Open Life Sci. 2019; 14: 628–637 Research Article Wei Hong*#, Jing Yang#, Yumei Cheng, Xiaolin Huang, Fengqin Rao, Ting Zhang, Pixiang Wang, Jian Liao, Xiaolan Qi, Zhizhong Guan, Zhenhong Chen, Guzhen Cui Bacteria co-colonizing with Clostridioides difficile in two asymptomatic patients https://doi.org/10.1515/biol-2019-0071 13/111) exhibited a high ratio in asymptomatic patients. Received March 11, 2019; accepted September 25, 2019 These isolates derived from two phyla: Firmicutes (51.3%, Abstract: Background: Clostridium difficile infection 57/111) and Proteobacteria (44.1%, 49/111). In addition, (CDI) is the leading cause of nosocomial diarrhea. co-colonization of human pathogens Fusobacterium Co-colonization of key bacterial taxa may prevent the nucleatum, Ralstonia pickettii, Klebsiella pneumoniae, transition from asymptomatic C. difficile colonization to Klebsiella quasipneumoniae and Clostridium tertium with CDI. However, little is known about the composition of key C. difficile was identified. To the best of our knowledge, bacterial taxa in asymptomatic patients. Methods: In the these pathogens have not been co-isolated with C. difficile present study, the culture method was used to examine previously. Conclusions: In summary, the present study the composition of stool microbiota in two asymptomatic identified the composition of fecal microbiota in two patients from Guizhou, China. Results: A total of 111 asymptomatic patients in Guizhou, China. These results strains were isolated and phylogenetic relationships suggested that co-infection with human pathogens may were determined by 16S ribosomal gene sequencing and be ubiquitous during CDI progression. Molecular Evolutionary Genetics Analysis version 7. The results demonstrated that Escherichia (33.3%, 37/111), Keywords: Clostridium difficile infection; asymptomatic Clostridium (24.3%, 27/111) and Enterococcus (11.7%, patients; co-colonization; microbial diversity; 16S rDNA sequencing *Corresponding authors: Wei Hong , Key Laboratory of Endemic and Ethnic Diseases, Guizhou Medical University, Ministry of Education, 1 Introduction Guiyang 55004, Guizhou, China, E-mail: [email protected] Fengqin Rao, Ting Zhang, Xiaolan Qi, Zhizhong Guan, Key Laboratory Clostridium difficile, recently renamed Clostridioides of Endemic and Ethnic Diseases, Guizhou Medical University, Ministry of Education, Guiyang 55004, Guizhou, China. difficile [1], is a gram-positive, rod-shaped and strictly Wei Hong, Fengqin Rao, Ting Zhang, Xiaolan Qi, Zhizhong Guan, Key anaerobic human pathogen. C. difficile infection (CDI) is Laboratory of Medical Molecular Biology, Guizhou Medical University, the leading cause of nosocomial diarrhea, which poses a Guiyang 55004, Guizhou, China. major threat to health care facilities, including long-term Guiyang Maternal and Child Health Hospital, Guiyang Jing Yang, care facilities, nursing homes and hospitals worldwide [2, 550004, Guizhou, China. Yumei Cheng, Department of Critical Care Medicine, the Affiliated 3]. The clinical symptoms of CDI range from mild diarrhea Hospital of Guizhou Medical University, Guiyang 550004, Guizhou, to pseudomembranous colitis, which may result in death. China. The mechanism of CDI onset is associated with Xiaolin Huang, Jian Liao, School/Hospital of Stomatology of Guizhou antibiotic usage. Antibiotics are used to treat bacterial Medical University, Guiyang 550004, Guizhou, China. infections; however, they disrupt the integrity of the Pixiang Wang, Department of Biosystems Engineering, Auburn Uni- intestinal microbiota in the human gut. The niche created versity, Auburn, Alabama 36849, United States Zhenhong Chen, Guzhen Cui, School of Basic Medical Science, by antibiotics provides a competing advantage to C. Guizhou Medical University, Guiyang, 550025, China Guiyang 550025, difficile against probiotics, thus leading to the propagation Guizhou, China of C. difficile and overproduction of toxin A and toxin B [4]. # These authors contributed equally to this work. Toxin A (enterotoxin) and toxin B (cytotoxin) induce cell ORCID: Wei Hong: 0000-0002-3317-9401; Guzhen Cui: 0000-0002- death, inflammation and the accumulation of neutrophils, 6500-039X which result in various symptoms of CDI [4]. Open Access. © 2019 Wei Hong et al. published by De Gruyter. This work is licensed under the Creative Commons Attribution 4.0 Public License. Bacteria co-colonizing with Clostridioides difficile in two asymptomatic patients 629 Following a course of antibiotic therapy for CDI, was more than 2 days; ii) patients who were willing to the recurrence of CDI has been described in 10-30% of participate in the study. We recorded age, sex, reason for patients after first infection and up to 60% after multi- admission, and receipt of