Antibiotic Resistance Genes in Water

Appl Microbiol Biotechnol (2009) 82:397414 DOI 10.
1007/s00253-008-1829-z
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 development of antibiotic resistance genes (ARGs) and bacteria which shade health risks to humans and animals. The emerging of ARGs in the water environment is becoming an increasing worldwide concern. Hundreds of various ARGs encoding resistance to a broad range of antibiotics have been found in microorganisms distributed not only in hospital wastewaters and animal production wastewaters, but also in sewage, wastewater treatment plants, surface water, groundwater, and even in drinking water. This review summarizes recently published information on the types, distributions, and horizontal transfer of ARGs in various aquatic environments, as well as the molecular methods used to detect environmental ARGs, including specific and multiplex PCR (polymerase chain reaction), real-time PCR, DNA sequencing, and hybridization based techniques. Keywords Antibiotic resistance gene . Environmental pollution . Gene transfer . Molecular detection method . Water environment
Introduction Antibiotics are widely used to protect the health of human and animals or to increase growth rate of animals as food additive. The majority of antibiotics are excreted unchanged into the environment. Thus, concerns about the potential impact of antibiotic residues in the aquatic environment keep growing in recent years (Sarmah et al. 2006; Wright 2007; Kemper 2008). In surface water, it is difficult to find an area where antibiotics cannot be detected, except for the pristine site in the mountains before the rivers or streams going through urban or agricultural areas (Yang and Carlson 2003). Some antibiotics can be found even in groundwater as deep as more than 10 m (Batt et al. 2006). Apart from chemical pollution caused by antibiotics themselves, the use of antibiotics may also accelerate the development of antibiotic resistance genes (ARGs) and bacteria, which shade health risks to humans and animals (Kemper 2008). These bacteria might be transmitted from environment to human via direct or indirect contact (Iversen et al. 2004; Kim et al. 2005; Rodrguez et al. 2006). Considering the growing evidences that clinical resistance is intimately associated with environmental ARGs and bacteria (Tatavarthy et al. 2006; Prabhu et al. 2007; Abriouel et al. 2008), it is quite clear that the research activities need to be expended to include nonpathogenic or environmental microorganisms. Currently, there are a number of publications relating to the occurrence of ARGs in different water environments, but few reviews have been done. This paper presents an overview of the latest information available in the literature on the types, distributions, and horizontal transfer of ARGs in various aquatic environments, as well as the molecular methods used to detect environmental ARGs.
X.-X. Zhang : T. Zhang (*) : H. H. P. Fang Environmental Biotechnology Lab, Department of Civil Engineering, The University of Hong Kong, Pokfulam Road, Hong Kong, SAR, China e-mail: zhangt@hkucc.hku.hk X.-X. Zhang Department of Environmental Science, Nanjing University, Nanjing, China
398
Appl Microbiol Biotechnol (2009) 82:397414
Types of environmental ARGs Applications of antibiotics in human, veterinary medicine, and agriculture for nearly 60 years have exerted a major impact on bacterial communities, resulting in various resistances to the
Table 1 Tetracycline resistance genes in water environments Gene Biological source
antibiotics, which is genetically controlled by ARGs. The use of antibiotics results in hundreds of ARGs being detected in various water environments (Tables 1, 2, 3, 4, and 5). These environmental ARGs are mainly created by the following mechanisms: (1) target bypass (dfrA1, A5, A7, A12, A15,
Environmental sourcea
Reference
Tetracycline efflux protein tetA Aeromonas, Alcaligenes, Arthrobacter, Comamonas, Escherichia, Listeria, Pseudomonas, Salmonella, and Vibrio; Plasmids pB10, pTB11 and pRSB101 tetA(41) tetB Serratia Afipia, Alcaligenes, Arthrobacter, Burkholderia, Escherichia, Pseudomonas, Serratia, Staphylococcus, and Vibrio Aeromonas, Alcaligenes, Arthrobacter, Brevibacterium, and Pseudomonas Aeromonas, Escherichia; microbial community Aeromonas, Pseudoalteromonas, and Vibrio
AS, DW, EW, NW, SD, SW, US
NW AS, DW, EW, NW, SW, US
tetC tetD tetE tetG tetH
AS, EW, SW, US AS, DW, EW, SW, US AS, EW, SD, SW, US AS, EW, SW, US SW SW SW SW
Pseudomonas; microbial community Aeromonas, Flavobacterium, Proteus, Pseudomona, Staphylococcus, and Wautersiella tetJ Pseudomonas tetY Acidiovorax, Acinetobacter, Comamonas, and Proteus tetZ Actinomycetales, Afipia, Brevibacterium, Burkholderia, Dietzia, Leucobacter, and Microbacterium Alcaligenes, Arthrobacter, and Pseudomonas tet33 tet39 Acinetobacter otrB Streptomycete Ribosomal protection protein tetB(P) Microbial community tetM Aeromonas, Bacillus, Escherichia, Lactococcus, Pseudoalteromonas, and Vibrio; microbial community tetO Paenibacillus, Pseudoalteromonas, Shewanella, Sporosarcina, and Vibrio; microbial community Microbial community Lactococcus and Vibrio; microbial community Microbial community Microbial community Streptomycete; microbial community
Szczepanowski et al. 2004; Agers and Sandvang 2005; Srinivasan et al. 2005; Tennstedt et al. 2005; Poppe et al. 2006; Rodrguez et al. 2006; Cernat et al. 2007; Dang et al. 2007; Macauley et al. 2007; Hu et al. 2008 Thompson et al. 2007 Agers and Sandvang 2005; Cernat et al. 2007; Dang et al. 2007; Jacobs and Chenia 2007; Kim et al. 2007; Kobashi et al. 2007, Macauley et al. 2007 Agers and Sandvang 2005; Akinbowale et al. 2007a; Macauley et al. 2007 Schmidt et al. 2001; Auerbach et al. 2007; Cernat et al. 2007 Schmidt et al. 2001; Dang et al. 2006; Agers and Petersen 2007 Auerbach et al. 2007; Macauley et al. 2007 Jacobs and Chenia 2007; Macauley et al. 2007 Macauley et al. 2007 Macauley et al. 2007 Kobashi et al. 2007; Macauley et al. 2007
SW SD, SW AS, NW, SW SD, SW AS, EW, NW, SD, SW, US
Agers and Sandvang 2005 Agers and Petersen 2007 Nikolakopoulou et al. 2005 Chee-Sanford et al. 2001; Pei et al. 2006 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 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 Smith et al. 2004; Auerbach et al. 2007; Mackie et al. 2006 Chee-Sanford et al. 2001; Kim et al. 2004; Auerbach et al. 2007; Suzuki et al. 2008 Chee-Sanford et al. 2001; Pei et al. 2006 Chee-Sanford et al. 2001; Mackie et al. 2006; Pei et al. 2006; Suzuki et al. 2008 Chee-Sanford et al. 2001; Nikolakopoulou et al. 2005
AS, EW, NW, SD, SW, US AS, EW, NW, SW, US AS, EW, SD, SW, US SD, SW SD, NW, SW AS, NW, SW
tetQ tetS tetT tetW otrA

