Recombinant DNA transfer to Escherichia coli of human faecal origin in vitro and in digestive tract of gnotobiotic mice

Transfer of the shuttle vector pRRI207 mediated by the helper plasmid pRK2013 from Escherichia coli to Bacteroides spp. was possible in vitro and in vivo in the digestive tract of axenic mice associated with Bacteroides uniformis 1004 or Bacteroides vulgatus of human origin. In vivo, transfer frequencies were nearly identical for B. uniformis (2U10 3U ) and B. vulgatus (4U10 3U ) and the transconjugant strains of B. uniformis and B. vulgatus became established in the digestive tract of mice at densities ranging from 10 P^1 0 Q to 10 R CFU g 3I of faeces, respectively. Transfer from E. coli to Bacteroides strains in gnotoxenic mice associated with human faecal flora (HFF) was not successful. Transconjugant-like clones appeared among the HFF of gnotoxenic mice after they were inoculated with B. uniformis TBUA, a transconjugant strain of B. uniformis 1004 obtained from triparental mating and which harboured the shuttle vector. Hybridisation showed that the shuttle vector pRRI207 was not present in these clones, and it is suggested that they could result from the transfer of a conjugative transposon ERL contained in B. uniformis 1004. Moreover, clones believed to have lost the shuttle vector hybridised with a specific probe to B. thetaiotaomicron and therefore did not originate from B. uniformis TBUA. z

Section: Discussionsupporting

confidence: 68%

Section: Discussionmentioning

confidence: 83%

See 1 more Smart Citation

Transfer of the shuttle vector pRRI207 between Escherichia coli and Bacteroides spp. in vitro and in vivo in the digestive tract of axenic mice and in gnotoxenic mice inoculated with a human microflora

Garrigues-Jeanjean

1999

“…It was found that our experimental conditions led to genetic transfers by conjugation, and mobilization (transfer of plasmid pCE325, in the presence of mob and tra elements). This mobilization had also been demonstrated in the digestive tract of axenic mice at the frequency of lo-" to lo-3 TC/R [31]. The non-detection of plasmid pCE328 transfer by conduction indicates the absence of specific IS sequences that enable the formation of a cointegrate with the conjugative plasmid R388, or else a low transfer frequency precluding the detection of this event in our conditions, Furthermore, these experiments showed the importance of plasmid content in donor bacteria for their establishment in biofilms composed of a recipient strain of E. cd'.…”

Section: Discussionmentioning

confidence: 61%

Recombinant plasmid DNA mobilization by activated sludge strains grown in fixed-bed or sequenced-batch reactors

Frank¹,

Simao-Beaunoir²,

Dollard³

et al. 1996

The possibilities of mobilizing recombinant DNA (rDNA) by strains from activated sludge have been studied. A mixture of ten bacterial strains isolated from activated sludge was grown on a support in a continuously fed fixed‐bed reactor or in suspension in a sequenced‐batch reactor with biomass recycling. After reaching steady state, the bacteria in both types of reactors were used as the recipient population for evaluating the dissemination of the non‐conjugative and non‐mobilizable recombinant plasmid pCE328. The dissemination of this plasmid, derived from pBR328 (tra−, mob−, oriT−), was studied in comparison with that of pCE325 (pBR325 derivative tra−, mob−, oriT+), and that of the natural broad host range conjugative plasmid R388 (IncW). Initially the mobilization properties of pBR type recombinant plasmids between two strains of Escherichia coli were determined in the fixed‐bed reactor. Only the mobilization of the recombinant plasmid bearing a transfer origin (pCE325) was observed. The survival of E. coli strains bearing either the non‐transferable plasmid pCE328 or the conjugative plasmid R388 and added to the activated sludge reactors was measured in the effluent of both types of reactors. In the fixed‐bed reactor effluent, the donor populations remained stable after three days of operation and reached 103 to 105 colony forming units (cfu) ml−1, whereas they decreased in the sequenced‐batch reactors: donors bearing the pCE328 plasmid decreased by 2 log units in 20 days, whereas those bearing the R388 plasmid decreased by less than 1 log unit. Mobilization of the pCE328 plasmid could not be detected in any case. Transfer of the conjugative plasmid R388, used as a positive propagation control, was detected in strains arising from activated sludge in both types of reactors. Nevertheless, establishment of transconjugants was observed only in the fixed biomass at the rate of 10−7 transconjugants per recipient.

“…Previous studies have examined plasmid transfer in laboratory media, and germ free or ex-germ free mice [20,21,23,24] or plasmid transfer under conditions that closely simulate gut environment [25], but the rate of plasmid transfer has not been compared in the different situations. In Schlundt et al [24], the transfer rate varied depending on the donor strain and the recipient strain used and also whether measured in vitro or in vivo.…”

Section: Discussionmentioning

confidence: 99%

Transfer of the plasmid RP1 in vivo in germ free mice and in vitro in gut extracts and laboratory media

Rang

Kennan

Midtvedt

et al. 1996

The frequency of conjugal transfer of the plasmid RP1 from two different Escherichia coli donor strains (HB101 and X7, RifR) to the same E. coli recipient strain (X7, NalR) was measured in vivo (germ free mice) and in vitro (intestinal extracts, caecal contents and laboratory liquid and agar media). The transfer frequencies of the plasmid from X7, RifR and HB101 in vivo were not significantly different from those obtained when using intestinal extracts or caecal contents. In contrast, compared to in vivo, a significant difference in frequency of transfer was obtained from one of the donors, X7, RifR when using laboratory liquid media (P < 0.05) and a significant difference in frequency was noted for donor HB101 when using laboratory agar (P < 0.01). When comparing the ratio of transfer frequency between the two different donors, the plasmid consistently transferred at a higher frequency in vivo and in in vitro caecal contents from X7, RifR than from HB101 (P < 0.05). The same pattern was observed when using in vitro intestinal extracts (P > 0.05). In contrast, when laboratory agar was used, the opposite occurred and the transfer was greater from HB101 than from X7, RifR (P < 0.05). The transfer frequency in laboratory liquid media was highly variable from donor X7, RifR and no significant difference (P > 0.05) could be seen between the two donors. We conclude that the intestinal extracts and caecal contents better reflect the natural environment than any of the laboratory media tested for the parameter investigated. Furthermore, it is shown that transfer using laboratory agar did not reflect in vivo conditions. The data supports the concept of using sub‐samples of an ecosystem as a microcosm for modelling the ecosystem.