Microbiome analysis in Lascaux Cave in relation to black stain alterations of rock surfaces and collembola.

Anthropization of Palaeolithic caves open for tourism may favour collembola invasion and result in the formation of black stains attributed to pigmented fungi. However, ecological processes underpinning black stain formation are not fully understood. Here, we tested the hypotheses that black stains from the Apse room of Lascaux Cave display a specific microbiota enriched in pigmented fungi, and that collembola thriving on the stains have the potential to consume and disseminate these black fungi. Metabarcoding showed that the microbiota of black stains and neighbouring unstained parts strongly differed, with in black stains a higher prevalence of Ochroconis and other pigmented fungi and the strong regression of Pseudomonas bacteria (whose isolates inhibited in vitro the growth of pigmented fungi). Isotopic analyses indicated that Folsomia candida collembola thriving on stains could feed on black stain in situ and assimilate the pigmented fungi they were fed with in vitro. They could carry these fungi and disseminate them when tested with complex black stains from Lascaux. This shows that black stain formation is linked to the development of pigmented fungi, which coincides with the elimination of antagonistic pseudomonads, and points towards a key role of F. candida collembola in the dynamics of pigmented fungi.

Microbiome Analysis of New, Insidious Cave Wall Alterations in the Apse of Lascaux Cave.

Lascaux Cave is a UNESCO site that was closed to the public following wall surface alterations. Most black stains that had formed on wall surface are stable or receding, but a new type of alteration visually quite different (termed dark zones) developed in Lascaux's Apse room in the last 15 years. Here, we tested the hypothesis that dark zones displayed a different microbial community than black stains previously documented in the same room, using metabarcoding (MiSeq sequencing). Indeed, dark zones, black stains and neighboring unstained parts displayed distinct microbial communities. However, similarly to what was observed in black stains, pigmented fungi such as Ochroconis (now Scolecobasidium) were more abundant and the bacteria Pseudomonas less abundant in dark zones than in unstained parts. The collembola Folsomia candida, which can disseminate microorganisms involved in black stain development, was also present on dark zones. Illumina sequencing evidenced Ochroconis (Scolecobasidium) in all collembola samples from dark zones, as in collembola from black stains. This study shows that the microbial properties of dark zones are peculiar, yet dark zones display a number of microbial resemblances with black stains, which suggests a possible role of collembola in promoting these two types of microbial alterations on wall surfaces.

Comparison of Actinobacteria communities from human-impacted and pristine karst caves.

Actinobacteria are important cave inhabitants, but knowledge of how anthropization and anthropization-related visual marks affect this community on cave walls is lacking. We compared Actinobacteria communities among four French limestone caves (Mouflon, Reille, Rouffignac, and Lascaux) ranging from pristine to anthropized, and within Lascaux Cave between marked (wall visual marks) and unmarked areas in different rooms (Sas-1, Passage, Apse, and Diaclase). In addition to the 16S rRNA gene marker, 441 bp fragments of the hsp65 gene were used and an hsp65-related taxonomic database was constructed for the identification of Actinobacteria to the species level by Illumina-MiSeq analysis. The hsp65 marker revealed higher resolution for species and higher richness (99% operational taxonomic units cutoff) versus the 16S rRNA gene; however, more taxa were identified at higher taxonomic ranks. Actinobacteria communities varied between Mouflon and Reille caves (both pristine), and Rouffignac and Lascaux (both anthropized). Rouffignac displayed high diversity of Nocardia, suggesting human inputs, and Lascaux exhibited high Mycobacterium relative abundance, whereas Gaiellales were typical in pristine caves and the Diaclase (least affected area of Lascaux Cave). Within Lascaux, Pseudonocardiaceae dominated on unmarked walls and Streptomycetaceae (especially Streptomyces mirabilis) on marked walls, indicating a possible role in mark formation. A new taxonomic database  was developed. Although not all Actinobacteria species were represented, the use of the hsp65 marker enabled species-level variations of the Actinobacteria community to be documented based on the extent of anthropogenic pressure. This approach proved effective when comparing different limestone caves or specific conditions within one cave.

Microbial ecology of tourist Paleolithic caves.

Microorganisms colonize caves extensively, and in caves open for tourism they may cause alterations on wall surfaces. This is a major concern in caves displaying Paleolithic art, which is usually fragile and may be irremediably damaged by microbial alterations. Therefore, many caves were closed for preservation purposes, e.g. Lascaux (France), Altamira (Spain), while others were never opened to the public to avoid microbial contamination, e.g. Chauvet Cave (France), etc. The recent development of high-throughput sequencing technologies allowed several descriptions of cave microbial diversity and prompted the writing of this review, which focuses on the cave microbiome for the three domains of life (Bacteria, Archaea, microeukaryotes), the impact of tourism-related anthropization on microorganisms in Paleolithic caves, and the development of microbial alterations on the walls of these caves. This review shows that the microbial phyla prevalent in pristine caves are similar to those evidenced in water, soil, plant and metazoan microbiomes, but specificities at lower taxonomic levels remain to be clarified. Most of the data relates to Bacteria and Fungi, while other microeukaryotes and Archaea are poorly documented. Tourism may cause shifts in the microbiota of Paleolithic caves, but larger-scale investigation are required as these shifts may differ from one cave to the next. Finally, different types of alterations can occur in caves, especially in Paleolithic caves. Many microorganisms potentially involved have been identified, but diversity analyses of these alterations have not always included a comparison with neighboring unaltered zones as controls, making such associations uncertain. It is expected that omics technologies will also allow a better understanding of the functional diversities of the cave microbiome. This will be needed to decipher microbiome dynamics in response to touristic frequentation, to guide cave management, and to identify the most appropriate reclamation approaches to mitigate microbial alterations in tourist Paleolithic caves.

