Ecological and biological determinants of fecal bacterial diversity in bats across four continents and phylogenetic screening for pathogen-affiliated bacterial lineages

Abstract

Objective: To characterize the diversity and composition of bacterial communities in bat fecal samples under distinct ecological and biological conditions and to investigate the phylogenetic relationships of selected bacterial lineages with clinically relevant taxa. Methods: Publicly available 16S rRNA gene sequencing data from 194 fecal samples covering 20 bat species from South America, Europe, Asia, and Africa were reanalyzed using DADA2 and the SILVA database. Alpha and beta diversity, taxonomic composition, and community differences according to host diet, habitat, sex, and geographic region were evaluated. Phylogenetic analyses were performed on amplicon sequence variants assigned to Pseudomonas, Staphylococcus, Clostridium, and Streptococcus. Results: A total of 3 778 110 reads passed quality filtering, representing 123 bacterial families and 203 genera. Proteobacteria (57%) and Firmicutes (40%) predominated, with Pseudomonas, Staphylococcus, and Carnobacterium among the most abundant genera. Microbial composition and diversity varied significantly according to diet, habitat, sex, and geographic region. Frugivorous bats showed the highest alpha diversity, whereas piscivorous bats showed the lowest, and Rousettus aegyptiacus exhibited the greatest bacterial richness. Bats inhabiting forest and cave habitats possessed higher bacterial richness, while communities from freshwater and coastal aquatic habitats were less diverse. Males showed higher relative abundance of Staphylococcus, whereas females were enriched in Pseudomonas. Distinct taxonomic signatures were linked to dietary guilds. Insectivorous bats were enriched in Gammaproteobacteria, Pseudomonadales, and Moraxellaceae; frugivorous bats were enriched in Lactobacillales and Streptococcus; and piscivorous bats were enriched in Firmicutes, including Staphylococcaceae and Clostridium. Sixty-two genera containing species with recognized pathogenic potential were identified. Phylogenetic analyses revealed amplicon sequence variants affiliated with Pseudomonas, Clostridium, and Streptococcus clustering with or related to clinically relevant lineages, such as Clostridioides difficile and Streptococcus pneumoniae. Conclusions: Bat fecal bacterial communities are highly diverse and vary significantly under different ecological and biological conditions. Detection of bacterial genera containing clinically relevant species and the phylogenetic proximity of selected amplicon sequence variants to known pathogens support further genomic, functional, and epidemiological studies to clarify their pathogenic and zoonotic relevance.

Keywords

Bat gut microbiome / Bacterial diversity / Ecological drivers / Zoonotic potential / Chiroptera / Gut microbiota / 16S rRNA sequencing / Bacterial pathogens / One Health

References

[1] Letko M, Seifert SN, Olival KJ, Plowright RK, Munster VJ. Bat-borne virus diversity, spillover and emergence. Nat Rev Microbiol 2020; 18(8): 461-471.

[2] Szentivanyi T, McKee C, Jones G, Foster JT. Trends in bacterial pathogens of bats: Global distribution and knowledge gaps. Transbound Emerg Dis 2023; 2023. doi: 10.1155/2023/9285855.

[3] Irving AT, Ahn M, Goh G, Anderson DE, Wang LF. Lessons from the host defences of bats, a unique viral reservoir. Nature 2021; 589(7842): 363-370.

[4] Dhivahar J, Parthasarathy A, Krishnan K, Kovi BS, Pandian GN. Bat-associated microbes: Opportunities and perils, an overview. Heliyon 2023; 9(12). doi: 10.1016/j.heliyon.2023.e22351.

[5] Veikkolainen V, Vesterinen EJ, Lilley TM, Pulliainen AT. Bats as reservoir hosts of human bacterial pathogen, Bartonella mayotimonensis. Emerg Infect Dis 2014; 20(6): 960-967. doi: 10.3201/eid2006.130956.

[6] Vengust M, Knapic T, Weese JS. The fecal bacterial microbiota of bats; Slovenia. PLoS One 2018; 13(5). doi: 10.1371/journal.pone.0196728.

[7] McKee CD, Bai Y, Webb CT, Kosoy MY. Bats are key hosts in the radiation of mammal-associated Bartonella bacteria. Infect Genet Evol 2021; 89. doi: 10.1016/j.meegid.2021.104719.

[8] Corduneanu A, Wu-Chuang A, Maitre A, Obregon D, Sándor AD, Cabezas-Cruz A. Structural differences in the gut microbiome of bats using terrestrial vs. aquatic feeding resources. BMC Microbiol 2023; 23: 93. doi: 10.1186/s12866-023-02836-7.

[9] Aizpurua O, Nyholm L, Morris E, Chaverri G, Herrera Montalvo LG, Flores-Martinez JJ, et al. The role of the gut microbiota in the dietary niche expansion of fishing bats. Anim Microbiome 2021; 3(1): 76. doi: 10.1186/s42523-021-00137-w.

