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Gut Microbiota Composition Related with Clostridium Difficile-Positive Diarrhea and C

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Gut Microbiota Composition Related with Clostridium Difficile-Positive Diarrhea and C bioRxiv preprint doi: https://doi.org/10.1101/126995; this version posted April 12, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 Title: Gut microbiota composition related with Clostridium difficile-positive diarrhea and C. 2 difficile type (A+B+, A-B+, and A-B-) in ICU hospitalized patients 3 Running title: Clostridium difficile-positive diarrhea 4 Anhua Wu1#, MD; Juping Duan1,2#, MD; Sidi Liu1, MM; Xiujuan Meng1, MD; Pengcheng Zhou1, 5 MD; Qingya Dou1, MM; Chunhui Li1*, MD 1 6 Infection Control Center, Xiangya Hospital, Central South University, Changsha, Hunan, China, 7 410008 8 2Changsha Hospital of Traditional Chinese Medicine, Changsha, Hunan, China, 410100 9 # Anhua Wu and Juping Duan should be regarded as co-first authors. 10 *Corresponding author: Chunhui Li 11 Tel: +86-073189753953 12 Fax: +86-073184327237 13 E-mail: [email protected] 14 Address: Infection Control Center, Xiangya Hospital, Central South University, NO.87th Xiangya 15 Road, Changsha, Hunan, China, 410008 16 Abstract 17 Background: Gut microbiota composition of intensive care unit (ICU) patients suffering from 18 Clostridium difficile-positive diarrhea (CDpD) is still poorly understood. This study aims to use 19 16S rDNA (and metagenome) sequencing to compare the microbiota composition of 58 (and 5) 20 ICU patients with CDpD (CDpD group), 33 (and 4) ICU patients with C. difficile negative 21 diarrhea (CDnD group), and 21 (and 5) healthy control subjects (control group), as well as CDpD 22 patients in A+B+ (N=34; A/B: C. difficile TcdA/B), A-B+ (N=7), and A-B- (N=17) subgroups. For bioRxiv preprint doi: https://doi.org/10.1101/126995; this version posted April 12, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 23 16S rDNA data, OTU clustering (tool: UPARSE), taxonomic assignment (tool: RDP classifier), 24 α-diversity and β-diversity analyses (tool: QIIME) were conducted. For metagenome data, 25 metagenome assembly (tool: SOAP), gene calling (tools: MetaGeneMark, CD-HIT, and 26 SoapAligner), unigene alignment (tool: DIAMOND), taxon difference analysis (tool: Metastats), 27 and gene annotation (tool: DIAMOND) were performed. 28 Results: The microbial diversity of CDpD group was lower than that of CDnD and control groups. 29 The abundances of 10 taxa (e.g. Deferribacteres, Cryptomycota, Acetothermia) in CDpD group 30 were significantly higher than that in CDnD group. The abundances of Saccharomycetes and 31 Clostridia were significantly lower in CDpD in comparison with control. A+B+, A-B+ and A-B- 32 subgroups couldn’t be separated in principal component analysis, while some taxa are + + - - 33 significantly different between A B and A B subgroups. 34 Conclusion: CDpD might relate to the decrease of beneficial taxa (i.e. Saccharomycetes and 35 Clostridia) and the increase of harmful taxa (e.g. Deferribacteres, Cryptomycota, Acetothermia) in 36 gut microbiota in ICU patients. C. difficile type might be slightly associated with gut microbiota 37 composition. 38 Keywords: 16S rDNA sequencing; Clostridium difficile-associated diarrhea; gut microbiota; 39 metagenome sequencing; TcdA/TcdB 40 Introduction 41 As a result of infection prior admission, illness-associated immunosuppression, and invasive 42 devices in intensive care unit (ICU), microbial infection is frequently developed in patients in 43 general ICU (Vincent et al. 2016). Thus, antibiotics are often used in ICU patients (Vincent et al. 44 2016), including Meropenem, Piperacillin/tazobactam, Imipenem/Cilastatin, Cefuroxime, bioRxiv preprint doi: https://doi.org/10.1101/126995; this version posted April 12, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 45 Ceftriaxone, Ceftazidime, Cefepime, Cefperazone-Sulbactam, Latamoxef, etc. However, antibiotic 46 treatment is the most crucial risk factor for the infection with Clostridium difficile (also known as 47 Peptoclostridium difficile) (Stevens et al. 2011; Yutin and Galperin 2013), as it can adversely 48 affect gut indigenous microbiota composition and decrease the colonization resistance to C. 49 difficile (Britton and Young 2012). Consequently, C. difficile-associated diarrhea happens in 9.45 50 cases per 10,000 patient-days in ICU (Lee et al. 2015). 51 Nevertheless, current antibiotic therapies for C. difficile infection (CDI) like vancomycin 52 have limited efficacy (Bagdasarian et al. 2015), and fecal microbiota transplantation (FMT) is 53 recommended as an alternative therapy in curing highly-recurrent CDI failed to respond to 54 vancomycin treatment (Kelly et al. 2015). The success of FMT emphasizes the importance of 55 understanding gut microbiome in CDI patients. Therefore, a better understanding of the 56 relationships between microbiota, C. difficile, and diarrhea in ICU patients may contribute to 57 identifying novel therapeutic targets for diarrhea in ICU patients. 