View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by RERO DOC Digital Library RESEARCH LETTER Construction and characterization of Enterococcus faecalis CG110/gfp/pRE25Ã, a tool for monitoring horizontal gene transfer in complex microbial ecosystems Martina C. Haug, Sabine A. Tanner, Christophe Lacroix, Leo Meile & Marc J.A. Stevens Laboratory of Food Biotechnology, Institute of Food, Nutrition and Health, ETH Zurich, Zurich, Switzerland Correspondence: Leo Meile, Laboratory of Abstract Food Biotechnology, Institute of Food, Nutrition and Health, ETH Zurich, Enterococci are among the most notorious bacteria involved in the spread of Schmelzbergstrasse 7, CH-8092 Zurich, antibiotic resistance (ABR) determinants via horizontal gene transfer, a process that Switzerland. Tel.: 141 44 632 33 62; fax: leads to increased prevalence of antibiotic-resistant bacteria. In complex microbial 141 44 632 14 03; e-mail: communities with a high background of ABR genes, detection of gene transfer is [email protected] possible only when the ABR determinant is marked. Therefore, the conjugative multiresistance plasmid pRE25, originating from a sausage-associated Enterococcus Received 6 August 2010; revised 8 September faecalis, was tagged with a 34-bp random sequence marker spliced by tet(M). The 2010; accepted 29 September 2010. plasmid constructed, designated pRE25Ã, was introduced into E. faecalis CG110/ Final version published online 28 October 2010. gfp, a strain containing a gfp gene as chromosomal marker. The plasmid pRE25Ã is DOI:10.1111/j.1574-6968.2010.02131.x fully functional compared with its parental pRE25, occurs at one to two copies per chromosome, and can be transferred to Listeria monocytogenes and Listeria innocua À6 À8 Editor: Andre´ Klier at frequencies of 6 Â 10 to 8 Â 10 transconjugants per donor. The markers on the chromosome and the plasmid enable independent quantification of donor and Keywords plasmid, even if ABR genes occur at high numbers in the background ecosystem. horizontal gene transfer; antibiotic resistance; Both markers were stable for at least 200 generations, permitting application of the pRE25. strain in long-running experiments. Enterococcus faecalis CG110/gfp/pRE25Ã is a potent tool for the investigation of horizontal ABR gene transfer in complex environments such as food matrices, biofilms or colonic models. Characterization of the human microbial community has Introduction revealed a vast diversity of resistance genes, indicating that Horizontal transfer of resistance genes and antibiotic- the human microbial community acts as a reservoir of ABR mediated selection pressure leads to a persistence and genes (Shoemaker et al., 2001; Sommer et al., 2009). So far, propagation of antibiotic-resistant bacteria in clinical envir- horizontal gene transfer (HGT) in the gut has mainly been onments, stock breeding, or in soil (Murray, 1990; Doucet- observed after ingestion of a donor and a defined recipient Populaire et al., 1991; Showsh & Andrews, 1992; Agerso & in the presence of a complex background flora or between Sandvang, 2005; Kazimierczak & Scott, 2007). Transfer of specific bacteria in gnotobiotic animals (Doucet-Populaire antibiotic resistance (ABR) determinants can cross the et al., 1991; Licht et al., 2002, 2003; Alpert et al., 2003; Avrain genus barrier and is mainly mediated by conjugative ele- et al., 2004; Mater et al., 2005, 2008; Hart et al., 2006; Lester ments such as transposons and plasmids (Shoemaker et al., et al., 2006; Jacobsen et al., 2007; Moubareck et al., 2007; 2001). Enterococci are Gram-positive, catalase-negative, Feld et al., 2008; Boguslawska et al., 2009). However, both oxidase-negative members of the functional related group experimental set-ups are limited in the selection of recipi- of lactic acid bacteria predominantly encountered in the ents against the microbial background and in the quantifica- gastrointestinal tract (GI-tract) of humans and animals. tion of gene transfer. MICROBIOLOGY LETTERS MICROBIOLOGY Enterococci harbor a variety of mobile genetic elements The 50-kb plasmid pRE25 from Enterococcus faecalis such as conjugative plasmids and transposons and therefore RE25 encodes resistances against the structural antibiotic the genus Enterococcus is supposed to be a main actor in the classes aminoglycosides, lincosamides, macrolides, chloram- spreading of ABR genes (Clewell, 1990). phenicol and streptothricin, and is transferrable to FEMS Microbiol Lett 313 (2010) 111–119 c 2010 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved 112 M.C. Haug et al. E. faecalis, Lactococcus lactis and Listeria innocua (Schwarz, Plasmid DNA from Escherichia coli was isolated using the 2001; Schwarz et al., 2001; Teuber et al., 2003). The plasmid Plasmid Midi Kit (Qiagen). Plasmids from E. faecalis were pRE25 belongs to the incompatibility group Inc18 of purified according to the preparative protocol for large-scale streptococcal plasmids, which replicate via the unidirec- plasmid isolation (Anderson & McKay, 1983) with slight tional y mechanism (Bruand et al., 1991; Ceglowski et al., modifications. 