Acinetobacter baumannii transformants expressing oxacillinases and metallo-ß-lactamases that confer resistance to meropenem: new tools for anti-Acinetobacter drug development and AMR preparedness.

Antimicrobial resistance (AMR) in Acinetobacter baumannii is an unmet medical need. Multiple drug-resistant/extremely drug-resistant strains of A. baumannii do not display growth well in in vivo models, and consequently, their response to antibacterial therapy is inconsistent. We addressed this issue by engineering carbapenem resistance motifs into the highly virulent genetic background of A. baumannii AB5075. This strain has a chromosomally encoded oxa-23 that was deleted (Δoxa-23), then plasmids expressing oxa-23, oxa-24/40, oxa-58, imp-1, vim-2, and ndm-1 were introduced to create the mutant strains. Each transformant was used as a challenge strain in a neutropenic murine thigh infection model and assessed for the extent of growth and response to meropenem 200 mg/kg subcutaneously every 6 h (q6h). Pharmacodynamic analyses were performed by transforming drug exposure from dose (mg/kg) to the fraction of the dosing interval; free meropenem concentrations were >minimum inhibitory concentration (MIC) (fT > MIC). AB5075 and the AB5075Δoxa-23 mutant had a MICs of 32 and 4 mg/L, respectively. The transformants harboring oxacillinases oxa-24/40 and oxa-58 had an MIC of 64 mg/L. The metallo-ß-lactamases imp-1, vim-2, and ndm-1 had MICs of 128, 64, and 64 mg/L, respectively. All vehicle-treated transformants displayed in vivo growth in the range of 0.75-1.4 log. The response to meropenem was consistent with the varying fT > MIC of the transformants and was readily described by an inhibitory sigmoid Emax relationship. Stasis was achieved with a fT > MIC of 0.36. These A. baumannii transformants are invaluable new tools for the assessment of anti-Acinetobacter compounds and provide a new pathway for AMR preparedness.

Multidrug-Resistant and Carbapenemase Gene-Carrying Acinetobacter colistiniresistens Isolated from Bloodstream Infection.

In this study, we investigated the antimicrobial susceptibility and molecular characteristics of antimicrobial resistance of Acinetobacter colistiniresistens strains isolated from the bloodstream using whole-genome sequencing. Clinical isolates identified as Acinetobacter baumannii and showing colistin resistance at the time of detection were collected. Antimicrobial susceptibility was determined using the VITEK2 system (bioMérieux) and Sensititre system (Thermo Fisher Scientific). Species identification and antimicrobial resistance gene searches were performed through whole-genome sequencing. Through whole-genome sequencing, three colistin-resistant strains from the bloodstream were identified as A. colistiniresistens. All three A. colistiniresistens strains were resistant to two or more antimicrobial agents except for colistin, and two of them were resistant to carbapenems. Genes involved in aminoglycoside [AAC(3)-Ⅱb, AAC(6')-Ⅰj, aadA2, ANT(3″)-Ⅱb, APH(3')-Ⅵa], macrolide (mphD, msrE), carbapenem and cephalosporin (OXA-420, VIM-2), fluoroquinolone and tetracycline (adeF), and sulfonamide (sul1, sul2) resistance were detected. We report multidrug-resistant A. colistiniresistens strains isolated from the bloodstream through whole-genome sequencing. Two strains carried carbapenemase genes, and this is the first report of VIM-2-producing A. colistiniresistens.

Emerging of multidrug-resistant Pseudomonas guariconensis with blaVIM-2 in an asymptomatic bacteriuria patient: A rare clinical presentation.

