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Abstract

Antibiotic resistance has become a major public health problem throughout the world. The presence of antibiotic-resistant bacteria such as and antibiotic resistance genes (ARGs) in hospital wastewater is a cause for great concern today. In this study, 276 isolates were recovered from hospital wastewater samples in Malaysia. All of the isolates were screened for susceptibility to nine different classes of antibiotics: ampicillin, ciprofloxacin, gentamicin, kanamycin, erythromycin, vancomycin, trimethoprim and sulfamethoxazole, chloramphenicol, tetracycline and nalidixic acid. Screening tests showed that 100 % of isolates exhibited resistance against kanamycin, vancomycin, trimethoprim and sulfamethoxazole and nalidixic acid. Additionally, 91, 87, 50, 43, 11 and 8.7 % of isolates showed resistance against erythromycin, gentamicin, ciprofloxacin, ampicillin, chloramphenicol and tetracycline, respectively. Based on these results, 100 % of isolates demonstrated multidrug-resistant (MDR) characteristics, displaying resistance against more than three classes of antibiotics. Of 276 isolates, nine exhibited resistance to more than nine classes of tested antibiotics; these were selected for antibiotic susceptibility testing and examined for the presence of conserved ARGs. Interestingly, a high percentage of the selected MDR isolates did not contain conserved ARGs. These results indicate that non-conserved MDR gene elements may have already spread into the environment in the tropics of Southeast Asia, and unique resistance mechanisms against several antibiotics may have evolved due to stable, moderate temperatures that support growth of bacteria throughout the year.

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2016-12-21
2020-01-29
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References

  1. Ahmed O. B., Elmekki M. A., Omer E. E., Elhassan M. M.. 2014; Molecular detection of methicillin resistant Staphylococcus aureus in patients with urinary tract infections in Khartoum State. J Sci Tech15:1–8
    [Google Scholar]
  2. Al-Marjani M., Kadhim A. A., Kinani Y. A.. 2015; Ciprofloxacin resistance in Staphylococcus aureus and Pseudomonas aeruginosa isolated from patients in Baghdad. Int J Pharm Sci Res6:382–385[CrossRef]
    [Google Scholar]
  3. Al-Mendalawi M. D.. 2010; Severe community-acquired infection caused by methicillin-resistant Staphylococcus aureus in Saudi Arabian children. Saudi Med J31:461–462[PubMed]
    [Google Scholar]
  4. Al-Saimary I. E. A.. 2011; Antibiogram and multidrug resistance patterns of Staphylococcus aureus (MDRSA) associated with post-operative wound infections in Basrah – Iraq. Med Pract Rev2:66–72
    [Google Scholar]
  5. Alam M. Z., Aqil F., Ahmad I., Ahmad S.. 2013; Incidence and transferability of antibiotic resistance in the enteric bacteria isolated from hospital wastewater. Brazilian J Microbiol44:799–806 [CrossRef]
    [Google Scholar]
  6. Ali R., Al-Achkar K., Al-Mariri A., Safi M.. 2014; Role of polymerase chain reaction (PCR) in the detection of antibiotic-resistant Staphylococcus aureus. Egypt J Med Hum Genet15:293–298 [CrossRef]
    [Google Scholar]
  7. Ba X., Harrison E. M., Edwards G. F., Holden M. T., Larsen A. R., Petersen A., Skov R. L., Peacock S. J., Parkhill J. et al. 2014; Novel mutations in penicillin-binding protein genes in clinical Staphylococcus aureus isolates that are methicillin resistant on susceptibility testing, but lack the mec gene. J Antimicrob Chemother69:594–597 [CrossRef][PubMed]
