isolates of Acinetobacter spp. collected in 14 hospitals in Spain in 2020. Relevantly, the MIC90 (i.e., minimum concentration at which 90% of isolates were inhibited) of antibiotics in combination with LN-1-255 decreased 4- to 8-fold for all of the Acinetobacter isolates. Considering only the carbapenem-resistant A. baumannii isolates, which produce carbapenem-hydrolyzing class D β-lactamases, the addition of LN-1-255 decreased the resistance rates from 95.1% to 0% for imipenem, from 100% to 9.8% for meropenem, from 70.7% to 7.3% for cefepime, and sulbactam resistance rates from 9.8% to 0% and intermediate susceptibility rates from 53.7% to 2.4%. The inhibitor also decreased the minimum inhibitory concentrations (MICs) when tested against non-carbapenem-resistant Acinetobacter spp. isolates. In conclusion, combining LN-1-255 with imipenem, meropenem, cefepime, and sulbactam to target A. baumannii, and especially carbapenem-resistant isolates, represents an attractive option that should be developed for the treatment of infections caused by this pathogen.

Mechanistic Basis of OXA-48-like β-Lactamases' Hydrolysis of Carbapenems.

Carbapenem-hydrolyzing class D β-lactamases (CHDLs) are an important source of resistance to these last resort β-lactam antibiotics. OXA-48 is a member of a group of CHDLs named OXA-48-like enzymes. On the basis of sequence similarity, OXA-163 can be classified as an OXA-48-like enzyme, but it has altered substrate specificity. Compared to OXA-48, it shows impaired activity for carbapenems but displays an enhanced hydrolysis of oxyimino-cephalosporins. Here, we address the mechanistic and structural basis for carbapenem hydrolysis by OXA-48-like enzymes. Pre-steady-state kinetic analysis indicates that the rate-limiting step for OXA-48 and OXA-163 hydrolysis of carbapenems is deacylation and that the greatly reduced carbapenemase activity of OXA-163 compared to that of OXA-48 is due entirely to a slower deacylation reaction. Furthermore, our structural data indicate that the positioning of the β5-β6 loop is necessary for carbapenem hydrolysis by OXA-48. A major difference between the OXA-48 and OXA-163 complexes with carbapenems is that the 214-RIEP-217 deletion in OXA-163 creates a large opening in the active site that is absent in the OXA-48/carbapenem structures. We propose that the larger active site results in less constraint on the conformation of the 6α-hydroxyethyl group in the acyl-enzyme. The acyl-enzyme intermediate assumes multiple conformations, most of which are incompatible with rapid deacylation. Consistent with this hypothesis, molecular dynamics simulations indicate that the most stable complex is formed between OXA-48 and imipenem, which correlates with the OXA-48 hydrolysis of imipenem being the fastest observed. Furthermore, the OXA-163 complexes with imipenem and meropenem are the least stable and show significant conformational fluctuations, which correlates with the slow hydrolysis of these substrates.

Testing of antibiotic combinations in NDM-1-producing nosocomial carbapenem resistant Acinetobacter baumannii

Carbapenem resistant Acinetobacter baumannii (CRAB) have become a serious problem in health care settings. Its high prevalence has been associated with nosocomial transmission, high mortality rates, drug resistance and massive economic loss. The most frequent mechanisms of resistance are carbapenem-hydrolyzing class D β-lactamases (CHDL), followed by class B metallo-β-lactamases (MBL). New Delhi metallo-β-lactamase-1(NDM-1) generated much global alarm and were labeled as superbugs which documented impossible to be treated. Preceding our study, eight different antibiotic combinations were evaluated using checkerboard method. This was based on the results obtained from antimicrobial susceptibility testing and phenotypic detection of metallo-β-lactamase production of 27 CRAB selected from our previous work. The existence of NDM-1 gene was tested using two different methods. All antibiotic combinations showed synergistic significant results and no antagonism activity was found. The percentage of NDM-1 gene in the first detection procedure was 12/27 (44.44%) were positive while the other negative 15 ones, 10/15 (66.67%) poses NDM-1were found when using the second method. PCR products were then verified by DNA sequencing. The final consensus sequences were analyzed and submitted to NCBI GenBank data base, representing accession numbers: (HQ6526091), (MK682763.1), (MK682764.1), (MK682767.1), (MN251665.1), (MN251666.1) and (MN251670.1). The alignments showed similarity ranged from 94%-100% nucleotides identity. We concluded that detection of CRAB using accurate, rapid methods and supplying hospital laboratories with molecular department for typing of pathogenic bacteria are essential. Each hospital should establish its own policies according to their antibiogram, national and international guidelines. Primary caretakers should comply with the implemented policies.