Gerwin C. Raangs

Extensive Set of 16S rRNA-Based Probes for Detection of Bacteria in Human Feces

ABSTRACT For the detection of six groups of anaerobic bacteria in human feces, we designed seven new 16S rRNA-based oligonucleotide probes. This set of probes extends the current set of probes and gives more data on the composition of the human gut flora. Probes were designed for Phascolarctobacterium and relatives (Phasco741), Veillonella (Veil223), Eubacterium hallii and relatives (Ehal1469), Lachnospira and relatives (Lach571), and Eubacterium cylindroides and relatives (Ecyl387), and two probes were designed for Ruminococcus and relatives (Rbro730 and Rfla729). The hybridization conditions for the new probes were optimized for fluorescent in situ hybridization, and the probes were validated against a set of reference organisms. The probes were applied to fecal samples of 11 volunteers to enumerate their target bacterial groups. The Phasco741 and Veil223 probes both detected average numbers below 1% of the total number of bacteria as determined with the bacterial kingdom-specific Bact338 probe. The Ecyl387 probe detected about 1.4%, the Lach571 and Ehal1469 probes detected 3.8 and 3.6%, respectively, and a combination of the Rbro730 and Rfla729 probes detected 10.3%. A set of 15 probes consisting of probes previously described and those presented here were evaluated in hybridization with the fecal samples of the same volunteers. Together, the group-specific probes detected 90% of the total bacterial cells.

Mapping the Pathways to Staphylococcal Pathogenesis by Comparative Secretomics

SUMMARY The gram-positive bacterium Staphylococcus aureus is a frequent component of the human microbial flora that can turn into a dangerous pathogen. As such, this organism is capable of infecting almost every tissue and organ system in the human body. It does so by actively exporting a variety of virulence factors to the cell surface and extracellular milieu. Upon reaching their respective destinations, these virulence factors have pivotal roles in the colonization and subversion of the human host. It is therefore of major importance to obtain a clear understanding of the protein transport pathways that are active in S. aureus. The present review aims to provide a state-of-the-art roadmap of staphylococcal secretomes, which include both protein transport pathways and the extracytoplasmic proteins of these organisms. Specifically, an overview is presented of the exported virulence factors, pathways for protein transport, signals for cellular protein retention or secretion, and the exoproteomes of different S. aureus isolates. The focus is on S. aureus, but comparisons with Staphylococcus epidermidis and other gram-positive bacteria, such as Bacillus subtilis, are included where appropriate. Importantly, the results of genomic and proteomic studies on S. aureus secretomes are integrated through a comparative “secretomics” approach, resulting in the first definition of the core and variant secretomes of this bacterium. While the core secretome seems to be largely employed for general housekeeping functions which are necessary to thrive in particular niches provided by the human host, the variant secretome seems to contain the “gadgets” that S. aureus needs to conquer these well-protected niches.

Effects of yogurt and bifidobacteria supplementation on the colonic microbiota in lactose-intolerant subjects

Aims:u2002 Colonic metabolism of lactose may play a role in lactose intolerance. We investigated whether a 2‐week supplementation of Bifidobacterium longum (in capsules) and a yogurt enriched with Bifidobacterium animalis could modify the composition and metabolic activities of the colonic microbiota in 11 Chinese lactose‐intolerant subjects.

Composition of faecal microbiota of elderly people

Elderly people are susceptible to intestinal dysbiosis such as constipation. In the present study, the gut microbiota of 15 elderly people was investigated by fluorescent in situ hybridization (FISH), denaturing gradient gel electrophoresis (DGGE), cloning and sequence analysis. Eighteen 16S rRNA-based oligonucleotide probes were applied to faecal samples from 15 healthy elderly volunteers (aged > 75 years) to enumerate the predominant bacterial groups. The results show that the composition of the faecal microbiota of these elderly people is different from that of adults. The Ruminococcus group (type species R. flavefaciens) becomes the most predominant group in the elderly, contributing 9.6% of the total cells. The percentages of Enterobacteriaceae, the Eubacterium cylindroides group and the Lactobacillus/ Enterococcus group are also higher in faecal samples from the elderly than in those from a group of healthy volunteers (aged 20-55 years). In contrast to this, the contributions of the three most predominant groups in adults, Bacteroides/Prevotella, the E. rectale/C. coccoides group and Faecalibacterium, are lower in elderly people. In some samples, <35% of the total hybridizable cells were identified. DGGE, cloning and sequence analysis of one of these samples showed that among the 12 possible predominant bacterial groups in this sample, 10 of them should react with our group-specific probes. The reasons why just small proportions of total bacteria are detected in some faecal samples with our probe set need further investigation.

Microbial detection and monitoring in advanced life support systems like the International Space Station

Potentially pathogenic microbes and so-called technophiles may form a serious threat in advanced life support systems, such as the International Space Station (ISS). They not only pose a threat to the health of the crew, but also to the technical equipment and materials of the space station. The development of fast and easy to use molecular detection and quantification methods for application in manned spacecraft is therefore desirable and may also be valuable for applications on Earth. In this paper we present the preliminary results of the SAMPLE experiment in which we performed molecular microbial analysis on environmental samples of the ISS as part of an ESA-MAP project.

