167 7 Mobil ity of Tylosin and Enteric Bacteria in Soil Columns Keri L. Henderson, Thomas B. Moorman, and Joel R. Coats 7.1 INTRODUCTION Theproductionofswine,cattle,andpoultryraisedforhumanconsumptionrepre- sents a signicant portion of the U.S. agricultural economy. To maximize produc- tion, producers regularly use antibiotics as supplements in animal feed and water to increase weight gain and prevent diseases among their livestock. In swine, for example, it is estimated that antibiotics are used for disease prevention and growth promotioninmorethan90%ofstarterfeeds,75%ofgrowerfeeds,50%ofnishing feeds,and20%ofsowfeeds,andequallyrelevantnumbersareseeninbeefcattle production(Hayesetal.,1999;USDAAPHISSwine2000andCOFE).Ithasbeen well documented that measurable quantities of these antibiotics are excreted, often inoriginalform,infecesandurineoflivestock(FAO/WHO,1991).Livestockwaste, containingantibiotics,isoftenusedasfertilizerforfarmeldsorpasturesandmay resultinnonpointsourcepollutionofgroundorsurfacewaters(Lokeetal.,2000). Although antibiotic residues have been studied extensively in tissues and excrement, we are only beginning to understand the environmental fate of antibiotics and their metabolites once the excreta reaches soil and water environments. Contents 7.1 Introduction 167 7.1.1 Tylosin 168 7.1.2 Enteric Bacteria 169 7.2 Mater ials and Metho ds 170 7.2.1 Preliminary Trial 170 7.2.2 Main Study 171 7.3 Results a nd Discussion 171 7.3.1 P reli mi na r y Tria l 171 7.3.2 Main Study 172 7.4 Conclusion 175 Ack nowledgments 175 References 176 © 2008 by Taylor & Francis Group, LLC 168 Fate of Pharmaceuticals in the Environment and in Water Treatment Systems Recently, antibiotics, including the veterinary antibiotic tylosin, which is describedinthisstudy,werefoundin48%of139streamwaterstestedin30states, accordingtotheU.S.GeologicalSurvey(Kolpinetal.,2002).Antibioticsenter- ing the environment could potentially alter bacterial populations and their activity in sediment and water, thus affecting biodegradation, nutrient cycling, and water quality. In addition, there is concern that antibiotics in the environment may induce antibiotic resistance, resulting in adverse human health effects. Certainly, there is signicant evidence for development of antibiotic resistance within animals and in the excretion of antibiotic-resistant bacteria in manure (Beaucage et al., 1979; Aar- estrupetal.,1997;Kelleyetal.,1998).Muchlessisknownabouttheabilityoflow concentrations of antibiotics to induce resistance in the environmental microbial population or to provide selective pressure for maintenance of antibiotic resistance genes among microorganisms, although the transfer of antibiotic resistance from agriculturalsettingstohumanshasbeenreported(Oppegaardetal.,2001). 7.1.1 TYLOSIN Tylosinisamacrolideantibioticwithactivityagainstgram-positiveandcertain gram-negative bacteria, including Staphylococcus, Listeria, Legionella, and Entero- coccus.Ithaslittleactivityagainstgram-negativeentericbacteriasuchasE. coli. Tylosin is used exclusively in veterinary applications and is closely related to eryth- romycin,whichhasanimportantroleinpublichealth.Tylosinconsistsoffourmajor factors:tylosinA,B,C,andD(Figure 7.1);eachofthefactorsisbiologicallyactive, withtylosinAbeingmostactiveandmostprevalentinmedicinalandfeedformula- tions (Teeter and Meyerhoff, 2003). Tylosin acts in bacteria by binding to the 50S ribosome subunit, which leads to inhibition of protein synthesis. Sensitive bacteria areinhibitedbyaslittleas500µg/L.Tylosinisusedasagrowthpromoterapplied inswinefeedandasatherapeuticproductinswineandcattle.Tylosinisacom- mon antibiotic used internationally in swine, cattle, and poultry production as both a therapeutic and a prophylactic (Massé et al., 2000; Rabølle and Spliid, 2000). In swineproduction,tylosinisamongthethreeantibioticsthataccountedforthemajor- ity(78.8%)ofdiseaseprevention.Tylosinwasthemostusedantibioticat31.3%of O O O OH N(CH 3 ) 2 O CH 3 O HO CH 3 OH CH 3 O H 3 C HO OR 2 OCH 3 O O R 1 CH 3 O H 3 C CH 3 H 3 C OH FIGURE 7.1 Chemical structure of tylosin including factors: A (R 1 =CHO, R 2 =CH 3 ); B (TYLAminusmycinose);C(R 1 =CHO, R 2 =H); and