Filomena De Leo

Sampling with adhesive tape strips: an easy and rapid method to monitor microbial colonization on monument surfaces

An easy and fast non-destructive method for sampling from monument and art object surfaces is proposed. The results obtained after sampling in regions of black spots and discoloration using adhesive tape strips showed that the method is useful for monitoring microbial colonization as well as for the identification of biodeteriogens. This technique is easy to apply, inexpensive and reproducible. The presence of fungi or algae on stone surfaces can be checked at an early stage of colonization or after cleaning procedures, without damage to the sampled surface. In addition, it is possible to obtain information on the morphology and taxonomy of microorganisms, and their relationships with the colonized material surfaces.

Microbial diversity in paleolithic caves: a study case on the phototrophic biofilms of the Cave of Bats (Zuheros, Spain)

The biological colonization of rocks in the Cave of Bats (Cueva de Los Murciélagos, Zuheros, Spain) was studied in order to reveal the diversity of microorganisms involved in the biofilm formation. The culturable, metabolically active fraction of biodeteriogens present on surfaces was investigated focusing on morphological, ultrastructural, and genetic features, and their presence related to the peculiar environmental conditions of the underground site. PCR-ITS analysis and 16S rDNA sequences were used to clusterize and characterize the isolated strains. The presence of bacterial taxa associated to the photosynthetic microflora and fungi within the biofilm contributed to clarify the relationships inside the microbial community and to explain the alteration observed at the different sites. These results will contribute to the application of more successful strategies for the preventive conservation of subterranean archaeological sites.

Chemoorganotrophic bacteria isolated from biodeteriorated surfaces in cave and catacombs

International Journal of Speleology 41(2) 125-136 Tampa, FL (USA) July 2012 Available online at scholarcommons.usf.edu/ijs/ & www.ijs.speleo.it International Journal of Speleology Official Journal of Union Internationale de Spéléologie Chemoorganotrophic bacteria isolated from biodeteriorated surfaces in cave and catacombs INTRODUCTION Any study of microbial communities colonizing biodeteriorated surfaces should involve a combination of several analytical techniques such as microscopy, culture techniques, biochemical tests and molecular tools, which are designed to give complementary results. Each individual technique while having its own limitations, contributes the necessary information to provide a better understanding of the microbial community as a whole and its role in the deterioration of inorganic and/or organic substrata (Urzì et al., 2003). Culture-based techniques are selective due to the limited choice of media used for the cultivation of microorganisms, and because the viable and culturable microflora (VCM) may be restricted to 1 to 5% of the whole population (Amann, 2000). However, the culture-based approach offers the possibility to isolate and thus analyze a great number of strains (Donachie et al.,2007). It is then possible to study the isolated strains, to cluster them into operational taxonomic units (OTUs), to compare different microbial communities on the basis of types of cultivable bacteria isolated in term of richness and the frequency of isolates, distribution or relative abundance of types. Furthermore, cultivation-based techniques in association with other complementary molecular techniques give a good idea of the “microorganisms in action” because, 1Department of Life Sciences, “M. Malpighi” University of Messina, Viale F. Stagno d’Alcontres 31, 98166 Messina, Italy; Corresponding author: Clara Urzì (clara.urzi@unime.it) 2Department of Physiology and Biochemistry, University of Malta, Biomedical Sciences Building, Msida, MSD2620, Malta. De Leo F., Iero A., Zammit G. and Urzì C. 2012. Chemoorganotrophic bacteria isolated from biodeteriorated surfaces in cave and catacombs. International Journal of Speleology, 41(2), 125-136 Tampa, FL (USA). ISSN 0392-6672. http://dx.doi.org/10.5038/1827-806X.41.2.1 The main objective of this work was the comparative analysis of a large number of bacterial strains isolated from biodeteriorated surfaces in three different sites, namely the catacombs of St. Callistus in Rome, Italy, the catacombs dedicated to St. Agatha in Rabat, Malta and the Cave of Bats in Zuheros, Spain. Our results showed that even considering only culturable chemoorganotrophic bacteria the variability is very high, reflecting the great variety of microhabitats present. Hence any strategies to prevent, control or eliminate the biofilm-embedded microbiota from an archeological surface should take into account a number of considerations as stipulated in our study.

