Virulence determinants and genetic diversity of adherent-invasive Escherichia coli (AIEC) strains isolated from patients with Crohn's disease.

BACKGROUND: Adherent invasive Escherichia coli (AIEC) are implicated in the pathogenesis of inflammatory bowel diseases (IBD) particularly Crohn's disease (CD). The aim of this study is to isolate, identify, genotype, and characterize the virulence factors and the clinical significance of AIEC strains. METHODS: Ileal and colonic biopsies from 24 active CD patients and 15 healthy controls (HC) were collected. E. coli strains were identified by standard biochemical tests and confirmed by MALDI-TOF (bioMerieux, France) system. The AIEC phenotypes were determined by the adhesion, invasion, and survival within macrophages assays. The genetic virulence factors and genotyping characteristics were determined by PCR and PFGE respectively. The abundance and the antibiogram profile of E. coli strains was determined by qPCR and VITEK®2 (bioMerieux, France) automated system respectively. RESULTS: E. coli strains from 17 CD patients and 14 HC were isolated, 10 (59%) and 7 (50%) of them were identified as AIEC strains, respectively. We found that chuA and ratA genes were the most significant genetic markers associated with AIEC compared to non-AIEC strains isolated from CD patients and HC p = 0.0119, 0.0094 respectively. The majority of E. coli strains obtained from CD patients showed antibiotic resistance (71%) compared to HC (29%) against at least one antibiotic. The AIEC-like strains were more resistant to antibiotics compared to non-AIEC-like strains (53%) and (21%) respectively. CONCLUSIONS: We have determined significant differences between AIEC strains and non-AIEC strains in terms of the prevalence of chuA and ratA virulence genes and the antibiotic resistance profiles. In addition, AIEC strains isolated from CD patients were found to be more resistant to penicillin/beta lactam and aminoglycoside antibiotics than AIEC strains isolated from HC 80%, 14% respectively.

The role of major virulence factors and pathogenicity of adherent-invasive Escherichia coli in patients with Crohn's disease.

Inflammatory bowel disease (IBD) is a term that describes Crohn's disease (CD) and ulcerative colitis (UC), and these two conditions are characterised by chronic inflammation of the gastrointestinal tract. Dysbiosis of intestinal microbiota has been consistently linked to patients with IBD. In the last two decades, the progressive implication of adherent-invasive Escherichia coli (AIEC) pathogenesis in patients with CD has been increasing. Here we discuss recent findings that indicate the role and mechanisms of AIEC in IBD. We also highlight AIEC virulence factor genes and mechanisms that suggest an important role in the severity of inflammation in CD patients. Finally, we emphasise data on the prevalence of AIEC in CD patients.

Polyethers isolated from the marine actinobacterium Streptomyces cacaoi inhibit autophagy and induce apoptosis in cancer cells.

Polyether compounds, a large group of biologically active metabolites produced by Streptomyces species have been reported to show a variety of bioactivity such as antibacterial, antifungal, antiparasitic, antiviral, and tumour cell cytotoxicity. Since some of these compounds target cancer stem cells and multi-drug resistant cancer cells, this family of compounds have become of high interest. In this study, three polyether-type metabolites (1-3), one of which was a new natural product (3), were isolated from the marine derived Streptomyces cacaoi via antimicrobial activity-guided fractionation studies. As several polyether compounds with structural similarity such as monensin have been linked with autophagy and cell death, we first assessed the cytotoxicity of these three compounds. Compounds 2 and 3, but not 1, were found to be cytotoxic in several cell lines with a higher potency towards cancer cells. Furthermore, 2 and 3 caused accumulation of both autophagy flux markers LC3-II and p62 along with cleavage of caspase-3, caspase-9 and poly (ADP-ribose) polymerase 1 (PARP-1). Interestingly, prolonged treatment of the compounds caused a dramatic downregulation of the proteins related to autophagasome formation in a dose dependent manner. Our findings provide insights on the molecular mechanisms of the polyether-type polyketides, and signify their potency as chemotherapeutic agents through inhibiting autophagy and inducing apoptosis.

Synthesis and Spectroscopic Studies of Phenanthroimidazole-Imine Derivatives and Evaluation of Their Antioxidant Activity.

The phenanthroimidazole derivatives are used in the drug industry in large scale. In this study four new phenanthroimidazole-imine derivatives were synthesized and their spectroscopic studies as well as their antioxidant behaviors were examined. Structural analyses were made by FT-IR, 1H-NMR, 13C-NMR, LC-MS spectroscopy techniques. UV-vis absorption and emission spectroscopy techniques have been used to determine the photophysical characteristics of four newly synthesized phenanthroimidazoles. The maximum absorption and emission wavelengths, molar extinction coefficients (ε), singlet energy levels (Es), Stokes' shift values (Δλ) of phenanthroimidazole-imine derivatives are given. Additionally, the antioxidant behavior of all compounds were investigated which the ascorbic acid used as standard molecule in present study.

