research article monitoring antibiotic residues and

Research ArticleMonitoring Antibiotic Residues and Corresponding AntibioticResistance Genes in an Agroecosystem

Yasser M Awad1 Kwon Rae Kim2 Sung-Chul Kim3 Kangjoo Kim4

Sang Ryong Lee5 Sang Soo Lee1 and Yong Sik Ok1

1Department of Biological Environment Kangwon National University Chuncheon 200-701 Republic of Korea2Department of Agronomy Oriental Medicine Resource Gyeongnam National University of Science and TechnologyJinju 660-758 Republic of Korea3Department of Bio-Environmental Chemistry Chungnam National University Daejeon 305-764 Republic of Korea4Department of Environmental Engineering Kunsan National University Kunsan 573-701 Republic of Korea5Department of Animal Biotechnology and Environment National Institute of Animal Science Rural Development AdministrationSuwon 441-706 Republic of Korea

Correspondence should be addressed to Sang Soo Lee sslee97kangwonackr and Yong Sik Ok soilokkangwonackr

Received 16 December 2014 Revised 13 February 2015 Accepted 22 February 2015

Academic Editor Athanasios Katsoyiannis

Copyright copy 2015 Yasser M Awad et alThis is an open access article distributed under the Creative CommonsAttribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Antibiotic resistance genes (ARGs) have been commonly reported due to the overuse worldwide of antibiotics Antibiotic overusedisturbs the environment and threatens public human healthThe objective of this study was tomeasure the residual concentrationsof veterinary antibiotics in the tetracycline group (TCs) including tetracycline (TC) and chlortetracycline (CTC) as well as those inthe sulfonamide group (SAs) including sulfamethazine (SMT) sulfamethoxazole (SMX) and sulfathiazole (STZ) We also isolatedthe corresponding ARGs in the agroecosystem Four sediment samples and two rice paddy soil samples were collected from sitesnear a swine composting facility along the Naerincheon River in Hongcheon Korea High performance liquid chromatography-tandem mass spectrometry (HPLC-MSMS) was employed with a solid-phase extraction method to measure the concentrationof each antibiotic ARGs were identified by the qualitative polymerase chain-reaction using synthetic primers SAs and theircorresponding ARGs were highly detected in sediment samples whereas TCs were not detected except for sediments sample 1ARGs for TCs and SAs were detected in rice paddy soils while ARGs for TCs were only found in sediment 2 and 4 Continuousmonitoring of antibiotic residue and its comprehensive impact on the environment is needed to ensure environmental health

1 Introduction

Veterinary antibiotics are generally used as additives tomaintain animal health and to promote animal growth Inthe USA approximately 12500 tons of antibiotics is usedfor livestock production every year [1 2] A large amount ofantibiotics in the form of active pharmaceutical ingredientshas been used in animal husbandry and on fish farms becauseof their high efficiency to promote growth or control disease[3] However gt 80 of the antibiotics used are excretedas active metabolites in feces and urine [2] Subsequentlythe excreted antibiotic residues are delivered to the sur-rounding environment resulting in elevating antibiotic con-centrations [4ndash6] The antibiotic residues of four antibiotic

groups including tetracyclines (TCs) sulfonamides (SAs)macrolides (MLs) and ionophores are detectable inwater andsediment near the mixed-landscape of the Cache La PoudreRiver watershed [7] The occurrence of antibiotic residuesalong a water system is more critical because they are highlymobile [7]

Release of antibiotics into the environment leads to thestrains of pathogenic antibiotic-resistant bacteria [8ndash11] Forexample TC resistance genes have been reported in watersamples collected from wastewater treatment plants nearswine production facilities in the USA [8 10] Sengeloslashvet al [12] also detected antibiotic resistance genes (ARGs)against TC MLs and streptomycin in bacteria isolated fromfive farmlands treated with swine manure slurry Rysz and

Hindawi Publishing CorporationJournal of ChemistryVolume 2015 Article ID 974843 7 pageshttpdxdoiorg1011552015974843

2 Journal of Chemistry

Table 1 Description of each sediment and soil sample

Site Site descriptionSediment 1 Site located 02 km away from a swine manure compositing facilitySediment 2 Site located 05 km away from a swine manure compositing facilitySediment 3 Site located 10 km away from a swine manure compositing facilitySediment 4 Site located 15 km away from a swine manure compositing facilitySoil 1 Rice paddy soil treated with swine manure and having a distance of 20 km from a swine manure composting facility

Soil 2 Rice paddy soil influencing antibiotics via irrigation and having a distance of 20 km from a swine manurecomposting facility

Alvarez [13] insisted that dissemination of ARGs severelydegrades environments biochemically and should be consid-ered a pollutant

Once antibiotic residues enter bacterial cells in theenvironment via passive diffusion they inhibit bacterialgrowth [14] TCs including TC chlortetracycline (CTC)oxytetracycline (OTC) doxycycline (DXC) andminocycline(MNC) inhibit protein synthesis inGram-positive andGram-negative bacteria by preventing the binding of aminoacyl-tRNA molecules to the 30S ribosomal subunit [14] Bacterialresistance to these antibiotics occurs by two mechanisms(i) the multiantibiotic-resistance pump and (ii) conferring ofbacterial resistance [14 15]

