antibiotic resistance genes in water

MINI-REVIEW

Antibiotic resistance genes in water environment

Xu-Xiang Zhang & Tong Zhang & Herbert H. P. Fang

Received: 25 October 2008 /Revised: 11 December 2008 /Accepted: 13 December 2008 / Published online: 8 January 2009# Springer-Verlag 2008

Abstract The use of antibiotics may accelerate the devel-opment of antibiotic resistance genes (ARGs) and bacteriawhich shade health risks to humans and animals. Theemerging of ARGs in the water environment is becomingan increasing worldwide concern. Hundreds of variousARGs encoding resistance to a broad range of antibioticshave been found in microorganisms distributed not only inhospital wastewaters and animal production wastewaters,but also in sewage, wastewater treatment plants, surfacewater, groundwater, and even in drinking water. Thisreview summarizes recently published information on thetypes, distributions, and horizontal transfer of ARGs invarious aquatic environments, as well as the molecularmethods used to detect environmental ARGs, includingspecific and multiplex PCR (polymerase chain reaction),real-time PCR, DNA sequencing, and hybridization basedtechniques.

Keywords Antibiotic resistance gene .

Environmental pollution . Gene transfer .

Molecular detection method .Water environment

Introduction

Antibiotics are widely used to protect the health of humanand animals or to increase growth rate of animals as foodadditive. The majority of antibiotics are excreted un-changed into the environment. Thus, concerns about thepotential impact of antibiotic residues in the aquaticenvironment keep growing in recent years (Sarmah et al.2006; Wright 2007; Kemper 2008). In surface water, it isdifficult to find an area where antibiotics cannot bedetected, except for the pristine site in the mountains beforethe rivers or streams going through urban or agriculturalareas (Yang and Carlson 2003). Some antibiotics can befound even in groundwater as deep as more than 10 m (Battet al. 2006).

Apart from chemical pollution caused by antibioticsthemselves, the use of antibiotics may also accelerate thedevelopment of antibiotic resistance genes (ARGs) andbacteria, which shade health risks to humans and animals(Kemper 2008). These bacteria might be transmitted fromenvironment to human via direct or indirect contact (Iversenet al. 2004; Kim et al. 2005; Rodríguez et al. 2006).Considering the growing evidences that clinical resistanceis intimately associated with environmental ARGs andbacteria (Tatavarthy et al. 2006; Prabhu et al. 2007;Abriouel et al. 2008), it is quite clear that the researchactivities need to be expended to include nonpathogenic orenvironmental microorganisms. Currently, there are anumber of publications relating to the occurrence of ARGsin different water environments, but few reviews have beendone. This paper presents an overview of the latestinformation available in the literature on the types,distributions, and horizontal transfer of ARGs in variousaquatic environments, as well as the molecular methodsused to detect environmental ARGs.

Appl Microbiol Biotechnol (2009) 82:397–414DOI 10.1007/s00253-008-1829-z

X.-X. Zhang : T. Zhang (*) :H. H. P. FangEnvironmental Biotechnology Lab,Department of Civil Engineering,The University of Hong Kong,Pokfulam Road,Hong Kong, SAR, Chinae-mail: [email protected]

X.-X. ZhangDepartment of Environmental Science, Nanjing University,Nanjing, China

Types of environmental ARGs

Applications of antibiotics in human, veterinarymedicine, andagriculture for nearly 60 years have exerted a major impact onbacterial communities, resulting in various resistances to the

antibiotics, which is genetically controlled by ARGs. The useof antibiotics results in hundreds of ARGs being detected invarious water environments (Tables 1, 2, 3, 4, and 5). Theseenvironmental ARGs are mainly created by the followingmechanisms: (1) target bypass (dfrA1, A5, A7, A12, A15,

Table 1 Tetracycline resistance genes in water environments

Gene Biological source Environmental sourcea Reference

Tetracycline efflux proteintetA Aeromonas, Alcaligenes, Arthrobacter,

Comamonas, Escherichia, Listeria,Pseudomonas, Salmonella, and Vibrio;Plasmids pB10, pTB11 and pRSB101

AS, DW, EW,NW, SD, SW,US

Szczepanowski et al. 2004; Agersø and Sandvang2005; Srinivasan et al. 2005; Tennstedt et al. 2005;Poppe et al. 2006; Rodríguez et al. 2006; Cernatet al. 2007; Dang et al. 2007; Macauley et al. 2007;Hu et al. 2008

tetA(41) Serratia NW Thompson et al. 2007tetB Afipia, Alcaligenes, Arthrobacter,

Burkholderia, Escherichia,Pseudomonas, Serratia,Staphylococcus, and Vibrio

AS, DW, EW,NW, SW, US

Agersø and Sandvang 2005; Cernat et al. 2007;Dang et al. 2007; Jacobs and Chenia 2007;Kim et al. 2007; Kobashi et al. 2007, Macauleyet al. 2007

tetC Aeromonas, Alcaligenes, Arthrobacter,Brevibacterium, and Pseudomonas

AS, EW, SW, US Agersø and Sandvang 2005; Akinbowale et al.2007a; Macauley et al. 2007

tetD Aeromonas, Escherichia;microbial community

AS, DW, EW,SW, US

Schmidt et al. 2001; Auerbach et al. 2007;Cernat et al. 2007

tetE Aeromonas, Pseudoalteromonas, and Vibrio AS, EW, SD,SW, US

Schmidt et al. 2001; Dang et al. 2006;Agersø and Petersen 2007

tetG Pseudomonas; microbial community AS, EW, SW, US Auerbach et al. 2007; Macauley et al. 2007tetH Aeromonas, Flavobacterium, Proteus,

Pseudomona, Staphylococcus, and WautersiellaSW Jacobs and Chenia 2007; Macauley et al. 2007

tetJ Pseudomonas SW Macauley et al. 2007tetY Acidiovorax, Acinetobacter, Comamonas,

and ProteusSW Macauley et al. 2007

tetZ Actinomycetales, Afipia, Brevibacterium,Burkholderia, Dietzia, Leucobacter, andMicrobacterium

SW Kobashi et al. 2007; Macauley et al. 2007

tet33 Alcaligenes, Arthrobacter, and Pseudomonas SW Agersø and Sandvang 2005tet39 Acinetobacter SD, SW Agersø and Petersen 2007otrB Streptomycete AS, NW, SW Nikolakopoulou et al. 2005Ribosomal protection proteintetB(P) Microbial community SD, SW Chee-Sanford et al. 2001; Pei et al. 2006tetM Aeromonas, Bacillus, Escherichia, Lactococcus,

Pseudoalteromonas, and Vibrio;microbial community

AS, EW, NW, SD,SW, US

Mackie et al. 2006; Akinbowale et al. 2007b;Auerbach et al. 2007; Dang et al. 2007;Kim et al. 2007; Nonaka et al. 2007; Hu et al.2008; Rahman et al. 2008; Suzuki et al. 2008

tetO Paenibacillus, Pseudoalteromonas, Shewanella,Sporosarcina, and Vibrio; microbialcommunity

AS, EW, NW, SD,SW, US

Chee-Sanford et al. 2001; Smith et al. 2004;Mackie et al. 2006; Pei et al. 2006;Auerbach et al. 2007; Nonaka et al. 2007

tetQ Microbial community AS, EW, NW,SW, US

Smith et al. 2004; Auerbach et al. 2007;Mackie et al. 2006

tetS Lactococcus and Vibrio; microbial community AS, EW, SD,SW, US

Chee-Sanford et al. 2001; Kim et al. 2004;Auerbach et al. 2007; Suzuki et al. 2008

tetT Microbial community SD, SW Chee-Sanford et al. 2001; Pei et al. 2006tetW Microbial community SD, NW, SW Chee-Sanford et al. 2001; Mackie et al. 2006;

Pei et al. 2006; Suzuki et al. 2008otrA Streptomycete; microbial community AS, NW, SW Chee-Sanford et al. 2001; Nikolakopoulou et al. 2005

a The antibiotic resistance genes were detected in the following water environments: SW special wastewater from hospital, animal production, andaquaculture area; US untreated sewage; AS activated sludge of sewage treatment plant; EW effluent water of sewage treatment plant; NW naturalwater; SD sediments; and DW drinking water

398 Appl Microbiol Biotechnol (2009) 82:397–414

A17, and 18; sulI, II, III, and A), inaccessibility of theantibiotics to their target enzyme by mutational changes orloss on the enzyme gene (Huovinen et al. 1995; Happi et al.2005); (2) efflux pumps (cmlA1 and A5; floR; otrB; tetA,A(41), B, C, D, E, G, H, J, Y, Z, 33 and 39), reduction ofintracellular concentrations of antibiotics by structural alter-ation of cellular membrane (Kumar and Schweizer 2005); (3)antibiotic inactivation (aacC1, C2, C3, and C4; aadA1, A2,A5, A13, and B; ampC; aphA1, D and (3″)-Ic; blaOXA-1,blaOXA-2, blaOXA-10, blaOXA-30, and blaPSE-1; mphA; nptII;sat1 and 2; strA and B), direct deactivation of antibioticmolecule (Wright 2005); or (4) target modification (ermA, B,C, E, F, T, V, and X; mecA; penA; otrA; tetB(P), M, O, Q, S,T, and W; vanA and B), modification of the action sites ofantibiotics (Lambert 2005). It is noteworthy that theresistance of certain antibiotic may be associated withdifferent ARGs based on more than one mechanism.

ARGs related to tetracycline

Tetracycline-resistant bacteria were found to emerge in theenvironments with the introduction of tetracycline (Danceret al. 1997). There have been at least 38 differenttetracycline resistance (tet) genes and three oxytetracyclineresistance (otr) genes characterized to date (Roberts 2005;Thompson et al. 2007). These genes include 23 genes,which code for efflux proteins (efflux pump mechanism),11 genes for ribosomal protection proteins (target modifi-cation mechanism), and three genes for an inactivatingenzyme and one gene with unknown resistance mechanism(Levy et al. 1999; Roberts 2005).

Among them, more than 22 tet or otr genes have beenfound in bacterial isolates from water environments(Table 1). Most environmental tet genes code for transportproteins, which pump the antibiotics out of the bacteria

Table 2 Aminoglycoside resistance genes in water environments

Gene Biological source Environmentalsourcea

Function Reference

aacA4 Plasmid pTB11 AS, NW Aminoglycoside-6′-N-acetyltransferase

Tennstedt et al. 2005; Mukherjeeand Chakraborty 2006

aacA29b Plasmid pTB11 AS Tennstedt et al. 2003aacC1 Microbial communities NW, SW, USaacC2 Microbial communities NW, SW, US Aminoglycoside-3-N-

acetyltransferaseLee et al. 1998; Heuer et al. 2002

aacC3 Microbial communities NW, SW, USaacC4 Microbial communities NW, SW, USaadA1 Aeromonas, Citrobacter and

Shigella; Plasmid pTB11AS, EW, NW,SW, US

Tennstedt et al. 2003; Henriques et al.2006a; Mukherjee and Chakraborty2006; Moura et al. 2007

aadA2 Aeromonas, Escherichia and Vibrio;Plasmids pB2, pB3 and pTB11

AS, NW, SD,SW, US

Aminoglycoside-3′-adenylyltransferase

Dalsgaard et al. 2000; Tennstedt et al.2003; Heuer et al. 2004;Taviani et al. 2008

aadA4 Plasmid pB8 AS Schlüter et al. 2005aadA5 Escherichia and Vibrio;

Plasmid pTB11AS, NW Park et al. 2003; Tennstedt et al. 2003;

Mohapatra et al. 2008aadA13 Aeromonas; Plasmid pTB11 SW Moura et al. 2007aadB Aeromonas AS Aminoglycoside-2″-

adenylyltransferaseTennstedt et al. 2003

aphA1 Salmonella DW, NW Aminoglycosidephosphoryltransferase

Cernat et al. 2007; Poppe et al. 2006

aphA2 Escherichia DW Cernat et al. 2007aphD Microbial communities NW, SW, US Heuer et al. 2002aph(3″)-Ic Mycobacterium - Ramón-García et al. 2006nptII Microbial communities NW Neomycin phosphotransferase Zhu 2007sat1 Aeromonas and Escherichia NW, SW Streptothricin acetyltransferase Henriques et al. 2006a; Moura et al. 2007sat2 Aeromonas and Escherichia NW, SW Henriques et al. 2006a; Moura et al., 2007strA Listeria, Salmonella and Vibrio;

Plasmids pB4 and pB10AS, NW, SW Streptothricin

phosphoryltransferaseTauch et al. 2003; Poppe et al. 2006;Jacobs and Chenia 2007; Mohapatraet al. 2008

strB Salmonella and Vibrio; PlasmidspB4 and pB10

AS, NW Tauch et al. 2003; Poppe et al. 2006;Mohapatra et al. 2008

a The abbreviations of environmental sources are the same as those in Table 1

Appl Microbiol Biotechnol (2009) 82:397–414 399

cell and keep the intercellular concentrations low to makeribosomes function normally (Roberts 2002). The effluxgenes of tetA, B, C, D, and E frequently appeared invarious environmental compartments including activatedsludge of sewage treatment plants (STPs) (Guillaume et al.2000), fish farming ponds (Schmidt et al. 2001; Dang etal. 2007), surface water (Poppe et al. 2006), and swinelagoon (Macauley et al. 2007). Recently, the tetracyclineresistance genes including tetM, O, S, Q, and W, codingfor ribosomal protection proteins, have also been detectedin microbial communities of sewage treatment systems(Auerbach et al. 2007), hospital or animal productionwastewaters (Kim et al. 2007; Nonaka et al. 2007), andeven in natural water environments (Mackie et al. 2006).

