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Background At the entry site of respiratory virus infections, the oropharyngeal microbiome has been proposed as a major hub integrating viral and host immune signals. Early studies suggested that infections with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are associated with changes of the upper and lower airway microbiome, and that specific microbial signatures may predict coronavirus disease 2019 (COVID-19) illness. However, the results are not conclusive, as critical illness can drastically alter a patient’s microbiome through multiple confounders. Methods To study oropharyngeal microbiome profiles in SARS-CoV-2 infection, clinical confounders, and prediction models in COVID-19, we performed a multicenter, cross-sectional clinical study analyzing oropharyngeal microbial metagenomes in healthy adults, patients with non-SARS-CoV-2 infections, or with mild, moderate, and severe COVID-19 (n = 322 participants). Results In contrast to mild infections, patients admitted to a hospital with moderate or severe COVID-19 showed dysbiotic microbial configurations, which were significantly pronounced in patients treated with broad-spectrum antibiotics, receiving invasive mechanical ventilation, or when sampling was performed during prolonged hospitalization. In contrast, specimens collected early after admission allowed us to segregate microbiome features predictive of hospital COVID-19 mortality utilizing machine learning models. Taxonomic signatures were found to perform better than models utilizing clinical variables with Neisseria and Haemophilus species abundances as most important features. Conclusions In addition to the infection per se, several factors shape the oropharyngeal microbiome of severely affected COVID-19 patients and deserve consideration in the interpretation of the role of the microbiome in severe COVID-19. Nevertheless, we were able to extract microbial features that can help to predict clinical outcomes.

The vitamin D receptor (VDR) is critical in regulating intestinal homeostasis and emerging evidence demonstrates that VDR deficiency is a critical factor in inflammatory bowel disease pathology. However, no clinical data exist regarding the intestinal expression of VDR in patients after allogeneic haematopoietic stem cell transplantation (HSCT). Analyzing intestinal biopsies from 90 patients undergoing HSCT with mortality follow-up, we demonstrated that patients with severe acute gastrointestinal graft versus host disease (GI-GvHD) showed significant downregulation of VDR gene expression compared to mild or no acute GI-GvHD patients (p = 0.007). Reduced VDR expression was already detectable at acute GI-GvHD onset compared to GvHD-free patients (p = 0.01). These results were confirmed by immunohistochemistry (IHC) where patients with severe acute GI-GvHD showed fewer VDR+ cells (p = 0.03) and a reduced VDR staining score (p = 0.02) as compared to mild or no acute GI-GvHD patients. Accordingly, low VDR gene expression was associated with a higher cumulative incidence of treatment-related mortality (TRM) (p = 1.6x10-6) but not with relapse-related mortality (RRM). A multivariate Cox regression analysis identified low VDR as an independent risk factor for TRM (p = 0.001, hazard ratio 4.14, 95% CI 1.78-9.63). Furthermore, VDR gene expression significantly correlated with anti-microbial peptides (AMPs) gene expression (DEFA5: r = 0.637, p = 7x10-5, DEFA6: r 0 0.546, p = 0.001). In conclusion, our findings suggest an essential role of the VDR in the pathogenesis of gut GvHD and the prognosis of patients undergoing HSCT.

Class 1 and 2 monoclonal antibodies inhibit SARS-CoV-2 entry by blocking the interaction of the viral receptor-binding domain with angiotensin-converting enzyme 2 (ACE2), while class 3 antibodies target a highly conserved epitope outside the ACE2 binding site. We aimed to investigate the plasticity of the spike protein by propagating wild-type SARS-CoV-2 in the presence of class 3 antibody S309. After 12 weeks, we obtained a viral strain that was completely resistant to inhibition by S309, due to successively evolving amino acid exchanges R346S and P337L located in the paratope of S309. The antibody lost affinity to receptor-binding domains carrying P337L or both amino acid exchanges, while ACE2 binding was not affected. The resistant strain replicated efficiently in human CaCo-2 cells and was more susceptible to inhibition of fusion than the original strain. Overall, SARS-CoV-2 escaped inhibition by class 3 antibody S309 through a slow, but targeted evolution enabling immune escape and altering cell entry. This immune-driven enhancement of infectivity and pathogenicity could play an important role in the future evolution of SARS-CoV-2, which is under increasing immunological pressure from vaccination and previous infections.