a
The antibiotic resistance genes were detected in the following water environments: SW special wastewater from hospital, animal production, and aquaculture area; US untreated sewage; AS activated sludge of sewage treatment plant; EW effluent water of sewage treatment plant; NW natural water; SD sediments; and DW drinking water
Appl Microbiol Biotechnol (2009) 82:397414 Table 2 Aminoglycoside resistance genes in water environments Gene Biological source Environmental Function sourcea AS, NW AS NW, SW, US NW, SW, US NW, SW, US NW, SW, US AS, EW, NW, SW, US Aminoglycoside-3adenylyltransferase Aminoglycoside-6-Nacetyltransferase Reference
399
aacA4 aacA29b aacC1 aacC2 aacC3 aacC4 aadA1
Plasmid pTB11 Plasmid pTB11 Microbial communities Microbial communities Microbial communities Microbial communities Aeromonas, Citrobacter and Shigella; Plasmid pTB11
Tennstedt et al. 2005; Mukherjee and Chakraborty 2006 Tennstedt et al. 2003 Lee et al. 1998; Heuer et al. 2002
Aminoglycoside-3-Nacetyltransferase
aadA2
Aeromonas, Escherichia and Vibrio; AS, NW, SD, Plasmids pB2, pB3 and pTB11 SW, US Plasmid pB8 Escherichia and Vibrio; Plasmid pTB11 Aeromonas; Plasmid pTB11 Aeromonas Salmonella Escherichia Microbial communities Mycobacterium Microbial communities Aeromonas and Escherichia Aeromonas and Escherichia Listeria, Salmonella and Vibrio; Plasmids pB4 and pB10 Salmonella and Vibrio; Plasmids pB4 and pB10 AS AS, NW SW AS DW, NW DW NW, SW, US NW NW, SW NW, SW AS, NW, SW
aadA4 aadA5 aadA13 aadB aphA1 aphA2 aphD aph(3)-Ic nptII sat1 sat2 strA
Aminoglycoside-2adenylyltransferase Aminoglycoside phosphoryltransferase
Tennstedt et al. 2003; Henriques et al. 2006a; Mukherjee and Chakraborty 2006; Moura et al. 2007 Dalsgaard et al. 2000; Tennstedt et al. 2003; Heuer et al. 2004; Taviani et al. 2008 Schlter et al. 2005 Park et al. 2003; Tennstedt et al. 2003; Mohapatra et al. 2008 Moura et al. 2007 Tennstedt et al. 2003 Cernat et al. 2007; Poppe et al. 2006
strB
a
AS, NW
Cernat et al. 2007 Heuer et al. 2002 Ramn-Garca et al. 2006 Neomycin phosphotransferase Zhu 2007 Streptothricin acetyltransferase Henriques et al. 2006a; Moura et al. 2007 Henriques et al. 2006a; Moura et al., 2007 Streptothricin Tauch et al. 2003; Poppe et al. 2006; phosphoryltransferase Jacobs and Chenia 2007; Mohapatra et al. 2008 Tauch et al. 2003; Poppe et al. 2006; Mohapatra et al. 2008
The abbreviations of environmental sources are the same as those in Table 1
A17, and 18; sulI, II, III, and A), inaccessibility of the antibiotics to their target enzyme by mutational changes or loss 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 of intracellular concentrations of antibiotics by structural alteration 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 antibiotic molecule (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 of antibiotics (Lambert 2005). It is noteworthy that the resistance of certain antibiotic may be associated with different ARGs based on more than one mechanism.
ARGs related to tetracycline Tetracycline-resistant bacteria were found to emerge in the environments with the introduction of tetracycline (Dancer et al. 1997). There have been at least 38 different tetracycline resistance (tet) genes and three oxytetracycline resistance (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 modification mechanism), and three genes for an inactivating enzyme and one gene with unknown resistance mechanism (Levy et al. 1999; Roberts 2005). Among them, more than 22 tet or otr genes have been found in bacterial isolates from water environments (Table 1). Most environmental tet genes code for transport proteins, which pump the antibiotics out of the bacteria
400 Table 3 Macrolide, chloramphenicol, and vancomycin resistance genes in water environments Gene Biological source Environmental sourcea Function
Reference
Macrolide resistance genes ermA Enterococcus ermB Bacillus and Enterococcus ermC Microbial community ermE Microbial community ermF Microbial community ermT Microbial community ermV Microbial community ermX Microbial community mphA Plasmid pRSB101 Chloramphenicol resistance genes cmlA1 Plasmid pB2 and pB3 cmlA5 Plasmid pTB11 catB2 Plasmid pTB11 catB3 Aeromonas catI Pseudomonas catII Vibrio catIII Pseudomonas catIV Vibrio and Pseudoalteromonas Listeria, Pseudoalteromonas floR Salmonella and Vibrio, Vancomycin resistance genes vanA Enterococcus and Staphylococci vanB
a
EW, EW, EW, SW EW, EW, SW EW, AS
SW SW SW SW SW SW
Erythromycin resistance methylase
Macrolide-2phosphotransferase Chloramphenicol efflux protein
Hayes et al. 2005; Chen et al. 2007 Hayes et al. 2005; Chen et al. 2007 Chen et al. 2007 Patterson et al. 2007 Chen et al. 2007 Chen et al. 2007 Patterson et al. 2007 Chen et al. 2007 Szczepanowski et al. 2004
AS AS AS SW NW SW NW SW DW, NW, SW
Chloramphenicol acetyltransferase
Florfenicol efflux protein Vancomycin resistance protein
Heuer et al. 2004 Tennstedt et al. 2003 Tennstedt et al. 2003 Jacobs and Chenia 2007 Dang et al. 2008 Dang et al. 2007 Dang et al. 2008 Dang et al. 2006 Srinivasan et al. 2005; Poppe et al. 2006; Dang et al. 2007 Schwartz et al. 2003; Volkmann et al. 2004; Messi et al. 2006 Iversen et al. 2002; Caplin et al. 2008
DW, EW, NW, SW, UW EW, NW, UW
Enterococcus
cell and keep the intercellular concentrations low to make ribosomes function normally (Roberts 2002). The efflux genes of tetA, B, C, D, and E frequently appeared in various environmental compartments including activated sludge of sewage treatment plants (STPs) (Guillaume et al. 2000), fish farming ponds (Schmidt et al. 2001; Dang et al. 2007), surface water (Poppe et al. 2006), and swine lagoon (Macauley et al. 2007). Recently, the tetracycline resistance genes including tetM, O, S, Q, and W, coding for ribosomal protection proteins, have also been detected in microbial communities of sewage treatment systems (Auerbach et al. 2007), hospital or animal production wastewaters (Kim et al. 2007; Nonaka et al. 2007), and even in natural water environments (Mackie et al. 2006). Many tet genes are located on nonmobile plasmids or incomplete 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 several environmental genera including Gram-negative and Grampositive species (Table 1). Recently, Agers and Petersen (2007) have found that tetE is often located on large horizontally transferable plasmids of Aeromonas spp. isolated from pond water of fish farm, and the gene has been
proved to be capable of interspecies transfer to Escherichia coli. tetA, D, and M can also be transferred horizontally by oxytetracycline resistance plasmid from environmental microorganisms to E. coli strains isolated from chicken, pig, and human, which indicates the potential environmental hazards caused by the tet ARGs (Akinbowale et al. 2007b). ARGs related to aminoglycoside Different from tetracycline resistance mechanisms mentioned above, the most major mechanism of aminoglycoside resistance is direct deactivation of this type of antibiotics by enzymatic modification (Shakil et al. 2008). More than 50 modification enzymes have been found so far (Vakulenko and Mobashery 2003; Ramn-Garca et al. 2006). These enzymes are divided into three groups based upon their biochemical actions on the aminoglycoside substrates, including acetyltransferases, phosphotransferases, and nucleotidyltransferases (adenylyltransferases), encoded by three types of genes, namely, aac, aph, and ant (aad), respectively. Different aminoglycoside-modifying enzymes have been reported in a broad range of bacteria isolated from patients or clinical environments (Filipova et al. 2006; Kelmani Chandrakanth et al. 2008).
Appl Microbiol Biotechnol (2009) 82:397414 Table 4 Sulphonamide and trimethoprim resistance genes in water environments Gene Biological source Environmental sourcea Reference
401
Dihydrofolate reductase encoding genes dfrA1 Aeromonas, Escherichia, and Salmonella dfrA5 Escherichia dfrA7 Escherichia dfrA12 Aeromonas, Escherichia, and Salmonella dfrA15 Vibrio dfrA17 Escherichia, Salmonella dfr18 Vibrio Dihydropteroate synthase encoding genes sulI Aeromonas, Escherichia, and Listeria; Plasmids pB2, pB3, pB8, and pB10; Microbial community sulII Acinetobacter, Escherichia, Salmonella, and Vibrio; Microbial community sulIII Escherichia; Microbial community sulA Microbial community
a
NW, SW NW NW DW, NW, SW EW, NW DW, NW NW AS, DW, NW, SD, SW
Henriques et al. 2006a; Mukherjee and Chakraborty 2006; Moura et al. 2007 Park et al. 2003; Mukherjee and Chakraborty 2006 Park et al. 2003 Moura et al. 2007; Antunes et al. 2006; Cernat et al. 2007 Park et al. 2003; Taviani et al. 2008 Park et al. 2003; Antunes et al. 2006; Cernat et al. 2007 Mohapatra et al. 2008 Heuer et al. 2004; Lin and Biyela 2005; Schlter et al. 2005; Srinivasan et al. 2005; Akinbowale et al. 2007a; Cernat et al. 2007; Hu et al. 2008 Pei et al. 2006; Agers and Petersen 2007; Cernat et al. 2007; Hu et al. 2008; Mohapatra et al. 2008; Pei et al. 2006; Hu et al. 2008 Pei et al. 2006
