The effect of selective decontamination on the intestinal microbiota as measured with IS-pro: a taxonomic classification tool applicable for direct evaluation of intestinal microbiota in clinical routine.

Selective decontamination of the digestive tract (SDD) is aimed at elimination of potential pathogenic microorganisms. In this study, the effect of SDD on gut microbiota was evaluated in a large homogenous group of elective colorectal cancer surgery patients. Rectal swabs were taken from 118 patients undergoing colorectal surgery. These patients were randomly assigned to receive perioperative SDD or to the control group (no SDD). Rectal swabs were taken prior to surgery, 3 days after commencing administration of SDD. Gut microbial profiles were obtained with the IS-pro technique, a standardized microbiota profiling assay applicable in clinical routine. Differences in abundance for different taxonomical groups and diversity between the groups were assessed. Unsupervised and supervised classification techniques were used to assess microbial signatures, differentiating between the SDD group and the control group. Patients in the SDD group had different gut microbial signatures than in the control group, also in phyla that are not a target for SDD. Escherichia coli, Sutterella spp., Faecalibacterium prausnitzii, and Streptococcus spp. were the species that differed the most between the two groups. The SDD group showed clustering into two subgroups. In one subgroup, a decrease in Proteobacteria was observed, whereas the other subgroup showed a shift in Proteobacteria species. This study shows that SDD not only decreases colonization of the gastrointestinal tract with potential pathogenic Gram-negative microorganisms, but also reduces the abundance of normal colonizers of our gastrointestinal system and leads to a shift in total microbiota composition.

The profiling of microbiota in vaginal swab samples using 16S rRNA gene sequencing and IS-pro analysis.

BACKGROUND: 16S rRNA gene sequencing is currently the most common way of determining the composition of microbiota. This technique has enabled many new discoveries to be made regarding the relevance of microbiota to the health and disease of the host. However, compared to other diagnostic techniques, 16S rRNA gene sequencing is fairly costly and labor intensive, leaving room for other techniques to improve on these aspects. RESULTS: The current study aimed to compare the output of 16S rRNA gene sequencing to the output of the quick IS-pro analysis, using vaginal swab samples from 297 women of reproductive age. 16S rRNA gene sequencing and IS-pro analyses yielded very similar vaginal microbiome profiles, with a median Pearson's R2 of 0.97, indicating a high level of similarity between both techniques. CONCLUSIONS: We conclude that the results of 16S rRNA gene sequencing and IS-pro are highly comparable and that both can be used to accurately determine the vaginal microbiota composition, with the IS-pro analysis having the benefit of rapidity.

The influence of age, gender and race/ethnicity on the composition of the human axillary microbiome.

OBJECTIVE: The microbial community plays an important role in the generation of human axillary odour by transforming odourless natural secretions into volatile odorous molecules. A limited number of traditional culturing methods and molecular based research have been performed to characterize the human axillary microbiome in small collection sample sizes. Moreover, only a few have considered the interpersonal variations across age, gender or race/ethnicity, and none have included all three variables within one single study. The aim of this study was to characterize the axillary microbiome of healthy subjects across different age groups, genders and races/ethnicities in a large sample size. METHODS: The underarm skin swab samples were collected from 169 healthy subjects. The axillary microbiome was analysed by IS-pro, a clinically validated high-throughput DNA fingerprinting technique. RESULTS: The results indicate that the senior subjects (55+) tend to have a higher number of total bacterial than younger adults (of a defined age). The diversity of odour causing bacteria, e.g. corynebacteria, increases with age. Among the three races/ethnicities studied, East Asians have a unique microbial composition compared to Caucasians and Hispanics, which may contribute to the different odour profiles observed among the races/ethnicities studied. CONCLUSION: Human axillary microbiome varies by age, gender and race/ethnicity. This study has provided an unprecedented fundamental knowledge about the axillary microbiota as a function of age, gender and race/ethnicity.