    [Google Scholar]
  8. Baquero F., Martínez J. L., Cantón R.. 2008; Antibiotics and antibiotic resistance in water environments. Curr Opin Biotechnol19:260–265 [CrossRef][PubMed]
    [Google Scholar]
  9. Bignardi G. E., Woodford N., Chapman A., Johnson A. P., Speller D. C.. 1996; Detection of the mec-A gene and phenotypic detection of resistance in Staphylococcus aureus isolates with borderline or low-level methicillin resistance. J Antimicrob Chemother37:53–63 [CrossRef][PubMed]
    [Google Scholar]
  10. Börjesson S., Melin S., Matussek A., Lindgren P.-E.. 2009; A seasonal study of the mecA gene and Staphylococcus aureus including methicillin-resistant S. aureus in a municipal wastewater treatment plant. Water Res43:925–932 [CrossRef]
    [Google Scholar]
  11. Bound J. P., Voulvoulis N.. 2004; Pharmaceuticals in the aquatic environment – a comparison of risk assessment strategies. Chemosphere56:1143–1155 [CrossRef][PubMed]
    [Google Scholar]
  12. Brown K. D., Kulis J., Thomson B., Chapman T. H., Mawhinney D. B.. 2006; Occurrence of antibiotics in hospital, residential, and dairy effluent, municipal wastewater, and the Rio Grande in New Mexico. Sci Total Environ366:772–783 [CrossRef][PubMed]
    [Google Scholar]
  13. Chambers H. F., Archer G., Matsuhashi M.. 1989; Low-level methicillin resistance in strains of Staphylococcus aureus. Antimicrob Agents Chemother33:424–428 [CrossRef][PubMed]
    [Google Scholar]
  14. Choi S. M., Kim S.-H., Kim H.-J., Lee D.-G., Choi J.-H., Yoo J.-H., Kang J.-H., Shin W.-S., Kang M.-W.. 2003; Multiplex PCR for the detection of genes encoding aminoglycoside modifying enzymes and methicillin resistance among Staphylococcus species. J Korean Med Sci18:631–636 [CrossRef][PubMed]
    [Google Scholar]
  15. Chopra I., Roberts M.. 2001; Tetracycline antibiotics: mode of action, applications, molecular biology, and epidemiology of bacterial resistance. Microbiol Mol Biol Rev65:232–260 [CrossRef][PubMed]
    [Google Scholar]
  16. CLSI 2015; Performance Standards for Antimicrobial Susceptibility Testing twenty-first informational supplement CLSI document M100-S21 Wayne, PA: Clinical and Laboratory Standards Institute;
    [Google Scholar]
  17. Cloney L., Marlowe C., Wong A., Chow R., Bryan R.. 1999; Rapid detection of mecA in methicillin resistant Saphylococcus aureus using cycling probe technology. Mol Cell Probe3:191–197[CrossRef]
    [Google Scholar]
  18. Collis C. M., Grammaticopoulos G., Briton J., Stokes H. W., Hall R. M.. 1993; Site-specific insertion of gene cassettes into integrons. Mol Microbiol9:41–52 [CrossRef][PubMed]
    [Google Scholar]
  19. de Kraker M., van de Sande-Bruinsma N.. 2007; Trends in antimicrobial resistance in Europe: update of EARSS results. Euro Surveill12:E070315-070313[PubMed]
    [Google Scholar]
  20. Delepierre A., Gayot A., Carpentier A.. 2012; Update on counterfeit antibiotics worldwide; public health risks. Med Mal Infect42:247–255 [CrossRef][PubMed]
    [Google Scholar]
  21. Denys G. A., Koch K. M., Dowzicky M. J.. 2007; Distribution of resistant Gram-positive organisms across the census regions of the United States and in vitro activity of tigecycline, a new glycylcycline antimicrobial. Am J Infect Control35:521–526 [CrossRef][PubMed]
    [Google Scholar]
  22. Dingwell R. T., Leslie K. E., Duffield T. F., Schukken Y. H., DesCoteaux L., Keefe G. P., Kelton D. F., Lissemore K. D., Shewfelt W. et al. 2003; Efficacy of intramammary tilmicosin and risk factors for cure of Staphylococcus aureus infection in the dry period. J Dairy Sci86:159–168 [CrossRef][PubMed]