DW, NW, SD, SW NW, SD SD
The abbreviations of environmental sources are the same as those in Table 1.
The aac, aph, and ant genes are widely distributed in various genera including Aeromonas, Escherichia, Vibrio, Salmonella, and Listeria spp. isolated from polluted or natural water environments (Table 2). The genes of aacC1, C2, C3, and C4, encoding aminoglycoside-3-N-acetyltransferase, were often detected in microbial communities or isolates from STPs (Heuer et al. 2002; Tennstedt et al. 2003, 2005), and the two adenylyltransferase genes, aadA1 and aadA2, were frequently reported all around the world in the isolates from aquaculture areas (Dalsgaard et al. 2000), river water (Park et al. 2003), STPs (Szczepanowski et al. 2004; da Silva et al. 2007), and surface urban water (Taviani et al. 2008). ARGs encoding resistances to other antibiotics in aminoglycoside group, for example, phosphotransferase genes encoding resistance to neomycin (nptII) and streptothricin (strAB), have also been detected in the river water of Canada (Zhu 2007) and Ganges river of India (Mohapatra et al. 2008). ARGs related to macrolidelincosamidestreptogramin, chloramphenicol, and vancomycin Although structurally unrelated to each other, the three antibiotics, macrolides, lincosamide, and streptogramin, are often investigated simultaneously for microbial resistance, since some macrolide resistance genes (erm) encode resistance to two or all three of these compounds (Roberts et al. 1999). Totally, more than 60 different genes confer resistance to one or more of the macrolidelincosamide streptogramin (MLS) antibiotics have been identified (Roberts 2008), including the genes associated with
ribosomal RNA (rRNA) methylation, efflux, and inactivation. MLS resistance is mostly mediated by rRNA methylases (encoded by erm genes), which methylate the adenine residues to prevent the three antimicrobials from binding to ribosomal protein (Roberts 2002; Cetin et al. 2008). The erm genes can easily be transferred from one host to another (Roberts 2003), since they are usually acquired and associated with mobile elements, such as plasmids (Liu et al. 2007) and transposons (Okitsu et al. 2005). Several erm genes have been detected in Enterococcus spp. isolated from poultry raising wastewaters (Hayes et al. 2005) and environmental DNA extracted from livestock manures (Chen et al. 2007; Table 3). Six classes of erm genes (A, B, C, F, T, and X) have been detected and quantified in the samples from animal production matures, lagoons, and a biofilter system treating hog house effluents (Chen et al. 2007). Among the macrolide resistance determinants, ermB is considered as the most prevalent gene in environmental microorganisms, especially in the strains of Enterococcus (Hayes et al. 2005) and Streptococcus spp. (Jensen et al. 2002). The mechanisms responsible for resistances to chloramphenicol and florfenicol include chloramphenicol acetyltransferases (encoded by cat genes), specific exporters (encoded by cml genes), and multidrug transporters (Schwarz et al. 2004). Of the chloramphenicol resistance genes known to date, several types of cat or cml genes have been reported to be of environmental origin (Table 3). Vancomycin resistance firstly emerged in enterococci, and recently, the resistance has also been detected in Staphylococcus aureus (Walsh and Howe 2002). So far, six types of vancomycin resistance
402 Table 5 -Lactam and penicillin resistance genes in water environments Gene ampC blaPSE-1 blaTEM-1 blaOXA-1 blaOXA-2 blaOXA10
Biological source Enterobacter, Salmonella
Environmental sourcea Function DW, NW, SW, US AmpC type -lactamase PSE-1-lactamase
Reference
Aeromonas, Salmonella and Vibrio EW, SD, SW, US Escherichia Plasmid pTB11 Aeromonas; Plasmids pB8, pB10 and pTB11 Plasmid pTB11 Salmonella Staphylococcus Listeria DW AS AS, EW, SW AS SW DW, NW, US DW, SW
Schwartz et al. 2003; Volkmann et al. 2004; Poppe et al. 2006 Dalsgaard et al. 2000; Jacobs and Chenia 2007; Taviani et al. 2008 TEM-type -lactamase Alpay-Karaoglu et al. 2007; Cernat et al. 2007 OXA-1 -lactamase Tennstedt et al. 2003 OXA-2 -lactamase Schlter et al. 2005; Tennstedt et al. 2005; Jacobs and Chenia 2007 OXA-10 -lactamase Tennstedt et al. 2003 OXA-30 -lactamase Penicillin-binding protein Antunes et al. 2006; Moura et al. 2007 Schwartz et al. 2003; Volkmann et al. 2004 Srinivasan et al. 2005
blaOXA30
mecA penA
a
genes (van) have been known (Messi et al. 2006), and vanA and vanB are the most prevalent ones in water environments (Table 3). ARGs related to sulfonamides and trimethoprim Sulfonamides are the first antibiotic developed for largescale introduction into clinical use, which target dihydropteroate synthase (DHPS). Trimethoprim competitively inhibits dihydrofolate reductase (DHFR), which is responsible for the reduction of dihydrofolate to tetrahydrofolate (Alekshun and Levy 2007). The two types of bio-enzymes are partly responsible for folate bio-synthesis, which is associated with thymine production and microbial growth (Nrochet et al. 2008). Resistances to sulfonamides and trimethoprim are often encoded by mutations located on highly conserved areas of DHPS genes (sul) and DHFR genes (dfr) (Skld 2000, 2001). Different types of mechanisms have been found to confer to sulfonamide resistance, mostly based on changes in the sul genes and mediation by mobile elements (Huovinen et al. 1995; Antunes et al. 2007). The most widespread trimethoprim resistance mechanism is the replacement of a trimethoprim-sensitive DHFR by a plasmid-, transposon-, or cassette-borne trimethoprim-resistant DHFR (Skld 2001; Blahna et al. 2006). Four kinds of sul genes (sulI, II, III, and A) have been found in the bacteria of environmental origin (Table 4). sulI and II have been detected in bacterial isolates from fecal slurry of dairy farms (Srinivasan et al. 2005), water or sediments of aquaculture areas (Akinbowale et al. 2007a; Agers and Petersen 2007), and even from the river or sea water without evidence of being polluted (Lin and Biyela 2005; Hu et al. 2008; Mohapatra et al. 2008). sulI, as a part
of class 1 integron, can be disseminated and transferred horizontally within and between bacterial species in wastewater (Tennstedt et al. 2003), river water (Mukherjee and 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; Didi et al. 2008), and several dfrA genes are commonly found in various environmental isolates (Table 4). The environmental habitats of these genes include urban wastewater treatment plants (da Silva et al. 2007), slaughterhouse wastewater treatment 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 located on class 2 integrons (Blahna et al. 2006), and dfr gene cassettes are frequently found in the variable regions of integrons and are often the only gene cassettes present in environmental isolates (Antunes et al. 2006; Mukherjee and Chakraborty 2006). ARGs related to -lactam -Lactams are the most widely used antibiotics, and resistance to these antibiotics is a severe threat because they have low toxicity and are used to treat a broad range of infections (Livermore 1996). The mechanisms of -lactam resistance include inaccessibility of the antibiotics to their target enzymes, modifications of target enzymes, and/or direct deactivation of the antibiotics by -lactamases (Walsh 2000; Li et al. 2007). In Gram-negative bacteria, the primary resistance mechanism is enzymatic inactivation through the cleavage of the -lactam ring by -lactamases. More than 400
403
