Type of Document Dissertation Author Brown, Mindy Gayle URN etd-11042008-161541 Title Antibiotic Resistance and Resistance Mechanisms in Bacteria Isolated from the Deep Terrestrial Subsurface Degree Doctor of Philosophy Department Biomedical Sciences, Department of Advisory Committee
Advisor Name Title David Balkwill Committee Chair Branko Stefanovic Committee Member Graham Patrick Committee Member Hank Bass Outside Committee Member Keywords
- Antibiotic Resistance
Date of Defense 2008-10-03 Availability unrestricted AbstractVarious natural environments have been examined for the presence of antibiotic-resistant bacteria and/or novel resistance mechanisms, but little is known about resistance in the terrestrial deep subsurface. This study examined two deep environments that differ in their known period of isolation from surface environments and the bacteria therein. One hundred and fifty-four strains of bacteria were isolated from sediments located 170-259 m below land surface at the U.S. Department of Energy Savannah River Site (SRS) in South Carolina and Hanford Site (HS) in Washington. Analyses of 16S rRNA gene sequences showed that both sets of strains were phylogenetically diverse and could be assigned to several genera in 3-4 phyla. All of the strains were screened for resistance to 13 antibiotics by plating on selective media and 90% were resistant to at least one antibiotic. 86% of the SRS and 62% of the HS strains were resistant to more than one antibiotic. Resistance to naladixic acid, mupirocin, or ampicillin was noted most frequently. The results indicate that antibiotic resistance is common among subsurface bacteria. The somewhat higher frequencies of resistance and multiple resistance at the SRS may, in part, be due to recent surface influence, such as exposure to antibiotics used in agriculture. However, the HS strains have never been exposed to anthropogenic antibiotics but still had a reasonably high frequency of resistance. Given their long period of isolation from surface influences, it is possible that they possess some novel antibiotic resistance genes and/or resistance mechanisms.
Seven of the strains from the HS that are resistant to tetracycline were examined for the presence of a novel antibiotic resistance gene. From these seven strains, a novel tetracycline resistance determinant was characterized. The predicted amino acid sequence shares only a 30% sequence similarity with TetA(Z), the most closely related previously described determinant. The new protein is a putative efflux pump with several characteristics in common with previously characterized efflux pumps including: a divergently transcribed TetR repressor, conserved GxxSDRxGRR motif, and transmembrane domains. The determinant has been assigned the name Tet 42.
Functional genes from another subset of 11 HS strains that are resistant to ciprofloxacin were sequenced for resistance-conferring mutations. The most common mechanism of resistance to this antibiotic is based on mutations in the functional genes for DNA gyrase (gyrA, gyrB) and topoisomerase II (parC, parE). Sequences for the genes gyrA, gyrB, and parC in resistant strains were compared to the same sequences from ciprofloxacin-sensitive strains from the HS and Escherichia coli. The strains grouped into three genera: Arthrobacter, Sphingomonas, and Pseudomonas. All of the resistant strains possessed some mutations in their gyrase and/or topoisomerase genes that result in the substitution of amino acids not seen in the gene products of E. coli and the sensitive strains. These mutations, some of which have not been reported previously, can be considered putative resistance-conferring mutations. The resistant subsurface strains were also grown in the presence of an efflux pump inhibitor, and a majority of the cultures did not grow when the inhibitor was added. Lack of growth in the presence of the inhibitor may indicate that ciprofloxacin resistance is due entirely or in part to an efflux pump. The presence of an efflux pump might also explain why some of the strains with a higher minimum inhibitory concentration (MIC) have fewer mutations in their gyrase and/or topoisomerase genes than do strains with a lower MIC. It is possible that, along with novel mutations that may play a role in resistance, these strains also posses an uncharacterized efflux pump.
A third approach used in this study to examine novel antibiotic resistance mechanisms was to look at differences in the entire proteome under normal and stressed conditions. The strain G887 is resistant to tetracycline and possesses the tetracycline resistance determinant Tet 42. Cultures of this strain were grown with tetracycline and without tetracycline. Protein extractions were performed from each culture and separated in the 1st dimension according to pI, on 4-7 Isoelectric Focusing Strips (IEF) strips and 6-11 IEF strips. After the 1st dimension separation, the proteins were separated by molecular weight on 12% acrylamide gels. The gels were stained with a fluorescent stain, imaged, and analyzed with spot analysis software. The gels run with the proteins from the tetracycline-treated culture indicated that several proteins visualized on both the 4-7 and 6-11 gels were upregulated in the presence of tetracycline. Some of these spots correspond to the molecular weight and pI for Tet A(42) or to those of several previously described general stress proteins.
This work demonstrates that there is a high frequency of antibiotic resistance in the deep terrestrial subsurface and that bacteria in this environment possess uncharacterized antibiotic resistance genes and mutations that confer resistance. Given the constant emergence of antibiotic-resistant pathogenic strains in clinical settings and the problems this creates with respect to the treatment of bacterial diseases, it becomes increasingly important to characterize antibiotic resistance genes that may exist in the environment but have not yet been transferred to clinically important species. Our ability to alter existing antibiotics or develop new drugs to counter novel resistance mechanisms will be dependent on such characterizations. It might also be worthwhile to investigate subsurface bacteria for the ability to produce antibiotics themselves. There is a real potential for novel antibiotic discovery, given the length of time these bacteria have been isolated from antibiotic-producing bacteria in surface environments.
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