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Diabetes mellitus is a chronic disease affecting glucose metabolism. The pathophysiological reactions underpinning the disease can lead to the development of late diabetes complications. The gut microbiota plays important roles in weight regulation and the maintenance of a healthy digestive system. Obesity, diabetes mellitus, diabetic retinopathy, diabetic nephropathy and diabetic neuropathy are all associated with a microbial imbalance in the gut. Modern technical equipment and advanced diagnostic procedures, including xmolecular methods, are commonly used to detect both quantitative and qualitative changes in the gut microbiota. This review summarises collective knowledge on the role of the gut microbiota in both types of diabetes mellitus and their late complications, with a particular focus on diabetic foot syndrome., (© 2023. Institute of Microbiology, Academy of Sciences of the Czech Republic, v.v.i.)

The family Bifidobacteriaceae constitutes an important phylogenetic group that particularly includes bifidobacterial taxa demonstrating proven or debated positive effects on host health. The increasingly widespread application of probiotic cultures in the twenty-first century requires detailed classification to the level of particular strains. This study aimed to apply the glutamine synthetase class I (glnAI) gene region (717 bp representing approximately 50% of the entire gene sequence) using specific PCR primers for the classification, typing, and phylogenetic analysis of bifidobacteria and closely related scardovial genera. In the family Bifidobacteriaceae, this is the first report on the use of this gene for such purposes. To achieve high-value results, almost all valid Bifidobacteriaceae type strains (75) and 15 strains isolated from various environments were evaluated. The threshold value of the glnAI gene identity among Bifidobacterium species (86.9%) was comparable to that of other phylogenetic/identification markers proposed for bifidobacteria and was much lower compared to the 16S rRNA gene. Further statistical and phylogenetic analyses suggest that the glnAI gene can be applied as a novel genetic marker in the classification, genotyping, and phylogenetic analysis of isolates belonging to the family Bifidobacteriaceae.

Melanoma is the least common form of skin tumor, but it is potentially the most dangerous and responsible for the majority of skin cancer deaths. We suggest that the skin microbiome might be changed during the progression of melanoma. The aim of this study is to compare the composition of the skin microbiota between different locations (skin and melanoma) of a MeLiM (Melanoma-bearing Libechov Minipig) pig model (exophytic melanoma). Ninety samples were used for PCR-DGGE analysis with primers specifically targeting the V3 region of the 16S rRNA gene. The profiles were used for cluster analysis by UPGMA and principal coordinate analysis PCoA and also to calculate the diversity index (Simpson index of diversity). By comparing the obtained results, we found that both bacterial composition and diversity were significantly different between the skin and melanoma microbiomes. The abundances of Fusobacterium and Trueperella genera were significantly increased in melanoma samples, suggesting a strong relationship between melanoma development and skin microbiome changes.

The order Lactobacillales represents a morphologically, metabolically, and physiologically diverse group of bacteria. Lactic acid bacteria represent the core of this phylogenetic group. They are a part of epiphytic microflora, fermented dairy, meat, fruit and vegetable products, and the digestive tract of humans and animals. Despite the fact that these bacteria form a phenotypically and genotypically heterogeneous group, their phylogenetic relationship enables to propose a common genetic marker usable in classification, typing, and phylogeny. By creation of consensus sequence based on available genomic sequences of some representatives of order Lactobacillales, a specific primer-pair binding variable region of aspS gene (length of 615 nts) encoding the aspartyl-tRNA synthetase was designed. This gene has not yet been used in classification and phylogeny of the order Lactobacillales, although it meets the requirements of molecular markers (distribution and single copy in bacterial genomes, functional constancy and genetic stability, sequence variability among taxonomic units, irreplaceable role in proteosynthesis). Primers were applied on 54 type and wild Lactobacillales strains. Obtained sequences allowed to provide alignments for purpose of phylogenetic tree reconstructions that uncovered particular phylogenetic clusters of vagococci/enterococci, obligately homofermentative and heterofermentative lactobacilli. Although a relatively short fragment of the aspS gene (approximately 33% of the complete gene sequence) was evaluated, much higher sequence variability (61.8% of pairwise identity) among strains examined compared with 16S rRNA gene (90.7%, length of 1318 nt) provides a relatively simple and effective tool for classification and typing of selected representatives of the order Lactobacillales.

