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University of Florida | Journal of Undergraduate Research | Volume 15, Issue 3 | Summer 2014 1 Identification of Microbial Diversity and Potential Pathogens in Bahamian Caves Melina G. Marte, Dr. David L. Reed, and J. Angel Soto-Centeno Florida Museum of Natural History University of Florid a The orga nic quality of caves allows for the presence of potentially pathogenic microorganisms, which can be transmitted to humans via contact with soils, water, or guano. The goal of this comparative study was to describe the microbial diversity, primaril y bacteri a and fungi, within cave substrates. The purpose seeks to increase awareness and minimize transmission of potential environment al pathogens for individuals entering Bahamian caves. The research hypothesis stated that high microbial diversity could potenti a lly pose a pathogenic threat to human health. Soil and wall sediment samples were obtained from two caves in Long Island, Bahamas. Two genes, 16S rRNA and 18S rRNA, were sequenced to identify bacteria and fungi, respectively. Empirical sequences were compared to known sequences using a threshold of <2% difference to provide an estimate of relative microbial diversity. Phylogenetic anal ysis revealed two distinct higher level bacterial and fungal groups related to many known pathogens. Rarefaction analysis de monstrated that a proportion of the microbial diversity is still underestimated in these caves. This was the first study attempting to d escribe the microbial diversity in Bahamian caves. High microbial diversity in the caves may reveal the influence of var ious factors altering this ecosystem. This may present a threat of transmittable pathogens to humans through inhalation or direct contact. Determining the extent of potential negative health outcomes when entering cave environments is important for maintai ning public health safety as well as for the preservation of this fragile ecosystem INTRODUCTION During the past decades, there has been an emergence of infectious diseases, which could signify global threat to human health ( Field, 2009). Field (2009) suggests that causal factors relating to emergence of these infectious diseases include interactions between wildlife, domestic animals, and human populations. Surveillance of novel zoonotic, animal borne pathogens provides new k nowledge and a strategy for countering emerging threats to human health, both of which are critical for decreasing disease transmission to humans. It is uncommon that pathogens responsible for infectious diseases in humans are confined to the human specie s (Roche & Gugan, 2011) Among these known human infectious diseases, approximately 58 to 61% have a nonhuman animal origin. Roughly 75% of all emerging infectious diseases that affect humans originate from animals (i.e. zoonosis ) and many have spilled o ver from wildlife reservoirs (Field, 2009). The crossing of species barriers by pathogens can be attributed to direct human contact or domestic animal transmission including worldwide travel, agricultural expansion, deforestation or habitat destruction, and urbanization (Field, 2009; Rupprecht, Wang, & Real, 2008). Accounting for about 20% of all mammalian species, bats are the second most abundant and diverse species after rodents. (Rupprecht, Wang, & Real, 2008; Van der Poel, Lina, & Kramps, 2006). Stud ying bats can be done directly or indirectly via methods involving their habitat/environment (Kunz et al., 2009). Many studies have shown that tourists, researchers who work with bats, and cave explorers are frequently exposed to many environmental pathoge ns (Jurado et al., 2010). Some studies even suggest that people entering or surrounding cave environments should be given recommendations for their health and safety (Jurado et al., 2010). It is important to take the cave environment into consideration whe n determining the factors that promote microorganism transmission. Microorganisms are found in all biological niches, including subterranean ones (Jurado et al., 2010). Caves, which are underground habitats, have a wide variety of organic material, minimu m lighting, constant temperature, and mineral surfaces (Jurado et al., 2010). These qualities of caves give rise to the presence of potentially pathogenic bacteria, fungi, and yeasts, which occur due to direct or indirect contact with soils, water, or bat feces (guano) (Jurado et al., 2010; Novakova, 2009). There are many countries that have programs in place to protect caves and their native biodiversity (Huppert, 1995). Some of these caves are visited as attractions featuring their ecological or cultural value (Jurado et al., 2010). This gives rise to the importance of caves as centers of mass tourism. Although some caves are visited about 500,000 times a year, others are more isolated. Roche and Gugan (2011) demonstrated that environmental habitat change s have affected biodiverstiy and disease emergence. With a greater number of people entering caves direct contact between bats and humans has increased providing
