Gehr, Ronald

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Engineering Conferences InternationalECI Digital ArchivesWastewater and Biosolids Treatment and Reuse:Bridging Modeling and Experimental Studies ProceedingsSpring 6-11-2014Wastewater disinfection with UV or PAA: Arethose surviving microbes really benign?Ronald GehrMcGill UniversityFollow this and additional works at: http://dc.engconfintl.org/wbtr_iPart of the Environmental Engineering CommonsThis Conference Proceeding is brought to you for free and open access by the Proceedings at ECI Digital Archives. It has been accepted for inclusion inWastewater and Biosolids Treatment and Reuse: Bridging Modeling and Experimental Studies by an authorized administrator of ECI Digital Archives.For more information, please contact franco@bepress.com.Recommended CitationRonald Gehr, "Wastewater disinfection with UV or PAA: Are those surviving microbes really benign?" in "Wastewater and BiosolidsTreatment and Reuse: Bridging Modeling and Experimental Studies", Dr. Domenico Santoro, Trojan Technologies and WesternUniversity Eds, ECI Symposium Series, (2014). http://dc.engconfintl.org/wbtr_i/21Wastewater disinfection with UV or PAA: Are those surviving microbes really benign?Ronald GehrDepartment of Civil Engineering and Applied Mechanics, McGill University, Montréal,Québec, H3A 0C3, CanadaWastewater and Biosolids Treatment and Reuse:  Bridging Modeling and Experimental Studies“Wastewater Treatment, Disinfection and Reuse” (Theme A)Otranto (Lecce), ITALYJune 11, 2014Introduction and context (1)• Wastewater (WW) disinfection does not aim to inactivate ALL microorganisms– typical indicator target levels: 200 – 1,000 CFU/100 mL (after dilution)• No information on – differential inactivation or selection of pathogenic/non-pathogenic microorganisms during WW disinfection, or – the effect of disinfection on antibiotic resistanceIntroduction and context (2)• Given the large number of pathogens, we use– indicator organisms (for convenience of testing) or – model organisms (as representative of pathogens)  • Fortunately, E. coli fit into both categories for bacteria:– easily isolated and cultured– large body of research on pathogenesis and genomics– includes pathogenic and non-pathogenic strainsObjective of the studyElucidate the dynamics of:A) pathogenic and non-pathogenic strains of E. coli, andB) their development or loss of antimicrobial resistance, following disinfection by PAA or UVWe will base our results on information obtained using microbial methods, i.e. at the genetic level.  Examples of legislation and guidelines for municipal effluent discharges affecting bathing watersApril, 2012April, 2012Which strains of E. coli should we use as model organisms?We based the selection on key pathotypes and virulence genesWhat cell characteristics create a particular pathotype? • Pathogenesis based on:– suitable number of virulence genes (VGs)– suitable combination of VGs – all encoding one or multiple virulence factors (VFs)Review of virulence, pathogenicity islands (PAIs) and integrons (coding for AMR)DNAProteinsCytoplasmicmembraneBacterial celli||il|ii||il|iPathogenicity island(PAI) codes for virulenceIntegron codes for AMRUV or PAA may induce repair mechanisms which then induce “jumping” of PAIs or integronsClassification of E. coli on the basis of clinical symptoms and phylogenetic groups.What cell characteristics create a particular pathotype? • Pathogenesis based on:– suitable number of virulence genes (VGs)– suitable combination of VGs – all encoding one or multiple virulence factors (VFs)• Our previous work: UPECs are the predominant pathotypes in WWTP effluents • A majority of UPEC virulence genes are clustered on pathogenicity islands (PAIs)Frigon, F., et