Prevalence, Molecular Characterization and Antibiogram Susceptibility Pattern of <i>Clostridioides difficile</i> from Food Samples in South Eastern Nigeria
DOI:
https://doi.org/10.26538/tjpps/v4i3.4Keywords:
South Eastern Nigeria, prevalence, Clostridioides difficile, Antibiogram, Molecular characterizationAbstract
Clostridioides difficile is a foodborne bacterium that causes severe gastrointestinal infections due to its virulence and antibiotic resistance. The major reason for this research is to ascertain Clostridioides difficile prevalence, molecular characterization, and antibiogram patterns in food samples from southeast Nigeria. A total of 440 food samples, including smoked fish and pork, were analyzed between June 2018 and December 2019. Enumeration of total anaerobes was performed using standard bacteriological techniques, while Clostridioides difficile isolation was carried out on selective differential agar. Biochemical identification was confirmed using molecular methods. The Kirby-Bauer disc diffusion was done to ascertain antibiogram susceptibility, and PCR activity was carried out to identify resistance gene (tetS, tetA, and ermB) and virulence (tcdA, tcdB, cdtA, and cdtB). Anaerobic bacterial counts varied across states, ranging from 1.85±0.12 log10 CFU/g in Enugu to 2.15±0.03 log10 CFU/g in Imo. Smoked fish and pork exhibited higher counts, with values between 5.16±0.01 and 5.36±0.01 log10 CFU/g. Identified anaerobes included Lysinibacillus macroides, Clostridium bolteae, Clostridium butyricum, and Clostridioides difficile. The prevalence of Clostridioides difficile was 2.00%, with isolates showing resistance to tetracycline (73.91%), erythromycin (73.91%), and ciprofloxacin (43.48%). Multiple antibiotic resistance was recorded at a rate of 0.44. Binary toxin genes (cdtA and cdtB) were found at low levels, 69.56% expressed tcdA, and all isolates of Clostridioides difficile carried the tcdB gene. Although rare in the area, binary toxin genes still pose a risk of severe Clostridioides difficile infections. This study emphasizes the significance of ongoing monitoring and controlling antibiotic resistance in foodborne bacteria.
References
Borji S, Kadivarian S, Dashtbin S, Kooti S, Abiri R, Motamedi H, Moradi J, Alvandi A. Global prevalence of Clostridioides difficile in 17,148 food samples from 2009 to 2019: a systematic review and meta-analysis. J Health Popul Nutr. 2023; (https://jhpn.biomedcentral.com/articles/10.1186/s41043-023-00369-3).
Lim SC, Collins DA, Imwattana K, Knight DR, Perumalsamy S, Hain-Saunders N, Riley TV. Whole-genome sequencing links Clostridium (Clostridioides) difficile in a single hospital to diverse environmental sources in the community. J ApplMicrobiol. 2022; 133(3): 1156-1168.
Khan MSA, Ahmad I. Pathogenic biofilms in environment and industrial setups and impact on human health. In: Understanding Microbial Biofilms. Academic Press; 2023. p. 587-604.
4. Liu X, Li W, Zhang W, Wu Y, Lu J. Molecular characterization of Clostridium difficile isolates in China from 2010 to 2015. Front Microbiol. 2018; (https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2018.00845/full)
Licciardi C, Primavilla S, Roila R, Lupattelli A, Farneti S, Blasi G, Petruzzelli A, Drigo I, Di RaimoMarrocchi E. Prevalence, molecular characterization, and antimicrobial susceptibility of Clostridioides difficile isolated from pig carcasses and pork products in Central Italy. Int J Environ Res Public Health. 2021;18, 11368. (https://www.mdpi.com/1660-4601/18/21/11368).
Gallo M, Ferrara L, Calogero A, Montesano D, Naviglio D. Relationships between food and diseases: What to know to ensure food safety. Food Res Int. 2020; 137:109414.
Adejumo AC, Adejumo KL, Pani LN. Risk and outcomes of Clostridium difficile infection with chronic pancreatitis. Pancreas. 2019; 48(8): 1041-1049.
Panhwar A, Abro R, Kandhro A, Khaskheli AR, Jalbani N, Gishkori KA, Qaisar S. Global water mapping, requirements, and concerns over water quality shortages. BMC Public Health. 2022;45(7):50-55.
Rapid Microbiology. Clostridioides difficile detection and identification methods. Available from: https://www.rapidmicrobiology.com/test-method/clostridium-difficile-detection-and-identification-methods
Borriello SP, Honour P. Detection, isolation and identification of Clostridium difficile. In: Borriello SP, editor.Antibiotic associated diarrhoea and colitis. Developments in Gastroenterology, vol 5. Dordrecht: Springer; 1984. p. 37-64.
European Centre for Disease Prevention and Control (ECDC). Laboratory procedures for diagnosis and typing of human Clostridium difficile infection [Internet]. Available from: https://www.ecdc.europa.eu/sites/default/files/documents/SOPs-Clostridium-difficile-diagnosis-and-typing.pdf
George WL, Sutter VL, Citron D, Finegold SM. Selective and differential medium for isolation of Clostridium difficile. J ClinMicrobiol. 1979; 9(2): 214-9. doi: 10.1128/jcm.9.2.214-219.1979. PMID: 429542; PMCID: PMC272994.
Bauer AW, Kirby MM, Sharis JL, Turck M. Antibiotic susceptibility testing by a standard single disk method. Am J ClinPathol. 1966; 45:493-6
Sambrook J, Russell DW. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press; 2001.
Clinical and Laboratory Standards Institute. Performance standards for antimicrobial susceptibility testing. 30th ed. CLSI supplement M100. Wayne (PA): Clinical and Laboratory Standards Institute; 2020.
