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Insights into the complete genomes of carbapenem-resistant Acinetobacter baumannii harbouring bla OXA-23, bla OXA-420 and bla NDM-1 genes using a hybrid-assembly approach.

ABSTRACT: Carbapenem resistance in Acinetobacter baumannii is due to bla OXA-23, which is endemic in India. Recently, the sporadic presence of bla OXA-58 as well as the occurrence of dual carbapenemases were observed. The mobility as well as the dissemination of these resistance genes were mainly mediated by various mobile genetic elements. The present study was aimed at characterizing the genetic arrangement of bla OXA-23, bla NDM-1 and bla OXA-58 identified in two complete genomes of carbapenem-resistant A. baumannii (CRAB). Complete genomes obtained using a hybrid-assembly approach revealed the accurate arrangement of Tn2006 with bla OXA-23, ISAba125 with bla NDM and ISAba3 with bla OXA-58. In addition, the association of IntI1 integrase with the bla CARB-2 gene and several virulence factors required for type-IV pili assembly, motility and biofilm formation have been identified. The current study provided deeper insight into the complete characterization of insertion sequences and transposons associated with the carbapenem-resistant genes using short reads of IonTorrent PGM and long reads of MinIon in A. baumannii .

SUBMITTER: Vijayakumar S

PROVIDER: S-EPMC7497828 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Insights into the complete genomes of carbapenem-resistant Acinetobacter baumannii harbouring bla OXA-23, bla OXA-420 and bla NDM-1 genes using a hybrid-assembly approach.

Vijayakumar Saranya S Wattal Chand C J K Oberoi O Bhattacharya Sanjay S Vasudevan Karthick K Anandan Shalini S Walia Kamini K Veeraraghavan Balaji B

Access microbiology 20200605 8

Carbapenem resistance in Acinetobacter baumannii is due to bla OXA-23, which is endemic in India. Recently, the sporadic presence of bla OXA-58 as well as the occurrence of dual carbapenemases were observed. The mobility as well as the dissemination of these resistance genes were mainly mediated by various mobile genetic elements. The present study was aimed at characterizing the genetic arrangement of bla OXA-23, bla NDM-1 ...[more]

PMID: 32974602

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Project description:BackgroundThe rapid emergence of carbapenem resistant Acinetobacter baumannii (CRAB) has resulted in an alarming situation worldwide. Realizing the dearth of literature on susceptibility of CRAB in genetic context in the developing region, this study was performed to determine the susceptibility profile against standard drugs/combinations and the association of in-vitro drug synergy with the prevalent molecular determinants.Methods and findingsA total of 356 clinical isolates of A. baumannii were studied. Confirmation of the isolates was done by amplifying recA and ITS region genes. Susceptibility against standard drugs was tested by Kirby Bauer disc diffusion. Minimum inhibitory concentration (MIC), MIC50 and MIC90 values against imipenem, meropenem, doripenem, ampicillin/sulbactam, minocycline, amikacin, polymyxin B, colistin and tigecycline was tested as per guidelines. Genes encoding enzymes classes A (bla GES, bla IMI/NMC-A, bla SME, bla KPC), B (bla IMP, bla VIM, bla NDM) and D (bla OXA-51, bla OXA-23 and bla OXA-58) were detected by multiplex polymerase chain reaction. Synergy against meropenem-sulbactam and meropenem-colistin combinations was done by checkerboard MIC method. Correlation of drug synergy and carbapenemase encoding genes was statistically analyzed.ResultsOf the total, resistance above 90% was noted against gentamicin, ciprofloxacin, levofloxacin, ceftazidime, cefepime, ceftriaxone, cotrimoxazole and piperacillin/tazobactam. By MIC, resistance rates from highest to lowest was seen against imipenem 89.04% (n=317), amikacin 80.33% (n=286), meropenem 79.49% (n=283), doripenem 77.80% (n=277), ampicillin/sulbactam 71.62% (n=255), tigecycline 55.61% (n=198), minocycline 14.04% (n=50), polymyxin B 10.11% (n=36), and colistin 2.52% (n=9). CRAB was 317 (89.04%), 81.46% (n=290) were multidrug resistant and 13.48% (n=48) were extensively drug resistant. All the CRAB isolates harboured bla OXA-51 gene (100%) and 94% (n=298) bla OXA-23 gene. The bla IMP gene was most prevalent 70.03% (n=222) followed by bla NDM, 59.62% (n=189). Majority (87.69%, 278) were co-producers of classes D and B carbapenemases, bla OXA-23 with bla IMP and bla NDM being the commonest. Synergy with meropenem-sulbactam and meropenem-colistin was 47% and 57% respectively. Reduced synergy (p= <0.0001) was noted for those harbouring bla OXA-51+blaOXA-23with bla NDM gene alone or co-producers.ConclusionPresence of bla NDM gene was a significant cause of synergy loss in meropenem-sulbactam and meropenem-colistin. In bla NDM endemic regions, tigecycline, minocycline and polymyxins could be viable options against CRAB isolates with more than one carbapenemase encoding genes.

