pathogens Article Association of Fungi and Archaea of the Gut Microbiota with Crohn’s Disease in Pediatric Patients—Pilot Study Agnieszka Krawczyk 1, Dominika Salamon 1,* , Kinga Kowalska-Duplaga 2 , Tomasz Bogiel 3,4 and Tomasz Gosiewski 1,* 1 Department of Molecular Medical Microbiology, Faculty of Medicine, Jagiellonian University Medical College, 31-121 Krakow, Poland; [email protected] 2 Department of Pediatrics, Gastroenterology and Nutrition, Faculty of Medicine, Jagiellonian University Medical College, 30-663 Krakow, Poland; [email protected] 3 Microbiology Department, Ludwik Rydygier Collegium Medicum in Bydgoszcz, Nicolaus Copernicus University in Torun, 85-094 Bydgoszcz, Poland; [email protected] 4 Clinical Microbiology Laboratory, University Hospital No. 1 in Bydgoszcz, 85-094 Bydgoszcz, Poland * Correspondence: [email protected] (D.S.); [email protected] (T.G.); Tel.: +48-(12)-633-25-67 (D.S.); +48-(12)-633-25-67 (T.G.) Abstract: The composition of bacteria is often altered in Crohn’s disease (CD), but its connection to the disease is not fully understood. Gut archaea and fungi have recently been suggested to play a role as well. In our study, the presence and number of selected species of fungi and archaea in pediatric patients with CD and healthy controls were evaluated. Stool samples were collected from children with active CD (n = 54), non-active CD (n = 37) and control subjects (n = 33). The prevalence and the number of selected microorganisms were assessed by real-time PCR. The prevalence of Candida Citation: Krawczyk, A.; Salamon, D.; tropicalis was significantly increased in active CD compared to non-active CD and the control group Kowalska-Duplaga, K.; Bogiel, T.; (p = 0.011 and p = 0.036, respectively). The number of Malassezia spp. cells was significantly lower in Gosiewski, T. Association of Fungi patients with active CD compared to the control group, but in non-active CD, a significant increase and Archaea of the Gut Microbiota was observed (p = 0.005 and p = 0.020, respectively). There were no statistically significant differences with Crohn’s Disease in Pediatric in the colonization by archaea. The obtained results indicate possible correlations with the course Patients—Pilot Study. Pathogens 2021, of the CD; however, further studies of the entire archeobiome and the mycobiome are necessary in 10, 1119. https://doi.org/10.3390/ order to receive a complete picture. pathogens10091119 Academic Editor: Ana Elena Pérez Keywords: fungi; archaea; gut microbiota; Crohn’s disease; gut dysbiosis Cobas Received: 4 August 2021 Accepted: 29 August 2021 1. Introduction Published: 1 September 2021 Crohn’s disease (CD) is included in the group of diseases known as inflammatory bowel diseases (IBD). It is believed that the incidence and severity of the course of the Publisher’s Note: MDPI stays neutral disease are conditioned by a combination of genetic, immunological, environmental and with regard to jurisdictional claims in microbiological factors (Figure1)[ 1–3]. The latter remain a constant interest of many published maps and institutional affil- research teams, due to the still unclear role of microorganisms in the pathogenesis of the iations. disease. The fact that microorganisms participate in causing CD is suggested by numerous studies and clinical observations, among others: exacerbation of the disease as a result of infection and food poisoning [4]; failure to induce enteritis in germ-free animals; alleviation Copyright: © 2021 by the authors. or amelioration of symptoms following the application of antibiotics, probiotics or colon Licensee MDPI, Basel, Switzerland. cleansing treatments [5,6], as well as achieving remission in patients who underwent a This article is an open access article transplant of gut microbiota from healthy people [7]. The majority of the studies to date distributed under the terms and have concerned the analysis of the bacteriobiome in IBD [8–14]. However, owing to the conditions of the Creative Commons recent development of molecular methods, more and more attention is also paid to other Attribution (CC BY) license (https:// microorganisms comprising the normal gut microbiota, i.e. fungi, viruses or archaea. creativecommons.org/licenses/by/ 4.0/). Pathogens 2021, 10, 1119. https://doi.org/10.3390/pathogens10091119 https://www.mdpi.com/journal/pathogens Pathogens 2021, 10, x FOR PEER REVIEW 2 of 11 Pathogens 2021, 10, 1119 2 of 11 other microorganisms comprising the normal gut microbiota, i.e. fungi, viruses or ar- chaea. Figure 1.FigureRisk factors 1. Risk for factors CD —Crohn’s for CD — Disease;Crohn’s NOD2—nucleotide-binding Disease; NOD2—nucleotide oligomerization-binding oligomerization domain containing do- 2; CARD15—caspasemain containing recruitment 2; CARD15 domain family,—caspase member recruitment 15; TLR—toll-like domain receptors;family, member OCTN—organic 15; TLR cation—toll transporter;-like re- ATG16L1—autophagy-relatedceptors; OCTN—organic protein 16cation like 1;transporter; LRRK2—leucine-rich ATG16L1 repeat—autophagy kinase 2; IRGM—immunity-related protein related16 like GTPase 1; M; STAT3—signalLRRK2— transducerleucine-rich and repeat activator kinase of transcription 2; IRGM— 3;immunity CLEC7A—C-Type related GTPase Lectin Domain M; STAT3 Containing—signal 7A, trans- IFN-γ— interferonducer gamma, and TNF- activatorα—tumor of necrosistranscription factor alpha;3; CLEC7A Th1—Type—C- 1Type helper Lectin T-cells; Domain Th17—T-helper Containing 17 cell, 7A, Il- IFN interleukin.