    [Google Scholar]
  23. Duran N., Ozer B., Duran G. G., Onlen Y., Demir C.. 2012; Antibiotic resistance genes & susceptibility patterns in staphylococci. Indian J Med Res135:89–96
    [Google Scholar]
  24. Everett M. J., Piddock L. J. V.. 1998; Mechanism of resistance to fluoroquinolones. In Quinolone Antibacterials pp.259–297 Edited by Kuhlmann J., Dahlhoff A., Zeiler H. J.. Berlin, Germany: Springer-Verlag KG;[CrossRef]
    [Google Scholar]
  25. Felmingham D., Reinert R. R., Hirakata Y., Rodloff A.. 2002; Increasing prevalence of antimicrobial resistance among isolates of Streptococcus pneumoniae from the PROTEKT surveillance study, and compatative in vitro activity of the ketolide, telithromycin. J Antimicrob Chemother50:25–37 [CrossRef][PubMed]
    [Google Scholar]
  26. Felmingham D., Cantón R., Jenkins S. G.. 2007; Regional trends in beta-lactam, macrolide, fluoroquinolone and telithromycin resistance among Streptococcus pneumoniae isolates 2001–2004. J Infect55:111–118 [CrossRef][PubMed]
    [Google Scholar]
  27. Fthenakis G. G.. 1998; Susceptibility to antibiotics of staphylococcal isolates from cases of ovine or bovine mastitis in Greece. Small Ruminant Res28:9–13 [CrossRef]
    [Google Scholar]
  28. Gao J., Ferreri M., Liu X. Q., Chen L. B., Su J. L., Han B.. 2011; Development of multiplex polymerase chain reaction assay for rapid detection of Staphylococcus aureus and selected antibiotic resistance genes in bovine mastitic milk samples. J Vet Diagn Invest23:894–901 [CrossRef][PubMed]
    [Google Scholar]
  29. Gavin D. R., Ruth M. H.. 1995; Gene cassettes: a new class of mobile element. Microbiol141:3015–3027[CrossRef]
    [Google Scholar]
  30. Giger W., Alder A. C., Gobel A., Golet E., Huber M., Joss A., Keller E., Kohler H. P., McArdell C. S. et al. 2007; Behaviour and removal of antibiotics in wastewater treatment. Presented at 1st EMCO (Emerging Contaminants) Workshop ‘Analysis and Removal of Contaminants From Wastewaters for the Implementation of the Water Framework Directive (WFD), European Union Sixth Framework Programme October 21–25, 2005. Dubrovnik, Croatia:
  31. Habibi S., Wig N., Agarwal S., Sharma S. K., Lodha R., Pandey R. M., Kapil A.. 2008; Epidemiology of nosocomial infections in medicine intensive care unit at a tertiary care hospital in northern India. Trop Doct38:233–235 [CrossRef][PubMed]
    [Google Scholar]
  32. Hartmann A., Alder A. C., Koller T., Widmer R. M.. 1998; Identification of fluoroquinolone antibiotics as the main source of umuC genotoxicity in native hospital wastewater. Environ Toxicol Chem17:377–382 [CrossRef]
    [Google Scholar]
  33. Hawraa W. A., Al-Dulaimi T., Al-Marzoqi A. H.. 2014; Phenotypic detection of resistance in Staphylococcus aureus isolates: detection of (mecA and femA) gene in methicillin resistant Staphylococcus aureus (MRSA) by polymerase chain reaction. J Nat Sci Res4:112–118
    [Google Scholar]
  34. Hiramatsu K., Kihara H., Yokota T.. 1992; Analysis of borderline-resistant strains of methicillin-resistant Staphylococcus aureus using polymerase chain reaction. Microbiol Immun36:445–453 [CrossRef]
    [Google Scholar]
  35. Holland T. L., Fowler V. G.. 2011; Vancomycin minimum inhibitory concentration and outcome in patients with Staphylococcus aureus bacteremia: pearl or pellet?. J Infect204:329–331 [CrossRef]
    [Google Scholar]
  36. Holmes A. J., Holley M. P., Mahon A., Nield B. S., Gillings M. R., Stokes H. W.. 2003; A distinctive and functional integron/gene cassette system present in soil bacterial communities associated with genomic diversity in Pseudomonas stutzeri. J Bacteriol185:918–928[CrossRef]
