Antibiotic bacteria in wastewater and WWTPs role in their spread into the environment. Manuela Antonelli

Transcription

1 Antibiotic bacteria in wastewater and WWTPs role in their spread into the environment Manuela Antonelli DICA - Dipartimento di Ingegneria Civile e Ambientale, 4 maggio 17

2 Antibiotic bacteria in wastewater and WWTPs role in their spread into the environment RESEARCH CONTEXT AND OBJECTIVES EXPERIMENTAL RESULTS CONCLUSIONS M. Antonelli 2/

3 Research context: antibiotic resistance Antibiotic resistant bacteria (ARB) are microorganisms able to survive to the exposure to one or more antibiotics in concentrations over the inhibition or toxicity thresholds Antibiotic resistance has been indicated as a worldwide sanitary issue by World Health Organization, producing periodic reports on ARB diffusion Several resistance mechanisms have been identified in many bacterial species, potentially determining the ineffectiveness of current antibiotic therapies and the increase in the health hazard posed by many infections Antimicrobial resistance: global report on surveillance (14) Antibiotic resistance propagate due to the enrichment of resistant bacteria or by the transfer of antibiotic resistance genes (ARGs) among members of a community M. Antonelli 3/

4 Research context: antibiotic resistance occurrence in wastewater Urban sewage are an important source of ARB, being wastewater treatment plants (WWTPs) hotspots for ARB and AR genes diffusion in the environment final effluent of WWTPs can determine the discharge of CFU of ARB per day per equivalent inhabitant (Rizzo et al., 13) Sewerage system and WWTPs are a favorable environment for AR transfer and enhancement, because of the presence of nutrients, antibiotics at sub-lethal concentrations and resistant bacteria Relevant evidences in literature, poor consensus and lack in homogeneity within different contamination sites due to the changing and complex nature of the phenomenon as well as to the difficulties in ARB detection Need for further research works assessing the influence of WWTPs on ARB, for protocol standardization and for the creation of a unique database M. Antonelli 4/

5 Research context: antibiotic resistance determination in wastewater Several methods were developed for determining the presence of ARB in environmental matrices, basically depending or not on bacteria cultivation: Cultivation-based methods: antibiotic resistance is the ratio between the number of bacteria growing in the presence and in the absence of antibiotic easier to implement even in WWTP labs Molecular methods: DNA probes and PCR techniques are used for detecting antibiotic resistance genes or genetic elements Method PROs CONs Culture-dependent Culture-independent Easy to perform and low costs Direct proof of bacteria activity Direct comparability with enforced regulations Precise and sensitive Independent from bacteria status and ability to growth on plate Time consuming Only cultivable bacteria ( 2-3%) High costs and difficult procedures Unstandardized and scarce literature Strong effect of matrix characterstics: need of sample preparation M. Antonelli 5/

6 Research context: role of wastewater treatment plants WWTPs play a significant role for antibiotic resistance: WWTPs are a prime location for monitoring antibiotics, ARB and ARGs in urban sewage Biological and disinfection processes are suspected to influence ARB selection and enhance ARGs spread Sewage Primary treatment BIOLOGICAL PROCESS Active and relevant growth of microbial population High bacterial concentration Long sludge retention time Exposure to sub-lethal concentrations of antibiotics (ng/l) DISINFECTION Key role in limiting bacteria spread surface water A few indications on the control of antibiotic resistance genes Different disinfecting actions based on chemical or physical disinfectants, potentially selecting ARB into Secondary treatment (biological process) Tertiary treatment No clear and unambiguous conclusions have been drawn so far Discharge in surface water M. Antonelli 6/

7 Research objective ARB monitoring in a urban area ARB monitoring in a densely populated urban area where anthropic pressure is high Milan metropolitan area selected as reference area for result generalisation ARB monitoring in the sewage: important anthropogenic pressure relevant presence of antibiotics in sewage THB@37 C and E. coli resistance to three widespread antibiotics (ampicillin, chloramphenicol, tetracycline) Assessing the influence of three locally operating WWTPs on ARB selection: different treatment schemes resistance at different sampling points of the three WWTPs M. Antonelli 7/

