Genetic diversity and epidemiology of accessory gene regulator loci in Clostridioides difficile

Abstract

Quorum sensing is known to regulate bacterial virulence, and the accessory gene regulator (agr) loci is one of the genetic loci responsible for its regulation. Recent reports examining Clostridioides difficile show that two agr loci, agr1 and agr2, regulate toxin production, but the diversity of agr loci and their epidemiology is unknown. In our study, in silico analysis was performed to research genetic diversity of agr, and C. difficile isolates from clinical samples underwent multilocus sequence typing (MLST) and PCR analysis of agr loci. To reveal the distribution of agr among different strains, phylogenetic analysis was also performed. In our in silico analysis, two different subtypes, named agr2R and agr2M, were found in agr2, which were previously reported. PCR analysis of 133 C . difficile isolates showed that 131 strains had agr1, 61 strains had agr2R, and 26 strains had agr2M; agr2R was mainly found in clade 1 or clade 2 organisms, whereas agr2M was only found in clade 4. With rare exception, agr1-negative sequence types (STs) belonged to clade C-Ⅰ and C-Ⅲ, and one clade 4 strain had agr2R. Our study revealed subtypes of agr2 not previously recognized, and the distribution of several agr loci in C. difficile . These findings provide a foundation for further functional and clinical research of the agr loci.

Keywords: Clostridioides difficile; accessory gene regulator; multilocus sequence typing; phylogenetic analysis; quorum sensing.

Fig. 1.

Genomic organization of (a) agr1, (b) agr2R and (c) agr2M loci. The targeted regions of the primer pairs in Table 1 are also shown, with the expected length of amplicons shown.

Fig. 2.

Multiple sequence alignment of Agr2R and Agr2M by Clustal Omega. Comparisons between amino acid sequences of AgrB, AgrD, AgrC and AgrA are shown. Symbols are as follows: an asterisk indicates identical, a colon means strongly similar, and a comma indicates weakly similar.

Fig. 3.

Visualization of PCR bands from agr1, agr2R and agr2M screening. 1: strain CD630 (laboratory strain); 2: strain R20291 (laboratory strain); 3: ST54/lade 1 (clinical isolate); 4: ST17/clade 1 (clinical isolate); and 5: ST81/clade 4 (clinical isolate).

Fig. 4.

Maximum-likelihood phylogenetic tree (model TN93, 1000 replicates) based on analysis by PhyML. STs found in our clinical samples; ST1 (corresponding to R20291 strain) and STs used in previous studies as references for clade C-Ⅰ, C-Ⅱ and C-Ⅲ underwent this analysis. Circles are drawn on branches with bootstrap value over 50. ●: agr1; ▽: agr1 +22R; □: agr1 +22M ; ◎: no agr locus; *: reference strains.