This case presents the clinical and genomic aspects of a rare and multidrug-resistant Pseudomonas guariconensis isolate carrying blaVIM-2 and highlights the need for heightened awareness in healthcare facilities. A 63-year-old woman underwent surgery for the diagnosis of a paraspinal abscess and infectious spondylitis. During hospitalization, the patient was diagnosed with heart failure exacerbation. The patient had no symptoms of urinary tract infection and met the criteria for asymptomatic bacteriuria. In urine culture, colonies of the organism grew >105 CFU/mL on blood agar and on MacConkey agar. The Bruker Biotyper mass spectrometry showed P. guariconensis. Based on the 16S rRNA gene sequence showed that a 99.79 % match with as P. guariconensis LMG 27394T. The average nucleotide identity with P. guariconensis LMG 27394T was 91.53 %. Antimicrobial susceptibility testing showed that the isolate was not susceptible to most of the antibiotics. Antimicrobial resistance genes identified were aph(6)-Id, aph(3″)-Ib, aac(6')-Ib3, aac(3)-If, gyrA mutation (T83I) and blaVIM-2.

Fluorinated captopril analogues inhibit metallo-ß-lactamases and facilitate structure determination of NDM-1 binding pose.

Bacterial resistance to the majority of clinically used ß-lactam antibiotics is a global health threat and, consequently, the driving force for the development of metallo-ß-lactamase (MBL) inhibitors. The rapid evolution of new MBLs calls for new strategies and tools for inhibitor development. In this study, we designed and developed a series of trifluoromethylated captopril analogues as probes for structural studies of enzyme-inhibitor binding. The new compounds showed activity comparable to the non-fluorinated inhibitors against the New Delhi Metallo-ß-lactamase-1 (NDM-1). The most active compound, a derivative of D-captopril, exhibited an IC50 value of 0.3 µM. Several compounds demonstrated synergistic effects, restoring the effect of meropenem and reducing the minimum inhibitory concentration (MIC) values in NDM-1 (up to 64-fold), VIM-2 (up to 8-fold) and IMP-26 (up to 8-fold) harbouring Escherichia coli. NMR spectroscopy and molecular docking of one representative inhibitor determined the binding pose in NDM-1, demonstrating that fluorinated analogues of inhibitors are a valuable tool for structural studies of MBL-inhibitor complexes.

CTX-M-15/27-positive Escherichia coli and VIM-2-producing Pseudomonas putida in free-living pigeons (Columba livia) in Tunisia.

OBJECTIVES: Wild birds are vectors of antimicrobial resistance. Birds living in close contact with humans or other animals, like feral pigeons (Columba livia), might be especially prone to acquire resistance genes such as those encoding extended-spectrum beta-lactamases (ESBLs) and carbapenemases. METHODS: Cloacal samples (n = 206) of free-living feral pigeons (C. livia) were collected in Sousse and Monastir, Tunisia. Antimicrobial susceptibility profiles were determined by disc-diffusion, and resistant isolates were short- and long-read whole-genome sequenced. Sequence analysis was performed using tools of the Centre for Genomic Epidemiology, and Phylogenetic analysis was performed based on the core-genome MLST. RESULTS: Fourteen (14/206, 6.8%) pigeons harboured Enterobacterales resistant to last-generations cephalosporins, of which 10 were CTX-M-15- or CTX-M-27-producers, while two (1.0%) carried a VIM-2-producing Pseudomonas putida. Positive pigeons lived on four different livestock farms. Three STs (ST206, ST5584, ST8149) were identified among E. coli, of which ST5584 and ST8149 were found in two different farms. Genetic diversity was also observed in Enterobacter cloacae and P. putida isolates. The blaCTX-M-27 genes were chromosomally encoded, while the blaCTX-M-15 genes were carried on highly similar IncF/F-:A-:B53 plasmids. The blaVIM-2 gene was located on a class 1 integron co-harbouring several resistance genes. CONCLUSION: Pigeons living on livestock farms carried clinically important resistance genes encoding ESBLs and carbapenemases. Our results evidenced that both clonal (ST8149 and ST5584) and plasmidic (IncF/F-:A-:B53) transfers played a role in the spread of resistance genes among pigeons. Further studies are needed to identify factors favouring the transfer and persistence of resistance genes within the pigeon communities.

Prevalence of carbapenemases among Gram-negative bacteria in Tunisia: first report of KPC-2 producing Acinetobacter baumannii.

