Present addresses: Saad Hannan, Department of Neuroscience, Physiology, and Pharmacology, University College London, London, WC1E 6BT, UK. Michelle Rogers, 4.28 Royal School of Mines, Department of Bioengineering, Imperial College London, South Kensington, London, SW7 2AZ, UK.
Correspondence: Adam P. Roberts, Department of Microbial Diseases, UCL Eastman Dental Institute, University College London, 256 Gray's Inn Road, London, WC1X 8LD, UK. Tel.: +44 207 915 1044; fax: +44 207 915 1127; e-mail: firstname.lastname@example.org
We demonstrate that live donor Veillonella dispar cells can transfer the conjugative transposon Tn916 to four different Streptococcus spp. recipients in a multispecies oral consortium growing as a biofilm in a constant depth film fermentor. Additionally, we demonstrate that purified V. dispar DNA can transform Streptococcus mitis to tetracycline resistance in this experimental system. These data show that transfer of conjugative transposon-encoded antibiotic resistance can occur by transformation in addition to conjugation in biofilms.
The usual mode of growth for many bacteria on our planet is as a biofilm (Costerton, 1995). A shared characteristic of bacterial cells growing as part of a biofilm is that they are usually embedded within a matrix of extracellular polymeric substances, which can include polysaccharides, proteins and nucleic acids (Flemming et al., 2007). This provides the cells within a biofilm the opportunity to remain in close proximity to one another in a stable environment over an extended period of time compared with planktonic cells. This situation has been shown to be conducive to gene transfer (Molin & Tolker-Nielsen, 2003).
Gene transfer is one of the main mechanisms of microbial adaptation and is responsible for the transfer of many different phenotypic traits including metabolic capabilities, virulence factors, heavy metal resistance and antibiotic resistance. Antibiotic resistance is currently one of the greatest challenges being faced by modern healthcare. Many of the phenotype-conferring genes, such as antibiotic resistance genes, are located on broad-host-range mobile genetic elements such as conjugative plasmids and conjugative transposons.
Conjugative transposons are mobile genetic elements capable of broad-host-range conjugative transfer between bacterial cells, often of different genera. The most extensively studied group of conjugative transposons are those of the Tn916 family (reviewed in Roberts & Mullany, 2009), which usually, but not exclusively, encode tetracycline resistance conferred by Tet(M). Members of this family of mobile genetic elements have been shown to transfer between various species of bacteria in different environments such as laboratory-based filter-matings, in vitro biofilm models (Roberts et al., 1999, 2001a; Ready et al., 2006) and in vivo rat and mouse gastrointestinal tracts (Doucet-Populaire et al., 1991; Alpert et al., 2003; Bahl et al., 2004). The transfer of Tn916 within biofilms has been hypothesized, but not proven, to occur by conjugation. In order to prove that conjugation, as opposed to transformation, is responsible for horizontal gene transfer, it is usual to include DNAse I in the experiment to digest all extracellular DNA (eDNA). The complexities of including DNAse I in a biofilm model have recently been compounded by the discovery that eDNA is central to the architecture and formation of some bacterial biofilms (Whitchurch et al., 2002). Therefore, the inclusion of this enzyme is likely to radically alter the biofilm system being investigated.
We have demonstrated previously that conjugation was responsible for the vast majority of transfer of Tn916 from Veillonella dispar strain 34.2A (Ready et al., 2006) to a number of Streptococcus spp. recipients during filter-mating experiments in the presence of DNAse I, suggesting that transfer in biofilms from this donor strain will likely be the result of conjugation. In order to investigate this further, we decided to see whether transformation could be responsible for the transfer of Tn916 within oral biofilms. Therefore, we have assayed the ability of V. dispar 34.2A to transfer the Tn916 element to members of a microbial consortium grown as a biofilm within a Constant Depth Film Fermentor (CDFF). In addition, we have also used donor DNA to ascertain whether this was able to transform any of the members of the biofilm to tetracycline resistance. Our results show that both V. dispar 34.2A live donor cells and purified V. dispar 34.2A DNA can transfer tetracycline resistance to Streptococcus spp. within the biofilm, indicating that transformation is likely to be involved in the dissemination of genetic information among the oral community.
