Effects of Antibiotics on Sewage Treatment Processes

1. Effects of Antibiotics on Sewage Treatment Processes Presented at Pharmaceutical Usage During an Influenza Pandemic – Implications for Sewage Treatment Plant Function March 3, 2009 Somerville College, University of Oxford, England Nancy G. Love, Ph.D. Professor and Chair Sudeshna Ghosh Postdoctoral Research Associate Department of Civil and Environmental Engineering University of Michigan

2. Biosolids Handling Effects of Antibiotics on Sewage Treatment Processes:Framing the Problem 10 Treatment 20 Treatment Disinfection Screenings Grit 10 Sludge 20 Sludge Effluent Residual

3. Biosolids Handling Effects of Antibiotics on Sewage Treatment Processes:Framing the Problem 10 Treatment 20 Treatment Disinfection Screenings Grit 10 Sludge 20 Sludge Effluent Pathogen protection occurs in multiple locations Residual

4. Biosolids Handling Effects of Antibiotics on Sewage Treatment Processes:Framing the Problem Baseline ~40-400 ng/L 10 Treatment 20 Treatment Disinfection Pandemic ~ 40–400 µg/L Screenings Grit 10 Sludge 20 Sludge Effluent Concentrations will be orders of magnitude higher than baseline, over several weeks Residual

5. Biosolids Handling Effects of Antibiotics on Sewage Treatment Processes:Framing the Problem Want biomass to be resistant to antibiotics so process functions 10 Treatment 20 Treatment Disinfection Screenings Grit 10 Sludge 20 Sludge Effluent Want effluents to be free of antibiotic resistant microorganisms to prevent introduction into environment Consider conflicting perspectives about antibiotic resistance Residual

6. Effects of Antibiotics on Sewage Treatment Processes:Framing the Problem – Anticipated Responses of Greatest Importance • Metabolic impact on the indigenous microbiology • Ecological implications for the indigenous microbiology • Consequences of microbial stress responses • Potential for spreading antibiotic resistance

7. Effects of Antibiotics on Sewage Treatment Processes:Framing the Problem – Anticipated Responses of Greatest Importance • Metabolic impact on the indigenous microbiology • Ecological implications for the indigenous microbiology • Consequences of microbial stress responses • Potential for spreading antibiotic resistance

8. Metabolic impact on the indigenous microbiology • Inhibition experiments have been done using ex situ standardized tests (e.g., Kümmererand colleagues) but connection back to the treatment process is tricky plus variations from plant to plant can generate contradictory results. • It is preferred to collect this data for target antibiotics using indigenous cultures and extant growth conditions to the degree possible. • Characteristics of primary substrates in wastewater is a key determinant in biotransformation of pharmaceuticals. • Growth environment is also a key determinant in biotransformation patterns

9. Metabolic impact on the indigenous microbiology • Inhibition experiments have been done using ex situ standardized tests (e.g., Kümmererand colleagues) but connection back to the treatment process is tricky plus variations from plant to plant can generate contradictory results. • It is preferred to collect this data for target antibiotics using indigenous cultures and extant growth conditions to the degree possible. • Characteristics of primary substrates in wastewater is a key determinant in biotransformation of pharmaceuticals. • Growth environment is also a key determinant in biotransformation patterns

10. Example - Trimethoprim is not inhibitory to activated sludge enrichment culture up to 10 mg/L Extant test with heterotrophic activated sludge enrichment culture Acetate-based feed Biomass concentration ~ 200 mg/L TSS No nitrifiers present 0.01 1,000 10,000 Control (µg/L) Khunjar, Love and Aga, 2009, in Progress

11. Metabolic impact on the indigenous microbiology • Inhibition experiments have been done using ex situ standardized tests (Kümmerer and colleagues) but connection back to the treatment process is tricky plus variations from plant to plant can generate contradictory results. • It is preferred to collect this data for target antibiotics using indigenous cultures and extant growth conditions to the degree possible. • Characteristics of primary substrates in wastewater is a key determinant in biotransformation of pharmaceuticals. • Growth environment is a key determinant in defining biotransformation patterns

