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Fusions involving CRAF (RAF1) are infrequent oncogenic drivers in pediatric low-grade gliomas, rarely identified in tumors bearing features of pilocytic astrocytoma, and involving a limited number of known fusion partners. We describe recurrent TRAK1::RAF1 fusions, previously unreported in brain tumors, in three pediatric patients with low-grade glial-glioneuronal tumors. We present the associated clinical, histopathologic and molecular features. Patients were all female, aged 8 years, 15 months, and 10 months at diagnosis. All tumors were located in the cerebral hemispheres and predominantly cortical, with leptomeningeal involvement in 2/3 patients. Similar to previously described activating RAF1 fusions, the breakpoints in RAF1 all occurred 5' of the kinase domain, while the breakpoints in the 3' partner preserved the N-terminal kinesin-interacting domain and coiled-coil motifs of TRAK1. Two of the three cases demonstrated methylation profiles (v12.5) compatible with desmoplastic infantile ganglioglioma (DIG)/desmoplastic infantile astrocytoma (DIA) and have remained clinically stable and without disease progression/recurrence after resection. The remaining tumor was non-classifiable; with focal recurrence 14 months after initial resection; the patient remains symptom free and without further recurrence/progression (5 months post re-resection and 19 months from initial diagnosis). Our report expands the landscape of oncogenic RAF1 fusions in pediatric gliomas, which will help to further refine tumor classification and guide management of patients with these alterations., (© 2023 The Authors. Brain Pathology published by John Wiley & Sons Ltd on behalf of International Society of Neuropathology.)

Examination of healthy and diseased human brain is essential to translational neuroscience. Protein-protein interactions play a pivotal role in physiological and pathological processes, but their detection is difficult, especially in aged and fixed human brain tissue. We used the in-situ proximity ligation assay (PLA) to broaden the range of molecular interactions assessable in-situ in the human neuropathology. We adapted fluorescent in-situ PLA to detect ubiquitin-modified proteins in human brains with Alzheimer's disease (AD), including approaches for the management of autofluorescence and quantification using a high-content image analysis system. We confirmed that phosphorylated microtubule-associated protein tau (Serine202, Threonine205) aggregates were modified by ubiquitin and that phospho-tau-ubiquitin complexes were increased in hippocampal and frontal cortex regions in AD compared to non-AD brains. Overall, we refined PLA for use in human neuropathology, which has revealed a profound change in the distribution of ubiquitin in AD brain and its association with characteristic tau pathologies., (© 2023. The Author(s).)

Recent studies suggest that the cross-talk between the gut microbiota and human immune system during the first year of life is an important regulator of the later development of atopic diseases. We explored the changes in the gut microbiota, blood regulatory T cells, and atopic sensitization in a birth-cohort of Estonian and Finnish children followed from 3 to 36 months of age. We describe here an infant Treg phenotype characterized by high Treg frequency, the maturation of Treg population characterized by a decrease in their frequency accompanied with an increase in the highly activated Treg cells. These changes in Treg population associated first with the relative abundance of Bifidobacterium longum followed by increasing colonization with butyrate producing bacteria. High bifidobacterial abundance in the neonatal microbiota appeared to be protective, while colonization with Bacteroides and E. coli was associated with later risk of allergy. Estonian children with lower risk of IgE mediated allergic diseases than Finnish children showed an earlier maturation of the gut microbiota, detected as earlier switch to an increasing abundance of butyrate-producing bacteria, combined with an earlier maturation of Treg cell phenotype and total IgE production. The children with established allergic diseases by age 3 showed a decreased abundance of butyrate producing Faecalibacterium . These results suggest that as well as the maintenance of a bifidobacterial dominated gut microbiota is important during the first weeks of life, the overtake by butyrate producing bacteria seems to be a beneficial shift, which should not be postponed., (Copyright © 2019 Ruohtula, de Goffau, Nieminen, Honkanen, Siljander, Hämäläinen, Peet, Tillmann, Ilonen, Niemelä, Welling, Knip, Harmsen and Vaarala.)

Objective: Investigation of bacterial invasion into the intestinal wall in necrotizing enterocolitis (NEC) specimens., Study Design: We compared 43 surgical NEC specimens with 43 age-matched controls. We used fluorescent in situ hybridization (FISH), a universal bacterial probe together with species-specific probes for Clostridium spp., Enterobacteriaceae, bacteroides and enterococci/lactobacilli. We used a FISH scoring system to reveal invasion of the intestinal wall, in which 1 represented no colonies and 4 invasion of the intestinal wall., Results: We observed invasion of the intestinal wall in 22/43 of the most affected NEC tissue samples as compared with 16/43 in the least affected NEC tissue samples (P=0.03). A FISH score of 4 was reached in 7/43 control cases. Enterobacteriaceae dominated the NEC specimens. Clostridium spp. were detected occasionally in NEC samples., Conclusion: Bacterial invasion of the intestinal wall is more present in most affected NEC tissue samples compared with least affected NEC tissue samples or controls. Enterobacteriaceae are prevalent in advanced NEC.

Emerging evidence suggests that the gut microbiota has a critical role in both the maintenance of human health and the pathogenesis of many diseases. Modifying the colonic microbiota using functional foods has attracted significant research effort and product development. The pioneering concept of prebiotics, as introduced by Gibson and Roberfroid in the 1990s, emphasized the importance of diet in the modulation of the gut microbiota and its relationships to human health. Increasing knowledge of the intestinal microbiota now suggests a more comprehensive definition. This paper briefly reviews the basics of the prebiotic concept with a discussion of recent attempts to refine the concept to open the door for novel prebiotic food ingredients, such as polyphenols, minerals and vitamins.

Background: Anastomotic leakage (AL) after colorectal surgery is a severe complication, resulting in morbidity, reinterventions, prolonged hospital stay and, in some cases, death. Some technical and patient-related aetiological factors of AL are well established. In many cases, however, none of these factors seem to explain the occurrence of AL. Recent studies suggest that the intestinal microbiome plays a role in wound healing, diabetes and Crohn's disease. The aim of this study was to compare the intestinal microbiota of patients who developed AL with matched patients with healed colorectal anastomoses., Methods: We investigated the microbiome in the doughnuts collected from 16 patients participating in the C-seal trial. We selected eight patients who developed AL requiring reintervention and eight matched controls without AL. We analysed the bacterial 16S rDNA of both groups with MiSeq sequencing., Results: The abundance of Lachnospiraceae is statistically higher (P = 0.001) in patient group who did develop AL, while microbial diversity levels were higher in the group who did not develop AL (P = 0.037). Body mass index (BMI) was also positively associated with the abundance of the Lachnospiraceae family (P = 0.022)., Conclusion: A correlation between the bacterial family Lachnospiraceae, low microbial diversity and anastomotic leakage, possibly in association with the BMI, was found. The relative abundance of the Lachnospiraceae family is possibly explained by the higher abundance of mucin-degrading Ruminococci within that family in AL cases (P = 0.011) as is similarly the case in IBD.