M ELINA G. M ARTE D R D AVID L. R EED & J. A NGEL S OTO C ENTENO University of Florida | Journal of Undergraduate Research | Volume 15, Issue 3 | Summer 2014 2 greater opportunities for pathogen transmission (Li et al., 2010). Thus highly visited caves where animals are abundant are reservoirs for pathogenic microorganisms. In another study, Jurado et al. (2010) aimed at determining the potential dangers to cave visitors especially with the increasing frequency of tourism to cave environments. They were interested in revealing the potential pathogenic microorganisms in caves and their reservoirs. The data showed that caves are a potential danger to visitors due to the presence of opportunistic microorganisms, whose existence and possible development in h umans are currently unknown (Jurado et al., 2010, pg. 15). Cave microorganisms frequently produce respiratory system infections; therefore it is imperative that humans are well informed of the potential hazards associated with caves (Jurado et al., 2010). Emerging infectious diseases with the ability to cause respiratory infections are of public health interest because of their potential rapid spread via aerosol (Chua et al. 2011). This study aimed at describing the diversity of pathogenic microorganisms, primarily bacteria and fungi, within certain cave environment substrates. The purpose was to comparatively study the presence of potential pathogens in bat infested areas versus non infested areas and between isolated caves and those that are actively vis ited by tourists. Based on previous research, it was hypothesized that frequently visited caves, as well as areas where bats are present, will present a greater assortment of pathogenic microorganisms. The results of this study intended to provide additional information about the microbial richness of caves in order to increase public diverse and widespread nature. Hamiltons cave was a privately owned cave reserved for tours. The soil in this cave was regularly cleared from debris, which represented a local ity with high human disturbance. Hillside cave was a partially hidden and relatively undisturbed cave with few signs of human visitation. Each cave had areas with and within each cave such that each independent site varied in temperature, relative humidity, and presence of bats (Table s 1& 2). Sites within each cave were selected 20 m of each other. Sterile cotton swabs were used to collect sediment samples by rubbing them in a linear pattern over a 20 cm2 area. Each sample contained from 1 5 grams o f soil/sediment and was placed in tubes of Phosphate buffered saline (PBS) for preservation. Samples were kept at 20 C for longterm storage in the lab awareness and minimize transmission of environmental infections to individuals entering Bahamian caves.without bat species present. The bat fauna included the following species: Tadaridabrasiliensis Eptesicusfuscus Macrotuswaterhousii Erophyllasezekorni and Nyctielluslepidus. This study did not involve direct manipulation of bats as it relied only on e nvironmental samples. Sediment samples were collected from three sites METHOD Materials Soil and wall sediment samples were collected in the summer months from Hamiltons Cave (N 23.11333 W 75.04404) and Hillside Cave (N 23.35314 W 75.12740) on Long Isl and, Bahamas using the sample flowchart (Figure 1) Bahamian caves were used because of their. Fungi Wall Site 1 Bacteria Fungi Soil Bacteria Fungi Wall Site 2 Bacteria Fungi Soil Bacteria Fungi Wall Site 3 Bacteria Fungi Soil Bacteria Figure 1 Cave sample flowchart Note. Each cave was sampled according to this flow chart that shows the types of samples obtained at each independent site. All environmental samples were further divided, isolated and amplified using general bacterial and fungal primer pairs Procedure The UltraClean Soil DNA Isolation Kit (MO BIO Laboratories, Carlsbad, CA) was used to perform extraction and purification of the samples using the manufacturers Alternative Protocol (for maximum DNA yields). An additional purification step consisting of dilution (3fold of DNA elution Solution 5, and 2fold of buffer Solution 3) and final elution was done after the last protocol step to remove remaining contaminants. To isolate families of bacteria and fungi, this study used general primers for 16S rRNA and 18S rRNA genes, respectively. Amplifications for both genes was obtained through Polymerase Chain Reaction (PCR) under the following thermal conditions: Initial denaturation at 94 C for 3 min; followed by 34 cycles of denaturation at 94 C for 30 sec., annealing at 50 C for 30 sec., elongation at 72 C for 30 sec ; and a final elongation at 72 C for 10 min. traction were added to