Project description:The Acinetobacter calcoaceticus-baumannii (ACB) complex, also known as ACB complex, consists of four bacterial species that can cause opportunistic infections in humans, especially in hospital settings. Conventional therapies for susceptible strains of the ACB complex include broad-spectrum cephalosporins, β-lactam/β-lactamase inhibitors, and carbapenems. Unfortunately, the effectiveness of these antibiotics has declined due to increasing rates of resistance. The predominant resistance mechanisms identified in the ACB complex involve carbapenem-resistant (CR) oxacillinases and metallo-β-lactamases (MBLs). This research, conducted at Kathmandu Model Hospital in Nepal, sought to identify genes associated with CR, specifically blaNDM-1, blaOXA-23-like, and blaOXA-24-like genes in carbapenem-resistant Acinetobacter calcoaceticus-baumannii (CR-ACB) complex. Additionally, the study is aimed at identifying the ACB complex through the sequencing of the 16s rRNA gene. Among the 992 samples collected from hospitalized patients, 43 (approximately 4.334%) tested positive for the ACB complex. These positive samples were mainly obtained from different hospital units, including intensive care units (ICUs); cabins; and neonatal, general, and maternity wards. The prevalence of infection was higher among males (58.14%) than females (41.86%), with the 40-50 age group showing the highest infection rate. In susceptibility testing, colistin and polymyxin B exhibited a susceptibility rate of 100%, whereas all samples showed resistance to third-generation cephalosporins. After polymyxins, gentamicin (30.23%) and amikacin (34.88%) demonstrated the highest susceptibility. A substantial majority (81.45%) of ACB complex isolates displayed resistance to carbapenems, with respiratory and pus specimens being the primary sources. Polymerase chain reaction (PCR) revealed that the primary CR gene within the ACB complex at this hospital was bla OXA-23-like, followed by bla NDM-1. To ensure the accuracy of the phenotypic assessment, 12 samples were chosen for 16s rRNA sequencing using Illumina MiSeq™ to confirm that they are Acinetobacter species. QIIME 2.0 analysis confirmed all 12 isolates to be Acinetobacter species. In the hospital setting, a substantial portion of the ACB complex carries CR genes, rendering carbapenem ineffective for treatment.

Project description:Carbapenem-resistant Acinetobacter baumannii (CRAB), which belonged to global clones 1 (GC1) or 2 (GC2), has been widely reported and become a global threat. However, non-GC1 and non-GC2 CRAB strains are not well-studied, especially for those with rare phenotype. Here, one pyomelanin-producing CRAB strain (A. baumannii DETAB-R21) was isolated from oral swab in the ICU. Antimicrobial susceptibility testing showed it was resistant to carbapenems, ceftazidime, levofloxacin, and ciprofloxacin. DETAB-R21 was ST164Pas and ST1418Oxf with KL47 and OCL5, respectively. Whole-genome sequencing (WGS) analysis revealed chromosome contained three copies of blaOXA-23 on three 4,805-bp Tn2006 composite transposons with various novel 9-bp target site duplications (TSD). A Tn125-like structure, including blaNDM-1, a novel 4,343 bp composite transposon encoding blaCARB-16, and three prophage regions were also identified. Importantly, hmgA was interrupted by a Tn2006 and contributed to pyomelanin production and further confirmed by hmgA overexpression. Furthermore, A. baumannii irradiated with UV light, DETAB-R21 showed a higher relatively survival rate compared to a control strain that did not produce pyomelanin. No effects of pyomelanin were observed on disinfectants susceptibility, growth, or virulence. In conclusion, pyomelanin-producing CRAB carrying the blaNDM-1 and blaOXA-23 genes embedded in the bacterial chromosome is of grave concern for health care settings, highlighting the need for effective measures to prevent further dissemination. IMPORTANCE Pyomelanin production is a quite rare phenotype in A. baumannii. Moreover, the mechanisms leading to the pyomelanin production was still unclear. Here, we for the first time, confirmed the mechanism of pyomelanin production, and further investigated the impact of pyomelanin on disinfectants susceptibility, growth, virulence, and UV irradiation. More importantly, many mobile genetic elements (MGEs), including three copies of Tn2006 composite transposons, one copy of blaNDM-1 on the Tn125-like structure and three prophage regions, were identified in the chromosome, demonstrated strong plasticity of A. baumannii genome. Our study provides important insights into the new rare ST164Pas A. baumannii strain with high level carbapenem resistance, which is of great threat for patients. These findings will provide important insights into the resistance gene transfer via transposition events and further spread in the clinic.