Background and Aims: Proton pump inhibitors (PPIs) are among the top 10 most widely used drugs in the world. PPI use has been associated with an increased risk of enteric infections, most notably Clostridium difficile. The gut microbiome plays an important role in enteric infections, by resisting or promoting colonisation by pathogens. In this study, we investigated the influence of PPI use on the gut microbiome., Methods: The gut microbiome composition of 1815 individuals, spanning three cohorts, was assessed by tag sequencing of the 16S rRNA gene. The difference in microbiota composition in PPI users versus non-users was analysed separately in each cohort, followed by a meta-analysis., Results: 211 of the participants were using PPIs at the moment of stool sampling. PPI use is associated with a significant decrease in Shannon's diversity and with changes in 20% of the bacterial taxa (false discovery rate <0.05). Multiple oral bacteria were over-represented in the faecal microbiome of PPI-users, including the genus Rothia (p=9.8×10(-38)). In PPI users we observed a significant increase in bacteria: genera Enterococcus, Streptococcus, Staphylococcus and the potentially pathogenic species Escherichia coli., Conclusions: The differences between PPI users and non-users observed in this study are consistently associated with changes towards a less healthy gut microbiome. These differences are in line with known changes that predispose to C. difficile infections and can potentially explain the increased risk of enteric infections in PPI users. On a population level, the effects of PPI are more prominent than the effects of antibiotics or other commonly used drugs., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/)

Background: The most frequent cause of voice prosthesis failure is microbial biofilm formation on the silicone valve, leading to destruction of the material and transprosthetic leakage. The Provox ActiValve valve is made of fluoroplastic, which should be insusceptible to destruction. The purpose of this study was to determine if fluoroplastic is insusceptible to destruction by Candida species., Methods: Thirty-three dysfunctional Provox ActiValves (collected 2011-2013). Biofilm analysis was performed with Illumina paired-end sequencing (IPES), assessment of biofilm-material interaction with fluorescence in situ hybridization (FISH), and confocal laser scanning microscopy (CLSM)., Results: IPES (n = 10) showed that Candida albicans and Candida tropicalis are dominant populations on fluoroplastic and silicone. Microbial diversity is significantly lower on fluoroplastic. Lactobacillus gasseri is the prevalent bacterial strain on most voice prostheses. FISH and CLSM (n = 23): in none of the cases was ingrowth of Candida species present in the fluoroplastic., Conclusion: Fluoroplastic material of Provox ActiValve seems insusceptible to destruction by Candida species, which could help improve durability of voice prostheses. © 2015 Wiley Periodicals, Inc. Head Neck 38: E432-E440, 2016., (© 2015 Wiley Periodicals, Inc.)

Faecalibacterium prausnitzii strain A2-165 was previously reported to have anti-inflammatory properties and prevent colitis in a TNBS model. We compared the immunomodulatory properties of strain A2-165 to four different F. prausnitzii isolates and eight abundant intestinal commensals using human dendritic cells (DCs) and mouse BMDCs in vitro. Principal component analysis revealed that the cytokine response to F. prausnitzii A2-165 is distinct from the other strains in eliciting high amounts of IL-10 secretion. The mouse DNBS model of relapsing IBD was used to compare the protective effects of F. prausnitzii A2-165 and Clostridium hathewayi, a low secretor of IL-10, on the Th1-driven inflammatory response to DNBS; attenuation of disease parameters was only observed with F. prausnitzii. In an in vivo mouse model of nasal tolerance to ovalbumin, F. prausnitzii A2-165 enhanced ovalbumin-specific T cell proliferation and reduced the proportion of IFN-γ(+) T cells in CLNs. Similarly, in vitro F. prausnitzii A2-165 stimulated BMDCs increased ovalbumin-specific T cell proliferation and reduced the number of IFN-γ(+) T cells. These mechanisms may contribute to the anti-inflammatory effects of F. prausnitzii in colitis and support the notion that this abundant bacterium might contribute to immune homeostasis in the intestine via its anti-inflammatory properties.

The microbiota in our gut performs many different essential functions that help us to stay healthy. These functions include vitamin production, regulation of lipid metabolism and short chain fatty acid production as fuel for epithelial cells and regulation of gene expression. There is a very numerous and diverse microbial community present in the gut, especially in the colon, with reported numbers of species that vary between 400 and 1500, for some those we even do not yet have culture representatives.A healthy gut microbiota is important for maintaining a healthy host. An aberrant microbiota can cause diseases of different nature and at different ages ranging from allergies at early age to IBD in young adults. This shows that our gut microbiota needs to be treated well to stay healthy. In this chapter we describe what we consider a healthy microbiota and discuss what the role of the microbiota is in various diseases. Research into these described dysbiosis conditions could lead to new strategies for treatment and/or management of our microbiota to improve health.

Most gut bacteria are obligate anaerobes and are important for human health. However, little mechanistic insight is available on the health benefits of specific anaerobic gut bacteria. A main obstacle in generating such knowledge is the lack of simple and robust coculturing methods for anaerobic bacteria and oxygen-requiring human cells. Here, we describe the development of a coculture system for intestinal Caco-2 cells and an anaerobic symbiont, Faecalibacterium prausnitzii, making use of 50 mL culture tubes. F. prausnitzii was grown in 40 mL YCFAG-agar with glass-adhered Caco-2 cells placed on top in 10 mL DMEM medium. Grown for 18-36 h in a humidified incubator at 37 °C and 5% CO2, coverslip-attached Caco-2 cells promoted growth and metabolism of F. prausnitzii, while F. prausnitzii suppressed inflammation and oxidative stress in Caco-2 cells. F. prausnitzii did not compromise Caco-2 cell viability. Exogenously added porcine mucin also promoted growth of F. prausnitzii, suggesting that it may be part of the mechanism of Caco-2-stimulated growth of F. prausnitzii. This 'Human oxygen-Bacteria anaerobic' (HoxBan) coculturing system uniquely establishes host-microbe mutualism of a beneficial anaerobic gut microbe in vitro and principally allows the analysis of host-microbe interactions of pure and mixed cultures of bacteria and human cells.

