Metagenomic identification of gut microbiota distribution on the colonic mucosal biopsy samples in patients with non-alcoholic fatty liver disease.

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) is known to be the most common liver disease in the world, and there are currently no approved pharmacological treatments to prevent or treat this condition. In addition to being associated with an increased risk of hepatocellular carcinoma and cirrhosis, NAFLD has now become the leading cause of liver failure-associated transplantation. The 16S rRNA gene which conserved regions can serve as universal primer binding sites for PCR amplification of gene fragments, while hypervariable regions contain significant sequence diversity useful for prokaryotic identification purposes. 16S rRNA gene sequences can be use by researchers to identify prokaryotic taxonomy found in clinical samples. As a result of increasing microbiota studies with developing technological developments, the role of intestinal microbiota in the pathogenesis of NAFLD is revealed in an important way. In this study, it was aimed to determine the clinical prognostic importance of gut microbiota in the pathogenesis of NAFLD and to determine the microbial composition with intestinal mucosal biopsy samples in NAFLD patients. MATERIAL AND METHOD: We included 20 patients diagnosed with NAFLD as a result of liver function tests, histological, ultrasonographic, biopsy evidence and 20 normal control groups created under exclusion criteria in this study. The healthy control group of the same age and gender as the patients were determined to be equal, and the age, gender, BMI, insulin resistance, AST, ALT levels of the individuals were recorded for analysis. Intestinal mucosal biopsy samples were taken from the individuals included in the study under sterile conditions. Microbial results were obtained as a result of 16S rRNA amplicon metagenomic processes. The region of approximately 1500 bp covering the V1-V9 region of the 16S rRNA gene was targeted to detect microbial diversity. The amplified regions were sequenced using next-generation sequencing. Operational Taxonomic Unit (OTU) value was obtained with bioinformatics software with the obtained sequence data. The analysis of the recorded parameters was done with the SPSS.19 statistical program. RESULTS: In the designed study, 16 phyla, 28 class, 56 order, 128 family, 415 genera, 1041 species microorganisms were analyzed taxonomically in a total of 40 individuals. In our study, Intestinal microbial diversity is lower in NAFLD patients compared to control group individuals. In addition, gram-negative bacteria were found to be more dominant in NAFLD patients. As a phylum, Proteobacteria increased in NAFLD group, Bacteroidetes and Actinobacteria in control group, while Firmicutes had equal distribution in both groups. BMI OR = 6.37, 95 %CI (0.39-0.40) p value was 0.001 in laboratory data, whereas Proteobacteria OR = 1.754, 95% CI (0.901-3.416), p value 0.05 in microbial profile. CONCLUSION: The 16S rRNA metagenomic study of intestinal microbiota using colonic mucosal biopsy samples in NAFLD disease was the first study in the Turkish population, and important data were obtained for other studies. In the data obtained, we think Proteobacteria, Ruminococcaceae, Escherichia coli and Bacilli are very important in both diagnostic and treatment options as a microbial profile in NAFLD.

The effect of PNPLA3 polymorphism as gain in function mutation in the pathogenesis of non-alcoholic fatty liver disease.

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) is often associated with metabolic syndrome (type 2 diabetes, hypertension, hypertriglyceridemia, insulin resistance, and obesity). NAFLD is multi-factorial in pathogenesis with some genetic predisposition. The variant patatin-like phospholipase domain-containing protein 3 (PNPLA3) is known to be an independent risk factor for hepatocellular cancer (HCC). The aim of this study was to investigate the role of PNPLA3 polymorphism as the risk factor for NAFLD. METHODOLOGY: Patients had histological, ultrasonographic, biopsy evidence of NAFLD (n=248) and 81 controls were studied for PNPLA3 polymorphism. PNPLA3 genotyping was done from peripheral blood DNA by real-time polymerase chain reaction (RT-PCR). RESULTS: PNPLA3 genotyping of the groups NAFLD (CC [n = 76], CG [n = 83], GG [n = 89]) and control (CC [n= 42], CG [n = 22], GG [n = 17]) was determined. In the patient group, the G allele was 261 (52.63%) and the C allele was 235 (47.37%), whereas in the control group, the G allele was 56 (34.54%) and the C allele was 106 (65.43%). In our study, 53 out of 174 women had GG allele and 54 out of 155 men had GG allele. CONCLUSION: The findings suggest that there is a predominant relationship between men with PNPLA3 I148M variant with NAFLD in women. Patients with NAFLD carrying PNPLA3 rs738409 G>C variant are at higher risk of NAFLD.

Degradation of 2,4,6-trinitrotoluene by P. aeruginosa and characterization of some metabolites.

