Dong et al. BMC Infectious Diseases (2018) 18:207 https://doi.org/10.1186/s12879-018-3111-z RESEARCH ARTICLE Open Access Effects of intestinal colonization by Clostridium difficile and Staphylococcus aureus on microbiota diversity in healthy individuals in China Danfeng Dong1†,QiNi1†, Chen Wang1, Lihua Zhang2, Zhen Li3, Cen Jiang1, EnqiangMao4* and Yibing Peng1* Abstract Background: Intestinal colonization by pathogenic bacteria is a risk factor for infection, and contributes to environmental contamination and disease dissemination. Alteration of gut microbiota also plays a pivotal role in the development of disease. Although Clostridium difficile and Staphylococcus aureus are well-recognized pathogens causing nosocomial and community infections, the intestinal colonization was not fully investigated. Herein, we explored their overall carriage rates in healthy adults from the community, and characterized the gut microbiomes of C. difficile and S. aureus carriers. Methods: Fecal samples were collected from 1709 healthy volunteers from communities in Shanghai, China, and tested for the presence of C. difficile,methicillin-sensitiveS. aureus (MSSA), and methicillin-resistant S. aureus (MRSA) using culture-based techniques. To explore differences in the gut microbiome, 16S rRNA gene sequencing was conducted using samples from non-carriers (CH), C. difficile carriers (CCD), MRSA carriers (CM), and MSSA carriers (CS). Results: Overall, we detected 12 C. difficile and 60 S. aureus isolates, accounting for 0.70% and 3.51% of total isolates, respectively. Eight isolates were determined to be MRSA, accounting for 13.3% of the S. aureus population. Sequencing data revealed that the microbial diversity and richness were similar among the four groups. However, at the phylum level, carriage of C. difficile or MRSA was associated with a paucity of Bacteroidetes and an overabundance of Proteobacteria compared with non-carriers. At the genus level, the prevalence of the genera Bacteroides, Prevotella, Faecalibacterium,andRoseburia was decreased in C. difficile-positive samples compared with the controls, while the proportion of Clostridium cluster XIVa species was increased. MRSA carriers exhibited a higher proportion of the genera Parasutterella and Klebsiella, but a decreased prevalence of Bacteroides. Compared with MSSA carriers, Klebsiella was the only genus found to be significantly enriched in MRSA carriers. Conclusions: In healthy adults, colonization by C. difficile or S. aureus did not significantly affect gut microbiota diversity. However, the alteration of the gut microbiota composition in C. difficile carriers could indicate a predisposition to further infection. Our study provides essential data on the prevalence and effects of C. difficile and S. aureus colonization on gut microbiota composition in healthy adults. Keywords: Clostridium difficile, Staphylococcus aureus, Intestinal colonization, Gut microbiota * Correspondence: [email protected]; [email protected] †Equal contributors 4Department of Emergency Intensive Care Unit, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, No.197 Ruijin ER Road, Shanghai 200025, China 1Department of Laboratory Medicine, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, No.197 Ruijin ER Road, Shanghai 200025, China Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Dong et al. BMC Infectious Diseases (2018) 18:207 Page 2 of 8 Background Methods Clostridium difficile and Staphylococcus aureus are well Study design and specimen collection known pathogens, causing both nosocomial and The present study was conducted in four communities in community-acquired infections. Although the clinical Shanghai, China, between May and August 2014. Healthy in- manifestations of these pathogens are diverse, their dividuals aged over 18 years without any acute or chronic colonization and subsequent dissemination from the in- gastrointestinal diseases were enrolled. Those who were ex- testinal tract show a commonality [1, 2]. C. difficile is posed to any hospital environment or antibiotics in the pre- widely accepted as the most common cause of vious 30 days before collection were excluded. Demographic antibiotic-associated diarrhea, and the incidence and se- data concerning the age and gender of the participants was verity of C. difficile infection has dramatically increased collected. A total of 1709 participants were enrolled in the over past decades [3]. Unlike