D (R 1 =CH 2 OH, R 2 =CH 3 ). © 2008 by Taylor & Francis Group, LLC Mobility of Tylosin and Enteric Bacteria in Soil Columns 169 swineproductionfacilitiessurveyed(BushandBiehl,2001).Ithasbeenshownthat tylosinistransformedintheanimalfromtylosinAtotylosinD,whichisachange fromanaldehydetoanalcoholonthemacrolidering.However,tylosinDmaybe convertedbacktoitsoriginalforminexcreta(FAO/WHO,1991).Concentrationsof tylosininswinefeedrangefrom10to100gtylosin/tonfeedforgrowthpromotion purposes (Elanco Animal Health Tylan ® Premix product label). Tylosinwaslistedinthetoptenmostfrequentlydetectedantibioticsinsurface waterfrom1999to2000(Kolpinetal.,2002).Boxalletal.(2003)identiedtylo - si nasakeypharmaceuticalofinterestintheenvironment.Severalstudieshave shownthatthisantibioticmayhaveanafnityforclayparticlesandorganicmatter insoil,aswellastheorganiccomponentsofmanure,whichcouldaffectitsability todegrade(RabølleandSpliid,2000;Kolzetal.,2005).Sorptiontosoilandmanure components may affect its bioavailability. Huang et al. (2001) described tylosin as oneofthemostlikelywatercontaminantsfromagriculturalrunoff.Duetoitssorp - tion characteristics, it is believed that tylosin would be transported with sediment duringarunoffevent(Davisetal.,2006).Veryfewstudieshaveevaluatedmobility anddegradationintheenvironmentinthepresenceofamanuresubstrate(Rabølle and Spliid, 2000; Kay et al., 2004; 2005); however, these studies assessed only total tylosinresiduesanddidnotquantifytylosinmetabolites.Sorptionofchemicalsonto solidphases,suchassoil,sediment,ormanure,isextremelyimportantbecauseit could affect the fate and impact of these substances in that environment. An under - st andingofthedegradationandfateofveterinaryantibioticsinsoilisimportant because of widespread use of the compounds in livestock production in the United States, and the concurrent application of manure to land. Agricultural lands typically contain subsurface tile drain networks, which may drain directly into streams and other surface water bodies. Based on the lack of data regarding the leaching ability of tylosin factors in soil, one objective of the present study was to address these data gapsbyquantifyingtylosinresidues(specicallytylosinAandtylosinD)inleachate fromtylosinappliedtosoilcolumnsinamanureslurry. 7.1. 2 E NTERIC BACTERIA Two genera of enteric bacteria were selected for use in the present study. Escherichia coli are g ram-negative, rod-shaped members of H-Proteobacteria. Enterococcus sp. are gram-positive cocci. Both E. coli and e nterococcus inhabit the gastrointestinal tractofmanymammals,includinglivestock,andareexcretedfromtheanimalsand found in manure (Schleifer and Kilpper-Balz, 1987; Schaechter, 2000). Both organ - is msarepotentiallypathogenicandcandevelopresistancetoantibioticsandhavethe potentialtotransferresistancegenestootherbacteria(Wegeneretal.,1999;Ochman etal.,2000).Thesebacteriaarealsousedasfecalindicatorspeciesforwaterqual - ity assessments (Molina, 2005). Because of these characteristics, it is important to understand the survival and mobility of these microorganisms in the environment. Severalresearchershavereported E. coli surviving up to 8 weeks, and enterococcus survivalrangingfrom35to>200dinsoils,dependingonsoiltexture,amountand type of manure applied, temperature, and competition with indigenous soil micro - or ganisms(Coolsetal.,2001;LauandIngham,2001;Andrewsetal.,2004;Entry et al., 2005; Johannessen et al., 2005). A study examining the mobility of enteric © 2008 by Taylor & Francis Group, LLC 170 Fate of Pharmaceuticals in the Environment and in Water Treatment Systems bacteria in soil indicated 2 to 6% of the inoculated enterococcus leached through soil columns; however, the bacteria were applied directly to the top of the soil rather than in a manure slurry (Celico et al., 2004). Soupir et al. (2006) reported enterococcus as beinghighlymobileinrunofffromasimulatedheavyrainfallevent;differenttypes ofmanureweretestedandcountsrangedfrom6000to187,000cfu/100mL. Asverylittleinformationisavailableonthefateofbacteriaexcretedinmanure once the manure is applied to soil, particularly in the presence of drug residues, another objective of the present study was to determine the survival, movement, and antibiotic resistance of enteric bacteria in undisturbed soil columns. 