OBJECTIF: La population microbienne joue un rôle important dans la génération de l'odeur axillaire par la transformation de sécrétions naturelles inodores en molécules odorantes et volatiles. Un nombre limité d'études par culture traditionnelle et de recherches moléculaires ont été réalisées pour caractériser le microbiome axillaire humain dans des échantillons de prélèvements de petite taille. En outre, seules quelques-unes de ces études ont tenu compte des variations interpersonnelles à travers l'âge, le sexe ou la race/l'origine ethnique, et aucune n'a inclus les trois variables dans une seule et même recherche. Le but de cette étude est de caractériser le microbiome de sujets sains est de réunir différents groupes d'âge, sexes et races/origines ethniques dans un échantillon important. MÉTHODES: Les échantillons de frottis cutanés de l'aisselle ont été recueillis sur 169 sujets sains. Le microbiome axillaire a été analysé par ISpro, une technique cliniquement validée d'empreinte ADN à haut débit. RÉSULTATS: Les résultats indiquent que les sujets séniors (55 ans et plus) ont tendance à présenter un plus grand nombre de bactéries que les adultes plus jeunes (d'un âge défini). La diversité des bactéries odorantes, par exemple, de type corynebacterium, augmente avec l'âge. Parmi les trois races/origines ethniques étudiées, les populations asiatiques présentent une composition microbienne unique par rapport aux populations caucasiennes et hispaniques, ce qui pourrait contribuer aux différents profils d'odeur observés dans les races/origines ethniques prises en compte. CONCLUSION: Le microbiome axillaire varie selon l'âge, le sexe et la race/l'origine ethnique. Cette étude fournit une connaissance fondamentale sans précédent sur la flore axillaire en fonction de l'âge, du sexe et de la race/l'origine ethnique.

The vaginal microbiome as a predictor for outcome of in vitro fertilization with or without intracytoplasmic sperm injection: a prospective study.