INTRODUCTION: The rapid evolution of the antibacterial resistance problem worldwide, including the Mediterranean countries, constitutes a real threat to public health. This study aims to characterize carbapenemase encoding genes among Gram-negative bacteria collected from some Tunisian hospitals. METHODOLOGY: Twenty-two clinical carbapenem-resistant Gram-negative bacteria were recovered, and identified by the matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) method. Antibiotic resistance was tested by disk diffusion method on Muller-Hinton Agar. The minimum inhibitory concentration (MIC) for imipenem was revealed by the E-test method. Carbapenemase encoding genes were screened by polymerase chain reaction (PCR). Genetic relatedness was performed by the pulsed field gel electrophoresis (PFGE) method. RESULTS: Our isolates, identified as K. pneumoniae (n = 7), P. mirabilis (n = 1), A. baumannii (n = 13), and P. aeruginosa (n = 1), presented high MIC values for imipenem. Enterobacerales were resistant to carbapenems due to OXA-48 production. Only, four K. pneumoniae harbored the blaNDM-1 gene. VIM-2 production was detected in P. aeruginosa. However, OXA-23 production was observed in A. baumannii isolates, one of which co-produced the KPC-2 enzyme that was identified for the first time in Tunisia in this species. A high genetic diversity was demonstrated by pulsed-field gel electrophoresis in K. pneumoniae and A. baumannii after XbaI and ApaI digestion respectively. CONCLUSIONS: Our findings highlight the spread of various unrelated clones of carbapenemase-producers in some Tunisian hospitals as well as the spread of several carbapenemase types.

Taxifolin as a Metallo-ß-Lactamase Inhibitor in Combination with Augmentin against Verona Imipenemase 2 Expressing Pseudomonas aeruginosa.

Among the various mechanisms that bacteria use to develop antibiotic resistance, the multiple expression of ß-lactamases is particularly problematic, threatening public health and increasing patient mortality rates. Even if a combination therapy-in which a ß-lactamase inhibitor is administered together with a ß-lactam antibiotic-has proven effective against serine-ß-lactamases, there are no currently approved metallo-ß-lactamase inhibitors. Herein, we demonstrate that quercetin and its analogs are promising starting points for the further development of safe and effective metallo-ß-lactamase inhibitors. Through a combined computational and in vitro approach, taxifolin was found to inhibit VIM-2 expressing P. aeruginosa cell proliferation at <4 µg/mL as part of a triple combination with amoxicillin and clavulanate. Furthermore, we tested this combination in mice with abrasive skin infections. Together, these results demonstrate that flavonol compounds, such as taxifolin, may be developed into effective metallo-ß-lactamase inhibitors.

The First Swedish Outbreak with VIM-2-Producing Pseudomonas aeruginosa, Occurring between 2006 and 2007, Was Probably Due to Contaminated Hospital Sinks.

Multidrug-resistant Pseudomonas aeruginosa is an increasing clinical problem worldwide. The aim of this study was to describe the first outbreak of a Verona integron-borne metallo-ß-lactamase (VIM)-2-producing P. aeruginosa strain in Sweden and its expansion in the region. A cluster of multidrug-resistant P. aeruginosa appeared at two neighbouring hospitals in 2006. The isolates were characterized by PCR, pulsed-field gel electrophoresis (PFGE), and whole-genome sequencing. Patient charts, laboratory records, and hygiene routines were reviewed, and patients, staff, and the environment were screened. The investigation revealed a clonal outbreak of a VIM-2-producing P. aeruginosa strain belonging to the high-risk clonal complex 111, susceptible only to gentamicin and colistin. No direct contact between patients could be established, but most of them had stayed in certain rooms/wards weeks to months apart. Cultures from two sinks yielded growth of the same strain. The outbreak ended when control measures against the sinks were taken, but new cases occurred in a tertiary care hospital in the region. In conclusion, when facing prolonged outbreaks with this bacterium, sinks and other water sources in the hospital environment should be considered. By implementing proactive control measures to limit the bacterial load in sinks, the waterborne transmission of P. aeruginosa may be reduced.

