Detection of mycobacteria in the environment of the Moravian Karst (Bull Rock Cave and the relevant water catchment area): the impact of water sediment, earthworm castings and bat guano H., Bartos M., Hribova P., Ulmann V., Hubelova D., Konecny O., Gersl M., Kudelka J., Voros D., Pavlik I. (2017): Detection of mycobacteria in the environment of the Moravian Karst (Bull Rock Cave and the relevant water catchment area): the impact of water sediment, earthworm castings and bat guano. Veterinarni Medicina, 62: 153-168.
download PDF
The presence of mycobacteria was studied in Bull Rock Cave (“Byci skala”) and the water catchment area of Jedovnice Brook (“Jedovnicky potok”) using direct microscopy after Ziehl-Neelsen (ZN) staining, culture examination and molecular techniques. Mycobacteria were detected in 47.1% of a total of 68 samples. The mycobacterial genes hsp65 and dnaA were detected and sequenced in 37 (74.0%) out of the 50 cave environmental samples and in 10 (55.6%) out of the 18 samples of water catchment sediments. Nine species of slowly growing mycobacteria (M. terrae, M. arupense, M. gordonae, M. lentiflavum, M. parascrofulaceum, M. parmense, M. saskatchewanense, M. simiae and M. xenopi) and two subsp. (M. avium subsp. avium and M. avium subsp. hominissuis) were detected. Fourteen species of rapidly growing mycobacteria (M. chelonae, M. chubuense, M. poriferae, M. flavescens, M. fortuitum, M. porcinum, M. rhodesiae, M. gilvum, M. goodii, M. peregrinum, M. mageritense, M. vanbaalenii, M. gadium and M. insubricum) were detected. The highest mycobacterial presence was documented by ZN staining and/or culture examinations in earthworm castings and bat guano (73.3% positivity out of the 15 samples) in the cave environment and in the water sediments collected under the outflow from the wastewater treatment plants (77.8% positivity out of nine samples). The highest total organic carbon (TOC) was detected in wooden material and earthworm castings with pH values between 5.0 and 7.7 in the cave environment and in water sediments collected under the outflow from the wastewater treatment plants with pH between 5.8 and 7.0. It could be concluded that the karst cave environment with its running surface water contaminated with different microorganisms or chemical substances creates favourable conditions not only for animals (especially earthworms) but also for mycobacteria. This fact is also demonstrated by the presence of these mycobacteria in the cave environment mainly in earthworm castings and bat guano.
Andreo Bartolomé, Goldscheider Nico, Vadillo Iñaki, Vías Jesús María, Neukum Christoph, Sinreich Michael, Jiménez Pablo, Brechenmacher Julia, Carrasco Francisco, Hötzl Heinz, Perles María Jesús, Zwahlen François (2006): Karst groundwater protection: First application of a Pan-European Approach to vulnerability, hazard and risk mapping in the Sierra de Líbar (Southern Spain). Science of The Total Environment, 357, 54-73
Blackwood KS, He C, Gunton J, Turenne CY, Wolfe J, Kabani AM (2000): Evaluation of recA sequences for identification of Mycobacterium species. Journal of Clinical Microbiology 38, 2846–2852.
Breitbart Mya, Hoare Ana, Nitti Anthony, Siefert Janet, Haynes Matthew, Dinsdale Elizabeth, Edwards Robert, Souza Valeria, Rohwer Forest, Hollander David (2009): Metagenomic and stable isotopic analyses of modern freshwater microbialites in Cuatro Ciénegas, Mexico. Environmental Microbiology, 11, 16-34
Brooks RW, Parker BC, Gruft H, Falkinham III JO (1984): Epidemiology of infection by non-tuberculous mycobacteria. V. Numbers in eastern United States soils and correlation with soil characteristics. American Review of Respiratory Diseases 130, 630–633.
Burkhardt R, Gregor VA, Novak K (1972): Exceptional flood in Bull Rock Cave (in Czech). Vlastivedne Zpravy z Adamova a Okoli 6, 4–8.
Butscher Christoph, Auckenthaler Adrian, Scheidler Stefan, Huggenberger Peter (2011): Validation of a Numerical Indicator of Microbial Contamination for Karst Springs. Ground Water, 49, 66-76
Cernosvitov L (1935): Monographie des Tschechoslovakischen Lumbriciden. Archiv pro Prirodovedecky Vyzkum Cech 19, 86 pp.
Coenye Tom, Vandamme Peter (2003): Intragenomic heterogeneity between multiple 16S ribosomal RNA operons in sequenced bacterial genomes. FEMS Microbiology Letters, 228, 45-49
CSO – Czech Statistical Office (2016a): Spatial analytical materials. Available at (Accessed July 21, 2016).