Many tet genes are located on nonmobile plasmids orincomplete transposons in the chromosome (Roberts 2005),but some genes encoding efflux enzymes (tetA, B, C, E, H,Y, Z, and 33) and ribosomal protection proteins (tetM and O)still have a broad host range and have been found in severalenvironmental genera including Gram-negative and Gram-positive species (Table 1). Recently, Agersø and Petersen(2007) have found that tetE is often located on largehorizontally transferable plasmids of Aeromonas spp. isolat-ed from pond water of fish farm, and the gene has been

proved to be capable of interspecies transfer to Escherichiacoli. tetA, D, and M can also be transferred horizontally byoxytetracycline resistance plasmid from environmentalmicroorganisms to E. coli strains isolated from chicken,pig, and human, which indicates the potential environmentalhazards caused by the tet ARGs (Akinbowale et al. 2007b).

ARGs related to aminoglycoside

Different from tetracycline resistance mechanisms men-tioned above, the most major mechanism of aminoglyco-side resistance is direct deactivation of this type ofantibiotics by enzymatic modification (Shakil et al. 2008).More than 50 modification enzymes have been found so far(Vakulenko and Mobashery 2003; Ramón-García et al.2006). These enzymes are divided into three groups basedupon their biochemical actions on the aminoglycosidesubstrates, including acetyltransferases, phosphotrans-ferases, and nucleotidyltransferases (adenylyltransferases),encoded by three types of genes, namely, aac, aph, and ant(aad), respectively. Different aminoglycoside-modifyingenzymes have been reported in a broad range of bacteriaisolated from patients or clinical environments (Filipovaet al. 2006; Kelmani Chandrakanth et al. 2008).

Table 3 Macrolide, chloramphenicol, and vancomycin resistance genes in water environments

Gene Biological source Environmental sourcea Function Reference

Macrolide resistance genesermA Enterococcus EW, SW Erythromycin resistance

methylaseHayes et al. 2005; Chen et al. 2007

ermB Bacillus and Enterococcus EW, SW Hayes et al. 2005; Chen et al. 2007ermC Microbial community EW, SW Chen et al. 2007ermE Microbial community SW Patterson et al. 2007ermF Microbial community EW, SW Chen et al. 2007ermT Microbial community EW, SW Chen et al. 2007ermV Microbial community SW Patterson et al. 2007ermX Microbial community EW, SW Chen et al. 2007mphA Plasmid pRSB101 AS Macrolide-2′-

phosphotransferaseSzczepanowski et al. 2004

Chloramphenicol resistance genescmlA1 Plasmid pB2 and pB3 AS Chloramphenicol efflux

proteinHeuer et al. 2004

cmlA5 Plasmid pTB11 AS Tennstedt et al. 2003catB2 Plasmid pTB11 AS Tennstedt et al. 2003catB3 Aeromonas SW Jacobs and Chenia 2007catI Pseudomonas NW Chloramphenicol

acetyltransferaseDang et al. 2008

catII Vibrio SW Dang et al. 2007catIII Pseudomonas NW Dang et al. 2008catIV Vibrio and Pseudoalteromonas SW Dang et al. 2006floR Listeria, Pseudoalteromonas

Salmonella and Vibrio,DW, NW, SW Florfenicol efflux

proteinSrinivasan et al. 2005; Poppe et al. 2006;Dang et al. 2007

Vancomycin resistance genesvanA Enterococcus and Staphylococci DW, EW, NW, SW, UW Vancomycin resistance

proteinSchwartz et al. 2003; Volkmann et al. 2004;Messi et al. 2006

vanB Enterococcus EW, NW, UW Iversen et al. 2002; Caplin et al. 2008



The aac, aph, and ant genes are widely distributed invarious genera including Aeromonas, Escherichia, Vibrio,Salmonella, and Listeria spp. isolated from polluted ornatural water environments (Table 2). The genes of aacC1,C2, C3, and C4, encoding aminoglycoside-3-N-acetyltrans-ferase, were often detected in microbial communities orisolates from STPs (Heuer et al. 2002; Tennstedt et al.2003, 2005), and the two adenylyltransferase genes, aadA1and aadA2, were frequently reported all around the worldin the isolates from aquaculture areas (Dalsgaard et al.2000), river water (Park et al. 2003), STPs (Szczepanowskiet al. 2004; da Silva et al. 2007), and surface urban water(Taviani et al. 2008).

ARGs encoding resistances to other antibiotics in amino-glycoside group, for example, phosphotransferase genesencoding resistance to neomycin (nptII) and streptothricin(strAB), have also been detected in the river water ofCanada (Zhu 2007) and Ganges river of India (Mohapatraet al. 2008).

ARGs related to macrolide–lincosamide–streptogramin,chloramphenicol, and vancomycin

Although structurally unrelated to each other, the threeantibiotics, macrolides, lincosamide, and streptogramin, areoften investigated simultaneously for microbial resistance,since some macrolide resistance genes (erm) encoderesistance to two or all three of these compounds (Robertset al. 1999). Totally, more than 60 different genes conferresistance to one or more of the macrolide–lincosamide–streptogramin (MLS) antibiotics have been identified(Roberts 2008), including the genes associated with

ribosomal RNA (rRNA) methylation, efflux, and inactiva-tion. MLS resistance is mostly mediated by rRNAmethylases (encoded by erm genes), which methylate theadenine residues to prevent the three antimicrobials frombinding to ribosomal protein (Roberts 2002; Cetin et al.2008). The erm genes can easily be transferred from onehost to another (Roberts 2003), since they are usuallyacquired and associated with mobile elements, such asplasmids (Liu et al. 2007) and transposons (Okitsu et al.2005).

Several erm genes have been detected in Enterococcusspp. isolated from poultry raising wastewaters (Hayes et al.2005) and environmental DNA extracted from livestockmanures (Chen et al. 2007; Table 3). Six classes of ermgenes (A, B, C, F, T, and X) have been detected andquantified in the samples from animal production matures,lagoons, and a biofilter system treating hog house effluents(Chen et al. 2007). Among the macrolide resistancedeterminants, ermB is considered as the most prevalent genein environmental microorganisms, especially in the strains ofEnterococcus (Hayes et al. 2005) and Streptococcus spp.(Jensen et al. 2002).

The mechanisms responsible for resistances to chloram-phenicol and florfenicol include chloramphenicol acetyltrans-ferases (encoded by cat genes), specific exporters (encodedby cml genes), and multidrug transporters (Schwarz et al.2004). Of the chloramphenicol resistance genes known todate, several types of cat or cml genes have been reported tobe of environmental origin (Table 3). Vancomycin resistancefirstly emerged in enterococci, and recently, the resistancehas also been detected in Staphylococcus aureus (Walsh andHowe 2002). So far, six types of vancomycin resistance

Table 4 Sulphonamide and trimethoprim resistance genes in water environments

Gene Biological source Environmental sourcea Reference

Dihydrofolate reductase encoding genesdfrA1 Aeromonas, Escherichia, and Salmonella NW, SW Henriques et al. 2006a; Mukherjee and Chakraborty 2006;

Moura et al. 2007dfrA5 Escherichia NW Park et al. 2003; Mukherjee and Chakraborty 2006dfrA7 Escherichia NW Park et al. 2003dfrA12 Aeromonas, Escherichia, and Salmonella DW, NW, SW Moura et al. 2007; Antunes et al. 2006; Cernat et al. 2007dfrA15 Vibrio EW, NW Park et al. 2003; Taviani et al. 2008dfrA17 Escherichia, Salmonella DW, NW Park et al. 2003; Antunes et al. 2006; Cernat et al. 2007dfr18 Vibrio NW Mohapatra et al. 2008Dihydropteroate synthase encoding genessulI Aeromonas, Escherichia, and Listeria;

Plasmids pB2, pB3, pB8, and pB10;Microbial community

AS, DW, NW, SD, SW Heuer et al. 2004; Lin and Biyela 2005; Schlüter et al. 2005;Srinivasan et al. 2005; Akinbowale et al. 2007a;Cernat et al. 2007; Hu et al. 2008

sulII Acinetobacter, Escherichia, Salmonella,and Vibrio; Microbial community

DW, NW, SD, SW Pei et al. 2006; Agersø and Petersen 2007; Cernat et al. 2007;Hu et al. 2008; Mohapatra et al. 2008;

sulIII Escherichia; Microbial community NW, SD Pei et al. 2006; Hu et al. 2008sulA Microbial community SD Pei et al. 2006

a The abbreviations of environmental sources are the same as those in Table 1.


genes (van) have been known (Messi et al. 2006), and vanAand vanB are the most prevalent ones in water environments(Table 3).

ARGs related to sulfonamides and trimethoprim

Sulfonamides are the first antibiotic developed for large-scale introduction into clinical use, which target dihydrop-teroate synthase (DHPS). Trimethoprim competitivelyinhibits dihydrofolate reductase (DHFR), which is respon-sible for the reduction of dihydrofolate to tetrahydrofolate(Alekshun and Levy 2007). The two types of bio-enzymesare partly responsible for folate bio-synthesis, which isassociated with thymine production and microbial growth(Nrochet et al. 2008). Resistances to sulfonamides andtrimethoprim are often encoded by mutations located onhighly conserved areas of DHPS genes (sul) and DHFRgenes (dfr) (Sköld 2000, 2001). Different types of mecha-nisms have been found to confer to sulfonamide resistance,mostly based on changes in the sul genes and mediation bymobile elements (Huovinen et al. 1995; Antunes et al. 2007).The most widespread trimethoprim resistance mechanism isthe replacement of a trimethoprim-sensitive DHFR by aplasmid-, transposon-, or cassette-borne trimethoprim-resis-tant DHFR (Sköld 2001; Blahna et al. 2006).

Four kinds of sul genes (sulI, II, III, and A) have beenfound in the bacteria of environmental origin (Table 4). sulIand II have been detected in bacterial isolates from fecalslurry of dairy farms (Srinivasan et al. 2005), water orsediments of aquaculture areas (Akinbowale et al. 2007a;Agersø and Petersen 2007), and even from the river or seawater without evidence of being polluted (Lin and Biyela2005; Hu et al. 2008; Mohapatra et al. 2008). sulI, as a part

of class 1 integron, can be disseminated and transferredhorizontally within and between bacterial species inwastewater (Tennstedt et al. 2003), river water (Mukherjeeand Chakraborty 2006), and sea water (Taviani et al. 2008).

More than 25 different resistant DHFR genes (dfr),subdivided into dfrA and dfrB, have been identified(Kehrenberg and Schwa 2005; Džidić et al. 2008), andseveral dfrA genes are commonly found in variousenvironmental isolates (Table 4). The environmentalhabitats of these genes include urban wastewater treatmentplants (da Silva et al. 2007), slaughterhouse wastewatertreatment plants (Moura et al. 2007), aquaculture systems(Jacobs and Chenia 2007), and river water (Park et al.2003; Mukherjee and Chakraborty 2006; Mohapatra et al.2008). dfrA1 is one of the static resistance genes locatedon class 2 integrons (Blahna et al. 2006), and dfr genecassettes are frequently found in the variable regions ofintegrons and are often the only gene cassettes present inenvironmental isolates (Antunes et al. 2006; Mukherjeeand Chakraborty 2006).