Background The implementation of quality of life (QoL) concepts in routine care, is still an open matter. We followed the Medical Research Council framework for complex interventions to implement a model of QoL diagnosis and therapeutic options, and investigated its effectiveness in patients with colorectal cancer. Methods This randomised, single-blind, multicentre, clinical trial enrolled patients diagnosed with primary colorectal cancer aged 18 years or older who were surgically treated in one of four recruiting hospitals in Germany. All patients received aftercare from one of 178 coordinating practitioners (CPs) who had access to 75 healthcare professionals providing tailored therapies. QoL was measured (EORTC QLQ-C30, QLQ-CR29) in all patients after surgery (baseline) and during aftercare (3, 6, 12, 18 months). Patients were randomised (1:1) into two groups: a care pathway, including QoL-profiles consisting of 13 QoL scales plus specific therapeutic recommendations forwarded to the patient’s CP or standard postoperative care adhering to the German national guideline for colorectal cancer (control). The primary endpoint was the proportion of patients in each group with a need for QoL therapy 12 months after surgery. Analyses were done in the intention-to-treat population. This trial is registered with ClinicalTrials.gov , number NCT02321813 and closed to accrual. Findings Between Jan 13, 2014, and Oct 28, 2015, 220 patients were enrolled and randomly assigned (n = 110 per group). At baseline (in hospital after surgery), a need for QoL therapy was diagnosed in 92/103 (89%) of intervention and 86/104 (83%) of control group patients. At 12 months (primary endpoint) the proportion of patients with a need for QoL therapy was 35/83 (42%; 95% CI 31–54%) in the intervention group versus 50/87 (57%; 95% CI: 46–68%) in the control group (p = 0·046, number needed to treat = 7; 95% CI 3–225). Interpretation Patients profited from the diagnosis of QoL deficits and tailored therapeutic options in their treatment of colorectal cancer. This trial confirmed the results of a previous RCT in breast cancer patients. The implementation of QoL concepts should become standard in treatment guidelines on cancer care. Funding Federal Ministry of Education and Research (BMBF; grant no. 01GY1339). Clinical trial information NCT02321813.

Recently, CEN/TS 17626:2021, the European pre-analytical standard for human specimens intended for microbiome DNA analysis, was published. Although this standard relates to diagnostic procedures for microbiome analysis and is relevant for in vitro diagnostic (IVD) manufacturers and diagnostic laboratories, it also has implications for research and development (R&D). We present here why standards are needed in biomedical research, what pre-analytical standards can accomplish, and which elements of the pre-analytical workflow they cover. The benefits of standardization for the generation of FAIR (findable, accessible, interoperable, reusable) data and to support innovation are briefly discussed.

The role of IL-22 in adult patients undergoing allogeneic stem cell transplantation (SCT) is of major interest since animal studies showed a protective and regenerative effect of IL-22 in graft versus host disease (GvHD). However, no clinical data exist on the tissue expression. Here we demonstrate that patients not suffering from transplant-related mortality (TRM) show significantly upregulated IL22 expression during histological and clinical GI-GvHD (p = 0.048 and p = 0.022, respectively). In contrast, in GvHD patients suffering from TRM, IL22 was significantly lower (p = 0.007). Accordingly, lower IL22 was associated with a higher probability of TRM in survival analysis (p = 0.005). In a multivariable competing risk Cox regression analysis, low IL22 was identified as an independent risk factor for TRM (p = 0.007, hazard ratio 2.72, 95% CI 1.32 to 5.61). The expression of IL22 seemed to be microbiota dependent as broad-spectrum antibiotics significantly diminished IL22 expression (p = 0.019). Furthermore, IL22 expression significantly correlated with G-protein coupled receptor (GPR)43 (r = 0.263, p = 0.015) and GPR41 expression (r = 0.284, p = 0.009). In conclusion, our findings reveal an essential role of IL22 for the prognosis of patients undergoing allogeneic SCT.

Deficiency in X-linked inhibitor of apoptosis protein (XIAP) is the cause for X-linked lymphoproliferative syndrome 2 (XLP2). About one-third of these patients suffer from severe and therapy-refractory inflammatory bowel disease (IBD), but the exact cause of this pathogenesis remains undefined. Here, we used XIAP-deficient mice to characterize the mechanisms underlying intestinal inflammation. In

Microbiota can exert immunomodulatory effects by short-chain fatty acids (SCFA) in experimental models of graft-versus-host disease (GvHD) after allogeneic hematopoietic stem cell transplantation (allo-SCT). Therefore we aimed to analyze the expression of SCFAs sensing G-protein coupled receptor GPR109A and GPR43 by quantitative PCR in 338 gastrointestinal (GI) biopsies obtained from 199 adult patients undergoing allo-SCT and assessed the interaction of GPR with FOXP3 expression and regulatory T cell infiltrates. GPR expression was strongly upregulated in patients with stage II-IV GvHD (p=0.000 for GPR109A, p=0.01 for GPR43) and at the onset of GvHD (p 0.000 for GPR109A, p=0.006 for GPR43) and correlated strongly with FOXP3 and NLRP3 expression. The use of broad-spectrum antibiotics (Abx) drastically suppressed GPR expression as well as FOXP3 expression in patients’ gut biopsies (p=0.000 for GPRs, FOXP3 mRNA and FOXP3+ cellular infiltrates). Logistic regression analysis revealed treatment with Abx as an independent factor associated with GPR and FOXP3 loss. The upregulation of GPRs was evident only in the absence of Abx (p=0.001 for GPR109A, p=0.014 for GPR43) at GvHD onset. Thus, GPR expression seems to be upregulated in the presence of commensal bacteria and associates with infiltration of FOXP3+ T regs, suggesting a protective, regenerative immunomodulatory response. However, Abx, which has been shown to induce dysbiosis, interferes with this protective response.

