Gold nanoparticles: An offer to control of vancomycin-resistant enterococci in wastewater

Document Type : Original Article


Department of Biology, Gorgan Branch, Islamic Azad University, Gorgan, Iran


Industrial wastewater is one of the most dangerous and important sources of bacterial pathogens. This study aimed to determine the frequency of vancomycin-resistant enterococci (VRE) in samples taken from wastewater plants of Golestan Province, Iran, and evaluate the antimicrobial effect of gold nanoparticles (AuNPs) in combination with vancomycin on the growth of isolates resistant to vancomycin. Samples were taken from three plants in Gorgan, Kordkuy and Bandar Turkoman. Enterococcal species were identified based on the most probable number (MPN), filtration, microbiological and biochemical tests. Susceptibility to six antibiotics with monitoring of vancomycin was investigated using the Kirby-Bauer method, according to the CLSI-2015 guidelines. The antibacterial effect of AuNPs was evaluated using agar well diffusion method. More than 60% of wastewater samples were positive for enterococcal species, 65% of which were found in raw effluent, while the remaining 35% were found in the treated effluent. Based on the results, 88.2% of the isolates were resistant to ampicillin. The frequency of vancomycin-resistant enterococci was 47.1%. Our findings indicate the presence of multi-drug resistant enterococci and high rate of vancomycin resistance in wastewater samples from the Golestan Province, Iran. Results show good antibacterial effects of AuNp s in combination with vancomycin in high densities against all the drug-resistant enterococci strains.


