Tuesday, March 3, 2009

Prevalence of tetracycline resistance genes in Greek seawater habitats

Theodora L. Nikolakopoulou, Eleni P. Giannoutsou, Adamandia A. Karabatsou, and Amalia D. Karagouni

Tetracyclines are antibiotics that have been used for over 40 years as a therapeutic agent in human and veterinary science and also as growth promoters in animal husbandry. Tetracyclines inhibit bacterial growth by interfering with protein synthesis. Overuse of such antibiotics has lead to the rapid spread of antibiotic resistance genes among bacteria. The most common mechanisms of resistance are tetracycline efflux, ribosome protection and tetracycline modification. Since these resistance genes are often found on mobile genetic elements such as transposons, they can be spread rapidly across bacterial species (Schmidt et al., 2001; Roberts, 2005).

The goal of the research presented in this paper was to analyze the presence of 12 tetracycline resistance (
tet A,B,C,D,E,G,H,K,L,M,O,T) genes in seawater sampled from different locations in Greece. The broader goal of this research is to study a complex ecosystem such as the marine environment, which acts like a reservoir of antimicrobial compounds and resistant bacteria (Aoki, 1992; Chee-Sanford et al., 2001).

Water was sampled from 4 different habitats: a) seawater near a wastewater treatment facility b) seawater near a fish farm c) sea water near a tourist spot and d) sea water from an uninhabited location. These water samples were plated onto a nutrient medium and then incubated. Colonies were picked and grown individually. Dilutions were made of these cultures and plated onto Agar containing tetracycline for 3-7 days at 20° C. Distinct colonies having different morphologies from each sample were isolated in pure culture. A total of 89 TcR colonies were picked: 36 from the fish farm, 23 from wastewater, 14 from the tourist place and 16 from the uninhabited location. These 89 colonies were analyzed for the presence of genes conferring TcR by polymerase chain reaction (PCR) using primers specific for the 12 kinds of TcR genes and then southern blotted. This showed that 60 colonies had more than one TcR (tet) gene and the remaining 29 had only
tetK which encodes a tetracycline efflux pump. Thus, tetK was clearly the most dominant of the 12 genes. Plasmid extraction from the 60 colonies that had more than one tet gene followed by gel electrophoresis revealed the presence of plasmids (around 40 kb in size) in 37 of the colonies. Ten of these 60 colonies had IncP-type plasmids as revealed by PCR using primers specific to sequences of IncP, IncQ, IncW and IncN plasmids. Thus, it is possible that the TcR genes were carried by these large plasmids. To confirm the presence of plasmids in all seawater samples, exogenous plasmid isolations were performed (Hills et al., 1996; Smalla et al., 2000) using tetracycline as a selective agent. TcR plasmids ranging in size between 40 and 80 kb were isolated from 80 transconjugants. 59 of 80 plasmids possessed one or more tet gene. The tetA gene was dominant as it was found in 36 plasmids as the only tet gene and was found with tetK and tetC on a smaller number of plasmids. PCR showed that 27 of the 59 plasmids belonged to the IncP group of broad host range plasmids.

To summarize, this study showed the presence of TcR bacteria in all seawater 4 samples. However, when the community composition of each sample was analyzed, the samples were found to vary from each other in the content of bacterial species. Thus despite the fact that TcR genes were found in these samples they were probably found on different bacteria. Of the 12 kinds of
tet genes, only 4 genes (tet K,A,M,C) were found in these samples while others (such as tet B, D, E, G, H, L, O, and T) were not found at all. This is the first study that reported the presence of tetK in the bacterial strains identified from the seawater samples. Moreover many of the tet genes were found on IncP- type broad host range plasmids. This suggests that the spread of TcR genes in marine environments could be because of their association with IncP-type broad host range plasmids.


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Roberts, M.C. 2005. Update on acquired tetracycline resistance genes. FEMS Microbiol. Lett. 245, 195-203.

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Chee-Sanford, J.C., R.I. Aminov, I.J. Krapac, N. Garrigues-Jeanjean, and R.I. Mackie. 2001. Occurrence and diversity of tetracycline resistance genes in lagoons and groundwater underlying two swine production facilities. Appl. Environ. Microbiol. 67, 1494-1502.

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Diya Sen
Graduate student, University of Idaho

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