different -lactamases encoded by hundreds of ARGs (bla) have been identified, and the enzymes are divided into four molecular classes, AD, mediating resistances to a broad range of -lactams including penicillins and cephalosporins (Li et al. 2007). A variety of bla genes (Table 5) have been identified in bacteria derived from fecal slurry and lagoon water of dairy farms (Srinivasan et al. 2005), water or sediments of aquaculture areas (Dalsgaard et al. 2000; Jacobs and Chenia 2007), STPs (Szczepanowski et al. 2004; Volkmann et al. 2004; Antunes et al. 2006; Taviani et al. 2008), and surface water (Schwartz et al. 2003; Poppe et al. 2006). The environmental compartments may further serve as reservoirs for -lactam resistance genes. The bla genes are often detected in animal-derived environmental pathogens including Aeromonas (Tennstedt et al. 2005; Jacobs and Chenia 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 -lactamases has been detected in the microbial isolates from wastewater, surface water, and even from drinking water films (Schwartz et al. 2003). mecA gene encoding methicillin resistance in staphylocci was observed to be prevalent in hospital wastewater biofilms (Schwartz et al. 2003). bla genes often coexist with other antimicrobial resistance determinants and can also be associated with mobile genetic elements, increasing the possibility of multidrug resistance and environmental dissemination (Tennstedt et al. 2003; Weldhagen 2004; Schlter et al. 2007b). The plasmids containing bla obtained from a wastewater treatment plant are frequently associated with transposons and integrons and often simultaneously carry other resistance determinants including aad (or aac) encoding aminoglycoside nucleotidyltransferase (or acetyltransferase), cml encoding chloramphenicol efflux protein, and cat encoding chloramphenicol acetyltransferase (Tennstedt et al. 2003).
DNA hybridization Molecular hybridization has been used to detect presence/ absence of specific ARGs for nearly 30 years (Mendez et al. 1980). Many improvements have been made on molecular hybridization, especially in probe design and synthesis, so that the technique, especially Southern blot, is still often applied to distinguish different ARGs in one group (i.e., tet genes) from each other (Roberts and Kenny 1986; Levy et al. 1999) or to identify presence of specific genes in certain environment (Agers and Petersen 2007; Malik et al. 2008). Southern hybridization and filter-mating experiments demonstrated that tet and class 1 integrons can be co-transferred from soil isolates to E. coli and/or Pseudomonas putida (Agers and Sandvang 2005). Using Southern blot or dot blot coupled with PCR method, Malik et al. (2008) found that ampC was frequently present in soil samples irrigated with wastewater. PCR-Southern blot assays showed that tet39 and sulII were common resistance genes in Acinetobacter spp. isolates from water and sediments of fish farms. With a number of nonradiolabeled systems becoming commercially available, radioactive labeling is no longer an option to label probes. As an important nonradiolabeled method, fluorescence in situ hybridization (FISH) has been established and implemented successfully for clinical detection of microbial resistances. The use of FISH technique has been described for the rapid identification of macrolide resistances caused by ribosomal mutations (Russmann et al. 2001). Recently, Werner et al. (2007) has performed a research to evaluate the reliability of FISH for clinical detection of linezolid-resistant enterococci and found that the FISH technique along with DNA probes containing locked nucleic acids with point mutation showed 100% sensitivity for the detection of phenotypic linezolid resistance and even allowed detection of a single mutated 23S rRNA gene allele in phenotypically linezolid-susceptible enterococci. Moosavian et al. (2007) developed and validated the FISH method for rapid detection of clarithromycin-resistant Helicobacter pylori in patients. Although FISH has been often used for clinical detection of antibiotic resistance, so far, few reports have been found about its use in identification of target bacteria harboring ARGs in environmental samples. PCR (simple and multiplex PCR) PCR assays have been widely used in both pure cultures and mixed environmental samples for detection of specific ARGs encoding resistances to aminoglycoside (Mohapatra et al. 2008; Taviani et al. 2008), chloramphenicol (Dang et al. 2008), -lactam (Taviani et al. 2008), macrolide (Chen et al. 2007; Patterson et al. 2007), penicillin
Molecular techniques for the detection and characterization of environmental ARGs Considering that ARGs are widespread in aquatic environments mentioned above, there is a need for the development and application of molecular methods to investigate the occurrence, transport, and fate of the environmental ARGs. So far, the methods used for detection, typing, and characterization of ARGs have covered, but not been limited to, specific and multiplex polymerase chain reaction (PCR), real-time PCR, DNA sequencing, and hybridization-based techniques including microarray.
404
(Srinivasan et al. 2005), sulphonamide (Agers and Petersen 2007), tetracycline (Jacobs and Chenia 2007), trimethoprim (Moura et al. 2007), and vancomycin (Caplin et al. 2008). Environmental target DNA or RNA at low concentrations can be amplified and detected by PCR-based methods. However, a false-positive result is often given in the PCR assay. Southern hybridization of PCR products labeled and used as DNA probes to plasmid or chromosome DNA samples from strains harboring target genes can avoid the false-positive PCR results (Ahmed et al. 2006; Akinbowale et al. 2007b). In addition to DNA hybridization, DNA sequencing is another common method used to verify the PCR products of certain ARGs (Thompson et al. 2007). In order to save time and effort, multiplex PCR methods have been developed and often used for simultaneous detection of more than one environmental ARG, including the genes encoding resistances to vancomycin (Bell et al. 1998), macrolide (Jensen et al. 2002), tetracycline (Ardic et al. 2005; Agers et al. 2007), sulfamethoxazole, and trimethoprim (Ramachandran et al. 2007). With various primer pairs in the same PCR reaction system, multiplex PCR can amplify the DNA fragments of several ARGs at the same time (Gilbride et al. 2006). The method saves considerable time and cost when different target regions are investigated simultaneously, but as a result of all the reactions taking place at the same conditions, some DNA amplifications can be inhibited and false-negative results are probably obtained. Another disadvantage of multiple PCR is that the dimer formation between primer pairs can disturb experimental results and lead to poor sensitivity (Markoulatos et al. 2002). Despite of the drawbacks mentioned above, multiplex PCR is still considered as a rapid and convenient method for the detection of multiple ARGs in isolated bacteria or environmental DNA (Agers et al. 2007; Gilbride et al. 2006). Quantitative PCR The quantitative real-time PCR (qRT-PCR) is usually used to quantify target DNA on the basis of the principle that initial concentration can be estimated according to the change of PCR product concentration with amplification cycles (Zhang and Fang 2006). Among the several fluorescent reagents developed for qRT-PCR, SYBR Green is the most common method used to quantify ARGs in bacterial isolates of clinical origin, including tet (Morsczeck et al. 2004), mef, and erm genes (Reinert et al. 2004). Recently, the technique has been frequently used to quantify ARGs in environmental samples, including tet genes in beef cattle farms (Yu et al. 2005), groundwater (Mackie et al. 2006), river sediments (Pei et al. 2006), and STPs (Auerbach et al. 2007), as well as sul genes in river sediments (Pei et al. 2006) and npt genes in river water (Zhu 2007).
TaqMan probe, another fluorescent reagent, has also been used to quantify tetO, tetW, and tetQ (Smith et al. 2004), as well as vanA, mecA, and ampC genes (Volkmann et al. 2004) in wastewater. Chen et al. (2007) validated TaqMan method for quantifying erm genes conferring resistance to MLS in the environmental samples from animal production areas. The qRT-PCR method is not only usually used for quantitative analysis on ARGs distribution in the environments but is also often applied to study the effects of environmental factors or treatment processes on removal of some ARGs, i.e., tet genes (Mackie et al. 2006; Auerbach et al. 2007), sul genes (Pei et al. 2006), and erm genes (Chen et al. 2007). Using qRT-PCR, Auerbach et al. (2007) investigated tet genes in Germany STPs and found that tetQ concentrations were highest in influent water while tetG concentrations were highest in activated sludge, and UV disinfection had no effects on reduction in the amount of detectable tet genes in wastewater effluent. In order to analyze the effect of river landscape on distributions of ARGs in sediments, some tet and sul genes in the sediments have been quantified using qRT-PCR, and higher resistance gene concentrations have been obtained at the impacted sites than at the pristine site (Pei et al. 2006). Using real-time PCR, Mackie et al. (2006) found that detection frequency of tetM, O, Q, and W genes was much higher in wells located closer to and down-gradient from swine lagoons than in wells more distant from the lagoons. With qRT-PCR, Chen et al. (2007) found that erm abundances in composted swine manure samples were significantly lower than those in swine manure, demonstrating that manure storage probably influences the persistence of the environmental genes. DNA Microarray Compared with other molecular methods, DNA microarray technique is a genomic analysis technique with highthroughput, high-speed and high-delicacy. For detection of antibiotic resistances, DNA microarray can provide detailed, clinically relevant information on the isolates by detecting the presence or absence of a large number of ARGs simultaneously in a single assay (Gilbride et al. 2006). Microarray allows detection of antibiotic resistance determinants within several hours and can be used as a time-saving and convenient tool supporting conventional resistance detection assays (Antwerpen et al. 2007). Microarray has been widely used to clinically detect antibiotic resistance of human pathogens E. coli (Zhu et al. 2007a), H. pylori (Chen et 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 genotypic resistance mechanisms of certain antibiotics (Chen et al. 2008b).
