key: cord-1008865-ty2fgrdr authors: Xu, R.; Liu, P.; Zhang, T.; Wu, Q.; Zeng, M.; Ma, Y.; Jin, X.; Xu, J.; Zhang, Z.; Zhang, C. title: Progressive worsening of the respiratory and gut microbiome in children during the first two months of COVID-19 date: 2020-07-17 journal: nan DOI: 10.1101/2020.07.13.20152181 sha: 7939f331bd1166a3bf27c163166f2f2d443d72bd doc_id: 1008865 cord_uid: ty2fgrdr Children are less susceptible to COVID-19 and manifests lower morbidity and mortality after infection, for which a multitude of mechanisms may be proposed. Whether the normal development of gut-airway microbiome is affected by COVID-19 has not been evaluated. We demonstrate that COVID-19 alters the respiratory and gut microbiome of children. Alteration of the microbiome was divergent between the respiratory tract and gut, albeit the dysbiosis was dominated by genus Pseudomonas and sustained for up to 25-58 days in different individuals. The respiratory microbiome distortion persisted in 7/8 children for at least 19-24 days after discharge from the hospital. The gut microbiota showed early dysbiosis towards later restoration in some children, but not others. Disturbed development of both gut and respiratory microbiomes, and prolonged respiratory dysbiosis in children imply possible long-term complications after clinical recovery from COVID-19, such as predisposition to an increased health risk in the post-COVID-19 era. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 17, 2020. . https://doi.org/10.1101/2020.07.13.20152181 doi: medRxiv preprint Study cohort 52 Nine COVID-19 children between 7-139 months old were enrolled in this study together with 53 14 age-matched healthy control children. A total of 103 specimens including 27 sets of paired 54 specimens (at least two of throat swab, nasal swab and feces) were collected from children with 55 COVID-19 ( Supplementary Fig. 1 ). The children were being followed between 25-58 days after 56 symptom onset. All samples were subjected to high-throughput sequencing of the V4-region of 57 bacterial 16S rRNA gene (Methods). Respiratory and gut microbiome dynamics in COVID-19 60 We analyzed the 16S-rRNA gene sequences of all specimens from three body sites, and obtained Table 1 ). Using the DMM method (Supplementary method), we identified 8 63 community types (Fig.1a) . The specimens from healthy children clustered into two community types, CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 17, 2020. . https://doi.org/10.1101/2020.07.13.20152181 doi: medRxiv preprint the gut and respiratory tract microbiomes of healthy children. In fact, the nasal cavity, throat and gut 73 still maintain similar microbial structures in adulthood. 74 Bacteria from stool specimens of COVID-19 children fell into three distinct community types 75 (COVID-GUT I-III), and those from nasal and throat swabs formed another three distinct types 76 (COVID-TN I-III) (Fig. 1a) . All COVID-19-related types are significantly separated from the type 77 H-MIX except COVID-TN-I that overlaps with H-MIX (Fig. 1b) . In particular, three respiratory tract-78 related types and three GUT-related types of COVID-19 children are also significantly separated from 79 each other, and distinctly different from healthy children. These results indicated that SARS-CoV-2 80 infection significantly changed the gut and respiratory tract microbiota of children, and the separation 81 of bacterial community structures between the gut and respiratory tracts suggested that the normal 82 development of the microbiota may be impaired. All COVID-19-related types showed lower richness and evenness than H-MIX, except for 84 COVID-GUT-I that has the most similarity to H-MIX and relatively normal microbiome structure. There was a gradual decrease from community type I to III for both gut and respiratory tract (Fig. 1c) , 86 indicating a progressive deterioration (dysbiosis) of the microbiome. Overall, the dysbiosis appeared 87 to be more severe in the respiratory tract than in the gut. To characterize eight microbial community types, we identified 35 indicator genera (Fig. 2a) . The H-MIX type was characterized by 11 genera, and the predominant commensal bacteria contained 92 Prevotella, Streptococcus, unclassified Pasteurellaceae, and Actinomyces (Fig. 2b) . Some of the 93 indicator bacteria in H-MIX were shared by the community types COVID-GUT-I (e.g. Prevotella, 94 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted July 17, 2020. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted July 17, 2020. . https://doi.org/10.1101/2020.07.13.20152181 doi: medRxiv preprint (H-MIX) or high-diversity community structures (COVID-TN-I) to late low-diversity dysbiosis 117 structure (COVID-TN-III), indicating a steady deterioration in composition and function of the 118 respiratory microbiome despite a fast clinical recovery (Fig. 3a) . Surprisingly, the respiratory 119 dysbiosis was sustained at least 19-24 days after discharge (i.e., 42-58 days after symptom onset) in 120 three children (CV01, CV02 and CV09). In contrast, the gut microbiome alternation varied greatly among these COVID-19 children. On the other hand, the presence of probiotic Bifidobacterium and the most important butyrate-138 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted July 17, 2020. . https://doi.org/10.1101/2020.07.13.20152181 doi: medRxiv preprint producing bacteria Faecalibacterium were inversely correlated with the existence of Pseudomonas 139 ( Fig. 3b and Supplementary Fig. 2 ), despite these beneficial bacteria presented at a very low relative 140 abundance and often decreased in late disease stage. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 17, 2020. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 17, 2020. . https://doi.org/10.1101/2020.07.13.20152181 doi: medRxiv preprint however, the dynamic changes of the microbiome were divergent between respiratory tract and gut, 178 and the microbiome appeared to be progressively worsening, especially in the respiratory tract for a 179 long period (25-52 days), substantially later than their clinical recovery (12-37 days) (Supplementary 180 Fig. 3 ). In this cohort, six children were older than 3 years of age, and should have relatively stable 181 adult-like microbiome. The dynamic characteristics of the microbiome during COVID-19 implied 182 that children's microbiome is still particularly vulnerable and less resilient than that of the adults even 183 after attaining a stable phase 7,9,16 . Importantly, the persistent worsening of the microbiomes caused 184 by COVID-19 might impart potential short-term and long-term health problems during childhood and 185 adulthood. 186 We and other have reported that altered respiratory microbiome with reduced bacterial diversity CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 17, 2020. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 17, 2020. . https://doi.org/10.1101/2020.07.13.20152181 doi: medRxiv preprint CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 17, 2020. is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 17, 2020. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 17, 2020. The PCR2 products were purified using the same Gel Extraction Kit and qualified using the Qubit® CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 17, 2020. The sOTU coverage of 87% was sufficient to capture microbial diversity. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 17, 2020. . https://doi.org/10.1101/2020.07.13.20152181 doi: medRxiv preprint scaling (NMDS) based on the Bray-Curtis distance under bacterial genus level. "The ANOSIM 378 statistic "R" compares the mean of ranked dissimilarities between groups to the mean of ranked 379 dissimilarities within groups. An R value close to "1.0" suggests dissimilarity between groups while 380 an R value close to "0" suggests an even distribution of high and low ranks within and between 381 groups 46 . ANOSIM p values lower than 0.05 suggest the higher similarity within sites. Richness 382 (Observed sOTUs) and Pielou's or Species evenness for each community type were calculated for 383 estimating the difference of alpha diversity. Those analyses described above were performed using R 384 package "vegan" v2.5-6. Using R package 'Pheatmap', the dynamic change of microbial community 385 types and compositions were visualized. Alpha diversity difference between groups were measured 386 using the Wilcoxon Rank Sum Test in R. is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 17, 2020. . https://doi.org/10.1101/2020.07.13.20152181 doi: medRxiv preprint Supplementary Table S1 . Fecal, nasal, and throat microbial abundances (phyla and genera). 463 The stars represent unclassified genera. shift of community type from early dysbiosis towards late incomplete restoration was found in both 474 respiratory and gut microbiomes within a short time. In children, however, the changes of the 475 community types were divergent between the respiratory tract and gut, possibly implying that the 476 "airway-gut axis" is still not established during the childhood. Moreover, children's respiratory 477 microbiome appeared to be progressively worsening for a long period despite a fast clinical recovery. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. 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