    [Google Scholar]
  37. Hooper D. C.. 1999; Mechanisms of fluoroquinolone resistance. Drug Resist Updat2:38–55 [CrossRef][PubMed]
    [Google Scholar]
  38. Islam Q.. 2011; Antimicrobial resistance: a man-made crisis. J Bangladesh Coll Phys Surg29:120–125
    [Google Scholar]
  39. Iversen A., Kühn I., Franklin A., Möllby R.. 2002; High prevalence of vancomycin-resistant enterococci in Swedish sewage. Appl Environ Microbiol68:2838–2842 [CrossRef][PubMed]
    [Google Scholar]
  40. Johnson A. P., Pearson A., Duckworth G.. 2005; Surveillance and epidemiology of MRSA bacteraemia in the UK. J Antimicrob Chemother56:455–462 [CrossRef][PubMed]
    [Google Scholar]
  41. Kaplan S. L., Hulten K. G., Gonzalez B. E., Hammerman W. A., Lamberth L., Versalovic J., Mason E. O. J.. 2005; Treatment of Staphylococcus aureus bacteremia in children. Clin Infect Dis40:1785–1791[CrossRef]
    [Google Scholar]
  42. Kehrenberg C., Schwarz S., Jacobsen L., Hansen L. H., Vester B.. 2005; A new mechanism for chloramphenicol, florfenicol and clindamycin resistance: methylation of 23S ribosomal RNA at A2503. Mol Microbiol57:1064–1073 [CrossRef][PubMed]
    [Google Scholar]
  43. Kemper N.. 2008; Veterinary antibiotics in the aquatic and terrestrial environment. Ecol Indic8:1–13 [CrossRef]
    [Google Scholar]
  44. Krishna S., Miller L. S.. 2012; Host–pathogen interactions between the skin and Staphylococcus aureus. Curr Opin Microbiol15:28–35 [CrossRef][PubMed]
    [Google Scholar]
  45. Krushna C. S.. 2012; Antibiotic resistance and environmental factors: focusing on the situation in Odisha, India. PhD thesis Karolinka Institute; Stockholm, Sweeden:
    [Google Scholar]
  46. Kruzel M. C., Lewis C. T., Welsh K. J., Lewis E. M., Dundas N. E., Mohr J. F., Armitige L. Y., Wanger A.. 2011; Determination of vancomycin and daptomycin MICs by different testing methods for methicillin-resistant Staphylococcus aureus. J Clin Microbiol49:2272–2273 [CrossRef][PubMed]
    [Google Scholar]
  47. Kümmerer K.. 2004; Resistance in the environment. J Antimicrob Chemother54:311–320 [CrossRef]
    [Google Scholar]
  48. Leclercq R.. 2002; Mechanisms of resistance to macrolides and lincosamides: nature of the resistance elements and their clinical implications. Clin Infec Dis34:482–492 [CrossRef]
    [Google Scholar]
  49. Leekha S., Diekema D. J., Perencevich E. N.. 2012; Seasonality of staphylococcal infections. Clin Microbiol Infect18:927–933 [CrossRef][PubMed]
    [Google Scholar]
  50. Ligozzi M., Rossolini G. M., Tonin E. A., Fontana R.. 1991; Nonradioactive DNA probe for detection of gene for methicillin resistance in Staphylococcus aureus. Antimicrob Agents Chemother35:575–578 [CrossRef][PubMed]
    [Google Scholar]
  51. Lim J.-A., Kwon A. R., Kim S. K., Chong Y., Lee K., Choi E. C.. 2002; Prevalence of resistance to macrolide, lincosamide and streptogramin antibiotics in Gram-positive cocci isolated in a Korean hospital. J Antimicrobial Chemother49:489–495 [CrossRef]
    [Google Scholar]
  52. Lowy F. D.. 2003; Antimicrobial resistance: the example of Staphylococcus aureus. J Clin Invest111:1265–1273 [CrossRef][PubMed]
    [Google Scholar]
  53. Martineau F., Picard F. J., Lansac N., Ménard C., Roy P. H., Ouellette M., Bergeron M. G.. 2000; Correlation between the resistance genotype determined by multiplex PCR assays and the antibiotic susceptibility patterns of Staphylococcus aureus and Staphylococcus epidermidis. Antimicrob Agents Chemother44:231–238 [CrossRef][PubMed]
    [Google Scholar]
