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Fluorescent probes targeted at 16S rRNA were designed for Peptostreptococcus anaerobius and Peptostreptococcus stomatis (Pana134), Parvimonas micra (Pamic1435), Finegoldia magna (Fmag1250), Peptoniphilus asaccharolyticus (Pnasa1254), Peptoniphilus ivorii (Pnivo731), Peptoniphilus harei (Pnhar1466), Anaerococcus vaginalis (Avag1280) and Anaerococcus lactolyticus (Alac1438), based on the 16S rRNA sequences of reference strains and 88 randomly chosen clinical isolates. These strains were also used for validation of the probes. Application of the probes to an additional group of 100 clinical isolates revealed that 87% of Gram-positive anaerobic cocci (GPAC) could be identified with this set of probes. The 16S rRNAs of 13 clinical isolates that could not be identified were sequenced. Most of these isolates were GPAC that were not targeted by the probes. No clinical isolates of Pn. asaccharolyticus were encountered. Near full-length sequences were obtained from 71 of 101 ( n = 88 + 13) sequenced clinical isolates. Of these, 25 showed <98% similarity with the homologues of the closest established species. The Fmag1250, Pamic1435, Pnhar1466, Pana134, Pnasa1254 and Pnivo731 probes allowed reliable identification and hybridised with all corresponding isolates. The Avag1280 and Alac1438 probes failed to hybridise with two isolates and one isolate, respectively, because of intra-species variation. However, overall, the set of probes yielded fast and reliable identification for the majority of clinical isolates. [ABSTRACT FROM AUTHOR]

Abstract: Objective: The study investigated whether impaired decision-making as measured by the Gambling Task and Sensation Seeking, on one hand, and nicotine-dependence and readiness to change, on the other hand, show mutual influences in cigarette smokers. Methods: Cigarette smokers were classified as dependent or non-dependent smokers. Assessment included stages of change (RCQ), decisional balance (DBS), Sensation Seeking Scale Form-V (SSS-V), and performance on the Gambling Task (GT). Results: With the exception of a significant higher score in the SSS-V subscale Experience Seeking in dependent smokers, correlations between nicotine-dependence and cognitive features were not significant. The directions of the non-significant differences were not consistent. No significant relationship was found between the SSS-V and the GT, on one hand, and the readiness to change smoking behaviour, on the other hand. Conclusions: The results of the present study suggest that impaired performance on the GT and high scores in Sensation Seeking do not play an important role in nicotine-dependence and readiness to change smoking behaviour or vice versa. [Copyright &y& Elsevier]

Background and aims: Multiple rodent models implicate resident intestinal bacteria in the pathogenesis of chronic immune mediated intestinal inflammation. Specific pathogen free (SPF) interleukin 10 gene deficient (IL-10-/-) mice develop colitis, which does not occur in the germ free (GF) state. We investigated whether broad or narrow spectrum antibiotics affect onset and progression of disease in various regions of IL-10-/- mice. Methods: Metronidazole, ciprofloxacin, vancomycin-imipenem (50 mg/kg/day), or water (control) was administered orally before (prevention) or two weeks after (treatment) colonisation of GF IL-10-/- mice with SPF bacteria. After four weeks, colonic histology scores and cytokine production by colonic explants were determined. Caecal and colonic contents were collected for quantitative bacterial analysis. Results: In the prevention study, all antibiotics decreased inflammation in the caecum and colon. However, in the treatment study, ciprofloxacin and vancomycin-imipenem decreased caecal inflammation, and reduced Escherichia coli and Enterococcus faecalis concentrations, whereas only vancomycin-imipenem lowered direct microscopic bacterial counts. In contrast, metronidazole and vancomycin-imipenem reduced colonic injury and eliminated anaerobic bacteria, including Bacteroides spp. Conclusions: Both narrow and broad spectrum antibiotics can prevent disease but treatment of established colitis is more selective. Ciprofloxacin is most effective in the treatment of caecal inflammation, metronidazole preferentially treats the colon, whereas vancomycin-imipenem definitively treats both regions. These results suggest that subsets of aerobic or anaerobic bacteria show regional differences in their capacity to mediate experimental colitis in lL-10-/- mice. [ABSTRACT FROM AUTHOR]