ARGs related to β-lactam

β-Lactams are the most widely used antibiotics, and resistanceto these antibiotics is a severe threat because they have lowtoxicity and are used to treat a broad range of infections(Livermore 1996). The mechanisms of β-lactam resistanceinclude inaccessibility of the antibiotics to their targetenzymes, modifications of target enzymes, and/or directdeactivation of the antibiotics by β-lactamases (Walsh 2000;Li et al. 2007). In Gram-negative bacteria, the primaryresistance mechanism is enzymatic inactivation through thecleavage of theβ-lactam ring byβ-lactamases. More than 400

Table 5 β-Lactam and penicillin resistance genes in water environments

Gene Biological source Environmental sourcea Function Reference

ampC Enterobacter, Salmonella DW, NW, SW, US AmpC typeβ-lactamase

Schwartz et al. 2003; Volkmann et al. 2004;Poppe et al. 2006

blaPSE-1 Aeromonas, Salmonella and Vibrio EW, SD, SW, US PSE-1β-lactamase Dalsgaard et al. 2000; Jacobs and Chenia 2007;Taviani et al. 2008

blaTEM-1 Escherichia DW TEM-type β-lactamase Alpay-Karaoglu et al. 2007; Cernat et al. 2007blaOXA-1 Plasmid pTB11 AS OXA-1 β-lactamase Tennstedt et al. 2003blaOXA-2 Aeromonas; Plasmids pB8,

pB10 and pTB11AS, EW, SW OXA-2 β-lactamase Schlüter et al. 2005; Tennstedt et al. 2005;

Jacobs and Chenia 2007blaOXA-10

Plasmid pTB11 AS OXA-10 β-lactamase Tennstedt et al. 2003

blaOXA-30

Salmonella SW OXA-30 β-lactamase Antunes et al. 2006; Moura et al. 2007

mecA Staphylococcus DW, NW, US Penicillin-bindingprotein

Schwartz et al. 2003; Volkmann et al. 2004

penA Listeria DW, SW Srinivasan et al. 2005



different β-lactamases encoded by hundreds of ARGs (bla)have been identified, and the enzymes are divided into fourmolecular classes, A–D, mediating resistances to a broadrange of β-lactams including penicillins and cephalosporins(Li et al. 2007).

A variety of bla genes (Table 5) have been identified inbacteria derived from fecal slurry and lagoon water of dairyfarms (Srinivasan et al. 2005), water or sediments ofaquaculture areas (Dalsgaard et al. 2000; Jacobs and Chenia2007), STPs (Szczepanowski et al. 2004; Volkmann et al.2004; Antunes et al. 2006; Taviani et al. 2008), and surfacewater (Schwartz et al. 2003; Poppe et al. 2006). Theenvironmental compartments may further serve as reser-voirs for β-lactam resistance genes. The bla genes are oftendetected in animal-derived environmental pathogens in-cluding Aeromonas (Tennstedt et al. 2005; Jacobs andChenia 2007), Enterobacter (Volkmann et al. 2004),Salmonella (Antunes et al. 2006; Moura et al. 2007),Staphylococcus (Schwartz et al. 2003; Volkmann et al.2004), and Vibrio spp. (Dalsgaard et al. 2000; Taviani et al.2008). ampC gene encoding β-lactamases has beendetected in the microbial isolates from wastewater, surfacewater, and even from drinking water films (Schwartz et al.2003). mecA gene encoding methicillin resistance instaphylocci was observed to be prevalent in hospitalwastewater biofilms (Schwartz et al. 2003).

bla genes often coexist with other antimicrobialresistance determinants and can also be associated withmobile genetic elements, increasing the possibility ofmultidrug resistance and environmental dissemination(Tennstedt et al. 2003; Weldhagen 2004; Schlüter et al.2007b). The plasmids containing bla obtained from awastewater treatment plant are frequently associated withtransposons and integrons and often simultaneously carryother resistance determinants including aad (or aac)encoding aminoglycoside nucleotidyltransferase (or ace-tyltransferase), cml encoding chloramphenicol effluxprotein, and cat encoding chloramphenicol acetyltransfer-ase (Tennstedt et al. 2003).

Molecular techniques for the detectionand characterization of environmental ARGs

Considering that ARGs are widespread in aquatic environ-ments mentioned above, there is a need for the developmentand application of molecular methods to investigate theoccurrence, transport, and fate of the environmental ARGs.So far, the methods used for detection, typing, andcharacterization of ARGs have covered, but not beenlimited to, specific and multiplex polymerase chain reaction(PCR), real-time PCR, DNA sequencing, and hybridiza-tion-based techniques including microarray.

DNA hybridization

Molecular hybridization has been used to detect presence/absence of specific ARGs for nearly 30 years (Mendezet al. 1980). Many improvements have been made onmolecular hybridization, especially in probe design andsynthesis, so that the technique, especially Southern blot, isstill often applied to distinguish different ARGs in onegroup (i.e., tet genes) from each other (Roberts and Kenny1986; Levy et al. 1999) or to identify presence of specificgenes in certain environment (Agersø and Petersen 2007;Malik et al. 2008). Southern hybridization and filter-matingexperiments demonstrated that tet and class 1 integrons canbe co-transferred from soil isolates to E. coli and/orPseudomonas putida (Agersø and Sandvang 2005). UsingSouthern blot or dot blot coupled with PCR method, Maliket al. (2008) found that ampC was frequently present in soilsamples irrigated with wastewater. PCR-Southern blotassays showed that tet39 and sulII were common resistancegenes in Acinetobacter spp. isolates from water andsediments of fish farms.

With a number of nonradiolabeled systems becomingcommercially available, radioactive labeling is no longer anoption to label probes. As an important nonradiolabeledmethod, fluorescence in situ hybridization (FISH) has beenestablished and implemented successfully for clinicaldetection of microbial resistances. The use of FISHtechnique has been described for the rapid identificationof macrolide resistances caused by ribosomal mutations(Russmann et al. 2001). Recently, Werner et al. (2007) hasperformed a research to evaluate the reliability of FISH forclinical detection of linezolid-resistant enterococci andfound that the FISH technique along with DNA probescontaining locked nucleic acids with point mutation showed100% sensitivity for the detection of phenotypic linezolidresistance and even allowed detection of a single mutated23S rRNA gene allele in phenotypically linezolid-suscep-tible enterococci. Moosavian et al. (2007) developed andvalidated the FISH method for rapid detection of clarithro-mycin-resistant Helicobacter pylori in patients. AlthoughFISH has been often used for clinical detection of antibioticresistance, so far, few reports have been found about its usein identification of target bacteria harboring ARGs inenvironmental samples.

PCR (simple and multiplex PCR)

PCR assays have been widely used in both pure culturesand mixed environmental samples for detection of specificARGs encoding resistances to aminoglycoside (Mohapatraet al. 2008; Taviani et al. 2008), chloramphenicol (Danget al. 2008), β-lactam (Taviani et al. 2008), macrolide(Chen et al. 2007; Patterson et al. 2007), penicillin


(Srinivasan et al. 2005), sulphonamide (Agersø andPetersen 2007), tetracycline (Jacobs and Chenia 2007),trimethoprim (Moura et al. 2007), and vancomycin (Caplinet al. 2008). Environmental target DNA or RNA at lowconcentrations can be amplified and detected by PCR-basedmethods. However, a false-positive result is often given in thePCR assay. Southern hybridization of PCR products labeledand used as DNA probes to plasmid or chromosome DNAsamples from strains harboring target genes can avoid thefalse-positive PCR results (Ahmed et al. 2006; Akinbowaleet al. 2007b). In addition to DNA hybridization, DNAsequencing is another common method used to verify thePCR products of certain ARGs (Thompson et al. 2007).

In order to save time and effort, multiplex PCR methodshave been developed and often used for simultaneousdetection of more than one environmental ARG, includingthe genes encoding resistances to vancomycin (Bell et al.1998), macrolide (Jensen et al. 2002), tetracycline (Ardic etal. 2005; Agersø et al. 2007), sulfamethoxazole, andtrimethoprim (Ramachandran et al. 2007). With variousprimer pairs in the same PCR reaction system, multiplexPCR can amplify the DNA fragments of several ARGs atthe same time (Gilbride et al. 2006). The method savesconsiderable time and cost when different target regions areinvestigated simultaneously, but as a result of all thereactions taking place at the same conditions, some DNAamplifications can be inhibited and false-negative resultsare probably obtained. Another disadvantage of multiplePCR is that the dimer formation between primer pairs candisturb experimental results and lead to poor sensitivity(Markoulatos et al. 2002). Despite of the drawbacksmentioned above, multiplex PCR is still considered as arapid and convenient method for the detection of multipleARGs in isolated bacteria or environmental DNA (Agersøet al. 2007; Gilbride et al. 2006).

Quantitative PCR

The quantitative real-time PCR (qRT-PCR) is usually used toquantify target DNA on the basis of the principle that initialconcentration can be estimated according to the change ofPCR product concentration with amplification cycles (Zhangand Fang 2006). Among the several fluorescent reagentsdeveloped for qRT-PCR, SYBR Green is the most commonmethod used to quantify ARGs in bacterial isolates ofclinical origin, including tet (Morsczeck et al. 2004), mef,and erm genes (Reinert et al. 2004). Recently, the techniquehas been frequently used to quantify ARGs in environmen-tal samples, including tet genes in beef cattle farms (Yu etal. 2005), groundwater (Mackie et al. 2006), river sedi-ments (Pei et al. 2006), and STPs (Auerbach et al. 2007), aswell as sul genes in river sediments (Pei et al. 2006) and nptgenes in river water (Zhu 2007).

TaqMan probe, another fluorescent reagent, has alsobeen used to quantify tetO, tetW, and tetQ (Smith et al.2004), as well as vanA, mecA, and ampC genes (Volkmannet al. 2004) in wastewater. Chen et al. (2007) validatedTaqMan method for quantifying erm genes conferringresistance to MLS in the environmental samples fromanimal production areas.

The qRT-PCR method is not only usually used forquantitative analysis on ARGs’ distribution in the environ-ments but is also often applied to study the effects ofenvironmental factors or treatment processes on removal ofsome ARGs, i.e., tet genes (Mackie et al. 2006; Auerbachet al. 2007), sul genes (Pei et al. 2006), and erm genes (Chenet al. 2007). Using qRT-PCR, Auerbach et al. (2007)investigated tet genes in Germany STPs and found that tetQconcentrations were highest in influent water while tetGconcentrations were highest in activated sludge, and UVdisinfection had no effects on reduction in the amount ofdetectable tet genes in wastewater effluent. In order toanalyze the effect of river landscape on distributions ofARGs in sediments, some tet and sul genes in the sedimentshave been quantified using qRT-PCR, and higher resistancegene concentrations have been obtained at the impacted sitesthan at the pristine site (Pei et al. 2006). Using real-timePCR, Mackie et al. (2006) found that detection frequency oftetM, O, Q, and W genes was much higher in wells locatedcloser to and down-gradient from swine lagoons than in wellsmore distant from the lagoons. With qRT-PCR, Chen et al.(2007) found that erm abundances in composted swinemanure samples were significantly lower than those in swinemanure, demonstrating that manure storage probably influ-ences the persistence of the environmental genes.

DNA Microarray

Compared with other molecular methods, DNA microarraytechnique is a genomic analysis technique with high-throughput, high-speed and high-delicacy. For detection ofantibiotic resistances, DNA microarray can provide detailed,clinically relevant information on the isolates by detectingthe presence or absence of a large number of ARGssimultaneously in a single assay (Gilbride et al. 2006).Microarray allows detection of antibiotic resistance determi-nants within several hours and can be used as a time-savingand convenient tool supporting conventional resistancedetection assays (Antwerpen et al. 2007). Microarray hasbeen widely used to clinically detect antibiotic resistance ofhuman pathogens E. coli (Zhu et al. 2007a), H. pylori (Chenet al. 2008a), Salmonella enterica (Guard-Bouldin et al.2007), and S. aureus (Zhu et al. 2007b; Spence et al. 2008).The technique can also be applied to analyze genotypicresistance mechanisms of certain antibiotics (Chen et al.2008b).