  54. Meshref A. A., Omer M. K.. 2011; Detection of (mecA) gene in methicillin resistant Staphylococcus aureus (MRSA) at Prince A/Rhman Sidery. J Med Genet Genomics3:41–45
    [Google Scholar]
  55. Murakami K., Minamide W., Wada K., Nakamura E., Teraoka H., Watanabe S.. 1991; Identification of methicillin-resistant strains of staphylococci by polymerase chain reaction. J Clin Microbiol29:2240–2244[PubMed]
    [Google Scholar]
  56. Ohlsen K., Ternes T., Werner G., Wallner U., Löffler D., Ziebuhr W., Witte W., Hacker J.. 2003; Impact of antibiotics on conjugational resistance gene transfer in Staphylococcus aureus in sewage. Environ Microbiol5:711–716 [CrossRef][PubMed]
    [Google Scholar]
  57. Olayinka B. O., Olayinka A. T., Obajuluwa. A. F., Onaolapo J. A., Olurinola P. F.. 2009; Absence of mecA gene in methicillin-resistant Staphylococcus aureus isolates. Afr J Infect Dis3:49–56
    [Google Scholar]
  58. Parth P. B.. 2011; Impact of Antimicrobial Resistance (AMR) in Developing Countries North South University;
    [Google Scholar]
  59. Pechère J.-C.. 2001; Macrolide resistance mechanisms in Gram-positive cocci. Int J Antimicrob Agents18:25–28 [CrossRef]
    [Google Scholar]
  60. Potz N. A., Hope R., Warner M., Johnson A. P., Livermore D. M.. London & South East ESBL Project Group 2006; Prevalence and mechanisms of cephalosporin resistance in Enterobacteriaceae in London and South-East England. J Antimicrob Chemother58:320–326 [CrossRef][PubMed]
    [Google Scholar]
  61. Ross J. I., Eady E. A., Cove J. H., Cunliffe W. J., Baumberg S., Wootton J. C.. 1990; Inducible erythromycin resistance in staphylococci is encoded by a member of the ATP-binding transport super-gene family. Mol Microbiol4:1207–1214 [CrossRef][PubMed]
    [Google Scholar]
  62. Rowe-Magnus D. A., Guerout A. M., Biskri L., Bouige P., Mazel D.. 2003; Comparative analysis of superintegrons: engineering extensive genetic diversity in the Vibrionaceae. Genome Res13:428–442 [CrossRef][PubMed]
    [Google Scholar]
  63. Schlüter A., Szczepanowski R., Pühler A., Top E. M.. 2007; Genomics of IncP-1 antibiotic resistance plasmids isolated from wastewater treatment plants provides evidence for a widely accessible drug resistance gene pool. FEMS Microbiol Rev31:449–477 [CrossRef]
    [Google Scholar]
  64. Schwartz T., Kohnen W., Jansen B., Obst U.. 2003; Detection of antibiotic-resistant bacteria and their resistance genes in wastewater, surface water, and drinking water biofilms. FEMS Microbiol Ecol43:325–335 [CrossRef][PubMed]
    [Google Scholar]
  65. Sedlak D. L., Pinkston K. E.. 2001; Factors affecting the concentrations of pharmaceuticals released to the aquatic environment. J Contemp Water Res Educ120:56–64
    [Google Scholar]
  66. Sharma R., Sharma C. L., Kapoor B.. 2005; Antibacterial resistance: current problems and possible solutions. Indian J Med Sci59:120–129 [CrossRef][PubMed]
    [Google Scholar]
  67. Sharpe M.. 2003; High on pollution: drugs as environmental contaminants. J Environ Monit5:43–46
    [Google Scholar]
  68. Shittu A. O., Okon K., Adesida S., Oyedara O., Witte W., Strommenger B., Layer F., Nübel U.. 2011; Antibiotic resistance and molecular epidemiology of Staphylococcus aureus in Nigeria. BMC Microbiol11:92 [CrossRef][PubMed]
    [Google Scholar]
  69. Song J. H.. 2015; Antimicrobial resistance control in Asia. AMR Control
    [Google Scholar]
  70. Stokes H. W., Hall R. M.. 1989; A novel family of potentially mobile DNA elements encoding site-specific gene-integration functions: integrons. Mol Microbiol3:1669–1683 [CrossRef][PubMed]