8 Experimental plan: research area Milan metropolitan area: more than inhabitants 22 hospitals and health care facilities WWTP3 7, PE INDUSTRIAL (34% COD) 5 HOSPITALS WWTP1 1,25, PE 12 HOSPITALS WWTP2 1,5, PE 5 HOSPITALS M. Antonelli 8/

9 Experimental plan: wastewater treatment plants WWTP1 WWTP2 WWTP3 Q = 432, m 3 /d Q = 345, m 3 /d Q = 177, m 3 /d PRELIMINARY TREATMENT PRELIMINARY TREATMENT PRELIMINARY TREATMENT IN IN BIOLOGICAL PROCESS FILTRATION F/M =.18 kg COD kg SSV 1 d 1 SRT = 28-3 d [Biomass] = 2.5 kg SSV /m 3 IN BIOLOGICAL PROCESS FILTRATION F/M = kg COD kg SSV 1 d 1 SRT = 3 d [Biomass] = 2.5 kg SSV /m 3 PRIMARY CLARIFIER OUT SED BIOLOGICAL PROCESS FILTRATION F/M = kg COD kg SSV 1 d 1 SRT = 9 d [Biomass] = 2.7 kg SSV /m 3 OUT BIO DISINFECTION (PAA) OUT DIS R REUSE (1 CFU/ ml as E. coli) D = 2 mg/l HRT = 45 min OUT BIO D 1 = 5- DISINFECTION (UV) mj/cm 2 D 2 = 15-3 OUT DIS S OUT DIS R mj/cm 2 SURFACE REUSE WATER OUT BIO DISINFECTION (NaOCl) OUT DIS S SURFACE WATER (5, CFU/ ml as E. coli) D = 1.8 mg/l HRT = 35 min M. Antonelli 9/

10 Experimental plan: analytical plan Sampling in sterile dark bottles 5 WWTP1 5 WWTP2 7 WWTP3 TSS COD disinfectant quenching in WWTP1 and WWTP3 254 nm ABS transport in refrigerated bag and processing in 4 h Chemical-physical analyses ARB ASSESSMENT ON THB@37 C and E. coli M. Antonelli 1/

11 Survival [%] Survival [%] Experimental plan: antibiotic resistance assessment 4 concentrations based on MIC (EUCAST) Ampicillin Chloramphenicol Tetracycline TOTAL # OF SAMPLES: 746 Colonies survived after the exposure to antibiotic A at the concentration C Survival A,C = THB A,C THB Colonies grown without exposure Resistance A = min S A,C when S A,C > NOT RESISTANT RESISTANT Antibiotic concentration (mg/l) Antibiotic concentration (mg/l) M. Antonelli 11/

12 Results: total heterotrophic bacteria (THB37 C) in wastewater Total heterotrophic bacteria is a wide class of microorganisms including all bacteria using organic matter for their metabolism Relevant differences in the characteristics of bacterial species aerobic or facultative aerobic, pathogenic or opportunistic, Cultivation method determines the emergence of certain species >37 C growth temperature: bacteria hosted by human and animals Microorganisms now not included in regulations on effluent discharge in surface water or to be reused in agriculture standards set only for faecal indicators PatríciaR (7) M. Antonelli 12/