12. Nonspecific oxygenase enzymes are helpful in the aerobic biotransformation of pharmaceuticals. Monooxygenases Dioxygenases Monooxygenases Ring Cleaving Dioxygenases TCA Cycle

13. Example – Presence of oxygenase-inducing substrates enhance pharmaceutical degradation Oxygenase-deficient activated sludge community Oxygenase-rich activated sludge community 17α-ethinylestradiol

14. Metabolic impact on the indigenous microbiology • Inhibition experiments have been done using ex situ standardized tests (Kümmerer and colleagues) but connection back to the treatment process is tricky plus variations from plant to plant can generate contradictory results. • It is preferred to collect this data for target antibiotics using indigenous cultures and extant growth conditions to the degree possible. • Characteristics of primary substrates in wastewater is a key determinant in biotransformation of pharmaceuticals. • Growth environment is a key determinant in defining biotransformation patterns

15. Batch growth Chemostat SRT = 7 day To evaluate the fate of pharmaceuticals by bacteria common to WWT processes, we use various cultures, feeds and reactor configurations • Ammonia oxidizing bacteria (N. europaea) – Monooxygenase • Heterotrophic culture OX-, No Oxygenases • Heterotrophic culture OX+, Mono/Di Oxygenases Pharmaceuticals in study: 17α-ethinylestradiol, carbamazepine, iopromide and trimethoprim

16. Metabolic fate of pharmaceuticals are a function of microorganism, growth condition, and substrate (data for 17α-ethinylestradiol)

17. Effects of Antibiotics on Sewage Treatment Processes:Framing the Problem – Anticipated Responses of Greatest Importance • Metabolic impact on the indigenous microbiology • Ecological implications for the indigenous microbiology • Consequences of microbial stress responses • Potential for spreading antibiotic resistance

18. Ecological implications for the indigenous microbiology • WWTPs provide a scenario where diverse bacteria are in close proximity (high suspended solid concentrations or biofilm). This physical setup is ideal for horizontal gene transfer. Dense, activated sludge floc Dense biofilm on membrane

19. Antibiotics Ecological implications for the indigenous microbiology • What happens to persistercells from patients? Can they proliferate in activated sludge communities? Susceptible bacteria will succumb or acquire resistance Resistant bacteria including pandemic strains will proliferate

20. Ecological implications for the indigenous microbiology • Will exposure to antibiotics over the time period of a pandemic shift the activated sludge microbial community to a more antibiotic resistant community?

22. Ecological implications for the indigenous microbiology • Community will be less diverse and more vulnerable to upset by other perturbations • Consider Critical Interdependencies: • During a pandemic, expect industrial wastewater inputs to be unpredictable and unprotected

24. Ecological implications for the indigenous microbiology • Community will be less diverse and more vulnerable to upset by other perturbations • Consider Critical Interdependencies: • During a pandemic, expect industrial wastewater inputs to be unpredictable and unprotected • Will WWTP failure amplify the public health crisis?

25. Ecological implications for the indigenous microbiology • Will ecological changes be permanent? • Plant may never return to the ecological state it was in before the pandemic, but… • there are multiple functional steady states that provide acceptable performance, even if the community structure has changed • If a higher proportion of antibiotic resistant cells exist in the bioreactor, then the risk of more antibiotic resistant cells being released into the environment increases.

26. Ecological implications for the indigenous microbiology • Will ecological changes be permanent? • Plant may never return to the ecological state it was in before the pandemic, but… • there are multiple functional steady states that provide acceptable performance, even if the community structure has changed • If a higher proportion of antibiotic resistant cells exist in the bioreactor, then the risk of more antibiotic resistant cells being released into the environment increases.

27. Ecological implications for the indigenous microbiology – will biomass in membrane bioreactors be more or less susceptible? Henriques, Holbrook, Kelly and Love. 2005. Water Research, 39:2559-2568.