Antibiotic research related to resistance genes has beenconfined to culturable bacteria isolated from pharmaceuti-cally originating wastewater The cultural isolation methodis the most commonly employed however only a fractionof actual microbiota in systems containing ARGs can bedetermined using this method [16] According to ARGoccurrence in environments affected by animal waste thepolymerase chain-reaction (PCR) method is highlighted toquantify genes conferring resistance to selective antibioticsSeveral studies have attempted to quantify ARGs by isolatingDNA [11 17] Knapp et al [18] showed that isolated DNAfrom five long-term soil series (over 60 years) was veryinformative regarding ARG abundance and their resistanceto antibiotics They also found that ARGs have increasedsharply in the environment from 1940 to 2008 Differentlydesigned primers are needed to detect antibiotic bacterialresistance Bacterial resistance to different types of antibioticswas primarily mediated by synthetic primers such as tet(A)-(E) tet(G) tet(M) tet(O) tet(Q) and tet(S) for TCs [11 19]and sul(I) and sul(II) for SAs [5]

To understand the relationship between antibiotics andcorresponding ARGs seasonal monitoring of veterinaryantibiotics is needed due to the variation of climatic featuresin Korea and the overused annual consumption of antibioticscompared to other countries [20] Korea has high-intensityrainfall and a large temperature difference between summerand winter seasons due to the geographical monsoon impact[21] This climate condition can lead to the mobilizationof antibiotics owing to the contamination of surroundingenvironment A continuous monitoring of antibiotics hasbeen performed near concentrated animal farming opera-tions (CAFOs) in Korea and antibiotics were detected inenvironment as mentioned in our previous studies [20 22]

This study was conducted to further evaluate the presence ofveterinary antibiotic residues released into the environmentand to identify ARGs in environmental components such assediment and soil possibly affected by a swine manure-basedcompost facility

2 Materials and Methods

21 Sampling Thesampling sites were located inHongcheonGangwon Province Korea which were assumed to beaffected by antibiotic release from a swine manure compost-ing facility (37∘ 341015840 2810158401015840 N 127∘ 521015840 2610158401015840 E) Specific descrip-tions of the sampling sites are provided in Table 1 Samplingwas done in March 2009The average temperature was 177∘Cand total precipitation was 958mm [23] Sediments weresampled based on the distance from the composting facilityof 02 05 1 and 15 km as sediment sample 1 2 3 and 4respectively along the Naerincheon River Paddy soils werecollected from sites (a) directly appliedwith swinemanure foragricultural purposes as soil 1 and (b) only irrigated using awater source from Naerincheon River as soil 2 Specificallysediment and soil samples were collected at a depth of 0ndash20 cm Four subsamples were collected from each site andthese subsamples formed a composite sample The sedimentand soil samples were air-dried and then passed through a2-mm sieve before analysis The current study is a part of acomprehensive monitoring (since April 2008) of antibioticsin water sediment and soil near swine composting facility[20 22]

22 Antibiotic Extraction and Quantification Antibioticresidues were extracted from the sediment and soil samplesusing the method described by Kim and Carlson [24] andOk et al [20] and were quantified by high performanceliquid chromatography-tandem mass spectrometry (HPLC-MSMS) (API 3000 Applied Biosystems Foster City CAUSA) Recovery and the limit of quantification were deter-mined Briefly to extract TCs and SAs 1 g of sedimentor soil sample was added to a 50mL polypropylene cen-trifuge tube with 20mL of McIlvaine buffer at pH 4 buffersolution and 200120583L of 5 Na

2EDTA followed by 20min

of shaking at 400 rpm before centrifugation for 15min at4000 rpm (Centrifuge FLETA 5 Hanil Science IndustrySeoul Korea) The supernatant was filtered through a 02 120583mglass fiber filter The extraction process was repeated andthe extracts were combined in a 40mL vial for solid-phase

Journal of Chemistry 3

Table 2 Conditions for high performance liquid chromatography-tandem mass (HPLC-MSMS) spectrometry

Equipment LC MSMS (TSQ Quantum Ultra Thermo)

LC condition

Column temp 15∘CColumn flow rate 300120583Lminminus1

Injection volume 20 120583L

Mobile phase A 999 water + 01 formic acidB 999 ACN + 01 formic acid

GradientA 96 + B 4 (0min)A 70 + B 30 (29min)A 96 + B 4 (30min)

MS condition

Ion source ESI positiveSpray voltage 4500VVaporizer temp 320∘CDrying gas flow 100 Lminminus1

Drying gas and nebulizer gas Nitrogen gasSheath gas pressure 40 psigAux gas pressure 20 psig

extraction (SPE) SPE was employed to retain antibioticson the cartridge so they could be effectively extracted withMeOH [25] Due to the wide range in pH hydrophilic-lipophilic balanced cartridges were used for the antibioticextraction and preextractants were purified on solid matrices[25] Electrospray ionization was also applied to quantifyantibiotic substances using HPLC-MSMS in positive modeThe detailed information and mobile phase conditions aresummarized in Table 2

23 Heterotrophic Plate Counts on Antibiotic-Selective MediaEach 1 g of moist sedimentsoil sample was diluted in ster-ilized water and agitated for 30min followed by a 100-fold serial dilution Aliquots (100 120583L) of the serially dilutedsample were spread directly onto the surface of R2A agarmedia (Difco Sparks MD USA) which contained variousantibiotics or no antibiotic as a control to enumerate andisolate resistant bacteria Specifically the media containedantibiotics of 30mg Lminus1 TC 7055mg Lminus1 CTC 4555mg Lminus1OTC 2818mg Lminus1 SMT 504mg Lminus1 SMX or 45mg Lminus1 STZA concentration that was five times greater than the reportedaverage LD