al."Biological and PhysicochemicalWastewater Treatment Processes Reduce the Prevalence of Virulent Escherichia coli".AEM 79, 3, 835- 844 (2013). Classification of E. coli on the basis of clinical symptoms and phylogenetic groups.Pathotyping rule for UPECsVirulence factorsVirulence genes No. of genes requiredAdhesins P-fimbriae: papA, papC, papG, pixA 2Capsules kpsM(II) , kpsM(III) 1Iron uptake systemsE. coli siderophore: iroNYersiniabactin: fyuA, irp(1), irp(2)Aerobactin: iucD, iutAABC Fe2+ transporter: sitA, sitD1Toxins Heamolysins: hlyA, vatCytotoxins/transporter:cnf(1), cnf(2),  sat1Our study: Need at least 5 of 19 virulence genes below for an isolate to be a UPEC.  (Rule from Frigon et al, AEM, 2013)Refining the questions regarding virulence, corresponding to our objectives• Are there changes in the proportions of UPEC E. coli when disinfecting with UV or PAA?• Do UV and PAA produce similar effects?• Will free-swimming populations (i.e. following filtration) respond differently than particle-associated populations? AntimicrobialsMode of action and resistance mechanism of various antimicrobialsLink between virulence and antimicrobial resistance (AMR)• E. coli can serve as vectors for dissemination of AMR genes• Positive co-occurrence of virulence and AMR genes has been demonstrated in UPECsFrigon, D.,et al. Impact of Wastewater TreatmentProcesses on Antimicrobial Resistance Genesand their Co-occurrence with Virulence Genes in Escherichia coli. Water Research, 50, 245-253 (2014)..Questions regarding antimicrobial resistance (AMR) genes• Are there changes in the number and classes of AMR genes in UPECs when disinfecting with UV or PAA?• Do UV and PAA produce similar effects?• Will free-swimming populations respond differently than particle-associated populations? Key methods - Virulence • Effluent samples from activated sludge (AS), biofilter (BF), and physicochemical (PC) plants• For some samples, particles removed by centrifugation and 20 µm filter• UV disinfection – collimated beam• PAA disinfection – 12% PAA, 30 or 60 min contact time; residuals by DPD• Target E. coli level 200 CFU/100 mL• Initial screening for 3 UPEC genes using Bioplex PCR• Major data source: microarrayKey methods - AMR • Same effluent samples and isolates as for virulence testing; same disinfection• For some samples, particles removed by centrifugation and 20 µm filter• The screen-positive isolates for the AS, BF1, BF2, and PC1 samples and all the isolates from the PC2 samples were genotyped by microarray• The microarray probed 70 AMR genes of 11 classes, and 8 mobile genetic element sequencesDNA microarray image of an E. coli isolate24Positive  control (lacY gene)Different colours of probes based on intensity ratioBuffer spotsBefore HybridizationAfter HybridizationResultsWastewater characteristicsParameter (unit)Wastewater treatment plantsAS BF BFa PC1 PC2Treatment processesConventional activated sludgeBiological filtration Physico-chemicalPhysico-chemicalpH 7.1 7.7 - 7.2 7.1UV T (%) 67.2 63.6 - 54.5 42.6SS (mg/L) 10 5.0 14 15 18COD (mg/L) 38 46 62 45 92E. coli (CFU/100 mL)7.8×103 1.1×105 3.0×105 1.6×105 9.9×105UV inactivation curves0.01.02.03.04.05.06.07.00 5 10 15 20 25 30 35 40Log E. coli (CFU/100 mL)UV fluence (mJ/cm2)AS, NFBF, NFPC1, NFPC2, NFPC2, FPlant UV fluence to reach 200 CFU/100 mLAS NF ~7BF NF ~15PC1 NF ~30PC2 NF ~30PC2 F ~15PAA inactivation curves0.01.02.03.04.05.06.00 20 40 60 80 100 120 140Log E. coli(CFU/100 mL)PAA dose, CT (mg-min/L)AS,T=30 min. NFPC1, T=30 min, NFBF, T=30 min, NFBFa, T=60 min, NFBFa, T=60 min, FPlant PAA  CT  (mg/l-min) to reach200 CFU/100 mLAS NF ~30BF NF 60 - 120BF F ~90PC1 NF ~55In the following slides:ND = non-disinfectedD = disinfectedImpact of UV on UPEC