Materials and methods
Plasmids, bacterial strains and growth conditions
The plasmid pAM120 was used as a control in the transformation experiments. pAM120 is derived from pGL101, which contains Tn916 in its entirety, together with flanking DNA on an EcoRI fragment (Gawron-Burke & Clewell, 1984). The V. dispar 34.2A and Enterococcus faecalis JH2-2 were grown on or in brain–heart infusion agar or broth (Oxoid Ltd, Basingstoke, UK). Escherichia coli DH5α containing pAM120 was grown in or on lysogeny broth (LB) or LB agar (Difco, Oxford, UK). Antibiotics were used at a concentration of 10 μg mL−1 (tetracycline) (Sigma, Poole, UK) unless stated otherwise.
Organisms for in vitro biofilm experiments
The V. dispar 34.2A live donor, V. dispar 34.2A genomic DNA or pAM120 DNA was added to a consortium of 21 tetracycline-susceptible bacteria. These recipient bacteria were Actinomyces meyeri (clinical isolate), Actinomyces odontolyticus (ATCC 17929) Actinomyces viscosus (NCTC 10951), Capnocytophaga gingivalis (ATCC 33624), Capnocytophaga granulosa (ATCC 51502), Capnocytophaga haemolytica (ATCC 51501), Capnocytophaga ochracea (ATCC 27872), Capnocytophaga sputigena (ATCC 33612), Fusobacterium nucleatum (NCTC 11326), Neisseria subflava (clinical isolate), Streptococcus anginosus (NCTC 10713), Streptococcus gordonii (NCTC 7865), Streptococcus intermedius (NCTC 2227), Streptococcus mitis (NCTC 12261), Streptococcus mutans (NCTC 10449), Streptococcus oralis (NCTC 11427), Streptococcus parasanguinis (NCTC 55898), Streptococcus pneumoniae (NCTC 7465), Streptococcus salivarius (NCTC 8618), Streptococcus sanguinis (NCTC 7863) and Streptococcus sobrinus (NCTC 12279).
Filter-mating and transfer experiments using an in vitro biofilm model
Filter-mating was carried out as described previously (Roberts et al., 2001b). The in vitro transfer experiments were carried out in a CDFF. The CDFF can generate large numbers of replicate biofilms (Wilson, 1999) and has been used previously to investigate antibiotic resistance and gene transfer in oral bacterial communities in vitro (Roberts et al., 1999, 2001a; Ready et al., 2002). Mating experiments were carried out on a bovine enamel substratum and the nutrient source for the experiments was mucin-containing artificial saliva (Pratten et al., 1998).
Anaerobic bacteria were grown for 48 h on Fastidious Anaerobic Agar (FAA) (LabM, Bury, UK) containing 5% defibrinated horse blood (E and O Laboratories, Bonnybridge, UK) in an anaerobic atmosphere (80% N2, 10% CO2, 10% H2) at 37 °C for 5 days. Facultative and obligate aerobes were grown on Columbia blood agar (Oxoid Ltd) containing 5% defibrinated horse blood in air supplemented with 5% CO2 at 37 °C for 2 days. All of the growth on a plate, after checking for purity, was harvested with a sterile swab and suspended in 1 mL of sterile phosphate-buffered saline. Each 1-mL suspension was subcultured to ensure the purity of the donor and recipients. The suspended bacteria were pooled and added to 500 mL of sterile artificial saliva and pumped into the CDFF at a flow rate of 2 mL min−1. Following this, the CDFF was fed with sterile artificial saliva at a rate of 0.5 mL min−1, corresponding to the resting salivary flow rate in humans (Pratten et al., 1998).
Selection and identification of transconjugants and transformants
Biofilms were removed from the fermentor at 24 and 48 h, serially diluted and inoculated onto FAA containing tetracycline (8 μg mL−1) and incubated aerobically and anaerobically. Following enumeration, bacteria were subcultured onto tetracycline-containing agar to ensure resistance and purity. Putative transconjugants were identified by partial 16S rRNA gene sequencing and further differentiation of the streptococcal species was achieved by carbohydrate fermentation and enzyme substrate utilization tests (Whiley & Beighton, 1998). The presence of the Tn916-like element was confirmed by PCR for the tet(M) gene and the integrase and excisionase (int and xis). The PCR results were confirmed by DNA sequencing of all of the amplicons.
