Ingestion of probiotics can modify gut microbiota and alter insulin resistance and diabetes development in rodents. We hypothesized that daily intake of Lactobacillus reuteri increases insulin ...sensitivity by changing cytokine release and insulin secretion via modulation of the release of glucagon-like peptides (GLP)-1 and -2.
A prospective, double-blind, randomized trial was performed in 21 glucose-tolerant humans (11 lean: age 49 ± 7 years, BMI 23.6 ± 1.7 kg/m(2); 10 obese: age 51 ± 7 years, BMI 35.5 ± 4.9 kg/m(2)). Participants ingested 10(10) b.i.d. L. reuteri SD5865 or placebo over 4 weeks. Oral glucose tolerance and isoglycemic glucose infusion tests were used to assess incretin effect and GLP-1 and GLP-2 secretion, and euglycemic-hyperinsulinemic clamps with 6,6-(2)H2glucose were used to measure peripheral insulin sensitivity and endogenous glucose production. Muscle and hepatic lipid contents were assessed by (1)H-magnetic resonance spectroscopy, and immune status, cytokines, and endotoxin were measured with specific assays.
In glucose-tolerant volunteers, daily administration of L. reuteri SD5865 increased glucose-stimulated GLP-1 and GLP-2 release by 76% (P < 0.01) and 43% (P < 0.01), respectively, compared with placebo, along with 49% higher insulin (P < 0.05) and 55% higher C-peptide secretion (P < 0.05). However, the intervention did not alter peripheral and hepatic insulin sensitivity, body mass, ectopic fat content, or circulating cytokines.
Enrichment of gut microbiota with L. reuteri increases insulin secretion, possibly due to augmented incretin release, but does not directly affect insulin sensitivity or body fat distribution. This suggests that oral ingestion of one specific strain may serve as a novel therapeutic approach to improve glucose-dependent insulin release.
In immunocompromised patients a colonisation with fungi carries the risk to develop serious invasive fungal infection. An early detection is therefore important, but not optimal hitherto. ...Fortunately, molecular genetic methods have increased the sensitivity of fungal detection and limited the time, until results are available. However, their success depends on an efficient extraction of genomic DNA from the fungal cell in the given diagnostic specimen.
To improve the routine DNA preparation method for yeasts and moulds, the impact of bead beating on fungal DNA release was evaluated. PBS, blood and respiratory rinse were spiked with Candida glabrata or Aspergillus fumigatus. DNA was extracted by mechanical bead beating in addition to the different steps of the DNA preparation protocol, which comprised liquid nitrogen treatment, proteinase K digestion and DNA isolation using the EZ1 DNA Tissue Kit and Workstation. In every method variant tested, treatment with liquid nitrogen did not improve the DNA release. Bead beating once followed by proteinase K digestion and EZ1-work-up led to the highest DNA release from fungus, spiked in PBS, and increased the extracted DNA amount of C. glabrata about 100-fold and of A. fumigatus about 10-fold in relation to sole EZ1-work-up. In fungus-spiked respiratory rinse and blood, highest increase in DNA release was measured after triple bead beating with simultaneous proteinase K digestion. Fungal DNA release of C. glabrata increased for >100-fold in respiratory rinse and for >1000-fold in blood and of A. fumigatus for >10-fold in respiratory rinse and about 5- to 10-fold in blood.
The data of this study clearly demonstrate that preparation of fungal DNA from human specimens is optimized by introduction of a bead beating step to the conventional DNA-preparation method without the necessity of a liquid nitrogen step.
Objective To characterize a potential pathogenic role of Mycoplasma salivarium and bacterial co-detection patterns on different implant augmentation types. Material and methods 36 patients were ...non-randomly assigned to autogenous lateral alveolar ridge augmentation with either cortical autogenous bone blocks, or healthy autogenous tooth roots or non-preservable teeth. Mucosal inflammation was assessed by probing pocket depth (PD) at all sampling sites and by bleeding on probing (BOP) in a subset of sampling sites, and standardized biofilm samples were obtained from the submucosal peri-implant sulcus and sulcus of a contralateral tooth at two times (t1 after implant placement; t2 after six months). Seven bacterial species were quantified using Taqman PCR. Results Mucosal inflammation did not differ between augmentation groups, but peri-implant sulci showed increased abundance of M. salivarium after augmentation with autogenous tooth roots lasting for at least six months (t1 p = 0.05, t2 p = 0.011). In M. salivarium-positive samples, Tannerella forsythia was correlated with PD (R = 0.25, p = 0.035) This correlation was not observed in M. salivarium-negative samples. Compared to all other samples, PD was deeper in co-detection (i.e., simultaneous M. salivarium and T. forsythia) positive samples (p = 0.022). No association of single or co-detection of bacteria with BOP was observed. Conclusion Presence of M. salivarium in peri-implant sulci varies with augmentation method and is associated with increased PD but not BOP. A potential causal role of M. salivarium in inflammation through a mechanism involving co-presence of T. forsythia requires further study.