Xylanase production from marine derived Trichoderma pleuroticola 08ÇK001 strain isolated from Mediterranean coastal sediments.

Xylanases constitutes one the most important enzymes with diverse applications in different industries such as bioethanol production, animal feedstock production, production of xylo-oligosaccharides, baking industry, paper and pulp industry, xylitol production, fruit juice, and beer finishing, degumming, and agriculture. Currently, industrial xylanases are mainly produced by Aspergillus and Trichoderma members. Since the marine environments are less studied compared to terrestrial environments and harbors great microbial diversity we aimed to investigate the xylanase production of 88 marine-derived filamentous fungal strains. These strains are semi-quantitatively screened for their extracellular xylanase production and Trichoderma pleuroticola 08ÇK001 xylanase activity was further characterized. Optimum pH and temperature was determined as 5.0 and 50 °C, respectively. The enzyme preparation retained 53% of its activity at pH 5.0 after 1 h and have found resistant against several ions and compounds such as K+ , Ba2+ , Na+ , ß-mercaptoethanol, Triton X-100 and toluene. This study demonstrates that marine-derived fungal strains are prolific sources for xylanase production and presents the first report about the production and characterization of xylanase from a marine derived T. pleuroticola strain. The characteristics of T. pleuroticola 08ÇK001 xylanase activity indicate possible employment in some industrial processes such as animal feed, juice and wine industries or paper pulping applications.

LuxCDE-luxAB-based promoter reporter system to monitor the Yersinia enterocolitica O:3 gene expression in vivo.

It is crucial to understand the in vitro and in vivo regulation of the virulence factor genes of bacterial pathogens. In this study, we describe the construction of a versatile reporter system for Yersinia enterocolitica serotype O:3 (YeO3) based on the luxCDABE operon. In strain YeO3-luxCDE we integrated the luciferase substrate biosynthetic genes, luxCDE, into the genome of the bacterium so that the substrate is constitutively produced. The luxAB genes that encode the luciferase enzyme were cloned into a suicide vector to allow cloning of any promoter-containing fragment upstream the genes. When the obtained suicide-construct is mobilized into YeO3-luxCDE bacteria, it integrates into the recipient genome via homologous recombination between the cloned promoter fragment and the genomic promoter sequence and thereby generates a single-copy and stable promoter reporter. Lipopolysaccharide (LPS) O-antigen (O-ag) and outer core hexasaccharide (OC) of YeO3 are virulence factors necessary to colonization of the intestine and establishment of infection. To monitor the activities of the OC and O-ag gene cluster promoters we constructed the reporter strains YeO3-Poc::luxAB and YeO3-Pop1::luxAB, respectively. In vitro, at 37°C both promoter activities were highest during logarithmic growth and decreased when the bacteria entered stationary growth phase. At 22°C the OC gene cluster promoter activity increased during the late logarithmic phase. Both promoters were more active in late stationary phase. To monitor the promoter activities in vivo, mice were infected intragastrically and the reporter activities monitored by the IVIS technology. The mouse experiments revealed that both LPS promoters were well expressed in vivo and could be detected by IVIS, mainly from the intestinal region of orally infected mice.

Investigation of enteropathogenic Escherichia coli and Shiga toxin-producingEscherichia coli associated with hemolytic uremic syndrome in Izmir Province, Turkey.

BACKGROUND/AIM: The purpose of this study was to investigate Shiga toxin-producing Escherichia coli (STEC) and enteropathogenic Escherichia coli (EPEC) strains originating from diarrheagenic patients. MATERIALS AND METHODS: A total of 102 patients with diarrhea between October 2012 and January 2013 were enrolled in this study. Multiplex and standard polymerase chain reactions were performed to detect and distinguish STEC and EPEC strains. O serotyping of EPEC was carried out by monovalent antisera. The O and H serotyping of STEC strains was performed at the Refik Saydam Institute, Ankara. RESULTS: A total of 5 (3.42%) strains were identified as STEC, and 3 strains (2.05%) were atypical EPEC. One of the STEC serotypes was O157:H7 carrying VT1, Stx1A, and escv genes. The other STEC strain was identified as O174:H21, which is associated with hemolytic uremic syndrome and consists of VT2 and Stx2A genes. One of the EPEC and three of the STEC serotypes were nontypeable. The serotypes of the atypical EPEC strains were identified as O114 and O26. CONCLUSION: To the best of our knowledge, this is the first report of O174:H21 from the Izmir region that was shown to be a Shiga toxin-producing non-O157 serotype of STEC.