13 Results: wastewater characteristics Low bacterial count: high dilution factor due to great water consumption and small brooks integrated in the sewage network Primary clarification is not effective in reducing C and E. coli presence Biological process reduces C and E. coli concentration by 2 and 3 orders of magnitude respectively PAA scarcely effective in THB inactivation, E. coli more sensitive to disinfectant Sampling point IN WWTP TSS (mg/l) COD (mg/l) UV254 (-) THB (CFU/mL) E. Coli (CFU/ ml) WWTP1 159± ± ±.35 (3±2.3) 1 6 (3±2.1) 1 6 WWTP2 98± ±3.1.14±.34 (4±3.6) 1 6 (3±1.2) 1 6 WWTP3 66± ± ±.35 (3±1.6) 1 6 (3±2.1) 1 6 SED WWTP3 76± ± ±.48 (3±1.5) 1 6 (3±2.) 1 6 OUT BIO OUT DIS WWTP1 2±1.4 21±3.3.7±.18 (2±3.5) 1 4 (5±6.9) 1 3 WWTP2 2±2. 21±4.5.8±.26 (2±3.4) 1 4 (5±5.9) 1 3 WWTP3 5±3.6 ±4.9.13±.7 (2±.7) 1 4 (2±1.5) 1 4 WWTP1 2±1.1 19±1.6.7±.2 (1±1.1) 1 4 < 1 WWTP2 (reuse) (2±.9) 1 2 < 1 1±1.1 18±3.4.7±.6 WWTP2 (discharge) (2±2.2) 1 4 (3±6.5) 1 2 WWTP3 5±3.6 21±4.9.14±.22 (5±3.4) 1 2 < 1 M. Antonelli 13/

14 Results: resistance to ampicillin general remarks WWTP1 IN WWTP2 IN WWTP3 IN WWTP3 SED WWTP1 OUT BIO WWTP2 OUT BIO WWTP3 OUT BIO THB@37 C WWTP1 OUT DIS R WWTP2 OUT DIS R WWTP2 OUT DIS WWTP3 OUT DIS 9 7 WWTP1 IN WWTP3 IN WWTP2 IN WWTP3 SED 9 7 WWTP1 OUT BIO WWTP2 OUT BIO WWTP3 OUT BIO 9 7 WWTP1 OUT DIS R WWTP2 OUT DIS R WWTP2 OUT DIS D WWTP3 OUT DIS D E. coli M. Antonelli 14/

15 Results: resistance to ampicillin general remarks 9 WWTP1 IN WWTP2 IN THB@37 C WWTP3 IN WWTP3 SED WWTP1 IN WWTP2 IN WWTP3 IN WWTP3 SED E. coli High variability of experimental data: relevant constraint of the method Ampicillin-resistant THB@37 C and E. coli in all sampling points No relevant changes at SED: primary clarification does not influence ARB No relation with the number of hospitals in the drainage area of each WWTP: other aspects should be considered, as length of the drainage network, or treatments performed to wastewater at the discharge into the sewage M. Antonelli 15/

16 Results: resistance to ampicillin - influent wastewater 9 WWTP1 IN WWTP2 IN THB@37 C WWTP3 IN WWTP3 SED Ampicillin-resistant THB@37 C in IN: 19% in WWTP1, 25% in WWTP2, 17% in WWTP3 Ampicillin-resistant E. coli in IN: 9 WWTP1 IN WWTP3 IN WWTP2 IN WWTP3 SED 25% in WWTP1, 47% in WWTP2, % in WWTP E. coli M. Antonelli 16/

17 Results: resistance to ampicillin general remarks 9 7 WWTP1 IN WWTP3 IN WWTP2 IN WWTP3 SED 9 7 WWTP1 OUT BIO WWTP2 OUT BIO WWTP3 OUT BIO 9 7 WWTP1 OUT DIS R WWTP2 OUT DIS R WWTP2 OUT DIS WWTP3 OUT DIS THB@37 C 9 WWTP1 IN WWTP3 IN WWTP2 IN WWTP3 SED 9 WWTP1 OUT BIO 9 WWTP1 OUT DIS R WWTP2 OUT BIO WWTP2 OUT DIS R 7 7 WWTP3 OUT BIO 7 WWTP2 OUT DIS D WWTP3 OUT DIS D E. coli M. Antonelli 17/