The gut microbiome is a diverse ecosystem, dominated by bacteria; however, fungi, phages/viruses, archaea, and protozoa are also important members of the gut microbiota. Exploration of taxonomic ...compositions beyond bacteria as well as an understanding of the interaction between the bacteriome with the other members is limited using 16S rDNA sequencing. Here, we developed a pipeline enabling the simultaneous interrogation of the gut microbiome (bacteriome, mycobiome, archaeome, eukaryome, DNA virome) and of antibiotic resistance genes based on optimized long-read shotgun metagenomics protocols and custom bioinformatics. Using our pipeline we investigated the longitudinal composition of the gut microbiome in an exploratory clinical study in patients undergoing allogeneic hematopoietic stem cell transplantation (alloHSCT; n = 31). Pre-transplantation microbiomes exhibited a 3-cluster structure, characterized by Bacteroides spp. /Phocaeicola spp., mixed composition and Enterococcus abundances. We revealed substantial inter-individual and temporal variabilities of microbial domain compositions, human DNA, and antibiotic resistance genes during the course of alloHSCT. Interestingly, viruses and fungi accounted for substantial proportions of microbiome content in individual samples. In the course of HSCT, bacterial strains were stable or newly acquired. Our results demonstrate the disruptive potential of alloHSCTon the gut microbiome and pave the way for future comprehensive microbiome studies based on long-read metagenomics.
Mycoplasma hominis is the second smallest facultative pathogen of the human urogenital tract. With less than 600 protein-encoding genes, it represents an ideal model organism for the study of ...host-pathogen interactions. For a comprehensive characterisation of the M. hominis action in infection a customized Mho microarray, which was based on two genome sequences (PG21 and LBD-4), was designed to analyze the dynamics of the mycoplasma transcriptome during infection and validated for M. hominis strain FBG. RNA preparation was evaluated and adapted to ensure the highest recovery of mycoplasmal mRNAs from in vitro HeLa cell infection assays. Following cRNA hybridization, the read-out strategy of the hybridization results was optimized and confirmed by RT-PCR. A statistically robust infection assay with M. hominis strain FBG enabled the identification of differentially regulated key effector molecules such as critical cytoadhesins (4 h post infection (pI)), invasins (48 h pI) and proteins associated with establishing chronic infection of the host (336 h pI). Of the 294 differentially regulated genes (>2-fold) 128 (43.5%) encoded hypothetical proteins, including lipoproteins that seem to play a central role as virulence factors at each stage of infection: P75 as a novel cytoadhesin candidate, which is also differentially upregulated in chronic infection; the MHO_2100 protein, a postulated invasin and the MHO_730-protein, a novel ecto-nuclease and domain of an ABC transporter, the function of which in chronic infection has still to be elucidated. Implementation of the M. hominis microarray strategy led to a comprehensive identification of to date unknown candidates for virulence factors at relevant stages of host cell infection.
Mycoplasma hominis is a facultative human pathogen primarily associated with bacterial vaginosis and pelvic inflammatory disease, but it is also able to spread to other sites, leading to arthritis ...or, in neonates, meningitis. With a minimal set of 537 annotated genes, M. hominis is the second smallest self-replicating mycoplasma and thus an ideal model organism for studying the effects of an infectious agent on its host more closely. M. hominis adherence, colonisation and invasion of HeLa cells were characterised in a time-course study using scanning electron microscopy, confocal microscopy and microarray-based analysis of the HeLa cell transcriptome. At 4 h post infection, cytoadherence of M. hominis to the HeLa cell surface was accompanied by differential regulation of 723 host genes (>2 fold change in expression). Genes associated with immune responses and signal transduction pathways were mainly affected and components involved in cell-cycle regulation, growth and death were highly upregulated. At 48 h post infection, when mycoplasma invasion started, 1588 host genes were differentially expressed and expression of genes for lysosome-specific proteins associated with bacterial lysis was detected. In a chronically infected HeLa cell line (2 weeks), the proportion of intracellular mycoplasmas reached a maximum of 10% and M. hominis-filled protrusions of the host cell membrane were seen by confocal microscopy, suggesting exocytotic dissemination. Of the 1972 regulated host genes, components of the ECM-receptor interaction pathway and phagosome-related integrins were markedly increased. The immune response was quite different to that at the beginning of infection, with a prominent induction of IL1B gene expression, affecting pathways of MAPK signalling, and genes connected with cytokine-cytokine interactions and apoptosis. These data show for the first time the complex, time-dependent reaction of the host directed at mycoplasmal clearance and the counter measures of this pestering pathogen.