Although health care professionals in The Netherlands are increasingly confronted with diverse immigrant groups, medical counselling and treatment of these groups has not been the subject of extensive research yet. From other studies it is well known that intercultural differences can have serious consequences for health care, e.g. in terms of risk of incorrect diagnoses or non-compliance. Eighty-seven autochthonous Dutch and immigrant (mainly from Turkey and Surinam) parents of child patients and their general practitioners (GPs) were recruited to investigate the influence of cultural differences on mutual understanding and patient compliance. Analyses of questionnaires and home interviews revealed that there is a relation between the cultural background of the patient and effectiveness of communication. Communication in consultations between GPs and persons from ethnic minorities is less effective than in consultations with Dutch persons: there is more misunderstanding, and also more non-compliance. In general, mutual understanding between GP and patient proves to be a strong predictor for patient compliance. These findings hold especially true for patients living in two worlds, i.e. a mixture of traditional and western cultures. The results are discussed in terms of methodological issues and practical implications for the health care providers. [ABSTRACT FROM AUTHOR]

Bacteria are involved in the onset and progression of periodontitis. A promising molecular technique, denaturing gradient gel electrophoresis (DGGE), to study microbial population dynamics in the subgingival pocket is presented. Twenty-three samples were taken from the subgingival pockets of nine patients and six healthy family members. From four periodontitis patients, 12 samples were evaluated before, 1 day after and 3 months after treatment. Part of the 16S rRNA gene of all bacteria was amplified by PCR and separated by DGGE, creating banding patterns representative of the community structure. Shifts in composition and diversity of the microbial population could be determined semiquantitatively, and this showed that treatment resulted in a decrease in the diversity of the population. After 3 months a microbial population 33–47% different from the population before treatment had re-established. Intense bands representing Exiguobacterium aurantiacum were present in 13 out of 25 samples, indicating that this species may play a role in periodontal disease. [ABSTRACT FROM AUTHOR]

Gastrointestinal mucositis is a complication of anticancer treatment, with few validated in vitro systems suitable to study the complex mechanisms of mucosal injury. Therefore, we aimed to develop and characterize a chemotherapeutic-induced model of mucositis using 3D intestinal organoids. Organoids derived from mouse ileum were grown for 7 days and incubated with different concentrations of the chemotherapeutic agent methotrexate (MTX). Metabolic activity, citrulline levels and cytokine/chemokine production were measured to determine the optimal dosage and incubation time. The protective effects of folinic acid on the toxicity of MTX were investigated by pre-treating organoids with (0.0005–50 µg/mL) folinic acid. The impact of microbial-derived short-chain fatty acids was evaluated by supplementation with butyrate in the organoid model. MTX caused a dose-dependent reduction in cell metabolic activity and citrulline production that was salvaged by folinic acid treatment. Overall, MTX causes significant organoid damage, which can be reversed upon removal of MTX. The protective effect of folinic acid suggest that the organoids respond in a clinical relevant manner. By using the model for intervention, it was found that prophylactic treatment with butyrate might be a valuable strategy for prophylactic mucositis prevention. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The relationship between fecal microbiota composition and frailty in the elderly was studied. Fecal samples from volunteers with high frailty scores showed a significant reduction in the number of lactobacilli (26-fold). At much higher population levels, both the Bacteroides/Prevotella (threefold) and the Faecalibacterium prausnitzii (fourfold) groups showed a significant reduction in percentage of total number of hybridizable bacteria in the elderly with high frailty scores. In contrast to this, the number of Enterobacteriaceae was significantly higher (sevenfold) in samples from very frail volunteers. [ABSTRACT FROM AUTHOR]

