Intestinal microbiota profiles in a genetic model of colon tumorigenesis correlates with colon cancer biomarkers.

Faecal (FM) and colon mucosal associated microbiota (MAM) were studied in a model of colorectal cancer (CRC), the Apc-mutated Pirc rats, and in age-paired wt F344 rats. Principal Coordinates Analysis indicated that samples' distribution was driven by age, with samples of young rats (1 month old; without tumours) separated from older ones (11-month-old; bearing tumours). Diversity analysis showed significant differences between FM and MAM in older Pirc rats, and between MAM of both Pirc and wt rats and the tumour microbiota, enriched in Enterococcus, Escherichia/Shigella, Proteus and Bifidobacteriaceae. In young animals, Pirc FM was enriched in the genus Delftia, while wt FM was enriched in Lactobacillus and Streptococcus. Some CRC biomarkers and faecal short chain fatty acids (SCFAs) were also measured. Colon proliferation and DClK1 expression, a pro-survival mucosal marker, were higher in Pirc than in wt rats, while the mucin MUC2, was lower in Pirc rats. Branched SCFAs were higher in Pirc than in wt animals. By Spearman analysis CRC biomarkers correlated with FM (in both young and old rats) and with MAM (in young rats), suggesting a specific relationship between the gut microbiota profile and these functional mucosal parameters deserving further investigation.

Allicin prevents the formation of Proteus-induced urinary crystals and the blockage of catheter in a bladder model in vitro.

Stone formation and catheter blockage are major complications of Proteus UTIs. In this study, we investigated the ability of allicin to inhibit P. mirabilis-induced struvite crystallization and catheter blockage using a synthetic bladder model. Struvite crystallization inhibition study was carried out using P. mirabilis lysate as urease enzyme source in synthetic urine (SU). Struvite productions were monitored by phase contrast light microscopy and measurements of pH, Mg2+ and Ca2+ precipitation and turbidity. A catheter blockage study was performed in a synthetic bladder model mimicking natural UTI in the presence of allicin at sub-MIC concentrations (MIC = 64 µg/ml). The results of crystallization study showed that allicin inhibited pH rise and consequently turbidity and precipitation of ions in a dose-dependent manner. The results of catheter blockage study showed that allicin at sub-MIC concentrations (2, 4, 8 µg/ml) significantly increased the time for catheter blockage to occur to 61, 74 and 92 h respectively compared to allicin-free control (48 h). In a similar way, the results showed that allicin delayed the increase of SU pH level in bladder model in a dose-dependent manner compared to allicin-free control. The results also showed that following the increase of allicin concentration, Mg2+ and Ca2+ deposition in catheters were much lower compared to allicin-free control, further confirmed by direct observation of the catheters' eyehole and cross sections. We conclude that allicin prevents the formation of Proteus-induced urinary crystals and the blockage of catheters by delaying pH increase and lowering Mg2+ and Ca2+ deposition in a dose-dependent manner.

Relationship between microbial diversity and nitrogenase activity of Stipagrostis pennata rhizosheath.

Nitrogen is the key factor for plant survival and growth, especially in the desert. Stipagrostis pennata, a sand born drought-resistant plant, could colonize pioneerly in Gurbantunggut Desert during revegetation. One strategy for their environment adaptation was the rhizosheath formatted by root-hair, mucilaginous exudates, microbial components, and soil particles, for which not only provides a favorable living microenvironment but also supplies essential nutrients. To understand the relationship between microorganisms living in rhizosheaths and the nitrogen nutrition supply, the microbial diversity and nitrogenase activity was estimated during the growth of S. pennata. Five samples of the rhizosheath, which based on the development periods of the plant, regreen, flowering, filling, seed maturating, and withering period, were collected. The nitrogenase activity was estimated by acetylene reduction and the microbial diversity was analyzed by 16S rRNA high-throughput sequencing. The results showed that the nitrogenase activity was increased slowly during regreen to flowering, while reached a peak rapidly at filling sample and then decreased gradually. A total of 274 operational taxonomic units (OTUs) were identified and significant differences in community structure and composition at each growth period. Among them, the main phyla included Actinobacteria and Proteus, which were the most abundant phyla in all periods. In addition, the microbial diversity in the grain filling period was higher than other periods in view of the analysis of alpha diversity and beta diversity. Furthermore, principal component analysis (PCA) analysis showed that the microbial communities in the filling period was low in similarity with other periods. Most importantly, the OTUs associated with nitrogen fixation is the most during the filling period, involving Phagecidae and Fucoraceae. Overall, the study not only revealed the differences in nitrogenase activity among different developmental periods in S. pennata, but also explored the potential bridges between it and community structure and diversity of bacteria.