50value was used for the water-soluble antibiotics

such as TC CTC and STZ whereas the maximum amountthat dissolved readily in water when added to melted agarwas used for the insoluble antibiotics such as SMX SMTand OTC [5] Each treated plate was incubated at 30∘C for48 h followed by incubation for 1 week in the dark at roomtemperature Colony forming units (CFUs) were enumeratedat the end of the culture period [26]

24 DNA Extraction and Purification DNA was extractedfrom 05 g of sediment or soil sample using a FastDNASPIN kit (QBiogene Carlsbad CA USA) The extractedDNA was purified using a Geneclean SPIN kit (QBiogene)to minimize PCR inhibition The concentration of DNAbeforeafter purification and recovery were determined

25 Primer Design Specific primers for nucleotide sequencesencoding theTC- and SA-resistant geneswere designed basedon the GenBank Database (httpwwwncbinlmnihgov)Seven sets of primers obtained from verifiable subjectedproducts were generated as shown in Table 3

26 Detection of ARGs Using Qualitative PCR PCR was per-formed to identify the TC and SA ARGs encoding ribosomalprotection We used a Bio-Rad kit (Hercules CA USA) ina reaction mixture with a final volume of 20120583L consistingeach of 2120583L of the times10 buffer 25mM dNTP mix 04120583Meach primer 175 units of Taq DNA polymerase and 50 pmolof DNA template (Takara Bio Shiga Japan) Amplificationwas conducted using a PTC-100 thermal cycler (Bio-Rad)to subject samples to conditions of initial denaturation at95∘C for 5min followed by 35 cycles of denaturation at 95∘Cfor 30 s 30 s annealing at 559∘C for SA genes 60∘C fortet(W) 503∘C for tet(O) 56∘C for tet(S) 439∘C for tetB(P)or 439∘C for tet(T) Extension was done at 72∘C for 30 swith a final extension at 72∘C for 7min The PCR productswere visualized on a 08 agarose gel using a Gel Doc 1000apparatus (Bio-Rad)

3 Results and Discussion

31 Antibiotic Concentrations The concentrations of TCsand SAs in the sediment and soil samples are shown inFigure 1 TC was only detected in sediment 1 (039 120583g kgminus1)(Figure 1(a)) TC and CTC antibiotic residues were detectedin both soils 1 and 2 and the maximum concentrationsof TC (093 120583g kgminus1) and CTC (600 120583g kgminus1) were observedin soil 1 This result shows that the antibiotic residues insoils are more long-lasting than those in sediment becauseof lower mobility in soils No OTC was found in anysample For instance stability of TCs is controlled by abioticand biotic factors with a range of 1ndash419-day half-lives in


Table 3 Polymerase chain-reaction (PCR) primers for tetracycline- (TC-) and sulfonamide- (SA-) resistant gene classes

Gene Primer Sequences Annealing temp (∘C) Amplicon size (bp)

tet(S) tetS-FWdagger GAAAGCTTACTATACAGTAGC 50 169tetS-RVDagger AGGAGTATCTACAATATTTAC

tet(T) tetT-FW AAGGTTTATTATATAAAAGTG 46 169tetT-RV AGGTGTATCTATGATATTTAC

otr(A) otrA-FW GGCATYCTGGCCCACGT 66 212otrA-RV CCCGGGGTGTCGTASAGG

sul(I) sulI-FW CGCACCGGAAACATCGCTGCAC 559 163sulI-RV TGAAGTTCCGCCGCAAGGCTCG

sul(II) sulII-FW TCCGGTGGAGGCCGGTATCTGG 608 191sulII-RV CGGGAATGCCATCTGCCTTGAG

sul(III) sulIII-FW TCCGTTCAGCGAATTGGTGCAG 60 128sulIII-RV TTCGTTCACGCCTTACACCAGC

sul(A) sulA-FW TCTTGAGCAAGCACTCCAGCAG 60 229sulA-RV TCCAGCCTTAGCAACCACATGG

daggerForwardDaggerReverse

0

2

4

6

8

Sed 1 Sed 2 Sed 3 Sed 4 Soil 1 Soil 2

TCCTC

Con

cent

ratio

n (

minus1)

120583gk

g

(a)

0

4

8

12

16

20


SMTSMX

STZ

Con

cent

ratio

n (

minus1)

120583gk

g

(b)

Figure 1 Average concentration of (a) tetracyclines (TCs) and (b) sulfonamides (SAs) in sediment and soil samples collected along theNaerincheon River downstream of a swine manure compositing facility

aquatic systems [27] In current study TCs were below thedetection limit in sediment samples and this might be due totheir strong sorption affinity to aluminum oxide Fe oxidesorganic carbon and clay particles in soil [20 27 28] Inparticular Al

2O3and Fe

2O3promote the dehydration of

TC to anhydrotetracycline (AHTC) epimerization of TCand formation of Al-TC and Fe-TC complexes [29ndash31] Inaddition Rubert [27] revealed that organic matter (humicand proteinaceous substances) can absorb TCs in soil andcan form complexation of TC in the presence of cations suchas Ca Cu Al and Fe Additionally sorption of TCs waspronounced in rice paddy soils due to high cation exchangecapacity by 924 and 890 cmol

