Introduction

Clostridioides difficile is an important pathogen of antimicrobial-associated diarrhea and nosocomial diarrhea in humans and has been ranked as the highest level of “Urgent threats” by the Centers for Disease Control and Prevention (CDC) in the USA (https://www.cdc.gov/).1 The clinical outcomes of C. difficile infection (CDI) range from self-resolving diarrhea to life-threatening pseudomembranous colitis and toxic megacolon, and even death.2 Two structurally similar toxins, toxin A (TcdA, an enterotoxin) and toxin B (TcdB, a cytotoxin), are the main virulence determinants associated with CDI.3 Furthermore, a third toxin, the binary toxin (CDT), has been linked to an increased severity of human infections, as evidenced by the hypervirulent C. difficile NAP1/BI/027 strain.4 Nevertheless, in practice, not all toxin-producing C. difficile strains produce the toxins mentioned above.

As a TcdA-negative and TcdB-positive strain, C. difficile sequence type (ST) 37 is regarded as one of the most prevailing C. difficile STs, which has resulted in widespread CDI and global outbreaks.5 In Asia, alongside C. difficile ST37, ST81 (also a TcdA-negative and TcdB-positive strain) emerges as another common genotype, notably associated with ribotype (RT) 369.6,7 In initial molecular epidemiological investigations, C. difficile ST81 was only found at a low prevalence and it was a predominantly endemic strain.8 However, recent studies have indicated that C. difficile ST81 has gradually replaced ST35, ST3, and ST37, becoming a prevalent strain in some regions throughout mainland China.6,9 For example, ST81 accounted for 26.4% of all isolated clinical toxigenic C. difficile in four tertiary hospitals in Beijing, China.6 Furthermore, using whole genome sequencing (WGS) to trace the origins of infection, C. difficile ST81 strains have been reported to cause outbreak in a tertiary hospital in Shanghai.10 It is interesting to note a report by Liu et al., in which the authors found that patients infected with C. difficile ST81 were more likely to have lower survival rates than those infected with non-ST81 strains.11 In order to elucidate the epidemiological characteristics of C. difficile ST81 isolates, several genomic studies have been carried out. One genome analysis study has revealed that C. difficile ST81 has a high-frequency amino acid mutation in the gyrA (Thr82Ile), which confers fluoroquinolone resistance.6 Compared with C. difficile ST37, C. difficile ST81 isolates exhibit an enhanced ability to transmit between hosts and survive in harsh environments with robust colonization, enhanced spore production, and slightly increased motility.12

However, given the potential for C. difficile ST81 transmission, high antimicrobial resistance and poor prognosis, research into the epidemiological characteristics of this strain remains limited. Compared with other STs, the molecular characteristics, such as the genetic diversity, the population structure, genome evolution, and pathogenicity in C. difficile ST81 in China need to be addressed. Here, we performed WGS and high-resolution phylogenomic analysis on C. difficile ST81 isolates in China with the aims to: 1) characterize the genetic diversity and population structure; 2) identify resistance mechanisms; and 3) analyze the phylogenetic relationships of C. difficile ST81 isolates. Overall, it is hoped that our data might provide a solid foundation for future in-depth investigations of functional determinants in C. difficile ST81 to inform novel strategies for improving infection prevention and control, as well as patient management.

Materials and Methods Collection of C. difficile Isolates

Between January 2010 and January 2021, stool samples were collected from adult- patients with diarrhea required for toxigenic C. difficile detection at five tertiary hospitals across different regions of China (HA, HB, HC, HD, HE). The stool sample was cultured anaerobically on cycloserine-cefoxitin-taurocholate agar (CCFA-TA; Oxoid) supplemented with 7% (v/v) sheep serum at 35°C for 48 hours before isolation. The suspected C. difficile colonies were identified using Brooke Matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF) (Bruker Daltonik GmbH, Bremen, Germany).