Bacteriocinogenic properties of Escherichia coli P2C isolated from pig gastrointestinal tract: purification and characterization of microcin V.

The aim of this study was to isolate and investigate the bacteriocinogenic and probiotic potential of new Gram-negative isolates. Of 22 bacterial isolates from pig intestine and chicken crops, ten isolates had demonstrated a good activity, and the most potent five strains were identified as four E. coli and one as Proteus sp. No virulence factors were detected for E. coli strains isolated from pig intestine. The semi-purified microcins proved to be resistant to temperature and pH variation, but sensitive to proteolytic enzymes. Of particular interest, strain E. coli P2C was the most potent, free of virulence genes and sensitive to tested antibiotics. Purification procedure revealed the presence of a single pure peak having a molecular mass of 8733.94 Da and matching microcin V (MccV). The sequence obtained by LC-MS/MS confirmed the presence of MccV. Purified MccV showed a good activity against pathogenic coliforms, especially E. coli O1K1H7 involved in avian colibacillosis. The present study provides evidence that E. coli strains isolated from pig intestine produce microcin-like substances. E. coli P2C is a safe MccV producer that could be a good candidate for its application as novel probiotic strain to protect livestock and enhance growth performance.

Analogues of Disulfides from Allium stipitatum Demonstrate Potent Anti-tubercular Activities through Drug Efflux Pump and Biofilm Inhibition.

Disulfides from Allium stipitatum, commonly known as Persian shallot, were previously reported to possess antibacterial properties. Analogues of these compounds, produced by S-methylthiolation of appropriate thiols using S-methyl methanethiosulfonate, exhibited antimicrobial activity, with one compound inhibiting the growth of Mycobacterium tuberculosis at 17 µM (4 mg L-1) and other compounds inhibiting Escherichia coli and multi-drug-resistant (MDR) Staphylococcus aureus at concentrations ranging between 32-138 µM (8-32 mg L-1). These compounds also displayed moderate inhibitory effects on Klebsiella and Proteus species. Whole-cell phenotypic bioassays such as the spot-culture growth inhibition assay (SPOTi), drug efflux inhibition, biofilm inhibition and cytotoxicity assays were used to evaluate these compounds. Of particular note was their ability to inhibit mycobacterial drug efflux and biofilm formation, while maintaining a high selectivity towards M. tuberculosis H37Rv. These results suggest that methyl disulfides are novel scaffolds which could lead to the development of new drugs against tuberculosis (TB).

Effect of probiotics on the basis of Bacillus subtilis and Bifidobacterium longum on the biochemical parameters of the animal organism.