28. Effects of Antibiotics on Sewage Treatment Processes:Framing the Problem – Anticipated Responses of Greatest Importance • Metabolic impact on the indigenous microbiology • Ecological implications for the indigenous microbiology • Consequences of microbial stress responses • Potential for spreading antibiotic resistance

29. Varies Source-effect studies identified relationships between chemicals and process upset modes The qualitative scale reflects the intensity of the effect for IC50-shocked reactors and the indicated NH3 and pH shock level, in comparison to a negative control. The intensity scale ranges from  (most intense process deterioration effect), 0 (no effect), and ++++ (most intense process improvement effect). X means inconclusive results Henriques et al., 2007. Water Environment Research

30. Corrective action plan matrices (CAPMs) are being developed and tested to guide operators in responding to process upsets. Nature of the Data Nature of the Contaminant Operational Flexibility Process Effects of Contaminant

31. Effects of Antibiotics on Sewage Treatment Processes:Framing the Problem – Anticipated Responses of Greatest Importance • Metabolic impact on the indigenous microbiology • Ecological implications for the indigenous microbiology • Consequences of microbial stress responses • Potential for spreading antibiotic resistance Antibiotics are related to oxidative stress, and oxidative stress influences WWTP performance. Keep this in mind!

32. What we know about microbial stress responses in activated sludge • Anti-oxidative stress mechanisms are active but we have linked them to performance upsets in activated sludge

33. Bactericidal antibiotics cause cellular death by oxidative stress Kohanskiet al. Cell 130, 797–810, September 7, 2007

34. Activated sludge deflocculates when exposed to oxidative (thiol-reactive) chemical stressors We linked the deflocculation mechanism to a glutathione-gated K+ efflux response directly resulting from exposure to oxidative stressors - can experience deflocculation within minutes of shock - can take days to weeks to recover via regrowth Control NEM CDNB

35. K+ K+ K+ K+ Electrophilic Chemical Stressor K+ K+ Potassium Efflux from Floc Deflocculated biomass Would glutathione-gated K+ efflux occur in response to a prolonged, concentrated dose of antibiotics? Bott, C. B. and Love, N. G. 2002. Investigating a mechanistic cause for activated sludge deflocculation in response to shock loads of toxic electrophilic chemicals. Water Environment Research, 74:306-315. Bott, C. B. and Love, N. G. 2004. Implicating the glutathione-gated potassium efflux system as a cause of electrophile-induced activated sludge deflocculation. Applied and Environmental Microbiology, 70(9):5569-5578.

36. What we know about microbial stress responses in activated sludge • Anti-oxidative stress mechanisms are active but we have linked them to performance upsets in activated sludge • Bacteriocidal antibiotics invoke oxidative stress • Activated sludge responds to oxidative stressors via glutathione-gated K+ efflux, which causes deflocculation • Deflocculation is particularly undesirable if a microbial community has a lot of antibiotic resistant strains • Similar to concern over Tamiflu detachment for biofilms (and flocs), but different mechanism. • Park and Choung (2007) showed in vitro that glutathione conjugates with ampicillin, tetracycline and sulfathiozole(Human and Ecological Risk Assessment, 13:1147-1155, 2007)

37. What we know about microbial stress responses in Pseudomonas aeruginosa • Activity of multidrug efflux pumps are activated by environmental toxins (not just antibiotics) • Activity of multidrug efflux pumps are reduced by anti-oxidants, making cells more susceptible to antibiotics

38. Pentachlorophenol induces mexB Ghosh, Fraga-Muller, Stevens and Love, in preparation Muller, J. F., Stevens, A. M., Craig, J. and Love, N. G. 2007. Transcriptome analysis reveals multi-drug efflux genes upregulated to protect Pseudomonas aeruginosa from pentachlorophenol stress. Applied and Environmental Microbiology 73(14):4550-4558.

