Environmental Microbiology (2004) 6 (9), 981–989 doi:10.1111/j.1462-2920.2004.00664.x© 2004 Blackwell Publishing Ltd Blackwell Science, LtdOxford, UKEMIEnvironmental Microbiology1462-2912Society for Applied Microbiology and Blackwell Publishing Ltd, 20046 9981989 Original Article Antibiotic resistance and uncultured bacteriaC. S. Riesenfeld, R. M. Goodman and J. Handelsman Received 28 October, 2003; revised 30 April, 2004; accepted 30 April,2004. *For correspondence. E-mail [email protected]; Tel. (+1)608 263 8783; Fax (+1) 608 265 5289. Uncultured soil bacteria are a reservoir of new antibiotic resistance genes Christian S. Riesenfeld, 1,2 Robert M. Goodman 1,2,3 and Jo Handelsman 1,2 * 1 Department of Plant Pathology, 2 Microbiology Doctoral Training Program, and 3 Gaylord Nelson Institute for Environmental Studies, University of Wisconsin-Madison, Madison, WI 53706, USA. SummaryAntibiotic resistance genes are typically isolated bycloning from cultured bacteria or by polymerase chainreaction (PCR) amplification from environmental sam-ples. These methods do not access the potential res-ervoir of undiscovered antibiotic resistance genesharboured by soil bacteria because most soil bacteriaare not cultured readily, and PCR detection of antibi-otic resistance genes depends on primers that arebased on known genes. To explore this reservoir, weisolated DNA directly from soil samples, cloned theDNA and selected for clones that expressed antibioticresistance in Escherichia coli . We constructed fourlibraries that collectively contain 4.1 gigabases ofcloned soil DNA. From these and two previouslyreported libraries, we identified nine clones express-ing resistance to aminoglycoside antibiotics and oneexpressing tetracycline resistance. Based on the pre-dicted amino acid sequences of the resistance genes,the resistance mechanisms include efflux of tetracy-cline and inactivation of aminoglycoside antibioticsby phosphorylation and acetylation. With one excep-tion, all the sequences are considerably different frompreviously reported sequences. The results indicatethat soil bacteria are a reservoir of antibiotic resis-tance genes with greater genetic diversity than previ-ously accounted for, and that the diversity can besurveyed by a culture-independent method.Introduction Antibiotic resistance is a concern for the management ofdisease in humans, animals and plants. The intenseresearch efforts to elucidate mechanisms of resistancehave focused on genes derived from a narrow range ofenvironments. Most of the known resistance determi-nants have been discovered in clinical and veterinarybacterial isolates, whereas other environmental reser-voirs of antibiotic resistance are not well characterized(Nwosu, 2001; Séveno et al ., 2002).Cultured microorganisms have been the source ofalmost all characterized antibiotic resistance genes;therefore, most previous studies have ignored the poten-tial reservoir of antibiotic resistance genes in unculturedbacteria. The majority of bacteria are not readily cul-tured on standard laboratory media (Giovannoni et al .,1990; Ward et al ., 1990; Amann et al ., 1995; Suzuki et al ., 1997; Hugenholtz et al ., 1998), and the diversityof the uncultured majority is vast (Head et al ., 1998;Torsvik et al ., 1998; Whitman et al ., 1998; Béjà et al .,2002). Despite recent progress in culturing methods(Connon and Giovannoni, 2002; Janssen et al ., 2002;Kaeberlein et al ., 2002), culture-independent techniquesare required to access the genetic diversity of mostbacteria.The polymerase chain reaction (PCR) is currentlyused for culture-independent isolation of antibiotic resis-tance genes from environmental samples (Waters andDavies, 1997; Smalla et al ., 2000; Aminov et al ., 2001;Frana et al ., 2001; Stokes et al ., 2001), but onlyaccesses genes that are similar to known sequencesand often does not recover complete genes. Because ofthe possibility of undiscovered gene families and undis-covered genetic diversity within known gene families, wecircumvented the limitations of both culturing and PCR-based methods by extracting and cloning DNA directlyfrom soil samples, thus constructing libraries thatinclude the genes of uncultured soil bacteria. Suchlibraries containing environmental DNA (Stein et al .,1996), or ‘metagenomic libraries’ (Rondon et al ., 2000),have been used previously to identify clones expressingvarious enzymes (Henne et al ., 2000; Rondon et al .,2000; Knietsch et al ., 2003) and antimicrobial activities(Brady and Clardy, 2000; Brady et al ., 2001; Gillespie et al ., 2002; Courtois et al ., 2003). Diaz-Torres and col-leagues (2003) recently constructed metagenomic librar-ies from the human oral cavity and identified a noveltetracycline resistance gene.982 C. S. Riesenfeld, R. M. Goodman and J. Handelsman © 2004 Blackwell Publishing Ltd, Environmental Microbiology , 6 , 981–989 Here, we describe the use of metagenomic libraries toisolate and identify antibiotic resistance genes from soil.We constructed libraries that contain fragments of clonedsoil DNA and selected for clones expressing antibioticresistance in Escherichia coli . Results Isolation of clones expressing antibiotic resistance We selected antibiotic-resistant clones from two bacterialartificial chromosome (BAC) libraries and four small-insertlibraries that collectively contain 5.4 gigabases of clonedsoil DNA (Table 1). In a preliminary screen, we detected10 unique clones expressing resistance to aminoglyco-side antibiotics or tetracycline (Table 2), two from the BAClibrary, SL2, and eight from the small-insert libraries. Nonalidixic acid-resistant clones were detected. Antibiotic susceptibility testing We measured the minimum inhibitory concentrations(MICs) of five different aminoglycosides on the nineclones that were selected on media containing ami-noglycoside antibiotics (Table 3). Clones 85C1, 171D10,CR6 and CR10 are more resistant to amikacin,butirosin, kanamycin and tobramycin than strains thatcontain a vector with no insert. Clones CR5 and CR11have the same resistance profile as 85C1, 171D10,CR6 and
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