Research in Microbiology 160 (2009) 38e40

www.elsevier.com/locate/resmic

Bacteria and free-living amoeba in the Lascaux Cave

Fabiola Bastian a, Claude Alabouvette a,*, Cesareo Saiz-Jimenez b
a

UMR INRA-Universite de Bourgogne, Microbiologie du Sol et de lEnvironnement, BP 86510, 21065 Dijon Cedex, France
b
Instituto de Recursos Naturales y Agrobiologia, CSIC, Apartado 1052, 41080 Sevilla, Spain
Received 26 September 2008; accepted 2 October 2008
Available online 17 October 2008

Abstract
The Lascaux Cave was discovered in 1940, and by 1960 it had received up to 1800 daily visitors. In 1963, the cave was closed and in 2001 it
was invaded by a Fusarium solani species complex which was treated for four years with benzalkonium chloride. However, Lascaux Cave
bacteria have only been poorly investigated. Here we show that the cave is now a reservoir of potential pathogenic bacteria and protozoa which
can be found in outbreaks linked to air-conditioning systems and cooling towers in community hospitals and public buildings.
2008 Elsevier Masson SAS. All rights reserved.
Keywords: Lascaux Cave; Benzalkonium chloride; Bacteria; Protozoa; Pathogens

1. Introduction
The conservation of Paleolithic paintings in caves is of
great interest due to their priceless cultural heritage. At
present, certain European caves are suffering from episodes of
biological contamination that threaten the conservation of the
paintings.
Recently, Zhou et al. [18] compared the bacterial diversity
in caves from Australia, China, Spain and USA. Phylogenetic
analyses revealed the presence of nine groups of the domain
Bacteria: Proteobacteria, Acidobacteria, Planctomycetes,
Chloroflexi, Bacteroidetes, Gemmatimonadetes, Nitrospirae,
Actinobacteria, Firmicutes, and some candidate divisions
(including OP3, GN08 and SBR1093). A general trend in
caves is that Proteobacteria is the largest group, representing
around 45% of DNA sequences, followed by Acidobacteria,
Actinobacteria and Firmicutes [18].
Lavoie and Northup [8] considered that Bacillus spp.,
Escherichia coli, and Staphylococcus aureus were indicators
of human impact in caves. Ikner et al. [5] reported that
diversity generally decreased as human impact increased and
that the degree of human impact was also reflected in the
* Corresponding author.
E-mail address: ala@dijon.inra.fr (C. Alabouvette).
0923-2508/$ - see front matter 2008 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.resmic.2008.10.001

phylogeny of the isolates recovered. Closest relatives for

isolates recovered from high-impact sites included Pseudomonas, Sphingomonas and Alcaligenes.
Data on pathogenic cave bacteria are scarce. A few reports
indicate that this should be taken into account. In fact, Aurantimonas altamirensis, a new species isolated from Altamira
Cave, was described by Jurado et al. [6]. Luong et al. [10]
reported the first cases of A. altamirensis recovered from
human clinical materials. Other pathogenic bacteria frequently
isolated from caves are E. coli, S. aureus [8], species of
Pseudomonas, Sphingomonas and Alcaligenes [5], Inquilinus
spp. (Leonila Laiz, data not published), etc.
The bacteria present in Lascaux Cave have rarely been
investigated and their ecology is unknown. Here we report
a study on the bacteria found in the cave and we discuss their
origin.
2. Materials and methods
We collected eleven samples between April 2006 and
January 2007 in different halls and galleries of Lascaux Cave.
The Painted Gallery, Great Hall of Bulls, Chamber of Felines
and Shaft of the Dead Man were selected as being representative of the different cave microenvironments. In the samples
we found white colonizations, black stains, areas not