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Although microarrays have been successfully used to assess the antibiotic resistances of clinical samples, few reports are found about using this technique to detect the ARGs in environmental samples. The first factor hampering its application in environmental samples is the low detection limit of the method, but microarray coupled with PCR method can enhance the detection limit for environmental ARGs (Gilbride et al. 2006). Patterson et al. (2007) developed a microarray system based on PCR amplification of 23 tet genes and 10 erm genes to screen environmental samples for the presence of these ARGs and found that tetW, O, and Q were the most abundant ARGs found in swine fecal samples and ermV, and E were the most common ones detected in farm and garden soil samples. Another reason for the poor applications of microarray in most environmental samples is the complexity of the samples and pretreatment. The presence of undesirable contaminants in environmental samples inhibits DNA extraction and/or target gene amplification, so the complicated pretreatment of environmental samples is necessary and crucial to get satisfactory detection results (Call 2005). Microarray technique can provide a detailed description of bacterial antibiotic resistance and can reveal global changes in ARG expression in response to environmental changes (Call et al. 2003; Gilbride et al. 2006). The information on gene expression provides insight into antibiotic resistance mechanisms and general genetic responses of ARGs to environment-related changes.
Geographical distribution of studying ARGs in water environment The geographical distribution of environmental ARGs has been indicated in the studies and detections on the genes all around the world (Fig. 1). In Europe, nearly all types of ARGs were frequently detected in aquatic environments of 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; Nikolakopoulou et al. 2005), Denmark (Schmidt et al. 2001; Agers and Sandvang 2005), and Greece (Heuer et al. 2002). Various water bodies in Europe have been found to contain some common ARGs, for example, vancomycin resistance genes van, which have been detected in dairy farm water of Italy (Messi et al. 2006), human-derived wastewater of England (Caplin et al. 2008), urban raw sewage, treated sewage and surface water of Sweden (Iversen et al. 2002), municipal wastewater, surface water, and drinking water biofilms of Germany (Schwartz et al. 2003; Volkmann et al. 2004).
In Northern America, tetracycline resistance genes were frequently detected in water environments, including lagoon water (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). Other environmental ARGs have also been found in the continent, encoding a wide resistance to aminoglycoside (Zhu 2007), -lactam (Srinivasan et al. 2005), chloramphenicol (Poppe et al. 2006), macrolide (Chen et al. 2007), and sulfonamide (Pei et al. 2006). However, environmental distribution of ARGs has seldom been reported in Southern America. In Asia, about 10 years ago, researchers began investigating 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 of Asian rivers, and tet genes were also found in marine aquatic 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 et al. 2008). Recently, several tet and sul genes have also been found in natural river basin of China (Hu et al. 2008). In India, ARGs occurring in river water confer resistances to aminoglycoside, sulfonamide, and trimethoprim (Mukherjee and Chakraborty 2006; Mohapatra et al. 2008). In Thailand, ARGs related to aminoglycoside and lactam resistances (Dalsgaard et al. 2000), as well as sulfonamide and tetracycline resistances (Agers and Petersen 2007) were detected in the sediments of fish or shrimp production areas. Investigations about environmental ARGs have also been carried out in Africa and Australia. Akinbowale et al. (2007a) found that Aeromonas containing resistance genes and class 1 integrons were present in sediments of fish farms of Australia. Plasmids and integrons carrying a variety of ARGs have been identified in bacteria isolated from South African aquaculture systems in the absence of antibiotic selection pressure (Jacobs and Chenia 2007). Class 1 integrons and integrating conjugative elements conferring resistances to trimethoprim, aminoglycoside, and -lactam were found in Vibrio strains isolated from surface urban water in Mozambique (Taviani et al. 2008).
Habitates of ARGs in water environment ARGs are prevalent in different water bodies, and the spread pathways of ARGs in various aquatic environments usually are complicated. Before learning about the fate and transport of ARGs in the environments, it is necessary to
406 Fig. 1 Detection of the antibiotic resistance genes in geographically isolated water environments, including the genes encoding resistance to aminoglycoside (red square), chloramphenicol (brown inverted triangle), -lactam (plus symbol), macrolide (sky blue triangle), sulfonamide (violet diamond), tetracycline (green circle) and trimethoprim (indigo star)
characterize the occurrence, and the first step in this endeavor is to identify major habitats of the ARGs in the environments. As a result of extensive use of human and veterinary antibiotics, hospital wastewater and livestock manure are considered as the major sources of environmental ARGs. ARGs can enter into aquatic environments by direct discharging of untreated wastewater or into STPs through wastewater collection systems and subsequently into the environments with effluents and discharged sludge (Auerbach et al. 2007). ARGs can be transferred into soils by amending farm land with animal manure and processed biosludge from STPs and then can leach to groundwater or be carried by runoff and erosion to surface water (Yang and Carlson 2003). Surface water and shallow groundwater are commonly used as source of drinking water; thus, ARGs can go though drinking water treatment facilities and enter into water distribution systems (Schwartz et al. 2003). Special wastewater from hospital, animal production, and aquaculture areas The broad use of human, veterinary, and aquaculture antibiotics may exert selective pressure on bacteria in the environments of hospital (Liu et al. 2007), animal production (Agers and Sandvang 2005), and fishery areas (Agers and Petersen 2007), which are thought to be main sources of ARGs distributing into the environments. Among all classes of ARGs, tet genes have the highest detection frequency, and about 20 types of tet genes have been 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 (Akinbowale et 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 (Agers and Sandvang 2005), tet39 (Agers and Petersen 2007), and otrA and B (Nikolakopoulou et al. 2005). Other ARGs frequently detected in these special wastewaters include methicillin resistance gene (mecA) in staphylococci isolated from hospital wastewater biofilms (Schwartz et al. 2003), chloramphenicol resistance genes (catII, IV and B3) in the aquaculture systems (Dang et al. 2006; Dang et al. 2007; Jacobs and Chenia 2007), and sulfonamide resistance genes (sulI, II, III, and A) in fish farms (Agers and Petersen 2007). Additionally, some types of ARGs including floR, penA, and strA (Srinivasan et al. 2005), as well as bla genes (Henriques et al. 2006b), were reported to occur in fecal slurry or lagoon of animal production areas. ARGs in these wastewaters are directly exposed to the environment and can eventually be transported to the nearby streams, rivers, lakes, or other aquatic bodies or leach downward through the soil during rainfall. Untreated sewage During the recent several years, various bacteria species isolated from untreated sewage were found to contain a variety of ARGs encoding resistances to aminoglycoside (da Silva et al. 2007; Taviani et al. 2008), -lactam (Schwartz et al. 2003; Volkmann et al. 2004; Antunes et al. 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 in environmental DNA extracted from municipal wastewater, tet genes (Auerbach et al. 2007) and aminoglycoside resistance genes (aacC1, C2, C3, C4, aadB, and aphD) (Heuer et al. 2002) were also found in sewage wastewaters.