(+)kgminus1 respectively for both

soils as mentioned in our previous findings [20 31ndash33]The maximum concentrations of SMT (1768 120583g kgminus1)

in sediment 4 SMX (1024 120583g kgminus1) in sediment 2 and

STZ (834 120583g kgminus1) in sediment 1 were found (Figure 1(b))Similar to the TCs a higher concentration of SAs wasobserved in soil 1 than those in soil 2 Additionally thedirect application of swine manure onto paddy soils (ie soil1) contributed to the longevity of antibiotic residues in soilscompared to that of indirect application via irrigation waterpossibly contaminated with antibiotics (ie soil 2)

These findings were in accordance with our previousstudy [20 22] that the concentrations of TCs includingCTC TC and OTC in sediment samples collected alongthe Naerincheon River are very low or below the detectionlimit We showed earlier that the antibiotic residues of SAsincluding SMT SMX and STZ are highly detectable in sed-iment indicating concentration levels of 3860ndash7032 891ndash1220 and 2368ndash4031 120583g kgminus1 respectively [20] Our resultsfor the trend of TCs and SAs concentrations in sediment and


Table 4 Antibiotic-resistant bacteria in CFUs isolated from sediment and soil samples cultured on R2A agar plates withwithout antibioticsafter a 24 h incubation at 30∘C

Plate counts of antibiotic-resistant bacteria (in CFU times 102)Sed 1 Sed 2 Sed 3 Sed 4 Soil 1 Soil 2

Control 21700ddagger

24100c

19300e

17900f

29800b

31600a

Tetracycline (TC) NDDagger 200a ND 033

b ND NDChlortetracycline (CTC) ND 067

a ND ND 100a ND

Sulfamethazine (SMT) 14533a

12667ab

9933b

5033c

3633c

3333c

Sulfamethoxazole (SMX) 5233b

8833a

3900cd

1433cd

1033d

1933cd

Sulfathiazole (STZ) 9933a

7933a

1367b

867b

2233b

1233b

daggerDifferent letters in each row indicate a significant difference at 005DaggerNot detected

Table 5 Polymerase chain-reaction (PCR) identification of antibiotic-resistant strains using different tetracycline (TC) and sulfonamide (SA)primers

Primer Sed 1 Sed 2 Sed 3 Sed 4 Soil 1 Soil 2tet(S) mdashdagger IDagger mdash I mdash mdashtet(T) mdash I mdash mdash I mdashotr(A) mdash mdash mdash I I mdashsul(I) mdash I I I I Isul(II) I I I I I Isul(III) I I I I I Isul(A) I I I I I IdaggerAbsentDaggerPresent

soil samples were quite similar to our previous study [20] butthe concentrations were much lower than their study Thisdiscrepancy may be explained by seasonal variations in pre-cipitation or temperature based on sampling season [24]Thepotential effects of rainfall during winter season and high-flow rate cause the dilution effect of the released veterinaryantibiotics from CAFOs Ok et al [20] and Kim and Carlson[24] found that the concentration levels of antibiotic residuesin water are strongly influenced by precipitation water levelflow conditions and water quality related to geographicconditions and type of antibiotic It was noteworthy that thefourth highest temperature record since 1973 was observedin March with 15∘C higher than normal mean temperaturewhile the annual precipitation inHongcheonwas 10004mmbased onweather information from theKoreaMeteorologicalAdministration [21 34] This also can be contributed to thedegradation of TCs in solid matrices (animal manure andsoil) Under soil acidic condition OTC was epimerized inswine manure and formed degradation products such as 4-epi-OT and epi-N-desmethyl-OT [27 35] Ingerslev et al [36]found that biodegradation of TCs was the main mechanismin sludge byAscomycetes fungi [37] and Streptomyces specieshowever sorption and transformation of TCs commonlyoccurred in soil [27 32] Our study confirmed that thetransformation and stability of TCs in sediment and soilare dependent on light temperature and physiochemicalproperties of the matrix [38]

The reason for the higher concentrations or mobilityof SAs compared to TCs is that SAs are likely moving afurther distance from the composting facility because of

lower organic carbon-normalized sorption coefficient (119870oc)and the lower hydrophobicity [24 31] Hu et al [39] alsoshowed that SAs have a range of distribution coefficients(119870119889) of 09ndash181mL gminus1 indicating high solubility in water

compared to other types of antibiotics

32 Antibiotic-Resistant Bacteria Total bacterial counts indi-cating antibiotic resistance (CFUstimes 102) are shown in Table 4Total bacterial counts in the sediment samples decreasedwithincreasing distance from the swine composting facility as arelease source of antibiotics This result indicates that theantibiotic-resistant bacteria were present close to the antibi-otic contamination source The total enumeration of CFUswas much higher without antibiotics than with antibioticsranging from 179 to 241 CFU times 102 gminus1 for sediments and 298to 316CFUtimes 102 gminus1 for soilsThe total bacterial count for SAswas 867ndash14533 CFU times 102 gminus1 for sediments and 1033ndash3633CFU times 102 gminus1 for soilsThese results indicate that the densityof culturable heterotrophic bacteria was generally higherin sediment than that in soil However no bacteria weredetected in any samples grown in the presence of TCs exceptin sediments 2 and 4 indicating a very low populationThisresult agrees with a study by Pepper and Gerba [26] showingthat SA-resistant bacteria are present in greater abundancethan TC-resistant bacteria

