Tetracycline Resistance-Encoding Plasmid from Bacillus sp. Strain #24, Isolated from the Marine Sponge Haliclona simulans

Abstract

Knowledge of the nature of resistance determinants in natural habitats is fundamental to increasing our understanding of the development of antibiotic resistance in clinical settings. Here we provide the first report of a tetracycline resistance-encoding plasmid, pBHS24, from a marine sponge-associated bacterium, Bacillus sp. strain #24, isolated from Haliclona simulans.The increased selection pressure created by the overuse and misuse of antibiotics in clinical and agricultural settings has undoubtedly driven the evolution and spread of resistance determinants (8, 11). While many resistance genes are believed to have their origin in natural ecosystems, their abundance, nature, and ecological role in such settings remain relatively obscure (2). It is nevertheless accepted that this knowledge is fundamental to tackle the ingenious ways that bacteria curtail the effectiveness of antimicrobial agents.Research on marine sponge-associated bacteria has increased exponentially in recent years, but this has focused predominantly on phylogeny and on the search for novel bioactive compounds (13,22). While much data are available on the microbial ecology of marine sponges (12, 22), comparatively little is known about the incidence and diversity of antibiotic resistance determinants and the often associated mobile elements among these bacteria.We have recently characterized the spore-forming population of the marine sponge Haliclona simulans, collected from Gurraig Sound, Kilkieran Bay, Galway, Ireland. One of the isolates was identified by partial 16S rRNA gene sequencing as a Bacillus sp., with 96% sequence identity to its closest relative, Bacillus lehensis. Bacillus sp. strain #24 was unable to grow in SYP (12) and LB media unless supplemented with either 50% artificial seawater (12) or 2% NaCl and was resistant to tetracycline and erythromycin (MICs of 75 g ml Ϫ1 and 3 mg ml Ϫ1 , respectively. MIC values reported in this study are defined as the minimal concentration of antibiotic able to inhibit the growth of the strains when spotted on Muller-Hinton medium plates containing increasing concentrations of the antibiotic). Screening of isolate #24 for plasmid DNA (6) was positive, with multiple bands visible on agarose gels. In order to ascertain its role in antibiotic resistance, total plasmid DNA purified from isolate #24 (QIAprep Spin miniprep kit optimized for Bacillus; Qiagen GmbH) was used to transform Bacillus subtilis 168 competent cells. This strain is sensitive to erythromycin and displays only a very low level of resistance to tetracycline (2 g ml Ϫ1 ) associated with the presence of a chromosomally carried tet(L) gene (9, 20). B. subtilis 168 is easily differentiated from isolate #24 by colony morphology (Fig. 1). A large number of transformants were obtained on LB medium-tetracycline plates, while no colonies grew on those containing erythromycin (both at 5 g ml Ϫ1 ). This implied that tetracycline resistance was associated with plasmid DNA, while erythromycin resistance was probably chromosomally encoded. A...

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“…While all three plasmids were 5,031 bp in size, pBSDMV46A had a single-base difference from pSU1 and pDMV2 (which were identical to each other). The pBSDMV46A, pSU1, and pDMV2 sequences were almost identical to those of three recently reported Tc r -encoding mobilizable plasmids, pMA67 (NC_010875) (39), pLS55 (NC_010375) (4), and pBHS24 (HM235948) (46). A detailed comparison of these plasmids, including pSU1, is available in reference 46.…”

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