Environmental Science and Pollution Research

, Volume 26, Issue 18, pp 18470–18483 | Cite as

Annual changes in the occurrence of antibiotic-resistant coliform bacteria and enterococci in municipal wastewater

  • Kristína LépesováEmail author
  • Petra Olejníková
  • Tomáš Mackuľak
  • Jozef Tichý
  • Lucia Birošová
Research Article


Wastewater contains subinhibitory concentrations of different micropollutants such as antibiotics that create selective pressure on bacteria. This phenomenon is also caused by insufficient wastewater treatment technology leading to the development and spread of antibiotic-resistant bacteria and resistance genes into the environment. Therefore, this work focused on monitoring of antibiotic-resistant coliform bacteria and enterococci in influent and effluent wastewaters taken from the second biggest wastewater treatment plant (Petržalka) in the capital of Slovakia during 1 year. Antibiotic-resistant strains were isolated, identified, and characterized in terms of susceptibility and biofilm production. All of 27 antibiotic-resistant isolates were identified mainly as Morganella morganii, Citrobacter spp., and E. coli. Multidrug-resistance was detected in 58% of isolated strains. All tested isolates could form biofilm; two strains were very strong producers, and 74% formed biofilm by strong intensity. The flow rate of the influent wastewater had a more significant impact on the number of studied bacteria than the temperature.

Graphical abstract


E. coli Coliform bacteria Wastewater treatment plant Multidrug-resistance Biofilm MALDI-TOF MS 


Funding information

This work was financially supported by the Scientific Grant Agency VEGA (Grant number VEGA 1/0096/17), by the Slovak Research and Development Agency (Grant number APVV-16-0171) and by a project for the building of infrastructure for the modern research of civilization diseases (ITMS 26230120006).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.