F. Bastian et al. / Research in Microbiology 160 (2009) 38e40

apparently colonized and therefore considered as references,

and an area cleaned with biocides in 2004, without apparent
colonization [2].
Microbial DNA was extracted from each sampling using
a method adapted to small soil quantities. Details are reported
elsewhere [1,2]. GenBank accession numbers of sequences of
the representative pathogenic microorganisms reported in this
paper are listed in Table 1.
3. Results and discussion
Microorganisms in Lascaux Cave grow as biofilms which
consist of assemblages containing many species of bacteria,
fungi, protozoa and other microorganisms. From the eleven
samples, we retrieved a total of 696 bacterial and 607 eukaryota clones. Clone coverage indicated that the major parts of
the diversity were detected. Here we will discuss those of
relevance for human health.
The most intriguing fact was the wide representation of
phylotypes related to pathogenic bacteria and protozoa
(Table 1), which amounted to 45.1%. In fact, the two most
abundant phylotypes in the cave were Ralstonia mannitolilytica
and Ralstonia pickettii, followed by Pseudomonas saccharophila, E. coli, Achromobacter xylosoxidans, and Pseudomonas
lanceolata. Relatively less abundant phylotypes were Afipia
spp., Legionella spp., Stenotrophomonas maltophilia and
Aquicella spp. A low number of clones corresponded to Pseudomonas fluorescens, Inquilinus spp., Escherichia albertii,
Bordetella ansorpii, and Sighella sonnei. These bacteria are
commonly found in water distribution systems of hospitals,
water supplies and cooling systems of public buildings, homes,
and factories, as well as being associated with protozoa
[3,12,13,16,17].
Table 1
Most representative pathogenic phylotypes found in Lascaux Cave
Representative Most closely
sequence
related taxon
S5-78
S5-88
S27-149
S5-49
S1-2
S5-117
S2-61
S2-6
S4-121
S26-17
S3-35
S1-36
S4-143
S4-48
S15-8

Ralstonia
mannitolilytica
Ralstonia pickettii
Pseudomonas
saccharophila
Escherichia coli
Achromobacter
xylosoxidans
Pseudomonas
lanceolata
Afipia spp.
Legionella spp.
Stenotrophomonas
maltophilia
Aquicella spp.
Inquilinus spp.
Pseudomonas
fluorescens
Escherichia albertii
Bordetella ansorpii
Sighella sonnei

Accession
number

Similarity Total no.

(%)
of clones (%)

EU770633 99

77 (11.1)

EU770634 99
EU770637 99

47 (6.8)
35 (5.0)

EU770632 99
EU770631 99

26 (3.7)
26 (3.7)

EU770635 99

26 (3.7)

EU789899 99
EU789859 96
EU790045 99

22 (3.1)
19 (2.7)
13 (1.9)

EU790294 97
EU789932 98
EU789833 99

10 (1.4)
4 (0.6)
5 (0.7)

EU790059 98
EU789995 97
EU790222 99

2
1
1
314

(0.3)
(0.1)
(0.1)
(45.1)