Monoculture transformation experiments
Each member of the consortia above was grown in a suitable broth to the stationary phase. These cultures were spun down and the supernatant was discarded. Next, the bacterial pellet was resuspended in 600 μL fresh broth and 50 μL of V. dispar 34.2A DNA was added, mixed and aliquots were spread on a 0.4-μm pore size nitrocellulose filter (Sartorious, Epsom, UK) resting on a suitable agar plate. These were incubated overnight in a suitable atmosphere. Following this, the cells from the filter were resuspended in fresh broth and dilutions of the cell suspension were aliquoted onto suitable agar plates containing tetracycline. These were incubated for 48 h, checking for growth at 24 and 48 h.
PCRs for amplification of the 16S rRNA gene, tet(M) and the integration excision regions of Tn916 was carried using primers as described previously (Ready et al., 2006). Sequencing reactions were carried out at the Department of Biochemistry, University of Cambridge. Genomic DNA isolations were carried using the Gentra Puregene Yeast/Bacteria DNA Isolation Kit (Qiagen, Crawley, UK). Plasmid DNA isolation from E. coli was carried out using the Qiagen Miniprep spin columns (Qiagen). Samples were checked by agarose (Bioline, London, UK) gel electrophoresis to ensure integrity and to determine the DNA concentration by comparison with known molecular weight markers (Bioline). Bioinformatic analysis was carried out using tools located on the NCBI server (http://www.ncbi.nlm.nih.gov/).
Antibiotic susceptibility testing
The antibiotic susceptibility of all isolates was determined using an agar dilution method. Tetracycline (range 0.125–512 μg mL−1) was incorporated into isosensitest agar supplemented with 5% v/v defibrinated horse blood. The inoculum was standardized and applied to the agar surface using a multipoint inoculator (Mast, Merseyside, UK). The plates were incubated for 24 h at 37 °C. The minimum inhibitory concentration (MIC) was defined as the lowest concentration of the antibiotic that completely inhibited visible growth (Andrews et al., 2008).
Transfer from V. dispar 34.2A live donor cells
At inoculation, the CDFF contained 21 tetracycline-susceptible species (MIC≤0.125 mg L−1; tet(M) negative) and one tetracycline-resistant donor strain of V. dispar (MIC 32 mg L−1; tet(M) positive) that was recovered from one subject of a previous study and shown to contain a transferable copy of Tn916 (Ready et al., 2006). After 24 h, tetracycline-resistant putative transconjugants of S. mitis (MIC 32 mg L−1), S. oralis (MIC 32 mg L−1), S. parasanguinis (MIC 32 mg L−1) and S. salivarius (MIC 64 mg L−1) were isolated from biofilms grown in the CDFF. PCR and sequence reaction analysis demonstrated that all four putative transconjugants contained the recombination regions (int and xis) of Tn916 and the 406-bp amplicon from the tet(M) gene, which was also present in the donor bacterium and absent in the recipients (Fig. 1). The donor and the putative transconjugants could be isolated from the biofilms at both time points.
Transfer from V. dispar 34.2A DNA in biofilms, but not pAM120 DNA
Transfer of tetracycline resistance from the V. dispar 34.2A genomic DNA was demonstrated to S. mitis during one of the three replicate fermentor experiments. The S. mitis was shown to contain the tet(M) gene and the integrase and excisionase region of Tn916 as described above (results not shown). Moreover, it had an MIC of 32 mg L−1 and was isolated at both time points. Transfer from this S. mitis transconjugant to E. faecalis JH2-2 was not detectable (detection limit of approximately 1 × 10−9 transfer events per recipient) in our experiments. No transformants could be detected when pAM120 was inoculated into the fermentor.
No transfer was detected during monoculture experiments
We carried out a control experiment whereby stationary-phase liquid monocultures of each member of the consortia were incubated on nitrocellulose filters with DNA from V. dispar 34.2A. No transformants were isolated from these experiments.