Although microarrays have been successfully used toassess the antibiotic resistances of clinical samples, fewreports are found about using this technique to detect theARGs in environmental samples. The first factor hamperingits application in environmental samples is the lowdetection limit of the method, but microarray coupled withPCR method can enhance the detection limit for environ-mental ARGs (Gilbride et al. 2006). Patterson et al. (2007)developed a microarray system based on PCR amplificationof 23 tet genes and 10 erm genes to screen environmentalsamples for the presence of these ARGs and found thattetW, O, and Q were the most abundant ARGs found inswine fecal samples and ermV, and E were the mostcommon ones detected in farm and garden soil samples.Another reason for the poor applications of microarray inmost environmental samples is the complexity of thesamples and pretreatment. The presence of undesirablecontaminants in environmental samples inhibits DNAextraction and/or target gene amplification, so the compli-cated pretreatment of environmental samples is necessaryand crucial to get satisfactory detection results (Call 2005).Microarray technique can provide a detailed description ofbacterial antibiotic resistance and can reveal global changesin ARG expression in response to environmental changes(Call et al. 2003; Gilbride et al. 2006). The information ongene expression provides insight into antibiotic resistancemechanisms and general genetic responses of ARGs toenvironment-related changes.

Geographical distribution of studying ARGs in waterenvironment

The geographical distribution of environmental ARGs hasbeen indicated in the studies and detections on the genesall around the world (Fig. 1). In Europe, nearly all typesof ARGs were frequently detected in aquatic environmentsof some countries, including Germany (Tennstedt et al.2003; Szczepanowski et al. 2004; Tennstedt et al. 2005;Nikolakopoulou et al. 2005), Portugal (Antunes et al.2006; da Silva et al. 2007; Moura et al. 2007), Belgium(Guillaume et al. 2000; Heuer et al. 2002; Nikolakopoulouet al. 2005), Denmark (Schmidt et al. 2001; Agersø andSandvang 2005), and Greece (Heuer et al. 2002). Variouswater bodies in Europe have been found to contain somecommon ARGs, for example, vancomycin resistancegenes van, which have been detected in dairy farm waterof Italy (Messi et al. 2006), human-derived wastewater ofEngland (Caplin et al. 2008), urban raw sewage, treatedsewage and surface water of Sweden (Iversen et al. 2002),municipal wastewater, surface water, and drinking waterbiofilms of Germany (Schwartz et al. 2003; Volkmannet al. 2004).

In Northern America, tetracycline resistance genes werefrequently detected in water environments, including lagoonwater (Chee-Sanford et al. 2001; Srinivasan et al. 2005;Macauley et al. 2007), surface water (Poppe et al. 2006;Thompson et al. 2007), and wastewater treatment systems(Mispagel and Gray 2005; Auerbach et al. 2007). Otherenvironmental ARGs have also been found in the continent,encoding a wide resistance to aminoglycoside (Zhu 2007),β-lactam (Srinivasan et al. 2005), chloramphenicol (Poppeet al. 2006), macrolide (Chen et al. 2007), and sulfonamide(Pei et al. 2006). However, environmental distribution ofARGs has seldom been reported in Southern America.

In Asia, about 10 years ago, researchers beganinvestigating ARGs distribution in aquatic environments(Lee et al. 1998). dfr (Park et al. 2003) and tet genes(Suzuki et al. 2008) were detected in water samples ofAsian rivers, and tet genes were also found in marineaquatic environments in Korea (Kim et al. 2004; Kim et al.2007) and Japan (Nonaka et al. 2007; Rahman et al.2008). In China, chloramphenicol (catI, II, III, and IV)and tetracycline resistance genes (tetA, B, D, E, and M)have been detected in aquaculture ponds (Dang et al.2006; Dang et al. 2007) and coastal marine water (Dang etal. 2008). Recently, several tet and sul genes have alsobeen found in natural river basin of China (Hu et al.2008). In India, ARGs occurring in river water conferresistances to aminoglycoside, sulfonamide, and trimeth-oprim (Mukherjee and Chakraborty 2006; Mohapatra et al.2008). In Thailand, ARGs related to aminoglycoside andlactam resistances (Dalsgaard et al. 2000), as well assulfonamide and tetracycline resistances (Agersø andPetersen 2007) were detected in the sediments of fish orshrimp production areas.

Investigations about environmental ARGs have also beencarried out in Africa and Australia. Akinbowale et al.(2007a) found that Aeromonas containing resistance genesand class 1 integrons were present in sediments of fishfarms of Australia. Plasmids and integrons carrying avariety of ARGs have been identified in bacteria isolatedfrom South African aquaculture systems in the absence ofantibiotic selection pressure (Jacobs and Chenia 2007).Class 1 integrons and integrating conjugative elementsconferring resistances to trimethoprim, aminoglycoside, andβ-lactam were found in Vibrio strains isolated from surfaceurban water in Mozambique (Taviani et al. 2008).

Habitates of ARGs in water environment

ARGs are prevalent in different water bodies, and thespread pathways of ARGs in various aquatic environmentsusually are complicated. Before learning about the fate andtransport of ARGs in the environments, it is necessary to


characterize the occurrence, and the first step in thisendeavor is to identify major habitats of the ARGs in theenvironments. As a result of extensive use of human andveterinary antibiotics, hospital wastewater and livestockmanure are considered as the major sources of environ-mental ARGs. ARGs can enter into aquatic environmentsby direct discharging of untreated wastewater or into STPsthrough wastewater collection systems and subsequentlyinto the environments with effluents and discharged sludge(Auerbach et al. 2007). ARGs can be transferred into soilsby amending farm land with animal manure and processedbiosludge from STPs and then can leach to groundwater orbe carried by runoff and erosion to surface water (Yang andCarlson 2003). Surface water and shallow groundwater arecommonly used as source of drinking water; thus, ARGscan go though drinking water treatment facilities and enterinto water distribution systems (Schwartz et al. 2003).

Special wastewater from hospital, animal production,and aquaculture areas

The broad use of human, veterinary, and aquacultureantibiotics may exert selective pressure on bacteria in theenvironments of hospital (Liu et al. 2007), animal produc-tion (Agersø and Sandvang 2005), and fishery areas(Agersø and Petersen 2007), which are thought to be mainsources of ARGs distributing into the environments.Among all classes of ARGs, tet genes have the highestdetection frequency, and about 20 types of tet genes havebeen found in these wastewaters around the world,including tetA (Srinivasan et al. 2005), tetB (Dang et al.2007), tetC (Akinbowale et al. 2007a), tetD, and tetE(Schmidt et al. 2001), tetG, J, Y, and Z (Macauley et al.2007), tetH (Jacobs and Chenia 2007), tetM (Akinbowaleet al. 2007b), tetO (Nonaka et al. 2007), tetQ (Smith et al.

2004), tetW (Mackie et al. 2006), tetS (Kim et al. 2004),tetB(P) and T (Chee-Sanford et al. 2001), tet33 (Agersø andSandvang 2005), tet39 (Agersø and Petersen 2007), andotrA and B (Nikolakopoulou et al. 2005).

Other ARGs frequently detected in these specialwastewaters include methicillin resistance gene (mecA)in staphylococci isolated from hospital wastewater bio-films (Schwartz et al. 2003), chloramphenicol resistancegenes (catII, IV and B3) in the aquaculture systems (Danget al. 2006; Dang et al. 2007; Jacobs and Chenia 2007),and sulfonamide resistance genes (sulI, II, III, and A) infish farms (Agersø and Petersen 2007). Additionally, sometypes of ARGs including floR, penA, and strA (Srinivasanet al. 2005), as well as bla genes (Henriques et al. 2006b),were reported to occur in fecal slurry or lagoon of animalproduction areas. ARGs in these wastewaters are directlyexposed to the environment and can eventually be trans-ported to the nearby streams, rivers, lakes, or otheraquatic bodies or leach downward through the soil duringrainfall.

Untreated sewage

During the recent several years, various bacteria speciesisolated from untreated sewage were found to contain avariety of ARGs encoding resistances to aminoglycoside(da Silva et al. 2007; Taviani et al. 2008), β-lactam(Schwartz et al. 2003; Volkmann et al. 2004; Antunes etal. 2006), trimethoprim (da Silva et al. 2007), tetracyclines(Auerbach et al. 2007), and vancomycin (Iversen et al.2002; Caplin et al. 2008). By direct PCR of ARGs inenvironmental DNA extracted from municipal wastewater,tet genes (Auerbach et al. 2007) and aminoglycosideresistance genes (aacC1, C2, C3, C4, aadB, and aphD)(Heuer et al. 2002) were also found in sewage wastewaters.

Fig. 1 Detection of the antibi-otic resistance genes in geo-graphically isolated waterenvironments, including thegenes encoding resistance toaminoglycoside (red square),chloramphenicol (browninverted triangle), β-lactam(plus symbol), macrolide (skyblue triangle), sulfonamide (vi-olet diamond), tetracycline(green circle) and trimethoprim(indigo star)


Sewage receives the bacteria previously exposed toantibiotics from private households and hospitals and isconsidered as a hotspot for ARGs. ARGs go into STPswith sewage water, and most of them cannot beeffectively removed with traditional treatment processbefore being released into the environments (Volkmannet al. 2004; Auerbach et al. 2007). Moreover, environmen-tal conditions of activated sludge or biofilms facilitatehorizontal transfer of the ARGs from one host to anotherbecause of the nutritional richness and high bacterialdensity and diversity (Tennstedt et al. 2003; Schlüter et al.2007b).

STP activated sludge or biofilms

Several previous studies have shown that STPs serve asimportant reservoirs for various ARGs (Smalla andSobecky 2002; Tennstedt et al. 2003; Schlüter et al.2007b). ARGs present in STPs encode a broad resistanceto antibiotics including aminoglycoside (Tennstedt et al.2005; Moura et al. 2007), tetracycline (Guillaume et al.2000; Mispagel and Gray 2005; Auerbach et al. 2007),quinolone (Bönemann et al. 2006), and β-lactam (Szcze-panowski et al. 2004; Taviani et al. 2008). STPs receivethe antibiotic-resistant bacteria with the inflow sewagewater originating from hospitals, private households,industry, and agriculture, so they play important roles inrecombination, exchange, and spread of environmentalARGs (Szczepanowski et al. 2004).

STPs are recognized as important interfaces betweendifferent water bodies, such as hospital wastewater,domestic wastewater, surface water, and groundwater,therefore may facilitate gene exchange and spread betweenthese environmental compartments (Schlüter et al. 2007b).Firstly, the presence of antibiotics in sewage selects for themaintenance of ARGs conferring resistance in activatedsludge (Kümmerer 2003). Secondly, high microbialdensity and diversity of biofilms and activated sludgemay facilitate genetic exchange in sewage treatmentbioreactors (Schlüter et al. 2007b); for example, some tetgenes preferentially migrate from wastewater to biofilm(Engemann et al. 2008). Additionally, various mobileelements at high density in STPs accelerate gene recom-bination and transfer that encode new or multipleantimicrobial resistances (Tennstedt et al. 2003; Schlüteret al. 2007a). Finally, many ARGs, for example, vanA andB, cannot be effectively removed by activated sludgeprocess widely used in STPs, the genes being found inboth influent and effluent water (Iversen et al. 2002;Caplin et al. 2008). ARGs enter into other water bodieswith effluent water and can be transferred horizontally tothe native bacteria in these aquatic environments(Schwartz et al. 2003).

STP effluent water

STP effluent and sludge application to agricultural fieldsare recognized as important sources of ARGs to surfacewaters and soils and subsequently into groundwater (Yangand Carlson 2003). Several reports have indicated thatbacteria harboring ARGs can be released from STPs intosurface waters (Tennstedt et al. 2005; Chen et al. 2007;Auerbach et al. 2007).

Some types of ARGs have been detected in STPeffluent water including van genes (Iversen et al. 2002),aac, aad, and oxa genes (Tennstedt et al. 2005), tet genes(Auerbach et al. 2007), erm genes (Chen et al. 2007), andbla and dfr genes (Taviani et al. 2008). Some aminoglyco-side and β-lactam resistance determinants in effluent waterare recombined into integrons horizontally transferred byplasmids or transposons (Tennstedt et al. 2005). Resistancedeterminants in bacteria have been detected from habitatsdownstream of STPs (da Silva et al. 2007; Taviani et al.2008), and antibiotic resistance regions can be extended,modified, recombined, and exchanged in and amongbacteria residing in these areas (Schlüter et al. 2007b).New resistance properties could be horizontally transferredto human pathogens, thus increasing the difficulties ofinfectious disease treatment and threatening public health(Iversen et al. 2004).

