Seville, L.; (2007) Study of tetracycline resistance determinants and their genetic supports in the oral and faecal metagenomes of six European countries. Doctoral thesis , University of London.

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Abstract

Investigations of the prevalence of antibiotic resistance genes and their genetic supports are essential for our understanding of the mechanisms of resistance, and their transfer. This study investigated the prevalence of tetracycline and erythromycin resistance in the Gram positive aerobic cultivable portion, and the total oral and faecal microbiota of six European countries. Only Gram positive isolates were investigated as they represent a distinct phylogenetic group. Furthermore, this project was part of a larger European-wide study on the biology of Gram positive organisms. A collection of 123 tetracycline and/or erythromycin resistant isolates was made, and through macroarray analysis the most common tet genes were found to be tet(W), let(O) and /<.'/(W) in the aerobic oral flora, and tet(M). tet(O) and tet(Q) in the aerobic faecal flora. Three isolates did not hybridise to any probes on the array. In order to investigate the contribution of the whole metagenome to antibiotic resistance, total extracted DNA was analysed on the macroarray and 12 BAG libraries were constructed. The most common let genes in the oral microbiota were tet( ), tet(Q) and /i7(30) and were /(//(W). tet(O). tet(Q) and /tV(32) in the faecal metagenome. The BAC libraries were evaluated for efficiency of cloning microbial DNA, and to ensure they were representative of each microbiota, by end-sequence analysis. The libraries were screened on tetracycline. 32 resistant clones were found, only four of these were stable. One. NFtetCl. contained tet(O). The entire insert was sequenced to determine its support, it was shown to contain orfs with similarity to tnpY from n445L and to or/6 from n916 and cppJ from the /e/(0)-harbouring Campylobacter coli plasmid pCC31. Clone SFtetCIO harboured tet( ) PCR analysis illustrated it was flanked by sequences with homology to those flanking tet(M) in Tn9/6 however, int from Jn9J6 did not amplify with specific primers. Clones IStetCl and FRStetCll did not hybridise to any probes on the array. These harbour either novel or rare tet genes. Clone IStetCl was subcloned and found to harbour a putatitive natural chimera of two tetracycline resistance plasmids: pRSB107 and pR64. This study thus provides further evidence of the prevalence of antibiotic resistant bacteria in the human Gastrointestinal (GI) tract, and the difference in prevalence of tetrcycline resistance determinants in the aerobic cultivable flora and total microbiota. Furthermore, it illustrates how antibiotic resistance genes are contained on mobile genetic elements which are mosaic in structure having undergone evolutionary changes in which functional modules are exchanged.

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