  1. Ahammad et al (2014) Increased waterborne blaNDM-1 resistance gene abundances associated with seasonal human pilgrimages to the upper ganges river. Environ Sci Technol 48:3014–3020CrossRefGoogle Scholar
  2. Auguet O, Pijuan M, Borrego CM, Rodriguez-Mozaz S, Triadó-Margarit X, Giustina SVD, Gutierrez O (2017) Sewers as potential reservoires of antibiotic resistance. Int Sci Total Environ 605-606:1047–1054CrossRefGoogle Scholar
  3. Aydin S, Ince B, Ince O (2015) Development of antibiotic resistance genes in microbial communities during long-term operation of anaerobic reactors in the treatment of pharmaceutical wastewater. Water Res 83:337–344CrossRefGoogle Scholar
  4. Azuma et al (2019) Environmental fate of pharmaceutical compounds and antimicrobial-resistant bacteria in hospital effluents, and contributions to pollutant loads in the surface waters in Japan. Sci Total Environ 657:476–484CrossRefGoogle Scholar
  5. Azzam MI, Ezzat SM, Othman BA, el-Dougdoug KA (2017) Antibiotics resistance phenomenon and virulence ability in bacteria from water environment. Water Sci 31:109–121CrossRefGoogle Scholar
  6. Badura A, Luxner J, Feierl G, Reinthaler FF, Zarfel G, Galler H, Pregartner G, Riedl R, Grisold AJ (2014) Prevalence, antibiotic resistance patterns and molecular characterization of Escherichia coli from Austrian sandpits. Environ Pollut 194:24–30CrossRefGoogle Scholar
  7. Baquero F, Martínez JL, Cantón R (2008) Antibiotics and antibiotic resistance in water environments. Curr Opin Biotechnol 19:260–265CrossRefGoogle Scholar
  8. Beenken KE, Blevins JS, Smeltzer MS (2003) Mutation in sarA in Staphylococcus aureus limits biofilm formation. Infect Immun 71(7):4206–4211CrossRefGoogle Scholar
  9. Berrazeg M et al (2014) New Delhi metallo-beta-lactamase around the world: an eReview using Google maps. Euro Surveill 19(20):1–14CrossRefGoogle Scholar
  10. Bhatt P, Tandel K, Shete V, Rathi KR (2015) Burden of extensively drug-resistant and pandrug-resistant Gram-negative bacteria at a tertiary-care centre. New Microbes New Infect 8:166–170CrossRefGoogle Scholar
  11. Birošova L et al (2014) Pilot study of seasonal occurrence and distribution of antibiotics and drug resistant bacteria in wastewater treatment plants in Slovakia. Sci Total Environ 490:440–444CrossRefGoogle Scholar
  12. Bouki CH, Venieri D, Diamadopoulos E (2013) Detection and fate of antibiotic resistant bacteria in wastewater treatment plants: a review. Ecotoxicol Environ Saf 91:1–9CrossRefGoogle Scholar
  13. Braga TM, Pomba C, Lopes MFS (2013) High-level vancomycin resistant Enterococcus faecium related to humans and pigs found in dust from pig breeding facilities. Vet Microbiol 161:344–349CrossRefGoogle Scholar
  14. Bratislava Water Company (2013) Press news. Modernization of the two largest wastewater treatment plants in Slovakia. (In Slovak) [ Online 20.3.2017]
  15. Cheng W, Li J, Wu Y, Xu L, Su C, Qian Y, Zhu YG, Chen H (2016) Behavior of antibiotics and antibiotic resistance genes in eco-agricultural systems: a case study. J Hazard Mater 304:18–25CrossRefGoogle Scholar
  16. Clark CG, Kruczkiewicz P, Guan C, McCorrister SJ, Chong P, Wylie J, van Caeseele P, Tabor HA, Snarr P, Gilmour MW, Taboada EN, Westmacott GR (2013) Evaluation of MALDI-TOF mass spectroscopy methods for determination of Escherichia coli pathotypes. J Microbiol Methods 94:180–191CrossRefGoogle Scholar
  17. Clinical Laboratory Standards Institute, CLSI (2014) M 100 – S 24 performance standards for antimicrobial susceptibility testing; twenty-fourth informational supplementGoogle Scholar
  18. Croxatto A, Prod'hom G, Greub G (2012) Applications of MALDI-TOF mass spectrometry in clinical diagnostic microbiology. FEMS Microbiol Rev 36:380–407CrossRefGoogle Scholar
  19. Dubey D, Padhy RN (2015) Infection dynamics of vancomycin and inducible clindamycin resistant Enterococcus faecalis in an Indian teaching hospital. Asian Pac J Trop Dis 5:127–132. CrossRefGoogle Scholar
  20. European Centre for Disease Prevention and Control, ECDC (2011) Surveillance of antimicrobial consumption in Europe 2011; Stockholm: 2014; 1–82Google Scholar
  21. European Centre for Disease Prevention and Control, ECDC (2017) Antimicrobial resistance surveillance in Europe 2016. Annual Report of the European Antimicrobial Resistance Surveillance Network (EARS-Net). Stockholm, Sweden: ECDC; 2017, ISBN 978–92–9498-099-1, 1–11Google Scholar