I DENTIFICATION OF M ICROBIAL D IVERSITY AND P OTENTIAL P ATHOGENS IN B AHAMIAN C AVES University of Florida | Journal of Undergraduate Research | Volume 15, Issue 3 | Summer 2014 3 Table 2. Environmental Descriptions: Hamiltons Cave Descriptions Site 1 Site 2 Site 3 Relative Humidity 84.30% 79.10% 83.70% Temperature 23.3C 27.1C 26.6C Bats Present Yes No Yes Species of Bat Present T. brasiliensis N/A N. lepidus, M. waterhousii Approximate Bat Count >500 N/A 750 1000, >30 Level of disturbance High High High Additional Observations Mossy walls, water puddles Dry soil, no obvious guano Obvious guano, insect remains, no breeze Note. Sites with bats present had obvious traces of guano and insect remains and each site varied in temperature and relative humid ity. There were more samples taken from sites with bats present. This cave had very high levels of disturbance due to active touri sm Prime, Gaithersburg, USA), reverse primer. PCR success was assessed using Gel Electrophoresis of fragmented DNA amplifications using a 1% agarose gel. Successful PCR samples were sent for sequencing using 96well plates to the University of Florida DNA Sequencing Core Lab at the Interdisciplinary Center for Biotechnology Research (ICBR). The software Geneious v.R6 (Biomatters, Ltd., NZ) was used to annotate and perform sequences alignment using the MUSCLE algorithm (Edgar, 2004). Sequences with fewest discrepancies and greatest matches were inserted into the Basic Local Alignment Search Tool (BLAST) in the National Center for Biotechnology Information (NCBI) database. Empirical sequences were compared to known sequences using a threshold of < 2% difference. For example, if the difference between two sequences was Table 1. Environmental Descriptions: Hillside Cave Descriptions Site 1 Site 2 Site 3 Relative Humidity 87.40% 75.90% 74.20% Temperature 27.7C 26.3C 26.9C Bats Present No Yes No Species of Bat Present N/A T. brasiliensis, E. fuscus N/A Approximate Bat Count N/A 750 1000, 1 N/A Level of disturbance Low Low Low A dditional Observations No obvious biofilm, mossy walls Clear walls, obvious guano Wet soil, far from entrance Note Sites with bats present had obvious traces of guano and each site varied in temperature and relative humidity. There were m ore samples taken from sites without bats present. This cave had relatively low levels of disturbance
M ELINA G. M ARTE D R D AVID L. R EED & J. A NGEL S OTO C ENTENO University of Florida | Journal of Undergraduate Research | Volume 15, Issue 3 | Summer 2014 4 greater than 2%, then they were considered different organisms. Rarefaction curves were created to determine the potential species richness of bacteria and fungi based on number of PCR samples. A phylogenetic approach was used to determine t he relationships of samples collected in caves with known samples obtained from BLAST, which allow ed preliminary identification of the potential pathogens found in each cave. Phylogenetic trees were generated using a Neighbor Joining (N J) algorithm and th e JukesCantor model of nucleotide evolution to determine the relationship between field collected samples and their closely related sequences from BLAST ( Saitou &Nei, 1987 ). RESULTS Using gel electrophoresis, single, double, and faint bands were classif ied as successful whereas clean PCRs were classified as having one visible band or collection of same size DNA fragments. Clean PCRs were selected and sent for sequencing and the remaining were discarded. Ratios of clean to successful PCRs for each cave were as follows: Hamiltons = 8/15; Hillside = 18/18. Aligned sequences were chosen for searching in the NCBI database using BLAST. Ratios of aligned to total sequences for each cave were as follows: Hamiltons = 8/15 (bacteria: 2/8, fungi: 6/7); Hillside cave = 25/36 (bacteria: 11/18, fungi: 14/18). Potential diversity could not be determined based on an individual cave due to the limited