Antibiotic susceptibility patterns of heavy metal-tolerant Gram-negative bacteria isolated from a major dumpsite in Ibadan, Nigeria

Abstract

Objective: To determine the antibiotic susceptibility patterns of heavy metal-tolerant Gram-negative bacteria isolated from a major dumpsite in Ibadan, Nigeria.
Methods: Gram-negative bacteria were isolated using MacConkey and eosin methylene blue agar and presumptive identification to species level was done using conventional methods. The isolates were screened for their tolerance to heavy metals. Antimicrobial susceptibility was tested by Kirby-Bauer disc diffusion method with the following antibiotics: tetracycline (10 µg), cotrimoxazole (25 µg), gentamicin (10 µg), cefuroxime (30 µg), chloramphenicol (10 µg), ceftriaxone (30 µg), cefotaxime (30 µg), ciprofloxacin (5 µg), amikacin (30 µg), ceftazidime (30 µg), and meropenem (10 µg). The zones of growth inhibition were interpreted based on the Clinical Laboratory Scientific Institute. Identification of the most metal-tolerant isolates was done using 16S rRNA.
Results: A total of 79 Gram-negative bacteria were isolated, 31 of which tolerated a minimum of three heavy metals (Pb at 10 350 µg/mL; Cu at 750 µg/mL; Cr at 750 µg/mL in the wet season and Pb at 2 350 µg/mL; Cu at 450 µg/mL; Cr at 300 µg/mL in the dry season). All 31 isolates showed varying degrees of multiple antibiotic resistance indexes. The isolates showed the highest resistance to cefuroxime and meropenem (100%) and the least resistance to chloramphenicol (51.6%). The most metal-tolerant isolates were identified as Aeromonas hydrophila and Pseudomonas aeruginosa.
Conclusions: This study revealed that the waste dumpsite contains significant amounts of heavy metals and antibiotic-resistant bacteria which may pose serious public health risks if they enter the food chain. The study highlighted the health risks of using untreated dumpsite soils for vegetable farming due to pathogen and heavy metal transfer, and it is unsuitable for agricultural use.

Keywords

Heavy metals / Dumpsites / Antibiotic resistance / Gram-negative bacteria

Conflict of interest statement

The authors declare that there is no conflict of interest.

Acknowledgement

The authors appreciate Prof. Olutoyin Fashae of the Department of Geography, University of Ibadan, Nigeria for assisting with developing the map using the cordinates from sample site.

Funding

This study receives no extramural funding.

Data availability statement

The data supporting the findings of this study are available from the corresponding author upon request.

Authors’ contributions

Nwadike BI and Barwa J conceptualized the study and designed the experimental frame work. Barwa J performed the laboratory experiments and Nwadike BI supervised data collection and analysis. Barwa J was involved in preliminary draft and Nwadike BI in critical review of manuscript. Nwadike BI and Barwa J read, revised and approved the final manuscript for publication.

References

[1] Tse AC, Adamu CI. Assessment of anthropogenic influence on quality of groundwater in hand-dug wells in parts of Makurdi metropolis, North Central Nigeria. Ife J Sci 2012; 14(1): 123-135.

[2] Cortez LAS, Ching JA. Heavy metal concentration of dumpsite soil and accumulation in Zea mays (corn) growing in a closed dumpsite in Manila, Philippines. Int J Environ Sci Dev 2014; 5(1): 77-80.

[3] Akmal T, Jamil F. Investigating correlations between illness and defensive behavior approach: A case of twin cities of Pakistan. Heliyon 2021; 7(6): e07327.

[4] Jannetto PJ, Cowl CT. Elementary overview of heavy metals. Clin Chem 2023; 69(4): 336-349.

[5] Olayiwola HA, Abudulawal L, Adewuyi GK, Azeez MO. Heavy metal contents in soil and plants at dumpsites: A case study of Awotan and Ajakanga dumpsite, Ibadan, Oyo State, Nigeria. J Environ Earth Sci 2017; 7(4): 11-24.

[6] Nath S, Paul P, Roy R, Bhattacharjee S, Deb B. Isolation and identification of metal-tolerant and antibiotic-resistant bacteria from soil samples of Cachar district of Assam, India. SN Appl Sci 2019; 1: 727.