C. difficile, S. aureus gut present study. Among them, 748 were female and 961 were colonization, which is a known risk factor for gastro- male. The average age was 63 (+/− 16) years old. To better intestinal infection [4], has not yet been well studied. document age-related kinetics, we categorized the partici- During the past decade, with the pandemic rise in the pants into three groups based on their ages: youth (18– incidence of methicillin-resistant S. aureus (MRSA) and 40 years), middle-aged (41–65 years), and elderly (> 65 years). methicillin-sensitive S. aureus (MSSA) infection in the Fresh fecal samples were collected into sterile containers and community and in healthcare facilities, fecal carriage of submitted for immediate culture. All samples were stored at S. aureus has attracted attention. For example, studies − 80 °C for subsequent DNA extraction. have shown that patients with intestinal colonization of To further characterize the intestinal microbiota of C. MSSA and/or MRSA have a higher frequency of diar- difficile-andS. aureus-positive individuals, we divided rhea and environmental contamination, as well as in- participants into four groups: 1) healthy individuals with- creased skin colonization [4, 5]. While many studies out any C. difficile or S. aureus colonization (group CH), have investigated the current status of C. difficile, MSSA, 2) individuals positive for C. difficile (group CCD), 3) indi- and MRSA colonization of high-risk populations and viduals positive for MRSA (group CM), and 4) those posi- healthcare facilities, there is limited data regarding the tive for MSSA (group CS). Due to the results that there prevalence and colonization of these pathogens in were fewer individuals positive for C.difficile and MRSA, healthy individuals in the community in China. all individuals meeting the criteria for the CCD and CM The diverse and abundant microbiota of the human groups were included in the study, with participants in the intestine plays a crucial role in protection against CH and CS groups randomly chosen and then age and pathogen colonization, as well as in overall human gender-matched with participants in the first two groups. health [6]. However, intestinal infection or colonization Detailed information regarding the selected participants is by pathogens such as S. aureus and C. difficile is associ- listed in Additional file 1: Table S1. ated with disruption of the gut microbiota [7]. Numer- ous studies have shown that the biodiversity and Detection of bacterial pathogens community structure of the gut microbiota is altered in To improve the sensitivity of C. difficile detection, we ali- patients infected with C. difficile or S. aureus,andthese quoted each stool sample into two parts. One aliquot was alterations may directly lead to diseases [6, 8]. In pretreated with ethanol and then directly plated onto cyclo- addition, gut dysbiosis caused by MRSA colonization is serine cefoxitin fructose agar (Oxoid Ltd., Basingstoke, more severe than that caused by MSSA, with reduced UK). The other was pre-cultured in cycloserine cefoxitin microbial diversity and a decreased prevalence of bene- fructose medium supplemented with taurocholic acid and ficial bacterial [9]. Fecal microbiota transplantation has lysozyme for 48 h to enrich the vegetative cells prior to been successfully used to treat C. difficile infection and plating on cycloserine cefoxitin fructose agar. Culturing MRSA-associated enterocolitis [10], indicating that was performed at 35 °C for 48 h in anaerobic condition. modulation of the gut microbiota may be a potentially Suspected C. difficile colonies were identified based on their useful approach to treat refractory infections. However, odor and appearance, and confirmed using a latex agglutin- whether the intestinal bacterial communities of asymp- ation test (C. difficile Agglutination Test Kit; Oxoid), gluD tomatic carriers are notably changed in individuals after gene detection, and a toxin A & B test using a VIDAS joining a community has not yet been studied and Immunoanalyzer (Biomerieux, Marcy-l’Etoile, France). needs to be clarified. To screen S. aureus isolates from stool samples, samples The main focus of this study was to estimate the were plated on mannitol salt agar (Becton Dickinson) and prevalence of C. difficile and S. aureus in the gut of cultured for 48 h at 35 °C in aerobic condition. Suspected healthy adults in the communities, and to investigate