Evaluation of the infB and rpsB gene fragments as genetic markers intended for identification and phylogenetic analysis of particular representatives of the order Lactobacillales.

Detailed differentiation, classification, and phylogenetic analysis of the order Lactobacillales are performed using molecular techniques that involve the comparison of whole genomes, multilocus sequence analysis, DNA-DNA hybridisation, and 16S rRNA sequencing. Despite the wide application of the latter two techniques, issues associated with them are extensively discussed. Although complete genomic analyses are the most appropriate for phylogenetic studies, they are time-consuming and require high levels of expertise. Many phylogenetic/identification markers have been proposed for enterococci, lactobacilli, streptococci, and lactobacilli. However, none have been established for vagococci and some genera within the order Lactobacillales. The objective of the study was to find novel alternative housekeeping genes for classification, typing, and phylogenetic analysis of selected genera within the order Lactobacillales. We designed primers flanking variable regions of the infB (504 nt) and rpsB (333 nt) genes and amplified and sequenced them in 56 strains of different genera within the order Lactobacillales. Statistical analysis and characteristics of the gene regions suggested that they could be used for taxonomic purposes. Phylogenetic analyses, including assessment of (in)congruence between individual phylogenetic trees indicated the possibility of using the concatenation of the two genes as an alternative tool for the evaluation of phylogeny compared with the 16S rRNA gene representing the standard phylogenetic marker of prokaryotes. Moreover, infB, rpsB regions and their concatenate were phylogenetically consistent with two widely applied alternative genetic markers in taxonomy of particular Lactobacillales genera encoding the 60 kDa chaperonin protein (GroEL-hsp60) and phenylalanyl-tRNA synthetase, alpha subunit (pheS).

Anticlostridial agent 8-hydroxyquinoline improves the isolation of faecal bifidobacteria on modified Wilkins-Chalgren agar with mupirocin.

UNLABELLED: The need for suitable selective cultivation media for the isolation of Bifidobacterium spp. continues to be a real concern in the field of intestinal microbiology. Isolation of bifidobacteria from human and animal faecal samples using selective agar plating may be problematic especially in samples with increased clostridial counts than bifidobacterial counts. Due to the absence of anticlostridial agents in existing selective media, clostridia can displace bifidobacteria resulting in incorrect estimation of their counts. Therefore, we supplemented the existing selective medium 'modified Wilkins Chalgren agar with mupirocin' (MWM) with 90 mg l(-1) of 8-hydroxyquinoline (8HQ), which was recently proved to act selectively against clostridia. The newly composed 'modified Wilkins-Chalgren agar with 8HQ' (MWMQ) was tested on pure bifidobacterial and clostridial strains, their mixtures, and using faecal samples of mammalian origin; its selectivity was evaluated by genus-specific identification of isolates. The results demonstrated that the presence of 8HQ in this agar eliminated the growth of nonbifidobacterial strains on MWMQ compared to that on MWM, whereas the recovery of bifidobacterial counts was at satisfactory levels. In conclusion, MWMQ could be recommended for bifidobacterial isolation from human and animal faeces especially when bifidobacteria are not numerically dominant and there are chances of clostridial contamination. SIGNIFICANCE AND IMPACT OF THE STUDY: Routine isolation of bifidobacteria from mammalian faeces does not use a reliable selective agar with an anticlostridial agent. Overgrowth of clostridia may result in incorrect estimation of bifidobacterial counts. Thus, in order to improve the selectivity of existing media for bifidobacterial isolation, we chose the modified Wilkins-Chalgren agar with mupirocin and supplemented it with 8-hydroxyquinoline (8HQ), a molecule that shows anticlostridial activity without affecting the growth of bifidobacteria. This newly composed medium showed enhanced selectivity and specificity compared to the original medium and therefore, can be recommended for the isolation of bifidobacteria from mammal faeces.

Lactobacillus bombi sp. nov., from the digestive tract of laboratory-reared bumblebee queens (Bombus terrestris).