18 Results: resistance to ampicillin - biological process 9 WWTP1 OUT BIO THB@37 C WWTP2 OUT BIO 7 WWTP3 OUT BIO WWTP1 WWTP2 WWTP3 IN OUT BIO IN OUT BIO IN OUT BIO WWTP1 OUT BIO WWTP2 OUT BIO WWTP3 OUT BIO E. coli THB@37 C E. coli M. Antonelli 18/

19 Results: resistance to ampicillin - biological process WWTP1 OUT BIO WWTP2 OUT BIO WWTP3 OUT BIO WWTP1 OUT BIO WWTP2 OUT BIO WWTP3 OUT BIO THB@37 C E. coli Survival trend change with respect to IN in WWTP1, unlike in WWTP2 and WWTP3 Biological process determines significant changes in microbial population, resulting in a modification of the bacterial resistance to ampicillin Conventional parameters for describing biological process, such as F/M ratio and SRT, are useless in defining the changes in bacterial antibiotic resistance M. Antonelli 19/

20 Results: resistance to ampicillin general remarks 9 7 WWTP1 IN WWTP3 IN WWTP2 IN WWTP3 SED 9 7 WWTP1 OUT BIO WWTP2 OUT BIO WWTP3 OUT BIO 9 7 WWTP1 OUT DIS R WWTP2 OUT DIS R WWTP2 OUT DIS WWTP3 OUT DIS THB@37 C WWTP1 IN WWTP3 IN WWTP2 IN WWTP3 SED 9 WWTP1 OUT BIO 9 WWTP1 OUT DIS R WWTP2 OUT BIO WWTP2 OUT DIS R 7 7 WWTP3 OUT BIO 7 WWTP2 OUT DIS D WWTP3 OUT DIS D E. coli M. Antonelli /

21 Results: resistance to ampicillin - disinfection WWTP1 OUT DIS R WWTP2 OUT DIS R WWTP2 OUT DIS WWTP3 OUT DIS WWTP1 OUT DIS R WWTP2 OUT DIS R WWTP2 OUT DIS D WWTP3 OUT DIS D THB@37 C E. coli Low number of E. coli in OUT DIS samples when reuse standard is enforced (small number of colonies in plates not exposed to antibiotics) hard to draw definite conclusions on the influence of the different disinfection processes UV treatment aimed to discharge into surface water (WWTP2 - OUT DIS) has not substantial impact in selecting ARB: resistance of about 19% comparable to OUT BIO M. Antonelli 21/

22 Results: resistance to ampicillin - disinfection 9 7 WWTP1 OUT DIS R WWTP2 OUT DIS R WWTP2 OUT DIS WWTP3 OUT DIS THB@37 C NaOCl showed a slightly decreasing trend with concentration: survival ratios at 23-34% The action of UV radiation was identical for resistant and non-resistant THB, irrespectively of UV dose, resulting in survival ratios comparable to biological process PAA acted selectively on ampicillin-resistant bacteria, unlike NaOCl and UV: it displayed a decreasing trend with concentration (survival ratios : 3-%) M. Antonelli 22/

23 Results: resistance to chloramphenicol WWTP1 IN WWTP3 IN Chloramphenicol (mg/l) WWTP2 IN WWTP3 SED THB@37 C WWTP1 OUT BIO WWTP2 OUT BIO WWTP3 OUT BIO Chloramphenicol (mg/l) WWTP1 OUT DIS R WWTP2 OUT DIS R WWTP2 OUT DIS WWTP3 OUT DIS Chloramphenicol (mg/l] WWTP1 IN WWTP3 IN Chloramphenicol (mg/l) WWTP2 IN WWTP3 SED Chloramphenicol (mg/l) WWTP1 OUT BIO WWTP2 OUT BIO WWTP3 OUT BIO E. coli WWTP1 OUT DIS R WWTP2 OUT DIS R WWTP2 OUT DIS D WWTP3 OUT DIS D Chloramphenicol (mg/l) Chloramphenicol acted strongly on THB@37 C and E. coli, determining very low survival ratios No significant bacterial resistance to chloramphenicol was detected M. Antonelli 23/

