Bacteria Co-colonizing with Clostridioides Difficile in Two Asymptomatic Patients

Abstract Background Clostridium difficile infection (CDI) is the leading cause of nosocomial diarrhea. Co-colonization of key bacterial taxa may prevent the transition from asymptomatic C. difficile colonization to CDI. However, little is known about the composition of key bacterial taxa in asymptomatic patients. Methods In the present study, the culture method was used to examine the composition of stool microbiota in two asymptomatic patients from Guizhou, China. Results A total of 111 strains were isolated and phylogenetic relationships were determined by 16S ribosomal gene sequencing and Molecular Evolutionary Genetics Analysis version 7. The results demonstrated that Escherichia (33.3%, 37/111), Clostridium (24.3%, 27/111) and Enterococcus (11.7%, 13/111) exhibited a high ratio in asymptomatic patients. These isolates derived from two phyla: Firmicutes (51.3%, 57/111) and Proteobacteria (44.1%, 49/111). In addition, co-colonization of human pathogens Fusobacterium nucleatum, Ralstonia pickettii, Klebsiella pneumoniae, Klebsiella quasipneumoniae and Clostridium tertium with C. difficile was identified. To the best of our knowledge, these pathogens have not been co-isolated with C. difficile previously. Conclusions In summary, the present study identified the composition of fecal microbiota in two asymptomatic patients in Guizhou, China. These results suggested that co-infection with human pathogens may be ubiquitous during CDI progression.


Introduction
Clostridium difficile, recently renamed Clostridioides difficile [1], is a gram-positive, rod-shaped and strictly anaerobic human pathogen. C. difficile infection (CDI) is the leading cause of nosocomial diarrhea, which poses a major threat to health care facilities, including long-term care facilities, nursing homes and hospitals worldwide [2,3]. The clinical symptoms of CDI range from mild diarrhea to pseudomembranous colitis, which may result in death.
The mechanism of CDI onset is associated with antibiotic usage. Antibiotics are used to treat bacterial infections; however, they disrupt the integrity of the intestinal microbiota in the human gut. The niche created by antibiotics provides a competing advantage to C. difficile against probiotics, thus leading to the propagation of C. difficile and overproduction of toxin A and toxin B [4]. Toxin A (enterotoxin) and toxin B (cytotoxin) induce cell death, inflammation and the accumulation of neutrophils, which result in various symptoms of CDI [4]. was more than 2 days; ii) patients who were willing to participate in the study. We recorded age, sex, reason for admission, and receipt of antibiotics. C. difficile infection (CDI) was defined as hospital-associated diarrhea (HAD) with a positive stool for C. difficile isolation. Asymptomatic patient was defined as a positive stool for C. difficile isolation without HAD [9].
Informed consent: Informed consent has been obtained from all individuals included in this study.

Ethical approval:
The research related to human use has been complied with all the relevant national regulations, institutional policies and in accordance the tenets of the Helsinki Declaration, and has been approved by the Human Ethics Committee of Guizhou Medical University (approval no. 2017-004).

Sample collection and processing
Stool samples were collected in 50 ml DNase & RNase-free NEST ® sample collection tube (Nest Scientific USA, Inc.) and transferred to the laboratory immediately on ice. The samples were soaked in the appropriate amount of fresh BHI medium for 10 min and vortexed for 10-20 sec. The mixed solution was serially diluted in fresh BHI medium and spread across BHI-blood or CCFA-blood (Cycloserine-Cefoxitin-Fructose Agar, Oxoid) agar [10]. The plates were incubated in an anaerobic chamber at 37˚C for 48 h [11]. Colonies were picked and further purified by re-streaking on a BHI-blood agar plate.