Degradation of 2,4,6-trinitrotoluene (TNT), a nitroaromatic explosive found in the soil and ground water, was investigated using Pseudomonas aeruginosa in in vitro experiments . Biodegradable abilitiy of this bacteria was performed with 50 and 75 mg L (-1) TNT concentrations in a defined liquid medium for 96 h time period. Treatment of TNT in supernatant samples taken at 0, 6, 12, 24, 48, 72 and 96 h from agitated vessels was followed by reverse-phase high-performance liquid chromatography (HPLC). In cultures supplemented with 50 and 75 mgL (-1) TNT, after 96 h of incubation 46% and 59% reduction were detected respectively. Two metabolites as degradation intermediates with nitrite release into the medium, 2,4-dinitrotoluene (2,4-DNT) and 4-aminodinitrotoluene (4-ADNT), were elucidated by thin layer chromatography (TLC) and gas chromatography-mass spectrometry (GC-MS). These findings clearly indicate that Pseudomonas aeruginosa can be used in bioremediation of TNT contaminated sites.

Degradation of 2,4,6-trinitrotoluene by P. aeruginosa and characterization of some metabolites

Degradation of 2,4,6-trinitrotoluene (TNT), a nitroaromatic explosive found in the soil and ground water, was investigated using Pseudomonas aeruginosa in in vitro experiments. Biodegradable abilitiy of this bacteria was performed with 50 and 75 mg L−1 TNT concentrations in a defined liquid medium for 96 h time period. Treatment of TNT in supernatant samples taken at 0, 6, 12, 24, 48, 72 and 96 h from agitated vessels was followed by reverse-phase high-performance liquid chromatography (HPLC). In cultures supplemented with 50 and 75 mgL−1 TNT, after 96 h of incubation 46% and 59% reduction were detected respectively. Two metabolites as degradation intermediates with nitrite release into the medium, 2,4-dinitrotoluene (2,4-DNT) and 4-aminodinitrotoluene (4-ADNT), were elucidated by thin layer chromatography (TLC) and gas chromatography-mass spectrometry (GC-MS). These findings clearly indicate that Pseudomonas aeruginosa can be used in bioremediation of TNT contaminated sites.

Aerobic biodegradation of 2,4,6-trinitrotoluene (TNT) by Bacillus cereus isolated from contaminated soil.

In this study, biological degradation of 2,4,6-trinitrotoluene (TNT) which is very highly toxic environmentally and an explosive in nitroaromatic character was researched in minimal medium by Bacillus cereus isolated from North Atlantic Treaty Organization (NATO) TNT-contaminated soils. In contrast to most previous studies, the capability of this bacteria to transform in liquid medium containing TNT was investigated. During degradation, treatment of TNT was followed by high-performance liquid chromatography (HPLC) and achievement of degradation was calculated as percentage. At an initial concentration of 50 and 75 mg L(-1), TNT was degraded respectively 68 % and 77 % in 96 h. It transformed into 2,4-dinitrotoluene and 4-aminodinitrotoluene derivates, which could be detected as intermediate metabolites by using thin-layer chromatography and gas chromatography-mass spectrometry analyses. Release of nitrite and nitrate ions were searched by spectrophotometric analyses. Depending upon Meisenheimer complex, while nitrite production was observed, nitrate was detected in none of the cultures. Results of our study propose which environmental pollutant can be removed by using microorganisms that are indigenous to the contaminated site.

Determination of microbial quality and plasmid-mediated multidrug resistant bacteria in fountain drinking water sources in Turkey.

The bacterial contamination as the total aerobic bacteria, coliform and fecal coliform numbers were determined and analyzed for temperature, pH, conductivity and dissolved oxygen in seasonally collected water samples from fifteen different stations placed in Adana-Tufanbeyli road line during March 2008 - January 2009. In addition, antibiotic resistance profiles of isolates were examined against frequently used antibiotics, and analyzed plasmid DNAof multiple antibiotic resistant (MAR) isolates. Total aerobic bacteria in fountain water samples was determined as 3 x 10(3) CFU ml(-1) and total and fecal coliforms were determined 460 MPN/100 ml. Results obtained from biochemical analysis showed that 121 of the isolates were Proteus vulgaris, 69 Escherichia coli, 51 Pseudomonas aeruginosa and 28 Citrobacter spp. According to these results, the existence of Vibrio parahaemolyticus in stations 2 and 10, and Streptococcus faecalis in stations 11 and 15 respectively were confirmed. Clostridium perfringens was not detected in water samples. A total of 273 isolates were tested for antimicrobial susceptibility by agar disc diffusion methods. A total of sixteen antibiotics were used for determination of antibiotic resistance of isolates. Resistance to bacitracin, vancomycine, cephalothin and ampicillin was detected in 77, 77, 63 and 50%, respectively. Multiple antibiotic resistance (MAR) value > or = 0.25 was determined in 68.4% of identified 273 isolates and meaning of this percentage were resistant to four and more antibiotics. Plasmid DNA was isolated from 22 isolates with multiple antibiotic resistance index ranged from 0.3 to 0.6 taken randomly by agarose-gel electrophoresis, some of them contain a high-molecular weight plasmid DNA. Highlight of our study that the appearance of potential antibiotic resistances in fountain drinking water requires increased surveillance for risk assessment and prevention strategies to protect public health.