Objective: Crohn's disease (CD) is caused by a complex interplay among genetic, microbial and environmental factors. ATG16L1 is an important genetic factor involved in innate immunity, including autophagy and phagocytosis of microbial components from the gut. We investigated the effect of inflammation on the composition of microbiota in the ileal mucosa of CD patients in relation to the ATG16L1 risk status., Design: Biopsies (n=35) were obtained from inflamed and non-inflamed regions of the terminal ileum of 11 CD patients homozygous for the ATG16L1 risk allele (ATG16L1-T300A) and 9 CD patients homozygous for the ATG16L1 protective allele (ATG16L1-T300). Biopsy DNA was extracted and the bacterial composition analysed by pyrosequencing. Intracellular survival rates of adherent-invasive Escherichia coli (AIEC) were analysed by determining colony forming units after exposure to monocytes isolated from healthy volunteers homozygous for the ATG16L1 risk or protective allele., Results: Inflamed ileal tissue from patients homozygous for the ATG16L1 risk allele contained increased numbers of Fusobacteriaceae, whereas inflamed ileal tissue of patients homozygous for the ATG16L1 protective allele showed decreased numbers of Bacteroidaceae and Enterobacteriaceae and increased Lachnospiraceae. The ATG16L1 allele did not affect the bacterial composition in the non-inflamed ileal tissue. Monocytes homozygous for the ATG16L1 risk allele showed impaired killing of AIEC under inflammatory conditions compared with those homozygous for the ATG16L1 protective allele., Conclusions: CD patients homozygous for the ATG16L1-T300A risk allele show impaired clearance of pathosymbionts in ileal inflammation indicating that ATG16L1 is essential for effective elimination of pathosymbionts upon inflammation., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.)

Purpose: Earlier, we showed in acute myeloid leukemia (AML) patients that the microbiota changes dramatically during anticancer treatment, coinciding with gastrointestinal mucositis: The commensal anaerobic populations reduce in favor of potential pathogens. Therefore, interventions targeting the microbiota during mucositis might be interesting but can better be tested in animals than in vulnerable mucositis patients. Here, we aimed to study the potential microbial changes during methotrexate (MTX)-induced gastrointestinal mucositis in a well-established rat model and to study whether this model can be used for future microbial intervention studies., Methods: After injection with MTX or saline (day 0), rats were sacrificed between days 2 and 11. Plasma citrulline level, jejunal histology, and the number and diversity of intestinal bacteria in feces (using fluorescence in situ hybridization (FISH)) were determined., Results: Mucositis was most severe on day 4 when food intake, plasma citrulline, and villus length were the lowest, compared with controls (P < 0.0125). At the same time, MTX-treated rats showed an overall decrease (705-fold) in most bacteria (using a universal probe), compared with controls (P < 0.125). Reduced bacterial presence was related with the presence of diarrhea and a reduced villus length (rho = 0.38, P < 0.05). At day 4, there was an absolute and relative decrease of anaerobes (13-fold and -58 %, respectively) and streptococci (296-fold and -1 %, respectively) but a relative increase of Bacteroides (+49 %), compared with controls (P < 0.125)., Conclusions: In the mucositis rat model, we found substantial decreases in the number and diversity of microbiota, resembling earlier findings in humans. The model therefore seems well suited to study the effects of different microbial interventions on mucositis, prior to performing human studies.

A decrease in the abundance and biodiversity of intestinal bacteria within the Firmicutes phylum has been associated with inflammatory bowel disease (IBD). In particular, the anti-inflammatory bacterium Faecalibacterium prausnitzii, member of the Firmicutes phylum and one of the most abundant species in healthy human colon, is underrepresented in the microbiota of IBD patients. The aim of this study was to investigate the immunomodulatory properties of F. prausnitzii strain A2-165, the biofilm forming strain HTF-F and the extracellular polymeric matrix (EPM) isolated from strain HTF-F. For this purpose, the immunomodulatory properties of the F. prausnitzii strains and the EPM were studied in vitro using human monocyte-derived dendritic cells. Then, the capacity of the F. prausnitzii strains and the EPM of HTF-F to suppress inflammation was assessed in vivo in the mouse dextran sodium sulphate (DSS) colitis model. The F. prausnitzii strains and the EPM had anti-inflammatory effects on the clinical parameters measured in the DSS model but with different efficacy. The immunomodulatory effects of the EPM were mediated through the TLR2-dependent modulation of IL-12 and IL-10 cytokine production in antigen presenting cells, suggesting that it contributes to the anti-inflammatory potency of F. prausnitzii HTF-F. The results show that F. prausnitzii HTF-F and its EPM may have a therapeutic use in IBD.

Background: A polymerase chain reaction (PCR)-based method has been used to identify oral anaerobic pathogens in biofilms on voice prostheses. The purpose of the present study was to determine the location of those pathogens inside the biofilms., Methods: Biofilms of 15 voice prostheses were sampled and used to identify the oral pathogens. Fluorescence in situ hybridization was applied on smears made on glass slides and on sections of intact biofilms visualized by confocal laser scanning microscopy (CLSM)., Results: Fusobacterium nucleatum (F. nucleatum) was the most frequently detected pathogen and the only tested species detected in microcolonies. The other microbes (Parvimonas micra [P. micra], Porphyromonas gingivalis [P. gingivalis], Tannerella forsythia [T. forsythia], and Treponema denticola [T. denticola]) were not detected or only detected as single cells. CLSM analysis showed that F. nucleatum resided on the biofilm surface., Conclusion: Although detectable, oral anaerobic pathogens seem to be no more than passers-by that adhere without further observed proliferation and apparently play no striking role in biofilm formation on voice prostheses., (© 2014 Wiley Periodicals, Inc.)