16S rDNA sequencing and phylogenetic analysis
The genomic DNA of purified strains were prepared using a TIANGEN ® bacterial genomic DNA extraction kit (DP302; TIANGEN Biotech, Beijing, China). Primers for 16s-V4-515F (5'-GTGCCAGCMGCCGCGGTAA-3') and 16S-V4-806R (5'-GGACTCHVGGGT-WTCTAAT-3') were used to amplify partial 16S rDNA of isolated strains according to Lianbing Lin et al. [12]. PCR amplification was performed in a GeneAmp ® PCR system 9700 (Applied Biosystems; Thermo Fisher Scientific, Inc., Waltham, MA, USA) using Q5 ® High-Fidelity Polymerase. The thermocycling conditions were as follows: 98°C for 30 sec; 30 cycles of 98°C for 10 sec, 55°C for 30 sec and 72°C for 10 sec; and a final extension at 72°C for 2 min. The PCR amplification products were recovered directly by TIANGEN ® PCR Following a course of antibiotic therapy for CDI, the recurrence of CDI has been described in 10-30% of patients after first infection and up to 60% after multiepisode infections [5]. Furthermore, recurrent CDI (RCDI) leads to increased morbidity and mortality [6], thus, the treatment of RCDI is still challenging. In recent years, fecal microbiota transplantation (FMT), which transfers healthy fecal microbiota from a healthy donor to a patient with RCDI, has been demonstrated to be effective in treating RCDI with an effective rate of ~90% [6,7]. These results suggested that the integrity of the gut microbiota be key for the treatment of CDI and RCDI.
By using whole metagenome shotgun sequencing, Vincent et al. demonstrated that co-colonization with key bacterial taxa may prevent the increased proliferation of C. difficle [8]. In clinical practice, a number of asymptomatic patients with C. difficile colonization do not develop CDI. 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 of C. difficile to CDI. Thus, these asymptomatic patients may be used as an appealing gut microbial homeostasis model in the nosocomial environment. Study of the composition of gut microbiota in this model may help develop treatments for CDI/RCDI. In addition, the intestinal microbial community in asymptomatic patients is easier to study compared with that of the healthy human fecal microbiome, as it contains lower bacterial diveristy and retains pivotal information.
Although the gut microbiota composition of C. difficile asymptomatic carriers may be important for finding new CDI/RCDI treatment strategies, limited information is 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 dietary habits are distinct from North America and Europe. The present study used the culture method to study the diversity of microbes in two C. difficile asymptomatic patients in Guizhou, China.

Selection criteria and ethics
Consecutive patients who were admitted to ICU wards of affiliated hospital of Guizhou Medical University between December 11, 2016 to August 25, 2017. These patients were screened for enrollment by following inclusion criteria, i) Patients were eligible for the study if they were receiving antimicrobial therapy and if their expected length of stay

Isolation of bacteria that co-colonize with C. difficile
The stool samples of asymptomatic patients were analyzed using BHIS-blood and CCFA-blood medium without antibiotics ( Figure 1) [15,16]. A total of 111 strains were isolated from the two fecal samples. The strains were purified, and the partial 16S ribosomal gene sequences were obtained and blasted against the 16SMicrobial database. The blast results are presented in Table 3. Among the strains, Escherichia species (E. marmotae and E. fergusonii; n=37; 33.3%) was the most abundant species co-colonizing with C. difficile. Enterococcus (E. saigonensis, E. faecalis, E. hirae; n=13; 11.7%) and Clostridium (C. clostridioforme, C. tertium; n=12; 10.8%) were ranked in the second and third place, respectively (Table 3).

Phylogenetic analyses
To determine the taxonomic associations of the isolated strains, a phylogenomic tree was constructed based on the values of nucleotide sequence pairwise similarity between the isolates. Strains from same species were clustered together ( Figure S1). To provide an in-depth view of the association between isolated species, duplicated strains were omitted and the actual number of isolated strains was subsequently marked ( Figure 2). A total of 22 types of strains were demonstrated to co-colonize with C. difficile. The strains were grouped into two major groups: Group 1 and group 2 ( Figure 2). The majority of strains in Group 1 belonged to the Firmicutes phylum, whereas the majority of strains in group 2 belonged to the Proteobacteria phylum. Bacteria from the Bacteroidete phylum was not isolated in the two samples [8]. This may have been caused by the bias of the medium or the novel gut microbiota structure of the patients, which may be affected by specific dietary habits and antibiotic use.
Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach and then selecting the topology with superior log-likelihood value. The tree is drawn to scale. The analysis involved 23 nucleotide sequences. All positions containing gaps and missing data were eliminated. A total of 230 positions were identified in the final dataset. Evolutionary analyses were conducted in MEGA7 [13].

Patient characteristics
A total of 51 patients were enrolled in the study during their hospitalization in the affiliated hospital of Guizhou Medical University from 11/12/2016 to 25/8/2017. Among these patients, one patient developed CDI; two patients were confirmed to exhibit asymptomatic C. difficile colonization (C. difficile was isolated from his/her stool samples, however, the patients did not develop any CDI symptoms, such as diarrhea and megacolon, Tables 1 and 2). The patient with CDI experienced diarrhea, which was not recurrent following antibiotic treatment. Patient characteristics are presented in Tables 1 and 2. The incident rate of CDI was nearly 2%, and incident rate of asymptomatic C. difficile colonization was nearly 4%. Reason for admission   asymptomatic patients with C. difficile colonization. A total of 111 strains were isolated from these samples, their partial 16S ribosome genes were sequenced, and NCBI-blast-2.7.1 and MEGA7 algorithms were used to determine the diversity and phylogenetic associations of these isolates. The isolates were derived from three phyla: Firmicutes, Proteobacteria and Fusobacteriia. Firmicutes (51.3%) and Proteobacteria (44.1%) were most abundant phyla. To the best of our knowledge, this is the first time the only isolated class. To the best of our knowledge, this was the first time that Fusobacteriia was co-isolated with C. difficile, which was not previously demonstrated in metagenomic research [8].