7.2 MATERIALS AND METHODS 7. 2.1 PRELIMINARY TRIAL TwentyintactsoilcoresofTamaseriessoilwerecollectedfromanagriculturaleld nearGrinnell,Iowa.Theeldhadnotreceivedmanureapplicationforover20years, thereby reducing the likelihood of background contamination of antibiotics in the present study. The soil was a loam, containing 46% sand, 36% silt, and 18% clay. Soilcores(10-cmdiameter×30-cmdepth)werecollectedusingaGiddingssoilcore apparatus(GiddingsMachineCo.,Windsor,Colorado).Soilcolumnswereimmedi - ately taken to the lab and were saturated from the bottom with 5mM CaSO 4 for 48 h. Soilcolumnswereplacedinshelvingunitsandrestedonfunnelspluggedwithglass wool and lled with washed sea sand (Fisher Scientic, Pittsburgh, Pennsylvania), so that the bottom of the soil column rested rmly on the sand. Soil columns were allowedtodrain,andthosewithdrainagetimesof24to48hwerechosenforthe experimentandwererandomlydividedamongtreatmentandcontrolgroups. Freshhogmanurewascollectedfromhogsonanantibiotic-freediet(IowaState University Swine Nutrition Farm, Ames, Iowa). Twelve-gram aliquots of manure werepreparedand10mLultrapurewaterwasaddedtoeachaliquottomakeaeld- representative manure slurry. The manure slurry was spiked with 60 μg tylosin tartrateinamethanolcarriertoreachatylosinconcentrationof5ppminmanure. Next,2×10 8 gfp-labeled ampicillin-resistant E. coli 0157:H7 B6914 were added to the slurry. These organisms were selected for the preliminary trial because of their ease of detection, relevance, and availability. The slurry was then poured onto the surfaceofthecolumns.Theveuntreated(control)columnsreceived20mLultra - p u rewater,equivalenttothemoistureadditionofthetreatedcolumns.Afterapplica- tionthetop2cmofthecolumnswererakedwithasterilizedspatulatosimulatethe incorporationofmanureintosoilthatwouldoccurduringmanureapplicationinthe eld.ThesemethodsaresimilartothosedescribedbySainietal.(2003). Sandwaswettedwithultrapurewaterpriortoleachingevents.Forty-eighthours afterapplication,a5-cm“rainfall”intheformof410mL5mMCaSO 4 was applied to thecolumndrop-wiseover2.5to3.5h.Leachatewascollectedfromthebottomofthe columns for 48 h, then immediately analyzed for tylosin and E. coli O157:H7 B 6914. Theconcentrationoftylosininleachatewasdeterminedusingenzyme-linked immunosorbent assay kits (ImmunoDiagnostic Reagents, San Diego, California) in which the concentration was correlated to absorbance at 405 nm using a THER - © 2008 by Taylor & Francis Group, LLC Mobility of Tylosin and Enteric Bacteria in Soil Columns 171 MOmaxmicroplatereaderwithSOFTmaxProV3.0software(MolecularDevices, Sunnyvale, California). The presence of gfp-labeled E . coli O157:H7 in leachate was measured using a most-probable number (MPN) technique (IDEXX, Westbrook, Maine). Total coli- fo rm bacteria were also enumerated using MPN. The remaining leachate was concentrated using solid-phase extraction cartridges (Waters Oasis ® HLB,Milford,Massachusetts).Cartridgeswereconditionedwith5 mLacetonitrilefollowedby5mLof10%acetonitrile.Followingsampleretention, cartridges were rinsed three times with 10 mL distilled water and 5 mL 10% aceto- ni trile. Tylosin was eluted from the cartridges with 2 mL of 98:2 acetonitrile:glacial aceticacid.Extractswereanalyzedfortotaltylosin,tylosinA,andtylosinDusing high performance liquid chromatography with tandem mass spectrometry (LC/MS/ MS )withagra dientofa mmoniumacetatepH4.0:acetonitrileover35minat40°C onaZorbaxSD-C184.6×250mmcolumn(AgilentTechnologies,SantaClara, California). Mass spectrometry was used for analysis of tylosin factors based on mass, as reference standards for each factor were not available. Mass spectrometry methods were similar to those described by Kolz et al. (2005). The limit of detection was 0.5 μg/L. 7. 2 . 