Natural water

Scores of ARGs have been found in the isolates ormicrobial communities in the natural waters, which werenot or slightly polluted (Jacobs and Chenia 2007; Mohapatraet al. 2008; Rahman et al. 2008). Several types ofaminoglycoside resistance genes have been detected in themicroorganisms isolated from surface water, including aac(Lee et al. 1998), aad (Park et al. 2003; Mukherjee andChakraborty 2006), aph (Poppe et al. 2006), npt (Zhu2007), and str (Mohapatra et al. 2008). The detectionfrequency is also high for sulfonamides resistance genes (sul)(Lin and Biyela 2005; Poppe et al. 2006; Mohapatra et al.2008) and dihydrofolate reductase genes (dfr) (Mukherjeeand Chakraborty 2006) in surface water. ampC in surfacewater biofilms (Schwartz et al. 2003) and bla genes inestuarine water have also been detected (Henriques et al.2006a).

ARGs in surface water and soils can leach to ground-water close to agriculture areas of animal production oraquaculture. Tetracycline resistance genes encoding bothribosomal protection proteins (tetO, Q, W, M, S, T, B(P),and otrA; Chee-Sanford et al. 2001) and efflux pumps(tetB, C, E, H, and Z; Aminov et al. 2002) have beendetected in the groundwater as far as 250 m downstreamfrom waste lagoons of swine farms. In a recent study, tetM,


O, Q, and W in wells near swine lagoons were quantified,and the genes were then detected in groundwater down-stream from manure lagoons (Mackie et al. 2006). Besidesin fresh waters, some ARGs associated with resistances toaminoglycoside (Heuer et al. 2002) and chloramphenicol(Dang et al. 2008) have also been detected in marine waterswith no evidence of being polluted.

Sediments

It is self-evident that ARGs in sediments are acquired fromwater environments or produced for selection by the anti-biotics present in the sediments. Sediments of aquaculturefarms are important antibiotic resistance regions wherevarious antimicrobials and ARGs are concentrated (Dalsgaardet al. 2000; Agersø and Petersen 2007).

Marine sediments perhaps can be considered asnatural reservoirs of tetracycline resistance gene tetM,which has been found in various bacterial species insediments of Tokyo Bay, Sagami Bay, and the openPacific Ocean (Rahman et al. 2008). It was found thatnumbers of oxytetracycline-resistant bacteria increased insediments around a marine aquaculture site after oxytet-racycline therapy, and tetM was evident in both Gram-positive and Gram-negative bacteria from various generain the sediments of the marine environment (Nonaka et al.2007).

Various ARGs have also been identified in river sedi-ments. Sulfonamide resistance genes including sulI, II, III,and A were detected in the microorganisms of river waterand sediments (Pei et al. 2006). In rivers running throughpristine, urban, and agriculturally influenced areas, ARGdetection frequency in sediments was enhancedcorresponding to the increases in concentrations of variousantibiotic compounds (Yang and Carlson 2003; Pei et al.2006). After a spatial monitoring of environmental bacteriaand genes, Suzuki et al. (2008) found that the detectionfrequency of ribosomal protection protein genes (tetM, S,and W) in sediments of the Mekong River watershed werepositively correlated with the occurrence rate of tetracy-cline-resistant bacteria in the same area.

Drinking water

Prevalence and resistance patterns of various microbialgenera isolated from drinking water distribution systemhave been recently reported (Koksal et al. 2007; Ram et al.2008). Multiple-antibiotic-resistant E. coli strains isolatedfrom drinking water was found to carry ARGs encodingresistances to aminoglycoside, β-lactam, tetracycline, andtrimethoprim-sulfamethoxazole (Alpay-Karaoglu et al.2007; Cernat et al. 2007), as well as class 1 integrons(Ozgumus et al. 2007).

In order to indicate possible ARGs transfer fromwastewater and surface water to the drinking water distribu-tion network, Schwartz et al. (2003) and Obst et al. (2006)investigated biofilms in hospital and municipal waste-water, as well as drinking water from river bank filtrate,and found that vanA and ampC genes occurred not onlyin wastewater biofilms but also in drinking water biofilms.Florfenicol resistance gene floR and penicillin resistancegene penA have also been found in Listeria monocyto-genes isolated from drinking water in dairy farms(Srinivasan et al. 2005). The appearance of potentialantibiotic resistances in drinking water distribution sys-tems of some nations or regions requires increasedsurveillance for risk assessment and prevention strategiesto protect public health.

ARGs horizontal transfer

ARGs emerge in aquatic environments as a direct result ofintensive use of antibiotics in hospitals, swine productionareas, and fish farms, and the genes in surface water andgroundwater around such areas can transfer antibioticresistance to the bacteria in drinking water or the foodchain (Chee-Sanford et al. 2001). Genetic mechanismsinvolved in horizontal transfer of ARGs among environ-mental bacteria may include the following: (1) conjugativetransfer by mobile elements including plasmids, trans-posons, and integrons on plasmids or transposons; (2)transformation by naked DNA, in the case of naturallycompetent state of some bacteria, or an environmentallyinduced competence such as the presence of calcium; and(3) transduction by bacteriophage. Antibiotic resistance inmost environmental bacteria is due to the acquisition ofnew genes, often associated with the mobile elements.

Plasmid is an initially discovered microbial mobile elementdistributed in water environment, and STPs are considered asimportant pools of the plasmids with transportable ARGs(Szczepanowski et al. 2004; Tennstedt et al. 2005). Manytypes of plasmids have been isolated from activated sludgeof STPs, which confer resistances to aminoglycoside(Tennstedt et al. 2003), quinolone (Bönemann et al. 2006),erythromycin (Schlüter et al. 2007a), as well as multipledrugs (Szczepanowski et al. 2005). Recently, Schlüter et al.(2007b) has reviewed the gene elements and functions ofIncP-1 plasmids isolated from wastewater treatment plants.These self-transmissible plasmids are capable of transferringto and replicating in a wide range of hosts and can encoderesistances to almost all types of clinically relevantantibiotics (Szczepanowski et al. 2005; Schlüter et al.2007b).

Among the mobile elements, transposon and integronalso play important roles in horizontal transfer of environ-


mental ARGs. Previous reports demonstrated that trans-posons and integrons carrying various ARGs often occurredin animal production or aquaculture areas (Schmidt et al.2001; Moura et al. 2007; Akinbowale et al. 2007a; Jacobsand Chenia 2007), STPs (Szczepanowski et al. 2004;Tennstedt et al. 2005; da Silva et al. 2007; Taviani et al.2008), surface waters (Poppe et al. 2006; Mukherjee andChakraborty 2006; Lin and Biyela 2005), and sediments(Dalsgaard et al. 2000). The elements are not self-replicating and must be carried by a phage or, moretypically, by a plasmid to move from one cell to another.Insertion sequences, a type of small transposons, encode noother functions but recombinase and transposase (Summers2006). Transposons and insertion sequences often jumprandomly and occasionally on genome or plasmid, resultingin new or multiple resistances (Naas 2007). Integron is notcapable of moving itself but can capture, integrate, andexpress resistance gene cassettes in their variable regionsand can be transmitted via transposons and conjugativeplasmids (Fluit and Schmitz 1999; Alekshun and Levy2007). Integrons with as many as nine ARGs, typically fouror five, are frequently found in clinical environments(Crowley et al. 2008; Labuschagne et al. 2008), agriculturalwastewaters (Jacobs and Chenia 2007), urban wastewaters(Tennstedt et al. 2003; da Silva et al. 2007), and even in thewaters not recently exposed to antibiotics (Park et al. 2003;Obst et al. 2006).

Some physicochemical factors can influence thedissemination of ARGs in aquatic environments. Thefirst factor contributing to the horizontal transfer ofARGs is the selective pressure from ever-increasingproduction and consumption of antibiotics for treatmentof disease and growth promotion. High selective pressurefacilitates the acquisition of ARGs, which may actuallyincrease the fitness of certain bacteria and allow the rapidemergence and dissemination on a worldwide scale (Enneet al. 2004; Luo et al. 2005). In addition, the presence ofantibiotics at low subinhibitory concentrations can acceler-ate horizontal transfer and dissemination of environmentalARGs (Kümmerer 2004). It was found that keepingantibiotic concentration at a subinhibitory level in themating medium significantly enhances conjugal transfermediated by plasmid or transposon in the environments(Ohlsen et al. 2003; Hecht et al. 2007). Additionally,Auerbach et al. (2007) found UV disinfection had no effecton removal of tet genes in wastewater effluent, but loss rateof tetM, O, P, and W in aquatic environments has asignificantly positive correlation to simulated sunlightexposure (Engemann et al. 2006).

Many studies revealed that the co-selection took place inthe various environmental bacteria with metal and antibioticresistance (Berg et al. 2005; Stepanauskas et al. 2005; Wrightet al. 2006). Bacteria in metal-contaminated environments

appeared to be easier to obtain antibiotic resistance pheno-types than in control areas (Baker-Austin et al. 2006).However, genetic mechanisms responsible for the co-resistances occurring in the environments are poorly under-stood, since few researches have been carried out toinvestigate ARGs in metal-contaminated environments,though the experimental results of molecular genetics mayhelp to explain these phenomena. Rasmussen and Sorensen(1998) found occurrence of conjugative plasmids carryingtetracycline and mercury resistance genes was increased in acontaminated site. Recently, a novel tetracycline resistancegene, tetA(41), has been found in Serratia marcescensisolated from a stream contaminated with heavy metals(Thompson et al. 2007), which provides indirect evidence ofco-resistance. Wright et al. (2008) found that class 1integrase gene was more abundant in the metal-exposedenvironments than in control, and the selective pressuresshaped the structure of the gene cassette pool, indicating thatrelative gene transfer potential is higher in the microbialcommunities of the contaminated environments.

ARGs as emerging environmental pollutants

It was suggested by Rysz and Alvarez (2004) that ARGsthemselves could be considered as environmental ‘pollu-tants’, since they are widely distributed in various environ-mental compartments, including wastewater and STPs,surface water, lagoon water of animal production areas,aquaculture water, sediments and soil, groundwater, anddrinking water. Recently, Pruden et al. (2006) has alsopointed out that ARGs may be thought as emerging‘contaminants’, for the public health problems resultedfrom the widespread dissemination of ARGs.

As many other chemical pollutants, for example,persistent organic pollutants and heavy metals, ARGs arewell-known ‘easy-to-get, hard-to-lose’ pollutants (Aminovand Mackie 2007). Usually, antibiotic resistance bacteriaand genes emerge in the environments under the selectionpressure of some antibiotics, but the ARGs cannot be easilyremoved from the polluted areas, even when the pressurehas disappeared (Salyers and Amabile-Cuevas 1997;Aminov and Mackie 2007). This may be one explanationwhy ARGs were often detected in antibiotic-free environ-ments (Rahman et al. 2008). Potential public healthconcerns for environmental ARGs carried by bacterialpathogens were reviewed by Heuer et al. (2006) and Zhouet al. (2007). Although the direct evidence about ARGstransfer from the environments to human bodies isunavailable, some studies still highlight the fact that ARGscan spread and be exchanged among environmental micro-organisms of different genera (Agersø and Sandvang 2005;Agersø and Petersen 2007) and the organisms even within


completely different kingdoms (Rodríguez et al. 2006),which is supposed to be a daunting public health risk(Seveno et al. 2002; Alpay-Karaoglu et al. 2007).

Some efforts have to be made to reduce the possibility ofARGs entering into and spread in the environments. Themost effective and direct approach is thought to be thereasonable use of antibiotics in health protection andagriculture production. New and effective wastewatertreatment processes are also needed to be developed toimprove removal efficiency of ARGs in STPs. Additionally,feasibility of agricultural application of sludge or irrigationwith reclaimed wastewater has to be discussed thoroughlyconsidering possible introduction of ARGs to soil andgroundwater. Researches on transfer and degradation path-ways of environmental ARGs and health risk assessment onthe genes may be performed to provide more scientificinformation for responsible authorities to make up regula-tory standards and guidelines to control environmentaldissemination of these “pollutants.”

Acknowledgements The authors wish to thank the Hong KongResearch Grants Council for the financial support of this study (HKU7129/05E and HKU 7195/06E).

