Phylogenetic characterization, antimicrobial susceptibilities, and mechanisms of resistance in bacteria isolates from a poultry waste-polluted river, southwestern Nigeria
Bacteria resistance to antibiotics has become a worldwide problem impacted partly by antibiotics use in livestock production. The present study investigated the pattern and mechanisms of resistance in 21 bacteria species isolated from a river used as a waste-dump by a poultry farm in Ogbomoso, southwestern Nigeria. Bacteria were isolated on nutrient agar supplemented with 50 µg/mL of tetracycline, their identities were determined by PCR amplification, and sequencing of their 16s rRNA genes and their susceptibility to 6 antimicrobials determined by broth dilution. The molecular basis of resistance among the isolates was also investigated by the PCR amplification of class 1 and 2 integrons, tetA, tetB, tetC, tetD, tetE, tetG, tetL, tetW, tetM, tet37, blaTEM, aadA1, strA, strB, sul2, sul3 and a 550bp gene fragment encoding a multidrug resistance efflux transporter of the resistance nodulation division (RND) family. All the isolates were resistant to the tested antimicrobials with MIC ranging from
Phylogenetic characterization, antimicrobial susceptibilities, and mechanisms of resistance in bacteria isolates from a poultry waste-polluted river, southwestern Nigeria
Bacteria resistance to antibiotics has become a worldwide problem impacted partly by antibiotics use in livestock production. The present study investigated the pattern and mechanisms of resistance in 21 bacteria species isolated from a river used as a waste-dump by a poultry farm in Ogbomoso, southwestern Nigeria. Bacteria were isolated on nutrient agar supplemented with 50 µg/mL of tetracycline, their identities were determined by PCR amplification, and sequencing of their 16s rRNA genes and their susceptibility to 6 antimicrobials determined by broth dilution. The molecular basis of resistance among the isolates was also investigated by the PCR amplification of class 1 and 2 integrons, tetA, tetB, tetC, tetD, tetE, tetG, tetL, tetW, tetM, tet37, blaTEM, aadA1, strA, strB, sul2, sul3 and a 550bp gene fragment encoding a multidrug resistance efflux transporter of the resistance nodulation division (RND) family. All the isolates were resistant to the tested antimicrobials with MIC ranging from
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- Witte W. Medical consequences of antibiotics use in agriculture. Science 297: 996-997, 1998.
- Wegener HC. Antibiotics in animal feed and their role in resistance development. Current Opinion in Microbiology 6: 439-445, 2003. 3. Pei R, Kim S, Carlson KH et al. Eff ect of river landscape on the sediment concentrations of antibiotics and corresponding antibiotic resistance genes (ARG). Water Research 40: 2427- 2435, 2006
- Aarestrup FM, Duran CO, Bruch DGS. Antimicrobial resistance in swine production. Animal Health Research Reviews 9: 135-148, 2008. 5. Smith SI, Aboaba OO, Odeigha P et al. Plasmid profi le of E. coli0157:H7 from apparently healthy animals. African Journal of Biotechnology 2: 322-324, 2003. 6. Kabir J, Umoh VJ, Audu–Okoh E et al. Veterinary drug use in poultry farms and determination of antimicrobial drug residues in commercial eggs and slaughtered chicken in Kaduna State, Nigeria. Food Control 15: 99-105, 2004.
- Parveen S, Lukasik J, Scott TM et al. Geographical variation in antibiotic resistance profi les of Escherichia coli isolated from swine, poultry, beef and dairy cattle farm water retention ponds in Florida. J Appl Microbiol 100: 50-57, 2006.
- Mannion C, Lynch PB, Egan J et al. Seasonal eff ects on the survival characteristics of Salmonella Typhimurium and Salmonella Derby in pig slurry during storage. J Appl Microbiol 103: 1386-1392, 2007.
- Kummerer K. Resistance in the environment. J Antimicrob Chemother 54: 311-320, 2004.
- Kummerer K. Signifi cance of antibiotics in the environment. J Antimicrob Chemother 52: 5-7, 2003.
- European Committee for Antimicrobial Susceptibility Testing. Determination of minimum inhibitory concentrations (mics) of antibacterial agents by broth dilution. Clin Microbiol Infect 9: 1-7, 2003.
- Adelowo OO, Fagade OE. Th e tetracycline resistance gene tet39 is present in both gram negative and gram positive bacteria from a polluted river, southwestern Nigeria. Lett Appl Microbiol 48: 167-172, 2009.
- Kado CI, Liu ST. Rapid procedure for detection and isolation of large and small plasmids. J Bacteriol 145: 1365-1373, 1981.
- Peirano G, Agerso Y, Aarestrup FM et al. Occurrence of integrons and antimicrobial resistance genes among Salmonella enterica from Brazil. J Antimicrob Chemother 58: 305-309, 2006.
- Agerso YA Sandvang D. Class 1 integrons and tetracycline resistance genes in Alcaligenes, Arthrobacter, and Pseudomonas spp. Isolated from pigsties and manured soil. Appl Environ Microbiol 71: 7941-7947, 2005.
- Meguro N, Kodama Y, Gallegos MT et al. Molecular characterization of resistance – nodulation – division transporters from solvent- and drug-resistant bacteria in petroleum contaminated soil. Appl Environ Microbiol 71: 580- 586, 2005.
- Th ompson JD, Higgins DG, Gibson TJ. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specifi c gap penalties and weight matrix choice. Nucleic Acids Res 22: 4673-4680, 1994.
- Saitou N, Nei M. Th e neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Bio Evol 4: 406-425, 1987.
- Kumar S, Tamura K, Nei M. MEGA3: Integrated soft ware for molecular evolutionary genetics analysis and sequence alignment. Briefi ngs in Bioinformatics 5: 150-163, 2004.
- Kumar A, Schweizer HP. Bacterial resistance to antibiotics: active effl ux and uptake. Advance Drug Delivery Review 57: 1486-1513, 2005.
- Pleydell EJ, Brown PE, Woodward MJ et al. Sources of variation in the ampicillin-resistant Escherichia coli concentration in the feces of organic broiler chickens. Appl Environ Microbiol 73: 203-210, 2007.
- Akinbowale OL, Peng M, Barton MD. Antimicrobial resistance in bacterial isolated from aquacultural sources in Australia. J Appl Microbiol 100: 1103-1113, 2006.
- Mazel D. Integrons: agents of bacterial evolution. Nature Reviews Microbiology, 4: 608-620, 2006.
- Poole K. Effl ux mediated multiresistance in gram negative bacteria. Clin Microbiol Infect 10: 12-26, 2004.
- Stavri M, Piddock LJV, Gibbons S. Bacterial effl ux pump inhibitors from natural sources. J Antimicrob Chemother 59: 1247-1260, 2006.
- Mahamoud A, Chevalier J, Alibert–Franco S et al. Antibiotic effl ux pumps in gram negative bacteria: the inhibitor response strategy. J Antimicrob Chemother 59: 1223-1229, 2007.
- Van Bambeke, F, Balzi E, Tulkens PM. Antibiotic effl ux pumps. Biochem Pharmacol 60: 457-470, 2000.
- Walsh C. Antibiotics: actions, origins, resistance. ASM Press, Washington D.C. 329pp, 2003.