[7] González HS, Ghneim-Herrera T. Heavy metals in soils and the remediation potential of bacteria associated with the plant microbiome. Front Environ Sci 2021; 9: 604216.

[8] Nnaji ND, Anyanwu CU, Miri T, Onyeaka H. Mechanisms of heavy metal tolerance in bacteria: A review. Sustainability 2024; 16(24): 11124.

[9] Ahemad M, Malik A. Prevalence of heavy metal and antibiotic resistance in bacterial isolates from metal polluted soils. Microbiol J 2014; 4(1): 12-21.

[10] Antimicrobial Resistance Collaborators. Global burden of bacterial antimicrobial resistance in 2019: A systematic analysis. Lancet 2022; 399: 629-655.

[11] Huang C, Tang Z, Xi B, Tan W, Guo W, Wu W, et al. Environmental effects and risk control of antibiotic resistance genes in the organic solid waste aerobic composting system: A review. Front Environ Sci Eng 2021; 15(6): 1-12.

[12] Tian M, He X, Feng Y, Wang W, Chen H, Gong M, et al. Pollution by antibiotics and antimicrobial resistance in livestock and poultry manure in China, and countermeasures. Antibiotics (Basel) 2021; 10(5): 539.

[13] Manyi-Loh C, Mamphweli S, Meyer E, Okoh A. Antibiotic use in agriculture and its consequential resistance in environmental sources: Potential public health implications. Molecules (Basel) 2018; 23(4): 795.

[14] Iwu CD, Korsten L, Okoh AI. The incidence of antibiotic resistance within and beyond the agricultural ecosystem: A concern for public health. Microbiol Open 2020; 9(9): e1035.

[15] Kraemer SA, Ramachandran A, Perron GG. Antibiotic pollution in the environment: From microbial ecology to public policy. Microorganisms 2019; 7(6): 180.

[16] Gomes MP. The convergence of antibiotic contamination, resistance, and climate dynamics in freshwater ecosystems. Water 2024; 16(18): 2606.

[17] Nwobodo DC, Ugwu MC, Anie CO, Al-Ouqaili MTS, Ikem JC, Chigozie UV, et al. Antibiotic resistance: The challenges and some emerging strategies for tackling a global menace. J Clin Lab Anal 2022; 36(9): e24655.

[18] Kumawat M, Nabi B, Daswani M, Viquar I, Pal N, Sharma P, et al. Role of bacterial efflux pump proteins in antibiotic resistance across microbial species. Microb Pathog 2023; 181: 106182.

[19] Wales AD, Davies RH. Co-selection of resistance to antibiotics, biocides, and heavy metals, and its relevance to foodborne pathogens. Antibiotics (Basel) 2015; 4(4): 567-604.

[20] Murray LM, Hayes A, Snape J, Gaze WH, Murray AK. Co-selection for antibiotic resistance by environmental contaminants. NPJ Antimicrob Resist 2024; 2(1): 1-13.

[21] Serwecińska L. Antimicrobials and antibiotic-resistant bacteria: A risk to the environment and to public health. Water 2020; 12(12): 3313.

[22] Courvalin P. Why is antibiotic resistance a deadly emerging disease? Clin Microbiol Infect 2016; 22(5): 405-407.

[23] Munita JM, Arias CA. Mechanisms of antibiotic resistance. Microbiol Spectr 2016; 4(2): 10.1128/microbiolspec.VMBF-0016-2015.

[24] Elfadadny A, Ragab RF, AlHarbi M, Badshah F, Ibáñez-Arancibia E, Farag A, et al. Antimicrobial resistance of Pseudomonas aeruginosa: Navigating clinical impacts, current resistance trends, and innovations in breaking therapies. Front Microbiol 2024; 1: 1374466.

[25] Turnbull PCB. Medical Microbiology. 4th edition. University of Texas Medical Branch: Galveston; 1996.

[26] Nirbhavane HM, Bagde US. Resistance by Enterobacter spp. towards several antimicrobial drugs and heavy metals: A review. Afr J Biotechnol 2017; 16(16): 826-841.

[27] Environmental Protection Agency. Acid Digestion of Sediments, Sludges, and Soils. [Online] Available from: https://www.epa.gov/sites/default/files/2015-06/documents/epa-3050b.pdf. [Accessed on 30 May 2024].