Transient expression of red and yellow fluorescent protein vectors in HCT-8 cells infected with Cryptosporidium parvum.

Electroporation in combination with the red and yellow fluorescent protein (RFP and YFP, respectively) reporter systems results in transient gene expression in HCT-8 cell lines infected and uninfected with Cryptosporidium parvum. Conditions are described allowing efficient electroporation of HCT-8 cell monolayers in a commercial electroporation device (180 kV/cm voltage, 250 microF capacitance, and 0 ohm (ohm) resistance resulting in a time constant of 0.4 ms.). Fluorescent microscopy showed that uninfected HCT-8 cell monolayers achieved higher gene transfer and expression efficiencies than HCT-8 cells infected with C. parvum under similar conditions. Real-time melting curve and quantitation analysis of reverse transcription polymerase chain reaction amplicons are presented for the detection of all mRNA sample levels. Our findings demonstrate that C. parvum infection of HCT-8 cells may negatively affect the transient expression of RFP and YFP plasmids. The focus of this study was to achieve transient expression of reporter genes in HCT-8 cell lines and provide the basis for further analyses of gene regulation in protozoan. These approaches may provide to understanding the feasibility of gene transfer and expression efficiencies HCT-8 cell cultures by RFP and YFP containing the CMV promoter; they could serve as tools for gene transfer in mammalian cell.

Resistances of antibiotics and heavy metals in Enterobacteriaceae spp. isolated from gills and intestines of Achanthobrama marmid (Heckel,1843) from Sir Dam lake Turkey.

A total of 94 bacteria, associated with wild Achanthobrama marmid (Heckel, 1843) in Sir Dam lake of Turkey identified. Subsequently selected isolates were characterized and identified to the genus level The 94 members of Enterobacteriaceae were isolated in the gills and intestines, and among the isolates, E. coli were represented at a rate of 55%, Shigella spp. at a rate of 21%, Salmonella spp. at a rate of 9%, Citrobacter spp. at a rate of 9%, Klebsiella spp. at a rate of 3% and Proteus spp., at a rate of 3%. A total of 94 bacteria resistant to antibiotics and heavy metals were isolated from total 47 of A. marmid samples and were investigated. Viable counts of antibiotic resistant bacteria isolated from gill and intestinal content samples showed high frequencies of resistance to Penicilline-G (KP) (68%), CZ (54%), FOX (48%), while the proportion of CRO (39%) and CTX (36%) resistance was low. In this research, heavy metal contamination in Sir Dam lake water samples and resistance frequency against heavy metals in isolated bacteria from gill and intestinal contents in A. marmid were investigated. Heavy metal contamination such as nickel (Ni), cadmium (Cd), copper (Cu) and chromium (Cr) determined diverse rate (except Mn) in water samples. The resistance frequency of the isolates was revealed different rate for the following heavy metals: Ni, Cd, Cu and Cr When the concentration of heavy metals increased, the resistance against heavy metals in diverse genus of isolates in different rate decreased.

Antibacterial agents and heavy metal resistance in Gram-negative bacteria isolated from seawater, shrimp and sediment in Iskenderun Bay, Turkey.

The aim of the present study was to determine the level of antibiotic resistance patterns and distribution of heavy metal resistance of bacterial isolates from seawater, sediment and shrimps, and to determine if there is a relationship between antibiotic and heavy metal resistance. We undertook studies in 2007 in the industrially polluted Iskenderun Bay, on the south coast of Turkey. The resistance of 236 Gram-negative bacterial isolates (49 from seawater, 90 from sediment and 97 from shrimp) to 16 different antibiotics, and to 5 heavy metals, was investigated by agar diffusion and agar dilution methods, respectively. A total of 31 species of bacteria were isolated: the most common strains isolated from all samples were Escherichia coli (11.4%), Aeromonas hydrophila (9.7%) and Stenotrophomonas maltophilia (9.3%). There was a high incidence of resistance to ampicillin (93.2%), streptomycin (90.2%) and cefazolin (81.3%), and a low incidence of resistance to imipenem (16.5%), meropenem (13.9%) and cefepime (8.0%). Some 56.8% of all bacteria isolated from seawater, sediment and shrimp were resistant to 7 or more antibiotics. Most isolates showed tolerance to different concentrations of heavy metals, and minimal inhibition concentrations ranged from 12.5 microg/ml to > 3200 microg/ml. The bacteria from seawater, sediment and shrimp showed high resistance to cadmium of 69.4%, 88.9%, and 81.1% respectively, and low resistance to manganese of 2%, 6.7% and 11.3% respectively. The seawater and sediment isolates which were metal resistant also showed a high resistance to three antibiotics: streptomycin, ampicillin and trimethoprim-sulphamethoxazole. In contrast, the shrimp isolates which were metal resistant were resistant to four antibiotics: cefazolin, nitrofurantoin, cefuroxime and ampicillin. Our results show that Iskenderun Bay has a significant proportion of antibiotic and heavy metal resistant Gram-negative bacteria, and these bacteria constitute a potential risk for public health.