33 PCR Assay for ARGs The occurrence of ARGs forTCs and SAs is shown in Table 5 and Figure 2 The resultsshowed that SA-resistant genes including sul(I) sul(II) and


1000 bp

700bp

200 bp100 bp

sul(I

)

Con

trol

tet(S

)

sul(A

)

tet(

T)

sul(I

I)

sul(I

II)

otr(

A)

Figure 2 Polymerase chain-reaction (PCR) identification of anti-biotic-resistant strains present in the sediment and soil samples

sul(A) were present in all sediment and soil samples exceptsul(I) which was not detected in sediment 1 However TC-resistant genes including tet(S) tet(T) and otr(A) were onlyfound in sediments 2 and 4 and soil 1 These findingsagree with the study by Pei et al [5] who quantified four SA-and five TC-resistant genes in sediments collected along theCache La Poudre River using both culture-based and PCRtechniques Auerbach et al [11] also reported a wide variety ofTC-resistant genes in different wastewater samples collectedin the USA

It was noteworthy that wastewater from CAFOs andthe application of compost or animal manure to rice paddysoils play a significant role in generating ARGs for SAsin both sediments and soils due to the accumulation ofveterinary antibiotics Similarly ARGs for TCs were foundin rice paddy soils Similar to our results previous studiesreported that ARGs were generated at higher levels nearCAFOs than background and associated with human andanimal diseases including different pathogenic bacteria suchas Salmonella and Shigella isolates [40] For example theyfound a significant correlation between the occurrence ofmethicillin-resistant Staphylococcus aureus (MRSA) in pigsand pig farmers in USA Canada and Europe [40 41] Thusthe findings of current study agree with previous studies[4 5 8 11 40 41] and demonstrate that antibiotic use inCAFOs is highly correlated with the fate and transport ofARGs in surrounding environment In a survey by Peak et al[42] a strong correlation between antibiotics and ARGs wasidentified

4 Conclusions

This study was conducted to investigate the residual concen-trations of selected TCs and SAs and to isolate correspondingARGs in the environment Higher concentrations of SAs insediment and soil samples were found compared to those ofTCs A culture-based technique and PCR were successfullyused to demonstrate TC- and SA-resistant genes in theenvironment Findings of current study revealed that thewidespread antibiotic use in CAFOs in Korea has the poten-tial to generate ARGs as emerging contaminants in solidenvironmental matrices Monitoring ARGs in surrounding

environments is encouraged to ensure public health Free-antibiotic swine industry in Korea is recommended to reducethe environmental risks of veterinary antibiotics

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors thank Dr Ruoting Pei at the University of Texasat San Antonio for a valuable review This research wassupported by the Ministry of Environment as ldquoThe GAIAProject (no 172-112-011)rdquo in Korea Instrumental analysis wassupported by a grant from the Institute of EnvironmentalResearch and the Central Laboratory at Kangwon NationalUniversity in Korea

References

[1] S B Levy ldquoThe challenge of antibiotic resistancerdquo ScientificAmerican vol 278 no 3 pp 46ndash53 1998

[2] K-R Kim G Owens S-I Kwon K-H So D-B Lee andY S Ok ldquoOccurrence and environmental fate of veterinaryantibiotics in the terrestrial environmentrdquo Water Air amp SoilPollution vol 214 no 1ndash4 pp 163ndash174 2011

[3] F C Cabello ldquoHeavy use of prophylactic antibiotics in aquacul-ture a growing problem for human and animal health and forthe environmentrdquo Environmental Microbiology vol 8 no 7 pp1137ndash1144 2006

[4] K Kummerer and A Henninger ldquoPromoting resistance bythe emission of antibiotics from hospitals and households intoeffluentrdquo Clinical Microbiology and Infection vol 9 no 12 pp1203ndash1214 2003

[5] R Pei S-C Kim K H Carlson and A Pruden ldquoEffect ofriver landscape on the sediment concentrations of antibioticsand corresponding antibiotic resistance genes (ARG)rdquo WaterResearch vol 40 no 12 pp 2427ndash2435 2006

[6] S I Kwon G Owens Y S Ok et al ldquoApplicability of the CharmII system for monitoring antibiotic residues in manure-basedcompostsrdquoWaste Management vol 31 no 1 pp 39ndash44 2011

[7] S C Kim Occurrence fate and transport of human and veteri-nary antibiotics in the watershed [PhD dissertation] ColoradoState University Fort Collins Colo USA 2006

[8] J C Chee-Sanford R I Aminov I J Krapac N Garrigues-Jeanjean and R I Mackie ldquoOccurrence and diversity of tetra-cycline resistance genes in lagoons and groundwater underlyingtwo swine production facilitiesrdquo Applied and EnvironmentalMicrobiology vol 67 no 4 pp 1494ndash1502 2001

[9] O Cars L D Hogberg M Murray O Nordberg C SLundborg and A D So ldquoMeeting the challenge of a concertedglobal response is needed to tackle rising rates of antibioticresistanceWithout it we risk returning to the pre-antibiotic erawarnrdquo British Medical Journal vol 337 pp 726ndash728 2008