References

Abriouel H, Omar NB, Molinos AC, López RL, Grande MJ,Martínez-Viedma P, Ortega E, Cañamero MM, Galvez A (2008)Comparative analysis of genetic diversity and incidence ofvirulence factors and antibiotic resistance among enterococcalpopulations from raw fruit and vegetable foods, water and soil,and clinical samples. Int J Food Microbiol 123:38–49

Agersø Y, Sandvang D (2005) Class 1 integrons and tetracyclineresistance genes in Alcaligenes, Arthrobacter, and Pseudomonasspp. isolated from pigsties and manured soil. Appl EnvironMicrobiol 71:7941–7947

Agersø Y, Petersen A (2007) The tetracycline resistance determinantTet 39 and the sulphonamide resistance gene sulII are commonamong resistant Acinetobacter spp. isolated from integrated fishfarms in Thailand. J Antimicrob Chemother 59:23–27

Agersø Y, Bruun MS, Dalsgaard I, Larsen JL (2007) The tetracyclineresistance gene tet(E) is frequently occurring and present on largehorizontally transferable plasmids in Aeromonas spp. from fishfarms. Aquaculture 266:47–52

Ahmed AM, Furuta K, Shimomura K, Kasama Y, Shimamoto T(2006) Genetic characterization of multidrug resistance inShigella spp. from Japan. J Med Microbiol 55:1685–1691

Akinbowale OL, Peng H, Barton MD (2007a) Class 1 integronmediates antibiotic resistance in Aeromonas spp. from rainbowtrout farms in Australia. Int J Antimicrob Agents 29:S113

Akinbowale OL, Peng H, Barton MD (2007b) Diversity of tetracy-cline resistance genes in bacteria from aquaculture sources inAustralia. J Appl Microbiol 103:2016–2025

Alekshun MN, Levy SB (2007) Molecular mechanisms of antibacte-rial multidrug resistance. Cell 128:1037–1050

Alpay-Karaoglu S, Ozgumus OB, Sevim E, Kolayli F, Sevimi A,Yesilgil P (2007) Investigation of antibiotic resistance profile andTEM-type beta-lactamase gene carriage of ampicillin-resistant

Escherichia coli strains isolated from drinking water. AnnMicrobiol 57:281–288

Aminov RI, Mackie RI (2007) Evolution and ecology of antibioticresistance genes. FEMS Microbiol Lett 271:147–161

Aminov RI, Chee-Sanford JC, Garrigues N, Teferedegne B, Krapac IJ,White BA, Mackie RI (2002) Development, validation, andapplication of PCR primers for detection of tetracycline effluxgenes of Gram-negative bacteria. Appl Environ Microbiol68:1786–1793

Antunes P, Machado J, Peixe L (2006) Characterization of antimicro-bial resistance and class 1 and 2 integrons in Salmonella entericaisolates from different sources in Portugal. J Antimicrob Chemo-ther 58:297–304

Antunes P, Machado J, Peixe L (2007) Dissemination of a new genecluster comprising sul3 (tnp-sul3-tnp) linked to class 1 integronswith an unusual 3’CS region (qacH) among Salmonella isolates.Int J Antimicrob Agents 29:S112–S113

Antwerpen MH, Schellhase M, Ehrentreich-Foerster E, Witte W,Nuebel U (2007) DNA microarray for detection of antibioticresistance determinants in Bacillus anthracis and closely relatedBacillus cereus. Mol Cell Probes 21:152–160

Ardic N, Ozyurt M, Sareyyupoglu B, Haznedaroglu T (2005)Investigation of erythromycin and tetracycline resistance genesin methicillin-resistant staphylococci. Int J Antimicrob Agents26:213–218

Auerbach EA, Seyfried EE, McMahon KD (2007) Tetracyclineresistance genes in activated sludge wastewater treatment plants.Water Res 41:1143–1151

Baker-Austin C, Wright MS, Stepanauskas R, McArthur JV (2006)Co-selection of antibiotic and metal resistance. Trends Microbiol14:176–182

Batt AL, Snow DD, Aga DS (2006) Occurrence of sulfonamideantimicrobials in private water wells in Washington County,Idaho, USA. Chemosphere 64:1963–1971

Bell JM, Paton JC, Turnidge J (1998) Emergence of vancomycin-resistant enterococci in Australia: phenotypic and genotypiccharacteristics of isolates. J Clin Microbiol 36:2187–2190

Berg J, Tom-Petersen A, Nybroe O (2005) Copper amendment ofagricultural soil selects for bacterial antibiotic resistance in thefield. Lett Appl Microbiol 40:146–151

Blahna MT, Zalewski CA, Reuer J, Kahlmeter G, Foxman B, MarrsCF (2006) The role of horizontal gene transfer in the spread oftrimethoprim-sulfamethoxazole resistance among uropathogenicEscherichia coli in Europe and Canada. J Antimicrob Chemother57:666–672

Bönemann G, Stiens M, Pühler A, Schlüter A (2006) MobilizableIncQ-related plasmid carrying a new quinolone resistance gene,qnrS2, isolated from the bacterial community of a wastewatertreatment plant. Antimicrob Agents Chemother 50:3075–3080

Call DR (2005) Challenges and opportunities for pathogen detectionusing DNA microarrays. Crit Rev Microbiol 31:91–99

Call DR, Borucki MK, Loge FJ (2003) Detection of bacterialpathogens in environmental samples using DNA microarrays. JMicrobiol Methods 53:235–243

Caplin JL, Hanlon GW, Taylor HD (2008) Presence of vancomycinand ampicillin-resistant Enterococcus faecium of epidemic clonalcomplex-17 in wastewaters from the south coast of England.Environ Microbiol 10:885–892

Cernat R, Balotescu C, Ivanescu D, Nedelcu D, Lazar V, Bucur M,Valeanu D, Tudorache R, Mitache M, Dragoescu M (2007)Mechanisms of resistance in multiple-antibiotic-resistant Escher-ichia coli strains isolated from drinking and recreational, sal-master waters. Int J Antimicrob Agents 29:S274

Cetin ES, Gunes H, Kaya S, Aridogan BC, Demirci M (2008)Macrolide-lincosamide-streptogramin B resistance phenotypes in clinicalstaphylococcal isolates. Int J Antimicrob Agents 31:364–368


Chee-Sanford JC, Aminov RI, Krapac IJ, Garrigues-Jeanjean N, MackieRI (2001) Occurrence and diversity of tetracycline resistance genesin lagoons and groundwater underlying two swine productionfacilities. Appl Environ Microbiol 67:1494–1502

Chen J, Yu ZT, Michel FC Jr, Wittum T, Morrison M (2007)Development and application of real-time PCR assays forquantification of erm genes conferring resistance to macrolides-lincosamides-streptogramin B in livestock manure and manuremanagement systems. Appl Environ Microbiol 73:4407–4416

Chen SH, Li YM, Yu CH (2008a) Oligonucleotide microarray: a newrapid method for screening the 23S rRNA gene of Helicobacterpylori for single nucleotide polymorphisms associated withclarithromycin resistance. J Gastroenterol Hepatol 23:126–131

Chen S, Zhang MJ, Ma HH, Saiyin H, Shen SQ, Xi JJ, Wan B, Yu L(2008b) Oligo-microarray analysis reveals the role of cyclophilinA in drug resistance. Cancer Chemother Pharmacol 61:459–469

Crowley D, Cryan B, Lucey B (2008) First detection of a class 2integron among clinical isolates of Serratia marcescens. Br JBiomed Sci 65:86–89

Dalsgaard A, Forslund A, Serichantalergs O, Sandvang D (2000)Distribution and content of class 1 integrons in different Vibriocholerae O-serotype strains isolated in Thailand. AntimicrobAgents Chemother 44:1315–1321

Dancer SJ, Shears P, Platt DJ (1997) Isolation and characterization ofcoliforms from glacial ice and water in Canada’s high arctic. JAppl Microbiol 82:597–609

Dang HY, Zhang XX, Song LS, Chang YQ, Yang GP (2006)Molecular characterizations of oxytetracycline resistant bacteriaand their resistance genes from mariculture waters of China. MarPollut Bull 52:1494–1503

Dang HY, Zhang XX, Song LS, Chang YQ, Yang GP (2007)Molecular determination of oxytetracycline-resistant bacteriaand their resistance genes from mariculture environments ofChina. J Appl Microbiol 103:2580–2592

Dang HY, Ren J, Song LS, Sun S, An LG (2008) Dominantchloramphenicol-resistant bacteria and resistance genes in coastalmarine waters of Jiaozhou Bay, China. World J Microb Biot 24(2):209–217

da Silva MF, Vaz-Moreira I, Gonzalez-Pajuelo M, Nunes OC, ManaiaCM (2007) Antimicrobial resistance patterns in Enterobacteriaceaeisolated from an urban wastewater treatment plant. FEMS Micro-biol Ecol 60:166–176

Džidić S, Šušković J, Kos B (2008) Antibiotic resistance mechanismsin bacteria: biochemical and genetic aspects. Food TechnolBiotech 46:11–21

Engemann CA, Adams L, Knapp CW, Graham DW (2006) Disap-pearance of oxytetracycline resistance genes in aquatic systems.FEMS Microbiol Lett 263:176–182

Engemann CA, Keen P, Knapp CW, Hall KJ, Graham DW (2008) Fateof tetracycline resistance genes in aquatic systems: Migrationfrom the water column to peripheral biofilms. Environ SciTechnol 42:5131–5136

Enne VI, Bennett PM, Livermore DM, Hall LM (2004) Enhancementof host fitness by the sul2-coding plasmid p9123 in the absenceof selective pressure. J Antimicrob Chemother 53:958–963

Filipova M, Bujdakova H, Drahovska H, Liskova A, Hanzen J (2006)Occurrence of aminoglycoside-modifying-enzyme genes aac(6′)-aph(2″), aph(3′), ant(4′) and ant(6) in clinical isolates ofEnterococcus faecalis resistant to high-level of gentamicin andamikacin. Folia Microbiol 51:57–61

Fluit AC, Schmitz FJ (1999) Class 1 integrons, gene cassettes,mobility, and epidemiology. Eur J Clin Microbiol Infect Dis18:761–770

Gilbride KA, Lee DY, Beaudette LA (2006) Molecular techniques inwastewater: understanding microbial communities, detecting patho-gens, and real-time process control. J Microbiol Methods 66:1–20

Guard-Bouldin J, Morales CA, Frye JG, Gast RK, Musgrove M(2007) Detection of Salmonella enterica subpopulations byphenotype microarray antibiotic resistance patterns. Appl Envi-ron Microbiol 73:7753–7756

Guillaume G, Verbrugge D, Chasseur-Libotte ML, MoensW, Collard JM(2000) PCR typing of tetracycline resistance determinants (Tet A–E)in Salmonella enterica serotype Hadar and in the microbialcommunity of activated sludges from hospital and urban wastewa-ter treatment facilities in Belgium. FEMS Microbiol Ecol 32:77–85

Happi CT, Gbotosho GO, Folarin OA, Akinboye DO, Yusuf BO,Ebong OO, Sowunmi A, Kyle DE, Milhous W, Wirth DT,Oduola AMJ (2005) Polymorphisms in Plasmodium falciparumdhfr and dhps genes and age related in vivo sulfaxine-pyrimeth-amine resistance in malaria-infected patients from Nigeria. ActaTrop 95:183–193

Hayes JR, Wagner DD, English LL, Carr LE, Joseph SW (2005)Distribution of streptogramin resistance determinants amongEnterococcus faecium from a poultry production environmentof the USA. J Antimicrob Chemother 55:123–126

Hecht DW, Kos IM, Knopf SE, Vedantam G (2007) Characterizationof BctA, a mating apparatus protein required for transfer of theBacteroides fragilis conjugal element BTF-37. Res Microbiol158:600–607

Henriques IS, Fonseca F, Alves A, Saavedra MJ, Correia A (2006a)Occurrence and diversity of integrons and β-lactamase genesamong ampicillin-resistant isolates from estuarine waters. ResMicrobiol 157:938–947

Henriques IS, Moura A, Alves A, Saavedra MJ, Correia A (2006b)Analysing diversity among β-lactamase encoding genes inaquatic environments. FEMS Microbiol Ecol 56:418–429

Heuer H, Krögerrecklenfort E, Wellington EMH, Egan S, van ElsasJD, van Overbeek L, Collard JM, Guillaume G, Karagouni AD,Nikolakopoulou TL, Smalla, K (2002) Gentamicin resistancegenes in environmental bacteria: prevalence and transfer. FEMSMicrobiol Ecol 42:289–302

Heuer H, Szczepanowski R, Schneiker S, Phler A, Top EM, SchlüterA (2004) The complete sequences of plasmids pB2 and pB3provide evidence for a recent ancestor of the IncP-1b groupwithout any accessory genes. Microbiology 150:3591–3599

