Biomedical subjects

P Pristas

Publications and source records attributed to P Pristas.

At least 19 recordsLinked to original sources

PubMed2006

Spreading and mutability of Selenomonas ruminantium plasmids.

Two small plasmids from Selenomonas ruminantium strain 19D were cloned in Escherichia coli and completely characterized. Sequence comparison indicated that the plasmids are similar to those reported in genetically vaguely related S. ruminantium strain S20. Small 1.4-kb plasmids pSRD191 and pONE430 are only distantly related (approximately 30 % for deduced Rep protein amino acid sequence) but possess a short highly conserved region outside rep gene. Larger plasmids pSRD192 and pONE429 possess large identical DNA regions in an otherwise dissimilar background. Recombination is proposed as an important mechanism of evolution and spreading of S. ruminantium plasmids.

Animals↗

PubMed2006

Limited genetic variability in Megasphaera elsdenii strains.

Levels of phenotypic and genotypic diversity among seven Megasphaera elsdenii strains recovered from rumen contents of cattle, sheep and lambs were determined by a combination of antibiotic-resistance analysis and PCR fingerprint techniques targeted both to the ribosomal RNA operon (ARDRA, RISA) and the whole genome (ERIC-PCR, RAPD-PCR). Despite exhibiting different antibiotic resistance profiles, the tested strains represent genetically nearly identical isolates. Close genetic relatedness was found among M. elsdenii isolates that originated from vastly different habitats worldwide, as revealed by the comparison of 16S rDNA sequences.

Animals↗

PubMed2006

New species of rumen treponemes.

Three strains of rumen treponemes were isolated and partially characterized. The strains differed significantly one from another in morphology, fermentation characteristics and plasmid profiles. Their genetic variability was assayed using DNA-based molecular approaches. Easily differentiated ARDRA (amplified ribosomal DNA restriction analysis) patterns indicated that the strains represent different bacterial species.

Animals↗

PubMed2006

Mercuric reductase gene transfer from soil to rumen bacteria.

Conjugal transfer between soil bacterial population and microorganisms isolated from the rumen of herbivores from mercury-polluted area was investigated. The transfer of merA encoding mercury-resistance plasmids from soil bacteria Enterobacter cloacae and Enterococcus durans into two ruminal isolates Citrobacter freundii and Bacillus subtilis was observed. Approximately the same frequency of mobilization in mating experiments was observed for both Gram-negative (approximately 2.5 x 10(-8), transconjugants-to-recipient ratio) and Gram-positive (approximately 1.3 x 10(-8)) bacteria.

Animals↗

PubMed2005

Seasonal dynamics of antibiotic-resistant Enterobacteriaceae in the gastrointestinal tract of domestic sheep.

Considerable variation in counts of antibiotic-resistant enterobacteria in the ovine gastrointestinal tract was observed. The occurrence of ruminal and fecal isolates resistant to ampicillin (Ap), kanamycin (Km) and tetracycline (Tc) culminated in summer months, followed by rapid decline in subsequent months. Using PCR the tem1bla (Apr), aphA1 (Kmr) and tetB (Tcr) genes were found to be predominant. Under in vitro conditions all resistance genes were transferable into laboratory Escherichia coli strain with relatively high frequency (10(-3) transconjugants per recipient).

Animals↗

PubMed2005

Occurrence of restriction-modification systems in ruminal butyrate-producing bacteria.

Thirty-five strains of ruminal bacteria belonging to the former Butyrivibrio fibrisolvens species were screened for the presence of site-specific restriction endonuclease and modification methyltransferase activities. Seven strains possessed endonuclease activities detectable in crude cell extracts. The recognition sequences and optimal reaction conditions for seven of them were determined. Five enzymes were found to be isoschizomers of type II endonucleases (EcoRV, NsiI, AseI (2x) and SauI), one was type IIS (FokI) and two remained unknown. The optimal reaction buffer was found to be a low ionic strength buffer and all enzymes possessed sufficient activity at 39 degrees C. The presence of DNA modification among all strains was also determined. Most of the methylation activities correlated with restriction activities, yet some strains possessed unaccompanied modification methyltransferases.

Journal Article↗

PubMed2004

A re-appraisal of the diversity of the methanogens associated with the rumen ciliates.

The diversity of methanogenic archaea associated with different species of ciliated protozoa in the rumen was analysed. Partial fragments of archaeal SSU rRNA genes were amplified from DNA isolated from single cells from the rumen protozoal species Metadinium medium, Entodinium furca, Ophryoscolex caudatus and Diplodinium dentatum. Sequence analysis of these fragments indicated that although all of the new isolates clustered with sequences previously described for methanogens, there was a difference in the relative distribution of sequences detected here as compared to that of previous work. In addition, many of the novel sequences, although clearly of archaeal origin have relatively low identity to the sequences in database which are most closely related to them.