39

Protozoa are nearly ubiquitous in aquatic environments,

water distribution systems and sewage water treatment
systems and they feed on microorganisms, including bacteria.
They were well represented in the cave, as shown by the 31
sequences detected belonging to the amoeba Nuclearia delicatula, mainly in the Painted Gallery and the Chamber of
Felines. Amoeba species are predominantly present at the
watereair interface where they are part of the biofilm
community [12]. Sequences of an endosymbiont of Acanthamoeba polyphaga, and of Candidatus Procabacter, an endosymbiont of Acanthamoeba sp. [4] were also identified.
Acanthamoebae and other free-living amoebae are potential
reservoirs and vectors for human infection, due in part to their
ubiquity in the environment, their resistant cyst stages [9] and
their potential to grow in the water supply, cooling and
humidification systems [11,13,17]. They share the habitat with
a variety of other bacteria and protozoa and are the reservoir of
several intracellular pathogens such as Legionella spp., Afipia
felis, R. pickettii, E. coli, Pseudomonas spp., Aquicella spp.,
etc. [3,17], which have also been found in the cave.
From the 22 Afipia sequences recorded, 20 corresponded to
Afipia genospecies 14 (95e99% similarity). This Afipia genospecies was also found in Altamira Cave (Leonila Laiz, data not
published) and has been shown to have similar axenic growth
requirements; it is able to parasitize free-living amoebae in the
same manner as Legionella [7]. Aquicella lusitana and Aquicella
siphonis sequences were found in the cave with similarities
ranging between 91 and 96%. Aquicella spp. are phylogenetically most closely related to the strictly intracellular uncultured
species of the genus Rickettsiella, which causes disease in
arthropods, and to the facultatively intracellular species of the
genus Legionella. Both Aquicella species proliferated in the
amoeba Hartmannella vermiformis. This suggests that Aquicella
spp. preferentially infect protozoans [13].
We found 19 sequences related to Legionella spp. grouped
into nine species, including the pathogenic Legionella pneumophila, Legionella jordanis and Legionella oakridgensis,
with similarities of 94, 88 and 86%, respectively. The 16S
rRNA similarities between members of the genus Legionella
were 90.2e99.1% [11]. It is suggested that these sequences
might arise from Legionella-like amoebic pathogens, and
further effort should be made to isolate and describe these
environmental Legionella-like species.
It should be noted that some samples showed the simultaneous
presence of sequences of Legionella, Afipia, and Aquicella (e.g.
samples from the Chamber of the Felines). Also in the Painted
Gallery and in the Great Hall of Bulls were found sequences from
endosymbionts of amoebae, together with an important number of
Afipia sequences. This supports the close relationship among these
bacteria and free-living amoebae in the entire cave.
What is the reason for the abundant presence of Ralstonia,
Pseudomonas, and other pathogenic bacteria? First at all, we
should keep in mind that an air-conditioning system was operating
inside the cave for decades. Then, in July 2001 the first evidence of
a Fusarium solani invasion and its associated bacterium Pseudomonas fluorescens appeared [1]. Shortly after this invasion, the first
treatments began, but due to rapid extension, intensive treatment

40

F. Bastian et al. / Research in Microbiology 160 (2009) 38e40

was begun in September based on BC solutions plus streptomycin

and polymyxin. The soils were treated with quicklime. Biocide
treatments, including quaternary ammonium derivatives, were
extensively carried out until 2004.
Quaternary ammonium compounds are widely used in
disinfection operations in the industry [15]. It is well known
that Gram-negative bacteria may adapt to survive against
higher concentrations of BC. This phenomenon is explained
by the protective role of biofilms, and bacteria grown in
a biofilm can be up to 1500 times more resistant to biocides
compared to the same bacteria growing in culture [14].
This study raises intriguing questions about the past,
present and future management of show caves. As a result of
years of BC treatments, the indigenous microbial communities
of Lascaux Cave have been replaced by microbial populations
selected for by biocide application. Lascaux Cave is now
a reservoir of potential pathogenic bacteria and protozoa,
which might be the result of many years of human impact (airconditioning system, biocide treatments). This resulted in
accumulation of all microorganisms found in outbreaks linked
to air-conditioning systems and cooling towers in community
hospitals and public buildings. To this was added a selection of
Ralstonia, Pseudomonas and amoebae hosting intracellular
bacteria resistant to BC.
In light of these data, it is necessary: i) to define the hazard
that represents, for staff and visitors, the presence of potential
pathogenic microorganisms and associated amoebae in a show
cave and to manage future visits if Lascaux Cave once again
allows visitors, taking into account this hazard; ii) to discuss
the suitability, in a cave, of both an air-conditioning system
and repeated BC treatments for combatting microbial invasion
that will not affect numerous bacteria and protozoa; and iii) to
clarify how this will interact with rock art conservation.
Acknowledgements
The first author and the research project MicrobiologyMicroclimate were supported by the Ministry of Culture and
Communication, France. Authors acknowledge support from
the Ministry of Culture and Communication and from project
CGL2006-07424/BOS, Spain. The collaboration of the Lascaux restoration team is highly appreciated. We thank Marisa
Chelius for valuable comments on the manuscript.
References
[1] Bastian, F., Alabouvette. Lights and shadows on the conservation of
a rock art cave: The case of Lascaux Cave. International Journal of
Speleology, in press.

