Tarmmykobiota, serum anti-Saccharomyces cerevisiae antikroppar och calprotectin koncentrationer hos patienter med inflammatorisk tarmsjukdom som får infliximab medicinering

Inflammatory bowel disease (IBD) is a globally increasing chronic disease, for which the pathogenesis still is unclear. The most common subtypes of IBD are Crohn’s disease (CD) and ulcerative colitis (UC). It is widely known that, in addition to the genetics, an altered immune response against the gut microbiome plays an important role in the development of the disease. For the IBD patients, to whom conventional medication is not sufficient, the TNF-α blocker infliximab, is given. However, about one third of the patients receiving infliximab treatment, do not respond to the drug, or lose response over time. Since there to this day are no reliable diagnostic markers available, the finding of such is of great importance. The goal of this study was to investigate possible markers for drug response in the gut mycobiota composition of IBD patients. The gut mycobiota composition of 72 IBD patients receiving infliximab was studied by MiSeq sequencing of fungal DNA from fecal samples, collected during one year. The sequencing data was analyzed using the mare package in R. In addition, anti-Saccharomyces cerevisiae antibody (ASCA) concentrations were measured from baseline serum samples by ELISA. Finally, calprotectin concentrations were measured from baseline and twelve weeks post infliximab serum samples by ELISA to study whether serum samples could be used instead of fecal samples for measuring calprotectin values. Results show an increase of the Candida and Spiromyces genera in the gut mycobiota of non-responding patients at baseline. At all timepoints, the Spiromyces genus was observed at a higher abundance, compared to the group of patients responding well or partially to the medication. Interestingly, the increase of Candida was seen only in Crohn’s disease patients, when looking at the composition at all timepoints. ASCA values did not differ between the response groups. The serum calprotectin values did not correlate with fecal calprotectin, and serum calprotectin can thus not be used as a marker of gut inflammation. In conclusion, the gut mycobiota can offer predictive markers for drug response prediction to infliximab in IBD patients, which can with further studies offer a clinical diagnostic tool for prediction of drug response.Inflammatorisk tarmsjukdom (IBD) är en kronisk inflammation i tarmarna som är allt mer förekommande i hela världen. De vanligaste typerna av IBD är Crohn’s sjukdom och ulcerös colit. En tydlig orsak till sjukdomen har inte blivit hittad, men man vet att IBD delvis orsakas av en ovanlig immunreaktion mot tarmens mikrobiom hos en genetiskt belagd person. Åt patienter, för vilka den vanliga inflammationshämmande medicineringen inte är tillräcklig, ges den biologiska medicinen infliximab. Upp till en tredjedel av patienterna som får infliximab får ingen läkemedelsrespons mot medicinen, antingen på grund av brist på respons från början, eller avfall av respons efter medicinering. Det är mycket viktigt att forska i prediktiva markörer, eftersom det i nuläget inte finns diagnostiska metoder för undersökning av respons mot infliximab. Målet med denna studie var att undersöka möjliga markörer för läkemedelsrespons i tarmmykobiotan hos IBD patienter. Tarmmykobiotans komposition undersöktes hos 72 IBD patienter, som får denna medicinering, genom MiSeq sekvensering av svamp DNA i avföringsprov tagna under ett år. Koncentrationen av anti-Saccharomyces cerevisiae antikroppar (ASCA) har mätts från serumprov tagna före medicineringen började genom ELISA. Slutligen mättes även koncentrationen av calprotectin från serumprov tagna före medicineringen började samt tolv veckor efter medicinering för att undersöka om dessa kunde användas istället för calprotectin värden mätta från avföring. I studien observeras en ökning av släkten Candida och Spiromyces i tarmmykobiotan hos gruppen av patienter för vilka infliximab inte fungerade i prover tagna innan påbörjad medicinering. En ökning av släktet Spiromyces var observerad även i prover tagna under medicinering, medan ökningen av Candida bara var observerad hos patienter som lider av Crohn’s sjukdom i prover tagna under medicineringen. Det observerades ingen korrelation mellan calprotectinvärden mätta från serum och avföring, vilket betyder att serum calprotectinvärden inte fungerar som inflammationsmarkör för tarmen. Slutligen observerades inga skildaktigheter i ASCA-värden i de olika grupperna. Sammanfattningsvis visar studien att tarmmykobiotan erbjuder möjliga prediktiva markörer för läkemedelsrespons mot infliximab hos IBD patienter som kan användas i diagnostiken