Three bacterial strains belonging to the genus Lactobacillus were isolated from the digestive tracts of laboratory-reared bumblebee queens (Bombus terrestris) using MRS agar under anaerobic conditions. The isolates were identified according to 16S rRNA gene sequence analysis as undescribed members of the genus Lactobacillus, with the highest 16S rRNA gene sequence similarity (96.9 %) to the uncharacterized bacterial strain Lactobacillus sp. Mboho2r2 isolated from the stomach of a European honeybee (Apis mellifera). Lactobacillus tucceti was found to be the closest related species with a validly published name, with 92.9 % 16S rRNA gene sequence similarity to the type strain. However, phylogenetic analyses based on different markers revealed that this species is phylogenetically very distant from the novel strains. The DNA G+C content of the proposed type strain BTLCH M1/2(T) is 37.8 mol%. The fatty acids C(19 : 1)ω6c and/or C(19 : 0) cyclo ω10c/19ω6, C(18 : 1)ω9c and C(16 : 0) were predominant in all strains. Diphosphatidylglycerol, phosphatidylglycerol, a phospholipid, seven glycolipids and two phosphoglycolipids were detected in the novel strains. Growth was observed at 47 °C. The peptidoglycan type A4α L-Lys-D-Asp was determined for strain BTLCH M1/2(T). Genotypic characteristics and phylogenetic analyses based on the phylogenetic markers hsp60, pheS, rpoA and tuf as well as phenotypic characteristics and the results of chemotaxonomic analyses confirmed that the new isolates belong to a novel species of the genus Lactobacillus, for which the name Lactobacillus bombi sp. nov. is proposed. The type strain is BTLCH M1/2(T) ( = DSM 26517(T) = CCM 8440(T)).

Vagococcus entomophilus sp. nov., from the digestive tract of a wasp (Vespula vulgaris).

Three unknown Gram-stain-positive, catalase-negative, facultatively anaerobic and coccus-shaped strains of bacteria were isolated from the digestive tracts of wasps (Vespula vulgaris). Analysis of 16S rRNA gene sequences revealed that these strains had identical sequences and showed that Vagococcus salmoninarum, with 96.2% sequence similarity, was the closest phylogenetic neighbour. Further analyses based on hsp60 and pheS gene sequences of representatives of the family Enteroccocaceae and genotypic and phenotypic characterization using (GTG)5-PCR fingerprintings, EcoRI ribotyping, DNA G+C content, whole-cell protein profiling, cellular fatty acid profiles analysis and extensive biotyping confirmed that the investigated strains were representatives of a novel bacterial species within the genus Vagoccocus for which the name Vagoccocus entomophilus sp. nov. is proposed. The type strain is VOSTP2(T) ( = DSM 24756(T) = CCM 7946(T)).

Lactobacillus rodentium sp. nov., from the digestive tract of wild rodents.

Three strains of regular, long, Gram-stain-positive bacterial rods were isolated using TPY, M.R.S. and Rogosa agar under anaerobic conditions from the digestive tract of wild mice (Mus musculus). All 16S rRNA gene sequences of these isolates were most similar to sequences of Lactobacillus gasseri ATCC 33323T and Lactobacillus johnsonii ATCC 33200T (97.3% and 97.2% sequence similarities, respectively). The novel strains shared 99.2-99.6% 16S rRNA gene sequence similarities. Type strains of L. gasseri and L. johnsonii were also most related to the newly isolated strains according to rpoA (83.9-84.0% similarities), pheS (84.6-87.8%), atpA (86.2-87.7%), hsp60 (89.4-90.4%) and tuf (92.7-93.6%) gene sequence similarities. Phylogenetic studies based on 16S rRNA, hsp60, rpoA, atpA and pheS gene sequences, other genotypic and many phenotypic characteristics (results of API 50 CHL, Rapid ID 32A and API ZYM biochemical tests; cellular fatty acid profiles; cellular polar lipid profiles; end products of glucose fermentation) showed that these bacterial strains represent a novel species within the genus Lactobacillus. The name Lactobacillus rodentium sp. nov. is proposed to accommodate this group of new isolates. The type strain is MYMRS/TLU1T (=DSM 24759T=CCM 7945T).

Alloscardovia macacae sp. nov., isolated from the milk of a macaque (Macaca mulatta), emended description of the genus Alloscardovia and proposal of Alloscardovia criceti comb. nov.