STUDY QUESTION: Is the presence or absence of certain vaginal bacteria associated with failure or success to become pregnant after an in vitro fertilization (IVF) or IVF with intracytoplasmic sperm injection (IVF-ICSI) treatment? SUMMARY ANSWER: Microbiome profiling with the use of interspace profiling (IS-pro) technique enables stratification of the chance of becoming pregnant prior to the start of an IVF or IVF-ICSI treatment. WHAT IS KNOWN ALREADY: Live-birth rates for an IVF or IVF-ICSI treatment vary between 25 and 35% per cycle and it is difficult to predict who will or will not get pregnant after embryo transfer (ET). Recently, it was suggested that the composition of the vaginal microbiota prior to treatment might predict pregnancy outcome. Analysis of the vaginal microbiome prior to treatment might, therefore, offer an opportunity to improve the success rate of IVF or IVF-ICSI. STUDY DESIGN, SIZE, DURATION: In a prospective cohort study, 303 women (age, 20-42 years) undergoing IVF or IVF-ICSI treatment in the Netherlands were included between June 2015 and March 2016. PARTICIPANTS/MATERIALS, SETTING, METHODS: Study subjects provided a vaginal sample before the start of the IVF or IVF-ICSI procedure. The vaginal microbiota composition was determined using the IS-pro technique. IS-pro is a eubacterial technique based on the detection and categorization of the length of the 16S-23S rRNA gene interspace region. Microbiome profiles were assigned to community state types based on the dominant bacterial species. The predictive accuracy of the microbiome profiles for IVF and IVF-ICSI outcome of fresh ET was evaluated by a combined prediction model based on a small number of bacterial species. From this cohort, a model was built to predict outcome of fertility treatment. This model was externally validated in a cohort of 50 women who were undergoing IVF or IVF-ICSI treatment between March 2018 and May 2018 in the Dutch division of the MVZ VivaNeo Kinderwunschzentrum Düsseldorf, Germany. MAIN RESULTS AND THE ROLE OF CHANCE: In total, the vaginal microbiota of 192 women who underwent a fresh ET could be analysed. Women with a low percentage of Lactobacillus in their vaginal sample were less likely to have a successful embryo implantation. The prediction model identified a subgroup of women (17.7%, n = 34) who had a low chance to become pregnant following fresh ET. This failure was correctly predicted in 32 out of 34 women based on the vaginal microbiota composition, resulting in a predictive accuracy of 94% (sensitivity, 26%; specificity, 97%). Additionally, the degree of dominance of Lactobacillus crispatus was an important factor in predicting pregnancy. Women who had a favourable profile as well as <60% L. crispatus had a high chance of pregnancy: more than half of these women (50 out of 95) became pregnant. In the external validation cohort, none of the women who had a negative prediction (low chance of pregnancy) became pregnant. LIMITATIONS, REASONS FOR CAUTION: Because our study uses a well-defined study population, the results will be limited to the IVF or IVF-ICSI population. Whether these results can be extrapolated to the general population trying to achieve pregnancy without ART cannot be determined from these data. WIDER IMPLICATIONS OF THE FINDINGS: Our results indicate that vaginal microbiome profiling using the IS-pro technique enables stratification of the chance of becoming pregnant prior to the start of an IVF or IVF-ICSI treatment. Knowledge of their vaginal microbiota may enable couples to make a more balanced decision regarding timing and continuation of their IVF or IVF-ICSI treatment cycles. STUDY FUNDING/COMPETING INTEREST(S): This study was financed by NGI Pre-Seed 2014-2016, RedMedTech Discovery Fund 2014-2017, STW Valorisation grant 1 2014-2015, STW Take-off early phase trajectory 2015-2016 and Eurostars VALBIOME grant (reference number: 8884). The employer of W.J.S.S.C. has in collaboration with ARTPred acquired a MIND subsidy to cover part of the costs of this collaboration project. The following grants are received but not used to finance this study: grants from Innovatie Prestatie Contract, MIT Haalbaarheid, other from Dutch R&D tax credit WBSO, RedMedTech Discovery Fund, (J.D.d.J.). Grants from Ferring (J.S.E.L., K.F., C.B.L. and J.M.J.S.S.), Merck Serono (K.F. and C.B.L.), Dutch Heart Foundation (J.S.E.L.), Metagenics Inc. (J.S.E.L.), GoodLife (K.F.), Guerbet (C.B.L.). R.K. is employed by ARTPred B.V. during her PhD at Erasmus Medical Centre (MC). S.A.M. has a 100% University appointment. I.S.P.H.M.S., S.A.M. and A.E.B. are co-owners of IS-Diagnostics Ltd. J.D.d.J. is co-owner of ARTPred B.V., from which he reports personal fees. P.H.M.S. reports non-financial support from ARTPred B.V. P.H.M.S., J.D.d.J. and A.E.B. have obtained patents `Microbial population analysis' (9506109) and `Microbial population analysis' (20170159108), both licenced to ARTPred B.V. J.D.d.J. and A.E.B. report patent applications `Method and kit for predicting the outcome of an assisted reproductive technology procedure' (392EPP0) and patent `Method and kit for altering the outcome of an assisted reproductive technology procedure' by ARTPred. W.J.S.S.C. received personal consultancy and educational fees from Goodlife Fertility B.V. J.S.E.L. reports personal consultancy fees from ARTPred B.V., Titus Health B.V., Danone, Euroscreen and Roche during the conduct of the study. J.S.E.L. and N.G.M.B. are co-applicants on an Erasmus MC patent (New method and kit for prediction success of in vitro fertilization) licenced to ARTPred B.V. F.J.M.B. reports personal fees from Advisory Board Ferring, Advisory Board Merck Serono, Advisory Board Gedeon Richter and personal fees from Educational activities for Ferring, outside the submitted work. K.F. reports personal fees from Ferring (commercial sponsor) and personal fees from GoodLife (commercial sponsor). C.B.L. received speakers' fee from Ferring. J.M.J.S.S. reports personal fees and other from Merck Serono and personal fees from Ferring, unrelated to the submitted paper. The other authors declare that they have no competing interests. TRIAL REGISTRATION NUMBER: ISRCTN83157250. Registered 17 August 2018. Retrospectively registered.

Randomized clinical trial of selective decontamination of the digestive tract in elective colorectal cancer surgery (SELECT trial).

