A total of 54 S. flexneri of six serotypes, including 1a (n=5), 2a (n=26), 2b (n=4), 4a (n=6), 6 (n=8), and Xv (n=5), were analyzed in this study. Based on the results of the biochemical reaction assays, we observed that all 54 S. flexneri isolates possessed 4 typical Shigella biochemical characteristics (Table 1). Among these BTs, BT4 (the ability to ferment glucose, mannitol, arabinose, and melibiose) was the predominant biotype, accounting for 70.37% (38/54) of all BTs. Furthermore, BT4 was widely found in each serotype, except serotype 6. S. flexneri 2a was distributed among all four biochemical phenotypes and mainly in BT4 (22/26, 84.62%). However, the other five serotype strains only had one or two biochemical phenotypes.
The frequencies of the virulence factor profiles in the S. flexneri isolates are listed in Fig 1. A total of seven virulence factors were detected in those isolates, including ipaH (100%), virA (100%), ipaBCD (92.59%), ial (77.78%), sen (79.63), set1A (48.15%) and set1B (48.15%). None of the studied strains possessed the stx gene. The Shigella enterotoxin genes set1A and set1B were only present in S. flexneri 2a, and all of these serotype isolates were positive for these two genes.
Regarding the differences in the distributions of the virulence factors, the 54 S. flexneri isolates fell into seven gene profile types (VT) (Table 2). Among these VTs, VT4 (positive for ipaH, virA, ipaBCD, ial, sen) and VT6 (positive for ipaH, virA, ipaBCD, ial, sen, set1A, set1B) were the most common, accounting for 29.63% and 44.44% of all VTs, respectively. Furthermore, 92.59% of the isolates carried two or more virulence factors. In addition, the virulence factor types were associated with the S. flexneri serotype. VT1 was only found in 4a, and VT4 was present in isolates from each serotype, except 2a. S. flexneri 2a major belonged to VT6 (24/26, 92.31%).
MLST-based genotype analysis
MLST was performed to analyze the genotypic diversity of S. flexneri isolates based on 15 housekeeping genes. The 54 isolates were divided into seven STs, including ST68, ST100, ST103, ST120, ST124, ST135 and ST227. Among them, ST227 was novel, while the six other STs have previously been reported. These seven STs belonged to several clonal complexes (CCs): CC10 (ST100 and ST103), CC26 (ST68), and others (ST120, ST124, ST135 and ST227). The clustering tree (Fig 2) based on the MLST data showed that ST68 was a singleton type and that the other six STs contained two or more isolates. The most common ST was ST100 (n=33, 61.11%), including isolates of serotypes 1a, 2a, and Xv. All the isolates of ST124 and ST227 belonged to S. flexneri 6 and 4a, respectively. The cluster tree indicated that isolates belonging to the same serotype closely clustered based on the province of isolation. In addition, according to the minimum spanning tree (MST) based on the allele, it was found that ST100, ST120 and ST135 had closer relationships and only differed in aspC, while ST68, ST124 and ST227 were very different from the other STs (Fig 3).
MLVA-based genotype analysis
MLVA based on eight VNTR loci was performed to further characterize the isolated S. flexneri strains. The copy numbers of the eight VNTR loci are listed in Fig 4. Overall, the 54 isolates based on the unique MLVA profiles were discriminated into 39 different MLVA types (MTs). Among them, twenty-eight MTs belonged to the singleton type, and the other ten MTs contained no more than three isolates. The MLVA cluster tree of the isolates showed that they were divided into five clusters, designated A to E, with a low coefficient of similarity from 20%-60% (Fig 4). Each cluster was further divided into many subclusters. MLVA can cluster different serotype strains separately and distinguish between the same serotype strains. The main cluster, cluster C, was observed clustering S. flexneri 2a isolates and further divided into 15 MTs. Additionally, clusters A (except GBSF1502176), D and E only clustered the Xv, 2b, and 6 serotype strains, respectively. The results showed differences based on the geographical origin and time span in the same serotype.
PFGE-based genotype analysis
The genotypes and genetic relatedness diversity of the 54 S. flexneri isolates were assessed by PFGE. NotI-digested S. flexneri chromosomal DNA generated 31 reproducible unique PFGE patterns (PTs), each with 11-16 bands (Fig 5). Eleven patterns were represented by more than one isolate, with PT20 (n = 8) containing the most isolates, followed by PT18 (n =5). The dendrogram of S. flexneri isolates showed low similarity (40%-60%) and could be classed into three gross clusters on the basis of their serotypes: clusters A, B and C. Isolates belonging to the same serotype but recovered in different years showed clear relatedness, as indicated by their grouping in the same clusters. The majority of serotype 2a isolates, with the exception of isolate QYSF1511395, grouped together in cluster B. The QYSF1511395 strain isolated from Qinghai Province clustered independently in cluster C. Isolates 1a, 2b and Xv clustered into cluster B and were closely related to the serotype 2a isolates. However, the isolates of serotypes 4a and 6 were assigned to cluster A with a relatively close relationship, but different serotype strains clustered separately.