Project description:BackgroundBetween January 2015 and July 2017, we investigated the frequency of carbapenem resistant Acinetobacter baumannii (CRAB) and carbapenem resistant Pseudomonas aeruginosa (CRPA) at the Mulago Hospital intensive care unit (ICU) in Kampala, Uganda. Carbapenemase production and carbapenemase gene carriage among CRAB and CRPA were determined; mobility potential of carbapenemase genes via horizontal gene transfer processes was also studied.MethodsClinical specimens from 9269 patients were processed for isolation of CRAB and CRPA. Drug susceptibility testing was performed with the disk diffusion method. Carriage of carbapenemase genes and class 1 integrons was determined by PCR. Conjugation experiments that involved blaVIM positive CRAB/CRPA (donors) and sodium azide resistant Escherichia coli J53 (recipient) were performed.ResultsThe 9269 specimens processed yielded 1077 and 488 isolates of Acinetobacter baumannii and Pseudomonas aeruginosa, respectively. Of these, 2.7% (29/1077) and 7.4% (36/488) were confirmed to be CRAB and CRPA respectively, but 46 were available for analysis (21 CRAB and 25 CRPA). Majority of specimens yielding CRAB and CRPA were from the ICU (78%) while 20 and 2% were from the ENT (Ear Nose & Throat) Department and the Burns Unit, respectively. Carbapenemase assays performed with the MHT assay showed that 40 and 33% of CRPA and CRAB isolates respectively, were carbapenemase producers. Also, 72 and 48% of CRPA and CRAB isolates respectively, were metallo-beta-lactamase producers. All the carbapenemase producing isolates were multidrug resistant but susceptible to colistin. blaVIM was the most prevalent carbapenemase gene, and it was detected in all CRAB and CRPA isolates while blaOXA-23 and blaOXA-24 were detected in 29 and 24% of CRAB isolates, respectively. Co-carriage of blaOXA-23 and blaOXA-24 occurred in 14% of CRAB isolates. Moreover, 63% of the study isolates carried class 1 integrons; of these 31% successfully transferred blaVIM to E. coli J53.ConclusionsCRAB and CRPA prevalence at the Mulago Hospital ICU is relatively low but carbapenemase genes especially blaVIM and blaOXA-23 are prevalent among them. This requires strengthening of infection control practices to curb selection and transmission of these strains in the hospital.