BACKGROUND: Infectious complications and anastomotic leakage affect approximately 30 per cent of patients after colorectal cancer surgery. The aim of this multicentre randomized trial was to investigate whether selective decontamination of the digestive tract (SDD) reduces these complications of elective colorectal cancer surgery. METHODS: The effectiveness of SDD was evaluated in a multicentre, open-label RCT in six centres in the Netherlands. Patients with colorectal cancer scheduled for elective curative surgery with a primary anastomosis were eligible. Oral colistin, tobramycin and amphotericin B were administered to patients in the SDD group to decontaminate the digestive tract. Both treatment and control group received intravenous cefazolin and metronidazole for perioperative prophylaxis. Mechanical bowel preparation was given for left-sided colectomies, sigmoid and anterior resections. Anastomotic leakage was the primary outcome; infectious complications and mortality were secondary outcomes. RESULTS: The outcomes for 228 patients randomized to the SDD group and 227 randomized to the control group were analysed. The trial was stopped after interim analysis demonstrated that superiority was no longer attainable. Effective SDD was confirmed by interspace DNA profiling analysis of rectal swabs. Anastomotic leakage was observed in 14 patients (6·1 per cent) in the SDD group and in 22 patients (9·7 per cent) in the control group (odds ratio (OR) 0·61, 95 per cent c.i. 0·30 to 1·22). Fewer patients in the SDD group had one or more infectious complications than patients in the control group (14·9 versus 26·9 per cent respectively; OR 0·48, 0·30 to 0·76). Multivariable analysis indicated that SDD reduced the rate of infectious complications (OR 0·47, 0·29 to 0·76). CONCLUSION: SDD reduces infectious complications after colorectal cancer resection but did not significantly reduce anastomotic leakage in this trial. Registration number: NCT01740947 ( https://www.clinicaltrials.gov).

Variability of core microbiota in newly diagnosed treatment-naïve paediatric inflammatory bowel disease patients.

BACKGROUND & AIMS: Intestinal microbiota is considered to play a crucial role in the aetiology of inflammatory bowel disease (IBD). We aimed to describe faecal microbiota composition and dynamics in a large cohort of children with de novo (naïve) IBD, in comparison to healthy paediatric controls (HC). METHODS: In this prospective study, performed at two tertiary centres, faecal samples from newly diagnosed, treatment-naïve paediatric IBD patients were collected prior to bowel cleansing for colonoscopy (t0) and 1, 3 and 6 weeks and 3 months after initiation of therapy. The microbial profiles of Crohn's disease (CD) and Ulcerative colitis (UC) patients were compared with HC and linked to therapeutic response. Microbiota composition was analysed by IS-pro technology. RESULTS: Microbial profiles of 104 new IBD-patients (63 CD, 41 UC, median age 14.0 years) were compared to 61 HC (median 7.8 years). IBD was mainly characterised by decreased abundance of Alistipes finegoldii and Alistipes putredinis, which characterize a healthy state microbial core. The classifier including these core species as predictors achieved an AUC of the ROC curve of .87. Core bacteria tended to regain abundance during treatment, but did not reach healthy levels. CONCLUSION: Faecal microbiota profiles of children with de novo CD and UC can be discriminated from HC with high accuracy, mainly driven by a decreased abundance of species shaping the microbial core in the healthy state. Paediatric IBD can therefore be characterized by decreased abundance of certain bacterial species reflecting the healthy state rather than by the introduction of pathogens.

Interpretation of microbiota-based diagnostics by explaining individual classifier decisions.

BACKGROUND: The human microbiota is associated with various disease states and holds a great promise for non-invasive diagnostics. However, microbiota data is challenging for traditional diagnostic approaches: It is high-dimensional, sparse and comprises of high inter-personal variation. State of the art machine learning tools are therefore needed to achieve this goal. While these tools have the ability to learn from complex data and interpret patterns therein that cannot be identified by humans, they often operate as black boxes, offering no insight into their decision-making process. In most cases, it is difficult to represent the learning of a classifier in a comprehensible way, which makes them prone to be mistrusted, or even misused, in a clinical environment. In this study, we aim to elucidate microbiota-based classifier decisions in a biologically meaningful context to allow their interpretation. RESULTS: We applied a method for explanation of classifier decisions on two microbiota datasets of increasing complexity: gut versus skin microbiota samples, and inflammatory bowel disease versus healthy gut microbiota samples. The algorithm simulates bacterial species as being unknown to a pre-trained classifier, and measures its effect on the outcome. Consequently, each patient is assigned a unique quantitative estimation of which species in their microbiota defined the classification of their sample. The algorithm was able to explain the classifier decisions well, demonstrated by our validation method, and the explanations were biologically consistent with recent microbiota findings. CONCLUSIONS: Application of a method for explaining individual classifier decisions for complex microbiota analysis proved feasible and opens perspectives on personalized therapy. Providing an explanation to support a microbiota-based diagnosis could guide decisions of clinical microbiologists, and has the potential to increase their confidence in the outcome of such decision support systems. This may facilitate the development of new diagnostic applications.