Bioactive Sheath/Core nanofibers containing olive leaf extract.

This study aimed at producing silk fibroin (SF)/hyaluronic acid (HA) and olive leaf extract (OLE) nanofibers with sheath/core morphology by coaxial electrospinning method, determining their antimicrobial properties, and examining release profiles of OLE from these coaxial nanofibers. Optimum electrospinning process and solution parameters were determined to obtain uniform and bead-free coaxial nanofibers. Scanning electron microscopy and transmission electron microscopy (TEM) were used to characterize the morphology of the nanofibers. The antimicrobial activities of nanofibers were tested according to AATCC test method 100. Total phenolic content and total antioxidant activity were tested using in vitro batch release system. The quality and quantity of released components of OLE were determined by high-performance liquid chromatography. The changes in nanofibers were examined by Fourier-transform infrared spectroscopy. Uniform and bead-free nanofibers were produced successfully. TEM images confirmed the coaxial structure. OLE-loaded nanofibers demonstrated almost perfect antibacterial activities against both of gram-negative and gram-positive bacteria. Antifungal activity against C. albicans was rather poor. After a release period of 1 month, it was observed that ∼70-95% of the OLE was released from nanofibers and it was still bioactive. Overall results indicate that the resultant shell/core nanofibers have a great potential to be used as biomaterials.

Cytosine-type nucleosides from marine-derived Streptomyces rochei 06CM016.

Rocheicoside A (3), a nucleoside analog possessing a novel 5-(hydroxymethyl)-5-methylimidazolidin-4-one substructure, was isolated from marine-derived actinomycete Streptomyces rochei 06CM016, together with a new (4) and three known compounds. Structures of the new metabolites were elucidated by one-dimensional ((1)H and (13)C) and 2D NMR (COSY, HMQC and HMBC) and HR-TOF-MS analyses. All the metabolites exhibited significant antimicrobial activity. A plausible mechanism was proposed for compound 3's formation from amicetin.

Isolation of pathogenic Yersinia enterocolitica strains from different sources in Izmir region, Turkey.

Yersinia enterocolitica is a foodborne pathogen that is very rarely encountered in Turkey. In this work, several human, porcine, and environmental samples collected from Izmir region in Turkey were examined for the presence of Y. enterocolitica using different cultivation and enrichment methods. A total of nine pathogenic Y. enterocolitica strains were isolated; five strains from pig stool and manure samples and four strains from waste water samples. On the other hand, no Y. enterocolitica was isolated from human diarrheal stool samples (n = 102) and from 12 gulf, canal, municipal pool, and well water samples. Biochemical and serological characterization of the nine Y. enterocolitica strains revealed that they belonged to three different bioserotypes: 4/O:3, 2/O:9, and 2/O:5,27. All the strains were deemed pathogenic based on virulence factor-specific PCR analysis. Detection of pathogenic Y. enterocolitica strains from the pig and waste water samples from the Izmir region indicates that Y. enterocolitica is a potential risk for public health.

Isolation strategies of marine-derived actinomycetes from sponge and sediment samples.

During the last two decades, discoveries of new members of actinomycetes and novel metabolites from marine environments have drawn attention to such environments, such as sediment and sponge. For the successful isolation of actinomycetes from marine environments, many factors including the use of enrichment and pre-treatment techniques, and the selection of growth media and antibiotic supplements should be taken into account. High-throughput cultivation is an innovative technique that mimics nature, eliminates undesired, fast-growing bacteria and creates suitable conditions for rare, slow-growing actinomycetes. This review comprehensively evaluates the traditional and innovative techniques and strategies used for the isolation of actinomycetes from marine sponge and sediment samples.

Bioassay-guided isolation of antibacterial and cytotoxic compounds from the mesophilic Actinomycete M-33-5.