Project description:Carbapenem-resistant Acinetobacter baumannii spp. are increasingly important pathogens with limited treatment options, and there is limited knowledge on the environmental factors contributing to their spread. We determined the occurrence of carbapenem-resistant A. baumannii in hospital wastewater and their phylogenetic relationships with clinical A. baumannii isolates. Grab samples of raw and treated hospital wastewater were collected monthly at the University College Hospital, Ibadan, Nigeria, between March 2021 and February 2022. Acinetobacter baumannii strains were selectively isolated and identified using VITEK2, and their whole genomes were sequenced on an Illumina platform. We performed antimicrobial susceptibility testing and in silico genomic characterization of the strains and determined their phylogenetic relationships to previously characterized clinical A. baumannii strains from Nigeria. A. baumannii complex isolates were recovered from wastewater throughout the study. Of the 82 isolates identified based on whole-genome sequences, 77 were A. baumannii. A. baumannii isolates had high resistance rates (≥48.1%) to 10 of 12 antimicrobials tested, and majority (42/77, 54.5%) were resistant to carbapenems, with bla NDM-1 being the most common (24/77, 31.2%) carbapenem resistance gene detected, followed by bla OXA-23 (n = 22, 28.6%). There was no statistically significant difference in carbapenem resistance rates or carbapenem gene carriage between the raw and treated wastewater isolates. Most of the isolates belonged to novel or sparsely described lineages, some of which were closely related to clinical isolates. The release of inadequately treated hospital wastewater into the environment may contribute to the increased spread of carbapenem-resistant and clinically important A. baumannii lineages in Ibadan, Nigeria. IMPORTANCE Acinetobacter baumannii is a leading cause of hospital-associated infections globally. A. baumannii reservoirs outside hospital settings are still unknown, and their occurrence in the environment is linked to clinical and anthropogenic activities. Although the risk of transmission of A. baumannii from environmental sources to humans is not fully understood, these sources pose significant risks for the continued dissemination of A. baumannii and their resistance traits. This study provides evidence that diverse and clinically relevant A. baumannii strains, many of which are resistant to carbapenems, are constantly being discharged into the environment through inadequately treated hospital wastewater. We further elucidate potential transmission routes between the environment and clinical infections and demonstrate the high prevalence of carbapenem resistance genes on highly mobile transposons among these strains. Our findings highlight the pressing need to address hospital wastewater as a crucial factor in curtailing the spread of carbapenem-resistant A. baumannii.

Project description:Purpose:A novel variant of OXA-23, named OXA-423, was identified in an Acinetobacter baumannii clinical isolate. The aim of this study was to analyse the resistance phenotype of OXA-423. Methods:The A. baumannii strain WY-0713 was isolated from an intensive care unit patient. PCR was used to detect the bla OXA-23-like genes. Amplifying, cloning and sequencing were performed for the complete bla OXA-23-like. The novel bla OXA-423 and its ancestor bla OXA-23 were cloned into the expression vector pET-28b(+), and transformed into E. coli Rosetta (DE3) for antibiotic susceptibility testing. SDS-PAGE, modified Hodge test and CarbaNP test were used for detecting the expression of OXA-423 and OXA-23. Results:PCR screening of A. baumannii WY-0713 was positive for bla OXA-23-like genes. Sequencing of the PCR product identi?ed a novel bla OXA-23-like, named bla OXA-423 which encoding OXA-423. OXA-423 differed from OXA-23 by a crucial amino acid substitution (Val128Ala). The V128A substitution was located at the conserved active-site motifs SAV of OXA-23. Antibiotic susceptibility testing performed using isogenic E. coli showed that the MICs of E. coli Rosetta (pET-OXA-423) for penicillins and carbapenems were lower (reduced MICs 4-fold to 16-fold) than that of E. coli Rosetta (pET-OXA-23). The MICs of cefotaxime, ceftazidime and aztreonam for both transformants remained the same as the acceptor strain. Moreover, OXA-423 was slightly inhibited by sulbactam, clavulanic acid and tazobactam. SDS-PAGE analysis showed that OXA-423 and OXA-23 were conspicuously expressed. Modified Hodge test and CarbaNP test were positive demonstrated both of them were functional. Conclusion:OXA-423, the first report of an amino acid substitution located at conserved active-site motifs of OXA-23, conferred lower MIC values of penicillins and carbapenems as compared with OXA-23, while without affecting the resistance pro?les of expanded-spectrum cephalosporins and aztreonam.

