In silico design and experimental evaluation of a new RNA FISH probe for detecting Streptomyces spp. in cultural heritage assets. (P100)

Abstract

Microbial colonization and biodeterioration is a growing problem in the preservation of indoor and outdoor patrimonial assets in Cultural Heritage (CH) [1]. Streptomyces species are involved in the biodeterioration of cultural heritage (CH) materials [2,3]. Streptomyces species are Gram-positive aerobic chemoorganotrophic and filamentous bacteria of the Actinobacteria phylum, that grows in several environments, with a filamentous form similar to fungi [4,5]. In CH, these microorganisms are oftentimes involved in the biomineralization processes and associated with the precipitation of calcite and barite forming superficial veils or also the production of biopigments that stain the colonized surfaces [3]. Additionally, Streptomyces spp. can produce bioactive secondary metabolites with interesting biological properties, like antifungals, antivirals, antitumor, anti-hypertensives, immunosuppressants, and especially antibiotics [5,6]. In fact, Actinomycetes account for over two-thirds of all-natural antibiotics, with the Streptomyces genus accounting for about 75 % [6]. Due to its biodeteriogenic potential and ability to produce secondary metabolites with specific properties it is very useful to develop rapid tools that can be used to detected/identified Streptomyces spp. This study aimed to design rRNA-FISH target probes to Streptomyces spp. and experimentally evaluate their specificity/performance to detect/identify these bacteria. The probes were designed in silico by using DECIPHER program. The calculation of hybridization efficiency and specificity was performed with the mathFISH program and blast nucleotide, respectively. Assays were performed with isolates of target Streptomyces spp. The stringency assay of hybridization was performed by variation the formamide concentration in the hybridization and washing buffers. Following a procedure previously reported, RNA FISH experiments were performed with Cy3 labelled probes [4]. Epifluorescence microscopy and flow cytometry were used to evaluate the results. The highest theoretical in silico hybridization efficiency (99.93 %) and specificity were observed, indicating excellent performance. In the absence of formamide, the streptomyces-Cy3 probe had a similar intensity signal to the positive control, but lower percentage of fluorescent cells. It was also possible to understand that the presence of formamide did not affect the signal of the streptomyces-Cy3 specific probe. Concluding, our results conduce to a new RNA FISH probe that can be used in a simple and quick way for detecting Streptomyces spp. in heritage assets