  22. European Committee on Antimicrobial Susceptibility Testing, EUCAST (2013) EUCAST guidelines for detection of resistance mechanisms and specific resistances of clinical and/or epidemiological importanceGoogle Scholar
  23. European Committee on Antimicrobial Susceptibility Testing, EUCAST (2015) Breakpoint tables for interpretation of MICs and zone diamteres; Version 5.0 valid from 2015Google Scholar
  24. Fernández M, Fernández M, Laca A, Laca A, Díaz M (2014) Seasonal occurrence of removal of pharmaceutical products in municipal wastewaters. J Environ Chem Eng 2:495–502CrossRefGoogle Scholar
  25. Feuerpfeil I, Sterzel W (1992) Presence of antibiotic-resistant coliform bacteria in the human intestinal flora. Bundesgesudheitsblatt 35:61–65Google Scholar
  26. Fraia D et al (2018) A novel energy assessment of urban wastewater treatment plants. Energ Convers Manage 163:304–313CrossRefGoogle Scholar
  27. Garrett JH (2015) An era of antimicrobial resistance: imminent threats of superbugs to vascular access clinicians and patients. JAVA 20(3):133–136. CrossRefGoogle Scholar
  28. Hamiwe T, Kock MM, Magwira CA, Antiabong JF, Ehlers MM (2019) Occurrence of enterococci harbouring clinically important antibiotic resistance genes in the aquatic environment in Gauteng, South Africa. Environ Pollut 245:1041–1049CrossRefGoogle Scholar
  29. Hrenovic et al (2017) The fate of carbapenem-resistant bacteria in wastewater treatment plant. Water Res 126:232–239CrossRefGoogle Scholar
  30. Huang JJ, Xi J, Hu HY, Li Y, Lu SQ, Tang F, Pang YC (2016) UV light tolerance and reactivation potential of tetracycline-resistant bacteria from secondary effluents of a wastewater treatment plant. J Environ Sci 41:146–153CrossRefGoogle Scholar
  31. Iweriebor BCH, Gaqavu S, Obi L, Nwodo U, Okoh A (2015) Antibiotic susceptibilities of Enterococcus species isolated from hospital and domestic wastewater effluents in Alice, eastern Cape Province of South Africa. Int J Environ Res Public Health 12:4231–4246. CrossRefGoogle Scholar
  32. Kay D, Crowther J, Stapleton CM, Wyer MD, Fewtrell L, Edwards A, Francis CA, McDonald AT, Watkins J, Wilkinson J (2008) Faecal indicator organism concentrations in sewage and treated effluents. Water Res 42(1):442–454CrossRefGoogle Scholar
  33. Khan HA, Ahmad A, Mehboob R (2015) Nosocomial infections and their control strategies. Asian Pac J Trop Biomed 5(7):509–514CrossRefGoogle Scholar
  34. Kumar A, Pal D (2018) Antibiotic resistance and wastewater: correlation, impact and critical human health challenges. JECE 6:52–58Google Scholar
  35. Laishram S, Sahni RD, Anandan S, Balaji V (2014) Evaluation of the antimicrobial activity of daptomycin and linezolid against vancomycin-resistant Enterococcus spp. isolates in south India. J Glob Antimicrob Re 2:194–197CrossRefGoogle Scholar
  36. Lamba M, Ahammad SZ (2017) Sewage treatment effluent in Delhi: a key contributor of β-lactam resistant bacteria and genes to the environment. Chemosphere 188:249–256CrossRefGoogle Scholar
  37. Lépesová K, Kraková L, Pangallo D, Medveďová A, Olejníková P, Mackuľak T, Tichý J, Grabic R, Birošová L (2018) Prevalence of antibiotic-resistant coliform bacteria, Enterococcus spp. and Staphylococcus spp. in wastewater sewerage biofilm. J Glob Antimicrob Res 14:145–151CrossRefGoogle Scholar
  38. Maal-Bared R, Bartlett KH, Bowie WR, Hall ER (2013) Phenotypic antibiotic resistance of Escherichia coli and E. coli O157 isolated from water, sediment and biofilms in an agricultural watershed in British Columbia. Sci Total Environ 443:315–323CrossRefGoogle Scholar
  39. Mackuľak T, Škubák J, Grabic R, Ryba J, Birošová L, Fedorova G, Špalková V, Bodík I (2014) National study of illicit drug use in Slovakia based on wastewater analysis. Sci Total Environ 494-495:158–165CrossRefGoogle Scholar
  40. Mackuľak T, Nagyová K, Faberová M, Grabic R, Koba O, Gál M, Birošová L (2015) Utilization of Fenton-like reaction for antibiotics and resistant bacteria elimination in different parts of WWTP. Environ Toxicol Pharmacol 40:492–497CrossRefGoogle Scholar