Anthropization level of Lascaux Cave microbiome shown by regional-scale comparisons of pristine and anthropized caves.

Limestone areas across the world develop karstic caves, which are populated by a wide range of macro- and microorganisms. Many of these caves display Paleolithic art or outstanding speleothems, and in the last century they have been subjected to anthropization due to touristic management and intense human frequentation. Despite their cultural importance and associated conservation issues, the impact of anthropization on cave biodiversity is not known. Here, we show that anthropization is associated with specific cave biota modifications. We compared diversity in four pristine caves, four anthropized show caves, and the iconic Lascaux Cave with even stronger anthropization. The predominant microbial higher taxa were the same in all caves, but the most anthropized cave (Lascaux) was unique as it differed from the eight others by a higher proportion of Bacteroidetes bacteria and the absence of Euryarchaeota and Woesearchaeota archaea. Anthropization resulted in lower diversity and altered community structure for bacteria and archaea on cave walls, especially in Lascaux, but with a more limited effect on microeukaryotes and arthropods. Our findings fill a key gap in our understanding of the response of karstic communities to anthropization, by revealing that tourism-related anthropization impacts on the prokaryotic microbiome rather than on eukaryotic residents, and that it shapes cave biota irrespective of cave natural features.

Rock substrate rather than black stain alterations drives microbial community structure in the passage of Lascaux Cave.

BACKGROUND: The World-famous UNESCO heritage from the Paleolithic human society, Lascaux Cave (France), has endeavored intense microclimatic perturbations, in part due to high touristic pressure. These perturbations have resulted in numerous disturbances of the cave ecosystem, including on its microbial compartment, which resulted in the formation of black stains especially on the rock faces of the passage. We investigated the cave microbiome in this part of Lascaux by sampling three mineral substrates (soil, banks, and inclined planes) on and outside stains to assess current cave microbial assemblage and explore the possibility that pigmented microorganisms involved in stain development occur as microbial consortia. METHODS: Microbial abundance and diversity were assessed by means of quantitative PCR and high-throughput sequencing (Illumina MiSeq) of several DNA and cDNA taxonomic markers. Five sampling campaigns were carried out during winter and summer to embrace potential seasonal effect in this somewhat stable environment (based on measurements of temperature and CO2 concentration). RESULTS: While the season or type of mineral substrate did not affect the abundances of bacteria and micro-eukaryotes on or outside stains, mineral substrate rather than stain presence appears to be the most significant factor determining microbial diversity and structuring microbial community, regardless of whether DNA or cDNA markers were considered. A phylogenetic signal was also detected in relation to substrate types, presence of stains but not with season among the OTUs common to the three substrates. Co-occurrence network analyses showed that most bacterial and fungal interactions were positive regardless of the factor tested (season, substrate, or stain), but these networks varied according to ecological conditions and time. Microorganisms known to harbor pigmentation ability were well established inside but also outside black stains, which may be prerequisite for subsequent stain formation. CONCLUSIONS: This first high throughput sequencing performed in Lascaux Cave showed that black stains were secondary to mineral substrate in determining microbiome community structure, regardless of whether total or transcriptionally active bacterial and micro-eukaryotic communities were considered. These results revealed the potential for new stain formation and highlight the need for careful microbiome management to avoid further cave wall degradation.

[New forms of health care coordination in geriatrics]. / Nouvelles formes de coordination paramédicale en gériatrie.

The global care of elderly patients leads to new forms of coordination between allied healthcare professionals. They are based on completely new ethical issues relating to the responsibility of all the healthcare professionals involved. These new practices call for the mobilisation of new skills, the development of new teaching and research into interprofessional collaboration.

Occurrence of multi-antibiotic resistant Pseudomonas spp. in drinking water produced from karstic hydrosystems.

Aquatic environments could play a role in the spread of antibiotic resistance genes by enabling antibiotic-resistant bacteria transferred through wastewater inputs to connect with autochthonous bacteria. Consequently, drinking water could be a potential pathway to humans and animals for antibiotic resistance genes. The aim of this study was to investigate occurrences of Escherichia coli and Pseudomonas spp. in drinking water produced from a karst, a vulnerable aquifer with frequent increases in water turbidity after rainfall events and run-offs. Water samples were collected throughout the system from the karstic springs to the drinking water tap during three non-turbid periods and two turbid events. E. coli densities in the springs were 10- to 1000-fold higher during the turbid events than during the non-turbid periods, indicating that, with increased turbidity, surface water had entered the karstic system and contaminated the spring water. However, no E. coli were isolated in the drinking water. In contrast, Pseudomonas spp. were isolated from the drinking water only during turbid events, while the densities in the springs were from 10- to 100-fold higher than in the non-turbid periods. All the 580 Pseudomonas spp. isolates obtained from the sampling periods were resistant (to between 1 and 10 antibiotics), with similar resistance patterns. Among all the Pseudomonas isolated throughout the drinking water production system, between 32% and 86% carried the major resistance pattern: ticarcillin, ticarcillin-clavulanic acid, cefsulodin, and/or aztreonam, and/or sulfamethoxazol-trimethoprim, and/or fosfomycin. Finally, 8 Pseudomonas spp. isolates, related to the Pseudomonas putida and Pseudomonas fluorescens species, were isolated from the drinking water. Thus, Pseudomonas could be involved in the dissemination of antibiotic resistance via drinking water during critical periods.