CSO – Czech Statistical Office (2016b): Public Database. Available at (Accessed July 21, 2016).
CSO – Czech Statistical Office (2016c): Population and Housing Census 2011. Available at (Accessed July 21, 2016).
De Mandal S, Panda AK, Lalnunmawii E, Bisht SS, Kumar NS (2015a): Illumina-based analysis of bacterial community in Khuangcherapuk Cave of Mizoram, Northeast India. Genome Data 5, 13–14.
De Mandal Surajit, Sanga Zothan, Senthil Kumar Nachimuthu (2015): Metagenome Sequencing Reveals Rhodococcus Dominance in Farpuk Cave, Mizoram, India, an Eastern Himalayan Biodiversity Hot Spot Region. Genome Announcements, 3, e00610-15-
Einsiedl Florian, Radke Michael, Maloszewski Piotr (2010): Occurrence and transport of pharmaceuticals in a karst groundwater system affected by domestic wastewater treatment plants. Journal of Contaminant Hydrology, 117, 26-36
Engbaek HC, Vergmann B, Weis Bentzon B (1967): The sodium lauryl sulphate method in culturing sputum for mycobacteria. Scandinavian Journal of Respiratory Diseases 48, 268–284.
Faimon Jiří, Štelcl Jindřich, Sas Daniel (2006): Anthropogenic CO2-flux into cave atmosphere and its environmental impact: A case study in the Císařská Cave (Moravian Karst, Czech Republic). Science of The Total Environment, 369, 231-245
Fischer O.A, Matlova L, Bartl J, Dvorska L, Svastova P, du Maine R, Melicharek I, Bartos M, Pavlik I (2003): Earthworms (Oligochaeta, Lumbricidae) and mycobacteria. Veterinary Microbiology, 91, 325-338
Hertod-Todtenfeldt JF (1669): Tartaro-Mastix Moraviae, per quem rariora & admiranda a natura in faecundo huius regionis gremio effusa, comprimis tartarus, illiusque effectus morbosi curiose examinantur, & cura tam therapeutica quam prophylactica proponitur. Vienna Austriae 263, 258.
Hill C. A., Polyak V. J. (): Karst piracy: A mechanism for integrating the Colorado River across the Kaibab uplift, Grand Canyon, Arizona, USA. Geosphere, 10, 627-640
Hrabe S (1929): Zwei neue lumbriculiden-arten, sowie einige bemerkungen zur systematik einiger bereits bekannter. Zoologischer Anzeiger 84, 9–21.
Hrabe S (1938): Trichodrilus moravicus und Claparedei, neue Lumbriculiden. Zoologischer Anzeiger 121, 73–85.
Hromas J (2009): Caves (in Czech). In: Mackovcin P, Sedlacek M (eds): Protected Areas in the Czech Republic. Agentura Ochrany Prirody a Krajiny CR a EkoCentrum Brno, Praha. 608 pp.
Hu Man, Wang Xiaohui, Wen Xianghua, Xia Yu (2012): Microbial community structures in different wastewater treatment plants as revealed by 454-pyrosequencing analysis. Bioresource Technology, 117, 72-79
Hubka V., Novakova A., Kola ik M., Jurjevi   eljko, Peterson S. W. (): Revision of Aspergillus section Flavipedes: seven new species and proposal of section Jani sect. nov.. Mycologia, 107, 169-208
Jurado Valme, Laiz Leonila, Rodriguez-Nava Veronica, Boiron Patrick, Hermosin Bernardo, Sanchez-Moral Sergio, Saiz-Jimenez Cesareo (2010): Pathogenic and opportunistic microorganisms in caves. International Journal of Speleology, 39, 15-24
Jurende CJ (1835): Die Wunder der Unterwelt. Jurende‘s Vaterlandischer Pilger, Brunn 22, 61–101.
Kapur V, Li LL, Hamrick MR, Plikaytis BB, Shinnick TM, Telenti A, Jacobs Jr WR, Banerjee A, Cole S, Yuen KY, Clarridge III JE, Kreiswirth BN, Musser JM (1995): Rapid Mycobacterium species assignment and unambiguous identification of mutations associated with antimicrobial resistance in Mycobacterium tuberculosis by automated DNA sequencing. Archives of Pathology and Laboratory Medicine 119, 131–138.
Kazda J (ed.) (2000): The Ecology of Mycobacteria. Kluwer Academic Publishers. 72 pp.
Kazda J, Pavlik I, Falkinham JO, Hruska K (eds) (2009): The Ecology of Mycobacteria: Impact on Animal’s and Human’s Health. 1st edn. Springer, New York. 520 pp.