Molecular Characterization and Antibiogram of Acinetobacter baumannii Clinical Isolates Recovered from the Patients with Ventilator-Associated Pneumonia.

A 2-year prospective study carried out on ventilator-associated pneumonia (VAP) patients in the intensive care unit at a tertiary care hospital, Hail, Kingdom of Saudi Arabia (KSA), revealed a high prevalence of extremely drug-resistant (XDR) Acinetobacter baumannii. About a 9% increase in the incidence rate of A. baumannii occurred in the VAP patients between 2019 and 2020 (21.4% to 30.7%). In 2019, the isolates were positive for IMP-1 and VIM-2 (31.1% and 25.7%, respectively) as detected by PCR. In comparison, a higher proportion of isolates produced NDM-1 in 2020. Here, we observed a high proportion of resistant ICU isolates towards the most common antibiotics in use. Colistin sensitivity dropped to 91.4% in the year 2020 as compared to 2019 (100%). Thus, the finding of this study has a highly significant clinical implementation in the clinical management strategies for VAP patients. Furthermore, strict implementation of antibiotic stewardship policies, regular surveillance programs for antimicrobial resistance monitoring, and screening for genes encoding drug resistance phenotypes have become imperative.

Extensively drug-resistant Acinetobacter baumannii co-producing VIM-2 and OXA-23 in intensive care units: Results of a one-day point prevalence in a Tunisian hospital.

OBJECTIVE: The main objective of this study was to identify carbapenem resistance mechanisms among clinical, commensal and environmental carbapenem-resistant Acinetobacter baumannii (CRAB) strains isolated in 5 Tunisian intensive care units (ICUs). MATERIALS AND METHODS: CRAB isolates were recovered from different sources: clinical specimens, rectal and environmental swabs. Bacterial identification was carried out using conventional methods and susceptibility testing according to EUCAST recommendations. Evaluation of phenotypic carbapenemase production of was performed using seven different methods, and molecular detection of carbapenemase-coding genes (blaOXA23, blaOXA24, blaOXA58, blaNDM, blaGES, blaOXA48, blaIMP, blaVIM and blaKPC) was done by PCR. The genetic relationships between CRAB isolates were analyzed by pulsed-field gel electrophoresis. RESULTS: All in all, 46 CRAB isolates were identified in clinical specimens (n = 26/26), rectal swabs (n = 17/36) and environmental swabs (n = 3/63). Most of them (n = 41/46) were clonally related and found in the different ICUs. All of the CARB isolates harbored blaOXA-51-like and blaOXA-23 genes, while blaVIM-2 gene was detected in 42 isolates (91.3 %). Phenotypic carbapenemase activity was found in all 46 strains, using the CIM-Tris test with 100 % sensitivity for OXA-23 and VIM-carbapenemase detection, thereby justifying its routine use. CONCLUSION: The emergence and diffusion of clonal CRAB strains inducing high mortality rates in ICUs is a major public health concern. Enhanced infection control practices and mandatory staff education are needed to control the spread of these multidrug-resistant bacteria.

Identification of a Stable Chromosomal Tandem Multicopy of blaVIM-63, a New blaVIM-2 Carbapenemase.

