Molecular Detection of Clostridium-Related DNA in Bats from Soppeng, Indonesia

  • Muhammad Erwan Arifin Department of Environmental Health, Faculty of Public Health, Universitas Hasanuddin Makassar, Sulawesi Selatan, Indonesia
  • Syamsuar Manyullei Department of Environmental Health, Faculty of Public Health, Universitas Hasanuddin Makassar, Sulawesi Selatan, Indonesia
  • Anwar Mallongi Department of Environmental Health, Faculty of Public Health, Universitas Hasanuddin Makassar, Sulawesi Selatan, Indonesia
  • Erniwati Ibrahim Department of Environmental Health, Faculty of Public Health, Universitas Hasanuddin Makassar, Sulawesi Selatan, Indonesia
  • Apik Indarty Moedjiono Department of Biostatics, Faculty of Public Health, Universitas Hasanuddin Makassar, Sulawesi Selatan, Indonesia
Keywords: Bats, Clostridium Spp., Zoonotic Disease, Health Promotion, 16S rRNA, One Health

Abstract

Introduction: Clostridium spp. are anaerobic, spore-forming bacteria widely distributed in soil, water, and the gastrointestinal tracts of humans and animals. Several species are associated with important human and veterinary diseases. Information regarding Clostridium-related bacteria in bats remains limited in Indonesia. This study aimed to generate preliminary molecular evidence of Clostridium-related bacterial DNA in bats from Soppeng Regency, South Sulawesi, Indonesia.

Methods: Bat samples were collected in Soppeng Regency, South Sulawesi, Indonesia. Ten fruit bats (Cynopterus brachyotis) were captured near roosting and foraging sites, and three intestinal tissue samples were selected for molecular analysis based on sample integrity and DNA preservation quality. Genomic DNA was extracted using a commercial extraction kit following aseptic sample processing. Molecular detection was performed using PCR targeting the bacterial 16S rRNA gene, followed by agarose gel electrophoresis. PCR-positive amplicons were subjected to Sanger sequencing and analyzed using BLAST against the NCBI/GenBank database based on sequence identity, query coverage, and E-value.

Results: All three analyzed samples produced positive amplification bands at the expected amplicon size of approximately 1,465 bp. BLAST analysis showed the closest sequence matches to Clostridium septicum (K1; 94% query cover; 97.57% identity; E=0.0), Paraclostridium sordellii (K3; 97% query cover; 96.64% identity; E=0.0), and Clostridium haemolyticum (K5; 95% query cover; 98.06% identity; E=0.0).

Conclusion: This study provides preliminary molecular evidence of Clostridium-related bacterial DNA in fruit bats from Soppeng Regency. However, DNA detection alone does not confirm bacterial viability, pathogenicity, transmission potential, or reservoir status. Further studies involving culture confirmation, toxin gene characterization, environmental sampling, and broader wildlife surveillance are needed to clarify the ecological and public health significance of these findings.

References

Daszak P, Cunningham AA, Hyatt AD. Emerging infectious diseases of wildlife--threats to biodiversity and human health. Science. 2000 Jan;287(5452):443–9. https://doi.org/10.1126/science.287.5452.443

Jones KE, Patel NG, Levy MA, Storeygard A, Balk D, Gittleman JL, et al. Global trends in emerging infectious diseases. Nature. 2008 Feb;451(7181):990–3. https://doi.org/10.1038/nature06536

Taylor LH, Latham SM, Woolhouse ME. Risk factors for human disease emergence. Philos Trans R Soc London Ser B, Biol Sci. 2001 Jul;356(1411):983–9. https://doi.org/10.1098/rstb.2001.0888

Calisher CH, Childs JE, Field HE, Holmes K V, Schountz T. Bats: important reservoir hosts of emerging viruses. Clin Microbiol Rev. 2006 Jul;19(3):531–45. https://doi.org/10.1128/cmr.00017-06

Brook CE, Dobson AP. Bats as “special” reservoirs for emerging zoonotic pathogens. Trends Microbiol. 2015 Mar;23(3):172–80. https://doi.org/10.1016/j.tim.2014.12.004

Hayman DTS. Bats as Viral Reservoirs. Annu Rev Virol. 2016 Sep;3(1):77–99. https://doi.org/10.1146/annurev-virology-110615-042203