[10] Dietrich M, Markotter W. Studying the microbiota of bats: Accuracy of direct and indirect samplings. Ecol Evol 2019; 9(4): 1730-1735. doi: 10.1002/ece3.4842.

[11] Callahan BJ, McMurdie PJ, Rosen MJ, Han AW, Johnson AJA, Holmes SP. DADA2: High-resolution sample inference from Illumina amplicon data. Nat Methods 2016; 13(7): 581-583. doi: 10.1038/nmeth.3869.

[12] Quast C, Pruesse E, Yilmaz P, Gerken J, Schweer T, Yarza P, et al. The SILVA ribosomal RNA gene database project: Improved data processing and web-based tools. Nucleic Acids Res 2013; 41: D590-D596. doi: 10.1093/nar/gks1219.

[13] Dhariwal A, Chong J, Habib S, King IL, Agellon LB, Xia J. MicrobiomeAnalyst: A web-based tool for comprehensive statistical, visual and meta-analysis of microbiome data. Nucleic Acids Res 2017; 45(W1): W180-W188. doi: 10.1093/nar/gkx295.

[14] Foster ZSL, Sharpton TJ, Grünwald NJ. Metacoder: An R package for visualization and manipulation of community taxonomic diversity data. PLoS Comput Biol 2017; 13(2). doi: 10.1371/journal.pcbi.1005404.

[15] Menéndez ML, Pardo JA, Pardo L, Pardo MC. The Jensen-Shannon divergence. J Franklin Inst 1997; 334(2): 307-318. doi: 10.1016/S0016-0032(96)00063-4.

[16] Katoh K, Rozewicki J, Yamada KD. MAFFT online service: Multiple sequence alignment, interactive sequence choice and visualization. Brief Bioinform 2019; 20(4): 1160-1166. doi: 10.1093/bib/bbx108.

[17] Hall TA. BioEdit: A user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp Ser 1999; 41: 95-98.

[18] Kumar S, Stecher G, Tamura K. MEGA7: Molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol Biol Evol 2016; 33(7): 1870-1874. doi: 10.1093/molbev/msw054.

[19] Letunic I, Bork P. Interactive Tree Of Life (iTOL) v5: An online tool for phylogenetic tree display and annotation. Nucleic Acids Res 2021; 49(W1): W293-W296. doi: 10.1093/nar/gkab301.

[20] Federici L, Masulli M, De Laurenzi V, Allocati N. An overview of bats microbiota and its implication in transmissible diseases. Front Microbiol 2022; 13. doi: 10.3389/fmicb.2022.1012189.

[21] Morales AE, Dong Y, Brown T, Baid K, Kontopoulos DG, Gonzalez V, et al. Bat genomes illuminate adaptations to viral tolerance and disease resistance. Nature 2025; 638(8050): 449-458. doi: 10.1038/s41586-024-08471-0.

[22] Dai W, Leng H, Li J, Li A, Li Z, Zhu Y, et al. The role of host traits and geography in shaping the gut microbiome of insectivorous bats. mSphere 2024; 9(4). doi: 10.1128/msphere.00087-24.

[23] Huang Y, Sun Y, Huang Q, Lv X, Pu J, Zhu W, et al. The threat of potentially pathogenic bacteria in the feces of bats. Microbiol Spectr 2022; 10(6). doi: 10.1128/spectrum.01802-22.

[24] Aziz SA, McConkey KR, Tanalgo K, Sritongchuay T, Low MR, Yong JY, et al. The critical importance of Old World fruit bats for healthy ecosystems and economies. Front Ecol Evol 2021; 9. doi: 10.3389/fevo.2021.641411.

[25] Santos RT, Flores RJD, Montecillo AD, De Leon MP. Mapping the bacterial diversity and functional landscape of the Philippine bat gut microbiome using a 16S-based metataxonomics approach. SciEnggJ 2025; 18(2): 275-294. doi: 10.54645/2025182HWH-76.

[26] Borges RM. Interactions between figs and gall-inducing fig wasps: Adaptations, constraints, and unanswered questions. Front Ecol Evol 2021; 9. doi: 10.3389/fevo.2021.685542.

[27] Pellón JJ, Medina-Espinoza EF, Lim BK, Cornejo F, Medellín RA. Eat what you can, when you can: Relatively high arthropod consumption by frugivorous bats in Amazonian Peru. Mamm Biol 2023; 103(1): 137-144. doi: 10.1007/s42991-022-00327-2.

[28] Ingala MR, Simmons NB, Wultsch C, Krampis K, Provost KL, Perkins SL. Molecular diet analysis of neotropical bats based on fecal DNA metabarcoding. Ecol Evol 2021; 11(12): 7474-7491. doi: 10.1002/ece3.7579.