Background Butyrogenic bacteria play an important role in gut microbiome homeostasis and intestinal epithelial integrity. Previous studies have demonstrated an association between administration of short-chain fatty acids like butyrate and protection from acute graft-vs-host disease (GvHD) after allogeneic stem cell transplantation (ASCT). Methods In the current study, we examined the abundance and butyrogenic capacity of butyrate-producing bacteria in 28 healthy donors and 201 patients after ASCT. We prospectively collected serial stool samples and performed polymerase chain reaction analysis of the butyrate-producing bacterial enzyme butyryl–coenzyme A (CoA):acetate CoA-transferase (BCoAT) in fecal nucleic acid extracts. Results Our data demonstrate a strong and prolonged suppression of butyrogenic bacteria early in the course of ASCT. In a multivariable analysis, early use of broad-spectrum antibiotics before day 0 (day of transplantation) was identified as an independent factor associated with low BCoAT copy numbers (odds ratio, 0.370 [95% confidence interval, .175–.783]; P = .009). Diminished butyrogens correlated with other biomarkers of microbial diversity, such as low 3-indoxylsulfate levels, reduced abundance of Clostridiales and low inverse Simpson and effective Shannon indices (all P Conclusions Our results are consistent with the hypothesis that alterations in the microbiome play an important role in GvHD pathogenesis and that microbial parameters such as BCoAT might serve as biomarkers to identify patients at high risk of lethal GI-GvHD.

Background Maintaining gastrointestinal (GI) microbiome diversity plays a key role during allogeneic stem cell transplantation (ASCT), and loss of diversity correlates with acute GI graft versus host disease (GvHD) and poor outcomes. Methods In this retrospective analysis of 161 ASCT patients, we used serial analyses of urinary 3-indoxyl sulfate (3-IS) levels and GI microbiome parameters within the first 10 days after ASCT to identify potential commensal microbiota–sparing antibiotics. Based on antibiotic activity, we formed 3 subgroups (Rifaximin without systemic antibiotics, Rifaximin with systemic antibiotics, and Ciprofloxacin/Metronidazole with/without systemic antibiotics). Results Mono-antibiosis with Rifaximin revealed higher 3-IS levels (P < .001), higher Clostridium cluster XIVa (CCXIVa) abundance (P = .004), and higher Shannon indices (P = .01) compared to Ciprofloxacin/Metronidazole with/without systemic antibiotics. Rifaximin followed by systemic antibiotics maintained microbiome diversity compared to Ciprofloxacin/Metronidazole with/without systemic antibiotics, as these patients showed still higher 3-IS levels (P = .04), higher CCXIVa copy numbers (P = .01), and higher Shannon indexes (P = .01). Even for this larger cohort of patients, the outcome was superior with regard to GI GvHD (P = .05) and lower transplant-related mortality (P < .001) for patients receiving Rifaximin plus systemic antibiotics compared to other types of systemic antibiotic treatment. Antibiosis with Ciprofloxacin/Metronidazole (n = 12, P = .01), Piperacillin/Tazobactam (n = 52, P = .01), Meropenem/Vancomycin (n = 16, P = .003), Ceftazidime (n = 10, P = .03), or multiple systemic antibiotics (n = 53, P = .001) showed significantly lower 3-IS levels compared to mono-antibiosis with Rifaximin (n = 14) or intravenous Vancomycin (n = 4, not statistically significant). Conclusions Different types of antibiotic treatments show different impacts on markers of microbiome diversity. The identification of antibiotics sparing commensal bacteria remains an ongoing challenge. However, Rifaximin allowed a higher intestinal microbiome diversity, even in the presence of systemic broad-spectrum antibiotics.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative motor neuron disease accompanied by both systemic and central nervous system-specific inflammation as well as deregulated energy metabolism. These potential pathogenetic factors have recently been found to mutually interact with the gut microbiota, raising the hypothesis of a link between microbiome alterations and ALS pathogenesis. The aim of our study was to assess whether ALS is associated with an altered composition of the fecal microbiota. We compared the fecal microbiota of 25 ALS patients with 32 age- and gender-matched healthy persons using 16S rRNA gene sequencing analysis. Confounding factors and secondary disease effects on the microbiome were minimized by selection of patients without dysphagia, gastrostomy, noninvasive ventilation, or reduced body mass index. Comparing the 2 carefully matched groups, the diversity and the abundance of the bacterial taxa on the different taxonomic levels as well as PICRUSt-predicted metagenomes were almost indistinguishable. Significant differences between ALS patients and healthy controls were only observed with regard to the overall number of microbial species (operational taxonomic units) and in the abundance of uncultured Ruminococcaceae. Conclusively, ALS patients do not exhibit a substantial alteration of the gut microbiota composition.