24 Results: resistance to tetracycline WWTP1 IN WWTP3 IN Tetracycline (mg/l) WWTP2 IN WWTP3 SED Tetracycline (mg/l) WWTP1 IN WWTP2 IN WWTP3 IN WWTP3 SED Tetracycline (mg/l) Tetracycline is the most effective antibiotic against the monitored indicators No bacterial resistance to tetracycline was detected THB@37 C WWTP1 OUT BIO WWTP2 OUT BIO WWTP3 OUT BIO Tetracycline (mg/l) Tetracycline (mg/l) WWTP1 OUT BIO WWTP2 OUT BIO WWTP3 OUT BIO E. coli WWTP1 OUT DIS R WWTP2 OUT DIS R WWTP2 OUT DIS WWTP3 OUT DIS Tetracycline (mg/l) WWTP1 OUT DIS R WWTP2 OUT DIS R WWTP2 OUT DIS D WWTP3 OUT DIS D M. Antonelli 24/

25 Results: statistical analyses for interaction assessment Antibiotic type, sampling point and WWTP were significant (p-value <.5) Initial colony number was not significant bacteria number seems not to be a key factor The significance of interaction terms confirms the presence of relevant differences in survival trends at various operating conditions Source df Mean Square F p-level Antibiotic concentration Initial THB concentration Antibiotic type Sampling point WWTP Antibiotic * sampling point Antibiotic * WWTP Sampling point * WWTP Antibiotic * sampling point * WWTP Analysis of Covariance (ANCOVA) - Data for THB@37 C M. Antonelli 25/

26 Conclusions: monitoring campaign The presence of a relevant quote of ampicillin-resistant bacteria in Milan area sewage was detected A direct relation between the discharge of hospitals in sewerage system and the occurrence of antibiotic resistance has not been highlighted A change in ampicillin-resistant bacteria fraction was determined by biological processes, having different extent in the three WWTPs and apparently not correlated to conventionally used operating parameters Peracetic acid acted selectively on ampicillin-resistant bacteria and limited the discharge of ARB in surface water Further studies are needed for UV disinfection, considering its specific mechanism action and implications on water quality Interaction between antibiotic resistance and WWTPs is a complex phenomenon, asking for accurate monitoring techniques and proper planning of treatment schemes M. Antonelli 26/

27 Additional investigation: resistance mechanisms Objectives Maintenance of antibiotic resistance genes (ARGs) at plasmid level has a fitness cost What is the effect of oxidative stress by disinfection on ARGs? Is the abundance of ARGs changing in a population as a consequence of PAA disinfection? Enterococcus faecium as bacterial indicator Investigation at laboratory scale in collaboration with CNR-ISE M. Antonelli 27/

28 Additional investigation: resistance mechanisms Experimental plan E. faecium transconjugant strain resistant to erythromycin and carrying ermb ARG Disinfection tests at two PAA doses: 7.5 and 25 mg L-1 min-1 Regrowth tests after PAA in two different conditions: 1. High nutrient limitation at C (survival test) 2. Favorable growing conditions in rich medium at 37 C (recovery test) M. Antonelli 28/

29 Additional investigation: resistance mechanisms Results and conclusion No significant variation in the abundance of ARG after disinfection in regrowth test Investigated ARGs are stable and the oxidative stress due to PAA disinfection at low doses does not induces a fitness cost resulting in ARG release Further investigation on resistance mechanisms are required to assess the influence of WWTPs and to identify best alternatives for ARGs control PAA dose mg L-1 min-1 M. Antonelli 29/

30 Thank you Antibiotic bacteria in wastewater and WWTPs role in their spread into the environment Manuela Antonelli

31 Advanced treatment for water and wastewater: an overview of research activity at POLIMI DICA Manuela Antonelli DICA - Dipartimento di Ingegneria Civile e Ambientale, 4 maggio 17