Korzeniewska Ewa (): Emission of bacteria and fungi in the air from wastewater treatment plants - a review. Frontiers in Bioscience, 3, 393-
Kriz M (1891): Die hohlen in den mahrischen Devonkalken und ihre vorzeit. I. Die slouperhohlen. Jahrbuch der Kaiserlich Koniglichen Geologischen Reichsanstalt, Wien 41, 443–570.
Kriz M (1892): Die Hohlen in den mahrischen Devonkalken und ihre Vorzeit II–IV. Jahrbuch der Kaiserlich Koniglichen Geologischen Reichsanstalt, Wien 42, 463–626.
LPSN – List of Prokaryotic Names with Standing in Nomenclature (2016): Available at (Accessed July 9, 2016).
Lukesova A, Novakova A (2009): Interactions between the soil micro-flora and invertebrates in Slovak and Moravian caves. In: 9th Central European Workshop on Soil Zoology, Ceske Budejovice, Czech Republic. April 17–20, 2007. 89–96.
Mayer J (ed.) (1781): Versuch einer Beschreibung der Gegend um Sluppe in Mahren. Schriften der Berlinischen Gesellschaft Naturforschender Freunde, Bd. II, Berlin. 56–65.
McNabb A., Eisler D., Adie K., Amos M., Rodrigues M., Stephens G., Black W. A., Isaac-Renton J. (): Assessment of Partial Sequencing of the 65-Kilodalton Heat Shock Protein Gene (hsp65) for Routine Identification of Mycobacterium Species Isolated from Clinical Sources. Journal of Clinical Microbiology, 42, 3000-3011
McNabb A., Adie K., Rodrigues M., Black W. A., Isaac-Renton J. (): Direct Identification of Mycobacteria in Primary Liquid Detection Media by Partial Sequencing of the 65-Kilodalton Heat Shock Protein Gene. Journal of Clinical Microbiology, 44, 60-66
Migon P (2011): Development of karst phenomena for geotourism in the Moravian Karst (Czech Republic). Geoturism, 3–24.
Mohammadi Zargham, Shoja Atefe (): Effect of annual rainfall amount on characteristics of karst spring hydrograph. Carbonates and Evaporites, , -
Moravkova M., Hlozek P., Beran V., Pavlik I., Preziuso S., Cuteri V., Bartos M. (2008): Strategy for the detection and differentiation of Mycobacterium avium species in isolates and heavily infected tissues. Research in Veterinary Science, 85, 257-264
Mukai Tetsu, Miyamoto Yuji, Yamazaki Toshio, Makino Masahiko (2006): Identification of Mycobacterium species by comparative analysis of the dnaA gene. FEMS Microbiology Letters, 254, 232-239
Nagel JA (1749): Beschreibung deren auf Allerhochsten Befehl Ihrer Rom. Kaas und Konigl. Maytt. Francisci I. untersuchten in dem Hertzogthum Crain befindlichen Seltenheiten der Natur. Manuscript in Vienna Court Library. 97 pp.
Nossa Carlos W (2010): Design of 16S rRNA gene primers for 454 pyrosequencing of the human foregut microbiome. World Journal of Gastroenterology, 16, 4135-
Nováková Alena (2009): Microscopic fungi isolated from the Domica Cave system (Slovak Karst National Park, Slovakia). A review. International Journal of Speleology, 38, 71-82
Parise M., Lollino P. (2011): A preliminary analysis of failure mechanisms in karst and man-made underground caves in Southern Italy. Geomorphology, 134, 132-143
Persing DH (ed.) (2011): Molecular Microbiology, Diagnostic Principles and Practice. 2nd edn. ASM Press, Washington, D.C. 936 pp.
Pizl V (2008): Are earthworms (Oligochaeta, Lumbricidae) regular or accidental dwellers of cave systems in the Czech Republic and Slovakia (in Czech). Acta Carsologica Slovaca 46, 197–201.
Prichystal A, Naplava M (1995): Mystery of Bull Rock Cave – the cave full of questions (in Czech). Amaprint, Trebic. 161 pp.
Roblickova M, Kana V (2013): New palaeontological research in Barova (Sobolova) Cave, Moravian Karst: Preliminary report. Acta Musei Moraviae Scientiae Geologicae 98, 111–127.
SAIF – State Agricultural Interventional Fund (2016): List of subsidy beneficiaries. Available at (Accessed July 20, 2016).