A novel bacterial strain, designated M8(T), was isolated from milk of a female macaque bred in captivity. The strain was Gram-stain-positive, anaerobic, irregular coccoid-rod-shaped without catalase activity. Analysis of 16S rRNA gene sequence similarity revealed that the isolate was most closely related to Alloscardovia omnicolens CCUG 31649(T) (96.4%) and Metascardovia criceti OMB105(T) (96.6%). Sequences of hsp60, fusA, and xfp genes also confirmed that the strain was most closely related to the type strains of A. omnicolens and M. criceti. The isolate produced fructose-6-phosphate phosphoketolase which is in agreement with classification within the family Bifidobacteriaceae. The major fatty acids were C18 : 1ω9c (35.8%), C16 : 1 (6.2 %) and C14 : 0 (5.7 %). Polar lipid analysis revealed five different glycolipids, two unidentified phospholipids and diphosphatidylglycerol. The peptidoglycan was of the type A4α l-Lys-d-Asp with the presence of d(l)-alanine, d-glutamine, d-asparagine and l-lysine. The DNA G+C content of strain M8(T) was 50.1 mol%. On the basis of genetic, phylogenetic and phenotypic data, strain M8(T) represents a novel species of the genus Alloscardovia for which the name Alloscardovia macacae sp. nov. is proposed. The type strain is M8(T) ( = DSM 24762(T) = CCM 7944(T)). In addition, our results also revealed that Alloscardovia omnicolens DSM 21503(T) and Metascardovia criceti DSM 17774(T) do not belong to different genera within the family Bifidobacteriaceae. We therefore propose to reclassify Metascardovia criceti as Alloscardovia criceti comb. nov. An emended description of the genus Alloscardovia is also provided.

Interference of Bifidobacterium choerinum or Escherichia coli Nissle 1917 with Salmonella Typhimurium in gnotobiotic piglets correlates with cytokine patterns in blood and intestine.

The colonization, translocation and protective effect of two intestinal bacteria - PR4 (pig commensal strain of Bifidobacterium choerinum) or EcN (probiotic Escherichia coli strain Nissle 1917) - against subsequent infection with a virulent LT2 strain of Salmonella enterica serovar Typhimurium were studied in gnotobiotic pigs after oral association. The clinical state of experimental animals correlated with bacterial translocation and levels of inflammatory cytokines [a chemokine, interleukin (IL)-8, a proinflammatory cytokine, tumour necrosis factor (TNF)-α and an anti-inflammatory cytokine, IL-10] in plasma and intestinal lavages. Gnotobiotic pigs orally mono-associated with either PR4 or EcN thrived, and bacteria were not found in their blood. No significant inflammatory cytokine response was observed. Mono-association with Salmonella caused devastating septicaemia characterized by high levels of IL-10 and TNF-α in plasma and TNF-α in the intestine. Di-associated gnotobiotic pigs were given PR4 or EcN for 24 h. Subsequently, they were infected orally with Salmonella and euthanized 24 h later. Pigs associated with bifidobacteria before Salmonella infection suffered from severe systemic infection and mounted similar cytokine responses as pigs infected with Salmonella alone. In contrast, EcN interfered with translocation of Salmonella into mesenteric lymph nodes and systemic circulation. Pigs pre-associated with EcN thrived and their clinical condition correlated with the absence of IL-10 in their plasma and a decrease of TNF-α in plasma and ileum.

AMNIOTIC MEMBRANE TRANSPLANTATION AT THE DEPARTMENT OF OPHTHALMOLOGY OF THE UNIVERSITY HOSPITAL BRNO.

BACKGROUND: The aim of the work is a discussion of amniotic membrane transplantation at the Eye Clinic of the University Hospital Brno and a retrospective evaluation of a group of patients for the period 2014-2019 who were treated for various indications. METHODS: Retrospective evaluation of the number and effectiveness of individual types of amniotic membrane in a group of patients after amniotic membrane transplantation (AMT) for various indications. A total of 134 patients were included in the study group, of which 68 were men and 66 were women. The median age was 70 years. The total number of amniotic membrane transplants performed during the selected six years was 139, with half the distribution using frozen (69 eyes) and lyophilized amniotic membrane (70 eyes). The AMT technique was chosen based on the initial finding and diagnosis. The type of amniotic membrane used (lyophilized vs. frozen) depended on the urgency of the procedure. RESULTS: The number of amniotic membrane transplantations was evaluated in a group of 134 patients (139 eyes) and their effectiveness in individual diagnoses was demonstrated. During the follow-up period, more transplantations of amniotic membranes were performed during hospitalization than in the outpatient department, both types of membranes (frozen and lyophilized). Amniotic membrane transplantation during hospitalization was performed in 89 eyes, in the outpatient mode in 50 eyes. Indications for amniotic membrane transplantation included microperforation and corneal perforation (30 eyes), non-healing corneal defects (21 eyes), descemetocele (19 eyes), neurotrophic defects (16 eyes), ablation of pterygium (12 eyes) and corneal lysis (11 eyes). eyes). Other conditions (conjunctival lesions, fornix reconstruction, burns, peripheral ulcerative keratitis, ocular cicatricial pemphigoid and keratectomy) were represented in smaller numbers. Despite the very diverse group of indications and the advanced age of the patients, a very good efficacy of the performed amniotic membrane transplantations was found. Some patients died during the follow-up period, so the limitation of work is short and unequal follow-up period. CONCLUSION: The success of the procedure depends not only on the correct timing, indication and technique of transplantation, but also on patient compliance and well-functioning cooperation of regional ophthalmologists.