  41. Magiorakos AP, Srinivasan A, Carey RB, Carmeli Y, Falagas ME, Giske CG, Harbarth S, Hindler JF, Kahlmeter G, Olsson-Liljequist B, Paterson DL, Rice LB, Stelling J, Struelens MJ, Vatopoulos A, Weber JT, Monnet DL (2012) Multidrug-resistant, extensively drug-resistant and pandrug-resistant bacteria: an international expert proposal for interim standard definitions for acquired resistance. Clin Microbiol Infect 18:268–281CrossRefGoogle Scholar
  42. Maheshwari M, Ahmad I, Althubiani AS (2016) Multidrug resistance and transferability of bla CTX-M among extended-spectrum β-lactamase-producing enteric bacteria in biofilm. J Glob Antimicrob Res 6:142–149CrossRefGoogle Scholar
  43. Metri BC, Jyothi P, Peerapur BV (2013) Antibiotic resistance in Citrobacter spp. isolated from urinary tract infection. Urol Ann 5(4):312–313CrossRefGoogle Scholar
  44. Naidoo S, Olaniran AO (2014) Treated effluent wastewater as a source of microbial pollution of surface water resources. Int J Environ Res Public Health 11:249–270. CrossRefGoogle Scholar
  45. Neudorf KD, Huang YN, Ragush CM, Yost CK, Jamieson RC, Truelstrup Hansen L (2017) Antibiotic resistance genes in municipal wastewater treatment systems and receiving waters in Arctic Canada. Sci Total Environ 598:1085–1094CrossRefGoogle Scholar
  46. Novo A, André S, Viana P, Nunes OC, Manaia CM (2013) Antibiotic resistance, antimicrobial residues and bacterial community composition in urban wastewater. Water Res 47:1875–1887CrossRefGoogle Scholar
  47. Olejníková P, Kurucová M, Švorc L, Marchalín Š (2012) Induction of resistance in Mycobacterium smegmatis. Can J Microbiol 59(2):126–129CrossRefGoogle Scholar
  48. Oravcová V et al (2017) Vancomycin-resistant enterococci with vanA gene in treated municipal wastewater and their association with human hospital strains. Sci Total Environ 609:633–643CrossRefGoogle Scholar
  49. Ory J, Bricheux G, Togola A, Bonnet JL, Donnadieu-Bernard F, Nakusi L, Forestier C, Traore O (2016) Ciprofloxacin residue and antibiotic-resistant biofilm bacteria in hospital effluent. Environ Pollut 214:635–645CrossRefGoogle Scholar
  50. Ou D, Chen B, Bai R, Song P, Lin H (2015) Contamination of sulfonamide antibiotics and sulfamethazine-resistant bacteria in the downstream and estuarine areas of Jiulong River in Southeast China. Environ Sci Pollut Res Int 22:12104–12113CrossRefGoogle Scholar
  51. Picot-Guéraud R, Batailler P, Caspar Y, Hennebique A, Mallaret MR (2015) Bacteremia caused by multidrug-resistant bacteria in a French university hospital center: 3 years of collection. Am J Infect Control 43:960–964CrossRefGoogle Scholar
  52. Qiao M, Ying GG, Singer AC, Zhu YG (2018) Review of antibiotic resistance in China and its environment. Environ Int 110:160–172CrossRefGoogle Scholar
  53. Ramos S, Chafsey I, Silva N, Hébraud M, Santos H, Capelo-Martinez JL, Poeta P, Igrejas G (2015) Effect of vancomycin on the proteome of the multiresistant Enterococcus faecium SU18 strain. J Proteome 113:378–387CrossRefGoogle Scholar
  54. Ranotkar S, Kumar P, Zutshi S, Prashanth KS, Bezbaruah B, Anand J, Lahkar M (2014) Vancomycin-resistant enterococci: troublemaker of the 21st century. J Glob Antimicrob Res 2:205–212CrossRefGoogle Scholar
  55. Rizzo L, Manaia C, Merlin C, Schwartz T, Dagot C, Ploy MC, Michael I, Fatta-Kassinos D (2013) Urban wastewater treatment plants as hotspots for antibiotic resistant bacteria and genes spread into the environment: a review. Sci Total Environ 447:345–360CrossRefGoogle Scholar
  56. Rodriguez-Mozaz S, Chamorro S, Marti E, Huerta B, Gros M, Sànchez-Melsió A, Borrego CM, Barceló D, Balcázar JL (2015) Occurrence of antibiotics and antibiotic resistance genes in hospital and urban wastewaters and their impact on the receiving river. Water Res 69:234–242CrossRefGoogle Scholar
  57. Ruelle V, Moualij BE, Zorzi W, Ledent P, Pauw ED (2004) Rapid identification of environmental bacterial strains by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Rapid Commun Mass Spectrom 18:2013–2019CrossRefGoogle Scholar
  58. Santos T, Capelo JL, Santos HM, Oliveira I, Marinho C, Gonçalves A, Araújo JE, Poeta P, Igrejas G (2015) Use of MALDI-TOF mass spectrometry fingerprinting to characterize Enterococcus spp. and Escherichia coli isolates. J Proteome 127:321–331CrossRefGoogle Scholar
  59. 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 Ecol 43:325–335CrossRefGoogle Scholar
  60. Sharma VK et al (2015) A review of the influence of treatment strategies on antibiotic resistant bacteria and antibiotic resistance genes. Chemosphere 150:702–714CrossRefGoogle Scholar