Diarrheagenic E. coli (DEC) are one of the most common causes for diarrhea worldwide, especially in children. We evaluated the rapid RIDA ® GENE (RG) real-time multiplex PCR assays (R-Biopharm, Darmstadt, Germany) for the detection of the most important diarrheagenic E. coli. Three hundred fifteen liquid or non-formed stool specimens were examined. The results of the RG multiplex assays were compared to specific PCR methods. The sensitivity and specificity of the RG PCRs were as follows, 100%/100% for the detection of EHEC, 96.3% and 99% for EPEC, 100% and 100% for the detection of EAEC, ETEC and EIEC, respectively. Overall, the RG real-time PCR system for the detection of DEC tested in this study provided reliable and rapid results and shows the ability as a useful addendum for the detection of diarrheagenic E. coli in the medical laboratory.

To the editor: Allogeneic bone marrow (BM) transplantation (alloBMT) is a unique curative therapy for diverse diseases. However, its use is limited by the development of severe treatment-related complications, most importantly, the occurrence of acute graft-versus-host-disease (aGVHD). Despite the

Clostridium (Clostridioides) difficile is a major cause of nosocomial diarrhoea. A first inter-laboratory ring trial was performed in four European countries to evaluate the genotyping and antibiotic susceptibility testing (AST) accuracy. Six C. difficile isolates representing the epidemiologic important ribotypes (RT), RT001, RT002, RT010, RT014, RT027, and RT078 were blinded and send to 21 participating laboratories. Participants tested the samples with their genotyping and AST methods in use for concordance with reference. A total of 21 genotyping- and 14 antimicrobial susceptibility data sets were obtained. Ribotyping (11 participants) correctly identified most RTs (median 91% concordance rate) except for RT002, which was misidentified in 4/11 reports. However, this isolate was correctly asserted to RT002 after an update of a publicly available ribotyping database. Multilocus sequence typing, surface layer sequence typing, DNA microarray based genotyping, and whole genome sequencing, which were used by 1–3 participants, identified all six isolates correctly. AST was done by epsilometry by the participants and compared to agar dilution data determined by the coordinating reference centre. Susceptibilities against metronidazole, moxifloxacin, and vancomycin were correctly identified in 235 of 237 cases and in accordance to agar dilution as the gold standard. Genotyping of the C. difficile test strains revealed a remarkable high concordance on the level of ribotypes with a wide variety of methods. Epsilometry appears to be a reliable method for AST of C. difficile isolates in routine clinical microbiology laboratories.

The composition of human as well as animal microbiota has increasingly gained in interest since metabolites and structural components of endogenous microorganisms fundamentally influence all aspects of host physiology. Since many of the bacteria are still unculturable, molecular techniques such as high-throughput sequencing have dramatically increased our knowledge of microbial communities. The majority of microbiome studies published thus far are based on bacterial 16S ribosomal RNA (rRNA) gene sequencing, so that they can, at least in principle, be compared to determine the role of the microbiome composition for host metabolism and physiology, developmental processes, as well as different diseases. However, differences in DNA preparation and purification, 16S rDNA PCR amplification, sequencing procedures and platforms, as well as bioinformatic analysis and quality control measures may strongly affect the microbiome composition results obtained in different laboratories. To systematically evaluate the comparability of results and identify the most influential methodological factors affecting these differences, identical human stool sample replicates spiked with quantified marker bacteria, and their subsequent DNA sequences were analyzed by nine different centers in an external quality assessment (EQA). While high intra-center reproducibility was observed in repetitive tests, significant inter-center differences of reported microbiota composition were obtained. All steps of the complex analysis workflow significantly influenced microbiome profiles, but the magnitude of variation caused by PCR primers for 16S rDNA amplification was clearly the largest. In order to advance microbiome research to a more standardized and routine medical diagnostic procedure, it is essential to establish uniform standard operating procedures throughout laboratories and to initiate regular proficiency testing.