1993; Le Chatelier et al., 1993). Sequence comparison of Construction of E. faecalis CG110/gfp/pRE25Ã pRE25 to other conjugative plasmids such as the Streptococ- cus agalactiae plasmid pIP501, the Staphylococcus plasmids In order to insert a 34-bp random sequence interspaced by pGO1 and pSK41, and the Lactococcus plasmid pMRC01 tet(M) into pRE25, the integration vector pMH401 was revealed that the modular organization of the transfer genes constructed (Fig. 1, Table 1). A 1-kb fragment directly region is well-conserved, indicating common transfer po- upstream of the stopcodon of the ermB gene was amplified tential of these plasmids (Grohmann et al., 2003). using the primer pair Ins_A2/B (Table 2), the proofreading Here, we describe the construction and features of a Phusion polymerase (Finnzymes, Espoo, Finland), and chromosomally tagged E. faecalis strain harboring the multi- DNA from L. lactis BuRE25 as template. Similarly, a 1-kb resistant conjugative plasmid pRE25Ã, a derivative of pRE25 fragment downstream of the stopcodon of the ermB gene carrying a unique DNA sequence downstream of the ery- was amplified using the primers Ins_C and Ins_D. The two thromycin resistance gene. The two markers allow distin- fragments were fused via splicing by overlap extension PCR guishing between donor strain and recipient bacteria and using the 34-bp overlapping region introduced in the the strain can therefore be used as a tool to monitor and primers Ins_B and Ins_C (Table 2, underlined). PCR was quantify horizontal ABR gene transfer in complex microbial then performed on the fused fragments using primers environments without defined recipients, such as the human Ins_A2 and Ins_D and 2 Â taq PCR Master Mix (Fermentas, GI-tract, food matrices, and biofilms. Le-Mont-sur-Lausanne, Switzerland). The resulting 2120- bp fragment was cloned into the cloning vector pGEMs-T Materials and methods Easy (Promega, Madison) according to the manufacturer’s instructions. The resulting plasmid was designated Bacterial strains and media pMH400, a plasmid containing the 1-kb up- and down- stream regions of the stopcodon of the ermB gene, inter- Bacterial strains and growth conditions used in this study spaced with a 34-bp random sequence. Subsequently, tet(M) are listed in Table 1. Chemicals were routinely obtained from was amplified from E. coli CG120/pAM120 DNA using Sigma-Aldrich (Buchs, Switzerland), except when stated primers HP14 and HP15 and Phusion DNA polymerase. otherwise. The 2678-bp fragment obtained was ligated into pMH400 linearized with SwaI. Correct plasmid construction was DNA isolation and manipulation checked by restriction analyses and by PCR targeting tet(M) DNA manipulations were essentially performed as described using primers HP14 and HP15 (Table 2). The obtained previously (Sambrook & Russell, 2001). Oligonucleotides plasmid was designated pMH401 and harbors the 1-kb up- were obtained from Microsynth (Balgach, Switzerland) and and downstream regions of the stopcodon of the ermB gene are listed in Table 2. DNA for PCR amplification was interspaced with tet(M) flanked by a 23-bp and an 11-bp extracted from single colonies using a trizol–lysozyme-based random sequence (Fig. 1). Plasmid pMH401 was transferred cell lysis and subsequent DNA isolation as described pre- into L. lactis BuRE25 (Table 1) by electroporation as viously (Goldenberger et al., 1995). DNA extraction for described previously (Holo & Nes, 1989) and primary quantitative PCR was performed as follows: cells from integrants were selected on streptococcal regeneration 2-mL cultures were harvested and resuspended in 400 mLof plates (Okamoto et al., 1983) containing 10 mgmLÀ1 tetra- TE buffer (10 mM Tris, 1 mM EDTA, pH 8.0). The suspen- cycline. A double-cross-over event results in integration sion was transferred to a screw cap tube containing 500 mLof of tet(M) flanked by the two random sequences downstream phenol : chloroform : isoamylalcohol (25 : 24 : 1) and 500 mg of the ermB gene in pRE25 (Fig. 1). Therefore, integrants of 0.1-mm zirconia/silica beads. Cells were disrupted by were streaked on brain–heart infusion (BHI) containing bead-beating four times 20 s at maximum speed in a 10 mgmLÀ1 erythromycin and after incubation for 48 h FastPrep device (MP Biomedicals, Illkirch, France), inter- at 30 1C, single colonies were checked for double-cross-over spaced by cooling on ice. The suspension was centrifuged at by PCR using the primer pairs Int401_A/F and Int401_G/D. 20 000 g and 4 1C for 10 min and the supernatant was An isolate showing correct PCR pattern was designated extracted once with chloroform to remove residual phenol. L. lactis BuRE25Ã,anL. lactis Bu2-60 derivative carrying The DNA was precipitated with isopropanol, washed with pRE25 tagged with tet(M) flanked by two 23- and 11-bp 70% ethanol, dried and resuspended in TE buffer. random sequences in its chromosome. Next, pRE25Ã was c 2010 Federation of European Microbiological Societies FEMS Microbiol Lett 313 (2010) 111–119 Published by Blackwell Publishing Ltd.