aroF and cm2: potential molecular markers for the detection of stone-inhabiting Actinobacteria on cultural heritage sites.

Tangible archeological sites and stone monuments are naturally decayed and deteriorated over time, providing substances that can sustain life, although they provide a complicated ecosystem characterized by low nutrition and desiccation. Stone-inhabiting bacteria (SIB) and especially members of the phylum Actinobacteria dominate such environments, particularly the members of the family Geodermatophilaceae. We used the published data of two confirmed SIB species to mine their genomes for specific molecular markers to rapidly survey the presence of SIB in cultural heritage material prior to further analysis. The search focused on the mycosporine-like amino acids (MAAs) synthesis pathway. MAAs are intracellular compounds biosynthesized by the shikimic acid pathway to synthesize aromatic amino acids and were found related to abiotic resistance features in microorganisms. Based on genome mining, the DAHP II (aroF) and a homolog of the Chorismate mutase gene (cm2) were found mostly in Actinobacteria and few other species. After calibration on five stone-inhabiting Actinobacteria (SIAb) species using conventional PCR, newly designed primers were successfully applied to environmental DNA extracted from two Egyptian pyramidal sites using a qPCR approach. This is the first report of aroF and cm2 as qPCR markers to detect SIAb from cultural heritage material prior to proceeding with further analysis (e.g., metagenomics and meta-barcoding analyses).

Occurrence of the black yeast Hortaea werneckii in the Mediterranean Sea.

The occurrence of cultivable fungi was investigated along the water column (25-2500 m depth) of four off-shore stations in the Mediterranean basin. An unexpected high abundance of fungi, accompanied by a scarce biodiversity, was observed up to 2500 m depth. The black yeast Hortaea werneckii, known to be one of the most salt tolerant eukaryotic organisms, was isolated for the first time from the Mediterranean Sea, and it was the dominant fungus present in seawater in almost all stations and depths, suggesting its ubiquitous distribution. Isolation of cultivable strains allowed their phylogenetic and taxonomic characterization, and demonstrated that almost all the retrieved fungal species should be considered of terrestrial origin, but well adapted to survive and reproduce at temperature and salinity conditions of the Mediterranean seawater.

Complex bacterial diversity in the white biofilms of the Catacombs of St. Callixtus in Rome evidenced by different investigation strategies.

Roman Catacombs are affected by different kinds of biofilms that were extensively investigated in the last 14 years. In particular, the areas far from the lamps are often covered by white biofilms of different extension, consistency and nature. The aim of this paper is to describe the profile of the microbial community present in two areas of the Ocean's Cubiculum (CSC13), characterized by similar alterations described as white biofilms, by using a multistep approach that included direct microscopy observations, culture-dependent and culture-independent methodologies through the extraction of DNA and RNA directly from the sampled areas. In addition to this, we extracted the DNA directly from the Petri dishes containing R2A and B4 media after incubation and growth of bacteria. Our results evidenced that a complex bacterial community (mainly constituted by filamentous Actinobacteria, as well as Firmicutes and Proteobacteria) colonizes the two different white biofilms, and its detection, quantitative and qualitative, could be revealed only by different approaches, each method giving different information that only partially overlap.

Description of Kribbella italica sp. nov., isolated from a Roman catacomb.

A novel actinobacterium, strain BC637(T), was isolated from a biodeteriogenic biofilm sample collected in 2009 in the Saint Callixstus Roman catacomb. The strain was found to belong to the genus Kribbella by analysis of the 16S rRNA gene. Phylogenetic analysis using the 16S rRNA gene and the gyrB, rpoB, relA, recA and atpD concatenated gene sequences showed that strain BC637(T) was most closely related to the type strains of Kribbella lupini and Kribbella endophytica. DNA-DNA hybridization experiments confirmed that strain BC637(T) is a genomic species that is distinct from its closest phylogenetic relatives, K. endophytica DSM 23718(T) (63 % DNA relatedness) and K. lupini LU14(T) (63 % DNA relatedness). Physiological comparisons showed that strain BC637(T) is phenotypically distinct from the type strains of K. endophytica and K. lupini. Thus, strain BC637(T) represents the type strain of a novel species, for which the name Kribella italica sp. nov. is proposed ( = DSM 28967(T) = NRRL B-59155(T)).

Kribbella albertanoniae sp. nov., isolated from a Roman catacomb, and emended description of the genus Kribbella.