  61. Smanthong N, Tavichakorntrakool R, Saisud P, Prasongwatana V, Sribenjalux P, Lulitanond A, Tunkamnerdthai O, Wongkham C, Boonsiri P (2015) Biofilm formation in trimethoprime/sulfamethoxazole-susceptible and trimethoprim/sulfamethoxazole-resistant uropathogenic Escherichia coli. Asian Pac J Trop Biomed 5(6):485–487CrossRefGoogle Scholar
  62. Sukmawinata E, Sato W, Uemura R, Sueyoshi M (2018) Antimicrobial resistant Enterococcus faecium, Enterococcus faecalis, and other Enterococcus species isolated from foal feces in Japan. J Equine Vet Sci 63:51–54CrossRefGoogle Scholar
  63. Tahrani L, Soufi L, Mehri I, Najjari A, Hassan A, van Loco J, Reyns T, Cherif A, Mansour HB (2015) Isolation and characterization of antibiotic-resistant bacteria from pharmaceutical industrial wastewaters. Microb Pathog 89:54–61CrossRefGoogle Scholar
  64. Taniguchi L, de Fátima Faria B, Rosa RT, de Paula e Carvalho A, Gursky LC, Elifio-Esposito SL, Parahitiyawa N, Samaranayake LP, Rosa EAR (2009) Proposal of a low-cost protocol for colorimetric semi-quantification of secretory phospholipase by Candida albicans grown in planktonic and biofilm phases. J Microbiol Methods 78(2):171–174. CrossRefGoogle Scholar
  65. Taučer-Kapteijn M, Hoogenboezem W, Heiliegers L, de Bolster D, Medema G (2016) Screening municipal wastewater effluent and surface water used for drinking water production for the presence of ampicillin and vancomycin resistant enterococci. Int J Hyg Environ Health 219:437–442CrossRefGoogle Scholar
  66. Tenaillon O, Skurnik D, Picard B, Denamur E (2010) The population genetics of commensal Escherichia coli. Nat Rev Microbiol 8:207–217. CrossRefGoogle Scholar
  67. Thenmozhi S et al (2014) Antibiotic resistance mechanisms of ESBL producing Enterobacteriaceae in clinical field: a review. Int J Pure Appl Biosci 2:207–224Google Scholar
  68. Tolouei S, Burnet JB, Autixier L, Taghipour M, Bonsteel J, Duy SV, Sauvé S, Prévost M, Dorner S (2019) Temporal variability of parasities, bacterial indicators, and wastewater micropollutants in a water resource recovery facility under various weather conditions. Water Res 148:446–458CrossRefGoogle Scholar
  69. Turolla A, Cattaneo M, Marazzi F, Mezzanotte V, Antonelli M (2018) Antibiotic resistant bacteria in urban sewage: role of full-scale wastewater treatment plants on environmental spreading. Chemosphere 191:761–769CrossRefGoogle Scholar
  70. Wan MT, Chou CHCH (2014) Spreading of β-lactam resistance gene (mecA) and methicillin-resistant Staphylococcus aureus through municipal and swine slaughterhouse wastewaters. Water Res 64:288–295CrossRefGoogle Scholar
  71. Wang Q, Wang P, Yang Q (2018) Occurrence and diversity of antibiotic resistance in untreated hospital wastewater. Sci Total Environ 621:990–999CrossRefGoogle Scholar
  72. Welker M, Moore ER (2011) Applications of whole-cell matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry in systematic microbiology. Syst Appl Microbiol 34:2–11CrossRefGoogle Scholar
  73. World Health Organization WHO (2016) Antibiotic resistance. (Online in 5.2.2016).
  74. Yang YX et al (2015) Molecular characterization of resistance, virulence and clonality in vancomycin-resistant Enterococcus faecium and Enterococcus faecalis: a hospital-based study in Biejing, China. Infect Genet Evol 33:253–260CrossRefGoogle Scholar
  75. Zhang S, Han B, Gu J, Wang C, Wang P, Ma Y, Cao J, He Z (2015) Fate of antibiotic resistant cultivable heterotrophic bacteria and antibiotic resistance genes in wastewater treatment processes. Chemosphere 135:138–145CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  • Kristína Lépesová
    • 1
    Email author
  • Petra Olejníková
    • 2
  • Tomáš Mackuľak
    • 3
  • Jozef Tichý
    • 3
  • Lucia Birošová
    • 1
  1. 1.Department of Nutrition and Food Quality Assessment, Faculty of Chemical and Food TechnologySlovak University of TechnologyBratislavaSlovakia
  2. 2.Institute of Biochemistry and Microbiology, Faculty of Chemical and Food TechnologySlovak University of TechnologyBratislavaSlovakia
  3. 3.Department of Environmental Engineering, Faculty of Chemical and Food TechnologySlovak University of TechnologyBratislavaSlovakia

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