39. Multidrug resistance efflux pumps and chlorinated phenols Martinez et al. FEMS Microbiol Rev 33, (2009) 430–449

40. What we know about microbial stress responses in Pseudomonas aeruginosa • Activity of multidrug efflux pumps are activated by environmental toxins (not just antibiotics) • Activity of multidrug efflux pumps are reduced by anti-oxidants, making cells more susceptible to antibiotics

41. Multidrug efflux pumps are upregulated in response to oxidative stress Oxidative stress may serve as a signal to activate the transcriptional regulator, MexR, of the mexAB–oprMmultidrug efflux operon in Pseudomonas aeruginosa Chen et al. PNAS 105, 13586–13591, September 9, 2008

42. Our data (unpublished): Pseudomonas aeruginosabecomes more susceptible to antibiotics in the presence of antioxidants Minimum inhibitory concentrations (µg/mL) of Pseudomonas aeruginosa PCP (120 mM pentachlorophenol), NAC (1 mM N-acetyl-L-cysteine), 3 mML-proline. NAC and proline are antioxidants MIC = more susceptible to antibiotic MIC = more resistant to antibiotic

43. Our data (unpublished): Pseudomonas aeruginosabecomes more susceptible to antibiotics in the presence of antioxidants Minimum inhibitory concentrations (µg/mL) of Pseudomonas aeruginosa PCP (120 mM pentachlorophenol), NAC (1 mM N-acetyl-L-cysteine), 3 mML-proline. NAC and proline are antioxidants MIC = more susceptible to antibiotic MIC = more resistant to antibiotic

44. Our data (unpublished): Pseudomonas aeruginosabecomes more susceptible to antibiotics in the presence of antioxidants Minimum inhibitory concentrations (µg/mL) of Pseudomonas aeruginosa PCP (120 mM pentachlorophenol), NAC (1 mM N-acetyl-L-cysteine), 3 mML-proline. NAC and proline are antioxidants MIC = more susceptible to antibiotic MIC = more resistant to antibiotic

45. Our data (unpublished): Pseudomonas aeruginosabecomes more susceptible to antibiotics in the presence of antioxidants Minimum inhibitory concentrations (µg/mL) of Pseudomonas aeruginosa PCP (120 mM pentachlorophenol), NAC (1 mM N-acetyl-L-cysteine), 3 mML-proline. NAC and proline are antioxidants MIC = more susceptible to antibiotic MIC = more resistant to antibiotic

46. Effects of Antibiotics on Sewage Treatment Processes:Framing the Problem – Anticipated Responses of Greatest Importance • Metabolic impact on the indigenous microbiology • Ecological implications for the indigenous microbiology • Consequences of microbial stress responses • Potential for spreading antibiotic resistance

47. Ecological implications – the fate of antibiotic resistance genes discharged to the natural environment correlate with urban and agricultural activity

48. Ecological implications – the fate of antibiotic resistance genes in digesters Thermophilic digestion is more effective than mesophilic digestion at eliminating tetracycline resistance genes WWTP industry’s recent move toward sustainable wastewater may promote a shift away from thermophilic digestion Ghosh, Firland LaPara, manuscript in preparation Western Lake Superior Sanitary District wastewater treatment facility (Duluth, MN) Samples were collected on different days over a one year period Data presented is from influent and digester samples collected on same day Similar patterns were observed on other sampling days tet(A), tet(O), tet(X) are tetracycline resistance conferring genes intI1 is integrase gene found on class 1 integron known to carry multiple antibiotic resistance conferring genes

49. Effects of Antibiotics on Sewage Treatment Processes:In Summary – Key Research Questions • Will WWTPs accumulate an ABR genotype? If so, will it be a permanent affect or is it reversible? • What is the risk and longevity of an increase in WWTP-enhanced ABR genotypes in the environment? • Will AB flux cause toxicity and performance failures at WWTPS? If so, can we apply strategies already learned to preemptively correct the operation of WWTPs under this scenario? • If there is toxicity due to AB flux during a pandemic, will it be an oxidative stress story?

50. Effects of Antibiotics on Sewage Treatment Processes:A Research Request • In designing experiments to evaluate answers to these questions, must ensure consideration for variables in WWT that will influence results (especially feed composition and reactor configuration). • Perform experiments with mixtures of antibiotics to determine synergistic effects.