Heuer OE, Hammerum AM, Collignon P, Wegener HC (2006) Humanhealth hazard from antimicrobial-resistant enterococci in animalsand food. Clin Infect Dis 43:911–916

Hu JY, Shi JC, Chang H, Li D, Yang M, Kamagata YC (2008)Phenotyping and genotyping of antihiotic-resistant Escherichiacoli isolated from a natural river basin. Environ Sci Technol42:3415–3420

Huovinen P, Sundstrom L, Swedberg G, Skold O (1995) Trimetho-prim and sulfonamide resistance. Antimicrob Agents Chemother39:279–289

Iversen A, Kühn I, Franklin A, Möllby R (2002) High prevalence ofvancomycin-resistant enterococci in Swedish sewage. ApplEnviron Microbiol 68:2838–2842

Iversen A, Kühn I, Rahman M, Franklin A, Burman LG, Olsson-Liljequist B, Torell E, Möllby R (2004) Evidence for transmis-sion between humans and the environment of a nosocomial strainof Enterococcus faecium. Environ Microbiol 6:55–59

Jacobs L, Chenia HY (2007) Characterization of integrons andtetracycline resistance determinants in Aeromonas spp. isolatedfrom South African aquaculture systems. Int J Food Microbiol114:295–306

Jensen LB, Agersø Y, Sengeløv G (2002) Presence of erm genesamong macrolide-resistant Gram-positive bacteria isolated fromDanish farm soil. Environ Int 28:487–491

Kehrenberg C, Schwa S (2005) dfrA20, a novel trimethoprimresistance gene from Pasteurella multocida. Antimicrob AgentsChemother 49:414–417


Kelmani Chandrakanth R, Raju S, Patil SA (2008) Aminoglycoside-resistance mechanisms in multidrug-resistant Staphylococcusaureus clinical isolates. Curr Mcirobiol 56:558–562

Kemper N (2008) Veterinary antibiotics in the aquatic and terrestrialenvironment. Ecol Indic 8:1–13

Kim SR, Nonaka L, Suzuki S (2004) Occurrence of tetracyclineresistance genes tet(M) and tet(S) in bacteria from marineaquaculture sites. FEMS Microbiol Lett 237:147–156

Kim SH, Wei CI, Tzou YM, An HJ (2005) Multidrug-resistantKlebsiella pneumoniae isolated from farm environments andretail products in Oklahoma. J Food Prot 68:2022–2029

Kim YH, Jun LJ, Park SH, Yoon SH, Chuang JK, Kim JC, Jeong HD(2007) Prevalence of tet(B) and tet(M) genes among tetracycline-resistant Vibrio spp002E in the aquatic environments of Korea.Dis Aquat Organ 75:209–216

Kobashi Y, Hasebe A, Nishio M, Uchiyama H (2007) Diversity oftetracycline resistance genes in bacteria isolated from variousagricultural environments. Microb Environ 22:44–51

Koksal F, Oguzkurt N, Samasti M, Altas K (2007) Prevalence andantimicrobial resistance patterns of Aeromonas strains isolated fromdrinking water samples in Istanbul, Turkey. Chemotherapy 53:30–35

Kumar A, Schweizer HP (2005) Bacterial resistance to antibiotics: activeefflux and reduced uptake. Adv Drug Deliv Rev 57:1486–1513

Kümmerer K (2003) Significance of antibiotics in the environment. JAntimicrob Chemother 52:5–7

Kümmerer K (2004) Resistance in the environment. J AntimicrobChemother 54:311–320

Labuschagne CDJ, Weldhagen GF, Ehlers MM, Dove MG (2008)Emergence of class 1 integron-associated GES-5 and GES-5-likeextended-spectrum-lactamases in clinical isolates of Pseudomonasaeruginosa in South Africa. Int J Antimicrob Agents 31:527–530

Lambert PA (2005) Bacterial resistance to antibiotics: Modified targetsites. Adv Drug Deliv Rev 57:1471–1485

Lee YJ, Han HS, Seong CN, Lee HY, Jung JS (1998) Distribution ofgenes coding for aminoglycoside acetyltransferases in gentamicinresistant bacteria isolated from aquatic environment. J Microbiol36:249–255

Levy SB, McMurry LM, Barbosa TM, Burdett V, Courvalin P, HillenW, Roberts MC, Rood JI, Taylor DE (1999) Nomenclature fornew tetracycline resistance determinants. Antimicrob AgentsChemother 43:1523–1524

Li XZ, Mehrotra M, Ghimire S, Adewoye L (2007) β-Lactamresistance and β-lactamases in bacteria of animal origin. VetMicrobiol 121:197–214

Lin J, Biyela PT (2005) Convergent acquisition of antibiotic resistancedeterminants amongst the Enterobacteriaceae sp. isolates of theMhlathuze River, KwaZulu-Natal (RSA). Water SA 31:257–260

Liu YF, Wang CH, Janapatla RP, Fu HM, Wu HM, Wu JJ (2007)Presence of plasmid pA15 correlates with prevalence ofconstitutive MLSB resistance in group A streptococcal isolatesat a university hospital in southern Taiwan. J AntimicrobChemother 59:1167–1170

Livermore DM (1996) Are all beta-lactams created equal? Scand JInfect Dis Suppl 101:33–43

Luo N, Pereira S, Sahin O, Lin J, Huang S, Michel L, Zhang Q (2005)Enhanced in vivo fitness of fluoroquinolone-resistant Campylo-bacter jejuni in the absence of antibiotic selection pressure. ProcNatl Acad Sci U S A 102:541–546

Macauley JJ, Adams CD, Mormile MR (2007) Diversity of tetresistance genes in tetracycline resistant bacteria isolated from aswine lagoon with low antibiotic impact. Can J Microbiol53:1307–1315

Mackie RI, Koike S, Krapac I, Chee-Sanford J, Maxwell S, AminovRI (2006) Tetracycline residues and tetracycline resistance genesin groundwater impacted by swine production facilities. AnimBiotechnol 17:157–176

Malik A, Çelik EK, Bohn C, Böckelmann U, Knobel K, Grohmann E(2008) Detection of conjugative plasmids and antibiotic resis-tance genes in anthropogenic soils from Germany and India.FEMS Microbiol Lett 279:207–216

Markoulatos P, Siafakas N, Moncany M (2002) Multiplex polymerasechain reaction: a practical approach. J Clin Lab Anal 16:47–51

Mendez B, Tachibana C, Levy SB (1980) Heterogeneity of tetracy-cline resistance determinants. Plasmid 3:99–108

Messi P, Guerrieri E, de Niederhäusern S, Sabia C, Bondi M (2006)Vancomycin-resistant enterococci (VRE) in meat and environ-mental samples. Int J Food Microbiol 107:218–222

Mispagel H, Gray JT (2005) Antibiotic resistance from wastewateroxidation ponds. Water Environ Res 77:2996–3002

Mohapatra H, Mohapatra SS, Mantri CK, Colwell RR, Singh DV(2008) Vibrio cholerae non-O1, non-O139 strains isolated before1992 from Varanasi, India are multiple drug resistant, containintSXT, dfr18 and aadA5 genes. Environ Microbiol 10:866–873

Moosavian M, Tajbakhsh S, Samarbaf-Zadeh AR (2007) Rapiddetection of clarithromycin-resistant Helicobacter pylori inpatients with dyspepsia by fluorescent in situ hybridization(FISH) compared with the E-test. Ann Saudi Med 27:84–88

Morsczeck C, Langendörfer D, Schierholz JM (2004) A quantitativereal-time PCR assay for the detection of tetR of Tn10 inEscherichia coli using SYBR Green and the Opticon. J BiochemBiophys Methods 59:217–227

Moura A, Henriques I, Ribeiro R, Correia A (2007) Prevalence andcharacterization of integrons from bacteria isolated from aslaughterhouse wastewater treatment plant. J Antimicrob Chemo-ther 60:1243–1250

Mukherjee S, Chakraborty R (2006) Incidence of class 1 integrons inmultiple antibiotic-resistant Gram-negative copiotrophic bacteriafrom the River Torsa in India. Res Microbiol 157:220–226

Naas T (2007) Insertion sequences, transposons and repeatedelements. Int J Antimicrob Agents 29:S1

Nikolakopoulou TL, Egan S, van Overbeek LS, Guillaume G, Heuer H,Wellington EMH, van Elsas JD, Collard JM, Smalla K, KaragouniAD (2005) PCR detection of oxytetracycline resistance genes otr(A) and otr(B) in tetracycline-resistant Streptomycete isolatesfrom diverse habitats. Curr Microbiol 51:211–216

Nonaka L, Ikeno K, Suzuki S (2007) Distribution of tetracyclineresistance gene, tet(M), in Gram-positive and Gram-negativebacteria isolated from sediment and seawater at a coastalaquaculture site in Japan. Microb Environ 22:355–364

Nrochet M, Couve E, Zouine M, Povart C, Glaser P (2008) Anaturally occurring gene amplification leading to sulfonamideand trimethoprim resistance in Streptococcus agalactiae. JBacteriol 190:672–680

Obst U, Schwartz T, Volkmann H (2006) Antibiotic resistantpathogenic bacteria and their resistance genes in bacterialbiofilms. Int J Artif Organs 29:387–394

Ohlsen K, Ternes T, Werner G, Wallner U, Loffler D, Ziebuhr W,Witte W, Hacher J (2003) Impact of antibiotics on conjugationalresistance gene transfer in Staphylococcus aureus in sewage.Environ Microbiol 5:711–716

Okitsu N, Kaieda S, Yano H, Nakano R, Hosaka Y, Okamoto R,Kobayashi T, Inoue M (2005) Characterization of ermB genetransposition by Tn1545 and Tn917 in macrolide-resistantStreptococcus pneumoniae isolates. J Clin Microbiol 43:168–173

Ozgumus OB, Celik-Sevim E, Alpay-Karaoglu S, Sandalli C, SevimA (2007) Molecular characterization of antibiotic resistantEscherichia coli strains isolated from tap and spring waters in acoastal region in turkey. J Microbiol 45:379–387

Park JC, Lee JC, Oh JY, Jeong YW, Cho JW, Joo HS, Lee WK, LeeWB (2003) Antibiotic selective pressure for the maintenance ofantibiotic resistant genes in coliform bacteria isolated from theaquatic environment. Water Sci Technol 47:249–253


Patterson AJ, Colangeli R, Spigaglia P, Scott KP (2007) Distributionof specific tetracycline and erythromycin resistance genes inenvironmental samples assessed by macroarray detection. Envi-ron Microbiol 9:703–715

Pei RT, Kim SC, Carlson KH, Pruden A (2006) Effect of riverlandscape on the sediment concentrations of antibiotics andcorresponding antibiotic resistance genes (ARG). Water Res40:2427–2435

Poppe C, Martin L, Muckle A, Archambault M, McEwen S, Weir E(2006) Characterization of antimicrobial resistance of SalmonellaNewport isolated from animals, the environment, and animalfood products in Canada. Can J Vet Res 70:105–114

Prabhu DIG, Pandian RS, Vasan PT (2007) Pathogenicity, antibioticsusceptibility and genetic similarity of environmental and clinicalisolates of Vibrio cholerae. Indian J Exp Biol 45:817–823

Pruden A, Pei R, Storteboom H, Carlson K (2006) Antibioticresistance genes as emerging contaminants: Studies in NorthernColorado. Environ Sci Technol 40:7445–7450

Rahman MH, Nonaka L, Tago R, Suzuki S (2008) Occurrence of twogenotypes of tetracycline (TC) resistance gene tet(M) in the TC-resistant bacteria in marine sediments of Japan. Environ SciTechnol 42:5055–5061

Ram S, Vajpayee P, Shanker R (2008) Contamination of potable waterdistribution systems by multiantimicrobial-resistant enterohemor-rhagic Escherichia coli. Environ Health Perspect 116:448–452

Ramachandran D, Bhanumathi R, Singh DV (2007) Multiplex PCRfor detection of antibiotic resistance genes and the SXT element:application in the characterization of Vibrio cholerae. J MedMicrobiol 56:346–351

Ramón-García S, Otal I, Martín C, Gómez-Lus R, Aínsa JA (2006) Novelstreptomycin resistance gene from Mycobacterium fortuitum.Antimicrob Agents Chemother 50:3920–3922

Rasmussen LD, Sorensen SJ (1998) The effect of long-term exposureto mercury on the bacterial community in marine sediment. CurrMicrobiol 36:291–297