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Sewage receives the bacteria previously exposed to antibiotics from private households and hospitals and is considered as a hotspot for ARGs. ARGs go into STPs with sewage water, and most of them cannot be effectively removed with traditional treatment process before being released into the environments (Volkmann et al. 2004; Auerbach et al. 2007). Moreover, environmental conditions of activated sludge or biofilms facilitate horizontal transfer of the ARGs from one host to another because of the nutritional richness and high bacterial density and diversity (Tennstedt et al. 2003; Schlter et al. 2007b). STP activated sludge or biofilms Several previous studies have shown that STPs serve as important reservoirs for various ARGs (Smalla and Sobecky 2002; Tennstedt et al. 2003; Schlter et al. 2007b). ARGs present in STPs encode a broad resistance to 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 (Bnemann et al. 2006), and -lactam (Szczepanowski et al. 2004; Taviani et al. 2008). STPs receive the antibiotic-resistant bacteria with the inflow sewage water originating from hospitals, private households, industry, and agriculture, so they play important roles in recombination, exchange, and spread of environmental ARGs (Szczepanowski et al. 2004). STPs are recognized as important interfaces between different water bodies, such as hospital wastewater, domestic wastewater, surface water, and groundwater, therefore may facilitate gene exchange and spread between these environmental compartments (Schlter et al. 2007b). Firstly, the presence of antibiotics in sewage selects for the maintenance of ARGs conferring resistance in activated sludge (Kmmerer 2003). Secondly, high microbial density and diversity of biofilms and activated sludge may facilitate genetic exchange in sewage treatment bioreactors (Schlter et al. 2007b); for example, some tet genes preferentially migrate from wastewater to biofilm (Engemann et al. 2008). Additionally, various mobile elements at high density in STPs accelerate gene recombination and transfer that encode new or multiple antimicrobial resistances (Tennstedt et al. 2003; Schlter et al. 2007a). Finally, many ARGs, for example, vanA and B, cannot be effectively removed by activated sludge process widely used in STPs, the genes being found in both influent and effluent water (Iversen et al. 2002; Caplin et al. 2008). ARGs enter into other water bodies with effluent water and can be transferred horizontally to the native bacteria in these aquatic environments (Schwartz et al. 2003).
STP effluent water STP effluent and sludge application to agricultural fields are recognized as important sources of ARGs to surface waters and soils and subsequently into groundwater (Yang and Carlson 2003). Several reports have indicated that bacteria harboring ARGs can be released from STPs into surface waters (Tennstedt et al. 2005; Chen et al. 2007; Auerbach et al. 2007). Some types of ARGs have been detected in STP effluent 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), and bla and dfr genes (Taviani et al. 2008). Some aminoglycoside and -lactam resistance determinants in effluent water are recombined into integrons horizontally transferred by plasmids or transposons (Tennstedt et al. 2005). Resistance determinants in bacteria have been detected from habitats downstream of STPs (da Silva et al. 2007; Taviani et al. 2008), and antibiotic resistance regions can be extended, modified, recombined, and exchanged in and among bacteria residing in these areas (Schlter et al. 2007b). New resistance properties could be horizontally transferred to human pathogens, thus increasing the difficulties of infectious disease treatment and threatening public health (Iversen et al. 2004). Natural water Scores of ARGs have been found in the isolates or microbial communities in the natural waters, which were not or slightly polluted (Jacobs and Chenia 2007; Mohapatra et al. 2008; Rahman et al. 2008). Several types of aminoglycoside resistance genes have been detected in the microorganisms isolated from surface water, including aac (Lee et al. 1998), aad (Park et al. 2003; Mukherjee and Chakraborty 2006), aph (Poppe et al. 2006), npt (Zhu 2007), and str (Mohapatra et al. 2008). The detection frequency 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) (Mukherjee and Chakraborty 2006) in surface water. ampC in surface water biofilms (Schwartz et al. 2003) and bla genes in estuarine water have also been detected (Henriques et al. 2006a). ARGs in surface water and soils can leach to groundwater close to agriculture areas of animal production or aquaculture. Tetracycline resistance genes encoding both ribosomal 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 been detected in the groundwater as far as 250 m downstream from waste lagoons of swine farms. In a recent study, tetM,
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O, Q, and W in wells near swine lagoons were quantified, and the genes were then detected in groundwater downstream from manure lagoons (Mackie et al. 2006). Besides in fresh waters, some ARGs associated with resistances to aminoglycoside (Heuer et al. 2002) and chloramphenicol (Dang et al. 2008) have also been detected in marine waters with no evidence of being polluted. Sediments It is self-evident that ARGs in sediments are acquired from water environments or produced for selection by the antibiotics present in the sediments. Sediments of aquaculture farms are important antibiotic resistance regions where various antimicrobials and ARGs are concentrated (Dalsgaard et al. 2000; Agers and Petersen 2007). Marine sediments perhaps can be considered as natural reservoirs of tetracycline resistance gene tetM, which has been found in various bacterial species in sediments of Tokyo Bay, Sagami Bay, and the open Pacific Ocean (Rahman et al. 2008). It was found that numbers of oxytetracycline-resistant bacteria increased in sediments around a marine aquaculture site after oxytetracycline therapy, and tetM was evident in both Grampositive and Gram-negative bacteria from various genera in the sediments of the marine environment (Nonaka et al. 2007). Various ARGs have also been identified in river sediments. Sulfonamide resistance genes including sulI, II, III, and A were detected in the microorganisms of river water and sediments (Pei et al. 2006). In rivers running through pristine, urban, and agriculturally influenced areas, ARG detection frequency in sediments was enhanced corresponding to the increases in concentrations of various antibiotic compounds (Yang and Carlson 2003; Pei et al. 2006). After a spatial monitoring of environmental bacteria and genes, Suzuki et al. (2008) found that the detection frequency of ribosomal protection protein genes (tetM, S, and W) in sediments of the Mekong River watershed were positively correlated with the occurrence rate of tetracycline-resistant bacteria in the same area. Drinking water Prevalence and resistance patterns of various microbial genera isolated from drinking water distribution system have been recently reported (Koksal et al. 2007; Ram et al. 2008). Multiple-antibiotic-resistant E. coli strains isolated from drinking water was found to carry ARGs encoding resistances to aminoglycoside, -lactam, tetracycline, and trimethoprim-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 from wastewater and surface water to the drinking water distribution network, Schwartz et al. (2003) and Obst et al. (2006) investigated biofilms in hospital and municipal wastewater, as well as drinking water from river bank filtrate, and found that vanA and ampC genes occurred not only in wastewater biofilms but also in drinking water biofilms. Florfenicol resistance gene floR and penicillin resistance gene penA have also been found in Listeria monocytogenes isolated from drinking water in dairy farms (Srinivasan et al. 2005). The appearance of potential antibiotic resistances in drinking water distribution systems of some nations or regions requires increased surveillance for risk assessment and prevention strategies to protect public health.