Genes encoding glycosyl hydrolase family 11 (GH11) xylanases and xylanases have been identified from Pseudobutyrivibrio xylanivorans. In contrast, little is known about the diversity and distribution of the GH10 xylanase in strains of P. xylanivorans. Xylanase and associated activities of P. xylanivorans have been characterized in detail in the type strain, Mz5. The aim of the present study was to identify GH10 xylanase genes in strains 2 and Mz5 of P. xylanivorans. In addition, we evaluated degradation and utilization of xylan by P. xylanivorans 2 isolated from rumen of Creole goats. After a 12-h culture, P. xylanivorans 2 was able to utilize up to 53% of the total pentose content present in birchwood xylan (BWX) and to utilize up to 62% of a ethanol-acetic acid-soluble fraction prepared from BWX. This is the first report describing the presence of GH10 xylanase-encoding genes in P. xylanivorans. Strain 2 and Mz5 contained xylanases which were related to GH10 xylanase of Butyrivibrio sp. Identifying xylanase-encoding genes and activity of these enzymes are a step toward understanding possible functional role of P. xylanivorans in the rumen ecosystem and contribute to providing an improved choice of enzymes for improving fiber digestion in ruminant animals, agricultural biomass utilization for biofuel production, and other industries.

Cronobacter spp. (formerly Enterobacter sakazakii) are emerging, opportunistic pathogens that are linked with food-borne infections in neonates and infants. In the present study, 291 samples of food, 36 samples from a dairy farm and 140 samples of dust from vacuum cleaners were examined for the presence of Cronobacter spp. using chromogenic media and biochemical tests. Altogether, 72 Cronobacter spp. strains were isolated in accordance with the reference standard ČSN P ISO/TS 22964 (2006). No Cronobacter spp. strains were detected in 10 samples of infant milk formula or in samples from a dairy farm. Twelve out of 20 positive food samples were dry products. The incidence of Cronobacter spp. in instant and powdered products and spices (12 positive isolates out of 82 samples) was significantly higher than that in other foods (P = 0.002), but lower than that in samples of dust (52 isolates; P < 0.001). The incidence of Cronobacter spp. in dust from restaurants, bars and hotels (13 positive isolates in 20 samples) was significantly higher than that in dust from households (P = 0.010). The polymerase chain reaction assay for the species-specific detection of the rpoB gene was performed in 49 isolates. Thirty-four Cronobacter spp. isolates were identified as Cronobacter sakazakii, nine isolates as Cronobacter malonaticus and one isolate as Cronobacter turicensis.

The qualitative and quantitative changes in the bacterial community composition in two mesophilic, commercially used biogas plants were monitored by denaturing gradient gel electrophoresis (DGGE) and real-time PCR. The main objective was to evaluate the influence of the co-substrate maize silage on total bacteria and some selected bacterial groups by comparing full-scale reactors fed solely with pig manure or additionally with maize silage. DGGE fingerprints reflected shifts in the bacterial community structure associated with maize silage as co-substrate and the real-time PCR results showed clear changes in the quantitative composition of the bacterial consortia of each fermenter. A clear dominance of Clostridia in all surveyed fermenters and considerably lower abundance of Bacteroidetes in the biogas plant fed with maize silage was shown.

16S rRNA sequences of ruminal uncultured bacterial clones from public databases were phylogenetically examined. The sequences were found to form two unique clusters not affiliated with any known bacterial species: cluster of unidentified sequences of free floating rumen fluid uncultured bacteria (FUB) and cluster of unidentified sequences of bacteria associated with rumen epithelium (AUB). A set of PCR primers targeting 16S rRNA of ruminal free uncultured bacteria and rumen epithelium adhering uncultured bacteria was designed based on these sequences. FUB primers were used for relative quantification of uncultured bacteria in ovine rumen samples. The effort to increase the population size of FUB group has been successful in sulfate reducing broth and culture media supplied with cellulose.

Twenty-five bifidobacteria were isolated from feces of calves. Isolates were identified, and their functional properties and antimicrobial activity were determined. From 10 strains with suitable properties rifampicin-resistant mutants (RRBs) were prepared and mixture of RRBs was administered to 2-d-old calves. These strains were identified by sequencing as Bifidobacterium animalis ssp. animalis (6 strains), B. thermophilum (2 strains), B. choerinum (1 strain) and B. longum ssp. suis (1 strain). The control group was without probiotic treatment. Survival ability of administered bifidobacteria was monitored in fecal samples by cultivation on modified TPY agar supplemented with mupirocin, acetic acid, and rifampicin. Administered bifidobacteria survived in gastrointestinal tract of calves for at least 60 d. Other bacteria were also determined after cultivation using fluorescence in situ hybridization (FISH). Bifidobacteria and lactobacilli dominated in fecal microflora. Significantly lower amounts of E. coli and higher amounts of bifidobacteria and total anaerobes were found in the treated group relative to the control group.

