Nanopore Sequencing and Bioinformatics for Rapidly Identifying Cultural Heritage Spoilage Microorganisms

Abstract

Microbiological methodologies allow understanding the causes that lead to the development of a certain microbial community colonizing an artistic surface, to characterize its composition and describe its role in the deterioration of the constituent materials. Metagenomics allows identifying microbial communities directly in their natural environments, bypassing the need for isolation and cultivation of individual species, thus providing a more comprehensive picture of the biodiversity present on a surface compared with standard cultivation methods. Furthermore, molecular analyses require small amounts of material, favoring the preservation of the artistic surface during sampling. Here, we verified the suitability of a protocol consisting in DNA extraction with micro-invasive sampling, using adhesive tape, PCR amplification with universal primers [bacteria (16S), fungi (ITS), and Viridiplantae (18S)], and amplicon sequencing by Oxford Nanopore Technologies (ONT) in the hypogeum of Basilica di San Nicola in Carcere Church (Rome, Italy). Sequence data were analyzed with a bioinformatic pipeline customized for pinpointing cultural heritage spoiling organisms, named "AmpLIcon SequencIng Analysis" (ALISIA). These data were integrated with traditional microbiology techniques that allowed the isolation of cultivable bacteria; three species were also characterized through their capability of biofilm formation and antibiotic resistance. Further, Fourier-transform infrared spectroscopy (FTIR) spectroscopy was performed to characterize the main products present on the masonry surface providing indications on the type of decay present. This novel biological workflow represents a powerful opportunity to investigate the microbial colonization of artistic surfaces aimed at implementing preservation strategies of cultural heritage from bio-spoilage.

The Microbiome of Leonardo da Vinci’s Drawings: A Bio-Archive of Their History

“…To date, the Nanopore sequencing technology has been scarcely applied in the field of cultural heritage, and only four studies have reported on the advantages of applying this state-of-the-art technology on valuable art objects. Three of these studies (Šoltys et al, 2019;Grottoli et al, 2020;Kisová et al, 2020) reported on the advantages of sequencing long DNA fragments when targeting ribosomal regions. However, all three studies were based on the target amplification approach that avoided inferring the relative proportion of the different phylogenetic groups.…”

The Bad and the Good—Microorganisms in Cultural Heritage Environments—An Update on Biodeterioration and Biotreatment Approaches

“…Selection of an appropriate methodology for microbiological research is usually based on conservation restrictions and study purpose, such as general contamination (biodeterioration and human health risk assessment) or targeted research on a given artwork/material [ 19 , 20 , 21 , 22 , 23 ]. For general contamination assessment, the air is the most popular study subject, therefore numerous sampling techniques have been developed with (i) impaction, (ii) filtration, and (iii) impingement as major methods [ 24 ].…”

“…When research on historic surfaces is conducted, only non-invasive or micro-invasive treatment methods can be applied using sterile scalpels, swabs, membrane filters or adhesive tapes [ 22 , 23 , 27 ]. However, it should be noted that the use of various sampling methods can provide different results.…”

“…Most commonly used short-read based technologies (<300 bp) enable reliable biodiversity investigation on the genus level at the maximum, while third-generation sequencing technologies make it possible to analyze the data on species or even the strain level due to longer sequencing reads [ 44 ]. Recent researches show that many species-level assignments could be done only with long-read sequences, thus indicating the importance of improved resolution for accurate assessment of degradation/pathogenicity potential of environmental samples, usually observed for certain species or strains, but not always on the genera level [ 23 , 45 ].…”

Natural sciences at the service of art and cultural heritage: an interdisciplinary area in development and important challenges

“…The practical application of this technology in the field of cultural heritage has been launched in the last two years and is still under development, but the few studies that exist so far on various materials, such as wax, stone, textiles and paintings on canvas-or paper-support, have already reported on the advantages of applying this cutting-edge technology to valuable art objects. Three of these studies, performed on a XVIII Century wax seal ( Soltys et al, 2019), the hypogeum of Basilica di San Nicola in Carcere Church in Rom (Grottoli et al, 2020), and on some XVII Century funeral textile items (Kisov a et al, 2020), have reported on the benefits of sequencing long DNA fragments when dealing with ribosomal regions. In other two studies, the Nanopore sequencing technology was applied for the first time together with a whole genome amplification protocol (WGA), either to make a rapid diagnosis of biological infection in canvas paintings of the XVIII and XIX Century (Piñar et al, 2020b) or to evaluate the complete microbiomes of some emblematic Leonardo da Vinci's drawings (Piñar et al, 2020c).…”