Acinetobacter baumannii is a nosocomial pathogen involved in various infections ranging from minor soft-tissue infections to more severe infections such as ventilator-associated pneumonia and bacteremia. The severity and the type of infections depend on the genetic and phenotypic variations of the strains. In this study, we compared the extent of biofilm formation and motility displayed by 60 multidrug-resistant A. baumannii clinical strains isolated from blood and sputum samples from patients from Southern India. Our results showed that isolates from the sputum samples formed significantly more robust biofilm compared to the blood isolates. On the other hand, we observed that the blood isolates were more motile than the sputum isolates. To the best of our knowledge, this is the first study that systematically evaluated the correlation between these two phenotypic traits and the nature of the isolates.

Acinetobacter species have emerged as one of the most clinically important pathogens. The phenotypic techniques which are currently available are insufficient in accurately identifying and differentiating the closely related and clinically important Acinetobacter species. Here, we discuss the advantages and limitations of the conventional phenotypic methods, automated identification systems, molecular methods and MALDI-TOF in the precise identification and differentiation of Acinetobacter species. More specifically, several species of this genus are increasingly reported to be of high clinical importance. Molecular characterization such as of bla OXA-51 -like PCR together with rpoB sequencing has high discriminatory power over the conventional methods for Acinetobacter species identification, especially within the Acinetobacter calcoaceticus–Acinetobacter baumannii complex.

IntroductionAcinetobacter baumannii is an important opportunistic pathogen responsible for causing nosocomial infections. Carbapenems are considered to be the drug of choice to treat infections caused by multidrug-resistant A. baumannii. The prevalent mechanism of carbapenem resistance in A. baumannii is enzymatic degradation by β-lactamases. Therefore, the aim of the study is to determine the prevalence and distribution of molecular determinants among the clinical isolates of carbapenem-resistant A. baumannii.MethodsA total of 103 consecutive, non-duplicate carbapenem-resistant A. baumannii isolated from blood and endotracheal aspirates (ETAs) were included in the study. The CarbAcineto NP test was performed for the screening of carbapenemase production. Polymerase chain reaction (PCR) was performed to detect extended spectrum β-lactamases (ESBLs), metallo-β-lactamases (MBLs) and oxacillinases (OXAs). PCR was done for the detection of ISAba1 elements, and mapping PCR was performed to identify the position of ISAba1 with respect to the OXA-23-like gene.ResultsAmong the 103 A. baumannii isolates, 94 were phenotypically identified as carbapenemase producers. blaPER was the most common among the ESBLs. Among MBLs, blaNDM was predominant followed by the blaVIM gene. blaOXA-51 and blaOXA-23 were the most common and present in all 103 isolates. Almost 80% of the isolates had ISAba1 upstream blaOXA-23 gene.ConclusionThe blaOXA-23 and blaNDM genes are the most common type of oxacillinases and metallo β-lactamases, respectively, and contribute to carbapenem resistance in clinical isolates of A. baumannii. The presence of ISAba1 upstream of the blaOXA-23 gene suggests that the insertion element acts as a promoter for its increased expression.FundingIndian Council of Medical Research, New Delhi, India (ref. no. AMR/TF/54/13ECDHII dated 23 October 2013).

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Emerging nosocomial strains of Acinetobacter baumannii are of recent concern as they are expressing extensive drug resistance (XDR). Using whole‐genome sequencing and molecular characterisation analysis, the current study reveals the presence of carbapenemase genes in 92.86% of studied Indian isolates. These included blaOXA‐51, blaOXA‐23, blaOXA‐58, and blaNDM genes, with over a third expressing dual carbapenemase genes. As per the MLST scheme, IC2Oxf/CC2Pas was the predominant clone, with 57.14% isolates belonging to this lineage. The presence of these carbapenemase genes resulted in sulbactam (SUL) resistance (MIC: 16–256 µg/ml) in all of the studied isolates. The efficacy of durlobactam (DUR), a novel β‐lactamase inhibitor that also inhibits PBP2 was assessed through in silico intermolecular interaction analysis. Several nonsynonymous single nucleotide polymorphisms were identified in PBP2 (G264S, I108V, S259T) and PBP3 (A515V, T526S) sequences. Minimal variations were recorded in the protein backbone dynamics in active‐site motifs of wild‐type and mutants, which correlated with negligible binding energy fluctuations for the PBP3‐SUL (−5.85 ± 0.04 kcal/mol) and PBP2‐DUR (−5.16 ± 0.66 kcal/mol) complexes. Furthermore, higher binding affinities and low inhibition constants were noted in OXA23‐DUR (−7.36 kcal/mol; 4.01 µM), OXA58‐DUR (−6.44 kcal/mol; 19.07 µM), and NDM‐DUR (−6.82 kcal/mol; 10.01 µM) complexes when compared with the conventional drugs avibactam and aztreonam. Stable interaction profiles of DUR with carbapenemases can possibly restore SUL activity against both PBP3WT and PBP3MTs. The study establishes the efficacy of the novel SUL–DUR combination as a successful treatment strategy in combating emerging XDR strains of A. baumannii.

The data shown highlight the wide difference in the molecular mechanisms of AMR profile between P. aeruginosa and A. baumannii. In P. aeruginosa, plasmid-mediated mechanisms are much lesser than the chromosomal mediated mechanisms. In A. baumannii, class D oxacillinases are more common than other mechanisms. Continuous surveillance to monitor the trends in AMR among MDR pathogens is important for implementation of infection control and to guide appropriate empirical antimicrobial therapy.

Background & objectives: Acinetobacter baumannii is an opportunistic pathogen responsible for causing nosocomial infections. A. baumannii develops resistance to various antimicrobial agents including carbapenems, thereby complicating the treatment. This study was performed to characterize the isolates for the presence of various β-lactamases encoding genes and to type the isolates to compare our clones with the existing international clones across five centres in India. Methods: A total 75 non-repetitive clinical isolates of A. baumannii from five different centres were included in this study. All the isolates were confirmed as A. baumannii by bla OXA-51-like PCR. Multiplex PCR was performed to identify the presence of extended spectrum β-lactamases (ESBL) and carbapenemases. Multilocus sequence typing was performed to find the sequence type (ST) of the isolates. e-BURST analysis was done to assign each ST into respective clonal complex. Results: bla OXA-51-like was present in all the 75 isolates. The predominant Class D carbapenemase was bla OXA-23-like followed by Class B carbapenemase, bla NDM-like . Class A carbapenemase was not observed. bla PER-like was the predominant extended spectrum β-lactamase. ST-848, ST-451 and ST-195 were the most common STs. Eight-novel STs were identified. e-BURST analysis showed that the 75 A. baumannii isolates were clustered into seven clonal complexes and four singletons, of which, clonal complex 208 was the largest. Interpretation & conclusions: Most of the isolates were grouped under clonal complex 208 which belongs to the international clonal lineage 2. High occurrence of ST-848 carrying bla OXA-23-like gene suggested that ST-848 could be an emerging lineage spreading carbapenem resistance in India.

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 .