Susceptibility of bifidobacteria to lysozyme as a possible selection criterion for probiotic bifidobacterial strains.

Resistance or susceptibility of bifidobacteria to lysozyme and growth of bifidobacteria in human milk were tested. Susceptible bifidobacterial strains stopped their growth almost immediately after the addition of lysozyme (400 microg/ml), moderately susceptible strains exhibited reduced growth rate, and growth curves of resistant strains were not affected. Strains of human origin were more resistant to lysozyme than animal strains. While strains of B. bifidum grew well in human milk samples, the growth B. animalis strains was inhibited after inoculation to human milk. The resistance to lysozyme seems to be a promising criterion for the selection of new probiotic bifidobacterial strains.

Auto-aggregation and co-aggregation ability in bifidobacteria and clostridia.

A total of 142 human and 88 calf bifidobacteria were isolated and identified; approximately 12 % of all isolated strains exhibited auto-aggregation (Agg) phenotype (Agg+). Properties considered to be predicting for their adhesion to intestine, i.e. auto-aggregation, and hydrophobicity were determined by xylene extraction in 18 human and 8 calf origin bifidobacteria. Co-aggregation of 8 human bifidobacteria with 8 clostridia was also evaluated. Agg varied between 16.3 and 96.4 %, hydrophobicity values ranged from 0 to 82.8 %. The strongest Agg and hydrophobicity were observed in B. bifidum and B. merycicum isolates. However, there were no statistically significant correlations between these two properties. Variability in the percentage of Agg and hydrophobicity was observed after cultivation of bifidobacteria on different carbon sources. All bifidobacteria showed co-aggregation ability with clostridia tested but there were remarkable differences depending on specific combinations of strains. The bifidobacterial strains with the highest ability to co-aggregate with clostridia were B. bifidum I4 and B. longum I10 isolated from infants; these strains gave also high values of Agg. Agg properties together with co-aggregation ability with potential pathogen can be used for preliminary selection of probiotic bacteria.

Comparison of intestinal microflora in healthy infants and infants with allergic colitis.

Twenty-eight exclusively breast-fed healthy infants and 16 infants also exclusively breast-fed with allergic colitis (aged 85 +/- 60 and 98 +/- 58 d, respectively) were screened for differences in fecal flora. Bifidobacteria were detected in 23 healthy infants and only in 4 fecal samples of infants with allergic colitis. 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. Infants with allergy colitis possessed significantly lower counts of bifidobacteria and total anaerobes and significantly higher counts of clostridia in their feces. In healthy infants, Bifidobacterium longum was the most frequently found species (54.5% of the samples), followed by B. adolescentis (20.0), B. breve (18.2), B. bifidum (16.4), B. dentium (10.9) and B. pseudocatenulatum (1.80). Bifidobacterial isolates from two babies with allergic colitis were identified as B. longum, one child from patients group contained species B. dentium and one baby B. adolescentis. Our results suggest that there are significantly lower counts of bifidobacteria in infants with allergic colitis than in healthy infants.

Implantation of posterior chamber phakic intraocular lens for myopia and hyperopia - long-term clinical outcomes.

