Disclosing a crypt: Microbial diversity and degradation activity of the microflora isolated from funeral clothes of Cardinal Peter Pázmány

Microbiological Research 168 (2013) 289–299

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Disclosing a crypt: Microbial diversity and degradation activity of the microflora isolated from funeral clothes of Cardinal Peter Pázmány Domenico Pangallo a,c,∗ , Lucia Kraková a , Katarína Chovanová a , Maria Buˇcková a , Andrea Puˇskarová a , ˇ Alexandra Simonoviˇ cová b a

Institute of Molecular Biology, Slovak Academy of Sciences, Bratislava, Slovakia Faculty of Natural Sciences, Commenius University, Department of Soil Science, Bratislava, Slovakia c Caravella, s.r.o., Bratislava, Slovakia b

a r t i c l e

i n f o

Article history: Received 3 October 2012 Received in revised form 1 December 2012 Accepted 2 December 2012 Available online 8 January 2013 Keywords: Crypt environment Textile materials Textile biodegradation Microbial characterization Keratin Fibroin

a b s t r a c t A crypt can be considered as a particular environment where different microbial communities contribute to decomposition of organic materials present inside during a long interval of time. The textile remains of the funeral clothes (biretta and tunic) of Cardinal Pázmány, an important historic figure dead in Bratislava the 19th March 1637, conserved in this kind of environment were subjected to microbial investigation. The sampling comprised three different approaches and the use of various kinds of cultivation media. Two different PCR-based clustering methods, f-ITS and f-CBH, were employed in order to select the bacterial and fungal microfloras which were identified in a second step by the 16S rRNA and ITS sequencing respectively. The isolated microflora was tested for its proteolytic, keratinolytic and cellulolytic activities and for its ability to grow on Fibroin agar medium. The combination of cultural, molecular and biodegradative assays was able to isolate and characterize a bacterial community composed mainly by members of the phyla Firmicutes and Actinobacteria. The fungal community appeared more diversified, together with several Penicillium and Aspergillus strains, members belonging to the species Beauveria bassiana, Eurotium cristatum, Xenochalara juniperi, Phialosimplex caninus and Myriodontium keratinophilum were isolated. Bacteria, especially the Bacillus members, showed their strong ability to degrade keratin and gelatin and a large portion of them was able to growth on Fibroin agar. The fungal isolates displayed a widespread cellulolytic activity and fibroin utilization, although they possessed a weaker and slower proteolytic and keratinolytic properties respect to bacterial counterpart. The present study can be considered perhaps as the first or among the few microbial investigations which treated the textile biodegradation from such unusual environment. © 2012 Elsevier GmbH. All rights reserved.

Introduction The tomb belonging to Cardinal Peter Pázmány (4th October 1570–19th March 1637) is situated in the crypt, near the main altar, inside the St. Martin Cathedral in Bratislava (Slovakia) (Hal’ko and Krampl 2011). Peter Pázmány was an important figure in the Austrian Monarchy during the XVII century. He was the soul of the Catholic Counter-Reformation in Hungary, in a period when the Hungarian Kingdom was occupied by the Ottoman Empire; in fact the capital was moved from Budapest to Bratislava (at that time Poszony or Pressburg), and when the protestant reformation gave origin to several insurrections against the Habsburg Monarchy.

∗ Corresponding author at: Institute of Molecular Biology, Slovak Academy of Sciences, Bratislava, Slovakia. E-mail address: [email protected] (D. Pangallo). 0944-5013/$ – see front matter © 2012 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.micres.2012.12.001

Cardinal Pázmány in 1619 founded a seminary for theological candidates at Nagyszombat (today: Trnava, Slovakia), then in 1635 he contributed for the foundation of the University in Nagyszombat (Trnava). Pázmány also built Jesuit colleges and schools at Pressburg (Bratislava) and Franciscan monasteries at Érsekújvár (now: Nové Zámky) and Körmöcbánya (now: Kremnica) (all in nowadays Slovakia; Fraknói 1886). By this short biography it is evident the importance of Cardinal Pázmány for Bratislava and for a big part of Slovakia. He died in Pressburg (Bratislava) in 1637 and he had asked in his last testament to be buried in the St. Martin Cathedral in unknown place. His grave was discovered for the first time during the cathedral reconstruction on 12th September 1859 by priest Ferdinand Knauz and others; the coffin was left opened and the crypt re-closed. In the beginning of the year 2010 the crypt was re-opened again for more accurate archeological and anthropological studies (Hal’ko and Krampl 2011). Our group was also involved in this investigation and our research was focused on the analysis of the microflora

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Fig. 1. Schematic plan of the presbytery and photos of the crypt of Cardinal Pázmány. (a) The plan by Dr. Michal Haviar showed the location of the crypt and the path for reaching it; (b) The hole found between the empty crypt and the Pázmány’s crypt, it is supposed that it was created during the first excursion organized by Ferdinand Knauz on 12th September 1859; (c) The Cardinal Pázmány crypt (dimension about 1.56 m × 2.89 m), on the left side there is the corpse of Cardinal Pázmány (died in 1637), on the right side there is the corpse of Archbishop Juraj Lippay (died in 1666). A desire of Archbishop Lippay was to be buried near Cardinal Pázmány.

present on the funeral clothes, biretta and tunic, of the famous Cardinal. It is already known that textile materials, such as wool, cotton, silk, can be deteriorated by different kinds of microorganisms which synergically contribute for the degradation of cellulose, keratin and fibroin (Szostak-Kotowa 2004). An interesting characteristic of our study is the environment of “conservation” of these kind of textile materials. Indeed, a crypt closed from more than three centuries represents a particular habitat for the isolated microflora. To our knowledge, until now, this kind of investigation was not performed, excluding some exception as for example Caretta and Piontelli (1998) where the fungal microflora isolated from the remains of a ninth century Longobard abbess was characterized. Some other literature record regards the microbial diversity of bulls of indulgence found inside a sepulcher in Spain (Jurado et al. 2010). Other recent publications paid attention on the deterioration and composition of textile materials in different archeological studies using chemical and

physical methods (Degano and Colombini 2009; Margariti et al. 2011). So, until now there are little information on microbial diversity and its textile degradation activities inside such particular environment as a tomb. In this study the microbial diversity of 9 different textile samples of the Cardinal’s funeral clothes was investigated by the combination of cultural and molecular methods. Moreover, the ability of the isolated microflora to degrade cellulose, gelatin, keratin and fibroin was also tested through specific assays. Materials and methods Sampling site The crypt is located down the presbytery of the Cathedral of Saint Martin in Bratislava (Fig. 1). In order to reach the Cardinal’s crypt it was necessary to go down through a hatchway placed on the floor of presbytery and to cross the crypt of one of the most influent