[2] Bastian, F., Alabouvette, C., Saiz-Jimenez, C. The impact of arthropods

on fungal community structure in Lascaux Cave. Journal of Applied
Microbiology, in press.
[3] Greub, G., Raoult, D. (2004) Microorganisms resistant to free-living
amoebae. Clinical Microbiology Reviews 17, 413e433.
[4] Horn, M., Fritsche, T.R., Linner, T., Gautum, R.K., Harzenetter, M.D.,
Wagner, M. (2002) Obligate bacterial endosymbionts of Acanthamoeba
spp. related to the b-Proteobacteria: proposal of Candidatus Procabacter
acanthamoebae gen. nov., sp. nov. International Journal of Systematic
and Evolutionary Microbiology 52, 599e605.
[5] Ikner, L.A., Toomey, R.S., Nolan, G., Neilson, J.W., Pryor, B.M.,
Maier, R.M. (2007) Culturable microbial diversity and the impact of
tourism in Kartchner Caverns, Arizona. Microbial Ecology 53, 30e40.
[6] Jurado, V., Gonzalez, J.M., Laiz, L., Saiz-Jimenez, C. (2006) Aurantimonas altamirensis sp. nov., a member of the order Rhizobiales
isolated from Altamira Cave. International Journal of Systematic and
Evolutionary Microbiology 56, 2583e2585.
[7] La Scola, B., Mallet, M.N., Grimont, P.A.D., Raoult, D. (2002)
Description of Afipia birgiae sp. nov. and Afipia massiliensis sp. nov. and
recognition of Afipia felis genospecies A. International Journal of
Systematic and Evolutionary Microbiology 52, 1773e1782.
[8] Lavoie, K.H., Northup, D.E. (2006) Bacteria as indicators of human
impact in caves. In G.T. Rea (Ed.), 17th National Cave and Karst
Management Symposium, Proceedings (pp. 40e47). Albany, NY:
NICKMS Steering Committee.
[9] Lloyd, D., Turner, I.N.A., Khunkitti, H.W., Hann, A.C., Furr, J.R.,
Russell, A.D. (2001) Encystation in Acanthamoeba castellanii: development of biocide resistance. Journal of Eukaryotic Microbiology 48,
11e16.
[10] Luong, M.-L., Bekal, S., Vinh, D.C., Lauzon, D., Leung, V.,
Al-Rawahi, G.N., Ng, B., Tamara Burdz, T., Bernard, K. (2008) First
report and characterization of Aurantimonas altamirensis in clinical
samples. Journal of Clinical Microbiology 46, 2435e2437.
[11] Newsome, A.L., Scott, T.M., Benson, R.F., Fields, B.S. (1998) Isolation
of an amoeba naturally harboring a distinctive Legionella species.
Applied and Environmental Microbiology 64, 1688e1693.
[12] Preston, T.M. (2003) The water-air interface: a microhabitat for
amoebae. European Journal of Protistology 39, 385e389.
[13] Santos, P., Pinhal, I., Rainey, F.A., Empadinhas, N., Costa, J., Fields, B.,
Benson, R., Verssimo, A., da Costa, M.S. (2003) Gamma-Proteobacteria
Aquicella lusitana gen. nov., sp. nov., and Aquicella siphonis sp. nov.
infect protozoa and require activated charcoal for growth in laboratory
media. Applied and Environmental Microbiology 69, 6533e6540.
[14] Sauer, K., Rickard, A.H., Davies, D.G. (2007) Biofilms and biocomplexity. Microbe 2, 347e353.
[15] Sundheim, G., Langsrud, S., Heir, E., Holck, A.L., Bessems, E.,
Terpstra, P.M.J. (1998) Bacterial resistance to disinfectants containing
quaternary ammonium compounds. International Biodeterioration &
Biodegradation 41, 235e239.
[16] Vaneechoutte, M., De Baere, T., Wauters, G., Steyaert, S., Claeys, G.,
Vogelaers, D., Verschraegen, G. (2001) One case each of recurrent
meningitis and hemoperitoneum infection with Ralstonia mannitolityca.
Journal of Clinical Microbiology 39, 4588e4590.
[17] Winiecka-Krusnell, J., Linder, E. (2001) Bacterial infections of freeliving amoebae. Research in Microbiology 152, 613e619.
[18] Zhou, J.P., Gu, Y.Q., Zou, C.S., Mo, M.H. (2007) Phylogenetic diversity
of bacteria in an earth-vave in Guizhou Province, Southwest of China.
Journal of Microbiology 45, 105e112.