The transfer of conjugative plasmids (Christensen et al., 1998; Molin & Tolker-Nielsen, 2003) and conjugative transposons (Roberts et al., 1999, 2001a; Ready et al., 2006) among bacteria growing in biofilms is now well documented. Additionally, the transformation of bacteria in biofilms by plasmid DNA has been demonstrated previously (Williams et al., 1996; Hendrickx et al., 2003). However, the ability of DNA to transform bacterial cells growing in oral biofilms is less well understood. With many species of bacteria inhabiting the oral cavity, including Streptococcus spp., Neisseria spp. and Actinobacillus spp., being naturally competent, it is likely that transformation of DNA is a major contributor to horizontal gene transfer in this environment.
In this study, we have shown that V. dispar 34.2A transfers Tn916 to four Streptococcus spp. in oral biofilms, and furthermore, the purified genomic DNA from V. dispar 34.2A can transform S. mitis to tetracycline resistance. Interestingly, pAM120 was unable to transform any of the biofilm consortia members to tetracycline resistance, indicating that the source of DNA in the biofilm may determine its transforming host range. This is certainly true for Neisseria meningitidis as suitable DNA for transformation must contain an uptake signal sequences (USS) (Goodman & Scocca, 1988). Various Gram-positive bacteria have no such USS requirement; however, only homologous DNA from related species is normally recombined into the cell's chromosome (Smith et al., 1999). It is possible that the surrounding regions of the Tn916 element in V. dispar 34.2A were similar enough to regions of the S. mitis chromosome to allow for homologous recombination and subsequent insertion of the element. Alternatively, the DNA containing Tn916 may have been taken up into the cytoplasm of the cell, followed by expression of the integration and excision genes, resulting in transposition from the incoming DNA molecule into the chromosome of the transformant. The lack of transfer from the S. mitis transformant suggests that only a fragment of Tn916 may have entered the S. mitis chromosome.
Another interesting observation from this study is that V. dispar 34.2A DNA was unable to transform any of the consortia members to tetracycline resistance when they were grown as a monoculture and incubated overnight on nitrocellulose filters with the purified V. dispar 34.2A DNA. This observation can be explained by the work of others who have demonstrated that, in the case of oral streptococci, competence is linked to biofilm formation through the production of the quorum-sensing molecule CSP and subsequent cell death, lysis and release of eDNA in a subpopulation of cells (Cvitkovitch et al., 2003; Perry et al., 2009). It is conceivable that stationary-phase cells spun down and spread onto a filter would not enter this competent phase of growth that is seen during biofilm formation and would therefore explain the lack of transformation.
While the predominant source of eDNA in biofilms is likely to be derived from dead bacterial cells (Kreth et al., 2005), there is recent evidence that some of the eDNA is actively transported from intact cells, as is the case for streptococci (Kreth et al., 2009). In addition to eDNA derived from dead and growing biofilm cells, we have recently shown, through metagenomic analysis of saliva-derived bacterial DNA BAC libraries, that we can isolate DNA from bacterial species, such as Phytoplasma sp., that are likely to be transient in the oral cavity (Seville et al., 2009). Furthermore, we have previously demonstrated transfer of tetracycline resistance from a transient Bacillus subtilis to a member of an oral biofilm (Roberts et al., 1999). The persistence of eDNA in the environment is dependent on many variables such as the presence of nucleases, but it has been demonstrated that eDNA can persist in various environments for a considerable length of time (reviewed in Nielsen et al., 2007). Therefore, it is likely that this source of DNA could also be a source of genetic information, which can be used by oral bacteria.
Our results provide more evidence that eDNA is likely to be an important source of usable genetic information for members of the oral microbial community. Furthermore, it is likely that both eDNA derived from the biofilm cells and also from transitory cells are involved in this process. Current work in our laboratory is now aimed at understanding the source of eDNA in oral biofilms and its contribution to horizontal gene transfer within this environment.
S.H. was funded by the Department of Biology, UCL Summer Studentship Scheme. A.S.J. is funded by the Malaysian Government. M.R. was funded by the Society for General Microbiology Vacation Studentship Scheme. Part of this work was undertaken at UCLH/UCL, which received a proportion of funding from the Department of Health's NIHR Biomedical Research Centres Funding Scheme, UK.