Animals↗

PubMed2004

Bacteriocin production and sensitivity.

Using an overlay test the production of bacteriocin-like activity and resistance was found in 6 of the total of 7 isolates (5 enterococcal and 2 streptococcal). The majority of strains were sensitive to all bacteriocin producers tested. After acetone precipitation, bacteriocin precipitates were tested for thermal stability. They exhibited high stability at 37 degrees C and some of them were active even after a treatment at 95 degrees C.

Anti-Bacterial Agents↗

PubMed2004

Occurrence of plasmid-mediated ampicillin resistance among enterobacteria from the ovine rumen.

Seasonal samplings of rumen and fecal populations of Enterobacteriacae from sheep digestive tract were done to elucidate potential occurrence and spreading of antibiotic resistance in the environment. Thus 350 rumen and fecal isolates were tested for ampicillin (Amp) resistance in single sampling. Low frequency of Amp resistance (from 0 to 15%) was observed. The occurrence of tem1 encoded Amp resistance confirmed by PCR was observed among both rumen and fecal isolates. The small tem1 carrying plasmid and its transfer (mobilization) was detected and partially characterized after conjugation to laboratory Escherichia coli strain.

Ampicillin↗

PubMed2004

GATC-specific restriction--modification systems in ruminal bacteria.

The GATC-specific restriction and modification activities were analyzed in 11 major bacterial representatives of ruminal microflora. Modification phenotype was observed in 13 out of 40 ruminal strains. MboI isoschizomeric restriction endonucleases were detected in 10 bacterial strains tested; three strains lacked any detectable corresponding endonuclease activity. The only examined strain of Mitsuokella multi-acida was found to possess a different type of endonuclease activity. This is the first report on restriction activity in ruminal treponemes M. multiacida and Megasphaera elsdenii.

Animals↗

PubMed2004

GATC-specific restriction and modification systems in treponemes.

AIMS: To investigate the presence of GATC-specific modification and restriction activities in rumen isolates of Treponema sp. METHODS: The presence of N6-methyladenine within GATC (Dam) sequences was analysed using isoschizomeric restriction endonucleases having different sensitivities to the methylation of the target sequence. A fast screening method was used for testing of site-specific endonuclease activities directly in crude cell extracts. Three out of six rumen isolates of Treponema sp. showed restriction activities. Restriction endonucleases were further purified by Heparin-Sepharose chromatography. Using PCR and specific primers, no sequence homologous to the T. pallidum dam gene was found. CONCLUSIONS: Three rumen treponemal strains were documented to possess MboI isoschizomeric restriction-modification systems. SIGNIFICANCE: This is the first report on restriction activity in rumen treponemes.

Adenine↗

PubMed2004

Assessment of ciliates in the sheep rumen by DGGE.

AIMS: This work was carried out to develop a rapid molecular profiling technique to screen ciliate populations in the rumen of sheep. METHODS AND RESULTS: DGGE was used to study the ciliate diversity in the rumen of sheep. There was considerable variation between sheep which were co-housed, and fed the same diet. However, no difference in the major banding patterns was detected, when samples were collected from a single sheep sampled at different points. Following dietary changes, use of a pair-wise comparison of lanes, demonstrated that although there was still diversity between the ciliate population of sheep, the effects as a result of dietary changes were greater. CONCLUSIONS: The technique generated molecular profiles which are sufficiently different to allow comparison between samples, and to permit molecular ecological studies on the rumen ciliate population. SIGNIFICANCE AND IMPACT OF THE STUDY: The outcome of this study means that ciliate diversity in the rumen may now be studied by those unfamiliar with morphological identification of these organisms.

Animal Feed↗

PubMed2002

Are ruminal bacteria protected against environmental stress by plant antioxidants?

AIMS: To investigate the activity response of the antioxidant enzymes superoxide dismutase (SOD) and glutathione peroxidase (GSHPx) of the rumen bacterium Streptococcus bovis following exposure to mercury(II) chloride (HgCl(2) in the presence of plant antioxidants. METHODS AND RESULTS: Streptococcus bovis was grown with 0 or 5 microg ml(-1) of HgCl(2) alone or together with antioxidant substances (AOS): seleno-l-methionine (Se), alpha-tocopherol (alpha toc), beta-carotene (beta car), melatonin (mel). The activities of SOD and GHPx were estimated in supernatants of disrupted bacterial cells. A significant decrease in the Strep. bovis SOD activity in the presence of HgCl(2) and tested AOS, except mel, was observed. The GSHPx activity of Strep. bovis was under the same cultivation conditions nonsignificantly changed and a significant decrease in the GSHPx activity was recorded only in the presence of beta car. CONCLUSIONS: The positive effect of Se, alpha toc and beta car on the elimination of environmental stress, evoked by mercury, in ruminal bacterium Strep. bovis in vitro was documented. SIGNIFICANCE AND IMPACT OF THE STUDY: The potential role of plant antioxidants in elimination of the environmental stress of ruminal bacteria evoked by heavy metals is discussed.