Detection of Toxin Genes by Polymerase Chain Reaction (PCR)

Following the 48 h of anaerobic blood agar culture, C. difficile isolates were suspended in distilled water in a microcentrifuge tube. Genomic DNA was then extracted using the simplified alkaline lysis method. All isolated strains were tested for tcdA, tcdB, and binary toxin genes by PCR, as previously described.13 Briefly, the primer pairs were NK9/NK11 for tcdA, NK104/NK105 for tcdB, cdtApos/cdtArev for cdtA, and cdtBpos/cdtBrev for cdtB. PCR amplification with primer pair NK9/NK11 was performed for 35 cycles, consisting of 95°C for 20s and 62°C for 120 s. The thermal profile for primer pairs NK104/NK105 was 35 cycles comprising 95°C for 20 s and 55°C for 120 s. Reactions for cdtA and cdtB genes were subjected to 30 cycles of 94°C for 45 s, 52°C for 60s and 72°C for 80 s. C. difficile strain ATCC BAA-1870 (ribotyping 027) was used as the positive template control of toxin genes.

Multi-Locus Sequence Typing (MLST)

To characterize genetic diversity and determining STs of C. difficile isolates in the study, MLST of seven housekeeping genes (adk, atpA, dxr, glyA, recA, soda, and tpi) was used to genotype all toxigenic isolates according to previously described protocols.14 Briefly, the amplification conditions of each housekeeping gene were 95°C for 15 min, followed by 35 cycles of 94°C for 30 s, 50°C for 40 s, and 72°C for 70 s. Allele designations were obtained through the C. difficile PubMLST batch profile query page to determine the ST, as previously described.8

Genome Sequencing and Assembly

Sequencing-quality genomic DNA of all C. difficile strains identified as ST81 were extracted using a DNeasy Blood and Tissue kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions, and quantitated using a Qubit 2.0 system. WGS was performed on an Illumina NovaSeq 6000 instrument (Illumina, San Diego, CA, USA), reaching a sequencing coverage of 200X. Before assembly, quality control of raw sequenced reads was performed using FastQC and adapter regions were trimmed using Trimmomatic.15 Trimmed reads were assembled de novo using SPAdes.16 The total assembly size of genome is approximately 4.2 Mb, with a scaffold N50 length of approximately 0.5Mb and the GC content of approximately 28.93%. Genomes were annotated using the Prokka.17

Phylogenetic and Evolutionary Analyses

To investigate population structure, a maximum-likelihood tree was constructed based on core-genome alignments obtained in Roary using MEGA 11 with 1000 bootstrap replicates and visualized using the Interactive Tree of Life web server.18,19 Further, to investigate the transmission events of C. difficile ST81, single nucleotide polymorphisms (SNPs) variants were called using Snippy (http://github.com/tseemann/snippy) with default parameters. The C. difficile M68 (NC_017175.1) chromosome served as the reference. The alignment file was filtered from variants with elevated densities of base substitutions as putative repetitive regions, mobile genetic elements (MGEs) and recombination events by Gubbins and used to calculate pairwise SNPs by SNP-dists (https://github.com/tseemann/snp-dists).20 The minimum spanning trees were constructed in PHYLOViZ based on the generated pairwise SNP tables.21

To explore the evolution of C. difficile ST81, Bayesian evolutionary analysis was performed using BEAST, which employs three clock models, two population models, and two site models, as described previously by Xu et al.22

Identification of Antimicrobial Resistance (AMR) Genes, Virulence Genes, and Transposons

To investigate the genetic characteristics of C. difficile ST81, AMR genes were detected based on the Comprehensive Antibiotic Resistance Database (CARD) (https://card.mcmaster.ca). Further, virulence genes were identified based on the local Virulence Factor Database (VFDB) (https://www.mgc.ac.cn/VFs/). For mutation analysis, gyrA and gyrB sequences were extracted and compared with reference sequences downloaded from the CARD using the Basic Local Alignment Search Tool (BLAST). Transposable elements were identified using Vrprofile2.23

