spo0A Gene Determination in Spore-Forming Bacteria Infections
Shler Ali Khorsheed 1*
Journal of Angiotherapy 8(6) 1-6 https://doi.org/10.25163/angiotherapy.869737
Submitted: 03 April 2024 Revised: 31 May 2024 Published: 04 June 2024
Abstract
Background: Endospore-forming Formicates (EFF) are prevalent in natural and man-made environments, posing significant contamination risks in hospitals and industrial facilities. Hospital-acquired infections often involve spore-forming bacteria, which are resistant to common disinfectants and treatments. Understanding and identifying these bacteria, particularly the genes responsible for spore formation, is crucial for infection control. The spo0A gene is a key regulator of endospore formation. Methods: This study was conducted from January 12, 2023, to May 12, 2023, at Kirkuk General Hospital, Iraq. A total of 200 postoperative wound samples were collected, cultured, and incubated to isolate pure cultures of spore-forming bacteria. DNA was extracted using the QIAamp DNA Mini Kit, and its concentration was measured with a NanoDrop spectrophotometer. PCR and quantitative PCR (qPCR) were employed to amplify the spo0A gene using specific primers. The PCR products were analyzed via gel electrophoresis, and the bacterial species were identified through sequencing. Results: Out of 200 samples, 100 tested positive for bacterial infection. The identified bacterial species included Escherichia coli, Enterobacter cloacae, Staphylococcus aureus, Streptococcus pneumoniae, Bacillus subtilis, and Clostridium perfringens. The presence of the spo0A gene was confirmed in Bacillus subtilis and Clostridium perfringens. Gel electrophoresis results demonstrated the high quality of the PCR products, facilitating further identification and sequencing of the bacterial species. Conclusion: This study showed the significance of the spo0A gene in spore formation and demonstrated the need for accurate and rapid diagnostic methods to control hospital-acquired infections.
Keywords: Spore-forming bacteria, PCR, Spo0A gene, Hospital-acquired infections, Bacterial contamination
References
Abdelaziz, A. A., & El-Nakeeb, M. A. (1988). Sporicidal activity of local anesthetics and their binary combinations. Journal of Clinical Pharmacy, 13(3), 249-256.
Agrawal, J., & Pradhan, G. (2009). Comparative study of post-operative wound infection following emergency lower segment caesarean section with and without the topical use of fusidic acid. Nepal Medical College Journal, 11(3), 189-191.
Alejandro, M. G., et al. (2020). Quantification of DNA through the NanoDrop spectrophotometer: Methodological validation using standard reference material and Sprague Dawley rat and human DNA. International Journal of Analytical Chemistry, 2020, Article ID 8896738, 9 pages.
Alves, M. A. V., et al. (2009). Principles and applications of polymerase chain reaction in medical diagnostic fields: A review. Brazilian Journal of Microbiology, 40(1), 1-11.
Anand, A. R., Madhavan, H. N., & Therese, K. L. (2000). Use of polymerase chain reaction (PCR) and DNA probe hybridization to determine the Gram reaction of the infecting bacterium in the intraocular fluids of patients with endophthalmitis. Journal of Infection, 41(3), 221-226.
Anand, A. R., Madhavan, H. N., & Therese, K. L. (2000). Use of polymerase chain reaction (PCR) and DNA probe hybridization to determine the Gram reaction of the infecting bacterium in the intraocular fluids of patients with endophthalmitis. Journal of Infection, 41(3), 221-226.
Barbut, F., et al. (2000). Epidemiology of recurrences or reinfections of Clostridium difficile-associated diarrhea. Journal of Clinical Microbiology, 38(6), 2386-2388.
Bej, A. K., Mahbubani, M. H., & Atlas, R. M. (1991). Amplification of nucleic acids by polymerase chain reaction (PCR) and other methods and applications. Critical Reviews in Biochemistry and Molecular Biology, 26(3-4), 301-334.
Bhuiya, F., Niska, R., & Xu, J. (2010). National Ambulatory Medical Care Survey: Emergency Department Summary. National Center for Health Statistics; Hyattsville, MD.
Boyce, J. M., & Pittet, D. (2002). Guideline for hand hygiene in health-care settings: Recommendations of the Healthcare Infection Control Practices Advisory Committee and the HICPAC/SHEA/APIC/IDSA Hand Hygiene Task Force. MMWR Recommendations and Reports, 51(RR-16), 1-45.
Boyce, J. M., & Pittet, D. (2002). Healthcare Infection Control Practices Advisory Committee, Hand Hygiene Task Force. Guideline for hand hygiene in health-care settings. Recommendations of the healthcare infection control practices advisory committee and the hand hygiene task force. MMWR Recommendations and Reports, 51(RR-16), 1-45.
Centers for Disease Control and Prevention. (2010). National Center for Health Statistics. National Hospital Ambulatory Medical Care Survey. Emergency Department Summary Tables. Available at: http://www.cdc.gov/nchs/data/nhamcs/web_tables.pdf. Accessed on: February 15, 2014.
Cheadle, W. G., & Qadan, M. (2009). Common microbial pathogens in surgical practice. Surgical Clinics of North America, 89(2), 295-310, vii.
