Masters Degrees (Microbiology)
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Browsing Masters Degrees (Microbiology) by Subject "Aeromonas"
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- ItemCharacterisation of plasmid p31T1 isolated from Aeromonas(Stellenbosch : Stellenbosch University, 2013-12) Laubscher, Inge; Rawlings, D. E.; Deane, S. M.; Stellenbosch University. Faculty of Science. Dept. of Microbiology.ENGLISH ABSTRACT: Plasmids are an integral part of the horizontal gene pool and, therefore, are the main vectors for the spread of antibiotic and heavy metal resistance genes in the environment. Functional and taxonomic characterization of novel plasmids is, therefore, central to our general understanding of plasmid biology and their contribution to microbial evolution. Two 14-kb mobilizable plasmids, p31T1 and p36T2, conferring resistance to tetracycline were isolated from the opportunistic fish pathogens Aeromonas sobria and Aeromonas hydrophila and were found to have indistinguishable restriction fragment length polymorphism (RFLP) patterns (Marx, MSc Thesis). DNA sequence analysis of the two isogenic plasmids (only p36T2 was sequenced) revealed the presence of 18 putative open reading frames (ORFs), of which the tetAR tetracycline resistance genes, associated with a truncated Tn1721, were the only ORFs with significant similarity to known sequences within the NCBI database. Putative functions were assigned to 10 of the ORFs based on their distant homology with proteins of known function. Six of the 18 ORFs, spanning 5.7-kb, were found to comprise the minimal region required for replication (minimal replicon) by means of deletion analysis using derivatives of p31T1. Of the six ORFs, ORF2 and ORF4 were found to be essential for plasmid replication. Inactivation of ORF3 resulted in an increase of plasmid copy number (PCN) from ~3 to ~7 plasmids per chromosome and a decrease in plasmid stability from ~80 % to 16 % over approximately 127 generations (7 days). Furthermore, by means of β-galactosidase promoter fusion assays it was shown that ORF3 autoregulated its own promoter. These results, therefore, suggested that although ORF3 was not essential for replication, it may be involved in plasmid copy number regulation and control. Host range analysis indicated that p31T1 was able to replicate in two other members of the γ-proteobacteria group (Escherichia coli and Pseudomonas putida) but was unable to do so in an α-proteobacterium strain, thus suggesting a limited host range. Furthermore, p31T1 was mobilized only at low frequencies (5.4 x 10-5 transconjugants per donor) by an IncP-1 conjugative system though it is possible that the mobilization system of these plasmids is adapted to function optimally with alternate conjugative systems. Given the unique PCN, stability, host range and mobilization characteristics determined for p31T1 and that no other plasmid replication and mobilization systems with significant sequence similarity to these plasmids have yet been identified, it is likely that these two plasmids are the first representative members of a new family of plasmids found within aquacultureassociated Aeromonas species and which are involved in the spread of tetracycline resistance.
- ItemCharacterization of plasmids isolated from Aeromonas spp. obtained from South African aquaculture systems(Stellenbosch : Stellenbosch University, 2007-03) Marx, Isa Jacoba; Chenia, H. Y.; Rawlings, D. E.; Stellenbosch University. Faculty of Science. Dept. of Microbiology.ENGLISH ABSTRACT: The plasmid content of 37 Aeromonas spp. isolates, obtained from South African tilapia, trout and koi aquaculture systems, was evaluated and 17 Aeromonas spp. isolates appeared to contain one or more plasmids ranging in size from 1 to 35 kb. Large plasmids (75-200 kb) were detected using S 1 nuclease pulsed-field gel electrophoresis in seven isolates. Twenty-three Escherichia coli transforrnants containing single/multiple plasmids from the 17 plasmid-containing isolates were characterized with respect to the transferred antimicrobial resistance phenotypes. Transforrnants displayed diverse phenotypes with the co-transfer of unrelated antibiotics. ~-lactam resistance was identified as the dominant resistance phenotype displayed by 13 transforrnants. Cotransfer of ampicillin, amoxycillin, augmentin, ceftriaxone, cefuroxime and the first generation quinolone, nalidixic acid occurred in 4 7 .8% of trans formants. Southern hybridization experiments with tetA, bla-TEM and strA-strB gene probes indicated the prevalence of the respective genes in 47.l %, 35.3% and 17.6% of the 17 plasmidcontaining Aeromonas spp., respectively. Low incidences of mobile genetic elements, i.e., Tnl 721 and inti were also detected. Analysis of the transformed plasmids' backbone DNA revealed the presence of possible broad-host-range plasmids based on their ability to replicate in the absence of host-encoded factors. Plasmids p3 l Tl and p36T2, isolated from A. sobria and A. hydrophila, respectively, were identified as potential lncQ-like plasmids based on positive hybridization signals with the repC gene from pRAS3.l, mobilization by the IncPa. plasmid RP4, and the relatively small size (14 kb). A 26.6 kb ColE-type plasmid isolated from A. sobria was characterized by the transferred resistance phenotype, endonuclease restriction mapping and partial sequencing. Failure to transfer the plasmid by conjugation indicated that this plasmid was not self-transmissible, while antibiotic susceptibility testing revealed indicated the transfer of resistance to 14 antibiotics to E. coli DH5a.. Overall, results indicated the high level of plasmid diversity and potential transfer of antibiotic resistance determinants between Aeromonas spp., emphasizing the role of plasmids and other mobile genetic elements in the dissemination of antibiotic resistance genes, especially in the aquaculture environment.