For the purpose of safe modulation of the intestinal microflora, probiotics have been increasingly used in recent years. In the present work, the effect of the probiotic sporobacterin (Bacillus subtilis 534) (I group) and soybean-bifidum (Bifidobacterium longum) (II group) on male rats of the Wistar line was evaluated. In assessing nonspecific immunity in vitro, there was an increase in the level of baseline level in the first and second groups (by 8.3 and 12.2% more control). The influence of probiotic preparations on the intestinal normoflora was assessed using PCR. Bifidumbacterin increased the normal microflora, in particular, Escherichia coli 1.55 times, Lactobacillus 1.26 times, Enterococcus 1.3 times as much control; the level of conditionally pathogenic microflora, in particular, Proteus spp. decreased by 1.3 times in comparison with the control. Sporobacterin also contributed to an increase in the amount of E. coli (1.55 times) and Lactobacillus (0.9 times). When a culture of Bifidobacterium longum was introduced, a selective reduction in the loss of chemical elements was observed against the background of the diet used. At the end of the experiment, the content of calcium in the body tissues of animals of group II exceeded this indicator in group I by 3.9%, phosphorus by 17.6%, copper by 28.5%, and zinc by 15.2%. The totality of the results obtained by us indicates that inclusion of Bifidumbacterium longum in the diet of animals makes the use of this preparation in the correction of mineral imbalance and improves the microflora of the intestines of animals by reducing the number of representatives of opportunistic microflora against the background of an increase in the number of basic representatives of normal microbiocenosis. Also, the use of probiotic drugs as additives leads to a slight increase in the level of nonspecific immunity, which increases the natural resistance of the organism.

Novel approach for the ammonium removal by simultaneous heterotrophic nitrification and denitrification using a novel bacterial species co-culture.

Agricultural activities lead excessive emission of ammonia nitrogen in the environment and can profoundly interfere the equilibrium of the natural ecosystems leading to their contamination. Actually, the biological purification of wastewaters is the most adopted technique thanks to its several advantages such as high performance and low energy consumption. For this reason, two novel strains of Alcaligenes sp. S84S3 and Proteus sp. S19 genus were isolated from an activated sludge and applied in the treatment of ammonium and nitrite in aqueous solution. Under the optimum operating conditions of temperature (30 °C), pH (7), carbon substrate (2 g/L of glucose) and duration of incubation time (69 h), the strain Alcaligenes sp. S84S3 could oxidize 65% of the ammonium as high as 272.72 mg-NH4(+)/L. Moreover, during 48 h, the nitrate reduction rate performed by the strain Proteus S19 was about 99 % without production of nitrite intermediate (negligible concentration). Moreover, the coculture of the strains Alcaligenes sp. S84S3 and Proteus sp. S19 could eliminate 65.83% of the ammonium ions without production of toxic forms of nitrogen oxides during a short time of incubation (118 h) at the same operational conditions with providing the aeration in the first treatment phase. The coculture of our isolated strains is assumed to have a good potential for nitrification and denitrification reactions applied in the treatment of wastewater containing ammonium, nitrite and nitrate. As a result, we can consider that the mixed culture is a practical method in the treatment of high-strength ammonium wastewater with reducing of sludge production.

Simultaneous nitrification and denitrification by novel heterotrophs in remediation of fish processing effluent.

Three isolates viz. Lysinibacillus sp. HT13, Alcaligenes sp. HT15 and Proteus sp. HT37 isolated from fish processing effluent and having a C/N ratio of 2, removed 218, 169, and 400 µg cell(-1) day(-1) NH4(+)-N, respectively without subsequent build up of nitrite or nitrate. Ability of the selected isolates in removing NH4(+)-N, NO2(-)-N, and NO3(-)-N was checked in the presence of four commonly reported and tested effluent carbon sources viz. pyruvate, glycerol, methanol, and acetate. Further, when supplemented to fish processing wastewater containing 234 ppm total Kjeldahl's nitrogen, Lysinibacillus sp. HT13, Alcaligenes sp. HT15, and Proteus sp. HT37 could remediate 95.74, 86.17, and 76.6% nitrogen, respectively in 48 h. This is the first report of a Lysinibacillus sp. carrying out aerobically the process of simultaneous nitrification and denitrification. The results demonstrate the potential of the isolates for use in treatment of fish processing effluents and demonstrating the efficient removal of ammonia.

[MICROBIAL LANDSCAPE OF BILE IN PATIENTS WITH AN ACUTE PURULENT CHOLANGITIS].