Chen, G., Kumar, A., Wyman, T. H., & Moran Jr, C. P. (2006). Spo0A-dependent activation of an extended-10 region promoter in Bacillus subtilis. Journal of Bacteriology, 188(4), 1411-1418.
Chomczynski, N., & Sacchi, P. (1987). Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Analytical Biochemistry, 162(1), 156-159.
Coates, G. A. J., Ayliffe, D., & Hoffman, P. N. (1984). Chemical disinfection in hospitals. Public Health Laboratory Service, London.
Cooper, G. M., Jones, J. C., Arbique, G. J., Flowerdew, G. J., & Forward, K. R. (2000). Intra and inter technologist variability in the quality assessment of respiratory tract specimens. Diagnostic Microbiology and Infectious Disease, 37(3), 231-235.
Cutting, S. M., & Hong, H. A. (2005). The use of bacterial spore formers as probiotics. FEMS Microbiology Reviews, 29(4), 813-835.
Eichenberger, P., De Hoon, M. J. L., & Vitkup, D. (2010). Hierarchical evolution of the bacterial sporulation network. Current Biology, 20(18), R735-R745.
Errington, J. (2001). Septation and chromosome segregation during sporulation in Bacillus subtilis. Current Opinion in Microbiology, 4(6), 660-666.
Errington, J. (2003). Regulation of endospore formation in Bacillus subtilis. Nature Reviews Microbiology, 1(2), 117-126.
Fagerlund, A., Stenfors Arnesen, L. P., & Granum, P. E. (2008). From soil to gut: Bacillus cereus and its food poisoning toxins. FEMS Microbiology Reviews, 32(4), 579-606.
Gallagher, S. R. (1994). Quantitation of DNA and RNA with absorption and fluorescence spectroscopy. Current Protocols in Human Genetics, 0(1), A.3D.1-A.3D.8.
Hariram, U., & Labbé, R. (2015). Spore prevalence and toxigenicity of Bacillus cereus and Bacillus thuringiensis isolates from U.S. retail spices. Journal of Food Protection, 78(3), 590-596.
Hugenholtz, P., Finegold, S. M., & Tringe, S. G. (2008). Renaissance for the pioneering 16S rRNA gene. Current Opinion in Microbiology, 11(5), 442-446.
Isibor, O. J., Oseni, A., & Eyaufe, A. (2008). Incidence of aerobic bacteria and Candida albicans in post-operative wound infections. African Journal of Microbiology Research, 2(12), 288-291.
Jongerius, E., Koopman, M. J., & Mossel, D. A. (1967). Enumeration of Bacillus cereus in foods. Applied Microbiology, 15(3), 650-653.
Kimura, Y., Sasahara, T., Ae, R., Watanabe, M., Yonekawa, C., Hayashi, S., & Morisawa, Y. (2016). Contamination of healthcare workers' hands with bacterial spores. Journal of Infection and Chemotherapy, 22(8), 521-525.
Kirkpatrick, F. H. (1991). Overview of agarose gel properties. In Electrophoresis of large DNA molecules: theory and applications (pp. 9-22).
Knutsson, R., Ehling-Schulz, M., & Scherer, S. (2011). Bacillus cereus. In S. Kathariou, P. Fratamico, & Y. Liu (Eds.), Genomes of Food- and Water-Borne Pathogens (pp. 147-164). ASM Press.
Knutsson, R., Ehling-Schulz, M., & Scherer, S. (2011). Bacillus cereus. In S. Kathariou, P. Fratamico, & Y. Liu (Eds.), Genomes of Food- and Water-Borne Pathogens (pp. 147-164). ASM Press.
Knutsson, R., Ehling-Schulz, M., & Scherer, S. (2011). Bacillus cereus. In S. Kathariou, P. Fratamico, & Y. Liu (Eds.), Genomes of Food- and Water-Borne Pathogens (pp. 147-164). ASM Press.
Koopman, M. J., Mossel, D. A., & Jongerius, E. (1967). Enumeration of Bacillus cereus in foods. Applied Microbiology, 15(3), 650-653.
Kubista, M., Andrade, J. M., Bengtsson, M., Forootan, A., Jonak, J., Lind, K., Sindelka, R., Sjoback, R., Sjogreen, B., Strombom, L., Stahlberg, A., & Zoric, N. (2006). The real-time polymerase chain reaction. Molecular Aspects of Medicine, 27(2-3), 95-125.
Lau, S. K., Woo, P. C., Teng, J. L., Tse, H., & Yuen, K. Y. (2008). Then and now: Use of 16S rDNA gene sequencing for bacterial identification and discovery of novel bacteria in clinical microbiology laboratories. Clinical Microbiology and Infection, 14(10), 908-934.
Lee, P. Y., Kim, Y. H., Costumbrado, J., & Hsu, C. Y. (2012). Agarose gel electrophoresis for the separation of DNA fragments. Journal of Visualized Experiments, (62), e3923.
Longo, M. C., Berninger, M. S., & Hartley, J. L. (1990). Use of uracil DNA glycosylase to control carryover contamination in polymerase chain reactions. Gene, 93(1), 125-128.
