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Transfer of a conjugative transposon, Tn5397 in a model oral biofilm

Adam P. Roberts, Jonathan Pratten, Michael Wilson, Peter Mullany
DOI: http://dx.doi.org/10.1111/j.1574-6968.1999.tb13714.x 63-66 First published online: 1 August 1999


A tetracycline resistance profile was established from a microcosm dental plaque in a constant depth film fermenter. The fermenter was inoculated with a Bacillus subtilis strain which contained the conjugative transposon, Tn5397, which confers tetracycline resistance upon its host. After 6 hour and 24 hour the tetracycline resistance profile of the biofilm was re-determined and a tetracycline resistant Streptococcus species was isolated. A molecular analysis of this strain confirmed that Tn5397 was present in the genomic DNA of the isolate. These data represent the first report, to our knowledge, of intergeneric transfer of a conjugative transposon in a mixed species biofilm and demonstrates the ability of conjugative transposons to disseminate antibiotic resistance genes in a mixed species environment.

  • Conjugative transposon
  • Tn5397
  • Oral biofilm
  • Gene transfer

1 Introduction

Tn5397 is a conjugative transposon originally isolated from Clostridium difficile strain 630 [1] (for a recent review on conjugative transposition see [2]) which confers tetracycline resistance (Tcr) upon its host via the tet(M) gene. Tn5397 can be transferred to, and from, Bacillus subtilis and C. difficile by filter mating [3]. Tn5397 is closely related to the well-studied conjugative transposon Tn916 in the regions concerned with conjugation. However, the region concerned with integration and excision is completely different. In Tn5397 the site specific recombinase, tndX, replaces the Tn916 genes int and xis [2,3]. We have obtained the complete sequence of tndX.

It has been suggested that the majority of bacteria exist, in their natural state, as part of a biofilm [4]. To determine if Tn5397 was capable of transfer in a biofilm we inoculated an established set of microcosm dental plaques with B. subtilis::Tn5397 and demonstrated transfer of Tn5397 in this environment.

2 Materials and methods

2.1 Bacterial strains

All bacterial strains used in this work are shown in Table 1. The strains were grown at 37°C. C. difficile strains were grown anaerobically (80% nitrogen, 10% hydrogen, 10% carbon dioxide). B. subtilis strains were grown aerobically. Both the C. difficile and B. subtilis strains were grown on Brain Heart Infusion (BHI) agar or in BHI broth, supplemented with tetracycline at a concentration of 10 mg ml−1 when required. B. subtilis strain BS6A was obtained from a filter mating between the TcrC. difficile strain 630 [1] and a tetracycline sensitive (Tcs) B. subtilis (CU2189) [5]. Tcr transconjugants arose at a frequency of 1×10−7 per donor. Filter mating was carried out as previously described [3]. That transfer of Tn5397 had taken place was verified by Southern blot analysis as done previously [3].

View this table:
Table 1

Strains of bacteria used in this work

StrainGenotype and/or phenotypeReference
C. difficile
630Tn5397 donor Tcr[1]
B. subtilis
CU2189Tcs recipient for filter mating experiments[5]
BS6Aresult of a filter mating between 630 and CU2189, Tcrthis work
S. acidominimusTn5397 containing transconjugant Tcrthis work

2.2 Production of biofilms

The constant depth film fermenter consists of a rotating turntable, which holds 15 polytetrafluoroethylene (PTFE) pans, located flush around its rim [6]. Each pan contains five cylindrical holes containing PTFE plugs. The biofilms were grown on bovine enamel discs, 5 mm in diameter, which sit on PTFE plugs of the same diameter and are recessed to a depth of 300 µm. Saliva was used as an inoculum to provide a multi-species biofilm consisting of organisms found in the oral cavity. The saliva was collected from 10 healthy individuals (who had not taken antibiotics within the last 12 months), equal amounts from each person were pooled and 1 ml of this pooled saliva was added to 500 ml of artificial saliva, mixed, and pumped into the CDFF for 8 h. After this time, the inoculum flask was disconnected and the CDFF fed from a medium reservoir of sterile artificial saliva as described previously [6].

2.3 Assessing the genera present as a proportion of the viable counts

Once the biofilm had reached a steady state (21 days) selective media were used to identify the four main genera present; Actinomyces spp. were isolated on cadmium fluoride/acriflavin/tellurite agar plates [7], Veillonella spp. on Veillonella agar (Difco), Streptococci on Mitis Salivarius agar (Difco) and Lactobacillus spp. on Rogosa agar (Oxoid). The total anaerobic count was performed on Wilkins-Chalgren agar (Oxoid) containing 8% horse blood. All the plates were incubated anaerobically for 4 days at 37°C. The total aerobic viable count was carried out on 8% blood agar (Oxoid) and incubated at 37°C aerobically.

2.4 Assessing the tetracycline resistance profile of the biofilm

To assess the tetracycline resistance profile of the biofilm, one pan was removed and the five discs on which the biofilms were formed were placed into individual aliquots of 100 µl sterile H2O. These were vortexed for 1 min and a 10−1 dilution prepared. Hundred µl of this dilution was spread onto blood agar containing 10 mg ml−1 tetracycline and onto blood agar plates containing no antibiotics. These were incubated in both aerobic and anaerobic conditions for 48 h checking growth at 24 h and 48 h. This was repeated with five replicate biofilms. Any Tcr organisms isolated after inoculation with BS6A were identified using API technology (Biomerieux, Basingstoke).

