Juridicidad de la conquista de Tenochtitlan 1519-

Prior to the inoculation o f B. subtilis BS6A, the Tc"^ profile and diversity o f the biofilm was established. The biofilm consisted o f streptococci (37 %), Actinomyces spp (16 %),

Lactobacillus spp (14 %) and Veillonella spp (4 %). A single Gram-negative species was

biofilms and grew both aerobically and anaerobically. Samples taken 6 h after inoculation o f the B. subtilis BS6A resulted in the isolation o f 3 morphologically distinct species of Tc"^ bacteria, the Gram-negative rod isolated before inoculation, the B. subtilis BS6A donor and a Gram-positive coccus, which was shown to be Streptococcus acidominimus.

The tetracycline resistant organisms isolated from the biofllms 24 h after inoculation with

B. subtilis BS6A were identical to the organisms isolated from the 6 h sample except that

the B. subtilis donor could not be isolated.

7.3.2 The gene tndX was present in the S. acidominimus genomic DNA

The PCR analysis o f the genomic DNA from the Tc'^ S. acidominimus showed that tndX

was present in this organism (Figure 7.2). Sequencing o f the PCR product showed that the gene was identical to tndX in the donor.

The diversity o f the biofilm (in terms o f the relative proportions o f streptococci,

Actinomyces spp, Lactobacillus spp and Veillonella spp.) was unaffected by the addition

o f B. subtilis BS6A to the biofilm. At sample times 0 h, 6 h and 24 h the biofllm

consisted o f Streptococci (37 %), Actinomyces spp (16 %), Lactobacillus spp (14 %) and

M 1 2 3 4 5 100 0 b p 667bp F i g u r e 7.2 . L a n e s M . M o l e c u l a r m a s s m a r k e r , 1 s h o w s P C R p r o d u c t u s i n g p r i m e r s R T 1 6 a n d R T 1 7 ( w h i c h a m p l i f y a p r o d u c t f r o m w i t h in tn d X ) on C. d iffic ile : : T n 5 3 9 7 ( F M I A ) c h r o m o s o m a l D N A . L a n e 2, 3 a n d 4 s h o w P C R p r o d u c t s u s i n g i d e n tic a l p r i m e r s o n th r e e d i f f e r e n t D N A p r e p a r a t i o n s o f T c ' S. a c id o m in im u s c h r o m o s o m a l D N A , L a n e 5 r e p r e s e n t s th e n e g a t i v e c o n tr o l (n o te m p l a t e ) . 7.4 Discussion

This work has demonstrated, for the first time, that horizontal gene transfer in a mixed species oral biofilm can occur. The conjugative transposon Tn5397 could be transferred within 6 hours o f inoculation o f the donor to the oral commensal Streptococcus

acidominimus, demonstrating that conjugative transposons can be transferred in biofilm

communities and that non-oral bacteria can transfer genes to oral commensals.

The fact that a low amount of tetracycline (1 pg ml'^ in the initial 100 ml inoculum) was present may have stimulated the transfer o f this element. Previous work (Doucet-

Populaire et a l, 1991) demonstrated tetracycline inducible transfer o f Tn7J^5 in the presence o f sub-inhibitory concentrations.

The B. subtilis strain used in this work could not be detected in the oral biofilms after 24

hours, this probably represents the time it took for the BHI medium to be diluted out by the artificial saliva medium. It does not mean however that the B. subtilis was dead. It may have been present in a non-cultivable state (e.g., as spores). Biofilms however, are resistant to colonisation from other non-oral species due to the synergistic and

antagonistic interactions that occur between the indigenous members o f the biofilm community (Costerton et a l, 1995). Nevertheless, the BS6A donor was present for sufficient time to allow 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 o f mobile genetic elements. This work extends that o f Doucet-Populaire et a l,{ } 9 9 \) to show that conjugative transposons are likely to be important in mediating gene transfer in the mouth as well as the gut.

7.5 Conclusions

Tn5397 can transfer from a B. subtilis host to a new host in a mixed species biofilm. The

B. subtilis donor was undetectable after 24 h indicating that organisms that are transient

Chapter 8