Characterization of dioxygenases and biosurfactants produced by crude oil degrading soil bacteria

ABSTRACT Role of microbes in bioremediation of oil spills has become inevitable owing to their eco friendly nature. This study focused on the isolation and characterization of bacterial strains with superior oil degrading potential from crude-oil contaminated soil. Three such bacterial strains were selected and subsequently identified by 16S rRNA gene sequence analysis as Corynebacterium aurimucosum, Acinetobacter baumannii and Microbacterium hydrocarbonoxydans respectively. The specific activity of catechol 1,2 dioxygenase (C12O) and catechol 2,3 dioxygenase (C23O) was determined in these three strains wherein the activity of C12O was more than that of C23O. Among the three strains, Microbacterium hydrocarbonoxydans exhibited superior crude oil degrading ability as evidenced by its superior growth rate in crude oil enriched medium and enhanced activity of dioxygenases. Also degradation of total petroleum hydrocarbon (TPH) in crude oil was higher with Microbacterium hydrocarbonoxydans. The three strains also produced biosurfactants of glycolipid nature as indicated d by biochemical, FTIR and GCMS analysis. These findings emphasize that such bacterial strains with superior oil degrading capacity may find their potential application in bioremediation of oil spills and conservation of marine and soil ecosystem.

The mazEF toxin-antitoxin system as a novel antibacterial target in Acinetobacter baumannii

Although analysis of toxin-antitoxin (TA) systems can be instructive, to date, there is no information on the prevalence and identity of TA systems based on a large panel of Acinetobacter baumannii clinical isolates. The aim of the current study was to screen for functional TA systems among clinical isolates of A. baumannii and to identify the systems’ locations. For this purpose, we screened 85 A. baumannii isolates collected from different clinical sources for the presence of the mazEF, relBE and higBA TA genes. The results revealed that the genes coding for the mazEF TA system were commonly present in all clinical isolates of A. baumannii. Reverse transcriptase-polymerase chain reaction analysis showed that transcripts were produced in the clinical isolates. Our findings showed that TA genes are prevalent, harboured by chromosomes and transcribed within A. baumannii. Hence, activation of the toxin proteins in the mazEF TA system should be investigated further as an effective antibacterial strategy against this bacterium.

Detection of metallo-β-lactamases producing Acinetobacter baumannii using microbiological assay, disc synergy test and PCR.

Background: One leading factor responsible for resistance in Acinetobacter baumannii, an important opportunist in health care institutions globally, is the production of carbapenamases like metallo-β-lactamases (MBLs), which hydrolyze a variety of β-lactams including penicillin, cephalosporins and carbapenems. However, neither any standard guidelines are available nor any method has been found to be perfect for their detection. Various methods have shown discordant results, depending upon the employed methodology, β-lactamase substrate and MBL inhibitor used. This study aims to evaluate two phenotypic methods against PCR as gold standard among carbapenem resistant A. baumannii for identifying MBL producers. Materials and Methods: A total of 130 A. baumannii were screened for imipenem and meropenem resistance by Kirby-Bauer disc diffusion method. Phenotypic expression of MBL was detected by EDTA-imipenem-microbiological (EIM) assay and extended EDTA disc synergy (eEDS) test and presence of bla-IMP and bla-VIM was detected by PCR in all the carbapenem resistant isolates. Results: Of the 43 imipenem and/or meropenem resistant A. baumannii isolates, 4 (9.3%) were found to be MBL producers by EIM and 3 (6.97%) by eEDS. Only bla-VIM gene was detected in 7 (16.28%) by PCR. In addition EIM detected 14 (32.56%) carbapenem resistant non-metallo enzyme producers. Conclusion: Of the two MBL genes targeted, bla-VIM was only detected and that too in isolates resistant to both imipenem and meropenem. Further, EIM was useful in differentiating MBL from non-metalloenzymes producers.

The genomic identification of Colombian Acinetobacter baumannii clinical isolates by RFLP-PCR analysis of the 16S-23S rRNA gene spacer region / Identificación genómica de aislamientos colombianos de Acinetobacter baumannii mediante RFLP-PCR de la región intergénica espaciadora de los genes 16S y 23S rRNA

The 16S-23S rRNA gene intergenic spacer (ITS) was analysed by RFLP in this study to identify A. baumannii from 139 isolates from four hospitals (identified as A, B, C and D). One hundred and twenty of these isolates (86.3%) belonged to the A. baumannii species; those identified as being A. baumannii were found to be polyclonal (19 clone groups) when determining the genetic relationships, 16 of them being found in hospital C. Hospitals A, B and D shared two clone groups isolated during different years. This study describes a rapid and easy method for genospecies identification of Acinetobacter baumannii.

Con el objeto de identificar la genomoespecie Acinetobacter baumannii, se estudiaron 189 aislamientos pertenecientes al Complejo Acinetobacter baumannii-Acinetobacter calcoaceticus provenientes de cuatro hospitales colombianos (denominados A,B,C,D) mediante el análisis por RFLP-PCR de la región intergénica espaciador (ITS) de los genes 16S y 23S rRNA. Se encontraron 120 aislamientos (86.3%) pertenecientes a la especie A. baumannii. La estructura de la población fue policlonal, con 19 grupos clonales, 16 de los cuales se hallaron en el hospital C. En los hospitales A,B y D se encontraron 2 grupos clonales aislados durante diferentes años. En este estudio se propone un método rápido y fácil para la identificación de Acinetobacter baumannii.