The Effect of Antibiotics on the Infant Gut Fungal Microbiota

Antibiotics are commonly used drugs in infants, causing disruptions in the developing gut microbiota with possible detrimental long-term effects such as chronic inflammatory diseases. The focus has been on bacteria, but research shows that fungi might have an important role as well. There are only a few studies on the infant gut fungal microbiota, the mycobiota, in relation to antibiotic treatment. Here, the aim was to investigate the impact of antibiotics on the infant gut mycobiota, and the interkingdom associations between bacteria and fungi. We had 37 antibiotic-naïve patients suffering from respiratory syncytial virus, of which 21 received one to four courses of antibiotics due to complications, and 16 remained antibiotic-naïve throughout the study. Fecal samples were collected before, during and after antibiotic treatment with a follow-up period of up to 9.5 months. The gut mycobiota was studied by Illumina MiSeq sequencing of the ITS1 region. We found that antibiotic use affected the gut mycobiota, most prominently seen as a higher relative abundance of Candida (p < 0.001), and a higher fungal diversity (p = 0.005–0.04) and richness (p = 0.03) in the antibiotic-treated infants compared to the antibiotic-naïve ones at multiple timepoints. This indicates that the gut mycobiota could contribute to the long-term consequences of antibiotic treatments

The gut fungal and bacterial microbiota in pediatric patients with inflammatory bowel disease introduced to treatment with anti-tumor necrosis factor-α

Publisher Copyright: © 2022, The Author(s).Pediatric inflammatory bowel disease (PIBD) is a globally increasing chronic inflammatory disease associated with an imbalanced intestinal microbiota and treated with several treatment options, including anti-tumor necrosis factor alpha (TNF-α), such as infliximab (IFX). Up to half of the patients do not respond to the drug and there are no methods for response prediction. Our aim was to predict IFX response from the gut microbiota composition since this is largely unexplored in PIBD. The gut microbiota of 30 PIBD patients receiving IFX was studied by MiSeq sequencing targeting 16S and ITS region from fecal samples collected before IFX and two and six weeks after the start of treatment. The response to IFX induction was determined by fecal calprotectin value < 100 µg/g at week six. The bacterial microbiota differed significantly between response groups, with higher relative abundance of butyrate-producing bacteria in responders compared to non-responders at baseline, validated by high predictive power (area under curve = 0.892) for baseline Ruminococcus and calprotectin. Additionally, non-responders had higher abundance of Candida, while responders had higher abundance of Saccharomyces at the end of the study. The gut microbiota composition in PIBD patients could predict response to IFX treatment in the future.Peer reviewe

Bacterial and Fungal Profiles as Markers of Infliximab Drug Response in Inflammatory Bowel Disease

