Biomedical subjects
Publications and source records attributed to V Kmet.
The effect of the oregano essential oil (EO) on lactobacilli and E. coli isolated from chicken was showed in the series of in vitro and in vivo experiments. Minimum inhibitory concentrations of amikacin, apramycin, streptomycin, neomycin against E. coli strains increased after oral application of EO.
Inhibitory effect of various lactobacilli against pathogenic strains of E. coli in model system Caco2 cells was determined by enumerating the number of adhering E. coli after pre-incubation (exclusion), post-incubation (displacement) or co-incubation (competition) with lactobacilli. Porcine E. coli strain F107 (F18ab, Stx2v) in the competition assay with porcine lactobacillus strain P10 gave bacterial counts 7.25 (log CFU per well); in the exclusion test it was only 7.05 while in displacement test it reached 7.29. The lowest E. coli counts adhering to Caco-2 cells were in exclusion assay (pre-incubation, Lactobacillus inoculated as the first). Pre-treatment of E. coli with our lactobacilli strains reduced the cultivable E. coli numbers.
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A total of 5 Bifidobacterium spp. isolated from pig and children' feces and 6 Lactobacillus spp. from chicken feces were examined for expression of aggregation, cell surface hydrophobicity (CSH) and adherence to intestinal mucin. Co-aggregation activity was seen in 3 strains of auto-aggregative bifidobacteria and 4 auto-aggregative strains of Lactobacillus spp. with 2 enterohemorrhagic E. coli (O157). CSH correlated with Lactobacillus auto-aggregating activity and adherence to mucin but the correlation between Bifidobacterium adherence to mucin and CSH was not confirmed.
The influence of cryptosporidial abomasitis on digestive anaerobic microflora in feedlot cattle with spontaneous Cryptosporidium andersoni colonization of abomasum was shown. Significant differences were found after the cultivation of abomasal content. Scopulariopsis brevicaulis was detected in damaged areas of infected abomasum. PCR analysis of ruminal fluid showed no differences between the two animal groups regarding qualitative composition of anaerobic and facultative anaerobic rumen microflora. The concentration of volatile fatty acids (acetate, propionate, butyrate) and ammonia in the rumen content showed that examined metabolic parameters were within normal limits except for ammonia content, which was higher in infected (291 mg/kg) than in healthy animals (203 mg/kg).
Thirty-three fully breast-fed infants aged between 1 and 12 weeks were screened for bifidobacteria in feces. Bifidobacteria counts in most fecal samples determined both by TPY agar and FISH procedure ranged from 10(8) to 10(11) CFU/g. Three infants did not contain any bifidobacteria in their fecal samples. One child was delivered by caesarean section and the other two by normal vaginal delivery. All bifidobacteria-free infants possessed Gram-positive regular rods as a major group of their fecal flora. These bacteria were identified as clostridia using genus-specific FISH probe. In bifidobacteria-positive samples, B. longum (57.9% of the samples) was the most frequently found species, followed by B. adolescentis (31.6%), B. bifidum (21.0%), B. breve (10.5%), B. pseudocatenulatum (5.3%), and B. dentium (5.3%).
A PCR-based method for rapid detection of food-borne thermotolerant campylobacters was evaluated through a collaborative trial with 12 laboratories testing spiked carcass rinse samples. The method showed an interlaboratory diagnostic sensitivity of 96.7% and a diagnostic specificity of 100% for chicken samples, while these values were 94.2 and 83.3%, respectively, for pig samples.
The effect of selected autoaggregative lactobacilli on enterohemorragic O157 Escherichia coli was studied. A total of 27 Lactobacillus sp. strains were examined for expression of autoaggregation and cell surface hydrophobicity. Autoaggregative isolates were obtained from the crops of chickens and vaginas of calves. Aggregative activity was seen between eight autoaggregative lactobacilli and six strains of enterohemorrhagic E. coli O157. The presence of aggregation-promoting factor (apf gene) was confirmed by polymerase chain reaction (PCR). We propose that autoaggregative lactobacilli that aggregate with enterohemorrhagic E. coli can express a class of APF proteins that exhibit the function of an aggregative mediator. The results of this study suggest that APF-producing lactobacilli could represent a further mechanism in the interaction of comensal microflora with enterohemorrhagic E. coli.