2 M AIN STUDY Fifty-six soil columns were collected at the same site previously described. These columnsweredividedamongseventreatmentgroups:control,manureonly,manure plus Enterococcus,m anure plus tylosin, tylosin plus Enterococcus,andmanureplus tylosin only, and Enterococcus . Ente rococcus waschosenforthemainstudybecause of its greater susceptibility to tylosin compared to E. coli,a soneoftheobjectivesof the present study was to evaluate development of resistance by manure-associated bacteriainthesoilcolumns.Similarmethodswereemployedasthosepreviously described, except the saturation and preleaching components were not performed, and all soil columns were utilized, resulting in eight replicates per treatment group. Additionally, we increased the concentration of tylosin and the amount of manure applied to better represent real-world applications. Thirty grams of fresh hog manure wasspikedwithtylosininanacetonecarriertoreachaconcentrationof50μg/g tylosininthemanure. Both enterococcus (which was inoculated) and E. coli (from m anure) were mea- suredinleachatewateraftereachrainevent,andresultsareexpressedascellsper 100mLofleachatewater.Afewsampleswerenotsufcientlydilutedandthus saturated the MPN panel, resulting in an MPN value that underestimates the true concentration. These values were used in the data analysis. Control columns (no manure)andcolumnstreatedwithmanureand Enterococcus l eached no E. coli. 7.3 RESULTS AND DISCUSSION 7. 3 .1 P RELIMINARY TRIAL Following a single rain event, analysis of leachate using LC/MS/MS revealed total tylosinresiduesupto2.8ng/mL,withameanconcentrationof0.8ng/mL(se= © 2008 by Taylor & Francis Group, LLC 172 Fate of Pharmaceuticals in the Environment and in Water Treatment Systems 0.3).Wefoundsimilarresultswhenusingimmunoassay;0.6ng/mLwasdetected inleachate.WhenexaminingspecictylosinfactorsusingLC/MS/MS,wefound thattylosinAaccountedforapproximately22%ofthetotaltylosinresidues;this correspondstoaconcentrationof0.2ng/mL.TylosinD,anothermajorfactor,was detectedat0.5ng/mL,or65%ofthetotalresidues.Thisresultisquiteinteresting consideringthecompositionofthetylosinappliedtothetopofthecolumn;tylosinD only accounted for approximately 10% in the formulation applied. Finding such dif- fe rent proportions in the leachate implies a differential metabolism or a differential mobilitybetweentylosinAandD.ItispossiblethattylosinDismorestableormore mobilethantylosinA.Furtherstudiesareneededtoelucidatethisphenomenon. Additionally,tylosinDhasonly35%oftheantibacterialactivityastylosinA, so the differences could have implications for low-level effects on soil microbial communities(TeeterandMeyerhoff,2003).Eachoftheothertylosinfactors(Band C), and even metabolites, can possess antimicrobial activity, which contributes to acomplexsituationinsoilandwaterwithrespecttobiologicalactivity.Likewise, some analytical methods, e.g.,ELISAr esiduequantication,alsoaredifferentially lesssensitivetosomefactorsormetabolitesandmoresensitivetoothers.Themajor advantagetothehighperformanceliquidchromatography(HPLC) orLC/MS method is the specicity, but the ELISA method is faster, cheaper, and can detect verylowconcentrationsinaqueoussamples(Huetal.,2006). FollowingMPNtestingofleachate,totalcoliformbacteriawerehighlyvari- ableinmanure-treatedcolumns,witharangeof1to644CFU/mLinthreeofve treatments.Onecontrolcolumnhad1cell/mLintheleachate,indicatingexternal sourcesofbacteria,whichcouldincludewildlifefromtheareainwhichthecolumns were collected. E. coli O157:H7 B6914 were detected in the leachate of two of the vetreatedcolumns;theyalsorangedfrom1to644cells/mL(thelimitofdetection for the assay). Although<0.1%ofthegfp-labeledcellsandtylosinresiduesappliedtothetopof thecolumnweredetectedintheleachateinthisstudy,theseresultsdoindicatethe abilityoftylosinandsomebacteriatomoveinanagronomicsoil. 7.3.2 MAIN STUDY Afterfourrainevents,lessthanone-thirdofthetreatedcolumnsleacheddetectable amountsoftylosin,withtheaverageconcentrationat<1ng/mLinleachate.These results were similar to those found in the preliminary trial. There were no apparent differences in the E. coli leaching from any of the manure treatments; therefore, the E. coli were averaged over these treatments for each leach- ingperiod(Figure 7.2). E. coli (from manure) were detected in all leachates, but the maximummeanconcentrationswereinthesecondandthirdleachates.Thedecline in E. coli concentrationseeninthefourthleachateislikelyduetodecreasingsur- vivalinthesoilandwashoutfromthecolumn. Enterococcus