Introduction: Since the discovery of Polymerase Chain Reaction and pioneering work on 16S ribosomal RNA genes in the 1980’s, the use of molecular techniques to study microbial ecosystems and pathogenic microorganisms has been increasing exponentially. Because microbial communities inhabiting various body sites such as the skin or the intestine can influence our physiology, there has been a massive interest in studying their diversity, functions, and role in the development of acute and chronic diseases.Area covered: In the present review, we gather knowledge on sequencing approaches to study microbial communities, especially human body microbiota, and give opinions on their potential and limitations, particularly with respect to clinical applications.Expert commentary: High-throughput sequencing delivered unprecedented views on complex microbial communities, but their popularization is accompanied by substantial technical hurdles that will need to be overcome for efficient implementation in routin...

Colorectal cancer (CRC) is associated with loss of epithelial barrier integrity, which facilitates the interaction of the immunological microenvironment with the luminal microbiome, eliciting tumor-supportive inflammation. An important regulator of intestinal inflammatory responses is IRAK-M, a negative regulator of TLR signaling. Here we investigate the compartment-specific impact of IRAK-M on colorectal carcinogenesis using a mouse model. We demonstrate that IRAK-M is expressed in tumor cells due to combined TLR and Wnt activation. Tumor cell-intrinsic IRAK-M is responsible for regulation of microbial colonization of tumors and STAT3 protein stability in tumor cells, leading to tumor cell proliferation. IRAK-M expression in human CRCs is associated with poor prognosis. These results suggest that IRAK-M may be a potential therapeutic target for CRC treatment.

Intestinal microbiota disruption is associated with acute gastrointestinal (GI) Graft-versus-Host Disease (GvHD) and poor outcome after allogeneic stem cell transplantation (ASCT). Here, in a retrospective analysis of 200 patients undergoing ASCT at the Regensburg University Medical Center, we assessed the relative expression of Paneth cell antimicrobial peptides (AMPs), Human Defensins (HD) 5 and 6 and regenerating islet-derived 3 alpha (Reg3 alpha), in 292 human intestinal biopsies as well as Reg3a serum levels in relation to acute GI GvHD. In the absence of GI GvHD, the relative expression of Paneth cell AMPs was significantly higher in the small intestine (duodenum to ileum) than in the stomach and large intestine (cecum to rectum) for Reg3a (p

The purpose of this review is to outline and evaluate the most recent literature on the role of the microbiome in urinary tract diseases.High throughput molecular DNA sequencing of bacterial 16S rRNA genes enabled the analysis of complex microbial communities inhabiting the human urinary tract. Several recent studies have identified bacterial taxa of the urinary microbiome to impact urinary tract diseases including interstitial cystitis, urgency urinary incontinence or calcium oxalate stone formation. Furthermore, treatment of urinary tract infections by antibiotics globally impacts community profiles of the intestinal microbiota and might indirectly influence human health. Alternative treatment options like application of probiotics for the treatment of urinary tract infections are currently under investigation.The urinary microbiome and its relationship to urinary tract diseases is currently under comprehensive investigation. Further studies are needed to shed light on the role of commensal microbiota for urinary tract infections.

Microbiological ecology and its ambition to describe the complete genome of complex living communities as a whole, have given us powerful tools to characterize the human gut microbiome on a genetic and, hence, taxonomic and abundance level; for a decade now, they have become sufficiently inexpensive, fast and feasible. Thus, opportunities arose to have a fresh and closer look at the microbiota-gut-brain-axis and its impact on human health; this axis comprises a complex multisystemic network of multidirectional interactions between brain and gut including influences beyond one generation. Gnotobiotic animal models have become essential for specific research targets. Combining gut microbiome analysis with observations on the hypothalamus-pituitary-adrenal axis and various aspects of inflammation helped to gain first insights into the role of the microbiota-gut-brain-axis in depressive disorders. Therapeutic endeavors with psychobiotics have not yet shown their value in clinical studies.

