Alterations of the salivary and fecal microbiome in patients with primary sclerosing cholangitis.

BACKGROUND: Primary sclerosing cholangitis (PSC) is a chronic, progressive liver disease known for its frequent concurrence with inflammatory bowel disease. PSC can progress to cirrhosis, end-stage liver disease, hepatobiliary cancer, and/or colorectal cancer. The etiopathogenesis of PSC remains poorly understood, and, as such, pharmacotherapy has yet to be definitively established. Little is known about the salivary microbiome in PSC and PSC-IBD. This study aimed to evaluate the oral microbiome of patients with PSC, with association to these patient's fecal microbial composition. METHODS: Saliva, fecal samples and Food Frequency Questionnaires were collected from 35 PSC patients with or without concomitant inflammatory bowel disease and 30 age- and BMI-matched healthy volunteers. 16S rRNA gene sequencing was performed using Illumina MiSeq platform. RESULTS: The salivary microbial signature of PSC was significantly altered as compared to healthy controls, independent of concomitant IBD, and was comprised of 19 significantly altered species, of which, eight species were consistently overrepresented in both fecal and saliva of patients with PSC, including Veillonella, Scardovia and Streptococcus. CONCLUSIONS: PSC is characterized by microbial dysbiosis in the gut and the salivary microbiome, independently from IBD. The PSC dysbiotic signature includes a reduction in autochthonous bacteria and an increased relative abundance of pathogenic bacteria, including an invasion of oral bacteria to the gut. PSC is a strong modulator of the microbial profile, in the gut and the oral microbiome. These results may lead to the development of biomarkers for screening and early diagnosis or the development of personalized medicine in PSC.

Molecular epidemiology of Campylobacter jejuni infection in Israel-a nationwide study.

The incidence of Campylobacter infection in Israel, particularly among children <2 years of age, has risen over the last decade and became one of the highest among industrialized countries. This study explored the molecular epidemiology of Campylobacter jejuni in Israel over a decade (2003-2012) using multilocus sequence typing (MLST) combined with demographic metadata. Representative clinical isolates (438) from a large national repository together with selected veterinary isolates (74) were subject to MLST. The distribution of age groups, ethnicity and clinical source across various genotypes was evaluated using Poisson modelling. The 512 studied isolates were assigned 126 distinct sequence types (STs) (18.8% novel STs) grouped into 21 clonal complexes (CCs). Most human, poultry and bovine STs clustered together in the leading CCs. Three dominant STs (ST21, ST6608, ST4766) were detected only since 2006. Patients infected with the leading CCs were similarly distributed along densely populated areas. The frequency of blood isolates was higher in patients infected with CC353 (relative rate (RR)=2.0, 95% CI 1.03-3.9, adjusted p value (adj.p) 0.047) and CC42 (RR=4.4, 95% CI 1.7-11.6, adj.p 0.018) and lower with CC257 (RR=0.3, 95% CI 0.1-0.9, adj. p 0.047). The distribution of age groups and ethnicity also varied across the leading CCs. In conclusion, C. jejuni isolates in a national sample appeared highly diverse with a high proportion of new STs. Phylogenic analysis was compatible with poultry and cattle as possible food sources of clinical infection. Demographic characteristics of the infected patients coupled with strain invasiveness across different genotypes revealed a complex epidemiology of C. jejuni transmission in Israel.

Development and performance of a quantitative PCR for the enumeration of Bdellovibrionaceae.

Quantification of Bdellovibrio-and-like organisms (BALOs) by microbial culturing has a number of substantial drawbacks. Therefore a quantitative PCR (qPCR) assay was designed for the culture-independent enumeration of the Bdellovibrionaceae. After optimization, the dynamic range of the qPCR assay was assessed, the specificity was evaluated and a comparison with quantitative microbial culturing was made. To evaluate the suitability of the qPCR assay for analysing environmental samples, fresh water samples were investigated by microbial culturing and by the qPCR assay. The results revealed a substantial difference between the two techniques and indicate that most Bdellovibrionaceae cells are left undetected in environmental samples when only current microbial culturing techniques are used. The application of this new technique is therefore likely to confirm the hitherto underestimated sizes and roles of predatory bacterial populations in nature.