[Carbapenem-resistant bacteria transferred to the Netherlands: patients colonised during hospitalisation in Romania]. / Carbapenemresistente bacteriën overgebracht naar Nederland.

Three patients were recently transferred to two Intensive Care Units (ICUs) in the Netherlands from two different ICUs in Bucharest, Romania. The patients appeared to be colonised with several, partly identical, carbapenemase-producing bacteria (CPB) after a short hospitalisation in Romania. In this article, we show that it is important to be aware of the possibility that patients are colonised with CPB after a short hospitalisation abroad. This has consequences for infection prevention measurements, but probably also for the discontinuation of selective bowel decontamination in the ICU.

Robust Microbiota-Based Diagnostics for Inflammatory Bowel Disease.

Strong evidence suggests that the gut microbiota is altered in inflammatory bowel disease (IBD), indicating its potential role in noninvasive diagnostics. However, no clinical applications are currently used for routine patient care. The main obstacle to implementing a gut microbiota test for IBD is the lack of standardization, which leads to high interlaboratory variation. We studied the between-hospital and between-platform batch effects and their effects on predictive accuracy for IBD. Fecal samples from 91 pediatric IBD patients and 58 healthy children were collected. IS-pro, a standardized technique designed for routine microbiota profiling in clinical settings, was used for microbiota composition characterization. Additionally, a large synthetic data set was used to simulate various perturbations and study their effects on the accuracy of different classifiers. Perturbations were validated in two replicate data sets, one processed in another laboratory and the other with a different analysis platform. The type of perturbation determined its effect on predictive accuracy. Real-life perturbations induced by between-platform variation were significantly greater than those caused by between-laboratory variation. Random forest was found to be robust to both simulated and observed perturbations, even when these perturbations had a dramatic effect on other classifiers. It achieved high accuracy both when cross-validated within the same data set and when using data sets analyzed in different laboratories. Robust clinical predictions based on the gut microbiota can be performed even when samples are processed in different hospitals. This study contributes to the effort to develop a universal IBD test that would enable simple diagnostics and disease activity monitoring.

Long Term Development of Gut Microbiota Composition in Atopic Children: Impact of Probiotics.

INTRODUCTION: Imbalance of the human gut microbiota in early childhood is suggested as a risk factor for immune-mediated disorders such as allergies. With the objective to modulate the intestinal microbiota, probiotic supplementation during infancy has been used for prevention of allergic diseases in infants, with variable success. However, not much is known about the long-term consequences of neonatal use of probiotics on the microbiota composition. The aim of this study was to assess the composition and microbial diversity in stool samples of infants at high-risk for atopic disease, from birth onwards to six years of age, who were treated with probiotics or placebo during the first year of life. METHODS: In a double-blind, randomized, placebo-controlled trial, a probiotic mixture consisting of B. bifidum W23, B. lactis W52 and Lc. Lactis W58 (Ecologic® Panda) was administered to pregnant women during the last 6 weeks of pregnancy and to their offspring during the first year of life. During follow-up, faecal samples were collected from 99 children over a 6-year period with the following time points: first week, second week, first month, three months, first year, eighteen months, two years and six years. Bacterial profiling was performed by IS-pro. Differences in bacterial abundance and diversity were assessed by conventional statistics. RESULTS: The presence of the supplemented probiotic strains in faecal samples was confirmed, and the probiotic strains had a higher abundance and prevalence in the probiotic group during supplementation. Only minor and short term differences in composition of microbiota were found between the probiotic and placebo group and between children with or without atopy. The diversity of Bacteroidetes was significantly higher after two weeks in the placebo group, and at the age of two years atopic children had a significantly higher Proteobacteria diversity (p < 0.05). Gut microbiota development continued between two and six years, whereby microbiota composition at phylum level evolved more and more towards an adult-like configuration. CONCLUSION: Perinatal supplementation with Ecologic® Panda, to children at high-risk for atopic disease, had minor effects on gut microbiota composition during the supplementation period. No long lasting differences were identified. Regardless of intervention or atopic disease status, children had a shared microbiota development over time determined by age that continued to develop between two and six years.