In allogeneic stem cell transplantation (ASCT), systemic broad-spectrum antibiotics are frequently used for treatment of infectious complications, but their effect on microbiota composition is still poorly understood. This retrospective analysis of 621 patients who underwent ASCT at the University Medical Center of Regensburg and Memorial Sloan Kettering Cancer Center in New York assessed the impact of timing of peritransplant antibiotic treatment on intestinal microbiota composition as well as transplant-related mortality (TRM) and overall survival. Early exposure to antibiotics was associated with lower urinary 3-indoxyl sulfate levels (P .001) and a decrease in fecal abundance of commensal Clostridiales (P = .03) compared with late antibiotic treatment, which was particularly significant (P = .005) for Clostridium cluster XIVa in the Regensburg group. Earlier antibiotic treatment before ASCT was further associated with a higher TRM (34%, 79/236) compared with post-ASCT (21%, 62/297, P = .001) or no antibiotics (7%, 6/88, P .001). Timing of antibiotic treatment was the dominant independent risk factor for TRM (HR, 2.0; P ≤ .001) in multivariate analysis besides increase age (HR, 2.15; P = .004), reduced Karnofsky performance status (HR, 1.47; P = .03), and female donor-male recipient sex combination (HR, 1.56; P = .02) A competing risk analysis revealed the independent effect of early initiation of antibiotics on graft-versus-host disease-related TRM (P = .004) in contrast to infection-related TRM and relapse (not significant). The poor outcome associated with early administration of antibiotic therapy that is active against commensal organisms, and specifically the possibly protective Clostridiales, calls for the use of Clostridiales-sparing antibiotics and rapid restoration of microbiota diversity after cessation of antibiotic treatment.

Objectives Aim of this study was to detect microorganisms in fetal membranes and placental tissue in preterm chorioamnionitis by combining fluorescence in situ hybridization (FISH) with broad range PCR. The combination of the two molecular techniques enables identification and localization of the microorganisms within the tissue, confirming their clinical relevance. Methods In a prospective cohort study, we compared 31 women with preterm premature rupture of membranes or preterm labour and preterm delivery by caesarean section with a control group of 26 women undergoing elective caesarean section at term. Fetal membranes and placental tissue were analysed by FISH and broad range 16S rRNA-gene PCR and sequencing. Results For 20 women in the preterm group, caesarean section was performed because of a clinical diagnosis of chorioamnionitis. Microorganisms were detected in the tissues by both molecular techniques in 11 out of 20 women. Among those, Ureaplasma spp. was most abundant, with five cases that remained culture-negative and would have been missed by routine diagnostic procedures. Other infections were caused by Staphylococcus aureus , Streptococcus mitis or Escherichia coli . FISH and PCR were negative for all women without suspected chorioamnionitis and for the control group. Conclusions Combination of FISH with broad-range PCR and sequencing permitted unambiguous identification of the causative microorganisms in chorioamnionitis. The high prevalence of Ureaplasma spp. should lead to a re-evaluation of its clinical significance and possible therapeutic consequences.

A carbapenemase-producing colistin-resistant Klebsiella oxytoca isolate was recovered from a blood culture of a female patient without previous report of risk factors to obtain multidrug-resistant Gram-negative bacilli. A combination of biochemical and molecular methods was used to identify the resistance mechanism of this isolate. Carbapenemase production was mediated by Verona integron-encoded metallo-β-lactamase (VIM)-2. Colistin resistance was not due to plasmid- borne mcr-1 gene, but we found an integration of IS5-like sequence in the mgrB gene of K. oxytoca. This gene is known to be an important regulator of the PhoPQ two-component system, and the disruption of this gene is most likely the cause of lipid A modification resulting in colistin resistance of our isolate. To the best of our knowledge this constitutes the first report of a carbapenemase-producing K. oxytoca with colistin resistance, a case that demonstrates the limited treatment options for infections with multidrug-resistant organisms.

Motivation In biomedicine, every molecular measurement is relative to a reference point, like a fixed aliquot of RNA extracted from a tissue, a defined number of blood cells, or a defined volume of biofluid. Reference points are often chosen for practical reasons. For example, we might want to assess the metabolome of a diseased organ but can only measure metabolites in blood or urine. In this case, the observable data only indirectly reflects the disease state. The statistical implications of these discrepancies in reference points have not yet been discussed. Results Here, we show that reference point discrepancies compromise the performance of regression models like the LASSO. As an alternative, we suggest zero-sum regression for a reference point insensitive analysis. We show that zero-sum regression is superior to the LASSO in case of a poor choice of reference point both in simulations and in an application that integrates intestinal microbiome analysis with metabolomics. Moreover, we describe a novel coordinate descent based algorithm to fit zero-sum elastic nets. Availability and Implementation The R-package “zeroSum” can be downloaded at https://github.com/rehbergT/zeroSum. Moreover, we provide all R-scripts and data used to produce the results of this manuscript as Supplementary Material. Supplementary information Supplementary material is available at Bioinformatics online.