Pangenome-Wide Association Study (Pan-GWAS) and Clusters of Orthologous Groups of Proteins (COG) Analysis

The Bayesian analyses identified two distinct C. difficile ST81 sub-lineages, namely sub-lineage I (SL I) and SL II. To determine significant genetic loci associated with each sub-lineage, a pan-GWAS of all C. difficile ST81 genomes was performed. Briefly, all C. difficile ST81 genome annotations were performed with Prokka.17 Roary was employed to estimate the size of the core and accessory genomes, and the results were used as an input for Scoary to identify the significant genetic loci associated with each sub-lineage.19,24 Moreover, genes were extracted from all genomes using an in-house Python script and uploaded to eggNOG-mapper (http://eggnog-mapper.embl.de/) to explore gene function.

Data Analysis

Statistical analyses were performed using SPSS version 23.0 (SPSS, Chicago, IL, USA).

Results Epidemiologic Analysis and Characterization of C. difficile Isolates

In total, 871 (9.0%) non-duplicate toxigenic C. difficile isolates were identified from 9675 patients suffering from diarrhea during the period of study. Among these isolates, a total of 59 STs were identified (data not shown), with ST81 accounting for 12.4% (108/871). Most C. difficile ST81 strains were isolated from inpatients (94.4%,102/108) while 5.6% (6/108) were isolated from outpatients. During this period, strains of C. difficile ST81 were isolated each year from the five participating hospitals, but the distribution of the C. difficile strains varied between the hospitals over time (Figure 1). In Hospital A (HA), the number of C. difficile ST81 isolates showed an increasing trend from 2016, peaking in 2020, while Hospital C (HC) had a significantly high number of isolates in 2015.

Figure 1 The distribution of 108 isolates in different hospitals and years.

Phylogenetic Analyses and Genomic Features of C. difficile ST81

All 108 WGS data were analyzed after sequence quality control and mapping to C. difficile M68 (NC_017175.1). A total of 11,950 genes were predicted to comprise the pangenome of C. difficile ST81. The core genes (ie, genes present in more than 99% of the genomes), which is usually used to evaluate the genomic diversity within species, represented 29.9% (3568/11,950) of the pangenome, while cloud genes, shell genes, and soft-core genes comprised 63.8% (7630/11,950), 5.8% (695/11,950), and 0.5% (57/11,950), respectively.

Seven AMR genes were identified while ermB and tetM were detected in all but one strain each (s15013004 and s13071105, respectively) (Figure 2A). The clbA, dfrF, and cfrB resistance genes were identified in a small number of strains. Furthermore, the majority of C. difficile ST81/RT369 isolates were found to have gyrA mutations (91.7%, 99/108), which manifested as a Thr82Ile amino acid mutation conferring fluoroquinolone resistance.

Figure 2 Phylogenetic analysis of 108 C. difficile ST81 strains based on single nucleotide polymorphisms (SNPs) in the core genome. (A) A phylogenetic tree was constructed, illustrating the relationships between strains and describing the strain number, antimicrobial resistance, transposons, and virulence genes in each hospital. (B) A Minimum spanning tree of C. difficile ST81 reveals formation of developmental clusters of clonal transmission based on core-genome single nucleotide polymorphisms.