Marine Antifouling for Underwater Archaeological Sites: TiO2 and Ag-Doped TiO2

Marine fouling plays a crucial role in the degradation of underwater archaeological sites. Limitation of fouling activity and its damages are one of the most critical issues for archaeologists and conservators. The common cleaning procedure, consisting in the manual removal of fouling, requires a continuous maintenance, while a proper inhibition of biological colonisation would provide a long-time protection against biofouling. On the other hand, the most used antifouling paints, especially for ship hulls, show considerable toxicity level. Since submerged archaeological sites are often included in environmental protected areas, more eco-friendly products must be used. We have explored the possibility to use titanium dioxide and Ag-doped titanium dioxide as antifouling agents. For this purpose, they have been synthetized by sol-gel method, and then XRD, XPS, and reflectance spectroscopy measurements have been carried out to gain structural information. The powders have been dispersed in a polymer and then applied to marble surface to evaluate the chromatic alteration induced by the treatments. By means of biological tests, it was possible to assess their behaviour as biofouling agents. Results show a decreasing of biofouling activity on treated stony surfaces.

Kribbella catacumbae sp. nov. and Kribbella sancticallisti sp. nov., isolated from whitish-grey patinas in the Catacombs of St Callistus in Rome, Italy

Several nocardioform actinomycetes were isolated from tufaceous surfaces with whitish-grey patinas in the catacombs of St Callistus in Rome, Italy. The morphology of the isolates and their chemotaxonomic characteristics such as LL-diaminopimelic acid in the cell-wall peptidoglycan, the major menaquinone of MK-9(H(4)), phosphatidylinositol, phosphatidylcholine, phosphatidylglycerol and diphosphatidylglycerol as the major polar lipids, as well as complex cellular fatty acid patterns with anteiso-C(15:0), iso-C(16:0) and iso-C(15:0) as predominating components, were in agreement with their classification as members of the genus Kribbella by 16S rRNA gene sequence analysis. The isolates fell into two clusters as revealed by their ribosomal intergenic spacer, RiboPrint and cellular fatty acid patterns and by their MALDI-TOF mass spectra. The two clusters were represented by the strains BC631(T) and BC633(T) which shared 97.9% 16S rRNA gene sequence similarity. Strain BC631(T) represented a cluster of yellow pigmented strains and was a phylogenetic neighbour of Kribbella koreensis DSM 17837(T) (gene sequence similarity 98.0%), while strain BC633(T) was related to Kribbella flavida DSM 17836(T) and Kribbella karoonensis DSM 17344(T) (gene sequence similarities of 98.8% and 98.6%, respectively). Strains BC631(T) and BC633(T) could be differentiated from each other and from their closest phylogenetic neighbours by phenotypic characteristics and DNA-DNA relatedness values far below 70%. It is concluded that the two new strains represent two novel species, for which the names Kribbella catacumbae sp. nov. (type strain BC631(T)=DSM 19601(T)=JCM 14968(T)) and Kribbella sancticallisti sp. nov. (type strain BC633(T)=DSM 19602(T)=JCM 14969(T)) are proposed.

Pathogenic microorganisms carried by migratory birds passing through the territory of the island of Ustica, Sicily (Italy)

Several studies have shown that migratory birds play an important role in the ecology, circulation and dissemination of pathogenic organisms. In October 2006, a health status evaluation was performed on a large population of migratory birds passing through the territory of Ustica (Italy), an island located on the migration route of many species of birds to Africa, and various laboratory tests were conducted. In total, 218 faecal swabs and the internal organs of 21 subjects found dead in nets were collected for bacteriological and virological examination, including avian influenza and Newcastle disease. In addition, 19 pooled fresh faecal samples were collected for mycological examination. The bacteriological analysis produced 183 strains belonging to 28 different species of the Enterobacteriaceae family. In particular, Salmonella bongori, Yersinia enterocolitica and Klebsiella pneumonia strains were isolated. Almost all of the isolates were susceptible to sulphamethoxazole/trimethoprime (99.4%), cefotaxime (98.9%), nalidixic acid (96.7%), chloramphenicol (95.6%), and tetracycline (93.4%). Alternatively, many strains were resistant to ampicillin (42.6%), amoxicillin-clavulanic acid (42.6%), and streptomycin (43.7%). According to reverse transcriptase-polymerase chain reaction analysis, all of the samples were negative for the M gene of avian influenza virus. Moreover, isolation tests conducted on specific pathogen free eggs were negative for avian influenza and Newcastle disease. Several hyphomycetes and yeasts belonging to different genera were present in the specimens, and Cryptococcus neoformans was observed in a pooled faecal sample. Antibiotic resistance in wildlife can be monitored to evaluate the impact of anthropic pressure. Furthermore, migratory birds are potential reservoirs of pathogenic agents; thus, they can be regarded as sentinel species and used as environmental health indicators.

