Onsite Wastewater Systems Differentially Reduce Antibiotic ...

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Onsite Wastewater Systems Differentially Reduce Antibiotic ...

Onsite Wastewater SystemsDifferentially ReduceAntibiotic Resistance andSurvival of E. coliStacy Pfluger 1 , Rene D. Massengale 2and Joe C. Yelderman, Jr.Baylor UniversityDepartments of Biology and Geology1 Presently at Angelina College, Lufkin, TX2 Presently at Harrisburg University, PAPREPARED IN COOPERATION WITH AND FINANCED BY THETEXAS ON-SITE WASTEWATER TREATMENT RESEARCH COUNCIL

Onsite Wastewater Treatment1/3 of US households use onsitesewage systems> 1 trillion gallons of wastewater passthrough these systems annuallyLarge source of discharge to surfaceand groundwater

Traditional Septic SystemsMay be impractical due to thin soils over bedrock,clay soils or seasonally high water tables.

Water Quality ConcernsFecal indicator bacteria are commonlyfound in surface waterE. coli, coliforms, Enterococcus spp.Origins of contaminants:– Animal feces, wild and domestic– Human sewage

Indicators used for WaterQuality AnalysisBifidobacterium Escherichia coli EnterococcusHere, we use E. coli and coliforms asindicators of wastewater treatment

Site Design and LayoutWastewaterTreatmentPlantReturnPumpDosingPumpAerobicSystemDosingPumpSepticDosingReturnSubmerged-bedWetland


Hypothesis: Some onsitesystems will better reduceE. coli concentrationsthan others.www.bd.com

Method: EPA Method 1604Membrane filtration of water samplesIncubated on MI agar– E. coli are blue– Coliforms fluoresce under UV

Log Reduction of E. coli by theWetland Varies SeasonallySeptic Wetland AerationSeptic Wetland AerationSeptic Wetland AerationSeptic Wetland Aeration

Aeration and Wetland ProvideSimilar Reduction AnnuallySeptic Wetland Aeration

Concentrations of E. coli from theWetland and Aeration are Similar

E. coli Reduction ConclusionsAeration and Septic + Wetlandreduce amounts of E. coli morethan septic alone.Quarterly: Aeration is constant;Septic + Wetland varies – leastreduction in winterAnnually: statistically similar

Significant Concentrations ofE. coli Remain7Log10(E. coli) per 100 mLMean(Log10(E. coli))6543210SepticRawB C D F HSepticTankSeptic +WetlandAerobicAerobicRaw

Significant Concentrations ofTotal Coliforms Remain7Log10(E. coli) per 100 mL6543210SepticRawSepticTankSeptic +WetlandAerobicAerobicRaw

E. coli variations 07-08

Hypothesis: Types of Soil willVary in Ability to ReduceBacterial Loads

Triplicate soil columns ofType 1b (sandy soils)andType III (clayey soils)

Type III Soils Reduce E.colibetter than Type Ib Soils

Soil Columns ReduceBacterial Load

Hypothesis: Antibioticresistance in E. coli will varybetween systems

Methods:Isolates plated on Tryptic Soy Agarwith 80µg/mL antibioticsGrowth

Antibiotic Resistance ProtocolMethod: plates stamped with isolates,incubated 36-48 hr at 37°C, growthcomparedControlAmpicillin 80µg/ml

Septic and Wetland may Increaseor Decrease the Percent of E. coliResistant to 80µg/mL Antibiotics

Septic and Wetland may Increaseor Decrease the Percent of E. coliResistant to 80µg/mL AntibioticsIncreased resistancein Wetland

Aeration Decreases the Percent E.coli Resistant to 80µg/mL Antibiotics

Aeration Effluent has the LowestPercentage E. coli Resistant to80µg/mL Antibiotics

Antibiotic Resistance ComparisonAeration: generally decreased thepercent of resistant E. coliSeptic: reduced percent resistant to 3antibiotics; increased the percentresistant to 4 antibioticsWetland: decreased percentresistant for 2 antibiotics; increasedpercent resistant to 5 antibiotics

Hypothesis: TreatmentSystems will Vary in Ability toReduce Pathogen Indicators

Methods:Commercially-available ELISA kits usedto detect proteins (indirect test forpathogensE. coli verotoxin – in some strainsE. coli O157:H7 – pathogenic strainClostridium dificile – pathogenic, diarrheaCrypotosporidium and Giardia – pathogenicprotozoansAdenovirus and Rotavirus – common virusescausing diarrheal disease

Septic, Septic+Wetland, andAeration Reduce PathogenSepticRawMarkersSepticSeptic +WetlandAerationRawAerationE. coli O157 33.33 0 0 20 0Clostridiumdifficile 16.67 0 16.67 20 20Cryptosporidium 0 0 0 20 20Giardia 100 100 66. 67 100 80•Adenovirus, Rotavirus, and E. coli verotoxin were not detected abovedetection limit in any sample.•** Small sample sizes

AcknowledgementsFunding provided by:Texas Onsite Wastewater Treatment ResearchCouncilBaylor University: Folmar and GardenerResearch AwardsLab Assistance:Tamara Strohm, Tiffany Gatchel, Casey Hill


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