1.Talebi M, Rahimi F, Katouli M, Kühn I, Möllby R, Eshraghi S,  et al. Prevalence and antimicrobial resistance of enterococcal species in sewage treatment plants in Iran. Water Air Soil Pollut 2007; 185: 111-9.
2. Carvalho Mda G, Steigerwalt AG, Morey RE, Shewmaker PL, Teixeira L M, Facklam R.   Characterization of three new enterococcal species, Enterococcus sp. nov. CDC PNS-E1, Enterococcus sp. nov. CDC PNS-E2, and Enterococcus sp. nov. CDC PNS-E3, isolated from human clinical specimens. J Clin Microbiol 2004; 42(3): 1192-8.
3. Martinez JL. Antibiotics and antibiotic resistance genes in natural environments. Science 2008; 321(5887): 365–7.
4.Emaneini M, Aligholi M, Aminshahi M. Characterization of glycopeptides, aminoglycosides and macrolide resistance among Enterococcus faecalis and Enterococcus faecium isolates from hospitals in Tehran. Pol J Microbiol 2008; 57(2): 173-8.
5. Cetinkaya Y, Falk P, Mayhall CG. Vancomycin-resistant enterococci. Clin Microbiol Rev 2000; 13(4): 686-707.
6. Sadowy E, Luczkiewicz A. Drug-resistant and hospital-associated Enterococcus faecium from wastewater, riverine estuary and anthropogenically impacted marine catchment basin. BMC Microbiol 2014; 14: 66.  
7. Iversen A, Kühn I, Franklin A, Möllby R. High prevalence of vancomycin-resistant enterococci in Swedish sewage. Appl Environ Microbiol 2002; 68(6): 2838-42.
8. Ryu H, Henson M, Elk M, Toledo-Hernandez C, Griffith J, Blackwood D, et al. Development of quantitative PCR assays targeting the 16S r RNA genes of Enterococcus spp. and their application to the identification of Enterococcus species in environmental samples. Appl Environ Microbiol 2013; 79(1): 196-204.
9. Ben Salah D, Besbes M, Boutiba I, Greco A, Ghozzi R, Mahjoubi F, et al. Enterococcus faecalis: A multicenter study on antibiotic resistance. Tunis Med 2003; 81(2): 109-12.
10. Borhani K, Ahmadi A, Rahimi F, Pourshafie MR, Talebi M.  Determination of vancomycin resistant Enterococcus faecium diversity in Tehran sewage using plasmid profile, biochemical fingerprinting and antibiotic resistance.  Jundishapur J Microbiol 2014; 7(2): e8951.
11. Bouki C, Venieri D, Diamadopoulos E. Detection and fate of antibiotic resistant bacteria in wastewater treatment plants: A review. Ecotoxicol Environ Saf 2013; 91: 1-9.
12. Baquero F, Martínez JL, Cantón R. Antibiotics and antibiotic resistance in water environments. Curr Opin Biotechnol 2008; 19(3): 260–5.  
13. Karden-Lilja M, Vuopio J, Koskela M, Tissari P, Salmenlinna S. Molecular typing of vancomycin-resistant Enterococcus faecium with an automated repetitive sequence-based PCR microbial typing system compared with pulsed-field gel electrophoresis and multilocus sequence typing. Scand J Infect Dis 2013; 45(5): 350-6.
14. Klare I, Fleige C, Geringer U, Witte W, Werner G. Performance of three chromogenic VRE screening agars, two Etest (R) vancomycin protocols, and different microdilution methods in detecting vanB genotype Enterococcus faecium with varying vancomycin MICs. Diagn Microbiol Infect Dis 2012; 74(2): 171-6.
15. Muscholl-Silberhorn A, Samberger E, Wirth R. Why does Staphylococcus aureus secrete an Enterococcus faecalis-specific pheromone? FEMS Microbiol Lett 1997; 157(2): 261-6.
16. Pereira M, Oliveira M M, Trindade T, Soares A M V M. Effects of gold nanoparticles on marine species. Paper presented at: 7th World Nano Conference. June 20-21, 2016 Cape Town, South Africa.
17. Belliraj TS, Nanda A, Ragunathan R. In-vitro hepatoprotective activity of Moringa oleifera mediated synthesis of gold nanoparticles. J Chem Pharm Res 2015; 7(2): 781–8.
18. Sharmila Sh, Zafar N, Riaz S, Sharif R, Nazir J, Naseem Sh. Gold nanoparticles: An efficient antimicrobial agent against enteric bacterial human pathogen. Nanomaterials 2016; 6(4): 71.
19. Bai X, Wang Y, Song Z, Feng Y, Chen Y, Zhang D, et al. The basic properties of gold nanoparticles and their applications in tumor diagnosis and treatment.Int J Mol Sci 2020; 21(7): 2480.
20. Burygin GL, Khlebtsov BN, Shantrokha AN, Dykman LA, Bogatyrev VA.  Khlebtsov NG. On the enhanced antibacterial activity of antibiotics mixed with gold nanoparticles. Nanoscale Res Lett 2009; 4(8): 794–801.
21. Mohammed Fayaz M, Girilal M, Mahdy SA, Somsundar SS, Venkatesan R, Kalaichelvan PT. Vancomycin bound biogenic gold nanoparticles: A different perspective for development of anti VRSA agents.Process Biochem 2011; 46(3): 636-41.
22. Eaton AD, Clesceri LS, Greenberg AE. Standard methods for the examination of water and wastewater, 21th Edition, American Public Health Association, Washington DC 1998; Section 9221.
23. Performance Standards for Antimicrobial Susceptibility Testing; Twenty-Fifth Informational Supplement. M100-S25. Wayne, PA 2015; 3(35): 56-8.
24. Elhani D, Klibi N, Dziri R, Ben Hassan M, Asli Mohamed S, Ben Said L, et al. vanA-containing E. faecium isolates of clonal complex CC17 in clinical and environmental samples in a Tunisian hospital. Diagn Microbiol Infect Dis 2014; 79(1): 60-3.
25. Lloret E, Pastor L, Martínez-Medina A, Blaya J, Pascual JA. Evaluation of the removal of pathogens included in the proposal for a European Directive on spreading of sludge on land during autothermal thermophilic aerobic digestion (ATAD). Chem Eng J 2012; 198-199: 171-9.
26. Aminov RI. The role of antibiotics and antibiotic resistance in nature. Environ Microbiol 2009; 11(12): 2970–88.
27. Kotzamanidis C, Zdragas A, Kourelis A, Moraitou E, Papa A, Yiantzi V, et al. Characterization of vanA-type Enterococcus faecium isolates from urban and hospital wastewater and pigs. J Appl Microbiol 2009; 107(3): 997–1005.
28. Kümmerer K. Resistance in the environment. J Antimicrob Chemother 2004; 54(2): 311–20.
29. Reinthaler FF, Posch J, Feierl G, Wust G, Haas D, Ruckenbauer G, et al. Antibiotic resistance of E. coli in sewage and sludge. Water Res 2003; 37(8): 1685-90.
30. Ferreira AE, Marchetti DP, De Oliveira LM, Gusatti CS, Fuentefria DB, Corcao G. Presence of OXA-23-Producing isolates of Acinetobacter baumannii in wastewater from hospitals in southern Brazil. Microb Drug Resist 2011; 17(2): 221-7.
31.Tremblay C L, Letellier A, Quessy S, Boulianne M, Daignault D, Archambault M. Multiple-antibiotic resistance of Enterococcus faecalis and Enterococcus faecium from cecal contents in broiler chicken and turkey flocks slaughtered in Canada and plasmid colocalization of tetO and ermB genes. J Food Prot 2011; 74(10): 1639-48.
32. Zhanel GG, Laing NM, Nichol KA, Palatinick LP, Noreddin A, Hisanaga T, et al. Antibiotic activity against urinary tract infection (UTI) isolates of vancomycin-resistant enterococci (VRE): Results from the 2002 North American Vancomycin Resistant Enterococci Susceptibility Study (NAVRESS). J Antimicrob Chemother 2003; 52(3): 382-8.
33. Williams DN, Ehrman SH, Holoman TRP. Evaluation of the microbial growth response to inorganic nanoparticles. J Nanobiotechnology 2006; 4: 3.
34. Das SK, Das AR, Guha AK. Gold nanoparticles: Microbial synthesis and application in water hygiene management. Langmuir 2009; 25(14): 8192–9.
35. Rai M, Yadav A, Gade A. Silver nanoparticles as a new generation of antimicrobials. Biotechnol Adv 2009; 27(1): 76-83.