Bacteriological analysis was conducted in 136 patients with an acute purulent cholangitis (APCH). The APCH causes were: choledocholithiasis--in 40 (29.9%) patients, coexistence of a common biliary duct stricture and choledocholithiasis--in 39 (28.7%), compression of external biliary ducts by the oedematous pancreatic head in secondary pancreatitis--in 15 (11%), pericholedocheal lymphadenitis--in 3 (2.2%).

In vitro cytotoxic effects of gold nanoparticles coated with functional acyl homoserine lactone lactonase protein from Bacillus licheniformis and their antibiofilm activity against Proteus species.

N-acylated homoserine lactonases are known to inhibit the signaling molecules of the biofilm-forming pathogens. In this study, gold nanoparticles were coated with N-acylated homoserine lactonase proteins (AiiA AuNPs) purified from Bacillus licheniformis. The AiiA AuNPs were characterized by UV-visible spectra, Fourier transform infrared spectroscopy (FTIR), transmission electron microscopy (TEM), and X-ray diffraction (XRD). The synthesized AiiA AuNPs were found to be spherical in shape and 10 to 30 nm in size. Treatment with AiiA protein-coated AuNPs showed maximum reduction in exopolysaccharide production, metabolic activities, and cell surface hydrophobicity and potent antibiofilm activity against multidrug-resistant Proteus species compared to treatment with AiiA protein alone. AiiA AuNPs exhibited potent antibiofilm activity at 2 to 8 µM concentrations without being harmful to the macrophages. We conclude that at a specific dose, AuNPs coated with AiiA can kill bacteria without harming the host cells, thus representing a potential template for the design of novel antibiofilm and antibacterial protein drugs to decrease bacterial colonization and to overcome the problem of drug resistance. In summary, our data suggest that the combined effect of the lactonase and the gold nanoparticles of the AiiA AuNPs has promising antibiofilm activity against biofilm-forming and multidrug-resistant Proteus species.

[ANTIMICROBIAL ACTION OF NOCARDIA VACCINII IMV B-7405 SURFACTANTS].

AIM: To study the effect of Nocardia vaccinii IMV B-7405 surfactants on some bacteria (including pathogens of genera Proteus, Staphylococcus, Enterobacter), yeast of Candida species and fungi (Aspergillus niger R-3, Fusarium culmorum T-7). METHODS: The antimi- crobial properties of surfactant were determined in suspension culture by Koch method and also by index of the minimum inhibitory concentration. Surfactants were extracted from supernatant of cultural liquid by mixture of chloroform and methanol (2:1). RESULTS: It is shown that the antimicrobial properties of N. vaccinii IMV B-7405 surfactant depended on the degree of purification (supernatant, solution of surfactant), concentration and exposure. Survival of Escherichia coli IEM-1 and Bacillus subtilis BT-2 (both vegetative cells and spores) after treatment for 1-2 hours with surfactants solution and the supernatant (the surfactant concentration 21 µg/ml) was 3-28%. Minimum inhibitory concentrations of N. vaccinii IMV B-7405 surfactants on studied bacteria, yeast and micromycetes were 11.5-85.0; 11.5-22.5 and 165.0-325.0 µ/ml respectively. CONCLUSIONS: Minimum inhibitory concentrations of N. vaccinii IMV B-7405 surfactants are comparable to those of the known microbial surfactants. The possibility of using the supernatant of culture liquid as an effective antimicrobial agent noticeably simplifies and reduces the cost of the technology of its obtaining.

[Oflomelid ointment: activity against hospital microbial strains].

Ofloxacin susceptibility was tested with the use of 100 hospital isolates by the disk diffusion method vs. the activity of Oflomelid ointment tested by the cavity method. It was shown that the hospital isolates of all the species were susceptible to the Oflomelid ointment, while susceptibility to ofloxacin was preserved in S. epidermidis, the susceptibility of Acinetobacter spp., E. coli, Proteus spp. being intermediate. Such a difference in the susceptibility of the isolates was evidently due to the properties of polyethylene glycols in the ointment composition.