The occurrence and species distribution of bifidobacteria in the digestive tract of important representatives of social insects such as ants, bees, wasps and bumblebees as well as the incidence of bifidobacteria in fecal samples of several species of vertebrates represented mainly by reptiles was assigned by culture-independent method based on DGGE and real time PCR. Bifidobacteria were present in the gut of most social insects--honey bees, wasps, cockroaches and bumblebees, except for ants. In honey bees, where the counts of bifidobacteria ranged from 2 to 8% of the total bacteria, the most common species seemed to be Bifidobacterium indicum. Proportion of bifidobacteria was found in broad range from 0.1 to 35-37% in wasps and cockroaches; the variance of bifidobacteria in bumblebees was lower, ranging from 1 to 7% of total bacterial count. Among studied vertebrates, the detectable presence of bifidobacteria was found only in trout (1.1%) and geckos (0.2%), but large amount of these bacteria was observed in Vietnamese box turtle, where bifidobacteria represented nearly one-fourth (22%) of total bacterial counts.

We isolated and amplified by PCR 16S rDNA from bacteria attached to the bovine rumen wall and analyzed it by denaturing gradient gel electrophoresis (DGGE) with subsequent sequence analysis. The attached bacterial community differed from the bacteria of rumen content; however, no differences were observed among the five epithelial sampling sites taken from each animal. The DGGE profile of the bacterial population attached to the rumen wall represented a high inter-animal variation.

A feeding study was performed to monitor the effect of chitosan intake on the fecal microbiota of ten healthy human subjects. Diversity of microflora was monitored during 8 weeks including 4 weeks of chitosan supplementations. Using denaturing gradient gel electrophoresis (DGGE) analysis of 16S rRNA gene amplicons and quantitative PCR method we revealed possible changes originating in the overall bacterial composition and also in the subpopulation of Bifidobacterium group. DGGE profiles displayed high complexity and individuality for each subject. Considerable variations in the composition of band patterns were observed among different persons. A raised level of fecal Bacteroides in response to chitosan intake was found in all samples. Bifidobacterium levels following chitosan intake increased or remain unchanged. Non-significant increase was, surprisingly, found in the numbers of butyrate-producing bacteria.

The diversity of anaerobic fungi was evaluated in cow semiliquid manure obtained from input homogenizing tank of biogas plant. Among three sets of tested primers, the combination of fungal universal ITS1F and Neocallimastigales specific Neo QPCR Rev primers was selected and used for the construction of clone library. Eighty-four new complete internal transcribed spacers (ITS1) and partial 5.8S rDNA sequences generated within this study were analyzed by Bayesian inference and assigned to an existing order of the Neocallimastigales. Sixty-seven% of sequences were affiliated with Cyllamyces, 24% with Piromyces, 7% with Anaeromyces, only 2% with Neocallimastix, and no sequences with Orpinomyces. According to Bayesian analysis the genus Caecomyces was polyphyletic and disappeared from the presented ITSbased phylogram. This study gave a first insight into the diversity of anaerobic fungi in cow manure, where the prevalence of fungi with bulbous morphology was indicated.

The presence of phytase activity was demonstrated in 26 strains of rabbit cecal bacteria. In 25 strains a low phytase activity, 0.10-0.62 micromol phosphate released per min per mg protein, was found. High activity (2.61 micromol/min per mg protein) was found in the strain PP2 identified as Enterococcus hirae. Phytase activity was cell-associated, being higher in the cell extract than in the cell walls. Extracellular phytase activity and cell-associated phosphatase activity were not detected. Phytase activity was optimal around pH 5.0, which is below the physiological cecal pH range. The K (m) determined using the Lineweaver-Burk plot was 0.19 micromol/mL. Cations Fe(3+), Cu(2+) and Zn(2+) at 0.5 mmol/L decreased phytase activity in sonicated cells of E. hirae by 99.4, 90.7 and 96.5 %, respectively. In contrast, Mg(2+) increased activity by 11.0 %. Characteristics of E. hirae phytase (pH optimum, K (m), cation sensitivity) were similar to those of other bacterial phytases reported in the literature. Other bacteria with a high phytase activity may be present in the rabbit cecum but remain to be identified.

The common feature of all chytridiomycetous fungi, aerobic as well as anaerobic, is an abundance of chitin in their cell wall. The genes coding for chitinases have therefore been widely used as phylogenetic markers in ascomycetes. As their utility for Chytridiomycetes has not been determined we chose the gene encoding an enzyme involved in chitin degradation and energy metabolism, the beta-(1,4)-N-acetylglucosaminidase (nag1). Primer pair Nag-forward and Nag-reverse was used to create PCR product from 5 strains of anaerobic and 7 strains of aerobic chytrids. However, Blast search of sequenced amplicons showed that these primers are specific only for fungus Emericella nidulans. Amino acid alignment of Nag1 proteins of fungal, protozoal and bacterial origin available in GenBank database was therefore performed. Five amino acid regions were found to be conserved enough to serve as a suitable domain for the design of a set of primers for the universal amplification of the nag1 gene in the Neocallimastigales fungi.

