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

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

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

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

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

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

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

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

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
-

-
ro
nment?” Much work is still needed to answer questions regarding the signicance
of pharmaceuticals in the environment.
ACKNOWLEDGMENTS
TheauthorswouldliketothankBethDouglass,USDA-ARS,forhertechnicalassis-
tancethroughouttheprojectandDingfeiHuforhisassistancewithHPLCanaly-
si
s. Funding for this project was provided in part by the Center for Health Effects
© 2008 by Taylor & Francis Group, LLC
176 Fate of Pharmaceuticals in the Environment and in Water Treatment Systems
ofEnvironmentalContaminationandaUSDA-CSREESNRIgrant.Thisworkis
part of the Iowa Agricultural and Home Economics Experiment Station Projects No.
5075 and 5091.
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