Purpose: Conditioning therapy with high-dose melphalan (HDM) is associated with a high risk of gut toxicity, fever and infections in haematopoietic stem cell transplant (HSCT) recipients. However, validated preclinical models that adequately reflect clinical features of melphalan-induced toxicity are not available. We therefore aimed to develop a novel preclinical model of melphalan-induced toxicity that reflected well-defined clinical dynamics, as well as to identify targetable mechanisms that drive intestinal injury. Methods: Male Wistar rats were treated with 4–8 mg/kg melphalan intravenously. The primary endpoint was plasma citrulline. Secondary endpoints included survival, weight loss, diarrhea, food/water intake, histopathology, body temperature, microbiota composition (16S sequencing) and bacterial translocation. Results: Melphalan 5 mg/kg caused self-limiting intestinal injury, severe neutropenia and fever while impairing the microbial metabolome, prompting expansion of enteric pathogens. Intestinal inflammation was characterized by infiltration of polymorphic nuclear cells in the acute phases of mucosal injury, driving derangement of intestinal architecture. Ileal atrophy prevented bile acid reabsorption, exacerbating colonic injury via microbiota-dependent mechanisms. Conclusion: We developed a novel translational model of melphalan-induced toxicity, which has excellent homology with the well-known clinical features of HDM transplantation. Application of this model will accelerate fundamental and translational study of melphalan-induced toxicity, with the clinical parallels of this model ensuring a greater likelihood of clinical success. [ABSTRACT FROM AUTHOR]

Climate change threatens many well‐preserved archaeological sites in the Arctic. The paper presents the first Arctic multi‐threat assessment focusing on the Nuuk region of Greenland. The results suggest that the majority of the 336 known archaeological sites are already exposed to impacts from microbial degradation, permafrost thaw and vegetation, and that these impacts will increase over the next 80 years. Additional impacts from coastal erosion are only noted at a limited number of sites due to a predominant consolidated and uplifting coast. The applied methods represent an important first step to identify threatened sites and emphasize important data limitations. [ABSTRACT FROM AUTHOR]

Background Gut microbiota and intestinal inflammation regulate the development of immune-mediated diseases, such as allergies. Fecal calprotectin is a biomarker of intestinal inflammation. Objective We evaluated the association of early-age fecal calprotectin levels to the later development of allergic diseases in children from farming and non-farming environments and further studied the effect of gut microbiota on the fecal calprotectin levels. Methods Fecal calprotectin was measured from 758 infants participating in the PASTURE study at the age of 2 months using the ELISA method. Serum-specific IgE levels were measured at 6 years of age. Data of environmental factors, doctor-diagnosed atopic dermatitis ( AD) and asthma were collected by questionnaire. Multivariate logistic regression models were used for analysis. The composition of fecal microbiota was analysed in a subgroup of 120 infants with 16S rRNA pyrosequencing. The effect of Escherichia coli lipopolysaccharide ( LPS) on in vitro monocyte IL-10 secretion was studied by flow cytometry. Results The infants with high fecal calprotectin levels at 2 months, that is above the 90th percentile, had an increased risk of developing AD and asthma/asthmatic bronchitis by the age of 6 years ( aOR 2.02 (1.06-3.85) and 2.41 (1.25-4.64), respectively). High fecal calprotectin levels correlated negatively with fecal Escherichia. LPS from E. coli stimulated production of IL-10 in monocytes. Conclusion and Clinical Relevance High degree intestinal inflammation at 2 months of age, detected as high fecal calprotectin, predicted asthma and AD by the age of 6 years and was linked to low abundance of fecal Escherichia. Impaired IL-10 activation due to the lack of colonization with E. coli could explain the intestinal inflammation associated high fecal calprotectin and later risk of asthma and AD. Our results have implications for the design of probiotic treatments and suggest that early intestinal colonization has long-term health effects. [ABSTRACT FROM AUTHOR]

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 and 4.8 × 10 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. [ABSTRACT FROM AUTHOR]