PURPOSE: To evaluate long-term refractive outcomes of implantable collamer lens (ICL) implantation and late postoperative complications. METHODS: We assessed outcomes of patients who underwent ICL implantation (type ICM V4 for myopia, ICH V3 for hyperopia, TICM V4 for astigmatism) in our department between 1998 and 2013. It comprised 62 eyes (40 myopic and 22 hyperopic). The average follow-up period was 10.5 years. We evaluated: uncorrected and best-corrected visual acuity (UCVA and BCVA), spherical equivalent (SE), ICL vault, endothelial cell density and late postoperative complications. RESULTS: In myopes, the average UCVA was 1.0±0.37 and BCVA 1.18±0.38, in hyperopes 0.78±0.19 and 1.14±0.18, respectively. The average SE in myopes, whose target refraction was emmetropia, was -0.6±0.83 Dsf, in hyperopes +0.73±0.93. Central ICL vault was 206.16µm±105.94, (range 10-427) in myopes, 195.5µm±109.09, (range 20-404) in hyperopes. The most common late postoperative complication was cataract formation. Three myopic eyes (7.5%) developed symptomatic anterior subcapsular opacities with loss of at least two lines of BCVA. Cataract significantly affecting visual acuity occurred in 5 myopic eyes (12.5%) and 2 hyperopic eyes (9.09%). In these eyes, ICL removal and cataract surgery was performed. CONCLUSIONS: In our experience, ICL implantation in moderate and high ametropia was effective and relatively safe. The most common late complication was cataract formation. This complication can be managed effectively surgically with good refractive outcomes without loss of BCVA.

Incidence of cataract following implantation of a posterior-chamber phakic lens ICL (Implantable Collamer Lens) - long-term results.

AIM: To evaluate late postoperative complications, especially cataract occurrence, its morphological type and factors affecting its development in patients implanted with ICL (Implantable Collamer Lens). METHODS: We analysed the results of ICL implantation in 34 patients (type ICM V4 for myopia, ICH V3 for hyperopia, TICM V4 for astigmatism) in our department between 1998 and 2013. The cohort comprised 62 eyes (40 myopic and 22 hyperopic). Seven eyes with a toric ICL implanted were included in these groups according to spherical equivalent (SE). The average follow-up period was 10.5 ± 3.5 years. We examined uncorrected and best-corrected visual acuity (UCVA and BCVA), SE, ICL vaulting (using anterior segment OCT) and occurrence of late postoperative complications, especially cataract formation in 2 groups of patients - myopes and hyperopes. RESULTS: Among the most common late postoperative complications were pigment dispersion syndrome in 27 eyes, 43.5% (12 myopic eyes and 15 hyperopic eyes) and cataract formation. Lens opacities, including opacities without loss of BCVA, were observed in 18 eyes (29%). Opacities affecting visual acuity were observed in 10 eyes (16.1%). Cataract significantly affecting visual acuity occurred in 7 eyes, i.e. 11.3% (5 myopic eyes and 2 hyperopic eyes). In these eyes, ICL removal and cataract surgery with implantation of posterior chamber intraocular lens (PC IOL) was performed. The most common morphological type of cataract were anterior subcapsular opacities (83.3%), the average time of onset was 3.4 ± 1.9 years after ICL implantation. We did not prove a statistically significant association either between cataract occurrence and age at the time of surgery, or between cataract occurrence and higher preoperative spherical equivalent. We did not prove a significant association between cataract occurrence and low vault, however in 7 eyes after cataract surgery and ICL removal we do not know the vault values. In one eye uveitis with cystoid macular oedema was observed, in two highly myopic eyes repeated ICL dislocation and cataract occurred. In both of these cases the ICL was removed. CONCLUSIONS: According to our experience, implantation of ICL in moderate and high ametropia was relatively safe upon assessing the long-term outcomes. Among the most common complications were pigment dispersion syndrome and anterior subcapsular cataract formation. Cataract can be managed effectively surgically with good refractive outcomes without loss of BCVA. However, loss of accommodation after cataract surgery and risk of vitreoretinal complications must be considered.Key words: ICL (Implantable Collamer Lens), posterior chamber phakic intraocular lens, refractive surgery, cataract occurrence.

Distribution of bifidobacteria in the gastrointestinal tract of calves.

Development of gastrointestinal microflora of calves with special reference to bifidobacteria was investigated; fecal bacteria were enumerated in calves aged 3 days to 7 weeks. Bacteria were detected by using selective media, bifidobacteria using modified TPY agar with an addition of mupirocin and acetic acid and by fluorescence in situ hybridization (FISH). Bifidobacteria were dominant group of fecal flora of calves after 7 d of life, constituting 10 % of total bacterial counts. The highest bacterial concentrations were observed in rumen, cecum, and colon, the lowest in abomasum and duodenum. Bifidobacteria and lactobacilli exhibited the highest survival ability during stomach passage and dominated in all parts of the digestive tract. Bifidobacteria counts determined by FISH were significantly higher than those provided by cultivation. Modified TPY agar was highly selective and suitable for bifidobacteria isolation but FISH was shown to be a more precise method for their enumeration. Our results show that gastrointestinal microflora of calves in the milk-feeding period is similar to breast-fed infants with respect to the occurrence of bifidobacteria as a dominant bacterial group. The use of Bifidobacterium strains offers a promising way for providing beneficial effectors for calves in the milk-feeding period.