This study characterizes a new genetic structure containing a multicopy of a blaVIM-2 variant with an A676C substitution, blaVIM-63. This gene was detected on the chromosome of two carbapenem-resistant clinical strains of Citrobacter freundii ST22 recovered from two patients, separated by a 6-month period, and previously in Pseudomonas aeruginosa ST2242 from the same hospital unit. Short-read sequencing was used to characterize the new variant in both species, and long-read sequencing was used to characterize the genome of C. freundii. On the P. aeruginosa chromosome, the blaVIM-63 gene was inserted between ISPsy 42-type sequences, flanked by an intl1 sequence, nearby aph(3')-VI, and sul1. On the C. freundii chromosome, the blaVIM-63 gene was inserted into a Tn6230-like transposon as a stable five-tandem-repeat multimer, flanked by the same intl1 as in P. aeruginosa. This structure was stable across subcultures and did not change in the presence of carbapenems. The blaVIM-63 gene was cloned into the pCR-Blunt plasmid to study antimicrobial susceptibility patterns and into pET29a for kinetic activity analysis. VIM-63 showed higher Km values than VIM-2 for ceftazidime and cefepime and higher kcat values for cefotaxime, ceftazidime, imipenem, and ertapenem, without differences in MIC values. This is the first study to describe this new variant, VIM-63, in two different species with a chromosomal location integrated into different mobile elements and the first to describe a stable multimer of a metallo-ß-lactamase. Despite the amino acid substitution, the susceptibility pattern of the new variant was similar to that of VIM-2. IMPORTANCE VIM group metallo-ß-lactamases are usually captured by IntI1 integrases. This work describes the detection for the first time of a novel, previously unknown variant of VIM-2, VIM-63. This carbapenemase has been found on the chromosome of two different species, Citrobacter freundii and Pseudomonas aeruginosa, from the same hospital. The adjacent genetic environment of the blaVIM-63 gene would indicate that the capture of this gene by IntI1 has occurred in two different genetic events in each of the species, and in one there has been a stable integration of tandem copies of this gene.

Characterization of imipenem-resistant Acinetobacter baumannii and Pseudomonas aeruginosa clinical isolates in a Moroccan hospital.

Acinetobacter baumannii and Pseudomonas aeruginosa are two pathogens with an important power of adaptation to antibiotics thus, both pose a real public health problem. Our study investigated epidemiological characteristics, antibiotic sensitivity profile and resistance genes of imipenem resistant A. baumannii and P. aeruginosa. This was a retrospective study carried out in the bacteriology laboratory of Mohammed V military training hospital, spanning from January 2018 to April 2021. Antibiotic susceptibility was studied by Mueller Hilton agar diffusion method with OXOID® type antibiotic discs and interpreted according to the recommendations of EUCAST 2021. Carbapenemase detection was performed by CarbaNP-test®. The molecular study was performed using conventional PCR. During the study period, we collected 1,072 imipenem-resistant isolates namely, 820 A. baumannii and 252 P. aeruginosa. The molecular study showed that out of 108 A. baumannii isolates 102 carried the bla OXA-51 and 100 isolates carried the bla OXA-23 gene. The coexistence of bla OXA-23 and bla NDM genes was detected in only 4 isolates. Altogether 50% of P. aeruginosa strains carried bla VIM-2. All investigated A. baumannii and P. aeruginosa strains were colistin susceptible in this study. Multiresistant bacterial infections are associated with longer hospitalization, higher hospital costs and higher mortality rates. Therefore, a collective action including the different actors of the healthcare system is necessary.

Pseudomonas putida group species as reservoirs of mobilizable Tn402-like class 1 integrons carrying blaVIM-2 metallo-ß-lactamase genes.

The Pseudomonas putida group (P. putida G) is composed of at least 21 species associated with a wide range of environments, including the clinical setting. Here, we characterized 13 carbapenem-resistant P. putida G clinical isolates bearing class 1 integrons/transposons (class 1 In/Tn) carrying blaVIM-2 metallo-ß-lactamase gene cassettes obtained from hospitals of Argentina. Multilocus sequencing (MLSA) and phylogenetic analyses based on 16S rDNA, gyrB and rpoD sequences distinguished 7 species among them. blaVIM-2 was found in three different cassette arrays: In41 (blaVIM-2-aacA4), In899 (only blaVIM-2), and In528 (dfrB1-aacA4-blaVIM-2). In41 and In899 were associated with complete tniABQC transposition modules and IRi/IRt boundaries characteristic of the Tn5053/Tn402 transposons, which were designated Tn6335 and Tn6336, respectively. The class 1 In/Tn element carrying In528, however, exhibited a defective tni module bearing only the tniC (transposase) gene, associated with a complete IS6100 bounded with two oppositely-oriented IRt end regions. In some P. putida G isolates including P. asiatica, P. juntendi, P. putida G/II, and P. putida G/V, Tn6335/Tn6336 were carried by pLD209-type conjugative plasmids capable of self-mobilization to P. aeruginosa or Escherichia coli. In other isolates of P. asiatica, P. putida G/II, and P. monteiliieilii, however, these blaVIM-2-containing class 1 In/Tn elements were found inserted into the res regions preceding the tnpR (resolvase) gene of particular Tn21 subgroup members of Tn3 transposons. The overall results reinforce the notion of P. putida G members as blaVIM-2 reservoirs, and shed light on the mechanisms of dissemination of carbapenem resistance genes to other pathogenic bacteria in the clinical setting.

