Invasive diarrhea due to Shigella species remains an important public health problem in developing countries.22 All four species of Shigella can cause shigellosis, but S. flexneri and S. sonnei are the most prevalent23. Present study confirmed the existence of diverse S. dysenteriae isolates in cows in local epidemiological studies. The rate of S. dysenteriae isolation from the diarrhea of cows was 1.14% (38/3321), and all the S. dysenteriae isolates were type 1.
Strain molecular characterization is important for epidemiological studies. In the present study, 38 S. dysenteriae isolates from cows were separated into 4 STs. Comparing the 15 housekeeping genes, only 4 allele genes (aspC, cyaA, icdA, and uidA) were identical. In our study, the majority of isolates (n = 13 and n = 15) belonged to new ST types ST228 and ST229. However, ST148, ST252, and ST1739 were previously reported in human isolates.24 PFGE is the most common typing procedure currently used with Shigella spp. because of its high discriminatory power.25 Taking PFGE as a reference epidemiological tool, the S. dysenteriae isolates in this study were heterogeneous and distributed into 28 PTs. The clustering of these diverse PTs allows us to learn more about the epidemiological characteristics of S. dysenteriae in specific geographical regions.
The ability of Shigella spp. to cause shigellosis is attributed to the expression of arrays of virulence genes associated with colonization, invasion/penetration and toxin-mediated disease.26 Virulence genes responsible for the pathogenesis of shigellosis are often multifactorial and coordinately regulated.27 Estimating the existence of virulence determinants in Shigella would help us better understand its pathogenicity.26 IpaH takes responsibility for the strain spread from cell to cell and the modification of host response to infection.28 All Shigella spp. were positive for ipaH as a typical marker because this gene exists in multiple copies on both the chromosome and the invasion plasmid. Additionally, in this study, most isolates simultaneously harbored the stx, ial and sen genes. The diversity of the observed virulence genes results in dysentery in hosts. Notably, these virulence genes are indistinguishable from human Shigella.
For shigellosis, antibiotic therapy can reduce the duration and severity of the illness.29 Based on previous reports, fluoroquinolones and third-generation cephalosporins are the best choice for empiric treatment for severe bacterial diarrheas caused by S. dysenteriae.30 However, resistance will emerge to any antimicrobial agent used intensively, and the differential selection pressures of antimicrobials lead to differences in drug resistance.31 These factors have contributed to Shigella strains acquiring resistance to the latest drug of choice, together with other effective drugs after therapy shigellosis. In addition, the resistance rate of S. dysenteriae 1 isolates to fluoroquinolones has achieved at an alarming rate, and the isolates have demonstrated multidrug resistance. Our current study indicated that continuous surveillance of the resistance pattern in pathogens would be essential for the choice of appropriate antimicrobial therapy and control the spread of Shigella.
The most common mechanism of the high level resistance to quinolone has been mostly attributed to accumulation of sequential mutations in DNA gyrase and DNA topoisomerase IV, typically of gyrA at codon 83 and/or 87, and of parC at codon 80.16,32 In addition, different mutation loci and different mutations at the same locus may result in different quinolone susceptibility levels.33,34 To date, limited studies have examined quinolone resistance mechanisms among Shigella isolates in animals. In the current study, we successfully investigated fluoroquinolone resistance-conferring mutations by sequencing QRDR of S. dysenteriae 1 isolated in different geographical locations. According to our results, only two main combinations of mutations were observed: gyrA codons 83 (Ser→Leu), and parC codons 83 (Ser→Leu) (55.17%, 16/29); gyrA codons 83 (Ser→Leu), 87 (Asp→Asn) and parC codons 80 (Ser→Ile), 83 (Ser→Leu) (41.38%, 12/29). This phenomenon of high resistance rate and single mutation pattern is likely related to the large-scale use of single quinolone antibiotics by veterinarians.
Generally, the PMQR determinants are located on mobile genetic elements, which may be associated with mobile or transposable elements among members of the Enterobacteriaceae family.35 Over the past few years, the dissemination of PMQR genes among quinolone-resistant Shigella isolates has been surveyed in some studies and has emerged as an important issue across the world.36–39 It has been reported that aac(6’)-Ib-cr significantly increases the frequency of selection of chromosomal mutants to reduce ciprofloxacin activity by N-acetylation at the amino nitrogen on its piperazinyl substituent.40 The aac(6’)-Ib-cr–positive Shigella was first isolated in 1998;41 however, the most striking finding of our previous and present study was the aac(6′)-Ib-cr allele wide penetration in all quinolone-resistant isolates.42,43 The qnr family could protect DNA gyrase against quinolones and confer low-level resistance to nalidixic acid and reduced susceptibility to fluoroquinolone, especially to ciprofloxacin.44 The qnr gene has occurred worldwide and contains a variety of subtypes, but it was not a common epidemic genotype according to our study. Otherwise, the plasmid-mediated qepA gene can encode a 14-transmembrane segment efflux pump and extrude several quinolones from bacteria, thereby conferring low levels of resistance to these antimicrobial agents and favoring the selection of new alterations that are able to induce full quinolone resistance.45,46 Compared with other PMQR determinants, qepA is a new resistance gene, first described in 2007,47 so it is not difficult to understand the lowest frequencies.
The QRDR mutation and PMQR determinants are both important to quinolone resistance. Compared with mutations in the QRDR, PMQR could not be the direct reason for quinolone and fluoroquinolone resistance.48 However, the presence of PMQR genes may facilitate the selection of QRDR mutations that result in higher levels of quinolone resistance.
In conclusion, this study described fluoroquinolone resistance and virulence genes among S. dysenteriae strains isolated from cows and the molecular characterization involved. To systematically understand S. dysenteriae, PFGE and MLST methods were applied to genetically characterize the 38 isolates. PFGE based on XbaI digestion divided the 38 isolates into 28 PTs, while MLST based on 15 housekeeping genes differentiated the 38 isolates into 4 STs. Although MLST provided suitable discrimination in S. dysenteriae subtyping, PFGE might exhibit a higher discriminatory ability. Overall, the data from this study will provide a useful typing resource, which will provide a scientific basis for addressing clinical and epidemiological issues regarding S. dysenteriae. Given this knowledge, continuous and extensive surveillance will be essential to explore and prevent the spread of the epidemic.