Discussion
The present study used the culture method to analyze the microbial diversity in the stool samples of two was consistent with previous research [19]. These species may serve as protective taxa against the transition from asymptomatic C. difficile colonization to CDI. For example, Clostridium spp. are potential protective bacterial taxa that may exert their protective effects through the production of secondary bile acids [8]; Lactobacillus paracasei strains have been demonstrated to exhibit health-promoting properties as probiotics [19,20]. Recently, Blautia producta, Ruminococcus, Lactobacillus paracasei and Escherichia have been used in a defined stool substitute mixture to treat antibiotic-resistant C. difficile colitis [21]. By contrast, species that are normally considered human pathogens were also identified to co-colonize with C. difficile, including Fusobacterium nucleatum, Bacillus cereus, Shigella dysenteriae, Ralstonia pickettii, Klebsiella pneumoniae, Klebsiella quasipneumoniae and Clostridium tertium. Fusobacterium nucleatum normally colonizes in the oral environment and has recently been demonstrated to be associated with intestinal tumorigenesis [22].
that Fusobacteriia (4.5%) was reported to co-colonize with C. difficile. Comparing with metagenomic research [8], Bacteroidete phylum was not identified in the present study, which may be due to the bias of screening medium and antibiotics used during the hospitalization of patients.
Although metagenomic sequencing approaches can provide abundant data for culturable and nonculturable microorganisms, culturomics has become increasingly important in recent years [17], as it may enable the design of a defined microbiota composition, which may be transferred to patients with CDI by FMT. Ann M.O'Hara et al have suggested that the microbial composition of the gut contributes to intestinal disorders and that the enhancement of beneficial bacteria may represent a promising therapeutic strategy against various diseases (e.g., CDI) caused by disruptions in the gut microbiota [18]. The present study demonstrated that Escherichia (33.3%), Clostridium (24.3%) and Enterococcus (11.7%) exhibited high ratios in the two tested samples, which  [14]. The isolated strain number for each species is indicated on the left. Green indicates strains that have been reported as normal human habitats and may act as protective taxa against CDI. Red represents human pathogens cocolonizing with C. difficile. Black represents strains of which the pathogenicity to humans is unknown (except C. difficile). The tree with the highest log likelihood (-2403.04) is presented.
Bacillus cereus is easily transferred through food and may cause emetic or diarrheal food-associated illness [23]. Shigella dysenteriae causes dysentery, which occurs most frequently in areas where poor sanitation and malnutrition are prevalent, especially in developing countries [24]. Klebsiella quasipneumoniae has been reported to cause pyogenic liver abscess [25]. Clostridium tertium commonly affects neutropenic patients with haematological malignancy [26]. In addition, to the best of our knowledge, Fusobacterium nucleatum, Ralstonia pickettii, Klebsiella pneumoniae, Clostridium tertium and Klebsiella quasipneumoniae have not been reported to co-infect with C. difficile [27]. These results suggested that co-infection may be ubiquitous during CDI progression. In this case, it could be associated to the special dietary habits in the Guizhou province, where pickled and spicy food is preferred. However, the underlying mechanism needs to be studied further.
In the two asymptomatic patients, C. difficile was detected by culturing method. Due to the number of asymptomatic patients in present study was limited and the microbiota composition is strongly influenced by their illness and medical treatment [8]. Therefore, we could not perform statistical analyses to assess general abundance of microbial taxa for asymptomatic patients. Furthermore, there are still two concerns should be carefully addressed in future studies. Firstly, the diversity of microbes was relatively low in the present study. For instance, Bacteroidete, Virus and Fungi were not identified; this may have been due to the bias of the screening medium and/ or antibiotic use of the patients during hospitalization. These problems should be carefully addressed in future studies. Secondly, co-colonization may also increase the potential for genetic transference of resistance, which results in the development of antibiotic-resistant pathogens [28]. However, weather horizontal transfer of antibiotic resistance-associated genes occurs among the isolated species is largely unknown.
In summary, the present study used the culture method to analyze stool samples from two patients with asymptomatic C. difficile colonization in Guizhou province. This is the first report of microbial diversity in C. difficile carriers in southwest China, where specific dietary habits are prevalent, with a preference for pickled and spicy food. The results of the present study may improve the awareness of CDI among clinicians and provide new options for CDI treatment in southwest China. Figure S1. Phylogenetic tree of the 111 isolated strains that co-colonized with C. difficile in asymptomatic patients. The evolutionary history was determined using the Maximum Likelihood method based on the Tamura-Nei model [14]. Green represents strains that have been reported as normal human habitats and may act as protective taxa against CDI. Red represents human pathogens co-colonizing with C. difficile. Black represents bacteria of which the pathogenicity to humans is unknown (except C. difficile).