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Fig. 2. Pictures showing the different sampling places of the tunic and biretta. (a) The tunic samples from B1 to B4 are evidenced, (b) the samples B5 and B6 after bone’s removal, (c) the samples recovered from the biretta A1–A3.

noble family of that age, the Pálffy dynasty. There is a theory that perhaps the crypt of Cardinal Pázmány was financed by this noble family and its location, very closed to the main altar, was chosen in order to attest the great importance of the Cardinal (Krampl, personal communication). The samples were exclusively recovered from the textile materials (tunic and biretta) inside the crypt (Fig. 2). The biretta appeared well conserved especially the external part made by wool, the tunic made by silk and linen (mainly silk) was more decayed and in some part fragmented (Birkuˇsová, personal communication). Many samples were picked up when the corpse of the Cardinal was still dressed with the tunic. The samples B5 and B6 are an exception, indeed they were recovered after moving the bone’s remains and the cranium. The samples from biretta were taken also inside the crypt and in the internal part (silk part) of biretta were present also some hair. So, it is not possible to exclude that several members of the isolated microflora have a human origin. The corpse remains of Cardinal Pázmány included only the cranium and some bones from the limbs and vertebral column. Hair and pieces of his beard were also found. Sampling, microflora isolation and microscopic observation The samples were recovered from the funeral biretta (composed inside by silk and outside by wool) and from the tunic (made mainly

by silk and linen). In total nine samples, three from the biretta (A1–A3) and six from the tunic (B1–B6), were taken by swabs and by the method of adhesive tape (MAT; Urzi and De Leo 2001). The swabs were treated in two different ways: (i) direct inoculation (we called this approach Direct) on Skim Milk Casein agar (SC; casein 0.5 g, glucose 0.1 g, skim milk 0.1 g, yeast extract 0.25 g, agar 2 g, distilled water 100 ml) for bacteria and Malt Extract Agar (MEA) for fungi; (ii) were brought to the laboratory suspended in physiological solution (we called this approach Swab), decimal diluted and plated in specific agar dishes for the growth of bacteria and fungi. The media used for bacterial isolation were: SC, R2A (Oxoid, Basingstoke, UK), MYP Agar Base (specific for the isolation of Staphylococci and Bacillus species; Biomark, Pune, India), MacConkey agar (MAC; Biomark), Pseudomonas Agar F base (PSF; Merck, Darmstadt, Germany), Brain Heart Infusion (BHI; Merck). For the isolation of fungi the following media were used: Dichloran Rose Bengal Chloramphenicol (DRBC), Malt Extract Agar (MEA), and Sabouraud Dextrose Agar (SAB), these media were purchased from Hi-Media (Bombay, India). The adhesive tapes, in laboratory, were cut and spread on BHI and SAB agar plates suitable for the growth of bacteria and fungi respectively. All the bacterial and fungal plates were incubated at room temperature (22–26 ◦ C) for about five days–2 weeks. All agar media were supplemented with either actidione (50 mg l−1 ; Fluka, Seelze,

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Germany) or chloramphenicol (50 mg l−1 ; Sigma–Aldrich, Seelze, Germany) in order to avoid the growth of fungi and bacteria respectively. After the selection of pure colonies, the fungi were maintained on SAB slants; the bacteria on plates of Tryptone-Soya Agar (TSA; Oxoid). The surface of a portion of tunic was observed by scanning electron microscope (SEM; Jeol JSM 6610, Tokyo, Japan) using the facilities offered by the Institute of Materials and Machine Mechanics of the Slovak Academy of Sciences. The textile sample prior observation was sputtered with gold ions. DNA extraction and PCR selection of isolated microflora Fresh bacterial colonies were collected from the plates of TSA, and their DNA was isolated by chaotropic solid-phase extraction using the SiMax Genome DNA kit (SBS Genetech, Beijing, China) following the instructions of the producer for bacterial cells. The fungal strains were inoculated in Sabouraud broth at 28 ◦ C until growth; then they were separated from the broth by filtration through sterile filter paper, after which the DNA was extracted by the DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany), according to enclosed protocol for animal and vegetable tissue. The bacterial and fungal strains were selected by fluorescence ITS PCR (f-ITS) and fluorescence cellobiohydrolase PCR (f-CBH) respectively, according to Kraková et al. (2012). The two PCR-based fluorescent methods amplified the bacterial internal transcribed spacer (ITS) between 16S and 23S rRNA gene and the fungal cellobiohydrolase gene. Then the fluorescently labeled PCR products were separated by capillary electrophoresis. Both PCR selection methods permitted to cluster the isolates in order to reduce them for the consequent identification through sequencing.