[28] Alnaimat S, Abushattal S. Laboratory manual in general microbiology for undergraduate students (short version). [Online] Available from: https://www.researchgate.net/publication/257380059_Laboratory_Manual_in_General_Microbiology_For_Undergraduate_Students_Short_Version. [Accessed on 23 May 2024].

[29] Fawole MO, Oso BA. Characterization of bacteria: Laboratory manual of microbiology. Spectrum Book Ltd.: Ibadan; 2004, p. 24.

[30] Singh V, Chauhan P, Kanta R, Dhewa T, Kumar V. Isolation and characterization of Pseudomonas resistant to heavy metals contaminants. Int J Pharm Sci Rev Res 2014; 3(2): 164-167.

[31] Hudzicki J. Kirby-Bauer disk diffusion susceptibility test protocol. [Online] Available from: https://asm.org/protocols/kirby-bauer-disk-diffusion-susceptibility-test-pro. [Accessed on 19 May 2024].

[32] Baccer R, Kirby M, Sherris J, Turek M. Antibiotic susceptibility testing by standard single disc diffusion method. Am J Clin Pathol 1966; 45: 493-496.

[33] Clinical and Laboratory Standards Institute. Performance standards for antimicrobial susceptibility testing-34th edition. [Online] Available from: https://webstore.ansi.org/standards/CLSI/clsim100ed34. [Accessed on 19 May 2024].

[34] Frank JA, Reich CI, Sharma S, Weisbaum JS, Wilson BA, Olsen GJ. Critical evaluation of two primers commonly used for amplification of bacterial 16S rRNA genes. Appl Environ Microbiol 2008; 74(8): 2461-2470.

[35] Sharma N, Singh G, Sudarsan Y, Singh SK, Malik M. An improved method for extraction of soil microbial DNA for metagenomics study. J Adv Res Agric Sci Technol 2019; 2(2): 1-4.

[36] Osmani M, Bani A, Hoxha B. Heavy metals and Ni phytoextraction in the metallurgical area soils in Elbasan. Albanian J Agric Sci 2015; 14(4): 414-419.

[37] National Environmental Standards and Regulations Enforcement Agency. First Eleven Gazetted Regulations Federal Republic of Nigeria Official Gazette. 2020. [Online] Available from: https://www.nesrea.gov.ng/wp-content/uploads/2020/02/Food_Beverages_and_Tobacco_Sector_Regulation%202009.pdf. [Accessed on 30 May 2024].

[38] World Weather Forecasts and Climate Information. Ibadan, Nigeria, weather and climate. [Online] Available from: https://weather-and-climate.com/average-monthly-Rainfall-Temperature-Sunshine,Ibadan,Nigeria. [Accessed on 30 May 2024].

[39] Magnano San Lio R, Favara G, Maugeri A, Barchitta M, Agodi A. How antimicrobial resistance is linked to climate change: An overview of two intertwined global challenges. Int J Environ Res Public Health 2023; 20(3): 1681.

[40] National Library of Medicine. Cadmium and cadmium compounds. [Online] Available from: https://www.ncbi.nlm.nih.gov/books/NBK304372/. [Accessed on 30 May 2024].

[41] Centers for Diseases Control and Prevention. National Biomonitoring Program 2021. [Online] Available from: https://www.cdc.gov/biomonitoring/Cadmium_FactSheet.html. [Accessed on 30 May 2024].

[42] Dogo RB, Tanko AA, Ajibade GA. Assessment of the heavy metals and physicochemical properties of soils, In dumpsites and adjacent farmlands in Kafanchan Town, Kaduna, Nigeria. J Environ 2022; 2(1): 39-55.

[43] Bassey IU, Brooks AA, Asikong BE, Andy IE. Environmental and public health aspects of solid waste management at the lemna dumpsite in Calabar, Cross River state, Nigeria. Int J Trop Dis Health 2015; 10(3): 1-13.

[44] Waturu G, Mutai W, Kiiru J, Musyoki M, Ochieng L. Antimicrobial resistance encountered in garbage collection areas and dumpsites in Nairobi, Kenya using Escherichia coli and Klebsiella as indicator species. Adv Microbiol 2017; 7: 653-665.

[45] Jiang ZX, Bian HF, Xu L, Li MX, He NP. Pulse effect of precipitation: Spatial patterns and mechanisms of soil carbon emissions. Front Ecol Evol 2021; 9: 673310.