Background Next-generation 16S ribosomal RNA gene sequencing is widely used to determine the relative composition of the mammalian gut microbiomes. However, in the absence of a reference, this does not reveal alterations in absolute abundance of specific operational taxonomic units if microbial loads vary across specimens. Results Here we suggest the spiking of exogenous bacteria into crude specimens to quantify ratios of absolute bacterial abundances. We use the 16S rDNA read counts of the spike-in bacteria to adjust the read counts of endogenous bacteria for changes in total microbial loads. Using a series of dilutions of pooled faecal samples from mice containing defined amounts of the spike-in bacteria Salinibacter ruber, Rhizobium radiobacter and Alicyclobacillus acidiphilus, we demonstrate that spike-in-based calibration to microbial loads allows accurate estimation of ratios of absolute endogenous bacteria abundances. Applied to stool specimens of patients undergoing allogeneic stem cell transplantation, we were able to determine changes in both relative and absolute abundances of various phyla, especially the genus Enterococcus, in response to antibiotic treatment and radio-chemotherapeutic conditioning. Conclusion Exogenous spike-in bacteria in gut microbiome studies enable estimation of ratios of absolute OTU abundances, providing novel insights into the structure and the dynamics of intestinal microbiomes. Electronic supplementary material The online version of this article (doi:10.1186/s40168-016-0175-0) contains supplementary material, which is available to authorized users.

Intestinal dysbiosis has been associated with acute gastrointestinal GvHD and poor outcome following allogeneic stem cell transplantation (ASCT). To assess the effect of a switch in 2012 from ciprofloxacin/metronidazole to rifaximin for gut decontamination on intestinal microbiota composition and ASCT outcome, we retrospectively analyzed 394 patients receiving ASCT from September 2008 through June 2015. In 131 and 90 patients, respectively, urinary 3-indoxyl sulfate levels and intestinal enterococcal load were measured before conditioning and weekly within the first 28 days after ASCT. The use of rifaximin correlated with lower enterococcal positivity (6.9 vs 21.9%, P=0.05) and higher urinary 3-indoxyl sulfate concentrations (10.5 vs 4.6 μmoL/mmoL crea, P

Analyzing human as well as animal microbiota composition has gained growing interest because structural components and metabolites of microorganisms fundamentally influence all aspects of host physiology. Originally dominated by culture-dependent methods for exploring these ecosystems, the development of molecular techniques such as high throughput sequencing has dramatically increased our knowledge. Because many studies of the microbiota are based on the bacterial 16S ribosomal RNA (rRNA) gene targets, they can, at least in principle, be compared to determine the role of the microbiome composition for developmental processes, host metabolism, and physiology as well as different diseases. In our review, we will summarize differences and pitfalls in current experimental protocols, including all steps from nucleic acid extraction to bioinformatical analysis which may produce variation that outweighs subtle biological differences. Future developments, such as integration of metabolomic, transcriptomic, and metagenomic data sets and standardization of the procedures, will be discussed.

Urinary 3-IS levels predict outcome after ASCT and are associated with antibiotics and NOD2/CARD15 variants.Publisher’s Note: There is an Inside Blood Commentary on this article in this issue.Indole, which is produced from l-tryptophan by commensal bacteria expressing tryptophanase, not only is an important intercellular signal in microbial communities, but also modulates mucosal barrier function and expression of pro- and anti-inflammatory genes by intestinal epithelial cells. Here, we hypothesized that decreased urinary excretion of 3-indoxyl sulfate (3-IS), the major conjugate of indole found in humans, may be a marker of gut microbiota disruption and increased risk of developing gastrointestinal (GI) graft-versus-host-disease. Using liquid chromatography/tandem mass spectrometry, 3-IS was determined in urine specimens collected weekly within the first 28 days after allogeneic stem cell transplantation (ASCT) in 131 patients. Low 3-IS levels within the first 10 days after ASCT were associated with significantly higher transplant-related mortality (P = .017) and worse overall survival (P = .05) 1 year after ASCT. Least absolute shrinkage and selection operator regression models trained on log-normalized counts of 763 operational taxonomic units derived from next-generation sequencing of the hypervariable V3 region of the 16S ribosomal RNA gene showed members of the families of Lachnospiraceae and Ruminococcaceae of the class of Clostridia to be associated with high urinary 3-IS levels, whereas members of the class of Bacilli were associated with low 3-IS levels. Risk factors of early suppression of 3-IS levels were the type of GI decontamination (P = .01), early onset of antibiotic treatment (P = .001), and recipient NOD2/CARD15 genotype (P = .04). In conclusion, our findings underscore the relevance of microbiota-derived indole and metabolites thereof in mucosal integrity and protection from inflammation.