ARGs horizontal transfer ARGs emerge in aquatic environments as a direct result of intensive use of antibiotics in hospitals, swine production areas, and fish farms, and the genes in surface water and groundwater around such areas can transfer antibiotic resistance to the bacteria in drinking water or the food chain (Chee-Sanford et al. 2001). Genetic mechanisms involved in horizontal transfer of ARGs among environmental bacteria may include the following: (1) conjugative transfer by mobile elements including plasmids, transposons, and integrons on plasmids or transposons; (2) transformation by naked DNA, in the case of naturally competent state of some bacteria, or an environmentally induced competence such as the presence of calcium; and (3) transduction by bacteriophage. Antibiotic resistance in most environmental bacteria is due to the acquisition of new genes, often associated with the mobile elements. Plasmid is an initially discovered microbial mobile element distributed in water environment, and STPs are considered as important pools of the plasmids with transportable ARGs (Szczepanowski et al. 2004; Tennstedt et al. 2005). Many types of plasmids have been isolated from activated sludge of STPs, which confer resistances to aminoglycoside (Tennstedt et al. 2003), quinolone (Bnemann et al. 2006), erythromycin (Schlter et al. 2007a), as well as multiple drugs (Szczepanowski et al. 2005). Recently, Schlter et al. (2007b) has reviewed the gene elements and functions of IncP-1 plasmids isolated from wastewater treatment plants. These self-transmissible plasmids are capable of transferring to and replicating in a wide range of hosts and can encode resistances to almost all types of clinically relevant antibiotics (Szczepanowski et al. 2005; Schlter et al. 2007b). Among the mobile elements, transposon and integron also play important roles in horizontal transfer of environ-
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mental ARGs. Previous reports demonstrated that transposons and integrons carrying various ARGs often occurred in animal production or aquaculture areas (Schmidt et al. 2001; Moura et al. 2007; Akinbowale et al. 2007a; Jacobs and 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 and Chakraborty 2006; Lin and Biyela 2005), and sediments (Dalsgaard et al. 2000). The elements are not selfreplicating and must be carried by a phage or, more typically, by a plasmid to move from one cell to another. Insertion sequences, a type of small transposons, encode no other functions but recombinase and transposase (Summers 2006). Transposons and insertion sequences often jump randomly and occasionally on genome or plasmid, resulting in new or multiple resistances (Naas 2007). Integron is not capable of moving itself but can capture, integrate, and express resistance gene cassettes in their variable regions and can be transmitted via transposons and conjugative plasmids (Fluit and Schmitz 1999; Alekshun and Levy 2007). Integrons with as many as nine ARGs, typically four or five, are frequently found in clinical environments (Crowley et al. 2008; Labuschagne et al. 2008), agricultural wastewaters (Jacobs and Chenia 2007), urban wastewaters (Tennstedt et al. 2003; da Silva et al. 2007), and even in the waters not recently exposed to antibiotics (Park et al. 2003; Obst et al. 2006). Some physicochemical factors can influence the dissemination of ARGs in aquatic environments. The first factor contributing to the horizontal transfer of ARGs is the selective pressure from ever-increasing production and consumption of antibiotics for treatment of disease and growth promotion. High selective pressure facilitates the acquisition of ARGs, which may actually increase the fitness of certain bacteria and allow the rapid emergence and dissemination on a worldwide scale (Enne et al. 2004; Luo et al. 2005). In addition, the presence of antibiotics at low subinhibitory concentrations can accelerate horizontal transfer and dissemination of environmental ARGs (Kmmerer 2004). It was found that keeping antibiotic concentration at a subinhibitory level in the mating medium significantly enhances conjugal transfer mediated 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 effect on removal of tet genes in wastewater effluent, but loss rate of tetM, O, P, and W in aquatic environments has a significantly positive correlation to simulated sunlight exposure (Engemann et al. 2006). Many studies revealed that the co-selection took place in the various environmental bacteria with metal and antibiotic resistance (Berg et al. 2005; Stepanauskas et al. 2005; Wright et al. 2006). Bacteria in metal-contaminated environments
appeared to be easier to obtain antibiotic resistance phenotypes than in control areas (Baker-Austin et al. 2006). However, genetic mechanisms responsible for the coresistances occurring in the environments are poorly understood, since few researches have been carried out to investigate ARGs in metal-contaminated environments, though the experimental results of molecular genetics may help to explain these phenomena. Rasmussen and Sorensen (1998) found occurrence of conjugative plasmids carrying tetracycline and mercury resistance genes was increased in a contaminated site. Recently, a novel tetracycline resistance gene, tetA(41), has been found in Serratia marcescens isolated from a stream contaminated with heavy metals (Thompson et al. 2007), which provides indirect evidence of co-resistance. Wright et al. (2008) found that class 1 integrase gene was more abundant in the metal-exposed environments than in control, and the selective pressures shaped the structure of the gene cassette pool, indicating that relative gene transfer potential is higher in the microbial communities of the contaminated environments.
ARGs as emerging environmental pollutants It was suggested by Rysz and Alvarez (2004) that ARGs themselves could be considered as environmental pollutants, since they are widely distributed in various environmental compartments, including wastewater and STPs, surface water, lagoon water of animal production areas, aquaculture water, sediments and soil, groundwater, and drinking water. Recently, Pruden et al. (2006) has also pointed out that ARGs may be thought as emerging contaminants, for the public health problems resulted from the widespread dissemination of ARGs. As many other chemical pollutants, for example, persistent organic pollutants and heavy metals, ARGs are well-known easy-to-get, hard-to-lose pollutants (Aminov and Mackie 2007). Usually, antibiotic resistance bacteria and genes emerge in the environments under the selection pressure of some antibiotics, but the ARGs cannot be easily removed from the polluted areas, even when the pressure has disappeared (Salyers and Amabile-Cuevas 1997; Aminov and Mackie 2007). This may be one explanation why ARGs were often detected in antibiotic-free environments (Rahman et al. 2008). Potential public health concerns for environmental ARGs carried by bacterial pathogens were reviewed by Heuer et al. (2006) and Zhou et al. (2007). Although the direct evidence about ARGs transfer from the environments to human bodies is unavailable, some studies still highlight the fact that ARGs can spread and be exchanged among environmental microorganisms of different genera (Agers and Sandvang 2005; Agers and Petersen 2007) and the organisms even within
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completely different kingdoms (Rodrguez 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 of ARGs entering into and spread in the environments. The most effective and direct approach is thought to be the reasonable use of antibiotics in health protection and agriculture production. New and effective wastewater treatment processes are also needed to be developed to improve removal efficiency of ARGs in STPs. Additionally, feasibility of agricultural application of sludge or irrigation with reclaimed wastewater has to be discussed thoroughly considering possible introduction of ARGs to soil and groundwater. Researches on transfer and degradation pathways of environmental ARGs and health risk assessment on the genes may be performed to provide more scientific information for responsible authorities to make up regulatory standards and guidelines to control environmental dissemination of these pollutants.
Acknowledgements The authors wish to thank the Hong Kong Research Grants Council for the financial support of this study (HKU 7129/05E and HKU 7195/06E).
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Antibiotic Resistance Genes in Water