[46] Umair M, Sun N, Du H, Hui N, Altaf M, Du B, et al. Bacterial communities are more sensitive to water addition than fungal communities due to higher soil K and Na in a degraded karst ecosystem of Southwestern China. Front Microbiol 2020; 11: 562546.

[47] Li X, Yan Y, Lu X, Fu L, Liu Y. Responses of soil bacterial communities to precipitation change in the semi-arid alpine grassland of Northern Tibet. Front Plant Sci 2022; 13: 1036369.

[48] Ibrahim UB, Kawo AH, Yusuf I, Yahaya S. Physicochemical and molecular characterization of heavy metal-tolerant bacteria isolated from soil of mining sites in Nigeria. J Genet Eng Biotechnol 2021; 19: 152.

[49] Zhang Y, Zheng J. Bioinformatics of metalloproteins and metalloproteomes. Molecules 2020; 25: 3366.

[50] Tchounwou PB, Yedjou CG, Patlolla AK, Sutton DG. Heavy metals toxicity and the environment. EXS 2012; 101: 133-164.

[51] Engwa GA, Ferdinand PU, Nwalo FN, Unachukwu MN. Mechanism and health effects of heavy metal toxicity in humans. Intech Open 2019. doi: 10.5772/intechopen.82511.

[52] Sitotaw B, Ayalew F, Girma A, Geta K, Kibret M. High prevalence of antibiotic resistance bacteria isolated from Bahir Dar city municipal solid waste dumpsite, North West Ethiopia. Environ Health Insights 2024; 18: 11786302241260508.

[53] Parmanik A, Das S, Kar B, Bose A, Dwivedi GR, Pandey MM. Current treatment strategies against multidrug-resistant bacteria: A review. Curr Microbiol 2022; 79(12): 388.

[54] Nguyen CC, Hugie CN, Kile ML, Navab-Daneshmand T. Association between heavy metals and antibiotic-resistant human pathogens in environmental reservoirs: A review. Front Environ Sci Eng 2019; 13: 46. doi.org/ 10.1007/s11783-019-1129-0.

[55] Joseph OJ, Ogunleye GE, Oyinlola KA, Balogun AI, Olumeko DT. Co-occurrence of heavy metals and antibiotics resistance in bacteria isolated from metal-polluted soil. Environ Anal Health Toxicol 2023; 38(4): e2023024-0.

[56] Hurst M, Lamb HM. Meropenem. Drugs 2000; 59(3): 653-680.

[57] Adesoji AT, Onuh JP, Palang IP, Liadi AM, Musa S. Prevalence of multi-drug resistant Pseudomonas aeruginosa isolated from selected residential sewages in Dutsin-Ma, Katsina State, Nigeria. J Public Health Afr 2023; 14(2): 2152.

[58] Hassuna NA, Darwish MK, Sayed M, Ibrahem RA. Molecular epidemiology and mechanisms of high-level resistance to meropenem and imipenem in Pseudomonas aeruginosa. Infect Drug Resist 2020; 13: 285-293.

[59] Sampah J, Owusu-Frimpong I, Aboagye FT, Owusu-Ofori A. Prevalence of carbapenem-resistant and extended-spectrum beta-lactamase-producing Enterobacteriaceae in a teaching hospital in Ghana. PloS One 2023; 18(10): e0274156.

[60] Rohana H, Hager-Cohen A, Azrad M, Peretz A. Trend of changes in chloramphenicol resistance during the years 2017-2020: A retrospective report from Israel. Antibiotics (Basel) 2023; 12(2): 196.

[61] Mackuľak T, Cverenkárová K, Vojs Staňová A, Fehér M, Tamáš M, Škulcová AB, et al. Hospital wastewater-source of specific micropollutants, antibiotic-resistant microorganisms, viruses, and their elimination. Antibiotics (Basel) 2021; 10(9): 1070.

[62] Larsson DGJ, Flach CF. Antibiotic resistance in the environment. Nat Rev Microbiol 2022; 20(5): 257-269.

[63] Zhu HS, Liang X, Liu JC, Zhong HY, Yang YH, Guan WP, et al. Antibiotic and heavy metal co-resistant strain isolated from enrichment culture of marine sediments, with potential for environmental bioremediation applications. Antibiotics 2023; 12(9): 1379.