To assess relevant parameters for the minimally invasive elimination of breast tumors by using a selective application of magnetite and exposure of the breast to an alternating magnetic field.The specific absorption rate (SAR) of different magnetite samples was determined calorimetrically. Temperature elevations based on magnetite mass (7-112 mg) and magnetic field amplitude (1.2-6.5 kA/m frequency, 400 kHz) were investigated by using human breast tissue. Parameter combinations (21 mg +/- 9 [SD], 242-second magnetic field exposure, 6.5-kA/m amplitude) were tested in 10 immunodeficient mice bearing human adenocarcinomas (MX-1 cells). Histologic sections of heated tumor tissue were analyzed.SAR data of different magnetite particle types ranged from 3 to 211 W/g. Temperature elevation (DeltaT) as a function of the magnetite mass increased linearly up to 28 mg; at higher masses, a saturation of DeltaT was observed at nearly 88 degrees C. The dependence of DeltaT on magnetic field amplitude (H) revealed a third-order power law: DeltaT = 0.26 degrees C/(kA/m)(3). H(3), with r(2) = 0.95. A mean temperature of 71 degrees C +/- 8 was recorded in the tumor region at the end of magnetic field exposure of the mice. Typical macroscopic findings included tumor shrinkage after heating. Histologically nuclear degenerations were observed in heated malignant cells.Magnetic heating of breast tumors is a promising technique for future interventional radiologic treatments.

Diffusion tensor imaging (DTI)-based tractography has become an integral part of preoperative diagnostic imaging in many neurosurgical centers, and other nonsurgical specialties depend increasingly on DTI tractography as a diagnostic tool. The aim of this study was to analyze the anatomic accuracy of visualized white matter fiber pathways using different, readily available DTI tractography software programs.Magnetic resonance imaging scans of the head of 20 healthy volunteers were acquired using a Siemens Symphony TIM 1.5T scanner and a 12-channel head array coil. The standard settings of the scans in this study were 12 diffusion directions and 5-mm slices. The fornices were chosen as an anatomic structure for the comparative fiber tracking. Identical data sets were loaded into nine different fiber tracking packages that used different algorithms. The nine software packages and algorithms used were NeuroQLab (modified tensor deflection [TEND] algorithm), Sörensen DTI task card (modified streamline tracking technique algorithm), Siemens DTI module (modified fourth-order Runge-Kutta algorithm), six different software packages from Trackvis (interpolated streamline algorithm, modified FACT algorithm, second-order Runge-Kutta algorithm, Q-ball [FACT algorithm], tensorline algorithm, Q-ball [second-order Runge-Kutta algorithm]), DTI Query (modified streamline tracking technique algorithm), Medinria (modified TEND algorithm), Brainvoyager (modified TEND algorithm), DTI Studio modified FACT algorithm, and the BrainLab DTI module based on the modified Runge-Kutta algorithm. Three examiners (a neuroradiologist, a magnetic resonance imaging physicist, and a neurosurgeon) served as examiners. They were double-blinded with respect to the test subject and the fiber tracking software used in the presented images. Each examiner evaluated 301 images. The examiners were instructed to evaluate screenshots from the different programs based on two main criteria: (i) anatomic accuracy of the course of the displayed fibers and (ii) number of fibers displayed outside the anatomic boundaries.The mean overall grade for anatomic accuracy was 2.2 (range, 1.1-3.6) with a standard deviation (SD) of 0.9. The mean overall grade for incorrectly displayed fibers was 2.5 (range, 1.6-3.5) with a SD of 0.6. The mean grade of the overall program ranking was 2.3 with a SD of 0.6. The overall mean grade of the program ranked number one (NeuroQLab) was 1.7 (range, 1.5-2.8). The mean overall grade of the program ranked last (BrainLab iPlan Cranial 2.6 DTI Module) was 3.3 (range, 1.7-4). The difference between the mean grades of these two programs was statistically highly significant (P0.0001). There was no statistically significant difference between the programs ranked 1-3: NeuroQLab, Sörensen DTI Task Card, and Siemens DTI module.The results of this study show that there is a statistically significant difference in the anatomic accuracy of the tested DTI fiber tracking programs. Although incorrectly displayed fibers could lead to wrong conclusions in the neurosciences field, which relies heavily on this noninvasive imaging technique, incorrectly displayed fibers in neurosurgery could lead to surgical decisions potentially harmful for the patient if used without intraoperative cortical stimulation. DTI fiber tracking presents a valuable noninvasive preoperative imaging tool, which requires further validation after important standardization of the acquisition and processing techniques currently available.

The therapeutic strategy for breast cancer is changing, especially for early tumor stages with good prognosis. One potential minimally invasive therapy modality consists in the accumulation of a well-tolerated magnetic material (iron oxides, particularly magnetite) in the target tissue. By applying an alternating magnetic field, energy is selectively absorbed and induces harmful heating of the tumor. The present review deals with the essential conditions and parameters as studied in vitro and in vivo in animal experiments. Extrapolations to the clinical situation are discussed, in particular, the heating potential of the magnetic material, the selection of the magnetic field parameters, the occurrence of eddy currents, the generation of localized heating spots and the expected temperature rises and their effects on the tumor area.