fractions0%4%8%12%16%20%24%ND D ND D ND D ND D ND DAS BF PC1 PC2 PC2Non-filtered Filtered% UPECAverage reduction of UPEC fractions: 55%For the PC plants, greater reduction in the free-swimming UPECs-80% -42% -22% -61% -71%Impact of PAA on UPEC fractions0%4%8%12%16%20%ND D ND D ND D ND DAS BF PC1 BFaNon-filtered Filtered% UPECAverage reduction of UPEC fractions: 52%-12% -100% -85% -11%Impact of UV on prevalence of antimicrobial resistance gene (ARG)-carrying E. coli inconsistentImpact of PAA on prevalence of antimicrobial resistance gene (ARG)-carrying E. coli inconsistentImpact of UV on occurrence of the mean number of antimicrobial resistance gene classes mainly downImpact of PAA on occurrence of the mean number of antimicrobial resistance gene classes all downMechanisms?  Reasons for different behaviour for UV and PAA?Different disinfection mechanisms for UV and PAADNAProteinsCytoplasmicMembraneUVBacterial CellPAADifferent disinfection mechanisms for UV and PAAAsk your favourite expertThe engineer How does the surviving cell respond?  (up-regulated genes)UV • Readily penetrates cells• Reacts mainly with DNA• Mainly DNA repair• Some protein expression• Some nucleotide metabolismPAA • Diffuses/reacts: outside → inside• Forms OH-radicals which react with proteins (oxidation of sulfur groups) and DNA• Oxidative stress response• Processing of sulfur amino acids• DNA repairNon-pathogenic strains can become pathogenic by inter-cellular mechanisms of horizontal gene exchange:39DNA of organismImported DNAVirulence genes can be assembled inside the cell in units by transposable elementsResult: assembly of Pathogenicity Islands (PAIs)→All genes for a pathotype can be transferred at oncePlasmid(moves between organisms)Transposon/Integrons(jumping DNA)Captured virulencegenes Organisms’ DNATransposable elements jump between DNA structuresSummary – impact of disinfection• Proportion of UPEC isolates relative to non-pathogenic isolates decreased by ~ 55% for UV and PAA• Although UV and PAA interact differently with cells, impact on virulence factors is similar• Inconsistent effects on prevalence of ARGs, but except for UV on AS effluents, mean number of ARG classes decreased• Filtration:– reduces UV fluence requirements, as expected– had little effect on PAA requirements: wastewater COD more important– impact on virulence: apparent reduction for UV– impact on AMR: reduction in all cases (PAA & UV; genes & classes)Explanations• Both UV and PAA disrupt DNA and genetic elements, but may also stimulate repair mechanisms, including gene transfer• “Importing” gene mechanisms can also function as “exporting” mechanisms.  If PAIs are exported out of the cell, they will not be detected and the cell will not be virulent or have AMRConsequences and future work• Virulence is rare, and the genetic requirements complex, hence loss of virulence is a reasonable first consequence of disinfection • Public health aspects: surviving microbes less likely to be virulent → standards may be conservative (good news!!)• Disinfection does not, in general, increase AMR (also good news!!)• Must examine repair in stressed and non-stressed environmentsAcknowledgements• Basanta Kumar Biswal, Dominic Frigon, Ramzi Khairallah, Luke Masson, Alberto Mazza• Funding from NSERC Strategic Grant # STPGP 35117-07 and Trojan Technologies Inc.• Bill Cairns for UV• Solvay/Interox for PAA• Plant operators at four WTPsThanks for listeniNg.Any questions?And don’t worry, we’ll survive this one too!

Wastewater disinfection with UV or PAA: Are those surviving microbes really benign?

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Wastewater disinfection with UV or PAA: Are those surviving microbes really benign?

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