Highly thermostable and alkaline alpha-amylase from a halotolerant-alkaliphilic Bacillus sp. AB68 / Alfa-amilase alcalina termoestável de Bacillus sp AB68 halotolerante-alcalifílico

An alkaliphilic and highly thermostable alpha-amylase producing Bacillus sp. was isolated from Van soda lake. Enzyme synthesis occurred at temperatures between 25ºC and 40ºC. Analysis of the enzyme by SDS-PAGE revealed a single band which was estimated to be 66 kDa. The enzyme was active in a broad temperature range, between 20ºC and 90ºC, with an optimum at 50ºC; and maximum activity was at pH 10.5. The enzyme was almost completely stable up to 80ºC with a remaining activity over 90 percent after 30 min pre-incubation. Thermostability was not increased in the presence of Ca2+. An average of 75 percent and 60ºC of remaining activity was observed when the enzyme was incubated between pH 5 and 9 for 1 h and for 2 h, respectively. The activity of the enzyme was inhibited by SDS and EDTA by 38 percent and 34 percent, respectively.

Bacillus sp AB68 alcalif¨ªlico produtor de alfa-amilase alcalina termoest¨¢vel foi isolado do lago Van soda. A s¨ªntese da enzima ocorreu entre 25ºC e 40ºC. A an¨¢lise da enzima por SDS-PAGE revelou uma ¨²nica banda estimada em 66 kDa. A enzima foi ativa em uma ampla faixa de temperatura, entre 20ºC e 90ºC, com um ¨®timo a 50ºC. A atividade m¨¢xima foi em pH 10,5. A enzima foi est¨¢vel at¨¦ 80ºC, mantendo 90 por cento de atividade ap¨®s 30 min de pr¨¦-incubação. A termoestabilidade não aumentou na presença de Ca2+. Quando incubada em pH entre 5 e 9 por 1h e por 2h, a enzima manteve 75 por cento e 60 por cento de atividade, respectivamente. SDS e EDTA causaram redução de 38 por cento e 34 por cento na atividade da enzima, respectivamente.

Highly thermostable and alkaline α-amylase from a halotolerant-alkaliphilic Bacillus sp. AB68.

An alkaliphilic and highly thermostable α-amylase producing Bacillus sp. was isolated from Van soda lake. Enzyme synthesis occurred at temperatures between 25°C and 40°C. Analysis of the enzyme by SDS-PAGE revealed a single band which was estimated to be 66 kDa. The enzyme was active in a broad temperature range, between 20°C and 90°C, with an optimum at 50°C; and maximum activity was at pH 10.5. The enzyme was almost completely stable up to 80°C with a remaining activity over 90% after 30 min pre-incubation. Thermostability was not increased in the presence of Ca(2+). An average of 75% and 60°C of remaining activity was observed when the enzyme was incubated between pH 5 and 9 for 1 h and for 2 h, respectively. The activity of the enzyme was inhibited by SDS and EDTA by 38% and 34%, respectively.

The reuse of dried activated sludge for adsorption of reactive dye.

Adsorption processes are alternative effective methods for removal of textile dyes from aqueous solutions. The adsorption ability of adsorbent affects by physico-chemical environment for this reason in this paper effect of initial pH, dye concentrations, temperature and dye hydrolyzation were determined in a batch system for removal of reactive dye by dried activated sludge. The Langmuir isotherm model was well described of adsorption reactive dye and maximum monolayer adsorption capacity (at pH 2) of activated sludge was determined as 116, 93 and 71mgg(-1) for 20 degrees , 35 degrees and 50 degrees C, respectively. Initial pH 2, 20 degrees C and 30min contact time are suitable for removal of reactive dyes from aqueous solutions. Activated sludge was characterized by FT-IR analysis and results showed that active sludge has different functional groups and functional groups of activated sludge are able to react with dye molecules in aqueous solution. The pseudo first-order, second-order and intraparticle diffusion kinetics were used to describe the kinetic data. The pseudo second-order kinetic model was fit well over the range of contact times and also an intra particle diffusion kinetic model was fit well but in the first 30min. The dye hydrolyzation was affected adsorption capacity of biomass and adsorption capacity of biomass decreased with dye hydrolyzation from 74 to 38mgg(-1).