The human gut microbiota plays a central role in human well-being and disease. In this study, we present an integrated, iterative approach of computational modeling, in vitro experiments, metabolomics, and genomic analysis to accelerate the identification of metabolic capabilities for poorly characterized (anaerobic) microorganisms. We demonstrate this approach for the beneficial human gut microbe Faecalibacterium prausnitzii strain A2-165. We generated an automated draft reconstruction, which we curated against the limited biochemical data. This reconstruction modeling was used to develop in silico and in vitro a chemically defined medium (CDM), which was validated experimentally. Subsequent metabolomic analysis of the spent medium for growth on CDM was performed. We refined our metabolic reconstruction according to in vitro observed metabolite consumption and secretion and propose improvements to the current genome annotation of F. prausnitzii A2-165. We then used the reconstruction to systematically characterize its metabolic properties. Novel carbon source utilization capabilities and inabilities were predicted based on metabolic modeling and validated experimentally. This study resulted in a functional metabolic map of F. prausnitzii, which is available for further applications. The presented workflow can be readily extended to other poorly characterized and uncharacterized organisms to yield novel biochemical insights about the target organism., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Aims/hypothesis: Recent studies indicate that an aberrant gut microbiota is associated with the development of type 1 diabetes, yet little is known about the microbiota in children who have diabetes at an early age. To this end, the microbiota of children aged 1-5 years with new-onset type 1 diabetes was compared with the microbiota of age-matched healthy controls., Methods: A deep global analysis of the gut microbiota composition was established by phylogenetic microarray analysis using a Human Intestinal Tract Chip (HITChip)., Results: Principal component analyses highlighted the importance of age when comparing age-matched pairs. In pairs younger than 2.9 years, the combined abundance of the class Bacilli (notably streptococci) and the phylum Bacteroidetes was higher in diabetic children, whereas the combined abundance of members of Clostridium clusters IV and XIVa was higher in the healthy controls. Controls older than 2.9 years were characterised by a higher fraction of butyrate-producing species within Clostridium clusters IV and XIVa than was seen in the corresponding diabetic children or in children from the younger age groups, while the diabetic children older than 2.9 years could be differentiated by having an increased microbial diversity., Conclusions/interpretation: The results from both age groups suggest that non-diabetic children have a more balanced microbiota in which butyrate-producing species appear to hold a pivotal position.

For some microbial species, such as Bacillus anthracis, the etiologic agent of the disease anthrax, correct detection and identification by molecular methods can be problematic. The detection of virulent B. anthracis is challenging due to multiple virulence markers that need to be present in order for B. anthracis to be virulent and its close relationship to Bacillus cereus and other members of the B. cereus group. This is especially the case in environments where build-up of Bacillus spores can occur and several representatives of the B. cereus group may be present, which increases the chance for false-positives. In this study we show the presence of B. anthracis-like bacteria and other members of the B. cereus group in a microbial community within the human environment of the International Space Station and their preliminary identification by using conventional culturing as well as molecular techniques including 16S rDNA sequencing, PCR and real-time PCR. Our study shows that when monitoring the microbial hygiene in a given human environment, health risk assessment is troublesome in the case of virulent B. anthracis, especially if this should be done with rapid, easy to apply and on-site molecular methods.

Background: Recent scientific developments have shed more light on the importance of the host-microbe interaction, particularly in the gut. However, the mechanistic study of the host-microbe interplay is complicated by the intrinsic limitations in reaching the different areas of the gastrointestinal tract (GIT) in vivo. In this paper, we present the technical validation of a new device--the Host-Microbiota Interaction (HMI) module--and the evidence that it can be used in combination with a gut dynamic simulator to evaluate the effect of a specific treatment at the level of the luminal microbial community and of the host surface colonization and signaling., Results: The HMI module recreates conditions that are physiologically relevant for the GIT: i) a mucosal area to which bacteria can adhere under relevant shear stress (3 dynes cm(-2)); ii) the bilateral transport of low molecular weight metabolites (4 to 150 kDa) with permeation coefficients ranging from 2.4 × 10(-6) to 7.1 × 10(-9) cm sec(-1); and iii) microaerophilic conditions at the bottom of the growing biofilm (PmO2 = 2.5 × 10(-4) cm sec(-1)). In a long-term study, the host's cells in the HMI module were still viable after a 48-hour exposure to a complex microbial community. The dominant mucus-associated microbiota differed from the luminal one and its composition was influenced by the treatment with a dried product derived from yeast fermentation. The latter--with known anti-inflammatory properties--induced a decrease of pro-inflammatory IL-8 production between 24 and 48 h., Conclusions: The study of the in vivo functionality of adhering bacterial communities in the human GIT and of the localized effect on the host is frequently hindered by the complexity of reaching particular areas of the GIT. The HMI module offers the possibility of co-culturing a gut representative microbial community with enterocyte-like cells up to 48 h and may therefore contribute to the mechanistic understanding of host-microbiome interactions.

The beneficial human gut microbe Faecalibacterium prausnitzii is a 'probiotic of the future' since it produces high amounts of butyrate and anti-inflammatory compounds. However, this bacterium is highly oxygen-senstive, making it notoriously difficult to cultivate and preserve. This has so far precluded its clinical application in the treatment of patients with inflammatory bowel diseases. The present studies were therefore aimed at developing a strategy to keep F. prausnitzii alive at ambient air. Our previous research showed that F. prausnitzii can survive in moderately oxygenized environments like the gut mucosa by transfer of electrons to oxygen. For this purpose, the bacterium exploits extracellular antioxidants, such as riboflavin and cysteine, that are abundantly present in the gut. We therefore tested to what extent these antioxidants can sustain the viability of F. prausnitzii at ambient air. The present results show that cysteine can facilitate the survival of F. prausnitzii upon exposure to air, and that this effect is significantly enhanced the by addition of riboflavin and the cryoprotectant inulin. The highly oxygen-sensitive gut bacterium F. prausnitzii can be kept alive at ambient air for 24 h when formulated with the antioxidants cysteine and riboflavin plus the cryoprotectant inulin. Improved formulations were obtained by addition of the bulking agents corn starch and wheat bran. Our present findings pave the way towards the biomedical exploitation of F. prausnitzii in redox-based therapeutics for treatment of dysbiosis-related inflammatory disorders of the human gut.

Objective: Pregnant individuals of many species, including humans, are more sensitive to various bacteria or their products as compared with non-pregnant individuals. Pregnant individuals also respond differently to different bacteria or their products. Therefore, in the present study, we evaluated whether the increased sensitivity of pregnant women to bacterial products and their heterogeneous response to different bacteria was associated with differences in whole blood cytokine production upon stimulation with bacteria or their products., Methods: Blood samples were taken from healthy pregnant and age-matched non-pregnant women and ex vivo stimulated with bacteria or LPS from Porphyromonas Gingivalis (Pg) or E-coli for 24 hrs. TNFα, IL-1ß, IL-6, IL-12 and IL-10 were measured using a multiplex Luminex system., Results: We observed a generally lower cytokine production after stimulation with Pg bacteria or it's LPS as compared with E-coli bacteria. However, there was also an effect of pregnancy upon cytokine production: in pregnant women the production of IL-6 upon Pg stimulation was decreased as compared with non-pregnant women. After stimulation with E-coli, the production of IL-12 and TNFα was decreased in pregnant women as compared with non-pregnant women., Conclusion: Our results showed that cytokine production upon bacterial stimulation of whole blood differed between pregnant and non-pregnant women, showing that the increased sensitivity of pregnant women may be due to differences in cytokine production. Moreover, pregnancy also affected whole blood cytokine production upon Pg or E-coli stimulation differently. Thus, the different responses of pregnant women to different bacteria or their products may result from variations in cytokine production.