The keratinolytic assay was performed using the Feather Broth: NH4 Cl 0.5 g, NaCl 0.5 g, K2 HPO4 0.3 g, MgCl 6H2 O 0.1 g, Yeast Extract 0.1 g, feathers, distilled water 1000 ml, pH 7.5. The medium without feathers was distributed in 100 ml flasks (each flask contained 50 ml of medium), after autoclaving few sterilized feathers were added in each flask. The flasks with Feather Broth were inoculated with bacterial and fungal isolates and incubated at 30 ◦ C for 3–14 days. The increasing turbidity of the broth indicated the feathers decay and therefore the keratinolytic ability of the inoculated microorganisms. The bacterial and fungal isolates were tested also for their ability to grow on Fibroin Agar. Fibrous fibroin (100 g) was prepared by heating the raw silk three times in 2 l of 8 M urea at 90 ◦ C for 20 min each time, followed by rinsing with distilled water and drying at 50 ◦ C for 6 h. Fibrous fibroin (10 g) was completely dissolved into 100 ml of 9 M LiBr. The solution was dialyzed against distilled water and the reconstituted fibroin was deposited at 4 ◦ C for 2 days. The Fibroin Agar was prepared suspending 1% of fibroin solution and 1.5% of agarose in 1000 ml of distilled water and then autoclaved. The bacterial strains were cultivated overnight in Nutrient Broth and then 10 ␮l of overnight culture were spot in the center of a Fibroin Agar plate. The fungal strains were grown in SAB slants, 5 ml of physiological solution was added in each tube, and then the slants were gentle shaken in order to suspend the fungal spores in solution. Ten ␮l of fungal suspension were spot in the center of a Fibroin Agar plate. This assay was performed in triplicate using 60 mm plates incubated at room temperature (22–26 ◦ C) until 10 days. Then, the colony diameter of growing bacteria was measured. For fungal strains the measurement of the diameter was not possible because the fungi tended to diffuse to the whole plate.

Microorganisms identification Results Fungal representatives (one or more) of each CBH fluorescence cluster were identified by the amplification of the internal transcribed spacer using the primers ITS1 and ITS4 (White et al. 1990). Bacterial isolates, selected on the basis of their f-ITS profiles, were identified by partially sequencing of 16S rDNA by the use of PCR method with primers 27F and 685R (Lane 1991). The fungal and bacterial PCR products were purified using ExoSAP-IT (Affymetrix, Cleveland, OH, USA) and sequenced for both strands by a commercial facility (Macrogen, Amsterdam, The Nederland). The sequences were compared directly with those in GenBank by BLAST search (http://blast.ncbi.nlm.nih.gov/Blast.cgi). The obtained sequences were deposited in the GenBank database under accession numbers JX869504–JX869572. Biodegradation assays The proteolytic activity of isolated microflora was tested by cultivation on Gelatin agar plates (R2A-Gel) which was prepared mixing autoclaved R2A Agar with 0.4% of sterilized gelatin (Sigma–Aldrich, Germany). After the growth of microorganism, in order to have a better visualization of the hydrolysis zone, a 10% tannin solution was flooded on the agar plates (Saran et al. 2007). This assay was performed in triplicate using 60 mm plates incubated at room temperature (22–26 ◦ C) generally from 3 until 7 days. The cellulolytic ability was checked using Czapek-Dox agar, without any carbon source, supplemented with 0.2% of hydroxyethylcellulose containing 13.5% (w/w) of covalently linked Ostazin Brilliant Red H-3B (OBR-HEC, Institute of Chemistry, Slovak Academy of Sciences, Bratislava, Slovakia) as suggested by Pangallo et al. (2007). The plates were incubated at room temperature (22–26 ◦ C) generally from 5 until 7 days.

Sampling and cultivation media The use of three different sampling approaches, two sampling by swab (direct and through the immersion of the swab in physiological solution) and by the method of adhesive tape, allowed the isolation of a larger amount of strains than if only one sampling approach was employed. This aspect is evident mainly for the isolation of fungal strains; in fact the 72% of fungal isolates (31 strains) were recovered by the adhesive tape method, the 16% (7 isolates) by the swab immersed in physiological solution and 12% of fungal strains (5) were isolated through the swab-direct approach. This data evidenced the MAT as the most effective sampling approach for the isolation of fungal strains. The swab approach demonstrated to be the most suitable sampling approach for bacteria, indeed the 78% (73 bacterial strains) of isolates were recovered by this method followed by the direct sampling with the 18% of isolates (17 bacteria) and by the MAT which was able to recover only the 4% of bacteria (4 strains). Sabouraud Dextrose Agar (SAB) permitted the isolation of the highest number of fungal strains, 34 over 43 isolates, but it is necessary to say that this agar was the only medium combined with the MAT sampling. Analogous situation was recorded by Skim Milk Casein Agar (SC), 36 bacterial strains were isolated using this medium, and it was the only one combined with the swab-direct sampling. If only the swab sampling approach is considered the SC medium resulted the most effective recovering 19 bacterial strains, followed by the PSF agar with 17 isolates, the R2A agar with 16, MacConkey agar with 9 isolates and MYP and BHI agar each with 6 isolated bacterial strains (Table 3).

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Table 1 Fluorescent ITS profiles and 16S rDNA sequences similarity of bacterial strains isolated from Cardinal’s clothes. f-ITS profiles (bp)

Number of isolates from funeral clothes

Number of cluster bacterial representatives sequenced

Identification on the basis of highest 16S rRNA similarity score

Ab (348; 441; 534)

27

10

Bb (216; 223; 232; 236; 245; 262) Cb (223; 242; 262)

2 8

1 5

Db (421; 430; 462; 469; 510)

5

2

9 2 6 1 2 16

3 1 3 1 1 8

1 1 1 5 1 1 1 4 1

1 1 1 2 1 1 1 2 1

Staphylococcus epidermidis AB617572 – 100%; EF522128 – 100%; FJ976549 – 100%; HQ203080 – 100% Bacillus megaterium GU124692 – 100% Bacillus sp. HQ711447 – 100%; HM567153 – 100%; HM567128 – 100% Staphylococcus pasteuri EF127830 – 100%; FR839669 – 100% Bacillus sp. GU595372 – 100% Bacillus simplex HQ284942 – 100% Bacillus aryabhattai GU563347 – 100% Micrococcus sp. HQ188562 – 99% Bacillus muralis GQ844961 – 100% Bacillus megaterium HQ842804 – 100%; CP001983 – 100%; HQ683934 – 100% Acinetobacter schindleri GU339299 – 100% Rathayibacter sp. DQ358658 – 100% Arthrobacter sp EF540497 – 100% Rothia terrae NR 043968 – 100% Staphylococcus warneri L37603 – 100% Staphylococcus capitis FJ357580 – 100% Brevibacterium luteolum AJ488509 – 99% Rothia sp. EF540463 – 98% Brachybacterium sp. FJ357630 – 99%