Tulehdukselliset suolistosairaudet (IBD) Crohnin tauti (CD) ja haavainen paksusuolentulehdus (UC) ovat maailmanlaajuisesti lisääntyviä sairauksia, joihin liittyy muuntunut suoliston mikrobisto. Infliksimabi (IFX) on TNF-alfan estäjä, jota käytetään IBD:n hoidossa, vaikkakin kolmas osa potilaista voi jäädä ilman hoitovastetta tai menettää sen. Luotettavia testejä hoitovasteen ennustamiseksi ei ole vielä saatavilla. Tutkimuksemme tavoitteena oli selvittää potilaiden ulosteen bakteerit, sienet ja hiivat infliksimabihoidon aikana sekä etsiä hoitovastetta ennustavia mittareita IBD potilailta. Tutkimuksessamme 72 IBD-potilasta (25 CD ja 47 UC) aloitti infliksimabihoidon ja heitä seurattiin vuoden ajan hoidon aloituksesta tai hoidon keskeytykseen saakka. Potilaiden ulostenäytteistä määritettiin sekvensointia hyödyntäen erikseen suoliston bakteerit (16S rRNA geenistä) sekä hiivat ja sienet (ITS1 geenin alueesta). Mikrobiston profiilit määritettiin ennen hoidon aloitusta, 2, 6, 12 viikkoa sekä 1 vuosi hoidon aloituksen jälkeen. IFX hoidon vaste määritettiin kolonoskopialla ja kliinisesti 12 viikon hoidon jälkeen. Tutkimuksessa havaittiin, että potilaiden ulosteen bakteerit sekä sienet ja hiivat erosivat merkittävästi toisistaan infliksimabihoitovasteen mukaan jo ennen hoidon aloitusta. Potilailla, jotka eivät saaneet vastetta hoidosta, oli vähemmän lyhytketjuisia rasvahappoja tuottavia bakteereja erityisesti Clostridia-luokasta sekä enemmän tulehdusta aiheuttavia bakteereja ja sieniä mm. Candida-suvusta. Tämä tulos testattiin bakteerien osalta myös ennustetestillä (ROC), joka erotti toisistaan ne potilaat, jotka saivat hyvän vasteen ja ne, jotka eivät saaneet vastetta (area under the curve > 0.8). Tulosten perusteella ulosteen bakteerit ja hiivat voisivat sopia ennustaviksi mittareiksi IBD-potilaiden infliksimabihoidon vasteen ennustamiseen.Peer reviewe

The Effect of Antibiotics on the Infant Gut Fungal Microbiota

Antibiotics are commonly used drugs in infants, causing disruptions in the developing gut microbiota with possible detrimental long-term effects such as chronic inflammatory diseases. The focus has been on bacteria, but research shows that fungi might have an important role as well. There are only a few studies on the infant gut fungal microbiota, the mycobiota, in relation to antibiotic treatment. Here, the aim was to investigate the impact of antibiotics on the infant gut mycobiota, and the interkingdom associations between bacteria and fungi. We had 37 antibiotic-naïve patients suffering from respiratory syncytial virus, of which 21 received one to four courses of antibiotics due to complications, and 16 remained antibiotic-naïve throughout the study. Fecal samples were collected before, during and after antibiotic treatment with a follow-up period of up to 9.5 months. The gut mycobiota was studied by Illumina MiSeq sequencing of the ITS1 region. We found that antibiotic use affected the gut mycobiota, most prominently seen as a higher relative abundance of Candida (p < 0.001), and a higher fungal diversity (p = 0.005–0.04) and richness (p = 0.03) in the antibiotic-treated infants compared to the antibiotic-naïve ones at multiple timepoints. This indicates that the gut mycobiota could contribute to the long-term consequences of antibiotic treatments

The Effect of Antibiotics on the Infant Gut Fungal Microbiota

Antibiotics are commonly used drugs in infants, causing disruptions in the developing gut microbiota with possible detrimental long-term effects such as chronic inflammatory diseases. The focus has been on bacteria, but research shows that fungi might have an important role as well. There are only a few studies on the infant gut fungal microbiota, the mycobiota, in relation to antibiotic treatment. Here, the aim was to investigate the impact of antibiotics on the infant gut mycobiota, and the interkingdom associations between bacteria and fungi. We had 37 antibiotic-naive patients suffering from respiratory syncytial virus, of which 21 received one to four courses of antibiotics due to complications, and 16 remained antibiotic-naive throughout the study. Fecal samples were collected before, during and after antibiotic treatment with a follow-up period of up to 9.5 months. The gut mycobiota was studied by Illumina MiSeq sequencing of the ITS1 region. We found that antibiotic use affected the gut mycobiota, most prominently seen as a higher relative abundance of Candida (p < 0.001), and a higher fungal diversity (p = 0.005-0.04) and richness (p = 0.03) in the antibiotic-treated infants compared to the antibiotic-naive ones at multiple timepoints. This indicates that the gut mycobiota could contribute to the long-term consequences of antibiotic treatments