The opportunistic pathogen Staphylococcus aureus is known to interfere with wound healing and represents a significant risk factor for wound infections and invasive disease. It is generally assumed that one individual is predominantly colonized by one S. aureus type. Nevertheless, patients with the genetic blistering disease epidermolysis bullosa (EB) often carry multiple S. aureus types. We therefore investigated whether different S. aureus types are present in individual wounds of EB patients and, if so, how they are spatially distributed. The staphylococcal topography in chronic wounds was mapped by replica-plating of used bandages and subsequent typing of S. aureus isolates. Individual chronic wounds of five patients contained up to six different S. aureus types. Unexpectedly, distinct S. aureus types formed micro-colonies that were located in close proximity and sometimes even overlapped. While some adjacent S. aureus isolates were closely related, others belonged to distinct molecular complexes. We conclude that the general assumption that one individual is predominantly colonized by one type of S. aureus does not apply to chronic wounds of EB patients. We consider this observation important, not only for EB patients, but also for other patients with chronic wounds in view of the potential risk for severe staphylococcal infections.

The role of the intestinal microbiota as a regulator of autoimmune diabetes in animal models is well-established, but data on human type 1 diabetes are tentative and based on studies including only a few study subjects. To exclude secondary effects of diabetes and HLA risk genotype on gut microbiota, we compared the intestinal microbiota composition in children with at least two diabetes-associated autoantibodies (n = 18) with autoantibody-negative children matched for age, sex, early feeding history, and HLA risk genotype using pyrosequencing. Principal component analysis indicated that a low abundance of lactate-producing and butyrate-producing species was associated with β-cell autoimmunity. In addition, a dearth of the two most dominant Bifidobacterium species, Bifidobacterium adolescentis and Bifidobacterium pseudocatenulatum, and an increased abundance of the Bacteroides genus were observed in the children with β-cell autoimmunity. We did not find increased fecal calprotectin or IgA as marker of inflammation in children with β-cell autoimmunity. Functional studies related to the observed alterations in the gut microbiome are warranted because the low abundance of bifidobacteria and butyrate-producing species could adversely affect the intestinal epithelial barrier function and inflammation, whereas the apparent importance of the Bacteroides genus in development of type 1 diabetes is insufficiently understood.

Invasive and biomaterial-associated infections in humans are often difficult to diagnose and treat. Here, guided by recent advances in clinically relevant optical imaging technologies, we explore the use of fluorescently labelled vancomycin (vanco-800CW) to specifically target and detect infections caused by Gram-positive bacteria. The application potential of vanco-800CW for real-time in vivo imaging of bacterial infections is assessed in a mouse myositis model and a human post-mortem implant model. We show that vanco-800CW can specifically detect Gram-positive bacterial infections in our mouse myositis model, discriminate bacterial infections from sterile inflammation in vivo and detect biomaterial-associated infections in the lower leg of a human cadaver. We conclude that vanco-800CW has a high potential for enhanced non-invasive diagnosis of infections with Gram-positive bacteria and is a promising candidate for early-phase clinical trials.

Faecalibacterium prausnitzii is one of the most abundant commensal microbes in the human gut. It is an important supplier of butyrate to the colonic epithelium, and low numbers of faecalibacteria have been associated with severe inflammatory bowel disease. Previous studies revealed that F. prausnitzii shuttles electrons extracellularly to oxygen in systems containing flavins and thiols. Since this electron shuttling to oxygen strongly stimulates growth, the present studies were aimed at elucidating the role of riboflavin as an extracellular electronophore of F. prausnitzii. We show that F. prausnitzii can use riboflavin as a mediator for extracellular electron transfer (EET) to the anode of microbial fuel cell systems. However, this bacterium relies on exogenous riboflavin, since it does not secrete this compound as shown by the analysis of a spent growth medium using cyclic voltammetry (CV). Importantly, CV showed that riboflavin can undergo fully reversible redox cycling under physiologically relevant conditions. Lastly, riboflavin is shown to mediate the electrochemical oxidation of the main bacterial reducing equivalent NADH. Based on our present observations, we hypothesize that riboflavin is of major importance as a redox mediator for bacterial EET and growth in the human gut.

Objective: Although the exact reason is not known, encapsulated gram-negative Porphyromonas gingivalis strains are more virulent than non-encapsulated strains. Since difference in virulence properties may be due to difference in cytokine production following recognition of the bacteria or their products by the host inflammatory cells, we compared cytokine production following stimulation with bacteria or lipopolysaccharides (LPS) of a non-encapsulated and an encapsulated P. gingivalis strain (K(-) and K1)., Design: Tumour necrosis factor-alpha (TNF-α) production following stimulation of the cell-line Mono Mac 6 with bacteria or LPS of both P. gingivalis strains was determined using flow cytometry. Furthermore, we investigated the effects of the two P. gingivalis strains or their LPS on TNF-α and Interleukin (IL-1β, IL-6, IL-12 and IL-10) production in whole blood using Luminex. In both experiments, Escherichia coli bacteria and LPS were used as a reference., Results: Both P. gingivalis strains induced lower cytokine production than E. coli with the exception of IL-6. P. gingivalis K1 bacteria elicited a higher overall cytokine production than P. gingivalis K(-). In contrast, P. gingivalis K1 LPS stimulation induced a lower cytokine production than P. gingivalis K(-) LPS., Conclusions: Our findings suggest that the encapsulated P. gingivalis K1 bacteria induce higher cytokine production than the non-encapsulated P. gingivalis K(-). This was not due to its LPS. The stronger induction of cytokines may contribute to the higher virulence of P. gingivalis K1., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Faecalibacterium prausnitzii is one of the most abundant bacteria in the human gut ecosystem and it is an important supplier of butyrate to the colonic epithelium. Low numbers of faecalibacteria have been associated with inflammatory bowel disease. Despite being extremely oxygen sensitive, F. prausnitzii is found adherent to the gut mucosa where oxygen diffuses from epithelial cells. This paradox is now explained on the basis of gas tube experiments, flavin-dependent reduction of 5,5'-dithiobis-2-nitrobenzoate and microbial fuel cell experiments. The results show that F. prausnitzii employs an extracellular electron shuttle of flavins and thiols to transfer electrons to oxygen. Both compounds are present in the healthy human gut. Our observations may have important implications for the treatment of patients with Crohn's disease, for example, with flavin- or antioxidant rich diets, and they provide a novel key insight in host-microbe interactions at the gut barrier.