[THE USE OF PROBIOTICS IN PATIENTS WITH RHEUMATOID ARTHRITIS].

The results of biological research of colon microbiota of patients with rheumatoid arthritis (RA) is in article presented. The signs of III degree dysbiosis, by reducing the concentration of Bacteroides spp., Bifidobacterium spp., Lactobacillus spp. populations, typical strain E. coli. But over growth of populations Klebsiella spp., Proteus spp., Staphylococcus spp., atypical forms of E. coli, Candida spp. The scheme for the correction of the colon microflora of patients with (RA) by was proposed bifiform. Increasing of populations concentration of Bifidobacterium spp., Bacteroides spp., Lactobacillus spp., typical E. coli, Enterococcus spp. and selective decontamination of Enterococcus (Hly+), Klebsiella spp., Proteus spp., Staphylococcus spp., lactosonegative and E.coli (Hly+) confirmed after using of this eubiotics.

[The formation of bio-films by opportunistic microorganisms isolated from patients with rheumatic diseases].

The study was carried out concerning capability of 194 strains of opportunistic microorganisms to form bio-films. It is established that bacteria ecizing organism of patients with rheumatic diseases have capacity to form microbial bio-films. The formation of bio-films is manifested with the same rate as in agents of inflammatory processes. At that, Escherichia coli, Staphylococcus haemolyticus and bacteria of genus Proteus isolated under rheumatic diseases have significantly higher capability to form biofilms that matters for development of comorbide infections.

Copper ion-stimulated McoA-laccase production and enzyme characterization in Proteus hauseri ZMd44.

The novel bioelectricity-generating bacterium of Proteus hauseri ZMd44 has been first identified to produce McoA-laccase (EC 1.10.3.2) induced by copper sulphate. The optimal concentration of copper is 3 mM as supplementation at the beginning of culture or early exponential growth phase, during which laccase is predominantly synthesized. Moreover, the whole cellular and intracellular activities of laccase increase in the degrees of inducible copper concentrations. A possible mechanism for this phenomenon is that copper ions enhance the laccase genetic transcription level during the laccase synthesis thus granting this strain in copper tolerance. McoA-laccase belongs to typical type 1 (T1) Cu site laccase by electron paramagnetic resonance (EPR) analysis of intracellular enzyme. From our results, the optimal temperature and pH are 60°C and pH 2.2, respectively. The kinetic profiles show that this enzyme is stable under 50°C and in the slightly acidic environment, making it a potentially useful enzyme in dye decolorization, paper-pulp bleaching and bioremediation industries.

[Taxonomic characteristics and mixed communities of wound infection agents in patients of reanimation and surgical departments of a hospital].

AIM: Study of taxonomical structure ofwound infection agents, prevalence of mixes, and detection of character of their possible connection with the results of various microorganisms population interaction in septic wounds. MATERIALS AND METHODS: A microbiological study of material from patients with wound infection (WI), 582 of those were cured in reanimation and intensive therapy departments (RITD; group 1) and 1455 - in surgical departments (SD; group 2), was performed. Taxonomic membership and ability to coexist was determined in 4129 microorganisms strains. Etiological role of the agents was evaluated by using values of consistency rate (CR). Species that were present in more than 50% of samples were considered consistent, in 25 to 50%--additional, and in less than 25%--random. Frequency rates (FR) were also determined, that is the fraction of a certain species (genus) of the microorganism (in %) from all the isolated cultures that correspond to 100%. For the determination of the significance of individual species of the agent in the structure of mixed microorganism populations, FR - their fraction (%) in mixed population from the number of strains of this species that correspond to 100% - was calculated. RESULTS: A significant part of the microorganisms strains, more frequently in reanimation department (65.5%), caused wound suppuration in populations mixed with other species of the agents. In reanimation and surgical departments consistent species of wound infection agents were not detected. A leading etiological role of Staphylococcus aureus (FR 19.2% and 23.9%) was determined, and FR of S. aureus strains in mixes was 64.6% in RITD and 46.8% in SD. The parameters ofotheragents of WI in the comparison groups were similar. However FR among mixes in RITD were significantly higher for streptococci that do not belong to S. pyogenes species (72,5%), and also nonfermentative microorganisms (67,2%), and in SD - in Klebsiella pneumoniae mixes. For agents of wound infection especially in RITD, low species diversity was characteristic and the number of mixes variants is significantly higher. In RITD mixed infections develop more frequently, and the ecological community of microorganisms reaches higher values than in SD. CONCLUSION: During the analysis of microbiologi-cal data in RITD and SD general patterns and specific features of taxonomical structure, prevalence of mixed populations and character of their ecological community in wound infection was determined.