Eb (242; 262; 472) Fb (241; 262; 404; 471) Gb (246; 263; 290; 310; 344; 431) Hb (245; 262; 348; 442; 472; 535) Ib (251; 260; 264; 371) Jb (262; 310; 343; 431) Kb (262; 348) Lb (261; 310; 348; 462; 522) Mb (262; 348; 761) Nb (417; 448) Ob (289; 462) Pb (327; 380; 416) Qb (294, 427, 437, 526, 591) Rb (409; 446) Sb (424; 467)

Fluorescence DNA fingerprints and microbial identification The DNA fluorescent fingerprints (f-ITS and f-CBH) helped in clustering the bacterial and fungal strains and therefore to choose the cluster representatives in order to be sequenced and identified. The f-ITS divided the 94 bacterial isolates in 19 f-ITS clusters (Ab–Sb). The biggest cluster is represented by 27 Staphylococcus epidermidis strains which produced the profile Ab (Table 1). The second biggest cluster is represented by 16 Bacillus megaterium strains (Jb). The Bacillus megaterium strains were separate by the f-ITS fingerprint in two different clusters, the Jb and the Bb (composed by only two strains). Also the Bacillus sp. isolates were divided in two clusters (Cb and Eb). The other bacterial groups produced their own typical f-ITS profiles. The numbers of peaks for the f-ITS profiles go from 2 (such as for the strains Acinetobacter schindleri, Rothia terrae, Staphylococcus warneri, Rothia sp. and Brachybacterium sp.) to 6 (such as for the strains Bacillus megaterium, Bacillus aryabhattai

and Microccus sp.), while the sizes of the peaks have a range from 216 to 761 bp (Table 1). The fluorescence profiles obtained by the amplification of the cbh gene were composed by a maximum of 4 peaks (such as for the strains Beauveria bassiana, Penicillium expansum, Ascomycota sp., Phialosimplex caninus and Alternaria sp.) to a minimum of one peak (for the strains Penicillium crociola, Eurotium cristatum, Penicillium crustosum and Aspergillus puniceus). The f-CBH permitted the formation of 19 fungal clusters (Af–Sf) which included a total of 43 fungal isolates; the biggest cluster is Pf where all the Penicillium roseopurpureum strains (11 isolates) are comprised (Table 2). Contrary to bacterial strains (where the members of Bacillus megaterium and Bacillus sp. groups displayed two different f-ITS profiles for each group), each fungal species is represented by a specific f-CBH profile. The majority of bacterial isolates belonged to the genus Bacillus or Staphylococcus, this two genera were recovered in all samples;

Table 2 Fluorescent CBH profiles and ITS sequences similarity of fungal strains isolated from Cardinal’s clothes. f-CBH profiles (bp)

Number of isolates from funeral clothes

Number of cluster fungal representatives sequenced

Identification on the basis of highest ITS similarity score

Af (539; 550; 648) Bf (381; 655) Cf (215; 545) Df (224; 546; 645) Ef (550; 578) Ff (552) Gf (622; 628) Hf (237; 500; 549; 893) If (265; 492; 552; 664) Jf (648) Kf (408; 550) Lf (301; 359; 547; 884) Mf (547; 663; 910) Nf (472; 550; 763) Of (549) Pf (551; 666)

2 2 1 3 4 2 1 1 3 1 1 2 2 1 3 11

1 1 1 1 2 1 1 1 1 1 1 1 1 1 1 4

Qf (406; 550; 701; 708) Rf (596; 640; 683; 699) Sf 540

1 1 1

1 1 1

Aspergillus pseudodeflectus EF634382 – 99% Penicillium commune HQ710540 – 100% Aspergillus sydowii HQ315849 – 100% Aspergillus tubingensis HQ315841 – 100% Penicillium brevicompactum HM469408 – 100% Penicillium crocicola EU427290 – 100% Penicillium spinulosum FR670336 – 100% Beauveria bassiana JN379808 – 100% Penicillium expansum HM469423 – 100% Eurotium cristatum GU784865 – 100% Myriodontium keratinophilum EU925387 – 99% Ascomycota sp. GU566286 – 94% Aspergillus fumigatus FM999061 – 100% Xenochalara juniperi DQ093775 – 99% Penicillium crustosum GU134895 – 100% Penicillium roseopurpureum AF034462 – 100%; GU566239 – 100%; GU944605 – 99% Phialosimplex caninus GQ169317 – 100% Alternaria sp. AY154686 – 100% Aspergillus puniceus EF652498 – 100%

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Table 3 Bacterial and fungal strains isolated from the clothes samples of Cardinal Pázmány. Sample

Bacterial isolates

Fungal isolates

A1/biretta Inside silk part

Staphylococcus pasteuri (MYP-A1X-SW); Bacillus aryabhattai (MAC-A1A-SW; MAC-A1C-SW; MAC-A1D-SW); Bacillus megaterium (MAC-A1B-SW); Rothia terrae (PSF-A1-SW); Rothia sp. (R2A-A1X-SW)