As essential mobile genetic elements, integrative and conjugative elements are responsible for horizontal gene transfer, driving increased genetic diversity, and the acquisition of exogenous genes.25 In these 108 isolates, only two kinds of transposons, Tn916, and Tn6189, were identified. With the exception of one, we identified Tn916 in most C. difficile ST81/RT369 isolates while 89.8% (97/107) of isolates contained Tn6189. Tn916, which encodes resistance to tetracycline and minocycline via Tet (M), is a major family of transposon reported in C. difficile, while Tn6189, described for the first time in 2019, is a carrier of ermB gene.26,27

To establish in-depth phylogeography of C. difficile ST81 in China, cgSNP calling was performed. The cgSNP alignment consisted of 1,589,277 bp. Pairwise cgSNP distance between the strains was on average 13 cgSNPs (range, 0–425 cgSNPs). For improved resolution of C. difficile ST81 phylogeny, we constructed a maximum-likelihood phylogenetic tree and a minimum spanning tree based on these cgSNPs (Figure 2B). Both methods indicated the occurrence of highly similar strains in very different regions in China.

Molecular Evolution and Transmission of C. difficile ST81/RT369 in China

To explore evolutionary patterns of C. difficile ST81, all genomes with details of collection dates were used to construct the Bayesian phylogeny tree. Based on the topology of the tree, the constructed Bayesian evolutionary tree indicated the presence of two genetically diverse sub-lineages, designated SL I and SL II (Figure 3A), and estimated that SL I emerged with the most recent common ancestors in ~2009 while SL II emerged in ~2010 (median estimates of 95% highest posterior density intervals were 2003 to 2009 for all strains of C. difficile ST81). Moreover, the Bayesian evolutionary tree also suggested that both sub-lineages emerged independently. The distribution of strains in SL I was non-hospital and non-time dependent while the strains in SL II were mostly isolated from Hospital A. Interestingly, all of the strains from SL II carried resistant genes ermB and tetM, the gyrA mutation, and Tn916 while the clbA, dfrF, and cfrB resistance genes were only detected in SL I.

Figure 3 Evolution of 108 C. difficile ST81 genomes over time. (A) A Bayesian phylogenetic tree was constructed, describing the strain number, antimicrobial resistance, transposons, and virulence genes in each hospital. Two major sub-lineages were defined: SL I and SL II. (B) A Manhattan plot of the associations of genes in the two sub-lineages based on genome-wide association analysis. Association of genes with phenotypes was determined using the multiple Kruskal–Wallis test. (C) Distribution of differentially expressed proteins among two C. difficile ST81 sub-lineages according to COG functional categories.

Pangenome Analyses

To investigate the variability between the two different C. difficile ST81 sub-lineages identified by Bayesian analysis, we conducted pangenome analysis of these isolates. Comparison of the two different C. difficile ST81 sub-lineages led to the identification of 2 and 15 unique genes in SL I and SL II isolates, respectively (Figure 3B). To gain an understanding of the function of these unique genes in C. difficile ST81 isolates, significant unique genes in both sub-lineages were assigned to COG functional categories. More unique genes in SL II were enriched in various COG functional categories compared with SL I (Figure 3C and Table 1). The largest proportion of unique genes belonged to function unknown, followed by transcription and defense mechanisms. It is important to note that signal transduction mechanisms, cell wall/membrane/envelope biogenesis, replication, recombination, and repair, as well as carbohydrate transport and metabolism genes in SL II isolates were much more abundant than those in SL I isolates, which may endow these isolates with the ability to defend against adverse stimuli, allowing them to use any extra energy to better adapt to the environment.

Table 1 Functional Annotation of Differentially Expressed Genes Between SL I to SL II

Discussion

It has been reported that the prevalence of CDI in mainland China was 14% while ST81 is among the common STs of C. difficile.28,29 In this longitudinal and systematic surveillance on the status of CDI in China, a multi-center study was undertaken with five large tertiary hospitals to improve our understanding of the molecular epidemiology of C. difficile ST81. Herein, we found that C. difficile ST81 accounted for 12.4% of the total toxigenic C. difficile isolates. Phylogenetic tree analyses indicated the occurrence of highly similar strains in very different regions in China. Two distinct sub-lineages of C. difficile ST81 were identified based on differences in genomic structure.