number of clean PCRs that could be sequenced. Instead, rarefaction curves represented the combined number of putative s pecies of bacteria and fungi sequenced in both caves (Figure 2). Fungi were more adequately sampled than bacteria, yet the potential diversity in both caves remains underestimated. Note. Rarefaction curves assessed the species richness from the results of sampling. Each line represented the number putative bacterial and fungal species sequenced in each PCR over time. The curves demonstrated that fungal sampling in both caves was more representative of putative species richness than bacterial sampling. Species richness based on cave could not be determined due to the limited number of successful and clean PCRs used, which may imply underestimation of diversity in these Bahamian caves. Phylogenetic analysis of empirical and BLAST sequences revealed two dist inct higher level groups for bacteria and fungi (Figures 3 & 4). Each phylogram had divergent bacterial and fungal samples that did not align well. Seven bacterial sequences formed a clade not closely related to BLAST sequences and five aligned within cert ain bacterial groups (Figure 3). Five fungal sequences could not be described; however, 13 samples clustered within certain fungal groups (Figure 4). One of the total aligned fungal sequences could not be categorized at any taxonomic level due to discrepancies within the clade. For Hamiltons Cave, one bacterial sequence was closely re lated to the Family Enterobacteriaceae (soil, bats present). No wall samples from Hamiltons Cave could be identified for bacteria. The closely related fungal sequences for Hamiltons Cave included: genus MicroascusFamily Microascaceae (soil, bats present) Family Trichomaceae (soil & wall, bats present), phylum Ascomycota (wall, bats present). Hillside Cave yielded related bacterial sequences to the following: genus Pseudomonas (soil, bats present), genus Bacillus (soil & wall, bats present), Family Microb acteriaceae (wall, bats present). Closely related fungal sequences for Hillside Cave included: genus MicroascusFamily Microascaceae (soil, bats present), genus Penicillium (wall, bats absent), family Nectriaceae (soil, bats present). DISCUSSION As the literature suggests, the organic nature of caves creates an ideal reservoir for potentially pathogenic agents. It is important to note that there are a myriad of contributing factors describing Bahamian caves as natural en vironments for harboring microorganisms in addition to the presence of bats, ecological conditions, and human disturbance. As indicated by multiple gel electrophoresis bands (fragmented DNA of variable sizes), general primers were amplifying many unknown s pecies especially for Hamiltons Cave. This greatly limited sequences that could be analyzed and consequently, rarefaction curves could not asses diversity based on each cave. Instead, curves demonstrated the total bacterial and fungal richness which conti nues to be underestimated in both caves. This has implications for public health because generally the greater Figure 2 Rarefaction c urve Note Rarefaction curves assessed the species richness from the results of sampling. Each line represented the number putative bacterial and fungal species sequenced in each PCR over time. The curves demonstrated that fungal sampling in both caves was more rep resentative of putative species richness than bacterial sampling. Species richness based on cave could not be determined due to the limited number of successful and clean PCRs used, which may imply underestimation of diversity in these Bahamian caves. 0 2 4 6 8 10 12 1 3 5 7 9 11 13 15 17 19 Putative Species PCR Number Bacteria Fungi
I DENTIFICATION OF M ICROBIAL D IVERSITY AND P OTENTIAL P ATHOGENS IN B AHAMIAN C AVES University of Florida | Journal of Undergraduate Research | Volume 15, Issue 3 | Summer 2014 5 Figure 3 Empirical and BLAST sequences phylogram: bacteria. Note. N J phylograms showing the relationships between empirical and related sequences from BLAST. Bacterial sequences were related to many known opportunistic pathogens
M ELINA G. M ARTE D R D AVID L. R EED & J. A NGEL S OTO C ENTENO University of Florida | Journal of Undergraduate Research | Volume 15, Issue 3 | Summer 2014 6 Figure 4 Empirical and BLAST sequences phylogram: fungi Note N J phylograms showing the relationships between empirical and related sequences from BLAST. Fungal sequences were related to many known opportunistic pathogens. One fungal sequence could not be identified due to taxonomic discrepancies.