Schön Georg, Göbel Ulf B., Schuppler Markus, Mertens Frank (1995): Molecular characterization of nocardioform actinomycetes in activated sludge by 16S rRNA analysis. Microbiology, 141, 513-521
Seman Milan, Gaálová Barbora, Cíchová Marianna, Prokšová Miloslava, Haviarová Dagmar, Fľaková Renáta (2015): The occurrence of coliform bacteria in the cave waters of Slovak Karst, Slovakia. Folia Microbiologica, 60, 269-278
Shabarova Tanja, Villiger Jörg, Morenkov Oleg, Niggemann Jutta, Dittmar Thorsten, Pernthaler Jakob (2014): Bacterial community structure and dissolved organic matter in repeatedly flooded subsurface karst water pools. FEMS Microbiology Ecology, 89, 111-126
Slana I., Kaevska M., Kralik P., Horvathova A., Pavlik I. (2010): Distribution of Mycobacterium avium subsp. avium and M. a. hominissuis in artificially infected pigs studied by culture and IS901 and IS1245 quantitative real time PCR. Veterinary Microbiology, 144, 437-443
Stastna P, Bezdek J, Kovarik M (eds) (2003): The Animal’s Species Described in Moravian Karst (in Czech). Obcanske Sdruzeni KORAX, Kulirov. 80 pp.
Tang YW, Sussman M, Dongyou L, Poxton I, Schwartzman J (eds) (2014): Molecular Medical Microbiology, Academic Press. 2216 pp.
Telenti A, Marchesi F, Balz M, Bally F, Bottger EC, Bodmer T (1993): Rapid identification of mycobacteria to the species level by polymerase chain reaction and restriction enzyme analysis. Journal of Clinical Microbiology 31, 175–178.
Terzić Josip, Marković Tamara, Lukač Reberski Jasmina (2014): Hydrogeological properties of a complex Dinaric karst catchment: Miljacka Spring case study. Environmental Earth Sciences, 72, 1129-1142
Thoen CO, Steele JH, Kaneene JB (eds) (2014): Zoonotic Tuberculosis: Mycobacterium bovis and Other Pathogenic Mycobacteria. 3rd edn. Wiley-Blackwell. 432 pp.
Turenne C. Y., Semret M., Cousins D. V., Collins D. M., Behr M. A. (): Sequencing of hsp65 Distinguishes among Subsets of the Mycobacterium avium Complex. Journal of Clinical Microbiology, 44, 433-440
Ulmann Vit, Kracalikova Anna, Dziedzinska Radka (2015): Mycobacteria in Water Used for Personal Hygiene in Heavy Industry and Collieries: A Potential Risk for Employees. International Journal of Environmental Research and Public Health, 12, 2870-2877
van der Wielen P. W. J. J., Heijnen L., van der Kooij D. (): Pyrosequence Analysis of the hsp65 Genes of Nontuberculous Mycobacterium Communities in Unchlorinated Drinking Water in the Netherlands. Applied and Environmental Microbiology, 79, 6160-6166
Varaine F, Rich ML (eds) (2014): Tuberculosis: Practical Guide for Clinicians, Nurses, Laboratory Technicians and Medical Auxiliarie. Medecins Sans Frontieres Parnters in Health, Paris. 303 pp.
Wankel H (1856): Ueber die Fauna der mahrischen Hohlen. Verhandlungen des Zoologisch-Botanischen Vereins in Wien, Wien 6, 467–470.
Wankel H (ed.) (1882): Bilder aus der Mahrischen Schweiz und ihrer Vergangenheit, Druck Verlag von Adolf Holzhausen, Wien; translated in Czech by Grolich V, Svoboda O, Urban J (1988). 2nd edn. Muzejni a Vlastivedna Spolecnost v Blansku, Brno. 307 pp.
Wayne LG, Kubica GP (1986): Genus Mycobacterium Lehmann and Neumann 1896, 363AL. In: Sneath PHA, Mair NS, Sharpe ME, Holt JG (eds.): Bergey’s Manual of Systematic Bacteriology. The Williams and Wilkins Co., Baltimore. 1436–1457.
Weisburg W G, Barns S M, Pelletier D A, Lane D J (1991): 16S ribosomal DNA amplification for phylogenetic study.. Journal of Bacteriology, 173, 697-703
Wilhartitz Inés C., Kirschner Alexander K. T., Brussaard Corina P. D., Fischer Ulrike R., Wieltschnig Claudia, Stadler Hermann, Farnleitner Andreas H. (2013): Dynamics of natural prokaryotes, viruses, and heterotrophic nanoflagellates in alpine karstic groundwater. MicrobiologyOpen, 2, 633-643
Zhang Ya, Kelly Walton R., Panno Samuel V., Liu Wen-Tso (2014): Tracing fecal pollution sources in karst groundwater by Bacteroidales genetic biomarkers, bacterial indicators, and environmental variables. Science of The Total Environment, 490, 1082-1090
download PDF

© 2021 Czech Academy of Agricultural Sciences | Prohlášení o přístupnosti