A2/biretta Outside wool part

Staphylococcus epidermidis (SC-A2A-SW; SC-A2B-SW); Bacillus sp. (SC-A2C-SW; MAC-A2B-SW); Staphylococcus pasteuri (SC-A2-D); Bacillus megaterium (PSF-A2A-SW; PSF-A2B-SW); Rathayibacter sp. (MAC-A2A-SW) Staphylococcus epidermidis (SC-A3X-SW); Bacillus simplex (SC-A3B-D); Micrococcus sp. (MYP-A3X-SW); Bacillus megaterium (SC-A3B-SW; SC-A3C-SW; R2A-A3-SW); Acinetobacter schindleri (SC-A3A-SW); Brachybacterium sp. (SC-A3A-D) Staphylococcus epidermidis (SC-B1X-D; R2A-B1C-SW); Bacillus megaterium (PSF-B1D-SW); Bacillus aryabhattai (SC-B1B-SW; SC-B1C-SW); Bacillus muralis (SC-B1A-SW); Rothia terrae (PSF-B1A-SW; PSF-B1B-SW); Staphylococcus capitis (R2A-B1B-SW); Rothia sp. (R2A-B1A-SW) Staphylococcus epidermidis (R2A-B2A-SW; R2A-B2B-SW; R2A-B2C-SW; R2A-B2D-SW; PSF-B2A-SW; PSF-B2B-SW; PSF-B2C-SW; PSF-B2D-SW; SC-B2X-SW; BHI-B2-2-SW; BHI-B2-3-SW; BHI-B2-4-SW; BHI-B2-6-SW; BHI-B2-7-SW; BHI-B2-8-SW; MYP-B2-SW); Bacillus sp. (SC-B2A-D; MAC-B2A-SW); Bacillus megaterium (SC-B2B-D); Staphylococcus warneri (SC-B2-SW) Staphylococcus epidermidis (R2A-B3A-SW); Bacillus sp. (SC-B3bA-D; SC-B3bC-D; R2A-B3C-SW); Bacillus simplex (R2A-B3D-SW); Bacillus megaterium (MYP-B3B-SW; SC-B3C-SW; SC-B3A-SW; SC-B3Bb-D; R2A-B3B-SW); Arthrobacter sp. (MYP-B3A-SW); Rothia sp. (SC-B3B-SW) Staphylococcus epidermidis (SC-B4X-SW); Bacillus sp. (R2A-B4A-SW; PSF-B4E-SW; MAC-B4B-SW); Staphylococcus pasteuri (PSF-B4C-SW); Bacillus sp. (SC-B4A-D; SC-B4B-SW; MAC-B4A-SW); Bacillus aryabhattai (PSF-B4B-SW); Bacillus muralis (PSF-B4D-SW); Bacillus megaterium (SC-B4A-SW; SC-B4C-SW); Rothia terrae (PSF-B4A-SW); Rothia sp. (SC-B4B-D) Staphylococcus epidermidis (R2A-B5X-SW); Bacillus sp. (BHI-B5F-T; PSF-B5B-SW; BHI-B5G-T); Staphylococcus pasteuri (SC-B5A-D); Bacillus megaterium (SC-B5B-D); Rothia terrae (PSF-B5A-SW)

Aspergillus pseudodeflectus (SAB-A1-SW); Ascomycota sp. (SAB-A1B-T; DRBC-A1A-SW); Aspergillus fumigatus (SAB-A1A-T); Penicillium roseopurpureum (DRBC-A1-SW); Phialosimplex caninus (DRBC-A1B-SW) Beauveria bassiana (DRBC-A2-SW); Eurotium cristatum (SAB-A2-T)

A3/biretta Outside wool part

B1/tunic White dust on surface near the abdominal part

B2/tunic Collar

B3/tunic Lower part near the legs

B4/tunic Sleeve part near the wrist

B5/tunic Part inside the tunic near the shoulders

B6/tunic More inside near sample B5

Staphylococcus epidermidis (BHI-B6G-T; SC-B6A-D; SC-B6B-D) Bacillus megaterium (R2A-B6-SW); Staphylococcus pasteuri (MYP-B6A-SW); Bacillus sp. (SC-B6D-D); Bacillus megaterium (SC-B6C-D); Brevibacterium luteolum (BHI-B6H-T)

Aspergillus pseudodeflectus (SAB-A3A-T)

Penicillium commune (SAB-B1A-T); Penicillium crocicola (SAB-B1B-T); Penicillium crustosum (MEA-B1C-D); Penicillium roseopurpureum (MEA-B1B-D; SAB-B1C-T); Aspergillus puniceus (MEA-B1A-D) Penicillium commune (SAB-B2A-T); Penicillium brevicompactum (SAB-B2D-T; SAB-B2E-T; MEA-B2-D); Penicillium crocicola (SAB-B2C-T); Penicillium spinulosum (SAB-B2B-T); Aspergillus fumigatus (SAB-B2-SW); Penicillium roseopurpureum (SAB-B2F-T) Aspergillus tubingensis (SAB-B3A-T; SAB-B3C-T); Penicillium brevicompactum (SAB-B3D-T); Penicillium expansum (SAB-B3E-T; SAB-B3F-T); Penicillium crustosum (SAB-B3B-T) Aspergillus tubingensis (SAB-B4-T); Myriodontium keratinophilum (MEA-B4-D)

Penicillium expansum (SAB-B5B-T); Xenochalara juniperi (MEA-B5-SW); Penicillium crustosum (SAB-B5A-T); Penicillium roseopurpureum (SAB-B5C-T; SAB-B5D-T; SAB-B5E-T) Aspergillus sydowii (SAB-B6B-T); Penicillium roseopurpureum (SAB-B6A-T; SAB-B6C-T; SAB-B6E-T; SAB-B6F-T); Alternaria sp. (SAB-B6D-T)

The prefix in each isolate code indicates the medium of isolation (SC; R2A agar; MYP agar; PSF agar; MAC; BHI; SAB; DRBC; MEA). The suffix in each isolate code indicates the sampling methods (D – Direct sampling; SW – Swab sampling; T – Tape sampling).

from sample B2 (tunic) only members of these two genera, especially Staphylococci strains, were isolated. The members of the genus Rothia represented the third most diffused bacterial genus, they were isolated from the sample A1 (silk part of the biretta) and from the tunic samples B1, B3, B4 and B5 (Table 3). The genus Penicillium is the most predominant group, only in three samples (A2, A3 and B4) members of this genus were not isolated. The ITS sequencing permitted the identification of different Penicillium species, such as P. commune, P. crocicola, P. crustosum, P. spinulosum, P. expansum, since the most predominant were the strains belonged to the species P. roseopurpureum and P. brevicompactum. The fungal portion exhibited a larger diversity respect the bacterial counterpart, indeed together with Penicillin different Aspergilli species and other kinds of fungal strains, such as Alternaria sp., Beauveria bassiana, Eurotium cristatum, Xenochalara juniperi, Phialosimplex caninus and Myriodontium keratinophilum were isolated (Table 3).

