J. Ent. Acar. Res..indd F. PALLA Characterization of microbial communities in pest colonized books by molecular biology tools Abstract - This work presents the identification of bacteria and fungi colonies in insect infesting books, by cultural-independent methodologies based on molecu- lar biology techniques. Microbial genomic DNA extraction, in vitro amplification of specific target sequences by polymerase chain reactions (PCR), sequencing and sequence analysis were performed. These procedures minimized the samples amount, optimized the diagnostic studies on bacteria and fungi colonization and al- lowed the identification of many species also in complex microbial consortia. The molecular techniques for sure accomplish and integrate the microbiological stan- dard methods (in vitro culture) and morphological analyses (OM, SEM, CLSM), in order to understand the role of microorganisms in bio-deterioration of cultural assets. This monitoring is also indispensable to shed light on the risk for visitors and/or professionals to contract potential illnesses within indoor environments. Key words: genomic DNA, paper biodeterioration, PCR, termites infestation. INTRODUCTION Insects and microorganisms play a significant role in the deterioration of organic substrates, their distribution and development is closely related to environmental pa- rameters (temperature, relative humidity, water activity) and chemical-physical prop- erties of materials (Camuffo, 1998; Valentin, 2003). Particular environments, such as museum, archives, libraries, basements are very often colonized by insects and micro- organisms due to the favorable micro-climate, characterized by water condensation phenomena and poor air exchange (Singh et al., 1995; Gallo et al., 1999; Else et al., 2003; Valentin, 2003). There is a huge literature on insects and microbial communities related to biodegradation of historic collections made by organic materials. Specifi- cally, insects as Thysanura (Lepismatidae), Isoptera (Rhinotermitidae, Kalotermidae), Coleoptera (Anobiidae, Cerambycidae, Lictidae, Dermestidae, Ptnidae), bacteria and/ or fungal species belonging respectively to Bacillus, Micrococcus, Streptomyces, Ac- tinomycetes, and/or Penicillum, Cladosporium, Aspergillus, Alternaria, Trichoderma, Rhizopus have been reported (Florian, 1994; Rust et al., 1996; Valentin, 2003), but many others have still to be identified. In this study a wide microbial colonization was revealed on books pages, must J. Ent. Acarol. Res. Ser. II, 43 (2): 61-67 30 September 2011 likely correlated to the termite infestation (Reticulitermes lucifugus Rossi) recognized not only on the collection but even in the wooden cabinets where the books were stored (Not & Guarneri, 2003). Since termites metabolic products (TMP) represent an excel- lent substrate for microbial growth, we hypothesize a close relationship between the TMP and the complexity of microbial consortia and consequently the clear biodete- rioration of books. Moreover by trophallaxis termites can transfer symbiotic bacteria and protozoa in the surrounding environment, where are also able to release chemical compounds that causes physiological changes and behavior in other individuals, usu- ally of the same species (Chiappini et al., 2001). As previously described we applied different molecular technologies for the characterization of bacteria and cyanobacteria colonizing cultural assets (Gargano et al., 2009; Palla et al., 2010). Results described the identification of bacterial species onto organic substrates. MATERIALS AND METHODS Sampling Sampling on the surfaces of books pages was performed by fragments (20 x 40 mm) of sterile Nylon membrane (Hybond membrane, Amersham-Biosciences), as de- scribed by Palla et al. (2006). This procedure allowed us to isolate single bacterial and fungal colonies. Colonizing biocenosis were analyzed by Scanning Electron Microscopy and bacte- ria through molecular protocols. Microbial DNA extraction Bacteria colonies isolated on nutrient agar: each colony was dissolved in 20 μl of 1X T.E. (10 mM TRIS-HCl pH 7,5 / 1 mM EDTA) and lysed at 94°C per 2 min. Aliquots (0.2 g) were utilized for DNA extraction by using QIAamp DNA stool kit (Qiagen); the yield was approximately 500 ng DNA per gram of sample. In vitro amplification of bacteria target sequences (PCR reactions) The ribosomal intergenic spacer was performed the polymerose chain reaction (ITS-PCR) using the oligonucleotides ITS1 = 5’-gTCgTAACAAggTAgCCgTA-3’ and ITS2 = 5’-gCCAAggCATCCACC-3’, as primers (Cardinale et al., 2004). Genomic DNA extracted from single bacterial colonies (2 μl) or directly from TMP (100 ng) was utilized as template in each reaction. Reaction mixture (up to 50 μl) consisted of: 0.5 Units of Taq DNA Polymerase (Invitrogen) in MgCl2 and salt conditions suggested by the company. The target sequence was amplified for 35 cycles as follows: denaturation for 1 min at 94°C; annealing for 1 min at 50°C; extension for 1 min at 72°C. A final extension step (7 min at 72°C) was added in order to allow the full-elongation of PCR products. PCR reaction products were resolved by electrophoresis on 2% agarose gel stained with SYBR Safe DNA gel stain (Invitrogen). Journal of Entomological and Acarological Research, Ser. II, 43 (2), 201162 Sequencing of ITS amplified fragments ITS-PCR products were purified by Qia-quick PCR purification kit (Qiagen) and sequenced by MWG-Biotech Custom Sequencing Services (http://www.mwg-biotech. com). The research for homology (16S-23S-ITS rDNA) was performed by nucleotide- nucleotide BLAST analyzer (Altschul et al., 1997; Pearson et al., 1988). Scanning Electron Microscopy Samples from books or termites metabolic products (TMP) were coated with gold particles (thickness ≈ 13 nm) by Agar-Auto-Spotter-Coater (B7341) and observed un- der high vacuum Leica Cambridge-Leo 420. RESULTS F. Palla: Characterization of microbial communities by molecular biology tools Fig. 1 - Fragment of wood cabinet infested by R. lucifugus. Fig. 2 - Bacteria colonies (A, B, C) growth on Nylon membrane fragments on Nutrien agar plate, after incubation at 30°C per 24 hours. A single fungal colony (F) is also recognizable. 63 Fig. 3 - SEM micrographs of book fragments colonized by bacteria (left) and actinomyces (right). Fig. 4 - Fragments of Termites Metabolic Products (left) utilized for the extraction of microbial DNA (right), analysed on 0.8% agarose gel, stained by SYBR safe (Invitrogen). M = λ Hind III DNA (molecular weight marker). Bar = 0.5 cm. Journal of Entomological and Acarological Research, Ser. II, 43 (2), 201164 Biodeterioration by termites R. lucifugus was clearly shown in both wood cabinet (Fig. 1) and in most of the books where a noticeable microbial colonization was also identified by a multidisciplinary approach. Bacteria and fungi colonies were revealed by in vitro culture (Nutrien agar, Difco) incubating the Petri dish for 24 hours at 30°C (Fig. 2), and scanning electron microscopy (Fig. 3). Bacterial colonization was char- acterized by ITS-PCR reactions, using as template the genomic DNA obtained from single colonies or extracted from TMP fragments (Fig. 4). Analysis of ITS-PCR prod- ucts (Fig. 5) allowed to identify Arthrobacter nicotianae, Micrococcus luteus, Kocuria rosea and Curtobacterium flaccumfaciens as predominant species. DISCUSSION AND CONCLUSION In this work we applied non-destructive sampling procedures, combining SEM analysis with in vitro culture and molecular biology techniques in order to characterize microbial, and particularly bacteria colonizing historic book collections. Furthermore, the study point out the potential relationship between termites infestation and microbial colonization. It is to be underlined that M. luteus and K. rosea are able to produce lytic enzymes and pigmented compounds (Gallo et al., 1999; Valentin, 2003; Palla et al., 2007) and the high cellulose degradation activity of C. flaccumfaciens (Lednicka et al., 2000) represents, for this typologies of organic substrates, an high potential risk of biodegradation. In this paper we set up protocols to revealing the presence of bacterial consortia over books surfaces and o TMP, which represent an extraordinary source for microorganisms. Fig. 5 - Electrophoretic analysis of amplified ITS (16S-23S) bacterial region. M= 100 bp DNA Ladder (Promega). F. Palla: Characterization of microbial communities by molecular biology tools 65 We want also point out that bacteria and fungi colonizing indoor environment are able to damage artifacts and release toxins detrimental to human health (Peltola et al., 2001; Nilsson et al., 2004). Molecular technologies could be used for characterizing these dangerous microbes, in order to establish suitable strategies for conservation and fruition of cultural assets. Finally, we are setting up molecular protocols for fungal identification by using oligonucleotide primers specific for ITS regions or tubulin gene (Glass et al., 1995; Palla et al., 2009). ACKNOWLEDGEMENTS The author wishes to acknowledge the Centro Regionale Progettazione e Restauro della Regione Siciliana for the support throughout the project. The author acknowledge C. Di Liberto for SEM analysis. REFERENCES ALTSCHUL S.F., MADDEN T.L., SCHÄFFER A.A., ZHANG J., ZHANG