Bacterial isolation and identification
During our routine surveillance of bacillary dysentery, 38 S. dysenteriae were only found on six farms in Gansu Province. Detailed information on the study isolates is listed in Table S3. Among them, 20 isolates were isolated from 3 beef cattle farms, and 18 isolates were isolated from 3 dairy farms. There are 14 and 10 S. dysenteriae isolates from the same farms of Zhangye and Jinchang, respectively.
All 38 S. dysenteriae isolates were type 1 according to the results of the serotype reactions. In addition, based on the typical biochemical characteristics of Shigella spp., analysis of biochemical reactions indicated the presence of 3 biotypes (BTs) among these isolates (Table 1, Table S1). Among these BTs, BT2 (ability to ferment glucose, arabinose, and melibiose) was the predominant biotype, accounting for 86.84% (33/38). Furthermore, BT2 was widely isolated from each locus, with the exception of Baiyin.
MLST-based genotype analysis
Thirty-eight S. dysenteriae isolates belonged to 4 MLST patterns (STs): ST57, ST191, ST228 and ST229. Among them, ST57 and ST191 were previously reported, while ST228 and ST229 were novel types. The allele number for each locus and the designation of the ST are listed in Fig 1 and shared on the EcMLST website. The most common STs identified were ST228 (n=13) and ST229 (n=15), accounting for 73.68% (28/38). ST228 and ST229 were major ST types for dairy cows (Jinchang and Wuwei) and beef cattle (Zhangye and Baiyin) farms, respectively. In addition, these two ST types were different by 3 loci: arcA, clpX, mutS. In this study, each farm has a fixed ST type isolates, except Zhangye which contains ST229 and ST57 types (Fig 1, Fig 2).
PFGE-based genotype analysis
The genotypes and genetic relatedness of the 38 isolates were further determined by using PFGE. PFGE patterns of these S. dysenteriae isolates were heterogeneous; however, multiple PFGE patterns were present among these strains (Fig 3). With approximately 80% similarity, XbaI-digested S. dysenteriae type 1 could be divided into 28 distinctive PFGE patterns (PT) and belonged to two major groups: A (A1-A4) and B (B1-B3), with 66% similarity. The cluster result of PFGE was similar with MLST, however, the same ST strain can be divided into several similar PT types. For example, the ST229 type is divided into 10 PT types, and the ST228 type is divided into 9 PT types. Interestingly, most strains in different geographical locations can be clustered individually, and strains in the farm can be divided into multiple PT types.
Prevalence of virulence genes
A total of five virulence genes were detected in those isolates involving ipaH, ipaBCD, ial, sen, and stx. The most frequently observed virulence genes are ipaH (100%), ipaBCD (92.11%), stx (73.68%), and ial (57.89%). The Shigella enterotoxin genes sen (28.95%) are occasionally present in Wuwei and Jinchang isolates. None of the studied strains possessed the set1A or set1B gene.
Regarding the differences in virulence gene distributions, the 38 S. dysenteriae isolates fell into 5 gene profile types (VT) (Table 2). Among these VTs, VT IV (n=17) and VT V (n=11) were the most common, accounting for 44.34% and 28.95%, respectively. One interesting finding was the presence of the same and/or similar VTs in the same locus. In addition, 92.11% of isolates carried two or more virulence genes. Three Linxia isolates (7.89%) belonged to VT I, which was only positive for ipaH.
Antimicrobial resistance profiles
The antimicrobial resistance profiles of the 20 antimicrobials for 38 S. dysenteriae are shown in Table 3. All S. dysenteriae isolates were uniformly multidrug resistant to at least 3 types of antimicrobial agents. Among them, resistance to E was the most common (36,94.74%), followed by AMP (35, 92.11%), KZ (33, 86.84%), CRO (32, 84.21%), CTX (32, 84.21%), TE (31, 81.58%), CN (31, 81.58%), ENR (26, 68.42%), LEV (25, 65.79%), CIP (25, 65.79%), NOR (15, 39.47%), OFX (15, 39.47%), C (15, 39.47%), and S (15, 39.47%). Fortunately, all 38 isolates were sensitive to AMC, FOX, FEP, MEM, IPM and AK. However, the resistance rate of fluoroquinolone antibiotics has reached 39.47% to 68.42%, which will narrow the choice of antibiotics.
Moreover, most of the isolates (29/38, 76.32%) were resistant to fluoroquinolone antibiotics. And 4 isolates were resistant to other fluoroquinolones other than CIP, including the resistance of SD020 to ENR and the resistance of SD002, SD026, SD036 to NOR and OFX. These resistant isolates were divided into 5 antimicrobial-resistance profile types (RT) (Table 4). Among these RTs, RT 4 (n=11) and RT 5 (n=12) were detected easily, accounting for 37.93% and 41.38%, respectively. All of the fluoroquinolone-resistant isolates were multidrug resistant (MDR). To be specific, 41.8% (12/29) of fluoroquinolone-resistant isolates were resistant to each class of antibiotics. Regarding the test for the virulence genes of ipaBCD, sen, stx, there are statistically significant difference between RT type and fluoroquinolone sensitive strains (P＜0.05) (Table 5).
In addition, the resistance profiles of strains which isolated from the same farms were very similar. And the isolates belonging to ST229 (A1 and A2 groups) and ST228 (B group) were resistant to fluoroquinolone antibiotics, while the isolates belonging to ST 57 (A4) and ST191 (A3) were sensitive to those antibiotics, except for SD020 isolated from Zhangye area with ST57 (PT15).
ARG analysis for fluoroquinolone-resistant S. dysenteriae isolates
To determine the molecular characterization in fluoroquinolone-resistant S. dysenteriae, both SNPs in QRDR of gyrA/B and parC/E genes and PMQR genes were analyzed. In 29 fluoroquinolone-resistant isolates, there was no strain that displayed mutations in the gyrB and parE genes, although some mutations in gyrA and parC were identified in each resistant isolate (Table 4). All resistant isolates in the present study carried common mutations in gyrA codon 83 (S→L) and parC codon 83 (S→L). Furthermore, the isolates with ST228 only carried the mutations in gyrA codon 83 (S→L) and parC codon 83 (S→L). While most of the isolates with ST229 carried all of the four mutated locuses, except isolates from Baiyin.
The PMQR genes qnr, aac(6’)-Ib-cr, and qepA occur worldwide and are increasingly detected in clinical isolates of Enterobacteriaceae [15,16]. In our study, aac(6’)-Ib-cr was the most common PMQR gene and harbored by each isolate; however, all isolates were negative for qepA, except SD001. Only seven (7/29, 24.14%) isolates harbored the qnr gene, and no isolate harbored qnr, aac(6’)-Ib-cr, and qepA simultaneously. Interestingly, the qnrB (n=2) gene and qnrS (n=5) were belonged into ST228 and ST229, respectively.