Occurrence of NDM-1 and VIM-2 Co-Producing Escherichia coli and OprD Alteration in Pseudomonas aeruginosa Isolated from Hospital Environment Samples in Northwestern Tunisia.

Hospital environments constitute the main reservoir of multidrug-resistant bacteria. In this study we aimed to investigate the presence of Gram-negative bacteria in one Northwestern Tunisian hospital environment, and characterize the genes involved in bacterial resistance. A total of 152 environmental isolates were collected from various surfaces and isolated using MacConkey medium supplemented with cefotaxime or imipenem, with 81 fermenter bacteria (27 Escherichia coli, and 54 Enterobacter spp., including 46 Enterobacter cloacae), and 71 non-fermenting bacteria (69 Pseudomonas spp., including 54 Pseudomonas aeruginosa, and 2 Stenotrophomonas maltophilia) being identified by the MALDI-TOF-MS method. Antibiotic susceptibility testing was performed by disk diffusion method and E-Test was used to determine MICs for imipenem. Several genes implicated in beta-lactams resistance were characterized by PCR and sequencing. Carbapenem resistance was detected among 12 isolates; nine E. coli (blaNDM-1 (n = 8); blaNDM-1 + blaVIM-2 (n = 1)) and three P. aeruginosa were carbapenem-resistant by loss of OprD porin. The whole-genome sequencing of P. aeruginosa 97H was determined using Illumina MiSeq sequencer, typed ST285, and harbored blaOXA-494. Other genes were also detected, notably blaTEM (n = 23), blaCTX-M-1 (n = 10) and blaCTX-M-9 (n = 6). These new epidemiological data imposed new surveillance strategies and strict hygiene rules to decrease the spread of multidrug-resistant bacteria in this area.

Sink drains as reservoirs of VIM-2 metallo-ß-lactamase-producing Pseudomonas aeruginosa in a Belgian intensive care unit: relation to patients investigated by whole-genome sequencing.

BACKGROUND: Hospital-acquired infections caused by VIM-encoded metallo-ß-lactamase-positive Pseudomonas aeruginosa are a major problem in intensive care units (ICUs) worldwide. A previous study conducted in the UZ Brussel hospital revealed that sink drains of the ICU were a possible source of various multidrug-resistant pathogenic bacteria. AIM: To investigate the presence and persistence of VIM P. aeruginosa in the sink drains of the four adult ICUs and their role in nosocomial infections, emphasizing sink-to-patient transmission. METHODS: Thirty-six sinks located in the ICUs of the UZ Brussel were sampled and screened for the presence of VIM P. aeruginosa in August and October 2019. Whole-genome sequencing (WGS) was performed on all positive sink drain isolates together with 61 isolates from patients who were retrospectively selected (ICU patients 2019-2020, N = 46; non-ICU patients 2019, N = 6). FINDINGS: Twenty sinks were found positive for P. aeruginosa at both sampling time-points. WGS revealed that the predominating environmental cluster belonged to sequence type ST111. Ten additional STs were identified. VIM-2 was detected among all ST17 (N = 2) and ST111 (N = 14) sink drain isolates. Based on whole-genome multi-locus sequence typing analysis of all genomes, 15 clusters of highly related isolates were identified, of which seven included both sink drain and clinical isolates. CONCLUSION: Our findings confirm that sink drains are a possible source of VIM-2 P. aeruginosa, probably after being contaminated with clinical waste from patients. Patients could be exposed to VIM-2 P. aeruginosa dispersed in their environment because of colonized sink drains.