Background: Biofilms on medical devices are a frequent reason for failure of the device. Voice prostheses in laryngectomized patients deteriorate within 3 to 4 months due to adhering biofilms, impeding proper functioning. Recently, we showed that these biofilms are dominated by Candida and lactobacilli. However, the early report of this finding lacked an in depth analysis of the species diversity and community structure., Methods: Denaturing gradient gel electrophoresis (DGGE) and sequence analysis of amplified rRNA genes was used to identify microorganisms. Fluorescence in situ hybridization (FISH) was used to analyze biofilm architecture., Results: Candida are the predominant fungi in the biofilms, while lactobacilli are the predominant bacteria in all investigated biofilms, followed by streptococci. FISH shows that lactobacilli and streptococci seem to have an important interaction with fungi., Conclusion: The results give a better understanding of biofilm formation and can be used in further development to prevent biofilm formation on voice prostheses., (Copyright © 2011 Wiley Periodicals, Inc.)

Enterococcus faecium belonging to the polyclonal subcluster CC17, with a typical ampicillin-resistant E. faecium (AREfm) phenotype, have become prevalent among nosocomial infections around the world. High-density intestinal AREfm colonization could be one of the factors contributing to the successful spread of these pathogens. We aimed to quantify the enterococcal intestinal colonization densities in stool samples from AREfm-colonized and non-colonized patients using fluorescent in situ hybridization (FISH). Stool samples were collected from AREfm-colonized (n = 8) and non-colonized (n = 8) patients. The relative number of Enterococcus faecalis and E. faecium was determined by FISH using specific 16S rRNA probes, while the total amount of bacterial cells was counted by staining the sample with 4',6-diamidino-2-phenylindole (DAPI). The median bacterial cell numbers in fecal samples, counted by DAPI staining, were 7.7 × 10(9) and 4.8 × 10(9) cells/g for AREfm-colonized and non-colonized patients, respectively (p = 0.34). The E. faecium densities in AREfm-colonized patients, accounting for 0.5-7% of all fecal bacterial cells, exceeded E. faecalis levels by over ten-fold. E. faecium was not detected in non-colonized patients. This study demonstrated high E. faecium cell densities in stool samples from patients colonized with AREfm. Increased cell densities may contribute to host-to-host transmission and environmental contamination, facilitating the spread of AREfm in the hospital setting.

In Crohn's disease (CD), chronic gut inflammation leads to loss of mucosal barrier integrity. Subsequent leakage of IgG to the gut could produce an increase of IgG coating of intestinal bacteria. We investigated if there is more IgG coating in patients than in volunteers and whether this is dependent on the host IgG response or on the gut bacteria. Fecal and serum samples were obtained from 23 CD patients and 11 healthy volunteers. Both the in vivo IgG-coated fecal bacteria and in vitro IgG coating after serum addition were measured by flow cytometry and related to disease activity. The bacterial composition in feces was determined using fluorescence in situ hybridization. The IgG-binding capacities of Escherichia coli strains isolated from feces of patients and volunteers were assessed. The results showed that the in vivo IgG-coated fraction of fecal bacteria of patients was slightly larger than that of volunteers but significantly larger after incubation with either autologous or heterologous serum. This was dependent on the bacteria and independent of disease activity or the serum used. The presence of more Enterobacteriaceae and fewer faecalibacteria in patient feces was confirmed. E. coli isolates from patients bound more IgG than isolates from volunteers (P < 0.05) after the addition of autologous serum. Together, these results indicate that CD patients have more IgG-binding gut bacteria than healthy volunteers. We showed that the level of IgG coating depends on the bacteria and not on the serum used. Furthermore, CD patients have a strong specific immune response to their own E. coli bacteria.

Faecalibacterium prausnitzii is one of the most abundant commensal bacteria in the healthy human large intestine, but information on genetic diversity and substrate utilization is limited. Here, we examine the phylogeny, phenotypic characteristics, and influence of gut environmental factors on growth of F. prausnitzii strains isolated from healthy subjects. Phylogenetic analysis based on the 16S rRNA sequences indicated that the cultured strains were representative of F. prausnitzii sequences detected by direct analysis of fecal DNA and separated the available isolates into two phylogroups. Most F. prausnitzii strains tested grew well under anaerobic conditions on apple pectin. Furthermore, F. prausnitzii strains competed successfully in coculture with two other abundant pectin-utilizing species, Bacteroides thetaiotaomicron and Eubacterium eligens, with apple pectin as substrate, suggesting that this species makes a contribution to pectin fermentation in the colon. Many F. prausnitzii isolates were able to utilize uronic acids for growth, an ability previously thought to be confined to Bacteroides spp. among human colonic anaerobes. Most strains grew on N-acetylglucosamine, demonstrating an ability to utilize host-derived substrates. All strains tested were bile sensitive, showing at least 80% growth inhibition in the presence of 0.5 μg/ml bile salts, while inhibition at mildly acidic pH was strain dependent. These attributes help to explain the abundance of F. prausnitzii in the colonic community but also suggest factors in the gut environment that may limit its distribution.

The shift in microbial diversity from young to mature plaque, related to caries activity on sound and restored surfaces, was studied using denaturing gradient gel electrophoresis. During a 20-week in situ study on caries progression 8 subjects wearing restored and unrestored dentin and enamel sections, biofilm was sampled after 1 and 20 weeks (young or mature plaque). A higher microbial diversity (mature plaque) was seen in caries-active compared to caries-free subjects. Rothia dentocariosa and Scardovia inopinata were absent from all caries-free sites, but appeared in 50% of the caries-active sites., (Copyright © 2012 S. Karger AG, Basel.)

Thermal stability is essential for the survival and well-being of preterm neonates. This is achieved in neonatal incubators by raising the ambient temperature and humidity to sufficiently high levels. However, potentially pathogenic microorganisms also can thrive in such warm and humid environments. We therefore investigated whether the level of microbial contamination (i.e., the bacterial load) inside neonatal incubators can be predicted on the basis of their average temperature and relative humidity settings, paying special attention to local temperature differences. Swab samples were taken from the warmest and coldest spots found within Caleo incubators, and these were plated to determine the number of microbial CFU per location. In incubators with high average temperature (≥ 34°C) and relative humidity (≥ 60%) values, the level of microbial contamination was significantly higher at cold spots than at hot spots. This relates to the fact that the local equilibrium relative humidity at cold spots is sufficiently high to sustain microbial growth. The abundance of staphylococci, which are the main causative agents of late-onset sepsis in preterm neonates, was found to be elevated significantly in cold areas. These findings can be used to improve basic incubator hygiene.

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.