The presence of the fedA (gene coding F18 fimbriae) and genes coding STa and LTI enterotoxins and verotoxin Stx2v was determined in 30 E. coli strains isolated from weaned pigs with postweaning diarrhea (PWD) and edema disease (ED). The fedA gene was detected in 22 strains (73.3%). It was mostly associated with the presence of ST gene determinant (14 from 22 fedA positive strains, 63.6%). Two strains possessed ST/Stx2v or LTI/Stx2v combination of genes for both toxins and two strains were negative for investigated toxin determinants. Among 8 fedA-negative strains, five strains without gene determinants for toxins were detected. All 30 E. coli strains were investigated for their binding to crude intestinal mucin of a weaned pig fixed in wells of microtitre plates. Positive mucin binding was observed in most of strains, however, great differences were shown between individual strains. Nineteen strains were classified as strongly adherent, 10 strains as weakly adherent, and only one nonadherent strain was found. Three E. coli strains, selected among the best mucin binders, bound to mucin in a concentration-dependent manner. A high mucin binding by E. coli strains was observed only after their cultivation on blood agar plates. Their cultivation in LB broth or on McConkey agar plates had negative effect on the mucin binding by these strains. The mucin binding is not restricted by the presence of fedA gene because the strains displaying very good binding are found either among fedA positive (1, 602/2, 4/3, 576/6) or fedA negative (DK 6, DK 8) E. coli strains. E. coli strains with the highest mucin binding ability belong to potential ST producents (strains 1, 602/2, 4/3, 6/2, 602/4) while the strains without genes coding toxin production displayed lower binding to mucin substratum with exception of the strains ZV5 and 13.
A total of 20 strains of lactobacilli were isolated from the oesophagus and vagina of 20 sows at the time of partus. Aggregation activity was seen between six homofermentative autoaggregative lactobacilli and three strains of pathogenic Escherichia coli with F4, F5 and F6 fimbriae. The highest aggregation activity was observed between vaginal Lactobacillus acidophilus PV 32 or oesophageal OE 2/1 and E. coli with F4 (K88). The presence of aggregation-promoting factor (APF) was confirmed by polymerase chain reaction (PCR) with primers of a specific fragment the apf gene derived from human L. gasseri 4B2 in one oesophageal L. acidophilus strain OE 2/1. We propose that autoaggregative lactobacilli that aggregate with diarrhoeagenic E. coli can express a class of APF proteins that exhibit the function of an aggregation mediator.
Lactobacillus gasseri 4B2 is a human isolate characterised by a strong autoaggregating phenotype mediated by APF (aggregation-promoting factor), a secreted protein. Two primer pairs were developed for simultaneous amplification of a specific fragment of the APF gene and a highly conserved region of the 16S rRNA gene. The specificity of this protocol was checked in DNA samples isolated from single and mixed cultures of Lactobacillus. The same amplification protocol was successfully used directly adding whole bacterial cells to PCR reaction tubes. The suitability of this method for in vivo studies was investigated through feeding L. gasseri 4B2 to mice and analysing colony forming units obtained by plating faecal samples on selective medium. The methodology allows a fast and reliable identification of the target strain without any DNA extraction procedure.
A total of 60 Lactobacillus sp. strains were examined for expression of auto-aggregation and cell surface hydrophobicity. Isolates were obtained from the vagina of healthy women (n = 20). The results obtained showed that the occurrence of cell surface hydrophobicity correlated with auto-aggregative activity in 12 homofermentative Lactobacillus sp. strains. The aggregation mechanism was mediated by the presence of an aggregation promoting factor (APF) in one Lactobacillus sp. strain, HV 142. APF was confirmed by DNA hybridisation with a 1.3 kb PstI DNA fragment of recombinant plasmid pFDS containing the reading open frame of the APF gene derived from Lactobacillus plantarum 4B2. Coaggregation activity was seen in three strains of auto-aggregative human vaginal lactobacilli and five strains of P-fimbriated uropathogenic Escherichia coli. Moreover, one of four Lactobacillus sp. strains (HV 389) aggregated with two of five E. coli tested. These results suggest that APF producing lactobacilli could represent a further mechanism in the interaction of commensal microflora with strains of uropathogenic E. coli.
