Characterization of plasmids isolated from Aeromonas spp. obtained from South African aquaculture systems

Marx, Isa Jacoba (2007-03)

Thesis (MSc)--University of Stellenbosch, 2007.

Thesis

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.

AFRIKAANSE OPSOMMING: Die plasmied inhoud van 37 Aeromonas sp. isolate, verkry vanaf Suid-Afrikaanse tilapia, koi en forel akwakultuur sisteme was vasgestel. Sewentien Aeromonas sp. isolate het die teenwoordigheid van een of meer plasmiede wat gewissel het in grootte vanaf 1 tot 35 kb getoon. Die teenwoordigheid van groot plasmiede (75-200 kb) was deur S 1 nuklease puts-veld gel elektroforese waargeneem in sewe isolate. Drie en twintig transformante wat Aeromonas sp. plasmiede bevat het, is gekarakteriseer op grond van die oordrag van antibiotika merkers. Die oordrag van antibiotika weerstandsfenotipes was baie divers, met die gesamentlike oordrag van weerstand teen onverwante anitbiotikas. B-Laktaam weerstand was ge"identifiseer as die dominante fenotipe, teenwoordig in 13 transformante. Die mede-oordrag van weerstand teen ampisillien, amoksisillien, amoksisillien te same met klavulaan suur, keftriaksoon, kefuroksiem en die eerste generasie quinoloon, naladiksiese suur was aanwesig in 47.8% van die transformante. Southern klad eksperimente met die tetA, bla-TEM en strA-strB gene het die voorkoms van plasmied gekodeered kopie van die onderskeie gene getoon in 47.1%, 35.3% en 17.6% van die 17 Aeromonas sp. wat plasmiede bevat het. Die vookoms van ander beweeglike genetiese elemente naamlik Tnl721 en int! was laag. Ontleding van die DNA van die getransformeerde plasmiede het die teenwoordigheid van plasmiede wat in 'n wye verskeidenheid van gashere kan repliseer getoon. Die replikasie vermoe was waargeneem in die afwesigheid van gasheer-gekodeerde faktore. Plasmiede p31T1 en p36T2, ge"isoleer vanuit A. sobria en A. hydrophila, onderskeidelik, was ge"identifiseer as potentiele IncQ-tipe plasmiede op grond van die positiewe hibridisasie syne met die repC geen van pRAS3.1, mobilisasie deur die lncPa plasmied, PR4, en die relatiewe klein plasmied grootte van 14 kb. Die 26.6 kb ColE-tipe plasmied ge"isoleer vanuit A. sobria was gekarakteriseer deur die oordrag van die weerstandsfenotipe, endonuklease snydingskartering and gedeeltelike DNA volgorede bepaling. Die onvermoe van hierdie plasmied om gedurended konjugasie oorgedra te word, was 'n aanduiding dat die plasmied nie self-oordragbaar is nie. Antibiotika sensitiwiteits toetsing het die oordrag van weerstand teen 14 antibiotika middels ge"identifiseer in die nuwe gasheer, E. coli DH5a. Algehele resultate het 'n hoe vlak van plasmied diversiteit en die potentiele oordrag van antibiotika weerstandsgene tussen Aeromonas sp. getoon. Dit beklemtoon weereens die rol van plasmiede en ander beweeglike genetiese elemente in die verspreiding van weerstandsgene in die akwakultuur omgewing.

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