Growth and aroma contribution of Microbacterium foliorum, Proteus vulgaris and Psychrobacter sp. during ripening in a cheese model medium.

The growth and aroma contribution of Microbacterium foliorum, Proteus vulgaris and Psychrobacter sp., some common but rarely mentioned cheese bacteria, were investigated in a cheese model deacidified by Debaryomyces hansenii during the ripening process. Our results show that these bacteria had distinct growth and cheese flavour production patterns during the ripening process. P. vulgaris had the greatest capacity to produce not only the widest variety but also the highest quantities of volatile compounds with low olfactive thresholds, e.g. volatile sulphur compounds and branched-chain alcohols. Such compounds produced by P. vulgaris increased after 21 days of ripening and reached a maximum at 41 days. The three bacteria studied exhibited various degrees of caseinolytic, aminopeptidase and deaminase activities. Moreover, P. vulgaris had a greater capacity for hydrolysing casein and higher deaminase activity. Our results show that P. vulgaris, a Gram-negative bacterium naturally present on the surface of ripened cheeses, could produce high concentrations of flavour compounds from amino acid degradation during the ripening process. Its flavouring role in cheese cannot be neglected. Moreover, it could be a useful organism for producing natural flavours as dairy ingredients.

Antimicrobial resistance and prevalence of canine uropathogens at the Western College of Veterinary Medicine Veterinary Teaching Hospital, 2002-2007.

Between January 2002 and June 2007, uropathogens were isolated from 473 of 1557 canine urine samples submitted to Prairie Diagnostic Services from the Western College of Veterinary Medicine Veterinary Teaching Hospital. Culture and susceptibility results were analyzed, retrospectively, to estimate the prevalence of common bacterial uropathogens in dogs with urinary tract infections and to identify changes in antimicrobial resistance. The most common pathogens identified were Escherichia coli, Staphylococcus intermedius, Enterococcus spp., and Proteus spp. Antimicrobial resistance increased during the study period, particularly among recurrent E. coli isolates. Using the formula to help select rational antimicrobial therapy (FRAT), bacterial isolates were most likely to be susceptible to gentamicin, fluoroquinolones, amoxicillin-clavulanic acid, and groups 4 and 5 (third generation) cephalosporins.

Antimicrobial activity of flavonoids from Pelargonium radula (Cav.) L'Hérit.

Flavonoids from Pelargonium radula (Cav.) L'Hérit were purified by column chromatography. Two fractions were obtained: F1 (main flavonoid isoquercitrin) and F2 (main flavonoid rutin). In vitro antimicrobial activity of F1 and F2 were tested against eleven species of bacteria and eleven species of fungi. Both fractions demonstrated strong inhibitory activity against Staphylococcus aureus, Proteus rettgeri, Candida tropicalis and Microsporum gypseum. Staphylococcus sp. (coagulase-negative) and Candida lusitaniae were strongly inhibited only by fraction F1 and Fusarium graminearum only by fraction F2.