[10] Y M Awad S S Lee S C Kim J E Yang and Y SOk ldquoNovel approaches to monitoring and remediation ofveterinary antibiotics in soil and water a reviewrdquo Korea Journalof Environmental Agriculture vol 29 no 4 pp 315ndash327 2010


[11] E A Auerbach E E Seyfried and K D McMahon ldquoTetracy-cline resistance genes in activated sludge wastewater treatmentplantsrdquoWater Research vol 41 no 5 pp 1143ndash1151 2007

[12] G Sengeloslashv Y Agersoslash B Halling-Soslashrensen S B Baloda JS Andersen and L B Jensen ldquoBacterial antibiotic resistancelevels in Danish farmland as a result of treatment with pigmanure slurryrdquo Environment International vol 28 no 7 pp587ndash595 2003

[13] M Rysz and P J J Alvarez ldquoAmplification and attenuation oftetracycline resistance in soil bacteria aquifer column experi-mentsrdquoWater Research vol 38 no 17 pp 3705ndash3712 2004

[14] D Schnappinger and W Hillen ldquoTetracyclines antibioticaction uptake and resistance mechanismsrdquo Archives of Micro-biology vol 165 no 6 pp 359ndash369 1996

[15] B Normander B B Christensen SMolin andN Kroer ldquoEffectof bacterial distribution and activity on conjugal gene transferon the phylloplane of the bush bean (Phaseolus vulgaris)rdquoApplied and Environmental Microbiology vol 64 no 5 pp1902ndash1909 1998

[16] R I Amann W Ludwig and K-H Schleifer ldquoPhylogeneticidentification and in situ detection of individual microbial cellswithout cultivationrdquo Microbiological Reviews vol 59 no 1 pp143ndash169 1995

[17] C S Riesenfeld R M Goodman and J Handelsman ldquoUncul-tured soil bacteria are a reservoir of new antibiotic resistancegenesrdquo Environmental Microbiology vol 6 no 9 pp 981ndash9892004

[18] C W Knapp J Dolfing P A I Ehlert and D W GrahamldquoEvidence of increasing antibiotic resistance gene abundancesin archived soils since 1940rdquo Environmental Science and Tech-nology vol 44 no 2 pp 580ndash587 2010

[19] V Burdett ldquoPurification and characterization of Tet(M) aprotein that renders ribosomes resistant to tetracyclinerdquo TheJournal of Biological Chemistry vol 266 no 5 pp 2872ndash28771991

[20] Y S Ok S-C Kim K-R Kim et al ldquoMonitoring of selectedveterinary antibiotics in environmental compartments near acomposting facility in Gangwon Province Koreardquo Environmen-tal Monitoring and Assessment vol 174 no 1ndash4 pp 693ndash7012011

[21] KMA ldquoKoreaMeteorological Administrationrdquo 2015 httpwebkmagokrengindexjsp

[22] YMAwad S-CKim SAMAbdEl-Azeemet al ldquoVeterinaryantibiotics contamination in water sediment and soil neara swine manure composting facilityrdquo Environmental EarthSciences vol 71 no 3 pp 1433ndash1440 2014

[23] KMA Monthly Weather Report Pub 11-1360000-000002-06 Korea Meteorological Administration Seoul Republic ofKorea 2009

[24] S-C Kim and K Carlson ldquoTemporal and spatial trends in theoccurrence of human and veterinary antibiotics in aqueous andriver sediment matricesrdquo Environmental Science and Technol-ogy vol 41 no 1 pp 50ndash57 2007

[25] S-C Kim and K Carlson ldquoLCndashMS2 for quantifying traceamounts of pharmaceutical compounds in soil and sedimentmatricesrdquo Trends in Analytical Chemistry vol 24 no 7 pp 635ndash644 2005

[26] I L Pepper and C P Gerba ldquoCultural methodsrdquo in Environ-mental Microbiology R M Maier I L Pepper and P C GerbaEds pp 173ndash188 Academic Press Elsevier Inc London UK2nd edition 2009

[27] K F Rubert Tetracycline Antibiotic Distribution and Transfor-mation in Aquatic Systems ProQuest 2008

[28] W-R Chen and C-H Huang ldquoAdsorption and transformationof tetracycline antibiotics with aluminum oxiderdquo Chemospherevol 79 no 8 pp 779ndash785 2010

[29] R A Figueroa and A A Mackay ldquoSorption of oxytetracyclineto iron oxides and iron oxide-rich soilsrdquo Environmental Scienceand Technology vol 39 no 17 pp 6664ndash6671 2005

[30] C Gu and K G Karthikeyan ldquoInteraction of tetracycline withaluminum and iron hydrous oxidesrdquo Environmental Science andTechnology vol 39 no 8 pp 2660ndash2667 2005

[31] J Tolls ldquoSorption of veterinary pharmaceuticals in soils areviewrdquo Environmental Science and Technology vol 35 no 17pp 3397ndash3406 2001

[32] S A Sassman and L S Lee ldquoSorption of three tetracyclinesby several soils assessing the role of pH and cation exchangerdquoEnvironmental Science and Technology vol 39 no 19 pp 7452ndash7459 2005

[33] A D Jones G L Bruland S G Agrawal and D VasudevanldquoFactors influencing the sorption of oxytetracycline to soilsrdquoEnvironmental Toxicology and Chemistry vol 24 no 4 pp 761ndash770 2005