[29] Adhikari BN, Paskey AC, Frey KG, Bennett AJ, Long KA, Kuhn JH, et al. Virome profiling of fig wasps (Ceratosolen spp.) reveals virus diversity spanning four realms. Virology 2024; 591. doi: 10.1016/j.virol.2024.109992.

[30] Dong Y, Zhang ZR, Mishra S, Wong ACN, Huang JF, Wang B, et al. Diversity and metabolic potentials of microbial communities associated with pollinator and cheater fig wasps in fig-fig wasp mutualism system. Front Microbiol 2022; 13. doi: 10.3389/fmicb.2022.1009919.

[31] Tuneu-Corral C, Puig-Montserrat X, Riba-Bertolín D, Russo D, Rebelo H, Cabeza M, et al. Pest suppression by bats and management strategies to favour it: A global review. Biol Rev Camb Philos Soc 2023; 98(5): 1564-1582. doi: 10.1111/brv.12967.

[32] Zhu D, Liu Y, Gong L, Si M, Wang Q, Feng J, et al. The consumption and diversity variation responses of agricultural pests and their dietary niche differentiation in insectivorous bats. Animals (Basel) 2024; 14(5). doi: 10.3390/ani14050815.

[33] Aihartza J, Vallejo N, Aldasoro M, García-Mudarra JL, Goiti U, Nogueras J, et al. Aerospace-foraging bats eat seasonably across varying habitats. Sci Rep 2023; 13: 19576. doi: 10.1038/s41598-023-46939-7.

[34] Merenstein C, Ward J, Allen D. Diplorickettsia bacteria in an Ixodes scapularis tick, Vermont, USA. Emerg Infect Dis 2020; 26(5): 1036-1038. doi: 10.3201/eid2605.191135.

[35] Gerbáčová K, Maliničová L, Kisková J, Maslišová V, Uhrin M, Pristaš P. The faecal microbiome of building-dwelling insectivorous bats (Myotis myotis and Rhinolophus hipposideros) also contains antibiotic-resistant bacterial representatives. Curr Microbiol 2020; 77(9): 2333-2344. doi: 10.1007/s00284-020-02095-z.

[36] Jackson RT, Lunn TJ, DeAnglis IK, Ogola JG, Webala PW, Forbes KM. Frequent and intense human-bat interactions occur in buildings of rural Kenya. PLoS Negl Trop Dis 2024; 18(2). doi: 10.1371/journal.pntd.0011988.

[37] Aizpurua O, Nyholm L, Morris E, Chaverri G, Herrera Montalvo LG, Flores-Martinez JJ, et al. The role of the gut microbiota in the dietary niche expansion of fishing bats. Anim Microbiome 2021; 3(1): 76. doi: 10.1186/s42523-021-00137-w.

[38] Liu Z, Zhao Y, Sossah FL, Okorley BA, Amoako DG, Liu P, et al. Characterization, pathogenicity, phylogeny, and comparative genomic analysis of Pseudomonas tolaasii strains isolated from various mushrooms in China. Phytopathology 2022; 112(3): 521-534. doi: 10.1094/PHYTO-12-20-0550-R.

[39] Savage PM, Savage TJ, Kruse DL, Stone PA. Leclercia adecarboxylata and Pseudomonas oryzihabitans infection after Achilles tendon repair: A case report and literature review. J Foot Ankle Surg 2023; 62(4): 742-745. doi: 10.1053/j.jfas.2022.11.004.

[40] Quiroz-Morales SE, García-Reyes S, Ponce-Soto GY, Servín-González L, Soberón-Chávez G. Tracking the origins of Pseudomonas aeruginosa phylogroups by diversity and evolutionary analysis of important pathogenic marker genes. Diversity 2022; 14(5). doi: 10.3390/d14050345.

[41] Nabi G, Wang Y, Lü L, Jiang C, Ahmad S, Wu Y, et al. Bats and birds as viral reservoirs: A physiological and ecological perspective. Sci Total Environ 2021; 754. doi: 10.1016/j.scitotenv.2020.142372.

[42] Das S, Jain D, Chaudhary P, Quintela-Tizon RM, Banerjee A, Kesavardhana S. Bat adaptations in inflammation and cell death regulation contribute to viral tolerance. mBio 2025; 16(3). doi: 10.1128/mbio.03204-23.

[43] Berman TS, Weinberg M, Moreno KR, Czirják GÁ, Yovel Y. In sickness and in health: The dynamics of the fruit bat gut microbiota under a bacterial antigen challenge and its association with the immune response. Front Immunol 2023; 14. doi: 10.3389/fimmu.2023.1152107.

[44] Clayton E, Munir M. Fundamental characteristics of bat interferon systems. Front Cell Infect Microbiol 2020; 10. doi: 10.3389/fcimb.2020.527921.

[45] Irving AT, Ahn M, Goh G, Anderson DE, Wang LF. Lessons from the host defences of bats, a unique viral reservoir. Nature 2021; 589(7842): 363-370. doi: 10.1038/s41586-020-03128-0.