I DENTIFICATION OF M ICROBIAL D IVERSITY AND P OTENTIAL P ATHOGENS IN B AHAMIAN C AVES University of Florida | Journal of Undergraduate Research | Volume 15, Issue 3 | Summer 2014 7 the microbial diversity, the greater the potential threat for pathogen exposure. Congruent with Jurado et al. (2010) both caves yielded a variety of similarly related sequences to bacteria and fungi known to cause opportunistic infections (Tabl es 3 & 4). For instance, fungal sequences appeared within a sister clade to the genus Penicillium or Aspergillus, Family Tricoco maceae (Figure 4 ). Members of this family are clinically significant given that some species cause infectious conditions such as Penicilliosis or Aspergillosis. These conditions result in skin lesions or respiratory impairments demonstrating the potential hazard of inhalation and direct contact with contaminated cave sediments. In addition, one sequence aligned well with a bacteria clade of the genus Pseudomonas (Figure 3) A species within this genus is known to cause an antibiotic resistant opportunistic i nfection which could be dangerous at the population level. Because this study was the first to explore microbiological diversity in Bahamian caves there were a number of limitations. General primers presented a major issue for PCR because they allowed for amplification of many unidentifiable microbial species. It was difficult to isolate one microorganism per sequence due to the similarities among various species in the BLAST database. In addition, logistic constrains prevented cloning, which would have fu rther identified species with more accuracy and certainty. Future studies should use specific primers to isolate various genes using cloning as verification in order to identify certain pathogenic species. Table 3 Samples and Pathogen Description: Hillside Cave Sample Cave Bats Type Identification Clinical Significance Wall Hillside Yes Bacterium Genus: Bacillus May cause ear infection, meningitis, urinary tract infections, and septicemia Wall Hillside No Fungus Genus : Penicillium May result in penicilliosis infection in immune compromised hosts Wall Hillside No Bacterium Family: Microbacteriaceae Genus causes diseases in mammals such as Tuberculosis and Leprosy Wall Hillside Yes Fungus Family: Trichocomaceae May result in infections such as penicilliosis or aspergillosis in immune compromised hosts Wall Hillside No Fungus Phylum: Ascomycota Includes genera that cause candidiasis and skin infections in immune compromised hosts Wall Hillside Yes Fungus Phylum: Ascomycota Includes genera that cause candidiasis and skin infections in immune compromised hosts Soil Hillside No Fungus Family: Microascaceae (Sister to Microascus ) Significant antibiotic resistant pathogenic agent especially in immune compromised hosts Soil Hillside No Bacterium Genus: Bacillus May cause ear infection, meningitis, urinary tract infections, and septicemia Soil Hillside No Bacterium Genus: Pseudomonas Includes species that is an emerging opportunistic pathogen causing infection Soil Hillside Yes Fungus Family: Nectriaceae Includes genus Fusarium which may cause opportunistic infections Soil Hillside No Fungus Not Identifiable N/A Note Hillside Cave yielded related and diverse bacteria and fungi that could lead to opportunistic infections especially to indi viduals who are immune compromised.