The transformation and subsequent regeneration of ruminal strain Streptococcus bovis AO24/85 protoplasts by plasmid DNA was studied. The best stabilizer for regeneration of protoplasted cells was 5% sucrose in the regeneration medium and in the agar plates. Optimal concentration of polyethylene glycol 6000 in the transformation medium was 25% for both plasmids tested. Addition of Ca2+ and Mg2+ ions (2.5 mmol l-1) to the transformation medium increased the proportion of regenerated cells. Transformation frequencies were 3 x 10(3) transformants per microgram of pNZ12 and 2.4 x 10(2) per microgram of pJK108, respectively.
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Autoaggregation was frequently encountered among intestinal lactobacilli isolated from weaned pigs. The aggregation mechanism was shown to be mediated by the production of a proteinaceous aggregation-promoting factor in two strains of Lactobacillus reuteri. A 32 kDa aggregation-promoting protein was detected in these strains by cross-reaction with rabbit polyclonal antibodies for Aggregation-Promoting Factor produced by the human isolate Lact. plantarum 4B2. Coaggregation reactions of Lact. reuteri strains with pathogenic and non-pathogenic Escherichia coli were detected.
Three Streptococcus bovis strains were tested in biotype assay and examined for the adherence to cells of rumen epithelium primoculture. The adherence pattern of ruminal streptococci in phosphate buffered saline at pH values ranging from 4.1 to 8.5 was determined. Our isolates of Streptococcus bovis strains adhered best at pH 7.0-7.3. To characterize the adhesive determinants, the bacterial cells were exposed to various treatments. Protease treatment dramatically decreased the adherence of all Streptococcus bovis strains, thus suggesting that the determinants responsible for the adherence are largely proteinaceous. Carbohydrates could be also significantly involved in the active sites of bacterial surface because metaperiodate-treated cells adhered much more poorly than control, sodium iodate-treated cells. Addition of carbohydrates (lactose, maltose and saccharose) had no significant effect on the adherence of Streptococcus bovis strains although a slight decrease in the adhesion was detected.
There is good evidence that the bacterial and fungal probiotics are effective in the manipulation of rumen development and function. The effects of adherent Streptococcus bovis preparations were manifested by significant increase of adherent S. bovis bacteria and alpha amylase activity in the rumen wall of young ruminants. Fungal probiotics based on Saccharomyces cerevisiae and Aspergillus oryzae are rapidly gaining acceptance as a means of improving productivity in adult ruminants. However, fungal probiotics may also have a complementary role to bacterial probiotics in young animals. Dietary yeast improved performance of calves and lambs in terms of feed intake and live weight gain and Aspergillus foetidus was effective in altering rumen fermentation in newly weaned lambs.
The occurrence and antibiotic-resistance spectrum of some strains of rumen streptococci were investigated in calves of four age categories in the milk diet period at 1-3, 4-7, 8-14 days of age and 6-8 weeks of age. More than 75% of the strains isolated from the calves of all age categories were resistant to more than one tested antibiotic (tetracycline, neomycin, erythromycin, streptomycin, chloramphenicol). The average number of streptococci isolated from the ingesta of calves during the period of observation was 10(5) per ml. The per cent of resistant strains showed an increasing trend from birth to the age of 8-14 days (Tab. I). The occurrence of resistant strains in 6-8 week calves was even lower than in the calves of the youngest age category. A four-marker type of resistance (41.6-50%) prevailed within 14 days of age. Two-marker types of resistance (42.6%) occurred most frequently in the calves of the oldest age category. Tab. II shows the spectrum of resistant strains. Tetracycline-resistant amylolytic strains of Streptococcus bovis were selected from among the isolated strains to be used as donor strains in conjugation experiments. Successful resistance transmission was observed in solid-surface conjugation. The transmission frequency ranged within the transconjugate values of 10(-3) to 10(-5) per donor cell within the species (Tab. III). A chromosomally rifampicin-resistant strain of Streptococcus bovis BM114 was used as a recipient as it did not contain any plasmids and was sensitive to the tested antibiotics. Agarose-gel electrophoresis was used to visualize the presence of plasmid DNA in donor strains (Fig. 1).