Microbial sensors of ultraviolet radiation based on recA'::lux fusions.

Escherichia coli strains containing plasmid-borne fusions of the recA promoter-operator region to the Vibrio fischeri lux genes were previously shown to increase their luminescence in the presence of DNA damage hazards, and thus to be useful for genotoxicant detection. The present study expands previous work by demonstrating and investigating the luminescent response of these strains to ultraviolet radiation. Several genetic variants of the basic recA'::lux design were examined, including a tolC modification of membrane efflux capacity, a chromosomal integration of the recA'::lux fusion, a different lux reporter (Photorhabdus luminescens instead of V. fischeri, allowing the assay to be run at 37 degrees C), and a different host bacterium (Salmonella typhimurium instead of E. coli). Generally, two modifications provided the fastest responses: the use of the S. typhimurium host or the P. luminescens lux reporter. Highest sensitivity, however, was demonstrated in an E. coli strain in which a single copy of the V. fischeri lux fusion was integrated into the bacterial chromosome.

Improved bacterial SOS promoter∷lux fusions for genotoxicity detection.

Escherichia coli strains containing plasmid-borne fusions of Vibrio fischeri lux to the recA promoter-operator region were previously shown to be potentially useful for detecting genotoxicants. In an attempt to improve past performance, the present study examines several modifications and variations of this design, singly or in various combinations: (1) modifying the host cell's toxicant efflux capacity via a tolC mutation; (2) incorporating the lux fusion onto the bacterial chromosome, rather then on a plasmid; (3) changing the reporter element to a different lux system (Photorhabdus luminescens), with a broader temperature range; (4) using Salmonella typhimurium instead of an E. coli host. A broad spectrum of responses to pure chemicals as well as to industrial wastewater samples was observed. Generally, fastest responses were exhibited by Sal94, a S. typhimurium strain harboring a plasmid-borne fusion of V. fischeri lux to the E. coli recA promoter. Highest sensitivity, however, was demonstrated by DPD3063, an E. coli strain in which the same fusion was integrated into the bacterial chromosome, and by DPD2797, a plasmid-bearing tolC mutant. Overall, the two latter strains appeared to perform better and seemed preferable over the others. The sensor strains retained their sensitivity following a 2-month incubation after alginate-embedding, but at the cost of a significantly delayed response.

Characterization of the rnc-97 mutation of RNAaseIII: a glycine to glutamate substitution increases the requirement for magnesium ions.

The rnc-97 mutation of the Escherichia coli double-stranded-RNA-specific ribonuclease III (RNAaseIII) was previously isolated by virtue of the lethal expression of RNAaseIII in Saccharomyces cerevisiae. Here we show that rnc-97 is a single point mutation causing the substitution of glycine 97 by glutamic acid. The mutation eliminates the lethal phenotype of RNAaseIII expression in yeast and reduces fourfold the effect of RNAaseIII expression on bacteriophage gy1 propagation in E. coli. Mutant RNAaseIII-G97E and wild-type RNAaseIII were purified according to published procedures. The apparent molecular masses of the two enzymes on SDS polyacrylamide gels are the same but they differ in pI (6.85 for RNAaseIII-G97E and 7.3 for RNAaseIII). Whereas the two enzymes (under standard assay conditions) do not show a great difference in activity towards double-stranded RNA and defined single-stranded RNAaseIII substrates, they differ dramatically (20-fold or more) under conditions of Mg2+ limitation. The hypothesis that limitation of Mg2+ ions in vivo is responsible for the phenotypes of the rnc-97 mutation in S. cerevisiae and E. coli is discussed.