Biodegradative assays evaluation Fungal and bacterial microfloras have been tested for their proteolytic, keratinolytic and cellulolytic properties and also for their

ability to growth on Fibroin Agar where fibroin, without sericine, was the only carbon source. Observing Table 4 it is possible to argue that the most frequent property of the bacterial isolates is the proteolytic one (Gelatin test), in fact it was displayed by 70 strains (74%) from 94 isolates. The proteolytic ability was generally jointed with the keratinolytic one; only 15 Staphylococcus epidermidis and 2 Rothia terrae strains were exclusively proteolytic. The keratinolytic and proteolytic abilities were very strong in many Bacillus strains; mainly Bacillus megaterium isolates which within only three days of incubation on Gelatin agar and in Feather Broth were able to produce a big zone around them and to degrade the feathers respectively. Only two strains (2% of the all isolates) presented cellulolytic activity: the Arthrobacter sp. MYP-B3A-SW and the Acinetobacter schindleri SCA3A-SW. Several Bacilli isolates, mainly Bacillus megaterium strains, were able to growth on Fibroin Agar, where formed colony with a diameter around 5–6 mm. Other two bacterial strains growing on Fibroin Agar were Rothia terrae and Brevibacterium luteolum, the latter produced a colony of 9 mm of diameter. The fungal strains displayed a potential ability to utilize fibroin; indeed thirty-two fungal isolates (74%) were able to growth on agar contained fibroin as the only carbon source (Table 5). The fungal isolates in Feather Broth exhibited three different reactions: many

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Table 4 Biodegradative characteristic of bacterial strains isolated from the Cardinal’s Pázmány funeral clothes. Bacteria isolates

Feather test

Gelatin test

Cellulose test

Fibroin test (diameter size, mm)

SC-A3A-D Brachybacterium sp. SC-A3B-D Bacillus simplex SC-B2A-D Bacillus sp. SC-B2B-D Bacillus megaterium SC-B3bA-D Bacillus sp. SC-B3bB-D Bacillus megaterium SC-B3bC-D Bacillus sp. SC-B4A-D Bacillus sp. SC-B5B-D Bacillus megaterium SC-B6C-D Bacillus megaterium SC-B6D-D Bacillus sp. SC-A2C-SW Bacillus sp. SC-A3A-SW Acinetobacter schindleri SC-A3B-SW Bacillus megaterium SC-A3C-SW Bacillus megaterium SC-B1A-SW Bacillus muralis SC-B1B-SW Bacillus aryabhattai SC-B1C-SW Bacillus aryabhattai SC-B2X-SW Staphylococcus epidermidis SC-B3A-SW Bacillus megaterium SC-B3C-SW Bacillus megaterium SC-B4A-SW Bacillus megaterium SC-B4B-SW Bacillus sp. SC-B4C-SW Bacillus megaterium R2A-A3-SW Bacillus megaterium R2A-B1C-SW Staphylococcus epidermidis R2A-B2A-SW Staphylococcus epidermidis R2A-B2B-SW Staphylococcus epidermidis R2A-B2C-SW Staphylococcus epidermidis R2A-B2D-SW Staphylococcus epidermidis R2A-B3B-SW Bacillus megaterium R2A-B3C-SW Bacillus sp. R2A-B4A-SW Bacillus sp. R2A-B5X-SW Staphylococcus epidermidis R2A-B6-SW Bacillus megaterium MYP-A1X-SW Staphylococcus pasteuri MYP-A3X-SW Micrococcus sp. MYP-B3A-SW Arthrobacter sp. MYP-B3B-SW Bacillus megaterium PSF-A1-SW Rothia terrae PSF-A2A-SW Bacillus megaterium PSF-A2B-SW Bacillus megaterium PSF-B1A-SW Rothia terrae PSF-B1B-SW Rothia terrae PSF-B1D-SW Bacillus megaterium PSF-B2B-SW Staphylococcus epidermidis PSF-B2D-SW Staphylococcus epidermidis PSF-B4A-SW Rothia terrae PSF-B4B-SW Bacillus aryabhattai PSF-B4D-SW Bacillus muralis PSF-B4E-SW Bacillus sp. PSF-B5A-SW Rothia terrae PSF-B5B-SW Bacillus sp. MAC-A1A-SW Bacillus aryabhattai MAC-A1B-SW Bacillus megaterium MAC-A1C-SW Bacillus aryabhattai MAC-A1D-SW Bacillus aryabhattai MAC-A2B-SW Bacillus sp. MAC-B2A-SW Bacillus sp. MAC-B4A-SW Bacillus sp. MAC-B4B-SW Bacillus sp. BHI-B2-2-SW Staphylococcus epidermidis BHI-B2-3-SW Staphylococcus epidermidis BHI-B2-4-SW Staphylococcus epidermidis BHI-B2-6-SW Staphylococcus epidermidis BHI-B2-7-SW Staphylococcus epidermidis BHI-B2-8-SW Staphylococcus epidermidis BHI-B6G-T Staphylococcus epidermidis BHI-B6H-T Brevibacterium luteolum BHI-B5F-T Bacillus sp. BHI-B5G-T Bacillus sp.

+ + + +++ + +++ + + +++ +++ + + − + + + + + + +++ + + + +++ + − − − − − +++ + + − +++ + + + +++ − +++ +++ − + +++ − − + +++ + + + + + +++ +++ +++ + + + + − − − − − − − +++ + +

+++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ − +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ + + + + + +++ +++ +++ + +++ + +++ + +++ + +++ +++ + + +++ + + + +++ +++ +++ + +++ +++ +++ +++ +++ +++ + +++ +++ + + + + + + + +++ +++ +++

− − − − − − − − − −

– (6) (5) (5) (6) (6) – (5) (5) (5) (5) (5) – (5) (5) (5) (4) (6) – (5) (5) (5) (5) (5) (5) – – – – – (5) (4) – – (4) – – – – – (6) (7) – – (6) – – (5) (6) (6) (5) – (6) – (4) – (6) (6) (4) (4) (4) – – – – – – – (9) (4) (4)

− + − − − − − − − − − − − − − − − − − − − − − − − − + − − − − − − − − − − − − − − − − − − − − − − − − − − − − − − − − −

−: no biodegradation activity; +: biodegradation activity; +++: strong biodegradation activity displayed within a maximum of 3 days of incubation.