Reinert RR, Franken C, van der Linden M, Lütticken R, Cil M, Al-Lahham A (2004) Molecular characterisation of macrolide resis-tance mechanisms of Streptococcus pneumoniae and Streptococcuspyogenes isolated in Germany, 2002-2003. Int J AntimicroblAgents 24:43–47

Roberts MC (2002) Resistance to tetracycline, macrolide-lincosamide-streptogramin, trimethoprim, and sulfonamide drug classes. MolBiotechnol 20:261–283

Roberts MC (2003) Acquired tetracycline and/or macrolide–lincosamides–streptogramin resistance in anaerobes. Anaerobe 9:63–69

Roberts MC (2005) Update on acquired tetracycline resistance genes.FEMS Microbiol Lett 245:195–203

Roberts MC (2008) Update on macrolide-lincosamide-streptogramin,ketolide, and oxazolidinone resistance genes. FEMS MicrobiolLett 282:147–159

Roberts MC, Kenny GE (1986) Dissemination of the tetM tetracyclineresistance determinant to Ureaplasma urealyticum. AntimicrobAgents Chemother 29:350–352

Roberts MC, Sutcliffe J, Courvalin P, Jensen LB, Rood J, Seppala H(1999) Nomenclature for macrolide and macrolide-lincosamidestreptogramin B antibiotic resistance determinants. AntimicrobAgents Chemother 43:2823–2830

Rodríguez C, Lang L, Wang A, Altendorf K, García F, Lipski A(2006) Lettuce for human consumption collected in Costa Ricacontains complex communities of culturable oxytetracycline-andgentamicin-resistant bacteria. Appl Environ Microbiol 72:5870–5876

Russmann H, Adler K, Haas R, Gebert B, Koletzko S, Heesemann J(2001) Rapid and accurate determination of genotypic clarithro-mycin resistance in cultured Helicobacter pylori by fluorescent insitu hybridization. J Clin Microbiol 39:4142–4144

Rysz M, Alvarez PJJ (2004) Amplification and attenuation oftetracycline resistance in soil bacteria: aquifer column experi-ments. Water Res 38:3705–3712

Salyers AA, Amabile-Cuevas CF (1997) Why are antibiotic resistancegenes so resistant to elimination? Antimicrob Agents Chemother41:2321–2325

Sarmah AK, Meyer MT, Boxall ABA (2006) A global perspective onthe use, sales, exposure pathways, occurrence, fate and effects ofveterinary antibiotics (VAs) in the environment. Chemosphere65:725–759

Schlüter A, Heuer H, Szczepanowski R, Poler SM, Schneiker S, PühlerA, Top EM (2005) Plasmid pB8 is closely related to the prototypeIncP-1b plasmid R751 but transfers poorly to Escherichia coli andcarries a new transposon encoding a small multidrug resistance(SMR) efflux protein. Plasmid 54:135–148

Schlüter A, Szczepanowski R, Kurz N, Schneiker S, Krahn I, Pühler A(2007a) Erythromycin resistance-conferring plasmid pRSB105,isolated from a sewage treatment plant, harbors a new macrolideresistance determinant, an integron-containing Tn402-like ele-ment, and a large region of unknown function. Appl EnvironMicrobiol 73:1952–1960

Schlüter A, Szczepanowski R, Pühler A, Top EM (2007b) Genomicsof IncP-1 antibiotic resistance plasmids isolated from wastewatertreatment plants provides evidence for a widely accessible drugresistance gene pool. FEMS Microbiol Rev 31:449–477

Schmidt AS, Bruun MS, Dalsgaard I, Larsen JL (2001) Incidence,distribution, and spread of tetracycline resistance determinantsand integron-associated antibiotic resistance genes among motileaeromonads from a fish farming environment. Appl EnvironMicrobiol 67:5675–5682

Schwartz T, Kohnen W, Jansen B, Obst U (2003) Detection ofantibiotic-resistant bacteria and their resistance genes in waste-water, surface water, and drinking water biofilms. FEMS Micro-biol Ecol 43:325–335

Schwarz S, Kehrenberg C, Doublet B, Cloeckaert A (2004) Molecularbasis of bacterial resistance to chloramphenicol and florfenicol.FEMS Microbiol Rev 28:519–542

Seveno NA, kallifidas D, Smalla K, van Elsas JD, Collard JM,Karagouni AD, Wellington EMH (2002) Occurrence and reser-voirs of antibiotic resistance genes in the environment. Rev MedMicrobiol 13:15–27

Shakil S, Khan R, Zarrilli R, Khan AU (2008) Aminoglycosidesversus bacteria—a description of the action, resistance mecha-nism, and nosocomial battleground. J Biomed Sci 15:5–14

Sköld O (2000) Sulfonamide resistance: mechanisms and trends. DrugResist Updat 3:155–160

Sköld O (2001) Resistance to trimethoprim and sulfonamides. Vet Res32:261–273

Smalla K, Sobecky PA (2002) The prevalence and diversity of mobilegenetic elements in bacterial communities of different environ-mental habitats: insights gained from different methodologicalapproaches. FEMS Microbiol Ecol 42:165–175

Smith MS, Yang RK, Knapp CW, Niu YF, Peak N, Hanfelt MM,Galland JC, Graham DW (2004) Quantification of tetracyclineresistance genes in feedlot lagoons by real-time PCR. ApplEnviron Microbiol 12:7372–7377

Spence RP, Wright V, Ala-Aldeen DAA, Turner DP, Wooldridge KG,James R (2008) Validation of virulence and epidemiology DNAmicroarray for identification and characterization of Staphylo-coccus aureus isolates. J Clin Microbiol 46:1620–1627

Srinivasan V, Nam HM, Nguyen LT, Tamilselvam B, Murinda SE,Oliver SP (2005) Prevalence of antimicrobial resistance genes inListeria monocytogenes isolated from dairy farms. FoodbornePathog Dis 2:201–211

Stepanauskas R, Glenn TC, Jagoe CH, Tuckfield RC, Lindell AH,McArthur JV (2005) Elevated microbial tolerance to metals and


antibiotics in metal-contaminated industrial environments. Envi-ron Sci Technol 39:3671–3678

Summers AO (2006) Genetic linkage and horizontal gene transfer, theroots of the antibiotic multi-resistance problem. Anim Biotechnol17:125–135

Suzuki S, Kobayashi T, Suehiro F, Tuyen CB, Tana TS (2008) Highoccurrence rate of tetracycline (TC)-resistant bacteria and TCresistance genes relates to microbial diversity in sediment ofMekong River main waterway. Microb Environ 23:149–152

Szczepanowski R, Krahn I, Linke B, Goesmann A, Pühler A, SchlüterA (2004) Antibiotic multiresistance plasmid pRSB101 isolatedfrom a wastewater treatment plant is related to plasmids residingin phytopathogenic bacteria and carries eight different resistancedeterminants including a multidrug transport system. Microbiol-ogy 150:3613–3630

Szczepanowski R, Braun S, Riedel V, Schneiker S, Krahn I, Pühler A,Schlüter A (2005) The 120 592 bp IncF plasmid pRSB107isolated from a sewage-treatment plant encodes nine differentantibiotic-resistance determinants, two iron-acquisition systemsand other putative virulence-associated functions. Microbiology151:1095–1111

Tatavarthy A, Peak K, Veguilla W, Reeves F, Cannons A, Amuso P,Cattani J (2006) Comparison of antibiotic susceptibility profilesand molecular typing patterns of clinical and environmentalSalmonella enterica serotype Newport. J Food Prot 69:749–756

Tauch A, Schlüter A, Bischoff N, Goesmann A, Meyer F, Pühler A(2003) The 79,370-bp conjugative plasmid pB4 consists of anIncP-1b backbone loaded with a chromate resistance transposon,the strA-strB streptomycin resistance gene pair, the oxacillinasegene blaNPS-1, and a tripartite antibiotic efflux system of theresistance-nodulation-division family. Mol Genet Genomics268:570–584

Taviani E, Ceccarelli D, Lazaro N, Bani S, Cappuccinelli P, ColwellRR, Colombo MM (2008) Environmental Vibrio spp., isolated inMozambique, contain a polymorphic group of integrative con-jugative elements and class 1 integrons. FEMS Microbiol Ecol64:45–54

Tennstedt T, Szczepanowski R, Braun S, Pühler A, Schlüter A (2003)Occurrence of integron-associated resistance gene cassetteslocated on antibiotic resistance plasmids isolated from awastewater treatment plant. FEMS Microbiol Ecol 45:239–252

Tennstedt T, Szczepanowski R, Krahn I, Pühler A, Schlüter A (2005)Sequence of the 68,869 bp IncP-1a plasmid pTB11 from a waste-water treatment plant reveals a highly conserved backbone, aTn402-like integron and other transposable elements. Plasmid53:218–238

Thompson SA, Maani EV, Lindell AH, King CJ, McArthur JV (2007)Novel tetracycline resistance determinant isolated from anenvironmental strain of Serratia marcescens. Appl EnvironMicrobiol 73:2199–2206

Vakulenko SB, Mobashery S (2003) Versatility of aminoglycosidesand prospects for their future. Clin Microbiol Rev 16:430–450

Volkmann H, Schwartz T, Bischoff P, Kirchen S, Obst U (2004)Detection of clinically relevant antibiotic-resistance genes inmunicipal wastewater using real-time PCR (TaqMan). J Micro-biol Methods 56:277–286

Walsh C (2000) Molecular mechanisms that confer antibacterial drugresistance. Nature 406:775–781

Walsh TR, Howe RA (2002) The prevalence and mechanisms ofvancomycin resistance in Staphylococcus aureus. Annu RevMicrobiol 56:657–675

Weldhagen GF (2004) Integrons and beta-lactamases-a novel perspec-tive on resistance. Int J Antimicrob Agents 23:556–562

Werner G, Bartel M, Wellinghausen N, Essig A, Klare I, Witte W,Poppert S (2007) Detection of mutations conferring resistance tolinezolid in Enterococcus spp. by fluorescence in situ hybridiza-tion. J Clin Microbiol 45:3421–3423

Wright GD (2005) Bacterial resistance to antibiotics: enzymaticdegradation and modification. Adv Drug Deliv Rev 57:1451–1470

Wright GD (2007) The antibiotic resistome: the nexus of chemical andgenetic diversity. Nat Rev Microbiol 5:175–186

Wright MS, Peltier GL, Stepanauskas R, McArthur JV (2006)Bacterial tolerances to metals and antibiotics in metal-contami-nated and reference streams. FEMS Microbiol Ecol 58:293–302

Wright MS, Baker-Austin C, Lindell AH, Stepanauskas R, Stikes HW,McArthur JV (2008) Influence of industrial contamination onmobile genetic elements: class 1 integron abundance and genecassette structure in aquatic bacterial communities. ISME J2:417–428

Yang S, Carlson K (2003) Evolution of antibiotic occurrence in a riverthrough pristine, urban and agricultural landscapes. Water Res37:4645–4656

Yu ZT, Michel FC Jr, Hansen G, Wittum T, Morrison M (2005)Development and application of real-time PCR assays forquantification of genes encoding tetracycline resistance. ApplEnviron Microbiol 71:6926–6933

Zhang T, Fang HHP (2006) Applications of real-time polymerasechain reaction for quantification of microorganisms in environ-mental samples. Appl Microbiol Biotechnol 70:281–289

Zhou QX, Luo Y, Wang ME (2007) Environmental residues andecotoxicity of antibiotics and their resistance gene pollution: areview. Asian J Ecotoxicol 2:243–251

Zhu B (2007) Abundance dynamics and sequence variation ofneomycin phosphotransferase gene (nptII) homologs in riverwater. Aquat Microb Ecol 48:131–140

Zhu LX, Wang D, Zhang GB, Jiang D, Zhang ZW, Zhang Q,Mitchelson K, Cheng J (2007a) Development of a base stackinghybridization-based microarray method for rapid identification ofclinical isolates. Diagn Microbiol Infect Dis 59:149–156

Zhu LX, Zhang ZW, Wang C, Yang HW, Jiang D, Zhang Q,Mitchelson K, Cheng J (2007b) Use of a DNA microarray forsimultaneous detection of antibiotic resistance genes amongstaphylococcal clinical isolates. J Clin Microbiol 45:3514–3521


antibiotic resistance genes in water

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environmental args

occurrence of args

emerging of args

horizontal transfer

use of antibiotics

hundreds of various

surface water

majority of antibiotics