Application of high-throughput sequencing methods for microbial biodiversity

analyses in research of archaeological textiles.

Probable cause of death of the people buried in the west crypt

of St. Anne Chapel in the Castle of the Teutonic Order in Malbork.

Magdalena Dyda1, Anna Makulec2, Monika Stachurska2, Andrzej Gołembnik3, Przemysław Decewicz1,4, Krzysztof Romaniuk4, Łukasz Dziewit4, Łukasz Drewniak1,5

Archaeological objects are often found in a very bad state of preservation, i.a.

due to long-term exposure to microbial activity. Although the presence of

microorganisms on such artifacts is generally recognized, their identification is

rarely performed. Disturbingly, the latest studies in this area indicate that many

of the investigated strains are potential human pathogens, constituting a health

risk for the conservators and archaeologists. Therefore, the identification of

microorganisms colonizing archaeological sites and objects should become a

standard procedure. However, to efficiently assess the biodiversity of such

communities, also with respect to the possible human pathogens, adaptation of

the available metagenomics methods may be required.

ResultsIntroduction

Fig. 1. Archaeological exploration of St. Anne’s Chapel,

textiles excavation and conservation

Materials and methods

• High-throughput sequencing may be a quasi-forensic method for

identification of pathogens that may have caused death of the people

buried centuries ago.

• Metagenomics analyses not only enable the characterization of

microbial communities, but they may also indicate the contamination

of archeological objects with potential pathogens.

• The use of next-generation sequencing technology allows for reliable

and complete identification of bacteria and fungi, even if they are

difficult to cultivate using conventional microbiological methods.

Conclusions

Aim

The aim of the study was to analyse the biodiversity of bacteria and fungi

in the textiles collected during archaeological works in the Castle of the

Teutonic Order in Malbork.

Microorganism identification

Samples were taken from the textiles found in two tombs in the west crypt of St. Anne

chapel in the Castle of the Teutonic Order in Malbork (Fig. 1 and 2). Total DNA was isolated

and purified from the collected samples, followed by amplification and sequencing of the

V3-V4 region of the 16S rRNA gene for Bacteria and ITS1 region of the ITS gene for Fungi.

Illumina MiSeq technology was used for sequencing of each amplicon.

1Research and Development for Life Sciences Ltd., Warsaw, Poland; *magdalena.dyda@rdls.pl2Academy of Fine Arts in Warsaw, Department of Conservation and Restoration of Textiles, Warsaw, Poland

3Incedo3D, Warsaw, Poland4University of Warsaw, Faculty of Biology, Department of Bacterial Genetics, Warsaw, Poland

5University of Warsaw, Faculty of Biology, Laboratory of Environmental Pollution Analysis Warsaw, Poland

Fig. 2. Biodiversity of Bacteria in samples of textiles collected from 2 graves.

0% 20% 40% 60% 80% 100%

Mycobacterium Chitinophaga Pedobacter Virgibacillus

Clostridium Sporanaerobacter Phenylobacterium Mesorhizobium

Sphingomonas Sphingopyxis Ralstonia Escherichia

Steroidobacter Rhodanobacter Other Unspecified

1st grave

2nd grave

Percentage composition of bacterial genus identified across samples in graves.

Data for genus which constituted more than 1% in overall compositions.

Two additional and artificial groups were created to include the genus with abundance

lower than the threshold (Other) and the ones with unspecified (i.e. ‘f__’) or uncertain (i.e. ‘Incertae_sedis’) taxonomic position assigned (Unspecified).

Percentage composition of fungal genus identified across samples in graves.

Data for genus which constituted more than 1% in overall compositions.

Two additional and artificial groups were created to include the genus with abundance

lower than the threshold (Other) and the ones with unspecified (i.e. ‘f__’) or uncertain (i.e. ‘Incertae_sedis’) taxonomic position assigned (Unspecified).

0% 20% 40% 60% 80% 100%

Aspergillus Penicillium Spiromastix Oidiodendron

Pseudogymnoascus Engyodontium Other Unspecified

1st grave

2nd grave

Fig. 3. Biodiversity of Fungi in samples of textiles collected from 2 graves.

The genetic analysis of archeological textiles were founded by the company RDLS Ltd. Research and Development for Life Sciences - spin-off company of the University of Warsaw.

Research and restoration works including conservation of archeological textiles were conducted within the framework of program “Conservation and building works in the complex of the Holy Virgin Church in the Castle Museum in Malbork“ co-financed by the European Economic Area (EEA) Financial Mechanism 2009 – 2014 and Norwegian Financial Mechanism

RDLS sp. z o.o. to pierwsza spółka typu spin-off Uniwersytetu

Warszawskiego, założona przez uznanych ekspertów z zakresu mikrobiologii

środowiskowej. Naszym celem jest znajdywanie komercyjnego zastosowania dla

wynalazków z zakresu bioremediacji i biodegradacji, a także prowadzenie

działalności usługowej związanej z monitoringiem środowiskowym. Dzięki

współpracy z Uniwersytetem Warszawskim możliwe jest wykorzystywanie

specjalistycznej aparatury badawczej oraz stały kontakt z wybitnymi specjalistami

z dziedziny mikrobiologii, genetyki oraz biochemii.

Jeden z działów spółki zajmuje się usługami dla muzeów oraz innych instytucji

zajmujących się ochroną obiektów zabytkowych, archiwaliów, konserwacją i

restauracją dzieł sztuki, archeologią. Dzięki interdyscyplinarnemu zespołowi

oferujemy pełen pakiet usług: od ustalenia przyczyny zmian obserwowanych na

obiekcie (w tym identyfikację mikroorganizmów czy owadów) po rekomendacje

dotyczące działań naprawczych lub stabilizujących. Współpracujemy z różnymi

instytucjami zarówno na zasadach komercyjnych jak i w ramach projektów

naukowych.

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02-096 Warszawa

tel.: 22 554 19 07 lub 786 28 44 96

e-mail: biuro@rdls.pl

RDLS Ltd. is spin-off company of the University of Warsaw,

established by the renown experts in environmental

microbiology. Our main goal is to commercialize innovative

solutions and invention in the fields of bioremediation and

biodegradation, as well as providing high quality services in

environmental monitoring. Thanks to close ties to the

University of Warsaw we stay in touch with cutting-edge

technology and seasoned experts in microbiology and

genetics.

For museums, art collectors and national heritage sites,

RDLS Ltd. offers solutions for biodeterioration detection and

preservation. We offer a complete array of services from the

microorganism identification to recommendation of possible

solution aimed at either recovery or suppression of the

biodegradation.

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