The Effect of Antibiotics on the Infant Gut Fungal Microbiota

Antibiotics are commonly used drugs in infants, causing disruptions in the developing gut microbiota with possible detrimental long-term effects such as chronic inflammatory diseases. The focus has been on bacteria, but research shows that fungi might have an important role as well. There are only a few studies on the infant gut fungal microbiota, the mycobiota, in relation to antibiotic treatment. Here, the aim was to investigate the impact of antibiotics on the infant gut mycobiota, and the interkingdom associations between bacteria and fungi. We had 37 antibiotic-na&iuml;ve patients suffering from respiratory syncytial virus, of which 21 received one to four courses of antibiotics due to complications, and 16 remained antibiotic-na&iuml;ve throughout the study. Fecal samples were collected before, during and after antibiotic treatment with a follow-up period of up to 9.5 months. The gut mycobiota was studied by Illumina MiSeq sequencing of the ITS1 region. We found that antibiotic use affected the gut mycobiota, most prominently seen as a higher relative abundance of Candida (p &lt; 0.001), and a higher fungal diversity (p = 0.005&ndash;0.04) and richness (p = 0.03) in the antibiotic-treated infants compared to the antibiotic-na&iuml;ve ones at multiple timepoints. This indicates that the gut mycobiota could contribute to the long-term consequences of antibiotic treatments

Biomarkers of the Gut Fungal and Bacterial Composition in Inflammatory Bowel Disease

