Appraisal of the internal transcribed spacer region as a phylogenetic marker for delineation of clinical Acinetobacter members of the Acb complex, and investigation of strain resistance to multiple antibiotics

Background: The Acinetobacter calcoaceticus-baumannii (Acb) complex contains two of the most clinically important nosocomial pathogens. Their ability to readily acquire antibiotic resistance determinants has added to their virulence. Indeed this virulence has meant that reliable methods of species identification are increasingly crucial. In this thesis I have examined the feasibility of using the internal transcribed spacer (ITS) region to identify individual strains of Acb complex members. The presence of plasmids and their potential role in antibiotic resistance was also explored. Methods: Forty-one putative A. baumannii isolates tentatively identified from ribotyping and pulsed-field gel electrophoresis data were used in this study. Their 16S-23S rrn ITS and 16S rRNA genes were sequenced and compared against all available ITS and 16SrRNA sequences from Acb strains. Antibiotic resistance profiles were determined for all strains. Plasmid screening was performed, with cloning and sequencing of selected plasmids. Results: Based on their ITS sequences, strains were assigned to species as follows: A.baumannii; 5 strains, A. nosocomialis; 31 strains. Three strains could not be placed as their ITS sequences were almost identical to the sequences of four different Acinetobacter species. The presence of indels (Maslunka, 2012) prevented confident identification of the final two strains using the ITS region. Two plasmids isolated from a multi-drug resistant Acinetobacter nosocomialis strain were sequenced and characterised. The plasmids, designated pRAY*-v3 and pAB49-v1, were6,078 bp and 2,200 bp in size, encoding five and two putative genes, respectively. Antibiotic resistance determinants were annotated for pRAY*-v3, explaining the hostorganism’s demonstrated resistance to aminoglycoside antibiotics. Conclusion: These results suggest that the ITS cannot be used always to differentiate between individual Acinetobacter species within the Acb complex. Furthermore, the detection of pRAY*-v3 and pAB49-v1 plasmids from strains of A. baumannii examined across different decades and continents in A. nosocomialis, suggests they may be disseminated widely among other members of the Acb complex.

Submission note: A thesis submitted in total fulfilment of the requirements for the degree of Master of Science to the School of Molecular Sciences, Faculty of Science Technology and Engineering, La Trobe University, Bundoora.