Class 1 Integron, Sulfonamide and Florfenicol Resistance Genes in Bacteria from Three Unsanitary Landfills, Ibadan, Nigeria

Objective: Municipal solid waste (MSW) landfills recently emerged as potential reservoirs of antibiotic resistance. However, little is known about the potentials of unsanitary landfill sites which are common in developing countries as reservoir of antibiotic resistance. In this study, we investigated the antibiotic resistance pattern and presence of selected resistance genes in bacteria from three MSW landfills in Ibadan, southwestern Nigeria. Methodology: Fifty six antibiotic- and metal-resistant bacteria isolated from leachate and leachate-contaminated surface and groundwater collected from three MSW landfills in Ibadan, Southwestern Nigeria, were searched for sulfonamides, florfenicol and beta-lactams resistance genes and integrons by PCR. Results: None of the 27 β-lactamase genes tested was detected in the isolates. sul1, sul2 and floR were however detected in 12 (21.4%), 2 (3.6%) and 12 (21.4%) bacteria species identified as Alcaligenes faecalis, Advenella kashmirensis, Brevundimonas spp. and Ralstonia pickettii. The 12 bacteria carrying sul1/floR gene combination were also positive for class 1 integron. Analysis of the gene cassettes in the variable regions of the class 1 integrons (>1.5kb) of the 12 intl1-positive isolates revealed the presence of aadB and unknown genes. The deduced amino acid sequence of the unknown portion of the gene cassettes shared 42-65% identity with sequences of Class D β-lactamase and OXA-2-like protein in the GenBank. Conclusions: Findings of this study suggest that the landfill ecosystem is a potential site for the evolution of novel resistance genes and are hence important reservoir of antibiotic resistant bacteria.  J Microbiol Infect Dis 2019; 9(1): 34-42.