OBJECTIVE: The aim of this study was to offer a comparative analysis of informal interpreters during medical consultations with both good and poor mutual understanding between general practitioners (GPs) and patients. METHODS: Sixteen video-registered medical interviews of Turkish immigrant patients were analysed. Stretches of discourse of eight interviews with good mutual understanding between patient and doctor were compared to eight interviews with poor mutual understanding. The discourse analysis focused on: (1) miscommunication and its causes; (2) changes in the translation; (3) side-talk activities. RESULTS: In the cases of 'poor mutual understanding', the instances of miscommunication far exceeded those in the 'good mutual understanding' group. Style of self-presentation, content omissions and side-talk activities seemed to hinder good mutual understanding. CONCLUSION: Alongside the evidence about problems with informal interpreting, sometimes the use of family interpreters can facilitate medical communication. PRACTICE IMPLICATIONS: Recommendations are given in order to increase physicians' awareness of the complex process of interpreting, as well as to empower informal interpreters and patients to effectively deal with this communicative triad. [ABSTRACT FROM AUTHOR]

Usually the choice of antibiotics for treatment of secondary peritonitis is based on the results of cultures of abdominal fluid. Despite adequate antimicrobial therapy residual or repeat infection occurs in approximately 30% of the cases. Culturing of abdominal fluid and peritoneum samples was performed by routine laboratory techniques. Fluorescence in situ hybridization was applied on human peritoneum samples to evaluate its usefulness in identifying bacteria in secondary peritonitis. Antibiotic therapy should be based on the results of the cultures of peritoneum samples rather than on the cultures of abdominal fluid.

Aims: 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. Methods and Results: The numbers of total cells, total bacteria and the Eubacterium rectale/Clostridium coccoides group in faeces as measured with fluorescent in situ hybridization and the faecal β-galactosidase activity increased significantly during supplementation. The number of Bifidobacterium showed a tendency to increase during and after supplementation. With PCR-denaturing gradient gel electrophoresis, in subjects in which B. animalis and B. longum were not detected before supplementation, both strains were present in faeces during supplementation, but disappeared after supplementation. The degree of lactose digestion in the small intestine and the oro-caecal transit time were not different before and after supplementation, whereas symptom scores after lactose challenge decreased after supplementation. Conclusions: The results suggest that supplementation modifies the amount and metabolic activities of the colonic microbiota and alleviates symptoms in lactose-intolerant subjects. The changes in the colonic microbiota might be among the factors modified by the supplementation which lead to the alleviation of lactose intolerance. Significance and Impact of the Study: This study provides evidence for the possibility of managing lactose intolerance with dietary lactose (yogurt) and probiotics via modulating the colonic microbiota. [ABSTRACT FROM AUTHOR]

It has been suggested that intestinal microbiota of allergic and non-allergic children differs in composition, and that microbiota–immune system interactions may predispose children to develop sensitization. Previous studies have examined fecal microbiota of allergic children with atopic dermatitis, but little is known about that of atopic wheezy children. To investigate the composition of the fecal microbiota of young sensitized wheezy and non-sensitized non-wheezy children, using molecular methods. Within the context of a prospective birth cohort, we carried out a nested case–control study of sensitized wheezy children (cases) and non-sensitized non-wheezy controls. Cases and controls were matched for age, sex, parental atopy, allergen exposure, and pet ownership. We evaluated the composition of fecal microbiota by nucleic acid-based methods (PCR combined with denaturing gradient gel electrophoresis and quantification of bifidobacteria by fluorescent in situ hybridization). Thirty-three case–control pairs (mean age 4.4 years) provided stool samples. Comparison of total bacterial community profiles showed that each child had a unique fecal microbiota (mean Dice's similarity coefficient 22%, range 3.3–60.8%). There was no difference between the groups in prevalence of Lactic Acid bacteria (12/33 vs. 11/33, P=0.8) or bifidobacteria (30/33 vs. 31/33, P=1.00, cases vs. controls). The bifidobacterial species detected were similar in both groups. The percentage of bifidobacteria in total fecal microflora was no different between cases (median 1.7%, range 0–20.8%) and controls (1.9%, 0–18.2%, P=0.7). However, cases with eczema had significantly fewer bifidobacteria (median 1.6%, range 0–4.8%) than their controls (4.0%, 1.9–18.2%, P=0.05). We found no differences in fecal microbiota composition between sensitized wheezy and non-sensitized, non-wheezy children aged 3–5 years using nucleic acid-based methods. Differences appear to be isolated to those allergic children with eczema. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]