    [Google Scholar]
  71. Strommenger B., Kettlitz C., Werner G., Witte W.. 2003; Multiplex PCR assay for simultaneous detection of nine clinically relevant antibiotic resistance genes in Staphylococcus aureus. J Clin Microbiol41:4089–4094 [CrossRef][PubMed]
    [Google Scholar]
  72. Subhankari P. S., Santanu K., Manjusri B., Somenath R.. 2011; Isolation and identification of vancomycin resistant Staphylococcus aureus from post operative pus sample. AJMS Al Ameen J Med Sci4:152–168
    [Google Scholar]
  73. Tenover F. C.. 2006; Mechanisms of antimicrobial resistance in bacteria. Am J Med34:64–73
    [Google Scholar]
  74. Thompson J. D., Higgins D. G., Gibson T. J.. 1994; clustal w: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res22:4673–4680 [CrossRef][PubMed]
    [Google Scholar]
  75. Thompson J. M., Gundogdu A., Stratton H. M., Katouli M.. 2013a; Antibiotic resistant Staphylococcus aureus in hospital wastewaters and sewage treatment plants with special reference to methicillin resistant Staphylococcus aureus (MRSA). J Appl Microbiol4:44–54[CrossRef]
    [Google Scholar]
  76. Thompson J. M., Gundogdu A., Stratton H. M., Katouli M.. 2013b; Antibiotic resistant bacteria in hospital wastewaters and sewage treatment plants. Science Forum and Stakeholder Engagement: Building Linkages, Collaboration and Science Quality225:229
    [Google Scholar]
  77. Varela A. R., André S., Nunes O. C., Manaia C. M.. 2014; Insights into the relationship between antimicrobial residues and bacterial populations in a hospital-urban wastewater treatment plant system. Water Res54:327–336 [CrossRef][PubMed]
    [Google Scholar]
  78. Wang Y., Wu C. M., Lu L. M., Ren G. W., Cao X. Y., Shen J. Z.. 2008; Macrolide-lincosamide-resistant phenotypes and genotypes of Staphylococcus aureus isolated from bovine clinical mastitis. Vet Microbiol130:118–125 [CrossRef][PubMed]
    [Google Scholar]
  79. Willmott C. J., Maxwell A.. 1993; A single point mutation in the DNA gyrase A protein greatly reduces binding of fluoroquinolones to the gyrase–DNA complex. Antimicrob Agents Chemother37:126–127 [CrossRef][PubMed]
    [Google Scholar]
  80. Xi C., Zhang Y., Marrs C. F., Ye W., Simon C., Foxman B., Nriagu J.. 2009; Prevalence of antibiotic resistance in drinking water treatment and distribution systems. Appl Environ Microbiol75:5714–5718 [CrossRef][PubMed]
    [Google Scholar]
  81. Yang C. M., Lin M. F., Liao P. C., Yeh H. W., Chang B. V., Tang T. K., Cheng C., Sung C. H., Liou M. L.. 2009; Comparison of antimicrobial resistance patterns between clinical and sewage isolates in a regional hospital in Taiwan. Lett Appl Microbiol48:560–565 [CrossRef][PubMed]
    [Google Scholar]
  82. Zapun A., Contreras-Martel C., Vernet T.. 2008; Penicillin-binding proteins and β-lactam resistance. FEMS Microbiol Rev32:361–385 [CrossRef]
    [Google Scholar]
  83. Zhang R., Eggleston K., Rotimi V., Zeckhauser R. J.. 2006; Antibiotic resistance as a global threat: evidence from China, Kuwait and the United States. Global Health2:6 [CrossRef][PubMed]
    [Google Scholar]
  84. Zhang X.-X., Zhang T., Fang H. H. P.. 2009; Antibiotic resistance genes in water environment. Appl Microbiol Biotechnol82:397–414 [CrossRef][PubMed]
    [Google Scholar]
  85. Zhu Y. G., Johnson T. A., Su J. Q., Qiao M., Guo G. X., Stedtfeld R. D., Hashsham S. A., Tiedje J. M.. 2013; Diverse and abundant antibiotic resistance genes in Chinese swine farms. Proc Natl Acad Sci USA110:3435–3440 [CrossRef][PubMed]
    [Google Scholar]
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