Animals↗

PubMed2001

Plasmids of Selenomonas ruminantium and development of host-vector system.

A high frequency of plasmids was detected in the rumen bacterium Selenomonas ruminantium. Plasmids 0.9-20 kb in size were detected in more than 50% tested strains. Densitometric analysis indicated that plasmid DNA could represents more than 25% of total cellular DNA. Up to six plasmids were detected in strain S. ruminantium 18. Two smallest cryptic plasmids pSRD181 and pSRD182 from this strain were cloned into Escherichia coli vector pBluescriptSK+ and partially characterized. The plasmid pSRD181 is 1.4 kb and pSRD182 is 2.0 kb. While computer analysis of pSRD181 sequence data showed high homology with replication protein of Staphylococcus aureus plasmids, the pSRD182 sequence showed no significant homology in GenBank data. Strain S. ruminantium 28 was successfully transformed with pJW1 derived plasmid pJ1B1 using ampicillin resistance gene as marker. This is the first report on transformation of selenomonads with foreign DNA.

Ampicillin Resistance↗

PubMed2001

Bacteriocins of ruminal bacteria.

Similar sequences of distribution of structural genes encoding enterocin A (isolated from the ruminal strain E. faecium BC25) and enterolysin A (isolated from the ruminal amylolytic strain S. bovis II/1) were demonstrated by PCR using oligonucleotide primers specific for these bacteriocins within the ruminal enterococcal and streptococcal strains. Variable occurrence of these bacteriocins was found within the populations of Gram-positive ruminal cocci.

Animals↗

PubMed2001

Restriction and modification systems of ruminal bacteria.

A high frequency of type II restriction endonuclease activities was detected in Selenomonas ruminantium but not in other rumen bacteria tested. Eight different restriction endonucleases were characterized in 17 strains coming from genetically homogeneous local population. Chromosomal DNA isolated from S. ruminantium strains was found to be refractory to cleavage by various restriction enzymes, implying the presence of methylase activities additional to those required for protection against the cellular endonucleases. The presence of Dam methylation was detected in S. ruminantium strains as well as in several other species belonging to the Sporomusa subbranch of low G + C Gram-positive bacteria (Megasphaera elsdenii, Mitsuokella multiacidus).

Animals↗

PubMed2001

Isolation and characterization of enterocin BC25 and occurrence of the entA gene among ruminal gram-positive cocci.

Enterocin BC25, a bacteriocin produced by Enterococcus faecium BC25 isolated from the rumen of cow was purified to homogeneity and sequenced. Twenty amino acids were identified in the peptide chain (TTHSGKYYGNGVYCT-KNKCT), identical to the N-terminal sequence of enterocin A. The DNA sequence of the enterocin BC25 structural gene and putative immunity protein exhibited high similarity to the entA gene. The occurrence of a 726 bp amplicon containing the enterocin A structural gene was studied among gram-positive ruminal cocci by PCR. Our results showed wide occurrence of the entA structural gene among ruminal enterococcal and streptococcal bacterial strains tested, and indicate variable ability to express bacteriocin production and resistance.

Amino Acid Sequence↗

PubMed2000

Characterisation of endoglucanases EGB and EGC from Fibrobacter succinogenes.

The enzymatic properties of two endoglucanases from Fibrobacter succinogenes, EGB and EGC, were analysed. EGB and EGC were purified from recombinant Escherichia coli cultures expressing their gene. The failure of purification of EGB by classical techniques led us to produce antipeptide antibodies that allowed immunopurification of the protein from E. coli as well as its detection in F. succinogenes cultures. Synthetic peptides were selected from the predicted primary structure of EGB, linked to bovine serum albumin and used as immunogens to obtain specific antibodies. One of the polyclonal antipeptide antisera was used to purify EGB. EGC was purified by affinity chromatography with Ni-NTA resin. The endo mode of action of the two enzymes on carboxymethyl-cellulose was different. The values of K(m) and V(max) were respectively 13.6 mg/ml and 46 micromol/min mg protein for EGB, and 7 mg/ml and 110 micromol/min mg protein for EGC. The reactivity of the antipeptide and the anti-EGC sera with F. succinogenes proteins of molecular mass different from that of EGB and EGC produced in E. coli suggested post-translational modification of the two enzymes in F. succinogenes cultures. Expression of endB and endC genes in F. succinogenes was confirmed by RT-PCR.

Animals↗