Chitanand MP, Kadam TA, Gyananath G, Totewad ND, Balhal DK. Multiple antibiotic resistance indexing of coliforms to identify high-risk contamination sites in aquatic environment. Indian J Microbiol. 2010; 50:216–220.
Chakravorty D, Helb D, Burday M, Connell N, Alland D. Use of an integrated MS-multiplexed MS/MS data acquisition strategy for high-coverage peptide mapping studies. Rapid Commun Mass Spectrom. 2007; 21(5): 730-744.
Smith JA, Brown PR. Amplification and detection of target genes using forward and reverse primers in agarose gel electrophoresis. J MolBiol Tech. 2022; 78(4):123–130.
Microsoft Corporation. Microsoft Excel (Computer Software). Redmond (WA): Microsoft Corporation; 2006.
Jenior ML, Leslie JL, Powers DA, Garrett EM, Walker KA, Dickenson ME, Petri WA Jr, Tamayo R, Papin JA. Novel Drivers of Virulence in Clostridioides difficile Identified via Context-Specific Metabolic Network Analysis. mSystems. 2021; 6(5): 00919-21. doi: 10.1128/msystems.00919-21.
Perez-Bou L, Gonzalez-Martinez A, Cabrera JJ, Juarez-Jimenez B, Rodelas B, Gonzalez-Lopez J, Correa-Galeote D. Design and Validation of Primer Sets for the Detection and Quantification of Antibiotic Resistance Genes in Environmental Samples by Quantitative PCR. Microb Ecol. 2024; 87:71. doi: 10.1007/s00248-024-02385-0.
Lund BM, Peck MW. A possible route for foodborne transmission of Clostridium difficile? Foodborne Pathog Dis. 2015; 12(3):183-189. doi: 10.1089/fpd.2014.1842.
Makinde OM, Ayeni KI, Sulyok M, Krska R, Adeleke RA, Ezekiel CN. Microbiological safety of ready-to-eat foods in low- and middle-income countries: A comprehensive 10-year (2009 to 2018) review. Compr Rev Food Sci Food Saf. 2020; 19:703-732.
Warriner K, Xu C, Habash M, Sultan S, Weese SJ. Dissemination of Clostridium difficile in food and the environment: Significant sources of C. difficile community-acquired infection? J ApplMicrobiol. 2016; 122(3):542-553.
Balsells E, Shi T, Leese C, Lyell I, Burrows J, Wiuff C, Campbell H, Kyaw MH, Nair H. Global burden of Clostridium difficile infections: a systematic review and meta-analysis. J Glob Health. 2018; 8(12):e010407. doi: 10.7189/jogh.09.010407.
European Centre for Disease Prevention and Control. Clostridioides difficile infections. In: ECDC. Annual epidemiological report for 2018−2020. Stockholm: ECDC; 2024.
Rodriguez C, Taminiau B, Bouchafa L, Romijn S, Van-Broeck J, Delmée M, Clercx C, Daube G. Clostridium difficile beyond stools: dog nasal discharge as a possible new vector of bacterial transmission. Heliyon. 2019; 5(5):1629.
Peng Z, Jin D, Kim HB, Stratton CW, Wu B, Sun X. Update on antimicrobial resistance in Clostridium difficile: resistance mechanisms and antimicrobial susceptibility testing. J ClinMicrobiol. 2017; 55(7):e02250-16. doi: 10.1128/jcm.02250-16.
European Centre for Disease Prevention and Control. Clostridioides (Clostridium) difficile infections. In: ECDC. Annual epidemiological report for 2016–2017. Stockholm: ECDC; 2022.
Spigaglia P, Barbanti F, Mastrantonio P, Brazier JS, Barbut F, Delmée M, Martin H, Kuijper EJ, O’Driscoll J, Allouch PY. Fluoroquinolone resistance in Clostridium difficile isolates from a prospective study of C. difficile infections in Europe. ClinMicrobiol Infect. 2016; 22(11):1027-1033. doi: 10.1016/j.cmi.2016.08.010.
Knight DR, Elliott B, Chang BJ, Perkins TT, Riley TV. Diversity and evolution in the genome of Clostridium difficile.ClinMicrobiol Rev. 2015; 28(3):721-741.
Rodriguez-Palacios A, Mo KQ, Shah BU, Msuya J, Bijedic N, Deshpande A, Ilic S. Global and Historical Distribution of Clostridioides difficile in the Human Diet (1981-2019): Systematic Review and Meta-Analysis of 21886 Samples Reveal Sources of Heterogeneity, High-Risk Foods, and Unexpected Higher Prevalence Toward the Tropic. Front Med (Lausanne). 2020; 7:1-22.
Surang C, Piyapong H, Amornrat A, Puriya N, Darunee C, Piriyaporn C, Tavan J. Evaluation of Multiplex PCR with Enhanced Spore Germination for Detection of Clostridium difficile from Stool Samples of the Hospitalized Patients. Biomed Res Int. 2013; 29(3):115-128.
Knetsch CW, Kumar N, Forster SC, Connor TR, Browne HP, Harmanus C, Sanders P, Harris SR, Lipman L, Keessen EC, Corver J, Lawley TD, Kuijper EJ. Zoonotic Transfer of Clostridium difficile Harboring Antimicrobial Resistance between Farm Animals and Humans. J ClinMicrobiol. 2018; 24(6):1053-106
Published
How to Cite
Issue
Section
License
Copyright (c) 2025 Tropical Journal of Phytochemistry and Pharmaceutical Sciences
This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.
