D Ferme’s research while affiliated with University of Ljubljana and other places

Two representative strains of Gram-negative rumen bacteria from the genus Prevotella were used as model organisms in order to evaluate the effect of cinnamaldehyde (the secondary metabolite found in extracts of the Cinnamomum family) vs. sodium monensin on growth, cell size and cell protein production. Prevotella bryantii B(1)4 was found to be remarkably more resistant to the action of both compounds than Prevotella ruminicola 23. The approximate IC(50) concentrations of sodium monensin influenced the increase in cell size of both strains during growth, which was much more pronounced in the case of the B(1)4 strain. A similar effect was observed in strain B(1)4 when 1.438 mmol/L cinnamaldehyde was added to the growth medium, indicating a possible interference with cell division. The action of cinnamaldehyde on P. bryantii B(1)4 was concentration-dependent, in contrast to the effect observed on P. ruminicola 23.

An in vitro study in dual-flow continuous-culture fermentors was conducted with two different concentrations of monensin, cinnamaldehyde or garlic extract added to 1:1 forage-to-concentrate diet in order to determine their effects on selected rumen bacterial populations. Samples were subjected to total DNA extraction, restriction analysis of PCR amplified parts of 16S rRNA genes (ARDRA) and subsequent analysis of the restriction profiles by lab-on-chip technology with the Agilent's Bioanalyser 2100. Eub338-BacPre primer pair was used to select for the bacteria from the genera Bacteroides, Porphyromonas and Prevotella, especially the latter representing the dominant Gram-negative bacterial population in the rumen. Preliminary results of HaeIII restriction analysis show that the effects of monensin, cinnamaldehyde and garlic extract on the BacPre targeted ruminal bacteria are somewhat different in regard to targeted populations and to the nature of the effect. Garlic extract was found to trigger the most intensive changes in the structure of the BacPre targeted population. Comparison of the in silico restriction analysis of BacPre sequences deposited in different DNA databanks and of the results of performed amplified ribosomal DNA restriction analysis showed differences between the predicted and obtained HaeIII restriction profiles, and suggested the presence of novel, still unknown Prevotella populations in studied samples.

Rumen bacteriophage-lyzed bacterial strains of the genus Prevotella were isolated and preliminarily characterized. The strain TCl-1 the species P. bryantii was the only prevotella strain successfully infected with filter sterilized rumen fluid from a black-and-white Holstein cow. Two types of plaques were observed, both rather small and turbid. Preliminary electron microscopy observation showed that several morphologically different bacteriophages were present in these plaques. The plaque eluates were further used for the infection of other prevotella strains. The plaques produced by the bacteriophages were observed with two strains, i.e. P. bryantii B(1)4 and P. brevis GA33. The bacteriophages from both strains were examined by transmission electron microscopy and several morphologically different bacteriophages were observed, among others also a large virion with an icosahedral head with the diameter of approximately 120 nm. The bacteriophage was identified in plaques of bacterial cells of the strain GA33 and has an approximately 800 nm long helical tail, which places it among the largest ruminal bacteriophages described to date. Other bacteriophages from the same indicator strain as well as from P. bryantii B(1)4 strain were smaller and tail structures were not observed in all of them.

Sampling, inoculation and extraction procedures were elaborated for the isolation of bacteriophages from cattle rumen fluid. Several strains from four different species of rumen bacterial genus Prevotella were used as indicator organisms as well as Escherichia coli, Salmonella sp. and Yersinia enterocolitica strains. Only one strain, i.e. P.bryantii TC1-1 was successfully infected with filter sterilised rumen fluid inoculum containing rumen bacteriophages. Two plaque morphotypes were observed, both being turbid and rather small but clearly different in size. The preliminary transmission electron microscopy analysis showed that all observed bacteriophages have presumably icosahedral symmetry. The infection trials of other Prevotella strains with isolated bacteriophages from the strain TC1-1 were successful only with type strains of species P.bryanti and P.brevis respectively. Bacteriophage purification and long term storage procedures were elaborated.

Sampling, inoculation and extraction procedures were elaborated for the isolation of bacteriophages from cattle rumen fluid. Several strains from four different species of rumen bacterial genus Prevotella were used as indicator organisms as well as Escherichia coli, Salmonella sp. and Yersinia enterocolitica strains. Only one strain, i.e. P.bryantii TC1-1 was successfully infected with filter sterilised rumen fluid inoculum containing rumen bacteriophages. Two plaque morphotypes were observed, both being turbid and rather small but clearly different in size. The preliminary transmission electron microscopy analysis showed that all observed bacteriophages have presumably icosahedral symmetry. The infection trials of other Prevotella strains with isolated bacteriophages from the strain TC1-1 were successful only with type strains of species P.bryanti and P.brevis respectively. Bacteriophage purification and long term storage procedures were elaborated.