M ELINA G. M ARTE D R D AVID L. R EED & J. A NGEL S OTO C ENTENO University of Florida | Journal of Undergraduate Research | Volume 15, Issue 3 | Summer 2014 8 Table 4. Samples and Pathogen Description: Hamiltons Cave Sample Cave Bats Type Identification Clinical Significance Soil Hamilton's Yes Fungus Family: Microascaceae (Sister to Microascus ) Significant antibiotic resistant pathogenic agent in immune compromised hosts; includes species that causes nail infection ( onchomycosis) Soil Hamilton's Yes Bacterium Family:Enterobacteriaceae Causes opportunistic infections, related to other common pathogens such as Salmonella and E. coli Soil Hamilton's Yes Fungus Family: Trichocomaceae May result in infections such as penicilliosis or aspergillosis in immune compromised hosts Wall Hamilton's No Fungus Phylum: Ascomycota Includes genera that cause candidiasis and skin infections in immune compromised hosts Wall Hamiltons Yes Fungus Family: Trichocomaceae May result in infections such as penicilliosis or aspergillosis in immune compromised hosts Wall Hamiltons Yes Fungus Family: Trichocomaceae May result in infections such as penicilliosis or aspergillosis in immune compromised hosts Wall Hamiltons Yes Fungus Phylum: Ascomycota Includes genera that cause candidiasis and skin infections in immune compromised hosts Note Hamiltons Cave yielded related and diverse opportunistic bacteria and fungi that could lead to opportunistic infections especially to individuals who are immunecompromised. REFERENCE S Bengis, R. G., Leighton, F. A., Fischer, J. R., Artois, M., & Mrner, T. (2004). The role of wildlife in emerging and re emerging zoonoses Recent emerging zoonoses Viral zoonoses. Africa 23(2), 497 511. Doctor Fungus. (2013)T he Fungi Descriptions. Retrieved from: http://www.doctorfungus.org/thefungi/Microascus.php Edgar, R.C. ( 2004 ) MUSCLE: Multiple sequence alignment with high accuracy and high throughput. Nucleic Acid Research 32(5):1792 1797. Field, H E. (2009). Bats and emerging zoonoses: henipaviruses and SA RS. Zoonoses and P ublic H ealth 56 (6 7), 278 84. doi:10.1111/j.1863 2378.2008.01218.x Huppert, G. N. ( 1995 ) Legal protection of caves in the United States. Environmental Geology 26 :121 123. Jlg, B., Elias, J., Zahn, A., Kppen, S., Becker Gaab, C., & Bogner, J. R. (2008). Bat associated histoplasmosis can be transmitted at entrances of bat caves and not only inside the caves. Journal of T ravel M edicine 15 (2), 133 6. doi:10.1111/j.1708 8305.2008.00193.x Jurado, Valme, Laiz, L., Rodriguez nava, V., Boi ron, P., Sanchezmoral, S., & Saiz jimenez, C. (2010). Pathogenic and opportunistic microorganisms in caves. International Journal Of Speleology 39 (January), 15 24. Kenyon College. (2011). Retrieved March 10, 2013 from the MicrobeWiki:http://microbewiki.k enyon.edu/index.php/MicrobeWiki Kunz, T. H., R. Hodgkison, & C. Weise. ( 2009 ) Methods f or capturing and handling bats. In T. H. Kunz & S. Parsons (Eds). Eological and behavioral methods for the study of bats ( 2nd ed) Location: The John Hopkins Universi ty Press. Li, L., Victoria, J. G., Wang, C., Jones, M., Fellers, G. M., Kunz, T. H., & Delwart, E. (2010). Bat guano virome: predominance of dietary viruses from insects and plants plus novel mammalian viruses. Journal of V irology 84 (14), 6955 65. doi:10.1128/JVI.00501 10 Novkov, A. (2009). Microscopic fungi isolated from the Domica Cave system ( Slovak Karst National Park Slovakia ). A review. International Journal Of Speleology 38 (January), 71 82. Roche, B., & Gugan, J.F. (2011). Ecosystem dynamics, biological diversity and emerging infectious diseases. Comptes R endus B iologies 334 (5 6), 385 92. doi:10.1016/j.crvi.2011.02.008 Rupprecht, C., Wang, L., & Real, L.A. (2008). Bat Zoonoses: The Realities. In G. Smith & A.M. Kelly (Eds.), Food Se curity in Global Economy: Veterinary Medicine and Public Health (pp.145 154). Location:University of Pennsylvania Press. Saitou, N. & Nei, M. (1987). The neighbor joining method: A new method for reconstructing phylogenetic trees. Molecular Biology and Evo lution. 4(4):406 425. Universal Protein Resource (UniProt). (2002 2013).Taxonomy. Retrieved f rom http://www.uniprot.org/taxonomy/85023 Van der Poel, W.H., Lina, & P.H., Kramps, J.A. (2006). Public health awareness of emerging zoonotic viruses of bats: a European perspective.