Discovery of novel marine enzymes for inhibition of biofilm formation of pathogen strains

Abstract

Microbial biofilms are unwanted assemblages in clinics and industries. Once established biofilms are difficult to treat and to remove and different approaches have been developed to address this problem. In this study, the focus was on the identification of novel anti-biofilm enzymes originating from the marine environment. To reach this target, enrichment cultures with five different coral species and water samples from a shark tank were prepared and supernatants were tested for their antibiofilm effects. The supernatant of enrichment cultures with the stony coral Montipora foliosa and with water from a shark tank (STW22) showed a clear and reproducible reduction of biofilms of the gram-negative pathogenic bacterium Stenotrophomonas maltophilia K279a by about 40 %. Confocal laser scanning microscopy and other test confirmed these findings. To further identify the responsible proteins, a metagenomic analysis of the microbial communities of the enrichment cultures was performed. This study led to the discovery of 83,679,656 for the M. foliosa and 87,355,173 total sequences for the STW22 sample. The metagenomes coded for 47,903 and 85,886 genes and proteins, respectively. In the data sets, a total of 2,643 and 4,858 potential antimicrobial enzymes were discovered for the M. foliosa and the STW22 samples, respectively. In the next step, the transcriptome of K279a biofilm grown cells was analyzed estimate the effect of the supernatants on biofilm growth. A clear stress response of the bacterial pathogen in response to the supernatants was observed. Many genes linked to metal resistance, antitoxins, transporter, and iron acquisition were strongly up-regulated indicating that the supernatants had profound effects on the metabolism of the bacterial pathogen. Further investigations of the supernatants via fast protein liquid chromatography (FPLC) technique identified a group of 338 potential hydrolases having impact on the bacterial pathogen. The specific analysis of two fractions and alignments with databases led to ColA a Bacillus cereus metalloprotease belonging to the MEROPS family M9. In biofilm prevention assays with K279a reduction of the biofilm density by these fractions was confirmed. In summary, enrichment cultures with marine material provided bioactive enzymes with strong antibiofilm activity. With the combination of metagenomic, transcriptomic, and proteomic approaches, a deep analysis of environmental samples and their effects on other pathogenic bacterial strains was successful. A foundation could be created for further investigations on anti-biofilm enzymes.