High Carbapenem Resistance Caused by VIM and NDM Enzymes and OprD Alteration in Nonfermenter Bacteria Isolated from a Libyan Hospital.

Acinetobacter baumannii and Pseudomonas aeruginosa are among the most prevalent pathogens causing a wide range of serious infections in hospitalized patients and contaminating intensive care units and inanimate surfaces. The purpose of this study was to investigate the mechanism of carbapenem resistance in clinical and hospital environmental isolates of A. baumannii and P. aeruginosa recovered from a Libyan hospital. From a total of 82 Gram-negative bacteria, 8 isolates of A. baumannii and 3 isolates of P. aeruginosa exhibited resistance to imipenem with minimum inhibitory concentrations ranging from 16 to >32 µg/mL. Five isolates of A. baumannii harbored blaOXA-23 gene, from which three isolates were collected from patients and two from hospital environment. Only one isolate harbored blaNDM-1 gene, which was responsible for carbapenem resistance in A. baumannii. The OprD gene seems to be disturbed by an insertion sequence (IS) in two isolates and affected by polymorphism in one isolate. Pulsed-field gel electrophoresis results showed high genetic diversity among carbapenemase producing A. baumannii. This study highlights the dissemination of blaOXA-23 and blaNDM-1 genes in a Libyan setting. Therefore, infection prevention and control practices, antimicrobial stewardship initiatives, and antimicrobial resistance surveillance systems should be implemented to prevent the wide spread of antimicrobial resistance.

Plasmids Carrying blaVIM-2 in Acinetobacter nosocomialis and A. seifertii Isolates from South Korea.

We identified nine Verona integron-encoded metallo-ß-lactamase-2 (VIM-2)-producing Acinetobacter nosocomialis (n = 8) and Acinetobacter seifertii (n = 1) isolates from South Korea and performed whole-plasmid sequencing for two A. nosocomialis isolates and one A. seifertii isolate. Genotyping, antibiotic resistance profiles, and whole plasmid sequences indicated clonal dissemination of the eight VIM-2-producing A. nosocomialis isolates. The plasmid-bearing blaVIM-2 in the A. seifertii isolate differed from those in the A. nosocomialis isolates.

Genomic context of the two integrons of ST-111 Pseudomonas aeruginosa AG1: A VIM-2-carrying old-acquaintance and a novel IMP-18-carrying integron.

Pseudomonas aeruginosa is an opportunist and versatile organism responsible for infections mainly in immunocompromised hosts. This pathogen has high intrinsic resistance to most antimicrobials. P. aeruginosa AG1 (PaeAG1) is a Costa Rican high-risk ST-111 strain with resistance to multiple antibiotics, including carbapenems, due to the activity of VIM-2 and IMP-18 metallo-ß-lactamases (MBLs). These genes are harbored in two class 1 integrons located inone out of the 57 PaeAG1 genomic islands. However, the genomic context associated to these determinants in PaeAG1 and other P. aeruginosa strains is unclear. Thus, we first assessed the transcriptional activity of VIM-2 and IMP-18 genes when exposed to imipenem (a carbapenem) by RT-qPCR. To select related genomes to PaeAG1, we implemented a pan-genome analysis to define and up-date the phylogenetic relationship among complete P. aeruginosa genomes. We also studied the PaeAG1 genomic islands content in the related strains and finally we described the architecture and possible evolutionary steps of the genomic regions around the VIM-2- and IMP-18-carrying integrons. Expression of VIM-2 and IMP-18 genes was demonstrated to be induced after imipenem exposure. In a subsequent comparative genomics analysis with 211 strains, the P. aeruginosa pan-genome revealed that complete genome sequences are able to separate clones by MLST profile, including a clear ST-111 cluster with PaeAG1. The PaeAG1 genomic islands were found to define a diverse presence/absence pattern among related genomes. Finally, landscape reconstruction of genomic regions showed that VIM-2-carrying integron (In59-like) is an old-acquaintance element harbored in the same known region found in other two ST-111 strains. Also, PaeAG1 has an exclusive genomic region containing a novel IMP-18-carrying integron (registered as In1666), with an arrangement never reported before. Altogether, we provide new insights about the genomic determinants associated with the resistance to carbapenems in this high-risk P. aeruginosa using comparative genomics.