2.5 Growth and inoculation of the donor

BS6A was grown overnight on BHI agar containing tetracycline at 10 mg ml−1 at 37°C aerobically. One colony from this plate was used to inoculate 10 ml of BHI broth containing tetracycline at 10 mg ml−1 and grown overnight in a shaking incubator 200 rpm, 37°C. This was diluted 1 in 10 with fresh, pre-warmed (37°C) BHI broth and left to grow shaking for 1 h at 37°C. This was then used to inoculate the CDFF over a period of 4 h at a constant temperature of 37°C.

2.6 Isolation of genomic DNA, PCR and sequencing

The genomic DNA of the transconjugant was isolated using a Puregene Gram-positive DNA Isolation Kit (Flowgen). PCR primers for the amplification of tndX were designed based on the sequence of this gene. Primers used were RT16 (5′ ACG AGA TGA TGG GTT GCA CA 3′) and RT17 (5′ TCT TTC CAA CCC AAT CAG TA 3′) which produced a 677 bp product. The following program was used: 94°C for 4 min, 25 cycles of 94°C for 1 min, 50°C for 1 min, 72°C for 1 min followed by a final extension of 72°C for 4 min and 4°C until analysed on a 1.0% agarose gel. DNA sequencing was carried out using the specific PCR primers, BigDye Terminator Mix and a 310 Genetic Analyser (Perkin Elmer).

3 Results

Prior to the inoculation of B. subtilis BS6A, the Tcr profile and diversity of the biofilm was established. The biofilm consisted of Streptococci (37%), Actinomyces spp. (16%), Lactobacillus spp. (14%) and Veillonella spp. (4%). One Gram-negative species was isolated which was Tcr at 10 mg ml−1, this was isolated from all biofilms and grew both aerobically and anaerobically. Samples taken 6 h after inoculation of the B. subtilis BS6A resulted in the isolation of three morphologically distinct species of Tcr bacteria. One was the Gram-negative rod isolated before inoculation; one was the B. subtilis donor and one was a Gram-positive coccus which was shown to be Streptococcus acidominimus. The tetracycline resistance organisms isolated from the biofilms 24 h after inoculation with the B. subtilis BS6A were identical to the organisms isolated from the 6 h sample except that the B. subtilis donor could not be isolated.

The PCR analysis of the genomic DNA from the TcrS. acidominimus showed that tndX was present in this organism (Fig. 1). The sequencing showed that the gene present was identical to tndX in the donor cell (data not shown).

Figure 1

PCR results showing the presence of tndX. Lane 1: DNA marker, the first band represents 2642 bp, the second bright band represents 1000 bp decreasing in 100 bp increments to 100 bp. Lane 2: B. subtilis BS6A DNA as template. Lane 3: S. acidominimus DNA as template. Lane 4: negative control, H2O as template.

The diversity of the biofilm (in terms of the relative proportions of Streptococci, Actinomyces spp., Lactobacillus spp. and Veillonella spp.) was unaffected by the addition of B. subtilis BS6A to the biofilm. At sample times 0 h, 6 h and 24 h the biofilm consisted of Streptococci (37%), Actinomyces spp. (16%), Lactobacillus spp. (14%) and Veillonella spp. (4%).

4 Discussion

In this study we have demonstrated, for the first time, horizontal gene transfer of a conjugative transposon in a mixed species oral biofilm. It was shown that the conjugative transposon Tn5397 could be transferred within 6 h of inoculation of the donor to the oral commensal S. acidominimus. These results demonstrate that conjugative transposons can be transferred in biofilm communities and that non-oral bacteria can transfer genes to oral commensals.

The B. subtilis strain used in this work could not be detected in the oral biofilms after 24 h, this probably represents the time it took for the BHI medium to be diluted out by the artificial saliva medium. Nevertheless, this was sufficient time for gene transfer to take place, indicating that bacteria that are transient in the oral cavity (e.g. in food) could be important in mediating the spread of mobile elements and the genes present on these elements, such as antibiotic resistance genes.

The work described in this paper represents the first demonstration of gene transfer in an oral biofilm, however other workers have shown that transfer of plasmid DNA can occur in natural and artificial biofilms. Demonstration of plasmid transfer in natural epilithic biofilms was shown by Williams et al. (1996) [8]. Lebaron et al. (1997) [9] demonstrated the transfer, by mobilisation, of a pBR derived plasmid from one Escherichia coli K12 strain to another in three different environments: the mouse gut, in sterile soil and in an aquatic biofilm environment. Furthermore, Christensen et al. (1998) [10] analysed the conjugational transfer of the TOL plasmid (pWWO) in a flow chamber biofilm community. The recipient organism in this case was Pseudomonas putida RI.

Conjugative transposons have been shown to have a vast host range with Tn916-like transposons being found naturally in, or having been introduced into, over 50 different species and 24 genera of bacteria [11]. Furthermore, they have also been shown to be capable of transfer between Enterococcus faecalis and Listeria monocytogenes in the digestive tract of gnotobiotic mice [12]. The data presented in this study show that transfer of these elements is likely to also be possible in the mouth. Thus conjugative transposons should be able to transfer in at least two of the body sites in which bacteria have the opportunity of coming into close contact. These observations underline the likely importance of these elements in mediating the spread of antibiotic resistance genes.


This work was supported by the Welcome Trust. A.P.R. was the recipient of a BBSRC studentship.


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