One hundred and twenty-six mesophilic Actinomycete cultures were isolated from the Aegean region of Turkey. The antimicrobial activities of pure isolates were tested using the agar-plaque method. Based on high antimicrobial activity against methicillin resistant Staphylococcus aureus (MRSA) and Escherichia coli O157-H7 (E. coli), the isolate M-33-5 was selected for bioactivity-guided isolation. Fermentation, followed by solvent partition (H2O-EtOAc, H2O-n-BuOH), showed that the highest activity was present in the EtOAc extract. By using chromatographic methods, two bioactive compounds were isolated and their structures were determined by spectral methods to be 4'-deacetyl griseusin A and griseusin A. The MIC values of griseusin A and 4'-deacetyl griseusin A against MRSA and E. coli were < or = 1.0 microg/mL. The cytotoxicities of the EtOAc extract and 4'-deacetyl griseusin A were also evaluated against two cancer cell lines (human servical cancer: HeLa; murine fibroblastic cells: L-929). The EtOAc extract showed strong cytotoxic activity against HeLa and L-929 lines with IC50 values of 1.57 and 2.43 microg/mL, respectively, whereas 4'-deacetyl griseusin A was very potent with IC50 values of 0.43 versus HeLa, and 0.12 microg/mL against L-929. The active strain M-33-5 was identified as Streptomyces griseus by 16SrDNA sequence data.

Metal biosorption capacity of the organic solvent tolerant Pseudomonas fluorescens TEM08.

Many kinds of biomass are being tested as a biosorption material for metal removal from the contaminated waters. In the present study the biosorption capacity of an organic solvent tolerant (OST) bacterium was investigated against Cr(VI) and Ni(II). The OST strain of Pseudomonas fluorescens TEM08 was isolated from an oil contaminated soil sample and grown in normal culture conditions (type I) and in the presence of the cyclohexane (type II). Two types of cells were used in the biosorption experiments to compare the organic solvent effect on the biosorption capacity. The biosorption equilibrium was described by Langmuir and Freundlich adsorption isotherms. The value of Q(0) was higher for type I cells (40.8 for Cr(VI); 12.4 for Ni(II)) then the type II (40.7 for Cr(VI); 11.2 for Ni(II)). The adsorption capacity constants (K(F)) of Freundlich model for type I cells and for type II cells were 10.87 and 8.78 for Ni(II) and 13.60 and 10.99 for Cr(VI), respectively.

A long-term effect of caries-related factors in initially caries-free children.

AIM: The aim of this study was to examine the rate and pattern of early childhood caries development in caries-free children based on their dental plaque accumulations, salivary Streptococcus mutans levels, maternal sharing, oral hygiene, and feeding attitudes at baseline and at 24-month follow-up period. DESIGN: A total of 92 children, aged between 15 to 35 months, comprised the study group. The children's dental examinations were first carried out at baseline, and 56 of them were re-examined 24 months later to determine the changes in dental status. The mothers were also interviewed at each examination based on a prepared questionnaire. RESULTS: The prevalence of caries incidence was 45% at 24-month evaluation period among initially caries-free children. The new caries formation was mostly observed on occlusal and aproximal surfaces of maxillary molars (28% and 26%) followed by mesial, distal and buccal surfaces of anterior teeth, respectively. Significant correlations between dental caries formation and maternal sharing, S. mutans levels, and plaque scores were found. CONCLUSION: The results indicated that early S. mutans colonization, high plaque accumulation, and maternal sharing were important factors on a child's caries development.

PCR-based identification of selected pathogens associated with endodontic infections in deciduous and permanent teeth.

OBJECTIVE: The aim of the present study was to evaluate the presence of the selected pathogens in samples from deciduous and permanent tooth root canals by using PCR method and to determine the association of these organisms with clinical symptoms. STUDY DESIGN: A total of 145 children, 5 to 13 years old, were involved in this study. The presence of selected pathogens (Actinomyces israelii, Candida albicans, Enterococcus faecalis, Fusobacterium nucleatum, Porphyromonas endodontalis, Porphyromonas gingivalis, Prevotella intermedia, Streptococcus intermedius, Treponema denticola, Parvimonas micra, Tannerella forsythensis, Enterococcus faecium, Prevotella melaninogenica) in infected root canals was studied using PCR. RESULTS: T. denticola (P = .012, .02) and E. faecalis (P = .012, .04) were highly associated with periapical radiolucency and previous pain, while P. gingivalis was associated with tenderness to percussion in both deciduous and permanent teeth (P = .01, .015). CONCLUSION: The results of the present study confirm that certain species of microorganisms are associated with clinical signs and symptoms of endodontic disease in both deciduous and permanent teeth.

Tryptamine derived amides with thiazole ring system from Thermoactinomyces strain TA66-2.

A moderately thermophilic actinomycete strain, which was identified as Thermoactinomyces strain TA66-2, was isolated from hot-spring water. Fermentation, followed by solvent partition and chromatographic separations, resulted in the isolation of two new and two known molecules. The structures of the new compounds were elucidated as 2-(1-Propionylaminoethyl)thiazole-4-carboxylic acid [2-(1H-indol-3-yl)ethyl]amide and 2-(1-Acetylaminoethyl)thiazole-4-carboxylic acid [2-(1H-indol-3-yl)-ethyl]amide by using spectral methods (1D-, 2D-NMR and LC-ESI-MS).

