Use of antibiotic resistance mutations to track strains of obligately anaerobic bacteria introduced into the rumen of sheep

Flint, Harry J.; Bisset, Jacqueline; Webb, Jennifer A.

doi:10.1111/j.1365-2672.1989.tb03393.x

Cited by 35 publications

(24 citation statements)

References 20 publications

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“…The ability of the NO4 to attach to feed particles has not been assessed, but this characteristic may allow decreased washout of the recombinant strain. Other speculations have been made in relation to the poor survival of newly introduced bacteria in the rumen 2,4,7,8,11) Results indicate that the sheep rumen is more favorable for the survival of NO4 compared to goat. The unidentified limiting factors may relate to the protozoa population, bacteria diversity, phages, trace nutrients and antibacterial compounds such as bacteriocins.…”

Section: Resultsmentioning

confidence: 99%

Survival of a Recombinant Rumen Bacterium in the Rumen of Sheep

Kobayashi

Yamada

Yamamoto

2001

Nihon Chikusan Gakkaiho

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Survival of the recombinant rumen bacterium Butyrivibrio fibrisolvens NO4, expressing an exogenous xylanase and erythromycin resistance, was assessed in vitro and in vivo. Selective enumeration of the recombinant strain from indigenous rumen bacteria was performed using an erythromycin-supplemented anaerobic medium.When incubated in vitro with mixed rumen bacteria and protozoa obtained from sheep, the numbers of the recombinant strain stabilised at 103/ml (48h). When incubated with mixed microbes from the goat rumen, strain NO4 disappeared 48h after inoculation. The presence of protozoa in the culture tended to decrease the numbers of strain NO4 ten-fold compared to bacteria alone. In vivo, numbers declined rapidly after inoculation, and the recombinant strain was no longer detectable in the sheep rumen after 144h.Animal Science Journal 72 (4) Recent developments in genetic engineering are applicable to rumen microbes, allowing improvements to their metabolic capabilities, e.g. fibrolysis, protein synthesis and detoxification6). One such application is close to be at the animal production stage3). Studies tracking Prevotellaruminicola2) and a Ruminococcus albus transconjugant8), inoculated into the rumen, suggested that rapid elimination or poor establishment occurs with bacteria that have been maintained in culture for a considerable time. However, Gregget al.3) observed that inoculated wild and recombinant Butyrivibrio fibrisolvens in the rumen of sheep were detectable by PCR even at 105-7 dilutions. Identification of factors important for survival and establishment of specific bacterial strains (including recombinants) in the rumen is required. The present paper describes the survival of the recombinant rumen anaerobic bacterium Butyrivibrio fibrisolvens NO4 in simple batch culture and the sheep rumen. Materials and MethodsButyrivibrio fibrisolvens NO45) expressing a Eubacterium ruminantium xylanase gene (xynA) and a was used. NO4 was anaerobically grown until mid logarithmic phase (approximately, OD600=0.5) in a reduced CCA medium1), and used directly for in vitro inoculation.A rumen cannulated sheep and goat (weighing 45 and 21kg) were used as rumen fluid donors for in vitro experiments. The animals were fed a cubed alfalfa hay and concentrate diet (400g:100g (sheep) and 200g:50g (goat) respectively) twice daily.Rumen fluid was collected prior to morning feeding, strained through 2 layers of surgical gauze and centrifuged of the rumen fluid was incubated with an acetate buffer at 37 were washed twice with an anaerobic dilution solution9), resuspended in McDougal's artificial saliva and combined if necessary (bacteria (B) and bacteria plus protozoa (BP)). Both B and BP were mixed with 2g ground alfalfa hay, for the simple batch culture (100ml in volume) experiments. Samples were taken at 0, 2, 4, 6, 8, 12, 24 and 48h after inoculation of strain NO4 to enumerate total bacteria , strain NO4 and protozoa.The donor sheep was used as the recipient for strain NO4 inoculation in vivo (managed according to M...

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Section: Resultsmentioning

confidence: 99%

Survival of a Recombinant Rumen Bacterium in the Rumen of Sheep

Kobayashi

Yamada

Yamamoto

2001

Nihon Chikusan Gakkaiho

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“…3a, b) showed that dosed organisms declined post-dosing, which is typical of dosing studies (Flint et al, 1989 ;Miyagi et al, 1995 ;Attwood et al, 1988), illustrating that ecological principles governing the persistence of bacterial inoculants in complex microbial communities are not well understood. It is, however, apparent that persistence is probably a consequence of community-level reproductive strategies in which community processes limit the relative abundance of individual organisms (Caldwell et al, 1997).…”

Section: Discussionmentioning

confidence: 97%

“…A second, and very significant, issue is whether introduced organisms can multiply and persist at levels in the rumen that are sufficient to improve fibre digestion. To address these questions, we increased the relative abundance of highly fibrolytic Ruminococcus by daily dosing over a period of 8 d. Introduced strains of ruminal bacteria often decline rapidly after dosing (Flint et al, 1989 ;Miyagi et al, 1995 ;Attwood et al, 1988), making it difficult to measure the effect of the dosed strains on fibre digestion. We hoped that our protocol would enable us to measure fibre digestion in the presence of high numbers of dosed strains and that repeated dosing would enable inoculants to establish in the rumen.…”

Section: Abbreviationsmentioning

confidence: 99%

Repeated ruminal dosing of Ruminococcus spp. does not result in persistence, but changes in other microbial populations occur that can be measured with quantitative 16S-rRNA-based probes

et al. 2001

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Digestibility of fibre in ruminants may be improved by the introduction of highly fibrolytic strains of ruminal bacteria. This approach may be feasible if, for example, strains of Ruminococcus that are significantly more fibrolytic than the normal population of Ruminococcus are used for inoculation purposes. Introduced strains of bacteria, irrespective of ecosystem, often decline after inoculation, and in this study, highly fibrolytic strains of Ruminococcus were continuously dosed to ensure that measurements of fibre digestion were made in the presence of significant numbers of the introduced bacteria. During dosing the total culturable count increased significantly (P T 005), but declined post-dosing. The level of dosed Ruminococcus, and total Ruminococcus, Fibrobacter succinogenes and eukaryotes measured by 16S rRNA probes increased significantly (P T 005) during the dosing period, but also declined post-dosing. When in vitro nylon bag digestibility, feed intake or whole-tract digestibility was measured, no improvement could be measured.

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“…These included rifampicin-resistant mutants of Bacteroides multiacidus strains 46/5(2) and FlOO, and of Selenomonas ruminantium strains 521C12 and FB314 (Flint et al, 1989a), and rifampicin-and erythromycin-resistant mutants of Ruminococcus albus strains 7 and 40 (Amino v et al, 1994;Miyagi et aI., 1995). These included rifampicin-resistant mutants of Bacteroides multiacidus strains 46/5(2) and FlOO, and of Selenomonas ruminantium strains 521C12 and FB314 (Flint et al, 1989a), and rifampicin-and erythromycin-resistant mutants of Ruminococcus albus strains 7 and 40 (Amino v et al, 1994;Miyagi et aI., 1995).…”

Section: Mutagenesis In Rumen Bacteriamentioning

confidence: 99%