Background: The gut is colonized by different microorganisms making up the gut microbiota. The bacteria are the most abundant and most studied member of the microbiota, but others, such as fungi might also be of importance. Using antibiotics in early life causes a disturbance of the developing gut microbiota, which might predispose to inflammatory bowel disease (IBD). IBD is a chronic inflammatory disease of the gut, divided into subtypes Crohn’s disease (CD) and ulcerative colitis (UC), and has a growing incidence and prevalence worldwide, particularly in Finland where currently over 1% of the population is diagnosed with IBD. The pathogenesis of IBD is still unknown, but genetics, environmental factors, and particularly the gut microbiota are known to be associated with the progression of the disease. There is no cure for IBD, but many medical treatment options including conventional therapy and biologics are available. Anti-tumor necrosis factor alpha (TNF-α) treatment is one of the most common biologics used to treat both CD and UC, and it is estimated that up to one-third of patients with IBD will eventually need it. It works by binding to both soluble and membrane-bound TNF-α, a pro-inflammatory cytokine, thereby blocking it from binding to its receptor. Infliximab (IFX) is an anti-TNF-α used to treat moderate to severe IBD successfully. However, up to half of the patients do not respond to IFX in the long term. Presently, there are no methods available to predict IFX response, which would be crucial, not only because of the high costs but also to avoid unnecessary pain from poor response and side effects, particularly in pediatric patients. Therefore, finding biomarkers that would improve disease characterization and personalization of health care would be of key importance. Aims: The present dissertation aimed to find markers to predict IFX treatment response from the gut fungal and bacterial microbiota composition, to study the changes in the gut bacterial composition during IFX treatment, and to study the pro-inflammatory characteristics upon antibiotic treatment on the antibiotic-naïve infant fungal gut microbiota. Methods: The differences in the gut fungal and bacterial microbiota composition between responders and non-responders to IFX were investigated in two separate prospective cohorts of adult and pediatric patients with IBD. The absolute abundances of the gut bacterial microbiota were also investigated during IFX treatment in adult patients with IBD. Finally, the effects of antibiotics were investigated in the antibiotic-naïve fungal gut microbiota by comparing antibiotic-treated and -naïve infants at multiple time points before, during, and after treatment. This was done by extracting DNA from fecal samples collected before, during, and after IFX treatment, and of infants before, during, and after antibiotic treatment. Additionally, DNA was extracted from biopsies collected from adult patients with IBD before, during, and after IFX treatment. The bacterial and fungal gut microbiota composition was characterized by MiSeq sequencing targeting the conserved 16S and internal transcribed spacer (ITS) regions. The absolute bacterial abundance in the fecal and mucosal microbiota in the adult IBD cohort was quantified by qPCR targeting 16S. Results: There was a significant difference in the fecal microbiota composition between response groups in both adult and pediatric patients with IBD predicting IFX response, further validated by high area under the curve (AUC) values (0.797–0.938), observed by responders having a higher relative abundance of butyrate-producing bacteria, particularly the class Clostridia. In adult patients with IBD, the mucosal healing in responders to IFX was characterized by an increased bacterial load in the mucosal and fecal bacterial microbiota, driven mostly by butyrate-producing bacteria. Finally, already one course of amoxicillin affected the gut fungal microbiota significantly, characterized by an increase in fungal diversity and richness and a higher relative abundance of Candida in the antibiotic-treated compared to the -naïve infants. Conclusions: First, the fungal microbiota might have an unpredicted role in the aberrant gut microbiota composition and therefore it should not be forgotten in microbiota research. Second, as antibiotics might predispose to disease by causing disruption in the gut microbiota, methods to restore the gut microbiota composition should be developed. Third, as the IFX response was predictable, and a shift in the gut bacterial microbiota was observed in the responders, but not in the non-responders to IFX, the possibility of restoring the gut microbiota should be included when evaluating treatment options in IBD.Bakgrund: Tarmen är koloniserad av olika mikroorganismer som tillsammans formar tarmmikrobiotan. Av dessa utgör bakterierna den största andelen och är samtidigt mest studerade. Även andra mikroorganismer, som t.ex. svampar, utgör ändå en betydande del av tarmmikrobiotans sammansättning. Störningar i tarmmikrobiotan som ett resultat av antibiotikabehandling i tidig barndom, under tarmmikrobiotans viktigaste utvecklingsfas, kan predisponera för inflammatorisk tarmsjukdom (IBD). IBD, med undergrupperna Crohn’s sjukdom (CD) och ulcerös kolit (UC) är en kronisk inflammatorisk sjukdom i tarmen som har en ökande incidens och prevalens globalt, och särskilt i Finland där över 1% av befolkningen diagnostiserats med IBD. Patogenesen för IBD är fortfarande okänd, men man vet att många gener, miljöfaktorer och särskilt tarmmikrobiotan är associerade med sjukdomens framskridande. Det finns inget botemedel för IBD, men många medicinska behandlingsalternativ, inklusive konventionell terapi och biologiska läkemedel finns tillgängliga. Anti-tumörnekrosfaktor alfa (TNF-α) är en av de biologiska läkemedel som används för att behandla både CD och UC, och det uppskattas att upp till en tredjedel av IBD-patienterna så småningom kommer att behöva anti-TNF-α. Detta fungerar genom att binda till både lösliga och membranbundna proinflammatoriska cytokinet TNF-α, och blockerar därmed bindningen till dess receptor. Infliximab (IFX) är en anti-TNF-α som används för att framgångsrikt behandla måttlig till svår IBD. Dock svarar upp till hälften av patienterna inte på IFX på lång sikt. Idag finns det inga tillgängliga metoder för att förutsäga responsen på IFX. Utvecklingen av dessa skulle vara essentiellt, inte bara på grund av att behandlingen är en ekonomisk belastning, utan framför allt för att undvika onödig smärta från dålig respons och biverkningar från behandlingen, särskilt hos barn. Därför vore det av stor betydelse att hitta biomarkörer som kunde hjälpa till vid karaktärisering av sjukdomen och individualisering av hälso- och sjukvården. Syften: Målen med denna forskning var att hitta prediktiva markörer för IFX-behandling i sammansättningen av tarmens svamp- och bakteriemikrobiota, att studera vad som händer med sammansättningen av tarmbakterier under IFX-behandling samt att studera de proinflammatoriska egenskaperna och effekten av antibiotika på den antibiotikanaiva svamp tarmmikrobiotan. Metoder: Skillnaderna i sammansättningen av svamp- och bakterie tarmmikrobiotan mellan IBD-patienter som hade god och dålig IFX respons undersöktes i två separata prospektiva kohorter bestående av vuxna och pediatriska IBD-patienter. Den absoluta sammansättningen av tarmens bakteriemikrobiota undersöktes också under IFX-behandling hos vuxna IBD-patienter. Slutligen undersöktes effekterna av antibiotika i den antibiotikanaiva svampiga tarmmikrobiotan genom att jämföra antibiotikabehandlade och -naiva spädbarn vid flera tidpunkter före, under och efter behandling. Detta gjordes genom att isolera DNA från avföringsprover som samlats in före, under och efter IFX-behandling och från spädbarn före, under och efter antibiotikabehandling. Dessutom isolerades DNA från biopsier som samlats in från vuxna IBD-patienter före, under och efter IFX-behandling. Tarmens bakterie- och svampmikrobiotasammansättning karakteriserades genom MiSeq-sekvensering genom att använda de bevarade 16S- och ITS-regionerna. Resultat: Det observerades en signifikant skillnad i tarmmikrobiotan mellan patienter med respons och icke-respons till IFX medicineringen hos både vuxna och pediatriska patienter med IBD. Responsen kunde även förutspås redan före behandlingen påbörjades, med högre andel av bakterier som producerar butyrat, speciellt de som tillhör klassen Clostridia, validerat med högt AUC värde. I vuxna patienter med IBD kunde slemhinnans tillfrisknande hos de patienter med respons identifieras med en växande bakteriemängd i både slemhinnans och avföringens bakteriesammansättning. Denna ökning drevs främst av bakterier som producerar butyrat. Slutligen observerades att redan en dos av amoxicillin påverkade svamp tarmmikrobiotan signifikant, karakteriserat av en ökning i diversitet och artrikedom, samt en högre relativ andel av Candida hos de antibiotikabehandlade spädbarnen i jämförelse med de antibiotika-naiva barnen. Slutsatser: Svamparna i tarmmikrobiotan bör beaktas i mikrobiotaforskning eftersom de kan ha en betydande roll i den rubbade tarmmikrobiotans sammansättning. Det har tidigare observerats att antibiotika kan predisponera för sjukdomar genom att orsaka störningar i tarmmikrobiotan. Därför borde metoder för att återupprätta mikrobiotans sammansättning utvecklas. Slutligen observerades det här att IFX responsen kunde förutsägas, samt att tarmmikrobiotan hos patienterna med bra IFX respons förändrades under behandlingen, vilket inte var fallet hos de som inte hade någon respons. På basis av detta borde man undersöka möjligheten att återupprätta och stöda förbättrandet av tarmmikrobiotan när man utvärderar möjliga behandlingar för IBD