Different utilization of glucose and raffinose in Bifidobacterium breve and Bifidobacterium animalis.

Growth of 2 strains of Bifidobacterium breve and 4 strains of Bifidobacterium animalis was evaluated with glucose, raffinose or a mixture of glucose and raffinose as substrates. All strains of B. animalis and one strain of B. breve grew more slowly on glucose than on raffinose or the mixture of glucose and raffinose; one strain of B. breve grew more rapidly on the mixture of glucose and raffinose than on raffinose while 2 strains of B. animalis grew faster on raffinose than on the mixture of raffinose and glucose. Both strains of B. breve utilized glucose and raffinose simultaneously. In contrast, all strains of B. animalis strains displayed atypical growth with rapid utilization of raffinose followed by slow utilization of glucose. The cell morphology of all strains of B. animalis was affected by the sugar used for cultivation: tiny and rather spherical cells were observed on glucose while on media with raffinose the cells were large and had the species-specific shape. Description of preferential utilization of various saccharides by bifidobacteria can contribute to the development of new synbiotic preparations and new cultivation media for bifidobacteria.

[Photodynamic therapy with visudyne in treatment of wet form of age-related macular degeneration--two years results]. / Fotodynamická terapie s preparátem visudyne v lécbe vlhké formy vekem podmínené makulární degenerace--dvouleté výsledky.

BACKGROUND: Photodynamic therapy with Visudyne represents a new method in treatment of the wet form of age-related macular degeneration. METHODS AND RESULTS: Effectiveness of photodynamic therapy with Visudyne was confirmed at our department in the group of 49 patients (13 men, 36 women) of mean age 72.7 years with predominantly classical and occult choroidal neovascular membrane in subfoveal localization in wet form of age-related macular degeneration. The observation period in all patients in this group was 24 month. During the observation period, 27 patients with predominantly classical choroidal neovascular membrane underwent 1 to 5 treatments (mean 1.8). Before the treatment the average best corrected visual acuity was 0.696 +/- 0.20 logMAR. At the end of observation period the average best corrected visual acuity was 0.985 +/- 0.39 logMAR. Best corrected visual acuity dropped by 2.88 lines of ETDRS (early treatment diabetic retinopathy study) visual charts. 22 patients with the genuine occult choroidal neovascular membrane underwent during the observation period 1 to 3 treatments (mean 1.5). Average best corrected visual acuity before the treatment was 0.755 +/- 0.25 logMAR. At the end of observation period the average best corrected visual acuity was 0.909 +/- 0.42 logMAR. We found out the decrease of average best corrected visual acuity by 1.55 lines of ETDRS charts. CONCLUSIONS: Decrease of the average best corrected visual acuity less than 3 lines at ETDRS charts is considered as stabilisation of the finding. In our group this goal was achieved in 2/3 of patients.

Detection of infant faecal bifidobacteria by enzymatic methods.

An enzyme-based assay was developed for the detection of bifidobacteria in infant faeces. Ninety-five samples from 51 breast-fed infants in the age between 3 and 276 days were investigated. Bifidobacteria and other bacterial groups were determined by cultivation and fluorescence in situ hybridisation (FISH). Faecal samples were examined for the activity of fructoso-6-phosphate phosphoketolase (F6PPK) and for other enzymatic reactions using the API-ZYM kit. Twenty-nine infants had high numbers of bifidobacteria (usually higher than 9 log CFU/g) in their faeces. Seventeen infants (35%) did not contain detectable amounts of bifidobacteria in their faecal samples. The remaining five individuals had low counts of bifidobacteria (3-6 log CFU/g). Most negative infants possessed major amounts of clostridia in their faecal flora. There were no significant differences among bifidobacterial counts obtained by cultivation and FISH, detection of F6PPK, alpha-galactosidase and alpha-glucosidase activities could routinely be used for the rapid and simple detection of bifidobacteria in infant faecal samples. Bifidobacterial colonies were identified using enzymatic tests and PCR procedure based on 16S rRNA gene sequences species-specific primers. In 14 samples, the identifications of individual isolates were compared with direct analyses of faeces using the nested PCR-denaturing gradient gel electrophoresis (nested DGGE) procedure. The results obtained in several cases are not identical. Bifidobacterium longum and Bifidobacterium breve were most frequently identified. Bifidobacteria-positive samples had high activities of alpha-galactosidase and alpha-glucosidase. On the contrary, negative samples missed either one or both of these enzymatic activities. While all positive samples tested showed distinctive fructose-6-phosphate phosphoketolase activity (F6PPK), none of the negative samples expressed F6PPK activity.