[34] J-C Yang H-S Hwang H-J Lee et al ldquoDistribution ofvascular plants along the altitudinal gradient of Gyebangsan(Mt) in Koreardquo Journal of Asia-Pacific Biodiversity vol 7 no1 pp e40ndashe71 2014

[35] B Halling-Soslashrensen A Lykkeberg F Ingerslev P Blackwelland J Tjoslashrnelund ldquoCharacterisation of the abiotic degradationpathways of oxytetracyclines in soil interstitial water using LC-MS-MSrdquo Chemosphere vol 50 no 10 pp 1331ndash1342 2003

[36] F Ingerslev L Torang M-L Loke B Halling-Sorensen andN Nyholm ldquoPrimary biodegradation of veterinary antibioticsin aerobic and anaerobic surface water simulation systemsrdquoChemosphere vol 44 no 4 pp 865ndash872 2001

[37] E Meyers and D A Smith ldquoMicrobiological degradation oftetracyclinesrdquo Journal of Bacteriology vol 84 no 4 pp 797ndash8021962

[38] T Soslasheborg F Ingerslev and B Halling-Soslashrensen ldquoChemicalstability of chlortetracycline and chlortetracycline degradationproducts and epimers in soil interstitial waterrdquo Chemospherevol 57 no 10 pp 1515ndash1524 2004

[39] X Hu Q Zhou and Y Luo ldquoOccurrence and source analysisof typical veterinary antibiotics in manure soil vegetables andgroundwater from organic vegetable bases northern ChinardquoEnvironmental Pollution vol 158 no 9 pp 2992ndash2998 2010

[40] A Pruden ldquoAntibiotic resistance associated with CAFOsrdquoin Hormones and Pharmaceuticals Generated by ConcentratedAnimal Feeding Operations Transport in Water and Soil LS Shore and A Pruden Eds vol 1 of Emerging Topics inEcotoxicology pp 71ndash83 Springer New York NY USA 2009

[41] T Khanna R Friendship C Dewey and J S Weese ldquoMethi-cillin resistant Staphylococcus aureus colonization in pigs andpig farmersrdquoVeterinary Microbiology vol 128 no 3-4 pp 298ndash303 2008

[42] N Peak C W Knapp R K Yang et al ldquoAbundance of sixtetracycline resistance genes in wastewater lagoons at cattlefeedlots with different antibiotic use strategiesrdquo EnvironmentalMicrobiology vol 9 no 1 pp 143ndash151 2007

Submit your manuscripts athttpwwwhindawicom

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Inorganic ChemistryInternational Journal of

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Analytical Methods in Chemistry

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Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Table 1 Description of each sediment and soil sample

Site Site descriptionSediment 1 Site located 02 km away from a swine manure compositing facilitySediment 2 Site located 05 km away from a swine manure compositing facilitySediment 3 Site located 10 km away from a swine manure compositing facilitySediment 4 Site located 15 km away from a swine manure compositing facilitySoil 1 Rice paddy soil treated with swine manure and having a distance of 20 km from a swine manure composting facility

Soil 2 Rice paddy soil influencing antibiotics via irrigation and having a distance of 20 km from a swine manurecomposting facility

Alvarez [13] insisted that dissemination of ARGs severelydegrades environments biochemically and should be consid-ered a pollutant

Once antibiotic residues enter bacterial cells in theenvironment via passive diffusion they inhibit bacterialgrowth [14] TCs including TC chlortetracycline (CTC)oxytetracycline (OTC) doxycycline (DXC) andminocycline(MNC) inhibit protein synthesis inGram-positive andGram-negative bacteria by preventing the binding of aminoacyl-tRNA molecules to the 30S ribosomal subunit [14] Bacterialresistance to these antibiotics occurs by two mechanisms(i) the multiantibiotic-resistance pump and (ii) conferring ofbacterial resistance [14 15]

Antibiotic research related to resistance genes has beenconfined to culturable bacteria isolated from pharmaceuti-cally originating wastewater The cultural isolation methodis the most commonly employed however only a fractionof actual microbiota in systems containing ARGs can bedetermined using this method [16] According to ARGoccurrence in environments affected by animal waste thepolymerase chain-reaction (PCR) method is highlighted toquantify genes conferring resistance to selective antibioticsSeveral studies have attempted to quantify ARGs by isolatingDNA [11 17] Knapp et al [18] showed that isolated DNAfrom five long-term soil series (over 60 years) was veryinformative regarding ARG abundance and their resistanceto antibiotics They also found that ARGs have increasedsharply in the environment from 1940 to 2008 Differentlydesigned primers are needed to detect antibiotic bacterialresistance Bacterial resistance to different types of antibioticswas primarily mediated by synthetic primers such as tet(A)-(E) tet(G) tet(M) tet(O) tet(Q) and tet(S) for TCs [11 19]and sul(I) and sul(II) for SAs [5]

To understand the relationship between antibiotics andcorresponding ARGs seasonal monitoring of veterinaryantibiotics is needed due to the variation of climatic featuresin Korea and the overused annual consumption of antibioticscompared to other countries [20] Korea has high-intensityrainfall and a large temperature difference between summerand winter seasons due to the geographical monsoon impact[21] This climate condition can lead to the mobilizationof antibiotics owing to the contamination of surroundingenvironment A continuous monitoring of antibiotics hasbeen performed near concentrated animal farming opera-tions (CAFOs) in Korea and antibiotics were detected inenvironment as mentioned in our previous studies [20 22]