The fecal short-chain fatty acids concentration was higher (154 +/- 46.9 mmol/L) in childhood patients than in healthy children (96.6 +/- 19.2 mmol/L). On the other hand, pH values were nonsignificantly lower in patients stool (6.78 +/- 0.75 vs. children 7.42 +/- 0.74). Using denaturing gradient gel electrophoresis specific for total bacteria, lactobacilli and bifidobacteria the microbial population was characterized in fecal samples and in duodenal biopsies. Bacteria adhering to duodenal biopsies were not dominating in stool samples. More than 50 % of detected bacterial species belonged to as yet uncultured strains.

The influence of geographic location, season, age, and part of the digestive tract on bacterial diversity was evaluated on intestinal microflora of honeybees, wasps, and cockroaches using DGGE analysis. PCR-DGGE analyses with universal bacterial primers targeting 200-bp region of the 16S rDNA gene afforded the profile of complex bacterial DNA; specific primers were used to determine the profile of bifidobacteria whose concentration in digestive tract was determined by real-time PCR. Selected PCR products were identified by sequencing. The microflora of the bees exhibited little variations among the hives from distant locations. Their bifidobacterial population formed 2.8-8.4 % of total bacteria and was very homogeneous. The total gut microflora of wasps was also homogeneous, only two samples being affected by the season or the location; on the other hand, wasp bifidobacterial population was very heterogeneous. Cockroaches showed the highest variations in microflora composition, the age and diet being the ultimate factors; bifidobacteria counts also varied among tested individuals (0.1-34.1 % of total bacteria). Our results suggest that nutrition habits are the strongest factor affecting the insect microflora, giving higher variations to omnivorous species.

The effect of gluten-free diet (GFD) and chitosan was evaluated in healthy individuals; GFD remarkably influenced the structure of the gut bacterial population and its metabolism. Administration of GFD and chitosan (3 g daily) significantly changed composition and metabolism of the bacterial population. Chitosan stimulated the counts of fecal chitinolytic bacteria and decreased the body mass of treated persons.

The influence of a host's diet on Butyrivibrio and Pseudobutyrivibrio populations was investigated by competitive PCR. Specific primers were designed and competitive PCRs developed for both groups. Results (from 4 cows with different diets) suggested that high-fiber intake essentially increases the Butyrivibrio amounts in the rumen, whereas high-energy food additives lead to its suppression. The Pseudobutyrivibrio concentration also changed during the experiment but without any significant relation to the host's diet.

The suitability of restriction fragment length polymorphism (RFLP) analysis of the ribosomal DNA cluster for discriminating two genera of anaerobic polycentric fungi, Orpinomyces and Anaeromyces, was determined. Three PCR-amplified DNA fragments--nuclear small subunit (SSU; 18S rDNA), the nuclear large subunit (LSU; 28S rDNA) and internal transcribed spacer (ITS)--were restricted with endonucleases AluI, DraI, HinfI and MboI. Although the SSU DNA fragment could be restricted successfully by all four enzymes, no differences were observed between restriction patterns of Orpinomyces and Anaeromyces. The most polymorphic restriction pattern between Orpinomyces and Anaeromyces resulted from cleavage of LSU rDNA fragments cut by AluI and HinfI and ITS fragment cut by DraI and HinfI. Genus-specific RFLP patterns were determined for Orpinomyces and Anaeromyces genera; the results showed that the PCR-RFLP analysis of rDNA offers an easy and rapid tool for differentiation of two polycentric genera of anaerobic fungi, which could be hardly separated on the basis of morphology.

The main representatives of bacteria in the human colon were investigated by specific PCR and denaturing gradient gel electrophoresis (DGGE). Prevalent in both cases were species of Bifidobacterium, Clostridium, Bacteroides, Faecalibacterium and Eubacterium. Simultaneously, cellulolytic bacteria were isolated from the human feces. The largest proportion was represented by ruminococcus-like isolates. Their presence was confirmed both by PCR and DGGE methods; the latter one was able to give more comprehensive data about the composition of bacterial population in the human colon chyme.

Competitive PCR method was developed for the detection and enumeration of Butyrivibrio fibrisolvens. Sequences of 16S rDNA were obtained from our isolates (serving as a source of data for primer design) and were distinguished into nine different groups of butyrivibria. Specific primers for two distinct groups were designed with the help of BioEdit program. These primers were tested with DNA of 20 strains of ruminal B. fibrisolvens isolates. Annealing temperature 58 degrees C showed a little specificity but a better selectivity was found after raising it up to 65 degrees C. A group 1 competitive fragment of 16S rDNA of different length was constructed using restriction cutting with MspI followed by ligation; the size of the resulting fragment was cut down by 75 bp. The fragment worked in the presence of the original 16S rDNA fragment of B. fibrisolvens JK 609.