also leached from the soil columns but in numbers far exceeding those observed for E. coli (Figure 7.3).Inaddition,thenumberoforganismswas dependent upon treatment. Soil treated with manure leached no Enterococcus in the rstraineventbutaveraged5199cells/100mLinthesecondleaching,thendeclined © 2008 by Taylor & Francis Group, LLC Mobility of Tylosin and Enteric Bacteria in Soil Columns 173 to less than 400 cells/100 mL in the third and fourth rain events. The manure plus tylosin(MT)treatmentwassimilarinmagnitudeandpatterntothemanure-only treatment in the leaching of Enterococcus, s uggestingthatthetylosinwasnotactive toward the Enterococcus in this soil/manure environment. Enterococcus added to manure(MB)alsoresultedinbacteriabeingleached,asdidtheMTBtreatment. Tylosin plu s Enterococcus w i thoutmanure(TB)treatmentresultedinmuchfewer Enterococcus be ingleached,reachingamaximumaverageof30cells/100mLat thethirdleaching.Itispossiblethatthetylosinwasmoreavailableto in hibit the microbes in this treatment compared with the similar manure-containing treatment (MTB). Enterococcus wa sdetectedinleachatefrom4of23untreatedsoilcolumns (controls) and in 2 of 12 leachates from columns treated only with tylosin, indicating minimal input of enterococcus from the soil. Leaching 0 1 2 3 4 5 E. coli (cells/100 mL) 0 300 600 900 1200 1500 1800 2100 FIGURE 7.2 Average E. coli in leachate water from intact soil columns treated with swine manure. Leaching 1 2 3 4 0 2000 4000 6000 8000 10000 MB MT MTB TB M FIGURE 7.3 Average Enterococcus in leachate water from soil columns treated with swine manure alone (M), manure plus Enterococcus (MB),manureplustylosin(MT),manureplus tylosin and Enterococcus (MTB), or tylosin and Enterococcus without manure (TB). © 2008 by Taylor & Francis Group, LLC Enterococcus (cells/100 mL) 174 Fate of Pharmaceuticals in the Environment and in Water Treatment Systems Putative tylosin-resistant Enterococcus werenotrecoveredinthecontrolsorthe tylosin treatment (no manure or Enterococcus a d ded), which was expected. Only tracelevels(2cells/100mL)oftylosin-resistantEnterococcus w e re recovered in the second leaching from the manure-treated columns. Resistant Enterococcus w e re recoveredinboththerstandsecondleachatesfromtheMBtreatmentatlevels of 1955 and 779 cells/100 mL. These detections were in the absence of exposure totylosinorotherantibioticsandmaybeexplainedbyanaturallevelofresistance in the population. Leaching of tylosin-resistant Enterococcus in the MT and MTB treatmentswasalsoobserved(3228cells/100mLinMTsecondleachingand137 cells/100 mL in MTB second leaching), but no tylosin-resistant bacteria were found in leachate from the TB columns. Thus, it can be concluded that tylosin-resistant bacteria were only leached from manure-treated columns, but that there was no obvi - ouseffectoftylosin.Itispossiblethoseresistantorganismswerepresentinthe manure or that some organisms in the manure developed resistance to tylosin during the study. The results of the tylosin plus Enterococcus t r eatment(TB)couldbedue to the poor survival of Enterococcus i n the absence of manure, the effect of tylosin, or both these factors. Thepatternofleachingwasthesameforbothbacteria( E. coli and Enterococ- cus) o ver time, with peak concentrations coming from the second and third leachings. Greater concentrations of Enterococcus w e re seen in column leachates compared to E. coli, p articularly at the second leaching. There was no obvious effect of tylosin on the prevalence of tylosin-resistant Enterococcus, b ut manure treatment resulted in elevated levels of resistant Enterococcus. T his could be due to the presence of indigenous tylosin-resistant Enterococcus a l ready present in the swine manure. The movement of both the indicator bacteria and antibiotics is likely due to macropores in this well-structured soil. The transport of these agents illustrates their mobility. Examination of current literature reveals a small number of comparable studies. RabølleandSpliid(2000)performedaleachingstudywithtylosininpackedsoil coresoftwosoiltypes:asandyloamandasandytype.TheK d values described for thosesoilswere128and10.8,respectively,anddesorptionwasreportedat13and 