___

  • 1. Pruden A, Joakim-Larsson DG, Amézquita A, et al. Management options for reducing the release of antibiotics and antibiotic resistance genes to the environment. Environ Health Perspect 2013; 121:878–885. 2. D’Costa VM, McGrann KM, Hughes DW, Wright GD. Sampling the antibiotic resistome. Science 2006; 311:374-377. 3. Finley RL, Collignon P, Joakim-Larsson DG, et al. The scourge of antibiotic resistance and the important role of the environment. Clin Infect Dis 2013; 57:704-710. 4. Knapp CW, McCluskey SM, Singh BK, Campbell CD, Hudson G, Graham DW. Basal antibiotic resistance gene abundances correlate with metal and geochemical conditions in Scottish soils. PLoS One 2011; 6:e27300. 5. Canton R. Antibiotic resistance genes from the environment: a perspective through newly identified antibiotic resistance mechanisms in the clinical setting. Clin Microbiol Infect 2009; 15 Suppl 1:20-25. 6. Baker-Austin C, Wright MS, Stepanauskas R, McArthur JV. Co-selection of antibiotic and metal resistance. Trends Microbiol 2006; 14:176–182. 7. Seiler C, Berendonk TU. Heavy metal driven co-selection of antibiotic resistance in soil and water bodies impacted by agriculture and aquaculture. Front Microbiol 2012; 3:1-10. 8. Pal C, Asiani K, Arya S, Rensing C, Stekel DJ, Joakim Larsson DG, Hobman JL. Metal resistance and its association with antibiotic resistance. Advances in Microbial Physiology 2017; 70:261-296. 9. Aruekwe A, Eggen T, Mödder M. Solid waste deposits as a significant source of contaminants of emerging concern to the aquatic and terrestrial environments- a developing country case study from Owerri, Nigeria. Sci Total Environ 2012; 438:94-102. 10. Efuntoye MO, Bakare AA, Sowunmi AA. Virulence factors and antibiotic resistance in Staphylococcus aureus and Clostridium perfringens from landfill leachate. Afr J Microbiol Res 2011; 5:3994-3997. 11. Threedeach S, Chiemchaisri W, Watanabe T, Chiemchaisri C, Honda R, Yamamoto K. Antibiotic resistance of Escherichia coli in leachates from municipal solid waste landfills: comparison between semi-anaerobic and anaerobic operations. Bioresour Technol 2012; 113:253-258. 12. Wang Y, Tang W, Qiao J, Song L. Occurrence and prevalence of antibiotic resistance in landfill leachate. Environ Sci Pollut Res 2015; 22:12525–12533. 13. Wu D, Huang Z, Yang K, Graham D, Xie B. Relationships between antibiotics and antibiotic resistance gene levels in municipal solid waste leachates in Shanghai, China. Environ Sci Technol 2015; 49:4122–4128. 14. Song L, Li L, Yang S, et al. Sulfamethoxazole, tetracycline, oxytetracycline and related antibiotic resistance genes in a large-scale landfill, China. Sci Total Environ 2016; 551-552:9-15. 15. Yu Z, He P, Shao L, Zhang H, Lü F. Co-occurrence of mobile genetic elements and antibiotic resistance genes in municipal solid waste landfill leachates: A preliminary insight into the role of landfill age. Water Research 2016; 106:583-592. 16. Zhang X-H, Xu Y-B, He X-L, et al. occurrence of antibiotic resistance genes in landfill leachate treatment plant and its effluent-receiving soil and surface water. Environmental Pollution 2016; 218:1255-1261. 17. American Public Health Association (APHA) (1998) Standard methods for the examination of water and wastewater. 20th Ed. American public Health Association Inc. Washington D.C. 18. National Environmental Standards and Regulations Enforcement Agency (NESREA) (2011) National Environmental (surface and groundwater quality control) Regulations. NESREA, Abuja, Nigeria. 19. Standards Organisation of Nigeria (SON) (2007) National Standards for Drinking Water Quality (NSDWQ). 20. CLSI. Performance standards for antimicrobial susceptibility testing; Twenty Fourth Informational Supplement (M100-S24). CLSI, 2014; Wayne, PA. USA. 21. Ünaldi Coral MN, Korkmaz H, Arikan B, Coral, G. Plasmid mediated heavy metal resistances in Enterobacter spp. Isolated from Sofulu landfill in Adana, Turkey. Ann Microbiol 2005; 55(3):175-179. 22. Peirano G, Agerso Y, Aarestrup FM, Falavina dos Reis EM, Rodirgues DP. Occurrence of integrons and antimicrobial resistance genes among Salmonella enterica from Brazil. J Antimicrob Chemother 2006; 58:305-309. 23. Wang N, Yang X, Jiao S, Zhang J, Ye B, Gao S. Sulfonamide-resistant bacteria and their resistance genes in soils fertilized with manures from Jiangsu Province, Southeastern China. PLoS One 2014; 9(11), e112626. 24. Adelowo OO, Fagade OE, Agerso YA. Antibiotic resistance and resistance genes in Escherichia coli from poultry farms, southwest Nigeria. J Infect Dev Ctries 2014; 8:1103-1112. 25. Trung NT, Hien TTT, Huyen TTT, et al. Simple multiplex PCR assays to detect common pathogens and associated genes encoding for acquired extended spectrum beta-lactamases (ESBL) or carbapenemases from surgical site specimens in Vietnam. Ann Clin Microbiol Antimicrob 2015; 14:23. 26. Voets GM, Fluit AC, Scharringa J, Stuart JC, Leverstein-van Hall MA. A set of multiplex PCRs for genotypic detection of extended-spectrum β-lactamases, carbapenemases, plasmid-mediated AmpC β-lactamases and OXA β-lactamases. Int J Antimicrob Agents 2011; 37:356-359. 27. Machado E, Canton R, Baquero F, et al. Integron content of extended-spectrum-β-lactamase-producing Escherichia coli strains over 12 years in a single hospital in Madrid, Spain. Antimicrob Agents Chemother 2005; 49(5):1823-1829. 28. Vinué L, Jove T, Torres C, Ploy MC. Diversity of class 1 integron gene cassette Pc promoter variants in clinical Escherichia coli strains and description of a new P2 promoter variant. Int J Antimicrob Agents 2011; 38:526-529. 29. Jové T, Da Re S, Denis F, Mazel D, Ploy MC. Inverse correlation between promoter strength and excision activity in class 1 Integrons. PLOS Genet 2010; 6(1): e1000793. 30. Huang L-N, Zhou H, Zhu S, Qu L-H. Phylogenetic diversity of bacteria in the leachate of a full-scale recirculating landfill. FEMS Microbiol Ecol 2004; 50:175-183. 31. Huang L-N, Zhu S, Zhou H, Qu L-H. Molecular phylogenetic diversity of bacteria associated with the leachate of a closed municipal solid waste landfill. FEMS Microbiol Lett 2005; 242:297-303. 32. Stamps BW, Lyles CN, Suflita JM, et al. Municipal solid waste landfills harbor distinct microbiomes. Front Microbiol 2016; 7:534. doi: 10.3389/fmicb.2016.00534 33. Allen SE, Boerlin P, Janecko N, et al. Antibiotic resistance in generic Escherichia coli isolates from wild small mammals living in swine farm, residential landfill, and natural environments in Southern Ontario, Canada. Appl Environ Microbiol 2011; 77:882-888. 34. Doublet B, Carattoli A, Whichard JM, et al. Plasmid-mediated florfenicol and ceftriaxone resistance encoded by the floR and blaCMY-2 genes in Salmonella enterica serovars Typhimurium and Newport isolated in the United States. FEMS Microbiol Lett 2004; 233:301-305. 35. Fernández-Alarcón C, Miranda CD, Singer RS, et al. Detection of the floR gene in a diversity of florfenicol resistant Gram-negative bacilli from freshwater salmon farms in Chile. Zoonoses Public Health 2010; 57:181-188.