This study was conducted to further evaluate the presence ofveterinary antibiotic residues released into the environmentand to identify ARGs in environmental components such assediment and soil possibly affected by a swine manure-basedcompost facility

2 Materials and Methods

21 Sampling Thesampling sites were located inHongcheonGangwon Province Korea which were assumed to beaffected by antibiotic release from a swine manure compost-ing facility (37∘ 341015840 2810158401015840 N 127∘ 521015840 2610158401015840 E) Specific descrip-tions of the sampling sites are provided in Table 1 Samplingwas done in March 2009The average temperature was 177∘Cand total precipitation was 958mm [23] Sediments weresampled based on the distance from the composting facilityof 02 05 1 and 15 km as sediment sample 1 2 3 and 4respectively along the Naerincheon River Paddy soils werecollected from sites (a) directly appliedwith swinemanure foragricultural purposes as soil 1 and (b) only irrigated using awater source from Naerincheon River as soil 2 Specificallysediment and soil samples were collected at a depth of 0ndash20 cm Four subsamples were collected from each site andthese subsamples formed a composite sample The sedimentand soil samples were air-dried and then passed through a2-mm sieve before analysis The current study is a part of acomprehensive monitoring (since April 2008) of antibioticsin water sediment and soil near swine composting facility[20 22]

22 Antibiotic Extraction and Quantification Antibioticresidues were extracted from the sediment and soil samplesusing the method described by Kim and Carlson [24] andOk et al [20] and were quantified by high performanceliquid chromatography-tandem mass spectrometry (HPLC-MSMS) (API 3000 Applied Biosystems Foster City CAUSA) Recovery and the limit of quantification were deter-mined Briefly to extract TCs and SAs 1 g of sedimentor soil sample was added to a 50mL polypropylene cen-trifuge tube with 20mL of McIlvaine buffer at pH 4 buffersolution and 200120583L of 5 Na

2EDTA followed by 20min

of shaking at 400 rpm before centrifugation for 15min at4000 rpm (Centrifuge FLETA 5 Hanil Science IndustrySeoul Korea) The supernatant was filtered through a 02 120583mglass fiber filter The extraction process was repeated andthe extracts were combined in a 40mL vial for solid-phase




LC condition





MS condition























0

2

4

6

8


TCCTC

Con

cent

ratio

n (

minus1)

120583gk

g

(a)

0

4

8

12

16

20


SMTSMX

STZ

Con

cent

ratio

n (

minus1)

120583gk

g

(b)



2O3and Fe












24100c

19300e

17900f

29800b

31600a



a ND ND 100a ND


12667ab

9933b

5033c

3633c

3333c


8833a

3900cd

1433cd

1033d

1933cd


7933a

1367b

867b

2233b

1233b











1000 bp

700bp

200 bp100 bp

sul(I

)

Con

trol

tet(S

)

sul(A

)

tet(

T)

sul(I

I)

sul(I

II)

otr(

A)




4 Conclusions





Acknowledgments


References





















































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of




LC condition





MS condition























0

2

4

6

8


TCCTC

Con

cent

ratio

n (

minus1)

120583gk

g

(a)

0

4

8

12

16

20


SMTSMX

STZ

Con

cent

ratio

n (

minus1)

120583gk

g

(b)



2O3and Fe












24100c

19300e

17900f

29800b

31600a



a ND ND 100a ND


12667ab

9933b

5033c

3633c

3333c


8833a

3900cd

1433cd

1033d

1933cd


7933a

1367b

867b

2233b

1233b











1000 bp

700bp

200 bp100 bp

sul(I

)

Con

trol

tet(S

)

sul(A

)

tet(

T)

sul(I

I)

sul(I

II)

otr(

A)




4 Conclusions





Acknowledgments


References





















































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of












0

2

4

6

8


TCCTC

Con

cent

ratio

n (

minus1)

120583gk

g

(a)

0

4

8

12

16

20


SMTSMX

STZ

Con

cent

ratio

n (

minus1)

120583gk

g

(b)



2O3and Fe












24100c

19300e

17900f

29800b

31600a



a ND ND 100a ND


12667ab

9933b

5033c

3633c

3333c


8833a

3900cd

1433cd

1033d

1933cd


7933a

1367b

867b

2233b

1233b











1000 bp

700bp

200 bp100 bp

sul(I

)

Con

trol

tet(S

)

sul(A

)

tet(

T)

sul(I

I)

sul(I

II)

otr(

A)




4 Conclusions





Acknowledgments


References





















































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of





24100c

19300e

17900f

29800b

31600a



a ND ND 100a ND


12667ab

9933b

5033c

3633c

3333c


8833a

3900cd

1433cd

1033d

1933cd


7933a

1367b

867b

2233b

1233b











1000 bp

700bp

200 bp100 bp

sul(I

)

Con

trol

tet(S

)

sul(A

)

tet(

T)

sul(I

I)

sul(I

II)

otr(

A)




4 Conclusions





Acknowledgments


References





















































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


1000 bp

700bp

200 bp100 bp

sul(I

)

Con

trol

tet(S

)

sul(A

)

tet(

T)

sul(I

I)

sul(I

II)

otr(

A)




4 Conclusions





Acknowledgments


References





















































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of











































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of

research article monitoring antibiotic residues and

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