Losick, R., & Stragier, P. (1996). Molecular genetics of sporulation in Bacillus subtilis. Annual Review of Genetics, 30, 297-341.
Ludwig, W., Krieg, N. R., Rainey, F. A., Schleifer, K.-H., & Whitman, W. B. (Eds.). (2009). Bergey’s manual of systematic bacteriology (2nd ed.). Springer.
Manzoor, S., Niazi, A., Asari, S., Bejai, S., Meijer, J., & Bongcam-Rudloff, E. (2014). Genome analysis of Bacillus amyloliquefaciens subsp. plantarum UCMB5113: A rhizobacterium that improves plant growth and stress management. PLoS ONE, 9(8), e104651.
Mazmanian, S. K., Debelius, J., Sharon, G., Garg, N., Knight, R., & Dorrestein, P. C. (2014). Specialized metabolites from the microbiome in health and disease. Cell Metabolism, 20(5), 719-730.
Medical Disability Guidelines. (2010). Wound infection, postoperative. Available at: http://www.mdguidelines.com/woundinfection-postoperative. Accessed on: June 24, 2010.
Meyer, T. E. (2002). Evolutionary analysis by whole-genome comparisons. Journal of Bacteriology, 184(8), 2260-2272.
Piggot, P. J., & Coote, J. G. (1976). Genetic aspects of bacterial endospore formation. Bacteriological Reviews, 40(4), 908-962.
Pittet, D., Allegranzi, B., & Boyce, J. (2009). World Health Organization World Alliance for Patient Safety First Global Patient Safety Challenge Core Group of Experts. The World Health Organization guidelines on hand hygiene in health care and their consensus recommendations. Infection Control & Hospital Epidemiology, 30(7), 611-622.
Raju, D., Sarker, M. R., Paredes-Sabja, D., & Torres, J. A. (2008). Role of small, acid-soluble spore proteins in the resistance of Clostridium perfringens spores to chemicals. International Journal of Food Microbiology, 122(3), 333-335.
Reith, S., & Richardson, J. F. (1993). Characterization of a strain of methicillin-resistant Staphylococcus aureus (EMRSA-15) by conventional and molecular methods. Journal of Hospital Infection, 25(1), 45-52.
Rhodehamel, E. J., Tallent, S. M., Harmon, S. M., & Bennett, R. W. (2012). Bacteriological analytical manual (BAM): Methods for specific pathogens. U.S. Food and Drug Administration. Chapter 14: Bacillus cereus.
Rhodehamel, E. J., Tallent, S. M., Harmon, S. M., & Bennett, R. W. (2012). Bacteriological analytical manual (BAM): Methods for specific pathogens. U.S. Food and Drug Administration. Chapter 14: Bacillus cereus.
Russell, A. D. (1990). Bacterial spores and chemical sporicidal agents. Clinical Microbiology Reviews, 3(2), 99-119.
Sambrook, J., & Russell, D. W. (2001). Molecular cloning: A laboratory manual (3rd ed.). Cold Spring Harbor Laboratory Press.
Setlow, P. (2006). Spores of Bacillus subtilis: Their resistance to and killing by radiation, heat, and chemicals. Journal of Applied Microbiology, 101(3), 514-525. https://doi.org/10.1111/j.1365-2672.2005.02736.x
Sogin, M. L., Relman, D. A., Dethlefsen, L., Huse, S. (2008). The pervasive effects of an antibiotic on the human gut microbiota, as revealed by deep 16S rRNA sequencing. PLoS Biology, 6(11), e280.
Strain, E. A., Kotewicz, K. M., Tallent, S. M., & Bennett, R. W. (2012). Efficient isolation and identification of Bacillus cereus group. Journal of AOAC International, 95(2), 446-451.
Sutter, V. L., Citron, D., & George, W. L. (1979). Selective and differential medium for isolation of Clostridium difficile. Journal of Clinical Microbiology, 9(2), 214-219.
Tripathi, D. K., Chauhan, D. K., Singh, V. P., & Kumar, D. (2012). Impact of exogenous silicon addition on chromium uptake, growth, mineral elements, oxidative stress, antioxidant capacity, and leaf and root structures in rice seedlings exposed to hexavalent chromium. Acta Physiologiae Plantarum, 34(1), 279-289.
Wagar, E. A. (1996). Direct hybridization and amplification applications for the diagnosis of infectious diseases. Journal of Clinical Laboratory Analysis, 10(6), 312-325.
Wolcott, M. J. (1992). Advances in nucleic acid-based detection methods. Clinical Microbiology Reviews, 5(4), 370-386.
Wunderlin, T., Roussel-Delif, L., Junier, T., Jeanneret, N., & Junier, P. (2013). Stage 0 sporulation gene A as a molecular marker to study diversity of endospore-forming Firmicutes. Environmental Microbiology Reports, 5(6), 911-924.
Yutin, N., & Galperin, M. Y. (2013). A genomic update on clostridial phylogeny: Gram-negative spore formers and other misplaced clostridia. Environmental Microbiology, 15(10), 2631-2641.
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