Analysis of penicillin-binding proteins (PBPs) in carbapenem resistant Acinetobacter baumannii.

Background & objectives : Acinetobacter baumannii is a Gram-negative, cocco-bacillus aerobic pathogen responsible for nosocomial infections in hospitals. In the recent past A. baumannii 0had developed resistance against β-lactams, even against carbapenems. Penicillin-binding proteins (PBPs) are crucial for the cell wall biosynthesis during cell proliferation and these are the target for β-lactams. Therefore, the present study was carried out to identify the PBPs in three (low, intermediate and high MICs) groups of carbapenem resistant isolates strains of A. baumannii. Methods: ATCC 19606 and 20 β-lactam resistant isolates of A. baumannii were obtained. Selective identification of the PBPs was done using Bocillin FL, a non-radioactive fluorescent derivative of penicillin. Results: The fluorescence emission from Bocillin-tag in SDS-PAGE gel of native strain identified eight PBPs, with apparent molecular weight of 94, 65, 49, 40, 30, 24, 22 and 17 kDa, however, these PBPs revealed alteration in carbapenem-resistant isolates. Interpretation & conclusions: A comparative analysis of PBPs in the resistant isolates with those of ATCC revealed a decreased expression of all PBPs except that of 65 and 17 kDa PBPs which were marginally downregulated, and simultaneous appearance of new 28 kDa PBP (in low and intermediate resistant isolates) and 36 kDa in high meropenem resistant group of A. baumannii. The present study indicated an association between alteration in PBPs and β-lactam resistance in A. baumannii.

Iron regulated outer membrane proteins (IROMPs) as potential targets against carbapenem-resistant Acinetobacter spp. isolated from a Medical Centre in Malaysia.

Background & objectives: Carbapenem-resistant Acinetobacter spp. have gained increasing significance as opportunistic pathogens in hospitalized patients. Carbapenem resistance is often associated with the loss and/or decrease in outer membrane proteins (OMP) and overexpression of multidrug efflux systems. However, carbapenem-hydrolysing β-lactamases of Ambler Class B (metallo-enzymes) and Ambler Class D (oxacillinases) have also been detected in Acinetobacter spp. In this study we have investigated the role of the iron regulated outer membrane protein (IROMPs) and the loss of a 29-kDa OMP in carbapenem resistance of Acinetobacter calcoaceticus. Methods: Carbapenem resistant clinical isolates (n=39) of Acinetobacter baumannii / calcoaceticus were used. Identification of Acinetobacter spp. at species level was done by amplified ribosomal DNA restriction analysis (ARDRA). MIC was evaluated using agar dilution method according to CLSI standards. Presence of outer membrane proteins were determined by SDS-PAGE. A representative strain of A. calcoaceticus, S26 with the loss of 29-kDa OMP was selected for further analysis as strain S26 had unique resistance mechanism, that is, the presence of IMP-4 metallo-β-lactamases. IROMPs were expressed under iron deficit conditions. Bands corresponding to IROMPs were excised from SDS-PAGE and used to immunize rabbits for the production of polyclonal antibodies. The antibodies raised against IROMPs were detected by an in-house ELISA and then used for bactericidal activity against carbapenem resistant A. baumannii / calcoaceticus. Results: All isolates were resistant to all antibiotics including imipenem and meropenem and had loss of a 29-kDa OMP. The polyclonal antibodies showed bactericidal effect against the organism tested and it specifically killed the bacteria grown in iron deficit medium. Interpretation & conclusions: In this study, a 29-kDa OMP has been identified to be the major outer membrane protein in A. baumannii / calcoaceticus and loss of this porin and overexpression of IROMPs have contributed to carbapenem resistance. Polyclonal antibodies raised against IROMPs may have a role in antimicrobial therapy in these isolates.

Bombas de expulsión multidrogas en Acinetobacter baumannii y resistencia a antimicrobianos / Multi-drug efflux pumps and antibiotic resistance in Acinetobacter baumannii

Los sistemas multidrogas bacterianos contribuyen al desarrollo del fenotipo de multi-resistencia presentado por cepas de Acinetobacter baumannü, patógeno intrahospi-talario, que durante los últimos años ha incrementado su importancia por la creciente resistencia a carbapenémicos. El fenotipo de multi-resistencia está otorgado por la combinación de varios mecanismos de resistencia entre los cuales se encuentran estos sistemas de bombas de expulsión. El sistema multidroga AdeABC se ha detectado en muchas de estas cepas multi-resistentes de A. baumannü y, se ha relacionado con resistencia a diversos grupos de antimicrobianos, incluidos tigeciclina y meropenem. La inhibición de dichos sistemas multidrogas permitiría aumentar la eficacia de la terapia antimicrobiana. La siguiente revisión se enfoca en las bombas de expulsión multidrogas presentes en A. baumannü, con particular énfasis en el sistema AdeABC.

Bacterial multi-drugs systems contribute to the development of multi-resistance patterns of Acinetobacter baumannii, a nosocomial pathogen of increasing importance due to its emerging resistance to carbapenems. The multi-resistance phenomena is generated by a combination of mechanisms, one of which the efflux pump system. Many of these multiresistant isolates of A. baumannii harbor genes for the AdeABC multi-drug efflux system, related with resistance to various groups of antibacterial agents, including tygecicline and meropenem. Inhibition of these systems would allow to increase the efficacy of this antimicrobial. This review focuses on the multi-drug efflux pump system oí A. baumanni with special emphasis in the AdeABC system.