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Table 5 Biodegradative characteristic of fungal strains isolated from the Cardinal’s Pázmány funeral clothes. Fungi

Feather testGrowth/Lysis

Gelatin test

Cellulose test

Growth on fibroin

MEA-B1A-D Aspergillus puniceus MEA-B1B-D Penicillium roseopurpureum MEA-B1C-D Penicillium crustosum MEA-B2-D Penicillium brevicompactum MEA-B4-D Myriodontium keratinophilum SAB-A1-SW Aspergillus pseudodeflectus SAB-B2-SW Aspergillus fumigatus DRBC-A1A-SW Ascomycota sp. DRBC-A1B-SW Phialosimplex caninus DRBC-A1-SW Penicillium roseopurpureum DRBC-A2-SW Beauveria bassiana MEA-B5-SW Xenochalara juniperi SAB-A1A-T Aspergillus fumigatus SAB-A1B-T Ascomycota sp. SAB-A2-T Eurotium cristatum SAB-A3A-T Aspergillus pseudodeflectus SAB-B1A-T Penicillium commune SAB-B1B-T Penicillium crocicola SAB-B1C-T Penicillium roseopurpureum SAB-B2A-T Penicillium commune SAB-B2B-T Penicillium spinulosum SAB-B2C-T Penicillium crocicola SAB-B2D-T Penicillium brevicompactum SAB-B2E-T Penicillium brevicompactum SAB-B2F-T Penicillium roseopurpureum SAB-B3A-T Aspergillus tubingensis SAB-B3B-T Penicillium crustosum SAB-B3C-T Aspergillus tubingensis SAB-B3D-T Penicillium brevicompactum SAB-B3E-T Penicillium expansum SAB-B3F-T Penicillium expansum SAB-B4-T Aspergillus tubingensis SAB-B5A-T Penicillium crustosum SAB-B5B-T Penicillium expansum SAB-B5D-T Penicillium roseopurpureum SAB-B6A-T Penicillium roseopurpureum SAB-B6B-T Aspergillus sydowii SAB-B6C-T Penicillium roseopurpureum SAB-B6D-T Alternaria sp. SAB-B6E-T Penicillium roseopurpureum SAB-B6F-T Penicillium roseopurpureum

−/− +/+ −/− −/− +/+ −/− +/+ +/− − +/+ −/− − −/− −/− −/− −/− −/− −/− −/− −/− −/− −/− −/− −/− −/− −/− −/− −/− −/− −/− −/− −/− −/− −/− − −/− −/− −/− −/− +/− −/−

+ + + + + + + + + − + − + + − + + + + + + + + + + + + + + + + + + + − + + + + + +

− − − − − + + − − − − − + + + + + + − + − − − − − − + − − + + − + + − + + − − − −

+ + + + + − + + + + + + − + + − + − − − + − + + + + + + − + + + + + + + + + − + +

−: no biodegradation activity; +: biodegradation activity.

of them did not grow, 2 of them only grew, and 4 strains (two Penicillium roseopurpureum, Aspergillus fumigatus SAB-B2-SW and Myriodontium keratinophilum MEA-B4-D) were able to grow and to degrade the feathers. Thirty-nine isolates displayed at least one of the following degradative properties: keratinolytic, proteolytic or cellulolytic. Only the Aspergillus fumigatus SAB-B2-SW produced positive results for all three over-mentioned degradative screenings. Similar to bacterial strains the proteolytic ability (Gelatin test) was frequently detected in fungal isolates (in 37 strains, 86% of the isolates), but contrary to bacterial counterpart the cellulolytic activity was more diffused and 16 fungal strains (37% of the total; Table 5) displayed this property. Discussion The deterioration of textile materials was treated in the past by different authors and it was also evident the role of microorganisms in this decay process (Seves et al. 1998; Forlani et al. 2000; Sanchez-Pinero and Bolivar 2004; Szostak-Kotowa 2004; AbdelKareem 2005; Kavkler et al. 2011). To our knowledge during the latest years few works were focused on the biodegradation of textile and on the investigation of the microbial diversity on this kind of materials. Moreover, very rare are the studies concerning such particular environment such as a crypt or a tomb and the microbial analysis of the textile remains (Caretta and Piontelli 1998; Jurado et al. 2010).

The combination of different sampling approaches and culture media showed a useful synergy and complementarity which consented the isolation of various kinds of microorganisms. The bacterial media were not so specific; indeed no Pseudomonas strains were isolated by the Pseudomonas agar (PSF), but this agar was useful for the isolation of several Rothia terrae strains. The MacConkey agar failed in the isolation of Enterobacteriaceae; only the MYP agar was able to isolate few Bacilli and Staphylococci strains, but also members of the genus Micrococcus and Arthrobacter. The two clustering methods f-ITS and f-CBH, for bacteria and fungi respectively, demonstrated again (Kraková et al. 2012; Pangallo et al. 2012) their ability to grouped the bacterial and fungal isolates in order to reduce the number of the strains which in a second step were identified at species level through bacterial 16S rDNA and fungal ITS sequencing. The f-CBH approach is used here for the first time in order to cluster the fungal microflora isolated from deteriorated textile materials. In addition, the amplification of the cbh permitted also to reveal the potential cellulolytic ability of the analyzed fungal isolates. The members of the phylum Firmicutes, represented by Bacillus and Staphylococcus strains, were isolated from all samples; these results evidenced the high concentration of this kind of bacteria in such kind of environment and materials. The predominant presence of Firmicutes strains perhaps hampered the isolation of other kinds of bacteria also when specific cultivation media were used. Staphylococci strains isolated during our investigation can be considered