Several studies of C. difficile have shown that C. difficile ST37/RT17, a TcdA-negative and TcdB-positive (A-B+) clone, is the most prevalent clone in Asia.5 However, other studies have also identified A-B+ genotype ST81 as the prevalent clone and causative agent of multiple outbreaks in China, since its identification in 2010.30 In the present study, although C. difficile ST81 was not the most common genotype, this ST accounted for 12.4% of the analyzed isolates, which was similar to data obtained in a Japanese multi-center surveillance study between April 2012 and March 2013.7 However, in recent years, several studies in China have shown that ST81 was the main common genotype responsible for C. difficile outbreaks.6,9 Although the exact cause remains unclear, some studies have suggested that this may be due to the increased resistance rates along with a higher production of treatment-resistant spores in this genotype.9,31 However, the composition of C. difficile ST81 strains varied in the studied hospitals.6 Similar trends were observed in this study, which suggests the need to establish a surveillance network as an important strategy for epidemiological monitoring of C. difficile, as well as for CDI transmission control.

AMR plays a significant role in the pathogenesis and spread of CDI, as it allows C. difficile to survive antimicrobial exposure in the host, while selective pressure allows the emergence and spread of AMR strains.32 Notably, fluoroquinolone resistance resulting from a gyrA (Thr82Ile) mutation in C. difficile RT027/ST1 is thought to have significantly facilitated its rapid expansion and dissemination in North America and Europe in the early 2000s.33C. difficile ST81 isolates have been shown to have the highest in vitro resistance rates for many antimicrobial classes, such as fluoroquinolones (ciprofloxacin, levofloxacin, and moxifloxacin), tetracyclines, macrolide-lincosamide-streptogramin B (MLSB).6 Although antimicrobial susceptibility tests were not performed for these C. difficile ST81 isolates in the present study, previous research has confirmed that the resistant phenotype and genotype showed good correlation.34 In this work, we found that most of the isolates carried the resistant genes (tetM, ermB) and mutations in gyrA were detected in almost all strains. It is interesting to note that the cdeA gene, described as a multidrug efflux transporter belonging to the multidrug and toxic compound extrusion family (MATE-family), has been shown to confer resistance not only to ethidium bromide and acriflavin but also to antibiotics, particularly fluoroquinolones and, in some cases, aminoglycosides. Remarkably, this gene was present in all isolates.35 However, in another study, this gene was found both in levofloxacin-resistant and susceptible strains.36 Further research into the role of the cdeA gene is required to elucidate the mechanisms behind this. Intriguingly, while resistance to rifamycin is characteristic of C. difficile ST37/RT17, none of rifamycin resistance genes were identified in isolates analyzed in this study.13 This phenomenon underscores the possibility that while C. difficile ST81 and C. difficile ST37/RT17 are the main genotypes of C. difficile clade 4, these two genotypes may have different molecular features.12 Another interesting note is that Tn916 and Tn6189 are found in most C. difficile ST81 isolates. These two transposons carry resistance genes (tetM and ermB, respectively), which may help explain why C. difficile ST81 harbors the aforementioned resistance genes. This also highlights the potential role of transposons in the horizontal transfer of resistance genes.