26%. After one “rain” event, approximately 70% of the tylosin was recovered in the top20cmofthe48-cmsoilcolumns,andnotylosinwasdetectedintheleachate fromeithersoiltype;however,itshouldbenotedthatthelimitofdetectionreported inthestudywas7μg/L,comparedto0.5μg/Lreportedinthisstudy.Usingthe sorption coefcient data from this study, Tolls (2001) stated tylosin would be low to slightly mobile in most soils, if comparing to pesticide sorption and mobility data; theseresultsaresimilartoourndingsinaloamsoil. Freundlich partition coefcients for tylosin in silty clay loam, sand, and manure rangedfrom1000to2000(Clayetal.,2005);desorptionwasfoundtobe<0.2%in the same soils. The concentrations used in these batch sorption studies were 23 to 200mg/kg,similartotheconcentrationsoftylosininthemanureappliedtooursoil columns. These sorption and desorption values may also be useful in a comparison to the conditions in our soil. Aeld-scalestudyperformedontylosinmobilityinaclayloamsoildetermined thatupto6%ofappliedtylosinwaspresentinrunoffwaterfromanagriculturalsoil (Oswaldetal.,2004).Thesamestudyalsoexaminedtheeffectofmanureontylosin © 2008 by Taylor & Francis Group, LLC Mobility of Tylosin and Enteric Bacteria in Soil Columns 175 mobilityandfoundincreasedrunoffpotentialoftylosin(upto23%)inmanured treatments; this was likely due to the greatly decreased inltration of the applied rainfall. Inltration was reduced by approximately 85% in manured treatments. This informationmaybeimportantinidentifyingfactorsthataffectedleachingoftylosin in our study. Finally,Sainietal.(2003)foundincreasedsurvivalofan E. coli strain when manure in which they were residing was incorporated into the soil. Additionally, they reported that most bacteria leached from soil columns after the rst rain event andthatincreasedtimebetweenapplicationofmanureandtherainfalleventresulted in decreased leaching of the bacteria, which could be a result of decreased survival. Theseresultsaredifferentfromourndingofhighernumbersofbacterialeaching fromthesecondandthirdrainevents.Sainietal.(2003)reported3.4to4.5log CFU/100mLintheleachatefromtherainevent16-dpostapplication;1to10%of theappliedinoculumwasdetectedintheleachate,regardlessoftimebetweenappli - cati on and rst rain event (Saini et al., 2003). Additionally, Recorbet et al. (1995) foundthatsurvivalofbacteriainsoilmaybeattributedtocolonizationofclayfrac - ti onsinsoil,whichcouldprovideprotectionfromstressors,includingenvironmental contaminants,andthatpreferentialowinsoilcolumnsmaybeextremelyimpor - ta nt, which is in agreement with our results. 7.4 CONCLUSION The goal of the present study was to evaluate the mobility and degradation of tylosin and the mobility of enteric bacteria in undisturbed agronomic soil columns. Results fromthepresentstudyindicatealowamountofmobilityoftylosininaloamsoil, with an average of 0.8 ng/mL total tylosin detected in the leachate from multiple simulated rainfall events. Tylosin D was the predominant factor present in the leach - ate . Microbiological analysis of the leachate revealed that enteric bacteria were fre- qu ently present in the leachate at numbers exceeding the suggested water quality criteria of 126 cells/100 mL for E. coli and 3 3 cells/100 mL for enterococcus (U.S. EPA,2003).Itislikelythatpreferentialowplayedaroleinthetransportoftylosin andbacteriathroughthesoilproleintheintactsoilcolumns. Numerous monitoring studies have now detected very low residues of antibiotics insurfacewater.Thecurrentstudyhasendeavoredtotakesomerststepstoward understandinghowthecompoundsmovetosurfacewater,howlongtheypersistin soil, and what transformation processes and products are evident in environmen - ta lmatrices.Manyquestionsremainunanswered,themostintriguingofwhichis “Whatisthesignicanceofthelowconcentrationsofantibioticresiduesintheenvi - ro nment?” Much work is still needed to answer questions regarding the signicance of pharmaceuticals in the environment. 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These detections were in the absence of exposure totylosinorotherantibioticsandmaybeexplainedbyanaturallevelofresistance in the population. Leaching of tylosin-resistant Enterococcus