A novel actinobacterium, strain BC640(T), was isolated from a biofilm sample collected in 2009 in the Saint Callistus Roman catacombs. Analysis of the 16S rRNA gene sequence showed that the strain belonged to the genus Kribbella. Phylogenetic analysis using the 16S rRNA gene and concatenated gyrB, rpoB, relA, recA and atpD gene sequences showed that strain BC640(T) was most closely related to the type strains of Kribbella yunnanensis and Kribbella sandramycini. Based on gyrB genetic distance analysis, strain BC640(T) was shown to be distinct from all Kribbella type strains. DNA-DNA hybridization experiments confirmed that strain BC640(T) represents a genomic species distinct from its closest phylogenetic relatives, K. yunnanensis DSM 15499(T) (53.5±7.8 % DNA relatedness) and K. sandramycini DSM 15626(T) (33.5±5.0 %). Physiological comparisons further showed that strain BC640(T) is phenotypically distinct from the type strains of K. yunnanensis and K. sandramycini. Strain BC640(T) ( = DSM 26744(T) = NRRL B-24917(T)) is thus presented as the type strain of a novel species, for which the name Kribbella albertanoniae sp. nov. is proposed.

Culturable and unculturable potential heterotrophic microbiological threats to the oldest pyramids of the Memphis necropolis, Egypt.

A large percentage of the world's tangible cultural heritage is made from stone; thus, it deteriorates due to physical, chemical, and/or biological factors. The current study explored the microbial community inhabiting two prehistoric sites with high cultural value in the Memphis necropolis of Egypt (Djoser and Lahun Pyramids) using amplicon-based metabarcoding and culture-dependent isolation methods. Samples were examined by epifluorescent microscopy for biological signs before environmental DNA extraction and in vitro cultivation. The metabarcoding analysis identified 644 bacterial species (452 genera) using the 16S rRNA and 204 fungal species (146 genera) using ITS. In comparison with the isolation approach, an additional 28 bacterial species (13 genera) and 34 fungal species (20 genera) were identified. A total of 19 bacterial and 16 fungal species were exclusively culture-dependent, while 92 bacterial and 122 fungal species were culture-independent. The most abundant stone-inhabiting bacteria in the current study were Blastococcus aggregatus, Blastococcus saxobsidens, and Blastococcus sp., among others. The most abundant rock-inhabiting fungi were Knufia karalitana and Pseudotaeniolina globosa, besides abundant unknown Sporormiaceae species. Based on previous reports, microorganisms associated with biodeterioration were detected on color-altered sites at both pyramids. These microorganisms are potentially dangerous as physical and chemical deterioration factors and require proper conservation plans from a microbiological perspective.

A Deep Insight into the Diversity of Microfungal Communities in Arctic and Antarctic Lakes.

We assessed fungal diversity in water and sediment samples obtained from five Arctic lakes in Ny-Ålesund (Svalbard Islands, High Arctic) and five Antarctic lakes on Livingston and Deception Islands (South Shetland Islands), using DNA metabarcoding. A total of 1,639,074 fungal DNA reads were detected and assigned to 5980 ASVs amplicon sequence variants (ASVs), with only 102 (1.7%) that were shared between the two Polar regions. For Arctic lakes, unknown fungal taxa dominated the sequence assemblages, suggesting the dominance of possibly undescribed fungi. The phylum Chytridiomycota was the most represented in the majority of Arctic and Antarctic samples, followed by Rozellomycota, Ascomycota, Basidiomycota, and the less frequent Monoblepharomycota, Aphelidiomycota, Mortierellomycota, Mucoromycota, and Neocallimastigomycota. At the genus level, the most abundant genera included psychrotolerant and cosmopolitan cold-adapted fungi including Alternaria, Cladosporium, Cadophora, Ulvella (Ascomycota), Leucosporidium, Vishniacozyma (Basidiomycota), and Betamyces (Chytridiomycota). The assemblages displayed high diversity and richness. The assigned diversity was composed mainly of taxa recognized as saprophytic fungi, followed by pathogenic and symbiotic fungi.

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.

Fungal outbreak in the Catacombs of SS. Marcellino and Pietro Rome (Italy): From diagnosis to an emergency treatment.