C. difficile ST81 has been reported to cause nosocomial transmission in a general hospital in China.10 The present study followed the approach defined by Eyre et al., in which a threshold of 0–2 cgSNPs was used to determine whether groups of two or more strains were clonally related.37 The constructed phylogenetic tree based on the cgSNPs provided a clear interpretation of the evolutionary relationship between all of the C. difficile ST81 isolates in this study, which showed that strains in the same clade are more closely related to each other and may have a common evolutionary ancestor. As the isolates were collected over time across China, and because no intersection of time and space was found after reviewing the clinical information, we could not conclude that this genotype caused spatial-temporal transmission in China. However, we have reason to believe that this genotype evolved more slowly while C. difficile ST81 showed low intra-ST cgSNP difference with a mean value of 13 cgSNPs. We also could not conclude that they likely had connections with the present outbreak or were exposed to a common source. These data indicate that isolates of C. difficile ST81 are genetically more closely related to each other compared with other ST lineages, and that related isolates can be identified regardless of geographical origin. The pangenome analyses showed an open pangenome, with the accessory genome comprising 70.1%. In comparison, other C. difficile lineages, like RT014 and ST11, have accessory genomes of 69.7% and 80.2%, respectively, which is similar to ST81.38,39 This finding necessitates additional research to understand the evolutionary causes and epidemiological implications of STs with open pangenomes. The low level of diversity found in this study suggested that ST81 was a persistent multi-province clone that was present in different provinces. Another study by Thiel et al. demonstrated that bacteria found in manure can escape into the atmosphere during agricultural land fertilization and be transported thousands of kilometers away.40,41 However, the true reasons for long-distance transmission or conservative core genome of C. difficile ST81 remain unclear and require further investigation.

Bayesian evolutionary analysis showed that C. difficile ST81 isolated from the present study formed two independent and stably transmitted sub-lineages based on the differences in structure. Although not exclusively containing strains from one region, the spread of C. difficile ST81 had occurred in ~2008, which was derived from the same most recent common ancestor. From this analysis, we inferred that the spread of C. difficile ST81 probably began with population movement in Hospital A before spreading to other hospitals in different regions. Although the real mechanism is unclear, given that the C. difficile ST81 genotype was first found in Japan, we hypothesized that as Hospital A is located in a major city with frequent exchanges involving Japanese people and international travelers, these interactions could have facilitated the transmission.7 Furthermore, there were significant difference in genomic characteristics in the two sub-lineages. Using GWAS analysis, we found that SL II isolates containing more unique genes belonged to signal transduction mechanisms and cell wall/membrane/envelope biogenesis, which may allow these isolates to better defend themselves against adverse stimuli and use any extra energy to better adapt to their environment. This further indicates that the SL II strain is isolated in only two regions.

It is important to acknowledge the limitations of this study. Although we collected isolates from patients with diarrhea in five different tertiary hospitals across mainland China, specific areas are needed for further research, such as expanding surveillance to more hospitals, and investigating environmental reservoirs. Secondly, we did not perform in vitro antimicrobial susceptibility tests for these C. difficile ST81 isolates, which meant that the lack of in vitro antimicrobial susceptibility testing limits the ability to correlate genetic resistance markers with actual resistance phenotypes. Thirdly, C. difficile ST81 is currently not a common genotype outside of China, and there is a lack of WGS data globally. Additionally, more in-depth functional studies on the unique genes found in SL II strains are necessary. Therefore, the representativeness in this work needs to be further studied by involving more isolates.

Conclusions

In this study, we investigated the genetic diversity of 108 C. difficile ST81 isolates with temporal and geographical variation. Our data suggests that C. difficile ST81 has high resistant rates to different antibiotics and is circulating in mainland China. Phylogeographic analyses of the cgSNPs identified through WGS of the isolates suggests that there are two main sub-lineages (SL I and SL II), which share ancestry, although SL II shows increased adaptability to the environment with relative genes. Considering the unusual antibiotic resistance and the conservation in core genes of C. difficile ST81, surveillance of this genotype needs to be further strengthened to improve disease control and reduce the risk of future outbreaks.

Data Sharing Statement

The genomic sequences of C. difficile ST81 isolates in this study were deposited in GenBank under accession number PRJNA432876. The data of this study are available by contacting the corresponding author upon reasonable request.

Ethics Approval

For this observational study, the need for patient consent was not required. Data were not identifiable back to the patients from whom they originated; an ethics approval was waived.

Funding

This study was supported by Basic Public Welfare Research Program of Zhejiang Province (No. LGF20H030004), National Nature Science Foundation of China (No. 82073609).

Disclosure

The authors report no conflicts of interest in this work.

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