Bacteriocins are attracting increased attention as an alternative to classic antibiotics in the fight against infectious disease and multidrug resistant pathogens. Bacillus subtilis strain MMA7 isolated from the marine sponge Haliclona simulans displays a broad spectrum antimicrobial activity, which includes Gram-positive and Gram-negative pathogens, as well as several pathogenic Candida species. This activity is in part associated with a newly identified lantibiotic, herein named as subtilomycin. The proposed biosynthetic cluster is composed of six genes, including protein-coding genes for LanB-like dehydratase and LanC-like cyclase modification enzymes, characteristic of the class I lantibiotics. The subtilomycin biosynthetic cluster in B. subtilis strain MMA7 is found in place of the sporulation killing factor (skf) operon, reported in many B. subtilis isolates and involved in a bacterial cannibalistic behaviour intended to delay sporulation. The presence of the subtilomycin biosynthetic cluster appears to be widespread amongst B. subtilis strains isolated from different shallow and deep water marine sponges. Subtilomycin possesses several desirable industrial and pharmaceutical physicochemical properties, including activity over a wide pH range, thermal resistance and water solubility. Additionally, the production of the lantibiotic subtilomycin could be a desirable property should B. subtilis strain MMA7 be employed as a probiotic in aquaculture applications.

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...

Aims:  Despite the frequent isolation of endospore‐formers from marine sponges, little is known about the diversity and characterization of individual isolates. The main aims of this study were to isolate and characterize the spore‐forming bacteria from the marine sponge Haliclona simulans and to examine their potential as a source for bioactive compounds. Methods and Results:  A bank of presumptive aerobic spore‐forming bacteria was isolated from the marine sponge H. simulans. These represented c. 1% of the total culturable bacterial population. A subgroup of thirty isolates was characterized using morphological, phenotypical and phylogenetic analysis. A large diversity of endospore‐forming bacteria was present, with the thirty isolates being distributed through a variety of Bacillus and Paenibacillus species. These included ubiquitous species, such as B. subtilis, B. pumilus, B. licheniformis and B. cereus group, as well as species that are typically associated with marine habitats, such as B. aquimaris, B. algicola and B. hwajinpoensis. Two strains carried the aiiA gene that encodes a lactonase known to be able to disrupt quorum‐sensing mechanisms, and various isolates demonstrated protease activity and antimicrobial activity against different pathogenic indicator strains, including Clostridium perfringens, Bacillus cereus and Listeria monocytogenes. Conclusions:  The marine sponge H. simulans harbours a diverse collection of endospore‐forming bacteria, which produce proteases and antibiotics. This diversity appears to be overlooked by culture‐dependent and culture‐independent methods that do not specifically target sporeformers. Significance and Impact of Study:  Marine sponges are an as yet largely untapped and poorly understood source of endospore‐forming bacterial diversity with potential biotechnological, biopharmaceutical and probiotic applications. These results also indicate the importance of combining different methodologies for the comprehensive characterization of complex microbial populations such as those found in marine sponges.

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A better understanding of the origin and natural reservoirs of resistance determinants is fundamental to efficiently tackle antibiotic resistance. This paper reports the identification of a novel 5.8 kb erythromycin resistance plasmid, from Bacillus sp. HS24 isolated from the marine sponge Haliclona simulans. pBHS24B has a mosaic structure and carries the erythromycin resistance gene erm(T). This is the first report of an erythromycin resistance plasmid from a sponge associated bacteria and of the Erm(T) determinant in the genus Bacillus.