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Effects of Antibiotics on Sewage Treatment Processes Presented at Pharmaceutical Usage During an Influenza Pandemic – Implications for Sewage Treatment.

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Presentation on theme: "Effects of Antibiotics on Sewage Treatment Processes Presented at Pharmaceutical Usage During an Influenza Pandemic – Implications for Sewage Treatment."— Presentation transcript:

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 Effects of Antibiotics on Sewage Treatment Processes: Framing the Problem ScreeningsGrit 1 0 Sludge Biosolids Handling Residual 1 0 Treatment 2 0 Treatment Disinfection Effluent 2 0 Sludge

3 Effects of Antibiotics on Sewage Treatment Processes: Framing the Problem ScreeningsGrit 1 0 Sludge Biosolids Handling Residual 1 0 Treatment 2 0 Treatment Disinfection Effluent 2 0 Sludge Pathogen protection occurs in multiple locations

4 Effects of Antibiotics on Sewage Treatment Processes: Framing the Problem ScreeningsGrit 1 0 Sludge Biosolids Handling Residual 1 0 Treatment 2 0 Treatment Disinfection Effluent 2 0 Sludge Concentrations will be orders of magnitude higher than baseline, over several weeks Baseline ~ ng/L Pandemic ~ 40–400 µg/L

5 Effects of Antibiotics on Sewage Treatment Processes: Framing the Problem ScreeningsGrit 1 0 Sludge Biosolids Handling Residual 1 0 Treatment 2 0 Treatment Disinfection Effluent 2 0 Sludge Consider conflicting perspectives about antibiotic resistance Want biomass to be resistant to antibiotics so process functions Want effluents to be free of antibiotic resistant microorganisms to prevent introduction into environment

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., 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 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., 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 also a key determinant in biotransformation patterns

10 Example - Trimethoprim is not inhibitory to activated sludge enrichment culture up to 10 mg/L 0.011,00010,000Control (µg/L) Khunjar, Love and Aga, 2009, in Progress Extant test with heterotrophic activated sludge enrichment culture Acetate-based feed Biomass concentration ~ 200 mg/L TSS No nitrifiers present

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 To evaluate the fate of pharmaceuticals by bacteria common to WWT processes, we use various cultures, feeds and reactor configurations 1)Ammonia oxidizing bacteria (N. europaea) – Monooxygenase 2)Heterotrophic culture OX-, No Oxygenases 3)Heterotrophic culture OX+, Mono/Di Oxygenases Batch growth Chemostat SRT = 7 day 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 17 α -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 flocDense biofilm on membrane

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

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?

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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

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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 Water Research, 39:

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 Source-effect studies identified relationships between chemicals and process upset modes The qualitative scale reflects the intensity of the effect for IC 50 -shocked reactors and the indicated NH 3 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 Varies Henriques et al., 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 Kohanski et al. Cell 130, 797–810, September 7, 2007

34 Activated sludge deflocculates when exposed to oxidative (thiol-reactive) chemical stressors ControlNEMCDNB 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

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

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: , 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 Transcriptome analysis reveals multi-drug efflux genes upregulated to protect Pseudomonas aeruginosa from pentachlorophenol stress. Applied and Environmental Microbiology 73(14):

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

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

42 PCPPCP + NACPCP + L-ProlineNACL-Prolilne Tetracycline Chloramphenicol Nalidixic Acid Carbenicillin Ciprofloxacin x2x-4xxx1/4-1/2x Minimum inhibitory concentrations (µg/mL) of Pseudomonas aeruginosa PCP (120 mM pentachlorophenol), NAC (1 mM N-acetyl-L-cysteine), 3 mM L-proline. NAC and proline are antioxidants Our data (unpublished): Pseudomonas aeruginosa becomes more susceptible to antibiotics in the presence of antioxidants MIC = more susceptible to antibiotic MIC = more resistant to antibiotic

43 PCPPCP + NACPCP + L-ProlineNACL-Prolilne Tetracycline Chloramphenicol Nalidixic Acid Carbenicillin Ciprofloxacin x2x-4xxx1/4-1/2x Minimum inhibitory concentrations (µg/mL) of Pseudomonas aeruginosa PCP (120 mM pentachlorophenol), NAC (1 mM N-acetyl-L-cysteine), 3 mM L-proline. NAC and proline are antioxidants Our data (unpublished): Pseudomonas aeruginosa becomes more susceptible to antibiotics in the presence of antioxidants MIC = more susceptible to antibiotic MIC = more resistant to antibiotic

44 PCPPCP + NACPCP + L-ProlineNACL-Prolilne Tetracycline Chloramphenicol Nalidixic Acid Carbenicillin Ciprofloxacin x2x-4xxx1/4-1/2x Minimum inhibitory concentrations (µg/mL) of Pseudomonas aeruginosa PCP (120 mM pentachlorophenol), NAC (1 mM N-acetyl-L-cysteine), 3 mM L-proline. NAC and proline are antioxidants Our data (unpublished): Pseudomonas aeruginosa becomes more susceptible to antibiotics in the presence of antioxidants MIC = more susceptible to antibiotic MIC = more resistant to antibiotic

45 PCPPCP + NACPCP + L-ProlineNACL-Prolilne Tetracycline Chloramphenicol Nalidixic Acid Carbenicillin Ciprofloxacin x2x-4xxx1/4-1/2x Minimum inhibitory concentrations (µg/mL) of Pseudomonas aeruginosa PCP (120 mM pentachlorophenol), NAC (1 mM N-acetyl-L-cysteine), 3 mM L-proline. NAC and proline are antioxidants Our data (unpublished): Pseudomonas aeruginosa becomes more susceptible to antibiotics in the presence of 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 Ghosh, Firl and 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 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

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.

51 Acknowledgements Dr. Sudeshna Ghosh Postdoctoral Research Associate Antibiotic resistance Professor Diana Aga University of Buffalo Environmental chemistry and pharmaceutical fate Wendell Khunjar Ph.D. Student, Virginia Tech Role of heterotrophic and autotrophic bacteria, and microbial physiology on pharmaceutical fate Dr. Charles Bott Summer 2009 – Director of Research, Hampton Roads Sanitation District Microbial stress responses to chemical shocks in WWTPs Dr. Joy Fraga Muller Post-Doc, Univ Washington Multidrug efflux pumps as a stress response

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