The development of fast and easy on-site molecular detection and quantification methods for hazardous microbes on solid surfaces is desirable for several applications where specialised laboratory facilities are absent. The quantification of bacterial contamination necessitates the assessment of the efficiency of the used methodology as a whole, including the preceding steps of sampling and sample processing. We used quantitative real-time polymerase chain reaction (qrtPCR) for Escherichia coli and Staphylococcus aureus to measure the recovery of DNA from defined numbers of bacterial cells that were subjected to three different DNA extraction methods: the QIAamp DNA Mini Kit, Reischl et al.'s method and FTA Elute. FTA Elute significantly showed the highest median DNA extraction efficiency of 76.9% for E. coli and 108.9% for S. aureus. The Reischl et al. method and QIAamp DNA Mini Kit inhibited the E. coli qrtPCR assay with a 10-fold decrease of detectable DNA. None of the methods inhibited the S. aureus qrtPCR assay. The FTA Elute applicability was demonstrated with swab samples taken from the International Space Station (ISS) interior. Overall, the FTA Elute method was found to be the most suitable to selected criteria in terms of rapidity, easiness of use, DNA extraction efficiency, toxicity, and transport and storage conditions.

The availability of water, which can be expressed in terms of water activity (a(w)), is one of the most important determinants for microbial homeostasis and growth on surface to air interfaces. Here we show, using an environmental control chamber containing a precisely controlled temperature/a(w) gradient in combination with a mathematical approach, that the environmental a(w) growth limit of a microorganism can be lower than its intracellular a(w) limit. This internal limit represents the point at which microbial cells cannot lower their internal a(w) any further in response to low external a(w) values without interfering with essential intracellular processes. To grow at external a(w) values below their internal limit, microbes need to generate more water metabolically than they lose to their environment. This internal a(w) limit can be calculated by measuring the a(w) growth limit of an organism at different water vapour diffusivities using barometric pressure as a variable. Fascinating morphological changes, such as rope-like superstructures formed by B. subtilis, are furthermore observed in response to low external a(w) values in particular around the calculated intracellular a(w) limit. The intracellular a(w) limit of an organism is a decisive parameter for water limitation-induced adaptations in cellular hydrophilicity and morphogenesis., (© 2011 Society for Applied Microbiology and Blackwell Publishing Ltd.)

The intestinal microbiota are a complex ecosystem influencing the immunoregulation of the human host, providing protection from colonising pathogens and producing SCFA as the main energy source of colonocytes. Our objective was to investigate the effect of dietary fibre exclusion and supplementation on the intestinal microbiota and SCFA concentrations. Faecal samples were obtained from healthy volunteers before and after two 14 d periods of consuming formulated diets devoid or supplemented with fibre (14 g/l). The faecal microbiota were analysed using fluorescent in situ hybridisation and SCFA were measured using GLC. There were large and statistically significant reductions in the numbers of the Faecalibacterium prausnitzii (P < or = 0.01) and Roseburia spp. (P < or = 0.01) groups during both the fibre-free and fibre-supplemented diets. Significant and strong positive correlations between the proportion of F. prausnitzii and the proportion of butyrate during both baseline normal diets were found (pre-fibre free r 0.881, P = 0.001; pre-fibre supplemented r 0.844, P = 0.002). A significant correlation was also found between the proportional reduction in F. prausnitzii and the proportional reduction in faecal butyrate during both the fibre-free (r 0.806; P = 0.005) and the fibre-supplemented diet (r 0.749; P = 0.013). These findings may contribute to the understanding of the association between fibre, microbiota and fermentation in health, during enteral nutrition and in disease states such as Crohn's disease.

Purpose: Selective digestive microbial decontamination (SDD) is hypothesized to benefit patients in intensive care (ICU) by suppressing Gram-negative potential pathogens from the colon without affecting the anaerobic intestinal microbiota. The purpose of this study was to provide more insight to the effects of digestive tract and oropharyngeal decontamination on the intestinal microbiota by means of a prospective clinical trial in which faecal samples were collected from ICU patients for intestinal microbiota analysis., Methods: The faecal samples were collected from ICU patients enrolled in a multicentre trial to study the outcome of SDD and selective oral decontamination (SOD) in comparison with standard care (SC). Fluorescent in situ hybridization (FISH) was used to analyze the faecal microbiota. The numbers of bacteria from different bacterial groups were compared between the three regimens., Results: The total counts of bacteria per gram faeces did not differ between regimens. The F. prausnitzii group of bacteria, representing an important group among intestinal microbiota, was significantly reduced in the SDD regimen compared to the SC and SOD. The Enterobacteriaceae were significantly suppressed during SDD compared to both SOD and SC; enterococci increased in SDD compared to both other regimens., Conclusions: The composition of the intestinal microbiota is importantly affected by SDD. The F. prausnitzii group was significantly suppressed during SDD. This group of microbiota is a predominant producer of butyrate, the main energy source for colonocytes. Reduction of this microbiota is an important trade-off while reducing gram-negative bacteria by SDD.

Aim: To test recolonization of periodontal lesions after full-mouth scaling and root planing (FM-SRP) or multiple session-SRP (MS-SRP) in a randomized clinical trial and whether FM-SRP and MS-SRP result in different clinical outcomes., Materials and Methods: Thirty-nine subjects were randomly assigned to FM-SRP or MS-SRP groups. At baseline and after 3 months, probing pocket depth (PPD), plaque index (PlI) and bleeding on probing (BoP) were recorded. At baseline, immediately after treatment, after 1, 2, 7, 14 and 90 days, paper point samples from a single site from the maxillary right quadrant were collected for microbiological analysis of five putative pathogens by polymerase chain reaction., Results: FM-SRP and MS-SRP resulted in significant reductions in PPD, BoP and PlI and the overall detection frequencies of the five species after 3 months without significant differences between treatments. Compared with MS-SRP, FM-SRP resulted in less recolonization of the five species, significantly for Treponema denticola, in the tested sites., Conclusion: FM-SRP and MS-SRP result in overall clinically and microbiologically comparable outcomes where recolonization of periodontal lesions may be better prevented by FM-SRP.