The present study reports a sudden fungal outbreak that occurred in the corridor near the entrance of the Catacombs of SS. Marcellino and Pietro in Rome (Italy) observed after 1 year of a restoration treatment that interested the walls of the entrance of the Catacombs and some artifacts placed in situ. The colonization was observed on the vault at the entrance and in correspondence with the restored marble pieces displayed on the left side of the corridor. No growth was observed on the right side where similarly treated marble slabs were placed. Samples taken in correspondence with fungal biofilm were analyzed through the combined use of microscopical, cultural, and molecular tools and showed that the vault and the left side of the corridor entrance were colonized by a complex fungal biofilm consisting mainly of Coniophora sp. and other genera, such as Hypomyces, Purpureocillium, Acremonium, Penicillium, and Alternaria, many of which are well known as responsible of biodeterioration of stone surfaces. Regarding the brown-rot basidiomycete Coniophora, it was able to form very large colonies on the substrata with a diameter of up to 57 cm. Although the direct observation under a light microscope evidenced the presence of abundant brown fungal conidia, several attempts to cultivate the microorganism failed, therefore only through DNA sequencing analyses, it was possible to identify and characterize this fungus. There is very little literature on the genus Coniophora which is reported as one of the causes of wet-rot decay of wood in buildings. A connection with calcium-containing materials such as bricks and mortars was demonstrated, but no data were available about the possible role of this species in the biodeterioration of stones. This study features the first finding of a strain related to the basidiomycetous genus of Coniophora in the order Boletales in association with evident phenomena of biodeterioration.

Design and Development of Fluorinated and Biocide-Free Sol-Gel Based Hybrid Functional Coatings for Anti-Biofouling/Foul-Release Activity.

Biofouling has destructive effects on shipping and leisure vessels, thus producing severe problems for marine and naval sectors due to corrosion with consequent elevated fuel consumption and higher maintenance costs. The development of anti-fouling or fouling release coatings creates deterrent surfaces that prevent the initial settlement of microorganisms. In this regard, new silica-based materials were prepared using two alkoxysilane cross-linkers containing epoxy and amine groups (i.e., 3-Glycidyloxypropyltrimethoxysilane and 3-aminopropyltriethoxysilane, respectively), in combination with two functional fluoro-silane (i.e., 3,3,3-trifluoropropyl-trimethoxysilane and glycidyl-2,2,3,3,4,4,5,5,6,6,7,7,8,8,9,9-hexadecafluorononylether) featuring well-known hydro repellent and anti-corrosion properties. As a matter of fact, the co-condensation of alkoxysilane featuring epoxide and amine ends, also mixed with two opportune long chain and short chain perfluorosilane precursors, allows getting stable amphiphilic, non-toxic, fouling release coatings. The sol-gel mixtures on coated glass slides were fully characterized by FT-IR spectroscopy, while the morphology was studied by scanning electron microscopy (SEM), and atomic force microscopy (AFM). The fouling release properties were evaluated through tests on treated glass slides in different microbial suspensions in seawater-based mediums and in seawater natural microcosms. The developed fluorinated coatings show suitable antimicrobial activities and low adhesive properties; no biocidal effects were observed for the microorganisms (bacteria).

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.

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.

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.

Whole Genome Sequencing and Comparative Genome Analysis of the Halotolerant Deep Sea Black Yeast Hortaea werneckii.

Hortaea werneckii, an extreme halotolerant black yeast in the order of Capnodiales, was recently isolated from different stations and depths in the Mediterranean Sea, where it was shown to be the dominant fungal species. In order to explore the genome characteristics of these Mediterranean isolates, we carried out a de-novo sequencing of the genome of one strain isolated at a depth of 3400 m (MC873) and a re-sequencing of one strain taken from a depth of 2500 m (MC848), whose genome was previously sequenced but was highly fragmented. A comparative phylogenomic analysis with other published H. werneckii genomes was also carried out to investigate the evolution of the strains from the deep sea in this environment. A high level of genome completeness was obtained for both genomes, for which genome duplication and an extensive level of heterozygosity (~4.6%) were observed, supporting the recent hypothesis that a genome duplication caused by intraspecific hybridization occurred in most H. werneckii strains. Phylogenetic analyses showed environmental and/or geographical specificity, suggesting a possible evolutionary adaptation of marine H. werneckii strains to the deep sea environment. We release high-quality genome assemblies from marine H. werneckii strains, which provides additional data for further genomics analysis, including niche adaptation, fitness and evolution studies.