Z., MILLER W., LIPMAN D.J., 1997 - Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Research, 25: 3389-3402. CAMUFFO D., 1998 - Microclimate for cultural heritage. Amsterdam and New York: Elsevier. CARDINALE M., BRUSETTI L., QUATRINI P., BORIN, S., PUGLIA A. M., RIZZI A., ZANARDINI E., SORLINI C., CORSELLI C., DAFFONCHIO D., 2004 - Comparison of different primer sets for use in au- tomated ribosomal intergenic spacer analysis of complex bacterial communities. Applied Environmental Microbiology, 70: 6147-6156. CHIAPPINI E., LIOTTA G., REGUZZI M.C., BATTISTI A., 2001 - Insetti e Restauro. Calderini Ed. agricole, Bologna. ELSE T.A., PANTLE C.R., AMY P.S., 2003 - Boundaries for biofilm formation: humidity and tem- perature. Applied Environmental Microbiology, 69: 5006-5010. FLORIAN M.L.E., 1994 - Heritage Eaters. Insects & Fungi in Heritage Collections. London: James & James. GALLO F., MAGGI O., PASQUARIELLO G., PERSIANI A.M., SCLOCCHI M.C., SCORRANO M., VALENTI P., 1999 - Aerobiological researches in book and archival heritage and graphic arts. In P. Tiano & C. Feroci (eds). International conference on microbiology and conservation: of Microbes and Art. Florence, 16-19 June 1999. CNR, Florence, Italy. GARGANO V., MANCUSO F.P., VITALE F., REALE S., CARACAPPA S., PALLA F., 2009 - La tecnologia del DNA-microarray per l’identificazione di specie microbiche su superfici e nell’aerosol di ambienti confinati. In: Sistemi Biologici e Beni Culturali, Convegno Nazionale AIAr, Orto Botanico - Palermo, 6-7 ottobre 2009, CRPR Sicilia - University of Palermo, Italy. GLASS L.N., DONALDSON G.C., 1995 - Development of primer sets designed for use with PCR to amplify conserved genes from filamentous Ascomycetes. Applied Environmental Microbi- ology, 61: 1323-1330. LEDNICKA D., MERGAERT J., CNOCKAERT M.C., SWINGS J., 2000 - Isolation and identification of cel- lulolytic bacteria involved in the degradation of natural cellulosic fibres. Systematic Applied Microbiology, 23: 292-299. Journal of Entomological and Acarological Research, Ser. II, 43 (2), 201166 NILSSON A., KIHLSTROM E., LAGESSON V., WESSEN B., LARSSON L., TAGESSON C., 2004 - Micro- organism and volatile organic compound airborne dust from damp residences. Indoor Air, 14: 74-82. NOT R., GUARNERI E., 2003 - Infestazione termitica nella Biblioteca comunale di Taormina (ME): proposte d’intervento. Atti della giornata studio: l’intervento di deacidificazione a libro in- tegro. Taormina (ME): 105-115. CRPR - Assessorato dei BBCC, Ambientali e P.I. Regione Siciliana. PALLA F., ANELLO L., MARINEO S., LOMBARDO G., 2006 - Characterization of bacterial community in indoor environment. In: Fort R., Alvarez De Buergo M., Gomez-Heras M. Vazquez-Cal- vo. Heritage, Weathering and Conservation. (1, pp. 361-365) Taylor & Francio, U.K. PALLA F., TARTAMELLA E., 2007 - Chromatic alteration on marble surfaces analysed by molecular biology tools. Conservation Sciences in Cultural Heritage, 7: 1-10. PALLA F., LANZA A., MANCUSO F.P., LOMBARDO G., SAITTA A., GARGANO M.L., 2009 - Definizione di un protocollo per la caratterizzazione morfologica e molecolare del genere Daldinia Ces. & De Not. (Ascomycota) in Sicilia. Congresso Nazionale della Società Botanica Italiana, 16-19 settembre 2009, University of Molise, Italy. PALLA F., BILLECI N., MANCUSO F.P., PELLEGRINO L., 2010 - Microscopy and molecular biology techniques to study biocenosis diversity in semi-confined environment. Conservation Sci- ence in Cultural Heritage, 10: 185-194. PEARSON W.R., LIPMAN D.J., 1988 - Improved tools for biological sequence comparison. Procee- dings of National Academy of Sciences - USA, 85: 2444-2448. PELTOLA J., ANDERSSON M.A., HAAHTELA T., MUSSALO-RAUHAMAA H., RAINEY F.A., KROPPENSTEDT M., SAMSON R.A., SALKINOJA-SALONEN S., 2001 - Toxic-metabolite-producing bacteria and fungus in an indoor environment. Applied Environmental Microbiology, 67: 3269-3274. RUST M., VINOD D., DRUZIK J., PRESSEUR F., 1996 – The feasability of using modified atmosphere to control insect pests in museum. Restaurator, 17: 43-60. SINGH A., GANGULI M., SING A.B., 1995 - Fungal spores are an important component of library air. Aerobiologia, 11: 231-237. VALENTIN N., 2003 - Microbial contamination in museum collections: Organic materials. In C. Saiz-Jimenez (ed.), Molecular Biology and Cultural Heritage: 85-91. Lisse/Abingdon/Ex- ton/Tokyo. A.A. Balkema Publishers. FRANCO PALLA, Dipartimento di Biologia Ambientale e Biodiversità, Università degli Studi di Palermo, via Archirafi 38, 90123 Palermo, Italy. E-mail: franco.palla@unipa.it F. Palla: Characterization of microbial communities by molecular biology tools 67