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Fig. 3. SEM microphotographies of two different places (A and B) of tunic surface. The circle on photo A1 and B1 indicates the areas which were gradually maximized on photos A2, A3, B2 and B3. On maximized photos is evident the presence of hypha and conidia on tunic surface.

as common skin and mucous commensal of humans (Chiller et al. 2001; Jain and Agarwal 2009) and the Bacillus strains are normally isolated from soil and other kinds of environments (Garbeva et al. 2003; Urzì et al. 2010; Sansinenea and Ortiz 2011; Pangallo et al. 2012). Some Bacillus isolates displayed 100% of similarity with recent identified species Bacillus muralis (Heyrman et al. 2005) and Bacillus aryabhattai (Shivaji et al. 2009). Jurado et al. (2010) have also isolated, during the analysis of paper bulls found with a corpse inside a sepulcher of the 16th in Spain, different members of the phylum Firmicutes, mainly Clostridium and Sporosarcina strains. The second most isolated bacterial phylum was represented by Actinobacteria, among them several strains were identified as Rothia terrae which were recently isolated for the first time from a soil sample in Taiwan (Chou et al. 2008). Other Actinobacteria isolates were represented by the strains Brachybacterium, Brevibacterium luteolum and Arthrobacter, members of these genera were frequently isolated or detected from mural and hypogean environments (Groth et al. 2001; Pepe et al. 2010; Pangallo et al. 2012; De Leo et al. 2012). A member of the genus Rathayibacter, which

are considered normal colonizers of different kinds of plants and grasses (Francis et al. 2010), was also isolated; perhaps this is connected with the use of different kinds of grasses (in this case mainly sprigs of rosemary) in order to fill the cushion placed under the head of the Cardinal. The presence of plants residues near the corpse of Cardinal and also the rests of the wooden coffin and various other wooden objects (pastoral staff, polychromatic chalice and paten) could explain also the isolation of several fungal isolates, such as Aspergillus pseudodeflectus (Peterson 2007), A. sidowi, A. tubingensis, Penicillium crustosum (Liu and Tian 2010), P. brevicompactum, P. expansum (Jang et al. 2011), P. spinulosum (Diguta et al. 2011), P. roseopurpureum (Houbraken et al. 2010), Eurotium cristatum (Yazdani et al. 2011) and Xenochalara juniperi (Menkis et al. 2006), which their ITS sequences were similar to strains related with wood or plants. The SEM photos (Fig. 3) showed the presence mainly of fungal hypha and conidia; therefore it is possible to suppose that this kind of microorganisms were lived on different places of tunic surface.

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The fungal analysis of textile materials from the remains of a ninth century abbess (Caretta and Piontelli 1998) concords with our findings exclusively for some members belonged to the genera Alternaria, Aspergillus and Penicillium. Unfortunately, we were not able to find in literature more studies which deal with the same topic; the microbial analysis of mummy surfaces, in some case, agreed with our results regarding the presence of Bacillus, ˇ ˇ Aspergillus and Penicillium strains (Cavka et al. 2010; Skrlin et al. 2011). Regarding the pathogenicity of present microflora it is possible to say that no very dangerous microorganisms were isolated, indeed many of them are associated with allergies and nosocomial infections which are critical mainly for immunocompromised patients (Simon-Nobbe et al. 2008; Fey and Olson 2010; Sydnor and Perl 2011). The keratinolytic and proteolytic abilities of Bacillus strains are confirmed by several authors (Brandelli et al. 2010) and recently Bacillus members, isolated from wool, were tested for the biodegradation of keratinous materials (Queiroga et al. 2012). Our results on fibroin utilization are in contrast with the past findings (Seves et al. 1998), where the bacterial isolated from a soil burial experiment with silk were members of the genus Pseudomonas and only one isolate was a Bacillus megaterium. In addition, Seves and collaborators did not isolate any fungal strains; this is another difference with our investigation which showed the presence of fungal strains and their quite common ability to grow on Fibroin Agar. Such ability was displayed by Beauveria bassiana, Eurotium cristatum, Myriodontium keratinophilum and many Penicillium and Aspergillus strains and by, above all, two fungal strains which showed a similarity of 99% and 100% with quite recent identified species, Xenochalara juniperi (Coetsee et al. 2000) and Phialosimplex caninus (Sigler et al. 2010) respectively. The comparison of our results with two recent reviews (Błyskal 2009; Brandelli et al. 2010) on biodegradation of keratinous materials displayed how the keratinolytic property of the isolates Myriodontium keratinophilum and Penicillium roseopurpureum are described in this study for the first time. Our biodegradative data described a certain degree of complementarity between the bacterial and fungal microflora. The bacteria through their strong ability to degrade keratin and protein could be considered as the first colonizers which prepare these substrates for the attack of fungal strains. Indeed, if on one hand our fungal isolates displayed a widespread cellulolytic activity and fibroin utilization, on the other hand they showed a weaker and slower proteolytic and mainly keratinolytic ability respect to bacterial counterpart. This study characterized the culturable portion of the microflora isolated from different textile materials conserved from a long time inside a crypt. The microbial diversity was explored by the combination of different sampling (Direct, Swab and MAT), cultivation and molecular approaches (f-ITS, f-CBH, 16S rDNA and ITS sequencing) which permitted the isolation of many “protagonists” of this kind of environment. To our knowledge our report is one of the few investigations focused on this unusual environment and mainly on textile materials in these kinds of conditions. Our findings will increase the knowledge regarding the ecology and properties of the microflora isolated from textile materials. Moreover, the biodegradation screening allowed also the selection of different microorganisms which could be exploited in different biotechnology processes, as for example the waste treatment through their ability to degrade protein and keratin residues (KorniłłowiczKowalska and Bohacz 2011; Rajkumar et al. 2011), but also for more sophisticated biorestoration approaches applied to the preservation of historical and artistic items (Ranalli et al. 2005; Hamed 2012).

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