antibiotics. C. difficile infection episode infections [5]. Furthermore, recurrent CDI (RCDI) (CDI) was defined as hospital-associated diarrhea (HAD) leads to increased morbidity and mortality [6], thus, the with a positive stool for C. difficile isolation. Asymptomatic treatment of RCDI is still challenging. In recent years, patient was defined as a positive stool for C. difficile fecal microbiota transplantation (FMT), which transfers isolation without HAD [9]. healthy fecal microbiota from a healthy donor to a patient with RCDI, has been demonstrated to be effective in Informed consent: Informed consent has been obtained treating RCDI with an effective rate of ~90% [6, 7]. These from all individuals included in this study. results suggested that the integrity of the gut microbiota be key for the treatment of CDI and RCDI. Ethical approval: The research related to human use has By using whole metagenome shotgun sequencing, been complied with all the relevant national regulations, Vincent et al. demonstrated that co-colonization with key institutional policies and in accordance the tenets of bacterial taxa may prevent the increased proliferation of C. the Helsinki Declaration, and has been approved by the difficle [8]. In clinical practice, a number of asymptomatic Human Ethics Committee of Guizhou Medical University patients with C. difficile colonization do not develop CDI. (approval no. 2017-004). The present study hypothesized that the presence of certain microbes in these patients may serve a pivotal role in preventing the transition of asymptomatic colonization 2.2 Sample collection and processing of C. difficile to CDI. Thus, these asymptomatic patients may be used as an appealing gut microbial homeostasis Stool samples were collected in 50 ml DNase & RNase-free model in the nosocomial environment. Study of the NEST® sample collection tube (Nest Scientific USA, Inc.) composition of gut microbiota in this model may help and transferred to the laboratory immediately on ice. The develop treatments for CDI/RCDI. In addition, the samples were soaked in the appropriate amount of fresh intestinal microbial community in asymptomatic patients BHI medium for 10 min and vortexed for 10-20 sec. The is easier to study compared with that of the healthy human mixed solution was serially diluted in fresh BHI medium fecal microbiome, as it contains lower bacterial diveristy and spread across BHI-blood or CCFA-blood (Cycloserine- and retains pivotal information. Cefoxitin-Fructose Agar, Oxoid) agar [10]. The plates were Although the gut microbiota composition of C. difficile incubated in an anaerobic chamber at 37˚C for 48 h [11]. asymptomatic carriers may be important for finding new Colonies were picked and further purified by re-streaking CDI/RCDI treatment strategies, limited information is on a BHI-blood agar plate. available regarding the bacteria that co-colonize with C. difficile in the asymptomatic patients. In developing countries, the awareness of CDI is insufficient, and the 2.3 16S rDNA sequencing and phylogenetic dietary habits are distinct from North America and Europe. analysis The present study used the culture method to study the diversity of microbes in two C. difficile asymptomatic The genomic DNA of purified strains were prepared patients in Guizhou, China. using a TIANGEN® bacterial genomic DNA extraction kit (DP302; TIANGEN Biotech, Beijing, China). Primers for 16s-V4-515F (5’-GTGCCAGCMGCCGCGGTAA-3’) and 16S-V4- 2 Methods 806R (5’-GGACTCHVGGGT-WTCTAAT-3’) were used to amplify partial 16S rDNA of isolated strains according to 2.1 Selection criteria and ethics Lianbing Lin et al. [12]. PCR amplification was performed in a GeneAmp® PCR system 9700 (Applied Biosystems; Consecutive patients who were admitted to ICU wards of Thermo Fisher Scientific, Inc., Waltham, MA, USA) affiliated hospital of Guizhou Medical University between using Q5® High-Fidelity Polymerase. The thermocycling December 11, 2016 to August 25, 2017. These patients were conditions were as follows: 98°C for 30 sec; 30 cycles of screened for enrollment by following inclusion criteria, i) 98°C for 10 sec, 55°C for 30 sec and 72°C for 10 sec; and a Patients were eligible for the study if they were receiving final extension at 72°C for 2 min. The PCR amplification antimicrobial therapy and if their expected length of stay products were recovered directly by TIANGEN® PCR 630 W. Hong, et al. purification kit (DP204, TIANGEN Biotech). A total of 20 3.2 Isolation of bacteria that co-colonize μl molecular grade water (heated to 60°C prior to applying with C. difficile to the column) was used to elute purified 16S rDNA. The DNA samples were stored at -20°C prior to sequencing The stool samples of asymptomatic patients were by GeneCreate Biotech (Wuhan,