In vitro antimicrobial effects of two antihalitosis mouth rinses on oral pathogens and human tongue microbiota

OBJECTIVESnThe aim of the study was to compare the antimicrobial activity of a mouth rinse containing chlorhexidine and cetylpyridinium chloride (MR1) with a stannous fluoride-based mouth rinse (MR2) in vitro.nnnMATERIALS AND METHODSnSamples of the tongues from 10 subjects with and 10 subjects without halitosis were inoculated on blood agar plates. The agar was perforated, and the cylindrical holes were filled either with mouth rinse MR1 or with mouth rinse MR2. After incubation, inhibition zones of the whole tongue microbiota and Fusobacterium nucleatum were measured. In addition, MR1 and MR2 were applied in a short interval killing test (SIKT) on four oral pathogens Porphyromonas gingivalis, Prevotella intermedia, F. nucleatum and Aggregatibacter actinomycetemcomitans. Total viable cell counts were made after two minutes of incubation with increasing concentrations of MR1 and MR2.nnnRESULTSnMR1 showed a significantly higher in vitro antimicrobial activity against the whole tongue microbiota and F. nucleatum than MR2 in both groups of subjects. In the SIK test, MR1 showed a significantly greater killing capacity than MR2. The results show that a mouth rinse with low concentrations of chlorhexidine and 0.05% cetylpyridinium chloride appears to be more effective in inhibiting growth of the human tongue microbiota in vitro than a fluoride/stannous fluoride-containing mouth rinse.nnnCONCLUSIONnThis in vitro observation supports the use of chlorhexidine and cetylpyridinium chloride in the treatment of oral halitosis.

Changes in oral microflora after full-mouth tooth extraction: a prospective cohort study.

AIMnThe aim of the study was to evaluate the effect of full-mouth tooth extraction on the oral microflora, with emphasis on the presence and load of Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis.nnnMATERIAL AND METHODSnAdult patients (nxa0=xa030), with moderate to advanced periodontitis and scheduled for full-mouth tooth extraction, were consecutively selected. Prior to and 1 and 3xa0months after full-mouth tooth extraction saliva, tongue, buccal and gingival mucosa and subgingival plaque/prosthesis samples were obtained. Aerobic and anaerobic culture techniques and quantitative real-time polymerase chain reaction (qPCR) were employed for the detection of oral pathogens.nnnRESULTSnFull-mouth tooth extraction resulted in reduction below detection level of A.xa0actinomycetemcomitans and P.xa0gingivalis in 15 of 16 and 8 of 16 previously positive patients using culture techniques and qPCR, respectively. Those patients remaining qPCR positive showed a significant reduction in load of these bacteria.nnnCONCLUSIONnFull-mouth tooth extraction significantly changes the oral microflora. These changes include reduction of A.xa0actinomycetemcomitans and P.xa0gingivalis, frequently to levels below detection threshold. In some patients, A.xa0actinomycetemcomitans and P.xa0gingivalis can persist in the edentulous oral cavity up to 3xa0months after full-mouth tooth extraction.

Metagenomic Characterization of the Human Intestinal Microbiota in Fecal Samples from STEC-Infected Patients

The human intestinal microbiota is a homeostatic ecosystem with a remarkable impact on human health and the disruption of this equilibrium leads to an increased susceptibility to infection by numerous pathogens. In this study, we used shotgun metagenomic sequencing and two different bioinformatic approaches, based on mapping of the reads onto databases and on the reconstruction of putative draft genomes, to investigate possible changes in the composition of the intestinal microbiota in samples from patients with Shiga Toxin-producing E. coli (STEC) infection compared to healthy and healed controls, collected during an outbreak caused by a STEC O26:H11 infection. Both the bioinformatic procedures used, produced similar result with a good resolution of the taxonomic profiles of the specimens. The stool samples collected from the STEC infected patients showed a lower abundance of the members of Bifidobacteriales and Clostridiales orders in comparison to controls where those microorganisms predominated. These differences seemed to correlate with the STEC infection although a flexion in the relative abundance of the Bifidobacterium genus, part of the Bifidobacteriales order, was observed also in samples from Crohns disease patients, displaying a STEC-unrelated dysbiosis. The metagenomics also allowed to identify in the STEC positive samples, all the virulence traits present in the genomes of the STEC O26 that caused the outbreak as assessed through isolation of the epidemic strain and whole genome sequencing. The results shown represent a first evidence of the changes occurring in the intestinal microbiota of children in the course of STEC infection and indicate that metagenomics may be a promising tool for the culture-independent clinical diagnosis of the infection.

Design and application of group-specific oligonucleotide probes for detecting and monitoring mouse clostridia

Clostridia dominate the rodent intestinal bacterial community and play an important role in physiological functions of the host. However, their ecology and diversity are still unclear. In our previous report, we showed that phylogenetically novel groups of Clostridia inhabit the mouse intestine and contribute to the normalization of germfree mice. In this study, five new oligonucleotide probes were designed and applied to detect these clostridial groups that are essential for the normalization of germfree mice. Faecal microbiota of conventional mouse strains and specific pathogen-free mice from different breeding colonies were analysed by fluorescence in situ hybridization using these five probes. Our results showed that the composition of Clostridia differed among mouse strains and also among mouse groups of the same inbred strain from different breeding colonies. These five new probes for mouse Clostridia were able to detect the difference in clostridial diversity in each mouse group. In addition to Clostridium, we also analysed Bacteroides and Lactobacillus using previously described probes and the number or the frequency of occurrence of Bacteroides was shown to be different among mouse groups analysed. The oligonucleotide probe set including our newly developed and previously described probes used in this study can be applied to monitoring of significant groups of mouse intestinal microbiota.