Isolation and identification of bacteria and fungi from ... - ictp

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C. Abrusci et al. / International Biodeterioration & Biodegradation 56 (2005) 58–68 63
formation (Jin et al., 2003), which has been associated
with biodeterioration processes (Azeredo and Oliveira,
2000). Bacillus spp.of course are ubiquitous, owing to
their wide range of physiological characteristics and
ability to degrade a wide range of substrates (Claus and
Berkeley, 1986), and the majority of them produce
endospores that are very resistant to environmental
extremes, antibiotics, disinfectants and other chemicals
and, are easily disseminated.
In the photographic film manufacturing process,
contamination by gelatin-degrading bacteria of the
genera Bacillus and Pseudomonas has been reported
(Stickley, 1986).More recently, it has been demonstrated
that many bacteria can colonize gelatin during
its production process.In addition to Bacillus, which are
predominant, non-sporulating bacterial strains identified
as diverse species in the genera Salmonella,
Kluyvera, Burkholderia, Pseudomonas, Yersinia, Brevundimonas,
Enterococcus, Staphylococcus and Streptococcus
have been found (De Clerck and De Vos, 2002), and
have the ability to liquefy gelatin. De Clerck et al.(2004)
reported that in semi-final gelatin extracts almost all
bacterial contaminants were Bacillus or other related
endospore formers (Brevibacillus, Geobacillus and Anoxybacillus),
although Enterobacter sakazakii and Staphylococcus;
S. epidermidis, S.lugdunensis and S.hominis
were also identified.These authors concluded that al
least some spores or vegetative cells were able to resist
the final UHT treatment used to prevent the potential
microbial contamination during gelatin manufacture.
Finally, the other important aspect is the presence of
bacteria in indoor environments, including archives.It is
known that vegetative cells or spores of endosporeforming
bacteria are common contaminants in indoor
environments, where other genera of bacteria, such as
Kocuria, Micrococcus, Staphylococcus, Aeromonas and
Pseudomonas have been isolated most frequently, at
least in Central and Eastern European countries (Gorny
and Dutkiewicz, 2002).
It can therefore be concluded that some cinematographic
films could have been contaminated during
manufacture with vegetative cells and especially spores
of bacteria that perhaps survive the manufacturing
process.Also, since some species of Staphylococci are
shed by humans, contamination may be a consequence
of handling during film copying or during maintenance
in the archives.Finally, airborne spores or vegetative
cells in indoor environments, including the archives are
possible contaminants.
3.2. Characterization and identification of filamentous
fungi and yeasts
A total of 17 strains of filamentous fungi and one
yeast were isolated from the films (Table 1).The most
frequent genera isolated (Fig.2) were Alternaria
(O’Donnell, 1993) Aspergillus (Klich, 2001) and Penicillium.
Other genera observed were Cladosporium,
Mucor, Trichoderma and Phoma (Fig.2).
Comparison of the ITS rDNA sequences of our
strains with the sequences stored in GenBank or in the
proprietary fungal DNA databases showed that the
strain HGC3N was 98% similar to Phoma glomerata
(AY183371); HGC2 was 100% similar to Trichoderma
longibranchiatum CBS446.95 (AY328039); HB1 and
HB41N were 100% similar to Alternaria alternata
isolate wb571 (AF455406); HB3A 100% similar to
Cladosporium cladosporioides strain ATCC 200941
(AF393691); and HM4 100% similar to Mucor racemosus
CBS 260.68 (AY213659).
When the two isolates corresponding to A. alternata
and the seven P. chrysogenum isolates were examined
using the microsatellite-primed PCR fingerprinting
technique (Bills et al., 1999), the A. alternata strains
had the same fingerprint (Fig.3) suggesting that they
were identical, so the cinematographic films B1 and B4
were contaminated with the same fungal strain.The
P. chrysogenum strains were very similar, although the
profile was only identical in the case of samples B6 and
B7 from the Barcelona archive indicating a common
source of P. chrysogenum contamination for the CTA
and CN films.
The most frequent fungal contaminants of the films
were airborne and soil micromycetes which have a
cosmopolitan distribution and can colonize many kinds
of surfaces (Florian and Manning, 2000; Lugauskas
et al., 2003). A. alternata is well adapted to cold
conditions, with a minimum growth temperature ranging
from 51 to 0 1C (Domsch et al., 1980). Penicillium
and Aspergillus spp.are ubiquitous taxa that can
produce numerous mitospores, or conidia, which are
easily dispersed by the air.Microorganisms associated
with the airborne route of exposure and indoor
environment may produce adverse human health effects
and therefore, they should be controlled (Gorny and
Dutkiewicz, 2002). Penicillium, Aspergillus spp.and
certain species of Alternaria are usually found as
contaminants or biodeterioration agents in many
different habitats and materials, including those considered
as representative of historical and cultural
heritage.As already mentioned, the only previous study
about airborne contamination and cinematographic
films stored in archives was undertaken in the Slovak
Republic (Opela, 1992), where in the summer months,
the airborne concentration increased remarkably.Also,
the microbial diversity in the air of the archives was
higher than on the stored cinematographic films.In
both, the taxa more frequently isolated were species of
Aspergillus, Penicillium and Cladosporium. At least six
species of filamentous fungi were isolated from the films,
including A. versicolor, C. cladosporioides, P. frequentens,
P. lanosum, T. viride and Mucor, all of which have