Preparation and characterization of photocatalytic Gd-doped TiO2 nanoparticles for water treatment.

In recent years, the photocatalytic process by using TiO2 nanoparticles (NPs) has produced a great interest in wastewater treatment due to its interesting features such as low-cost, environmental compatibility, and especially capacity to eliminate persistent organic compounds as well as microorganisms in water. In the present work, the photocatalytic activity of Gd-doped TiO2 nanopowders, with different doping amount 0.1, 1, and 5 mol% synthesized by the sol-gel method, was studied under UV/Visible irradiation for water treatment application. The Gd-doped TiO2 nanoparticles were investigated for their photocatalytic degradation of methylene blue (MB) dye and antibacterial activities against two bacterial strains namely Stenotrophomonas maltophilia (S. maltophilia) and Micrococcus luteus (M. luteus). MB dye was used as a pollutant model to estimate reactive oxygen species (ROS) generation and to correlate killing action of nanoparticles with the generation of ROS. Scanning electron microscopy (SEM), energy-dispersive X-ray spectroscopy (EDS), X-ray diffraction (XRD), and Raman spectroscopy were used to characterize the as-synthesized nanomaterials. Photocatalytic, as well as antibacterial tests, showed that doping with an appropriate amount of Gd could reduce the radiative recombination process of photogenerated electron-hole pairs in TiO2 and induce a significant enhancement in photocatalytic and consequently antibacterial activity. The experimental sequence of bactericidal activity and photocatalytic degradation efficiency exhibited by the different gadolinium-doped nanoparticles was the following: 0.1 mol% Gd-doped TiO2 > 1 mol% Gd-doped TiO2 > 5 mol% Gd-doped TiO2 > pure titania.

The extremely halotolerant black yeast Hortaea werneckii - a model for intraspecific hybridization in clonal fungi.

The polymorphic black yeast Hortaea werneckii (Capnodiales, Ascomycota) is extremely halotolerant (growth from 0 to 30% [w/v] NaCl) and has been extensively studied as a model for halotolerance in Eukaryotes for over two decades. Its most frequent sources are hypersaline environments and adjacent sea-water habitats in temperate, subtropical and tropical climates. Although typically saprobic, H. werneckii can also act as a commensal coloniser on human skin, causing tinea nigra on hands and soles. Here, we report that addition of NaCl to culture media expands the growth range of H. werneckii to 37 °C, which explains its colonisation of human skin, with its increased salinity. The morphological and physiological plasticity/ versatility of H. werneckii indicate that a species complex might be involved. This was investigated in this polyphasic taxonomic analysis based on the global diversity of H. werneckii strains collected from hypersaline environments, and from humans and animals. Analysis of D1/D2domains of 28S and internal transcribed spacer rDNA revealed 10 and 17 genotypes, respectively, that were not always compliant. The genotypes have global distributions. Human and environmental strains with the same genotypes are intermingled. Due to the limited number of phylogenetically informative characters in the ribosomal DNA dataset, the partial genes encoding for ß-tubulin (BTB) and mini-chromosome maintenance protein (MCM7) were also sequenced. The use of these genes was hampered by ambiguous sequences obtained by Sanger sequencing, as a consequence of the diploid and highly heterozygous genome of many H. werneckii strains. Analysis of the BTB and MCM7 genes showed that in some cases two copies of the gene from the same genome are positioned in distant phylogenetic clusters of the intraspecific gene tree. Analysis of whole-genome sequences of selected H. werneckii strains generally confirmed the phylogenetic distances estimated on the basis of ribosomal genes, but also showed substantial reticulation within the phylogenetic history of the strains. This is in line with the hypothesis that the diploid genomes of H. werneckii were formed by hybridizations, which have sometimes occurred between relatively divergent strains.

Global Molecular Diversity of the Halotolerant Fungus Hortaea werneckii.

A global set of clinical and environmental strains of the halotolerant black yeast-like fungus Hortaea werneckii are analyzed by multilocus sequencing and AFLP, and physiological parameters are determined. Partial translation elongation factor 1-α proves to be suitable for typing because of the presence/absence of introns and also the presence of several SNPs. Local clonal expansion could be established by a combination of molecular methods, while the population from the Mediterranean Sea water also responds differently to combined temperature and salt stress. The species comprises molecular populations, which in part also differ physiologically allowing further diversification, but clinical strains did not deviate significantly from their environmental counterparts.

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.

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.