Schountz T. Immunology of bats and their viruses: challenges and opportunities. Viruses. 2014 Dec;6(12):4880–901. https://doi.org/10.3390/v6124880

Luis AD, Hayman DTS, O’Shea TJ, Cryan PM, Gilbert AT, Pulliam JRC, et al. A comparison of bats and rodents as reservoirs of zoonotic viruses: are bats special? Proceedings Biol Sci. 2013 Apr;280(1756):20122753. https://doi.org/10.1098/rspb.2012.2753

Lotfy WM. Bats (Chiroptera) as Vectors of Diseases and Parasites: Facts and Myths: A Book Review. Open Parasitol J. 2020;8(1):10–1. http://dx.doi.org/10.2174/1874421402008010010

Schloss PD, Handelsman J. Status of the microbial census. Microbiol Mol Biol Rev. 2004 Dec;68(4):686–91. https://doi.org/10.1128/mmbr.68.4.686-691.2004

Songer JG. Clostridial enteric diseases of domestic animals. Clin Microbiol Rev. 1996 Apr;9(2):216–34. https://doi.org/10.1128/cmr.9.2.216

Stevens DL, Aldape MJ, Bryant AE. Life-threatening clostridial infections. Anaerobe. 2012 Apr;18(2):254–9. https://doi.org/10.1016/j.anaerobe.2011.11.001

Popoff MR, Bouvet P. Clostridial toxins. Future Microbiol. 2009 Oct;4(8):1021–64. https://doi.org/10.2217/fmb.09.72

Shrestha A, Uzal FA, McClane BA. Enterotoxic Clostridia: Clostridium perfringens Enteric Diseases. Microbiol Spectr. 2018 Sep;6(5). https://doi.org/10.1128/microbiolspec.gpp3-0003-2017

Rusman R, Ainun NR, Mannyullei S, Amqam H. Identification of Ectoparacite and Endoparacite in Rat at Tamangapa Landfill Makassar City. Hasanuddin J Public Heal [Internet]. 2021 Feb 28;2(1 SE-Articles):32–41. Available from: https://journal.unhas.ac.id/index.php/hjph/article/view/12191

Untari H, Setiyono A, Handharyani E, Padaga M, Astuti D, Setiyaningsih S. Molecular and histological detection of Coxiella burnetii in ruminants in East Java, Indonesia. Iraqi J Vet Sci. 2024;38(2):285–92 https://doi.org/10.33899/ijvs.2023.142008.3153.

Clarridge JE 3rd. Impact of 16S rRNA gene sequence analysis for identification of bacteria on clinical microbiology and infectious diseases. Clin Microbiol Rev. 2004 Oct;17(4):840–62, table of contents. https://doi.org/10.1128/cmr.17.4.840-862.2004

Janda JM, Abbott SL. 16S rRNA gene sequencing for bacterial identification in the diagnostic laboratory: pluses, perils, and pitfalls. J Clin Microbiol. 2007 Sep;45(9):2761–4. https://doi.org/10.1128/jcm.01228-07

Woo PCY, Lau SKP, Teng JLL, Tse H, Yuen K-Y. Then and now: use of 16S rDNA gene sequencing for bacterial identification and discovery of novel bacteria in clinical microbiology laboratories. Clin Microbiol Infect Off Publ Eur Soc Clin Microbiol Infect Dis. 2008 Oct;14(10):908–34 https://doi.org/10.1111/j.1469-0691.2008.02070.x.

Gentles AD, Guth S, Rozins C, Brook CE. A review of mechanistic models of viral dynamics in bat reservoirs for zoonotic disease. Pathog Glob Health. 2020 Dec;114(8):407–25. https://doi.org/10.1080/20477724.2020.1833161

Allam TA, Abdel-kader F, Kadry M. Isolation, toxin gene profiling, and phylogenetic analysis of Clostridium perfringens in Egyptian fruit bats: public health and epidemiological implications. Sci Rep [Internet]. 2025;15(1):40354. Available from: https://doi.org/10.1038/s41598-025-26288-3

Bandelj P, Knapi? T, Rousseau J, Podgorelec M, Presetnik P, Vengust M, et al. Clostridioides difficile in bat guano. Comp Immunol Microbiol Infect Dis. 2019 Aug; 65:144–7. https://doi.org/10.1016/j.cimid.2019.05.016

