Sponge Microbiota Are a Reservoir of Functional Antibiotic Resistance Genes

Abstract

Wide application of antibiotics has contributed to the evolution of multi-drug resistant human pathogens, resulting in poorer treatment outcomes for infections. In the marine environment, seawater samples have been investigated as a resistance reservoir; however, no studies have methodically examined sponges as a reservoir of antibiotic resistance. Sponges could be important in this respect because they often contain diverse microbial communities that have the capacity to produce bioactive metabolites. Here, we applied functional metagenomics to study the presence and diversity of functional resistance genes in the sponges Aplysina aerophoba, Petrosia ficiformis, and Corticium candelabrum. We obtained 37 insert sequences facilitating resistance to D-cycloserine (n = 6), gentamicin (n = 1), amikacin (n = 7), trimethoprim (n = 17), chloramphenicol (n = 1), rifampicin (n = 2) and ampicillin (n = 3). Fifteen of 37 inserts harbored resistance genes that shared <90% amino acid identity with known gene products, whereas on 13 inserts no resistance gene could be identified with high confidence, in which case we predicted resistance to be mainly mediated by antibiotic efflux. One marine-specific ampicillin-resistance-conferring β-lactamase was identified in the genus Pseudovibrio with 41% global amino acid identity to the closest β-lactamase with demonstrated functionality, and subsequently classified into a new family termed PSV. Taken together, our results show that sponge microbiota host diverse and novel resistance genes that may be harnessed by phylogenetically distinct bacteria.

Keywords: antibiotic resistance; functional metagenomics; microbiota; resistance gene; sponge.

Figure 1

Characterization of the 26 antibiotic resistance genes that were identified with high confidence (E < 1.0E-7). (A) Amino acid identity distribution of the resistance genes that were obtained from the library of 31 sponge bacteria with their best hit (bitscore sorted) in NCBI's database. (B) Amino acid identity distribution of the resistance genes that were obtained from libraries based on DNA from sponge tissue with their best hit (bitscore sorted) in NCBI's database. (C) The mechanisms of action of all 26 resistance genes. (D) The taxonomic assignments of the resistance genes that were obtained from the library of 31 sponge bacteria.

Figure 2

Maximum Likelihood Tree based on protein sequences of: the novel ß-lactamase (bla_PSV-1) discovered in Pseudovibrio ascidiaceicola DN64_1D03 (blue), the closest homologs of the novel ß-lactamase in the NCBI non-redundant protein sequences database (red), and the closest homologs of the novel ß-lactamase in the CBMAR database (green) (the two closest homologs were picked for each of the two closest families: bla_LEN and bla_SHV). In this figure, only the proteins in green and the novel ß-lactamase (bla_PSV-1) have demonstrated functionality. The tree was constructed in MEGA using 1000 iterations of bootstrapping. Bootstrap values <50 are not shown. The horizontal bar indicates the number of substitutions per site.