The Effect of Antibiotics on the Infant Gut Fungal Microbiota

Antibiotics are commonly used drugs in infants, causing disruptions in the developing gut microbiota with possible detrimental long-term effects such as chronic inflammatory diseases. The focus has been on bacteria, but research shows that fungi might have an important role as well. There are only a few studies on the infant gut fungal microbiota, the mycobiota, in relation to antibiotic treatment. Here, the aim was to investigate the impact of antibiotics on the infant gut mycobiota, and the interkingdom associations between bacteria and fungi. We had 37 antibiotic-naïve patients suffering from respiratory syncytial virus, of which 21 received one to four courses of antibiotics due to complications, and 16 remained antibiotic-naïve throughout the study. Fecal samples were collected before, during and after antibiotic treatment with a follow-up period of up to 9.5 months. The gut mycobiota was studied by Illumina MiSeq sequencing of the ITS1 region. We found that antibiotic use affected the gut mycobiota, most prominently seen as a higher relative abundance of Candida (p < 0.001), and a higher fungal diversity (p = 0.005–0.04) and richness (p = 0.03) in the antibiotic-treated infants compared to the antibiotic-naïve ones at multiple timepoints. This indicates that the gut mycobiota could contribute to the long-term consequences of antibiotic treatments.publishedVersionPeer reviewe