Colonization and vertical transmission of Streptococcus mutans in Turkish children.

The aim of the study was to establish the colonization of Streptococcus mutans and to determine the possibility of intra-familial transmission in a group of Turkish children and their parents. A total of 56 children participated in the study together with their parents (20 fathers and 49 mothers). Saliva samples were collected from the individuals and cultivated on S. mutans selective TYCSB agar. The typical isolates of S. mutans were identified by using classical microbiological methods, as well as molecular typing of S. mutans clones which was performed by using AP PCR with OPA5 primer for the detection of transmission. The vertical transmission of salivary S. mutans was detected among 14 mother-father-child, 35 mother-child (one twins) and 6 father-child combinations. The homologies of strain types were recorded as 24% and 16.6% for mother-child and father-child combinations, respectively. A significant positive correlation (p<0.001) was found between the infected children and their parents with high S. mutans counts.

The correlation between serum immunoglobulin A and immunoglobulin G levels and the presence of Treponema denticola in human periapical lesions.

The aim of this study was to compare the serum immunoglobulin A (IgA) and immunoglobulin G (IgG) levels and the presence of Treponema denticola in the root canals in a group of teeth with/without periapical lesion. A total of 66 children aged 8 to 13 years old were involved in this study. Five milliliters of blood samples were taken to detect the serum IgA and IgG levels. Sixty-six endodontic samplings were also obtained to determine the presence of T. denticola by polymerase chain reaction. The presence of T. denticola between the groups with/without periapical lesion was statistically significant (p = 0.026). A significant negative correlation was found between serum IgG and IgA levels and the presence of T. denticola (p = 0.023 and 0.038, respectively). This study may support the hypothesis that the presence of T. denticola in the root canals is mainly related to the periapical lesions, and the higher levels of serum IgG and IgA levels may protect against T. denticola.

Detection of Enterococcus faecalis in Necrotic Teeth Root Canals by Culture and Polymerase Chain Reaction Methods.

OBJECTIVES: The aim of this study was to investigate the presence of Enterococcus faecalis in endodontic infections in both deciduous and permanent teeth by culture and polymerase chain reaction (PCR) methods. METHODS: A total of 145 children aged 5-13 years old were involved in this study. The presence of E. faecalis in necrotic deciduous and permanent teeth root canals was studied using culture and polymerase chain reaction methods. RESULTS: Among 145 molar teeth, 57% (n=83) presented necrotic asymptomatic pulp tissues and were included in this study. Culture and PCR methods detected the test species in 18 and 22 of 83 teeth involved, respectively. E. faecalis was cultured from 8 (18%) of 45 necrotic deciduous teeth and from 10 (26%) of 38 necrotic permanent teeth. PCR detection identified the target species in 10 (22%) and 12 (32%) of necrotic deciduous and permanent teeth respectively. Statistically significant difference in the presence of E. faecalis in deciduous and permanent teeth was found by culture and PCR methods (P=0.03 and 0.02, respectively). The difference in the presence of E. faecalis between two different methods was not statistically significant (P>.05). CONCLUSIONS: The results of the present study confirm that both culture and PCR methods are sensitive to detect E. faecalis in root canals.

Association of maternal-child characteristics as a factor in early childhood caries and salivary bacterial counts.

UNLABELLED: The aim of this study was to investigate the caries-related microorganisms in saliva and the prevalence of Early childhood caries (ECC) in 15- to 35-month-old Turkish children and their associations with the characteristics of the mothers, socioeconomic criteria, and feeding habits of the children. METHODS: Saliva samples of 101 children were studied to determine the numbers of Streptococcus mutans, lactobacilli, and Candida albicans. A questionnaire regarding the characteristics of the mothers, socioeconomic criteria, and feeding habits of the children was carried out before the dental examinations of the mother-child pairs. RESULTS: The regression analyses revealed DMFS scores of the mothers as an impact factor for the children's caries experience. The prolonged usage of feeding bottle with sweetened milk, pacifier use, and maternal sharing were strongly associated with the colonization of S. mutans, lactobacilli, and C. albicans, respectively. A significant correlation was also found between maternal education and S. mutans. CONCLUSION: Data indicated that the mother's DMFS scores, education, and feeding habits were strong risk indicators for the colonization of caries-related micro-organisms and ECC.