32 Advanced Water and Wastewater Treatment THE RESEARCH GROUP 1 ASSISTANT PROFESSOR 1 POST DOC RESEARCHER 1 FELLOW RESEARCHER 4 PHD STUDENTS 11 MSC STUDENTS 4 BSC STUDENTS M. Antonelli 32/

33 Advanced Water and Wastewater Treatment MAIN RESEARCH LINES Innovative sensors and management strategies for resilient drinking water networks Removal of micropollutants and emerging contaminants by activated carbon and innovative materials Disinfection of drinking water and wastewater: suitability, optimization and modelling Process modelling, optimization and control for wastewater reuse ONGOING PhDs Stochastic modelling of the fate of emerging contaminants Advanced oxidation processes: innovative technologies and modelling Microbial resistance to antibiotics M. Antonelli 33/

34 THE DRINKABLE PROJECT Development of systems for real-time on-line monitoring and management of drinking water distribution networks Combination of: miniaturized sensors CFD modelling process analysis Local detection of water parameters (temperature, ph, conductivity, fouling, THMs, ) Implications: early warning for contamination, process optimization M. Antonelli 34/

35 AN INNOVATIVE SENSOR FOR DETECTION OF FOULING IN PIPES Miniturized sensors for the measurement of biofilm and inorganic scale Inter-digitated commercial microelectrodes with Arduino microcontroller Correlation between impedance signal and fouling thickness High accuracy (5 µm) and discrimation of slime nature M. Antonelli 35/

36 REMOVAL OF POLLUTANTS BY ACTIVATED CARBON Complex and competitive process: lab and pilot experiments Process modelling aimed at feasibility and economic assessment Characterisation of activated carbon to correlate: adsorbent removal capacity towards specific pollutants adsorbent characteristics adsorbent lifetime Assessment of BAC (Biological Activated Carbon) process which couples adsorption and biological activity of attached biomass Guidelines for process design being GAC adsorption the BAT M. Antonelli 36/

37 DISINFECTION BY PERACETIC ACID (PAA) Emerging disinfectant whose mechanisms are still partially unknown: decay, inactivation Potential combination with UV for a additive/synergic effect Lab testing (DoE, Design of Experiments) and monitoring combined with modelling (regression, analysis of uncertainty, simulations, machine learning) Development of predictive kinetic models for optimization and process control M. Antonelli 37/

38 MODELLING OF ENVIRONMENTAL REACTORS BY CFD (Computational Fluid Dynamics) Simulation tool for the local description of chemical-physical phenomena in multiphysical systems Equations solved in each element of computational domain concurrently Fluid dynamics Radiation transfer Chemical reactions Design of optimized reactors, process intensification, reactor scale-up M. Antonelli 38/

39 MODELLING OF ENVIRONMENTAL REACTORS BY CFD (Computational Fluid Dynamics) Simulation tool for the local description of chemical-physical phenomena in multiphysical systems INLET Equations solved in each element of computational domain concurrently Fluid dynamics Radiation transfer Chemical reactions OUTLET Design of optimized reactors, process intensification, reactor scale-up M. Antonelli 39/

40 MECHANISTIC ASSESSMENT OF AOPs (Advanced Oxidation Processes) Investigation of involved reaction mechanisms: lab testing and modelling Established and innovative AOPs: UV coupled to H 2 O 2 or O 3 Ozonation Heterogeneous photocatalysis on TiO 2 Heterogeneous photocatalysis on hematite Determination of reactive species (OH radicals) generation as a function of operating parameters and estimation of indicators for process performance Design of optimized processes and comparison among AOPs M. Antonelli /

41 INFLUENCE OF DISINFECTION ON MICROBIAL COMMUNITIES Assessment of the role of disinfection in modifying microbial communities and antibiotic resistance Selective action of PAA against resistant bacteria in comparison to non-resistant, unlike for NaClO Occurrence of defense strategies in microbial communities: aggregation Important sanitary implications and guidelines for process design M. Antonelli 41/