Project description:BackgroundCarbapenem resistant Acinetobacter species have caused great difficulties in clinical therapy in the worldwide. Here we describe an Acinetobacter johnsonii M19 with a novel blaOXA-23 containing transposon Tn6681 on the conjugative plasmid pFM-M19 and the ability to transferand carbapenem resistance.MethodsA. johnsonii M19 was isolated under selection with 8 mg/L meropenem from hospital sewage, and the minimum inhibitory concentrations (MICs) for the representative carbapenems imipenem, meropenem and ertapenem were determined. The genome of A. johnsonii M19 was sequenced by PacBio RS II and Illumina HiSeq 4000 platforms. A homologous model of OXA-23 was generated, and molecular docking models with imipenem, meropenem and ertapenem were constructed by Discovery Studio 2.0. Type IV secretion system and conjugation elements were identified by the Pathosystems Resource Integration Center (PATRIC) server and the oriTfinder. Mating experiments were performed to evaluate transfer of OXA-23 to Escherichia coli 25DN.ResultsMICs of A. johnsonii M19 for imipenem, meropenem and ertapenem were 128 mg/L, 48 mg/L and 24 mg/L, respectively. Genome sequencing identified plasmid pFM-M19, which harbours the carbapenem resistance gene blaOXA-23 within the novel transposon Tn6681. Molecular docking analysis indicated that the elongated hydrophobic tunnel of OXA-23 provides a hydrophobic environment and that Lys-216, Thr-217, Met-221 and Arg-259 were the conserved amino acids bound to imipenem, meropenem and ertapenem. Furthermore, pFM-M19 could transfer blaOXA-23 to E. coli 25DN by conjugation, resulting in carbapenem-resistant transconjugants.ConclusionsOur investigation showed that A. johnsonii M19 is a source and disseminator of blaOXA-23 and carbapenem resistance. The ability to transfer blaOXA-23 to other species by the conjugative plasmid pFM-M19 raises the risk of spread of carbapenem resistance. The carbapenem resistance gene blaOXA-23 is disseminated by a conjugative plasmid containing the novel transposon Tn6681 in Acinetobacter johnsonii M19.

Project description:Background and Objective:The emergence of carbapenem-resistant K. pneumoniae (CRKP) continues to escalate and is alarming because of the emergence of pan drug-resistant strains. The objective of this study was to investigate the existence of 12 carbapenemase genes among CRKP clinical isolates. Methods:Ninety-six Klebsiella spp. clinical isolates were collected. The isolates were identified phenotypically and genotypically. These isolates were screened for susceptibility to 24 different antibiotics. The modified Hodge test (MHT) and the Carba Nordmann/Poirel (NP) test were used to phenotypically screen carbapenem-resistant strains for carbapenemase production. Phenotypic characterization of carbapenemases was performed using the combined disk synergy test (CDST). Additionally, the presence of 12 carbapenemase genes in CRKP isolates was investigated. The DNA sequence of bla NDM and bla GES genes was determined. The BOX-PCR technique was used to determine the clonal relationship between CRKP isolates. Results:All carbapenem-resistant isolates were related to K. pneumoniae. Susceptibility testing showed that 19.79% (19/96) of the collected isolates were carbapenem-resistant. Of the CRKP isolates, 68.42% (13/19) tested positive for the MHT and Carba NP test. CDST showed that 42.11% (8/19), 63.16% (12/19), 47.37% (9/19), and 73.68% (14/19) of the CRKP isolates tested positive for the inhibitory effect of clavulanic acid, sulbactam, phenylboronic acid, and tazobactam, respectively, while 84.21% (16/19) and 68.42% (13/16) tested positive for the inhibitory effect of EDTA and mercaptopropionic acid, respectively. It was found that 10.53% (2/19) of the isolates tested positive for the inhibitory effect of sodium chloride. Molecular investigation of carbapenemases showed that 26.32% (5/19), 73.68% (14/19), 21.05% (4/19), 10.53% (2/19), and 5.26% (1/19) of the isolates tested positive for bla NDM, bla OXA-48, bla OXA-181, bla OXA-51, and bla OXA-23, respectively. None of the isolates tested positive for bla OXA-40 and bla OXA-58. Two allelic variants of bla NDM (bla NDM-1 and bla NDM-25) were detected. BOX-PCR revealed high clonal relatedness between CRKP isolates. Conclusion:MHT was more sensitive than Carba NP test for evaluating carbapenemase production and class D carbapenemase genes were the most prevalent of the 12 carbapenemase genes that were evaluated.

Dataset Information

Insights into the complete genomes of carbapenem-resistant Acinetobacter baumannii harbouring bla OXA-23, bla OXA-420 and bla NDM-1 genes using a hybrid-assembly approach.

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Insights into the complete genomes of carbapenem-resistant <i>Acinetobacter baumannii</i> harbouring <i>bla</i> <sub>OXA-23,</sub> <i>bla</i> <sub>OXA-420</sub> and <i>bla</i> <sub>NDM-1</sub> genes using a hybrid-assembly approach.

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