Emergence of clinical isolates of Pseudomonas asiatica and Pseudomonas monteilii from Japan harbouring an acquired gene encoding a carbapenemase VIM-2.

Pseudomonas asiatica and Pseudomonas monteilii, belonging to the Pseudomonas putida phylogenetic group, are occasionally isolated from clinical samples, partly because they are often misidentified as P. putida in clinical laboratories. There are five reports describing carbapenem-resistant clinical isolates of these species. Carbapenem-resistant strains of P. asiatica and P. monteilii were isolated from stool samples. These isolates were sequenced using Illumina MiSeq and reidentified using average nucleotide identity (ANI) based on comparisons of their whole-genome sequences using the OrthoANI algorithm. The clonal relatedness of the isolates was assessed by pulse-field gel electrophoresis (PFGE). The size of plasmids conveying bla VIM-2 was examined by Southern blotting. A total of six carbapenem-resistant clinical isolates of P. asiatica (two isolates) and P. monteilii (four isolates) were obtained from stool samples from five patients in a Japanese hospital. All isolates harboured blaVIM-2. The two isolates of P. asiatica had a different pattern in the PFGE analysis, with both having a 23 kb plasmid. Of the four isolates of P. monteilii with similar patterns in the PFGE analysis, three had 320 kb plasmids and one had a 240 kb plasmid. The genetic environments of the 320/240 kb and 23 kb plasmids differed. The results strongly indicated that carbapenem-resistant P. asiatica and P. monteilii producing metallo-ß-lactamase are emerging in Japan. This is the first report of carbapenem-resistant P. asiatica and P. monteilii in Japan.

Probing the mechanisms of inhibition for various inhibitors of metallo-ß-lactamases VIM-2 and NDM-1.

To probe the mechanism of inhibition of several previously-published metallo-ß-lactamase (MBL) inhibitors for the clinically-important MBL Verona integron-encoded metallo-ß-lactamase 2 (VIM-2), equilibrium dialyses with metal analyses, native state electrospray ionization mass spectrometry (ESI-MS), and UV-Vis spectrophotometry were utilized. The mechanisms of inhibition were analyzed for ethylenediaminetetraacetic acid (EDTA); dipicolinic acid (DPA) and DPA analogs 6-(1H-tetrazol-5-yl)picolinic acid (1T5PA) and 4-(3-aminophenyl)pyridine-2,6-dicarboxylic acid (3AP-DPA); thiol-containing compounds, 2,3-dimercaprol, thiorphan, captopril, and tiopronin; and 5-(pyridine-3-sulfonamido)-1,3-thiazole-4-carboxylic acid (ANT-431). UV-Vis spectroscopy and native-state ESI-MS results showed the formation of ternary complexes between VIM-2 and 1T5PA, ANT-431, thiorphan, captopril, and tiopronin, while a metal stripping mechanism was shown with VIM-2 and EDTA and DPA. The same approaches were used to show the formation of a ternary complex between New Delhi Metallo-ß-lactamase (NDM-1) and ANT-431. The studies presented herein show that most of the inhibitors utilize a similar mechanism of inhibition as previously reported for NDM-1. These studies also demonstrate that native mass spectrometry can be used to probe the mechanism of inhibition at lower enzyme/inhibitor concentrations than has previously been achieved.