58 In recent years, high-throughput deep-sequencing of 16S rDNA and metagenome has been 59 applied to investigating microbiota composition. Milani et al. used 16S rDNA and metagenome 60 sequencing to study the gut microbiota compositions of three groups of elderly (age≥65) 61 hospitalized patients, involving 30 CDI-negative patients not exposed to antibiotics (AB- group), + 62 29 CDI-negative patients exposed to antibiotics (AB group), and 25 CDI-positive patients (CDI 63 group) (Milani et al. 2016). The microbial diversity of CDI-positive group was significantly lower 64 than that of CDI-negative group. CDI was found to relate with decrease in gut commensals like 65 Bacteroides, Alistipes, Lachnospira, and Barnesiella, while antibiotic treatment in CDI-negative 66 patients might associate with the depletion of commensals like Alistipes (Milani et al. 2016). bioRxiv preprint doi: https://doi.org/10.1101/126995; this version posted April 12, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 67 Schubert et al. utilized 16S rDNA sequencing to study the gut microbiota compositions of CDI 68 cases, diarrheal controls, and non-diarrheal controls (Schubert et al. 2014). Statistical models were 69 developed for CDI and diarrhea by incorporating clinical and demographic data with microbiome 70 data, and loss of several species in Ruminococcaceae, Lachnospiraceae, Bacteroides, and 71 Porphyromonadaceae might associate with CDI (Schubert et al. 2014). Generally, only C. difficile 72 that produces toxin A (an intestinotoxin, TcdA) and/or toxin B (a cytotoxin, TcdB) can cause 73 gastrointestinal diseases in humans. However, precise microbiome alterations related with diarrhea 74 and C. difficile-positive diarrhea (CDpD) have not been completely elucidated in ICU patients. 75 Also, relationships between C. difficile type (i.e. A+B+, A-B+, and A-B-) and gut microbiota 76 composition have not been investigated. 77 In this study, we utilized 16S rRNA and metagenome deep-sequencing to characterize the gut 78 microbiota composition in healthy subjects and ICU patients stratified for C. difficile, as well as + + - + - - 79 the gut microbiota composition in ICU patients with A B CDpD, A B CDpD, and A B CDpD. 80 Our results may help to understand the mechanisms underlying diarrhea and CDpD development 81 in ICU patients and identify potential candidates for curative or preventive microbe therapy. 82 Results 83 Clinical features of subjects 84 For Result-1, subjects in CDpD group (N=58), CDnD group (N=33), and control group 85 (N=21) were similar for gender, and subjects in CDpD and CDnD groups were similar for age, 86 gender, inpatient days, diabetes, cancer, hematopathy, respiratory failure, renal insufficiency, 87 tuberculosis, surgery, and exposure to immunosuppressant and glucocorticoids (Table 1). 88 bioRxiv preprint doi: certified bypeerreview)istheauthor/funder.Allrightsreserved.Noreuseallowedwithoutpermission. 89 Table 1. Demographic and clinical characteristics of the population in each experimental group https://doi.org/10.1101/126995 Result-1 Result-2 Result-3 CDpD CDnD Control P-val A+B+ A-B+ A-B- P-val CDpD CDnD Control P-value ue ue N 58 33 (100%) 21 34 7 17 5 4 (100%) 5 (100%) ; (100%) (100%) (100%) (100%) (100%) (100%) this versionpostedApril12,2017. Age Mean 55.3 58.8 36.6 56.8 54.4 52.6 54.2 72.0 38.8 SD 15.9 15.6 9.5 <0.00 17.3 10.1 15.3 0.684 21.4 11.7 13 0.035 1 CDpD vs CDnD 0.276 A+B+ vs A-B+ 0.728 CDpD vs CDnD 0.132 CDpD vs Control <0.00 A+B+ vs A-B- 0.392 CDpD vs Control 0.164 The copyrightholderforthispreprint(whichwasnot 1 CDnD vs Control <0.00 A-B+ vs A-B- 0.806 CDnD vs Control 0.011 1 Gender Fema 14 14 (42.4%) 8 0.162 10 0 (0%) 4 0.253 3 (60%) 2 (50%) 0 (0%) 0.151 le (24.1%) (38.1%) (29.4%) (23.5%) bioRxiv preprint doi: Male 44 19 (57.6%) 13 24 7 13 2 (40%) 2 (50%) 5 (100%) certified bypeerreview)istheauthor/funder.Allrightsreserved.Noreuseallowedwithoutpermission. https://doi.org/10.1101/126995 (75.9%) (61.9%) (70.6%) (100%) (76.5%) Inpatient days Mean 29.4 27.2 31.1 27.4 26.9 0.738 23.4 39.3 SD 18.9 17.2 20.4 11.4 18.6 19.8 7.7 CDpD vs CDnD 0.577 A+B+ vs A-B+ 0.649 CDpD vs CDnD 0.157 A+B+ vs A-B- 0.471 ; this versionpostedApril12,2017. A-B+ vs A-B- 0.955 Comparison between CDpD and CDnD Comparison between CDpD and CDnD Diabetes Yes 9 2 (6.06%) 0.183 6 0 (0%) 3 0.481 0 (0%) 0 (0%) NA (15.5%) (17.6%) (17.6%) Cancer Yes 6 4 (12.1%) 0.794 3 1 2 0.844 0 (0%) 1 (25%) 0.444 (10.3%) (8.82%) (14.3%) (11.8%) The copyrightholderforthispreprint(whichwasnot Hematopathy Yes 2 0 (0%) 0.533 2 0 (0%) 0 (0%) 0.650 0 (0%) 0 (0%) NA (3.45%) (5.88%) Resp.
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