Airborne fungal spores colonising marbles exposed in the terrace of Messina Museum, Sicily

Studies were carried out on the air and on Carraramarble blocks located in the terrace of MessinaMuseum, in order to know the likelihood of airbornefungal spores coming into contact with and colonisingtridimensional objects. Our results showed there were not significantdifferences between airborne fungi circulating inspring and in autumn; Aspergillus, Penicillium,Fusarium, Alternaria, Cladosporium,Ulocladium, Aureobasidium, Phoma were themost common isolates. However, only few species wereable to settle on the marble surfaces as demonstratedby their isolation after 2 and 6 years of exposition.

Analysis and comparison of the microflora isolated from fresco surface and from surrounding air environment through molecular and biodegradative assays

The aim of this study was to find a correlation among the environmental isolated microflora and the fresco colonizators through the investigation of their biodegradative abilities and DNA characteristics. A molecular technique named RAMP (Random Amplified Microsatellite Polymorphisms) was utilized in order to analyze the DNA diversity of bacterial and fungal species isolated from fresco as well as from air samples. The RAMP-PCR results were combined with the screening of some biodegradative properties obtained through the use of specific agar plate assays detecting the proteolytic, solubilization and biomineralization abilities of the isolated microflora. This comparative analysis showed that only in few cases a direct link among the fresco and airborne isolates of specific microbial group existed. The investigation clearly evidenced that colonization of surface of Ladislav’s fresco occurred in different time and by different strains than those observed at the moment of sampling campaign. Furthermore, the microflora investigation permitted the identification of taxonomically interesting bacteria with particular biodegradative properties, which had been less studied until now.

Adherence ability of Candida africana: a comparative study with Candida albicans and Candida dubliniensis

In this study, we compared the adherence ability to human Hela cells and biofilm formation of three closely related Candida yeast. In our experiments, Candida africana showed poor adhesion ability to human Hela cells and the absence of biofilm formation on polyvinyl chloride strips. Conversely, Candida albicans and Candida dubliniensis formed mature biofilms and stable attachment to Hela cells. To our knowledge, this is the first comparative study reporting data on biofilm formation and adherence to human Hela cells by C. africana.

Effects of biocide treatments on the biofilm community in Domitilla's catacombs in Rome

Different types of biofilms are widespread on lithic faces of the Catacombs of Domitilla (Rome, Italy) due to the favorable microclimatic conditions (temperature, high RH% and low irradiance). The biofilm, once established, becomes particularly dangerous due to the coverage of valuable surfaces causing spoilage, softening of materials and mineral precipitation. It is common practice to treat these surfaces with biocides in order to eradicate the microorganisms present. The aim of the present research was to compare the changes occurring to the microbial community present in the biofilm in one site of the Catacombs of Domitilla (CD15) before and after a biocide treatment (a mixture of quaternary ammonium compounds and octylisothiazolone, OIT), applied for a one month period. A multistep approach was followed, based on microscopy, cultural methods and molecular techniques (f-ITS and 16S rDNA sequencing), for the phenotypic and genetic analysis of the culturable microbial population. Our results highlighted that the biocide treatments had little effect against cyanobacteria, while the bacterial population increased in numbers but changed drastically in terms of diversity. In fact, some bacteria proliferate at the expense of the organic matter released by dead microorganisms as demonstrated by laboratory tests. Further, our data describe how the microbial interaction can have different responses depending on the favorable conditions for one kind of microorganism in respect to the others. This study exemplifies the real risks of applying biocide treatments on complex microbial communities and pinpoints the necessity of subjecting treatments to monitoring and reassessment. Moreover, the work showed the potential of bacteria isolated after the treatment for use, under controlled conditions, in combatting unwanted microbial growth in that they possess a positive tropism toward stressed microorganisms and high hydrolytic enzymatic activity against cell components (e.g. cellulose, chitin and pectin). A tentative protocol is proposed.