Ee, Chan Shiao
(2017)
Development of a dna-based method for simultaneous detection of acinetobacter baumannii, antimicrobial resistance genes and its genotypes by dna fingerprinting.
PhD thesis, Universiti Sains Malaysia .
Abstract
Acinetobacter species have emerged as important healthcare associated pathogens worldwide. Of the described Acinetobacter, A. baumannii constitutes as the most significant causative agent implicating various severe infections, especially ventilated associated pneumonia and bloodstream infections, associated high mortality and morbidity in patients. Current routine identification systems are unable to identify Acinetobacter to the species level. Hereof, this study aimed to develop a reliable tool for simultaneous detection of Acinetobacter genus, A. baumannii and four genes encoding the carbapenem-hydrolyzing class D β-lactamases, and to investigate the molecular epidemiology of carbapenem susceptible and non-susceptible A. baumannii clinical isolates in a hospital setting. A thermostabilized multiplex PCR assay was developed with primers designed on specific sequence regions of 16S rRNA gene, 33-36 kDa outer membrane protein and four carbapenem-hydrolyzing class D β-lactamase genes to achieve the goal of this study. An internal control was incorporated to validate reliability and robustness of the assay. Specific detection of Acinetobacter genus, A. calcoaceticus-A. baumannii complex and A. baumannii on pure bacterial cultures and spiked blood cultures was demonstrated using the developed assay. The assay yielded detection limits of 100 pg of purified DNA and 106 CFU/ml with DNA thermolysates prepared from either bacterial cultures or spiked human whole blood specimens. The assay was capable to detect at least one CFU of bacterial cells in a pre-enriched medium. The assay detected Acinetobacter with its resistance genes in three hours with high specificity and sensitivity (100%). Accelerated stability evaluation of thermostabilized multiplex PCR reagents demonstrated that vacuum-dried mixes were stable at room temperature for approximately 232 days. The developed thermostabilized multiplex PCR assay enabled simultaneous bacterial identification and detection of its resistance gene which would be useful in rapid diagnosis to reduce morbidity and mortality of patients with Acinetobacter infections. Speciation on Acinetobacter isolates recovered from 115 blood specimens collected over 24-month period with amplified ribosomal DNA restriction analysis was performed. A. baumannii (60.87%; 70 isolates) was found to be the predominant Acinetobacter genomic species followed by A. nosocomialis (19.13%; 22 isolates). Of the total 115 Acinetobacter isolates, 46.09% and 5.22% of A. baumannii and Acinetobacter species, respectively, were carbapenems-resistant. Pulsed-field gel electrophoresis was performed to ascertain genetic relatedness of carbapenem susceptible and non-susceptible A. baumannii clinical isolates. All the carbapenem non-susceptible A. baumannii isolates were consistently found to harbour β-lactamase gene, blaOXA-51-like (100%; all 70 isolates) and blaOXA-23-like (34.29%; 24 isolates). Two predominant clusters contained of mostly carbapenems non-susceptible A. baumannii isolates were observed in each year of study. Based on the multiple-locus variable-number tandem-repeat analysis, the characterized A. baumannii isolates mostly belonged to the international clonal lineage II, of which is worldwide distributed. Finding of this study further demonstrated the emergence of carbapenem resistance in members within the Acinetobacter genus (other than A. baumannii), emphasizing the importance of wisely prescribed antimicrobial agents and stringent implementing infection control measures to reduce further the development of resistance phenotypes and clonal spreading in clinical settings.
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