RESUMO
Nosocomial infections are major clinical threats to hospitalised patients and represent an important source of morbidity and mortality. It is necessary to develop rapid detection assays of nosocomial pathogens for better prognosis and initiation of antimicrobial therapy in patients. In this study, we present the development of molecular methods for the detection of six common nosocomial pathogens including Escherichia coli, Staphylococcus aureus, Streptococcus pneumoniae, Klebsiella pneumoniae, Pseudomonas aeruginosa and Acinetobacter spp. Conventional multiplex PCR and SYBR Green based real time PCR assays were performed using genus and species specific primers. Blind testing with 300 clinical samples was also carried out. The two assays were found to be sensitive and specific. Eubacterial PCR assay exhibited positive results for 46 clinical isolates from which 43 samples were detected by real time PCR assay. The sensitivity of the assay is about 93.7 percent in blind test isolates. The PCR results were reconfirmed using the conventional culture method. This assay has the potential to be a rapid, accurate and highly sensitive molecular diagnostic tool for simultaneous detection of Escherichia coli, Staphylococcus aureus, Streptococcus pneumoniae, Klebsiella pneumoniae, Pseudomonas aeruginosa and Acinetobacter spp. This assay has the potential to detect nosocomial pathogens within 5 to 6 hours, helping to initiate infection control measures and appropriate treatment in paediatric and elderly (old aged) patients, pre-and post surgery patients and organ transplant patients and thus reduces their hospitalization duration .
RESUMO
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.