-γ— interferon gamma, TNF-α—tumor necrosis factor alpha; Th1—Type 1 helper T-cells; Th17—T- helper 17 cell, Il- interleukin.Fungi are organisms that belong to the domain Eukaryota. Despite the fact that they are a smaller population in the human microbiota than bacteria, their interactions with Fungi are immuneorganisms cells that or the belong production to the of domain metabolites Eukaryota are relevant. Despite to human the healthfact that and they disease [15]. are a smaller populationThe studies in to datethe human indicate microbiota that the course than of bacteria, CD may betheir associated interactions with thewith impact of mycobiome on GALT (gut-associated lymphoid tissue). The presence of antibodies against immune cells or the production of metabolites are relevant to human health and disease Saccharomyces cerevisiae (ASCA—anti-Saccharomyces cerevisiae antibodies) recognizing cell [15]. The studieswall to mannansdate indicate of yeast that is the a clinical course biomarker of CD may of patients be associated with CD with [16,17 the]. impact of mycobiome on GALTWith regard (gut- toassociated their general lymphoid structure, tissue). archaea The resemble presence bacteria, of antibodies while genetically, against Saccharomycesthey are cerevisiae more closely (ASCA related—anti to eukaryotes.-Saccharomyces They cerevisiae belong to theantibodies) domain Archaea recog-, among nizing cell wallwhich mannans three of basic yeast groups is a clinical can be distinguished,biomarker of whichpatients are with halophilic, CD [16,17]. methanogenic and With regardthermophilic to their general archaea. structure, Methanogens archaea are the resemble group that bacteria, most frequently while colonizesgenetically, the human they are more closelygastrointestinal related tract. to eukaryotes. The importance They of belong the archaea to the presence domain in the Archaea human, gastrointestinalamong which three basictract groups is not fully can understood.be distinguished, Until now, which strong are emphasis halophilic, has beenmethanogenic placed on studyingand the role of bacteria in the course of CD, while the role of those microorganisms is so far less thermophilic archaea. Methanogens are the group that most frequently colonizes the hu- taken into account by researchers. man gastrointestinalTherefore, tract. The the importance objective of thisof the study archaea was an presence assessment in ofthe the hum prevalencean gastro- of the most intestinal tract commonis not fully representatives understood. of ArchaeaUntil now,and yeaststrong fungi emphasis in children has and been teenagers placed withon CD at studying the roledifferent of bacteria stages ofin thethe disease. course Moreover,of CD, while the number the role of of these those microorganisms microorganisms in individual is so far less takengroups into was account determined. by researchers. Therefore, the objective of this study was an assessment of the prevalence of the most 2. Results common representatives of Archaea and yeast fungi in children and teenagers with CD at 2.1. Characteristics of the Subjects different stages of the disease. Moreover, the number of these microorganisms in individ- ual groups was determined.There were 124 children included in the study: 54 patients with active CD, 37 pa- tients in remission and 33 healthy individuals for the control group. The age of each group of patients was similar: average 13.15 (±2.86) years for patients with active CD vs. 2. Results 12.86 (±3.95) years for patients with non-active CD vs. 11.39 (±3.99) for healthy children 2.1. Characteristics of the Subjects There were 124 children included in the study: 54 patients with active CD, 37 patients in remission and 33 healthy individuals for the control group. The age of each group of Pathogens 2021, 10, x FOR PEER REVIEW 3 of 11 patients was similar: average 13.15 (±2.86) years for patients with active CD vs. 12.86 (±3.95) years for patients with non-active CD vs. 11.39 (±3.99) for healthy children from Pathogens 2021, 10, 1119 the control group. The group of patients with active CD consisted of 21 girls (39%). In3 ofthe 11 group of patients with non-active CD, there were 17 girls (46%). The control group in- cluded 19 girls (58%). The average Pediatric Crohn’s Disease Activity Index (PCDAI) value in the group of patients with active CD amounted to 33.23 pts (±13.30) while in the from the control group. The group of patients with active CD consisted of 21 girls (39%). group of patients in remission, 3.7 pts (±3.65). In the group of patients with non-active CD, there were 17 girls (46%).