Mucositis, also referred to as mucosal barrier injury, is one of the most debilitating side effects of radiotherapy and chemotherapy treatment. Clinically, mucositis is associated with pain, bacteremia, and malnutrition. Furthermore, mucositis is a frequent reason to postpone chemotherapy treatment, ultimately leading towards a higher mortality in cancer patients. According to the model introduced by Sonis, both inflammation and apoptosis of the mucosal barrier result in its discontinuity, thereby promoting bacterial translocation. According to this five-phase model, the intestinal microbiota plays no role in the pathophysiology of mucositis. However, research has implicated a prominent role for the commensal intestinal microbiota in the development of several inflammatory diseases like inflammatory bowel disease, pouchitis, and radiotherapy-induced diarrhea. Furthermore, chemotherapeutics have a detrimental effect on the intestinal microbial composition (strongly decreasing the numbers of anaerobic bacteria), coinciding in time with the development of chemotherapy-induced mucositis. We hypothesize that the commensal intestinal microbiota might play a pivotal role in chemotherapy-induced mucositis. In this review, we propose and discuss five pathways in the development of mucositis that are potentially influenced by the commensal intestinal microbiota: 1) the inflammatory process and oxidative stress, 2) intestinal permeability, 3) the composition of the mucus layer, 4) the resistance to harmful stimuli and epithelial repair mechanisms, and 5) the activation and release of immune effector molecules. Via these pathways, the commensal intestinal microbiota might influence all phases in the Sonis model of the pathogenesis of mucositis. Further research is needed to show the clinical relevance of restoring dysbiosis, thereby possibly decreasing the degree of intestinal mucositis.

Periodontitis and caries are infectious diseases of the oral cavity in which oral biofilms play a causative role. Moreover, oral biofilms are widely studied as model systems for bacterial adhesion, biofilm development, and biofilm resistance to antibiotics, due to their widespread presence and accessibility. Despite descriptions of initial plaque formation on the tooth surface, studies on mature plaque and plaque structure below the gum are limited to landmark studies from the 1970s, without appreciating the breadth of microbial diversity in the plaque. We used fluorescent in situ hybridization to localize in vivo the most abundant species from different phyla and species associated with periodontitis on seven embedded teeth obtained from four different subjects. The data showed convincingly the dominance of Actinomyces sp., Tannerella forsythia, Fusobacterium nucleatum, Spirochaetes, and Synergistetes in subgingival plaque. The latter proved to be new with a possibly important role in host-pathogen interaction due to its localization in close proximity to immune cells. The present study identified for the first time in vivo that Lactobacillus sp. are the central cells of bacterial aggregates in subgingival plaque, and that Streptococcus sp. and the yeast Candida albicans form corncob structures in supragingival plaque. Finally, periodontal pathogens colonize already formed biofilms and form microcolonies therein. These in vivo observations on oral biofilms provide a clear vision on biofilm architecture and the spatial distribution of predominant species.

Background: Normally, humans are protected against infections by their anaerobic intestinal microorganisms providing colonization resistance. In immunocompromised patients, the endogenous intestinal gram-positive and gram-negative pathogens often cause infectious complications. Therefore, we analyzed the effect of chemotherapy treatment and antimicrobial prophylaxis on intestinal bacterial populations (microbiota) among pediatric patients with acute myeloid leukemia who are prone to intestinal mucositis and infections., Methods: During 36 chemotherapy cycles, fecal samples were collected from pediatric patients with acute myeloid leukemia. Fecal bacterial populations were analyzed by polymerase chain reaction denaturing gradient gel electrophoresis fingerprinting. Fluorescent in situ hybridization analysis with specific bacterial oligonucleotide probes was used to quantify the fecal bacteria., Results: During chemotherapy treatment, the total number of bacteria in fecal samples was 10(9) per gram of dry weight feces, which was 100-fold lower than than in healthy control samples. Fluorescent in situ hybridization analysis showed that this decrease was the result of an up to 10,000-fold decrease in anaerobic bacteria, partly compensated for by a 100-fold increase in potentially pathogenic enterococci. Additional experiments showed that both prophylactic and therapeutic use of antibiotics could not sufficiently explain the tremendous changes in intestinal microbial composition. In vitro tests showed a direct bacteriostatic effect of chemotherapeutics., Conclusions: Patients with acute myeloid leukemia treated with chemotherapy and prophylactic antibiotics are unable to maintain colonization resistance because of a decrease in anaerobic bacteria and an increase in potentially pathogenic aerobic enterococci. We hypothesize that this disturbance in the balance between anaerobic and aerobic bacteria will further increase the risk of gram-positive aerobic infections among immunocompromised patients with cancer.

Viridans group streptococci (VGS) are a well-known cause of infections in immunocompromised patients, accounting for severe morbidity and mortality. Streptococcus mitis group species (Streptococcus mitis, Streptococcus pneumoniae, Streptococcus oralis) are among the VGS most often encountered in clinical practice. Identifying the portal of entry for S. mitis group strains is crucial for interventions preventing bacterial translocation. Unfortunately, tracking the source of S. mitis group strains is dependent on a combination of extremely laborious and time-consuming cultivation and molecular techniques (enterobacterial repetitive intergenic consensus-PCR [ERIC-PCR]). To simplify this procedure, a PCR analysis with newly designed primers targeting the household gene glucose kinase (gki) was used in combination with denaturing gradient gel electrophoresis (DGGE). This gki-PCR-DGGE technique proved to be specific for S. mitis group strains. Moreover, these strains could be detected in samples comprised of highly diverse microbiota, without prior cultivation. To study the feasibility of this new approach, a pilot study was performed. This confirmed that the source of S. mitis group bacteremia in pediatric patients with acute myeloid leukemia could be tracked back to the throat in five out of six episodes of bacteremia, despite the fact that throat samples are polymicrobial samples containing multiple S. mitis group strains. In contrast, using the classical combination of cultivation techniques and ERIC-PCR, we could detect these strains in only two out of six cases, showing the superiority of the newly developed technique. The new gki-PCR-DGGE technique can track the source of S. mitis group strains in polymicrobial samples without prior cultivation. Therefore, it is a valuable tool in future epidemiological studies.

The response of different bacterial species to reduced water availability was studied using a simple relative humidity gradient technique. Interestingly, distinct differences in morphology and growth patterns were observed between populations of the same species growing at different relative humidity. Gram-positive cocci increased in cell size as they approached humidity growth limits and staphylococcal species started growing in tetrad/cubical formations instead of their normal grape-like structures. Gram-negative rods displayed wave-like patterns, forming larger waves as they became increasingly filamentous at low humidity. In contrast, cells of the Gram-positive bacterium Bacillus subtilis became shorter, curved, and eventually almost coccoid. Moreover, B. subtilis started to sporulate at low humidity. The altered morphology and/or growth patterns of bacteria growing at low humidity might be more ecologically relevant than their textbook appearance at high humidity since their natural habitats are often dry. Transmission electron microscopic analyses revealed that staphylococci grown at low humidity have significantly thickened cell walls, which may explain why these cells displayed increased resistance to vancomycin. We conclude that our relative humidity gradient technique is widely applicable for investigating effects of relative humidity on microbial survival, growth and competitive success at solid-air interfaces, making it a versatile tool in microbial ecology.