Antibacterial screening of some Peruvian medicinal plants used in Callería District.

Nine ethanol extracts of Brunfelsia grandiflora (Solanaceae), Caesalpinia spinosa (Caesalpiniaceae), Dracontium loretense (Araceae), Equisetum giganteum (Equisetaceae), Maytenus macrocarpa (Celastraceae), Phyllanthus amarus (Euphorbiaceae), Piper aduncum (Piperaceae), Terminalia catappa (Combretaceae), and Uncaria tomentosa (Rubiaceae), medicinal plants traditionally used in Calleria District for treating conditions likely to be associated with microorganisms, were screened for antimicrobial activity against nine bacterial strains using the broth microdilution method. Among the plants tested, Phyllanthus amarus and Terminalia catappa showed the most promising antibacterial properties, inhibiting all of the strains tested with minimum inhibitory concentrations (MICs) ranging from 0.25 to 16 mg/ml. The extract from aerial part of Piper aduncum was significantly more active against Gram-positive (MICs ranging from 1 to 2 mg/ml) than against Gram-negative bacteria (MICs > 16 mg/ml).

Utilization of mathematical processing of retinal images with use of adaptive contrast control (ACC) method to detect quantity of vascular endings.

PURPOSE: The authors investigated whether the method of mathematical processing of retinal images with the use of a computer can be used to evaluate ocular background figures of patients with physiologic retinal findings. This method is based on identification of vascular endings in an examined retinal area. When the authors mention vascular endings, they do not refer to factual endings, but recognizable vascular endings; there are no endings in a vascular system. METHODS: Adaptive contrast control (ACC) method was used to determine a number of vascular endings. The method is based on mathematical processing of a digitized retina picture with the use of a computer. On a digitized retinal picture, the vascular system is identified with the use of the conditional erosion methodology, and the number of vascular endings is then determined. The ACC method was used to process a file of retinal pictures of 38 patients (76 eyes) with physiologic retinal findings. RESULTS: Based on the results of statistical analysis, the authors detected that the number of vascular endings showed a normal curve (Gaussian distribution, p=0.05). A tight correlation between quantities of vascular endings in the right and the left eyes was also detected, which means that the quantity of vascular endings in the right and the left eyes is in a very close correlation (p=0.05). CONCLUSIONS: The authors highlight that the curve of the number of vascular endings of patients with physiologic retinal findings shows a Gaussian distribution.

[Trends in indications of perforating keratoplasty at the Department of Ophthalmology, Faculty Hospital, Brno, Czech Republic, E.U., during the period 2008-2012]. / Trendy v indikáciách k perforujúcej keratoplastike na Ocnej klinike FN Brno v rokoch 2008-2012.

INTRODUCTION: The aim of the study is retrospective analysis of perforating keratoplasty (PKP) indications at the Department of Ophthalmology, Faculty Hospital, Masaryk University, Brno, Czech Republic, E.U., during the period of 5 years, from January 1st, 2008 to December 31st, 2012. MATERIAL AND METHODS: We performed the PKP indications retrospective analysis at the Department of Ophthalmology, Faculty Hospital, Masaryk University, Brno, Czech Republic, E.U., during the period of 5 years, from January 1st, 2008 to December 31st, 2012, with complex evaluation of demographic and clinical data. The clinical diagnoses indicating the perforating keratoplasty were divided into 6 groups (keratoconus, bulous keratopathy, keratitis, corneal dystrophies, injuries, corneal transplant failure, and others) according to Cunningham et al. 2011 (2) and Boimer et al. 2012(1) methods. RESULTS: Our results correspond to data in the literature published abroad, where the majority of recently published papers refer significant increase of corneal transplant failure in the last years of follow-up.Key words: perforating keratoplasty, indications.