Soto-López JD, Fernández-Soto P, Muro A. Bacterial Composition Across Bat Species: A Human Health Perspective. Vol. 15, Animals. 2025. p. 3126. https://www.mdpi.com/2076-2615/15/21/3126#

Klimpel S, Mehlhorn H. Bats (Chiroptera) as vectors of diseases and parasites. Springer; 2016. https://doi.org/10.1007/978-3-642-39333-4

Luna N, Páez-Triana L, Ramírez AL, Muñoz M, Goméz M, Medina JE, et al. Microbial community dynamics in blood, faeces and oral secretions of neotropical bats in Casanare, Colombia. Sci Rep [Internet]. 2024;14(1):25808. Available from: https://doi.org/10.1038/s41598-024-77090-6

Bennett AJ, Suski CD, O’Keefe JM. Molecular epidemiology of Eimeria spp. parasites and the faecal microbiome of Indiana bats (Myotis sodalis): a non-invasive, multiplex metabarcode survey of an endangered species. Microb genomics. 2025 Feb;11(2). https://doi.org/10.1099/mgen.0.001358

Jian Z, Ying L, Tao G, Jingzhu Z, Fengming C, Shijun L. Investigating the gut bacteria structure and function of hibernating bats through 16S rRNA high-throughput sequencing and culturomics. mSystems [Internet]. 2025 Apr 9;10(5): e01463-24. Available from: https://doi.org/10.1128/msystems.01463-24

Satilmis S, Ulger Toprak N, Ilg?n C, Soyletir G. Evaluation of direct 16S rRNA PCR from clinical samples for bacterial detection in normally sterile body sites. J Infect Dev Ctries. 2019 Nov;13(11):978–83. https://doi.org/10.3855/jidc.11732

Bouchiat C, Ginevra C, Benito Y, Gaillard T, Salord H, Dauwalder O, et al. Improving the Diagnosis of Bacterial Infections: Evaluation of 16S rRNA Nanopore Metagenomics in Culture-Negative Samples. Front Microbiol. 2022;13(July):1–13. https://doi.org/10.3389/fmicb.2022.943441

Han D, Gao P, Li R, Tan P, Xie J, Zhang R, et al. Multicenter assessment of microbial community profiling using 16S rRNA gene sequencing and shotgun metagenomic sequencing. J Adv Res. 2020 Nov; 26:111–21. https://doi.org/10.1016/j.jare.2020.07.010

Corthals A, Martin A, Warsi OM, Woller-Skar M, Lancaster W, Russell A, et al. From the Field to the Lab: Best Practices for Field Preservation of Bat Specimens for Molecular Analyses. PLoS One [Internet]. 2015 Mar 23;10(3): e0118994. Available from: https://doi.org/10.1371/journal.pone.0118994

Youssef N, Boutros CF, Dakroub F, Akl F, Reslan L, Finianos M, et al. The clinical impact of 16S ribosomal RNA PCR and sequencing in the identification of bacterial infections: a 7-year report from a Lebanese tertiary care center. Front Cell Infect Microbiol. 2025;15(August):1–16. https://doi.org/10.3389/fcimb.2025.1619640

Mu X, Guo J, Wang H, Li Y, Yuan K, Xu H, et al. Establishment and preliminary application of PCR-RFLP genotyping method for Giardia duodenalis in goats. BMC Vet Res [Internet]. 2024;20(1):527. Available from: https://doi.org/10.1186/s12917-024-04386-0

John D V., Aryalakshmi B, Deora H, Purushottam M, Raju R, Mahadevan A, et al. Identification of microbial agents in culture-negative brain abscess samples by 16S/18S rRNA gene PCR and sequencing. Trop Biomed. 2022;39(4):489–98. https://doi.org/10.47665/tb.39.4.002

Shaw P, Cao H, Lo Y. Authentication of Chinese Medicinal Materials By Dna Technology: Techniques and Applications. Second Edi. London, UK: World Scientific Publishing; 2023. https://doi.org/10.1142/4700

Published
2026-07-16
How to Cite
Muhammad Erwan Arifin, Manyullei, S., Anwar Mallongi, Erniwati Ibrahim, & Apik Indarty Moedjiono. (2026). Molecular Detection of Clostridium-Related DNA in Bats from Soppeng, Indonesia. Media Publikasi Promosi Kesehatan Indonesia (MPPKI), 9(7), 1417-1425. https://doi.org/10.56338/mppki.v9i7.11276
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