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Appl Microbiol Biotechnol (2009) 82:397414 DOI 10.

Antibiotic resistance genes in water environment

Appl Microbiol Biotechnol (2009) 82:397414

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

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

tetC tetD tetE tetG tetH

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

tetQ tetS tetT tetW otrA

aacA4 aacA29b aacC1 aacC2 aacC3 aacC4 aadA1

Aminoglycoside-2adenylyltransferase Aminoglycoside phosphoryltransferase

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

Appl Microbiol Biotechnol (2009) 82:397414

EW, EW, EW, SW EW, EW, SW EW, AS

Erythromycin resistance methylase

Macrolide-2phosphotransferase Chloramphenicol efflux protein

Florfenicol efflux protein Vancomycin resistance protein

DW, EW, NW, SW, UW EW, NW, UW

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

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

DW, NW, SD, SW NW, SD SD

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

Appl Microbiol Biotechnol (2009) 82:397414

Biological source Enterobacter, Salmonella

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

Appl Microbiol Biotechnol (2009) 82:397414

Appl Microbiol Biotechnol (2009) 82:397414

Appl Microbiol Biotechnol (2009) 82:397414

Appl Microbiol Biotechnol (2009) 82:397414

Appl Microbiol Biotechnol (2009) 82:397414

Appl Microbiol Biotechnol (2009) 82:397414

Appl Microbiol Biotechnol (2009) 82:397414

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