Background/Objectives: In oral and maxillofacial surgery, the reconstruction of defects often involves the transfer of skin tissue into the oral cavity utilizing microvascular grafts. This study ...investigates postoperative changes in microbial colonization following intraoral microvascular transplantation, as well as potential influencing factors. Methods: In 37 patients undergoing intraoral reconstructions, pre- and postoperative swabs were taken from the donor and recipient regions to quantify the seven selected marker bacteria using TaqMan PCRs. Patient-specific factors and clinical data were also recorded. Results: The infection-associated Acinetobacter baumannii tended to decrease postoperatively, while the infectious pathogens Pseudomonas aeruginosa, Enterococcus faecalis and the family of Enterobacteriaceae showed a postoperative increase without being directly associated with a clinical infection. Streptococcus mitis showed a significant postoperative decrease on buccal mucosa and increase on the graft surface (oral dysbiosis) and was significantly reduced or displaced by other bacteria (e.g., Mycoplasma salivarium, positive selection) when treated with ampicillin/sulbactam. Conclusions: The cutaneous microbiome of the graft adapts to the local intraoral environment. Postoperative shifts in oral bacterial colonization and an increase in infection-relevant bacteria were observed. These perioperative changes in colonization are also influenced by the administration of ampicillin/sulbactam. Consequently, single doses of antibiotics appear to be more beneficial compared to longer-term preventive use.
Ureaplasma
species (spp.) are considered commensals of the adult genitourinary tract, but have been associated with chorioamnionitis, preterm birth, and invasive infections in neonates, including ...meningitis. Data on mechanisms involved in
Ureaplasma
-driven neuroinflammation are scarce. The present study addressed brain inflammatory responses in preterm lambs exposed to
Ureaplasma parvum
(UP) in utero. 7 days after intra-amniotic injection of UP (
n
= 10) or saline (
n
= 11), lambs were surgically delivered at gestational day 128–129. Expression of inflammatory markers was assessed in different brain regions using qRT-PCR and in cerebrospinal fluid (CSF) by multiplex immunoassay. CSF was analyzed for UP presence using
ure
B-based real-time PCR, and MRI scans documented cerebral white matter area and cortical folding. Cerebral tissue levels of atypical chemokine receptor (ACKR) 3, caspases 1-like, 2, 7, and C–X–C chemokine receptor (CXCR) 4 mRNA, as well as CSF interleukin-8 protein concentrations were significantly increased in UP-exposed lambs. UP presence in CSF was confirmed in one animal. Cortical folding and white matter area did not differ among groups. The present study confirms a role of caspases and the transmembrane receptors ACKR3 and CXCR4 in
Ureaplasma
-driven neuroinflammation. Enhanced caspase 1-like, 2, and 7 expression may reflect cell death. Increased ACKR3 and CXCR4 expression has been associated with inflammatory central nervous system (CNS) diseases and impaired blood–brain barrier function. According to these data and previous in vitro findings from our group, we speculate that
Ureaplasma
-induced caspase and receptor responses affect CNS barrier properties and thus facilitate neuroinflammation.
Helicobacter pylori infection is strongly associated with gastritis, gastroduodenal ulcer disease and gastric carcinoma. The virulence of H. pylori strains increases with the presence of the ...pathogenicity island PAI, which encodes a Type 4 Secretion System and the oncoprotein CagA. Two major CagA types can be distinguished by differences in the repetitive EPIYA region in the C-terminal sequence; the more virulent East Asian CagA type with EPIYA-A, -B, and -D motifs and the Western CagA type with EPIYA-A, -B, and C motifs, the virulence of which is associated with the multitude of EPIYA-C motifs.
In this study, the cagA gene was characterized in H. pylori strains isolated from Mongolians suffering from gastritis (80%) or ulcer (20%). The EPIYA region of 53 isolates was determined by PCR-amplification of overlapping cagA regions and subsequent Sanger sequencing. Only one H. pylori isolate carried the East Asian type (ABD) and 52 isolates the Western type of CagA, thereof 30 the EPIYA type ABC, 19 the ABCC type and one each of type ABCCCC, AAABC and AAAAB. An amino acid exchange from EPIYA-B to EPIYT-B was predominantly found in CagA proteins in strains with < 2 EPIYA-C copies (n = 25/32; p = 0.015) including the two EPIYA-A enriched CagA proteins, which have not been described to date. Due to the amino acid triplet preceding the EPIYA motif and strength of predicted phosphorylation, the multiple EPIYA-A motifs A2, A3 and A4 were shown to cluster with EPIYA-B and EPIYT-B with the unique feature of amino acid E in position − 4 to Y of EPIYA. It has been described that tyrosine-phosphorylated EPIYA-A and -B motifs counteract the EPIYA-C-driven signaling towards host cell transformation and malignancy. Thus, Mongolian H. pylori strains carrying CagA proteins not only with a few EPIYA-C segments but also with multiplied EPIYA-A segments are probably less virulent; a thesis that needs further investigation at the protein level.
To determine prevalence of lymphogranuloma venereum among men who have sex with men in Germany, we conducted a multicenter study during 2009-2010 and found high rates of rectal and pharyngeal ...infection in men positive for the causative agent, Chlamydia trachomatis. Many infections were asymptomatic. An adjusted C. trachomatis screening policy is justified in Germany.
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