Fecal microbiome analysis as a diagnostic test for diverticulitis.

Disease-specific variations in intestinal microbiome composition have been found for a number of intestinal disorders, but little is known about diverticulitis. The purpose of this study was to compare the fecal microbiota of diverticulitis patients with control subjects from a general gastroenterological practice and to investigate the feasibility of predictive diagnostics based on complex microbiota data. Thirty-one patients with computed tomography (CT)-proven left-sided uncomplicated acute diverticulitis were included and compared with 25 control subjects evaluated for a range of gastrointestinal indications. A high-throughput polymerase chain reaction (PCR)-based profiling technique (IS-pro) was performed on DNA isolates from baseline fecal samples. Differences in bacterial phylum abundance and diversity (Shannon index) of the resulting profiles were assessed by conventional statistics. Dissimilarity in microbiome composition was analyzed with principal coordinate analysis (PCoA) based on cosine distance measures. To develop a prediction model for the diagnosis of diverticulitis, we used cross-validated partial least squares discriminant analysis (PLS-DA). Firmicutes/Bacteroidetes ratios and Proteobacteria load were comparable among patients and controls (p = 0.20). The Shannon index indicated a higher diversity in diverticulitis for Proteobacteria (p < 0.00002) and all phyla combined (p = 0.002). PCoA based on Proteobacteria profiles resulted in visually separate clusters of patients and controls. The diagnostic accuracy of the cross-validated PLS-DA regression model was 84 %. The most discriminative species derived largely from the family Enterobacteriaceae. Diverticulitis patients have a higher diversity of fecal microbiota than controls from a mixed population, with the phylum Proteobacteria defining the difference. The analysis of intestinal microbiota offers a novel way to diagnose diverticulitis.

IS-pro: high-throughput molecular fingerprinting of the intestinal microbiota.

The human intestinal microbiota is known to play an important role in human health and disease, and with the advent of novel molecular techniques, disease-specific variations in its composition have been found. However, analysis of the intestinal microbiota has not yet been applicable in large-scale clinical research or routine diagnostics because of the complex and expensive nature of the techniques needed. Here, we describe a new PCR-based profiling technique for high-throughput analysis of the human intestinal microbiota, which we have termed IS-pro. This technique combines bacterial species differentiation by the length of the 16S-23S rDNA interspace region with instant taxonomic classification by phylum-specific fluorescent labeling of PCR primers. We validated IS-pro in silico, in vitro, and in vivo, on human colonic biopsies and feces, and introduced a standardized protocol for data analysis. IS-pro is easy to implement in general clinical microbiological laboratories with access to capillary gel electrophoresis, and the high-throughput nature of the test makes analysis of large numbers of samples feasible. This combination renders IS-pro ideally suited for use in clinical research and routine diagnostics.

Vibrio cholerae non-O1 non-O139 infection in an immunocompromised patient returning from Spain, July 2009.

We describe a severe gastroenteritis with non-O1, non-O139 Vibrio cholerae in an immunocompromised patient returning from a holiday in Spain in July 2009. Predisposing factors and possible cholera enterotoxin production could explain the unusually grave symptomatology. Patient recovered after doxycyclin treatment.

The Dienes phenomenon: competition and territoriality in Swarming Proteus mirabilis.

When two different strains of swarming Proteus mirabilis encounter one another on an agar plate, swarming ceases and a visible line of demarcation forms. This boundary region is known as the Dienes line and is associated with the formation of rounded cells. While the Dienes line appears to be the product of distinction between self and nonself, many aspects of its formation and function are unclear. In this work, we studied Dienes line formation using clinical isolates labeled with fluorescent proteins. We show that round cells in the Dienes line originate exclusively from one of the swarms involved and that these round cells have decreased viability. In this sense one of the swarms involved is dominant over the other. Close cell proximity is required for Dienes line formation, and when strains initiate swarming in close proximity, the dominant Dienes type has a significant competitive advantage. When one strain is killed by UV irradiation, a Dienes line does not form. Killing of the dominant strain limits the induction of round cells. We suggest that both strains are actively involved in boundary formation and that round cell formation is the result of a short-range killing mechanism that mediates a competitive advantage, an advantage highly specific to the swarming state. Dienes line formation has implications for the physiology of swarming and social recognition in bacteria.