Shigella spp. are a group of enteropathogenic bacteria consisting of four major species: Shigella dysenteriae, Shigella flexneri, Shigella boydii and Shigella sonnei. Despite the improvement in personal hygiene and heightened awareness of people about the importance of preventing infections, the global burden of shigellosis remains considerable. Based on data from the Chinese Center for Disease Control and Prevention, shigellosis is the third most commonly reported infectious disease in China, calling for urgent attention. Over years, a significant shift has occurred in the species of Shigella contributing to clinical disease, especially in developing countries, i.e. S. sonnei has become the prevalent species [Reference Qu1, Reference Filliol-Toutain2]. For instance, Chang et al. reported a significantly higher rate of S. sonnei in eastern, northern, and northeast regions of China, probably due to unbalanced economic growth [Reference Chang3].

Fluoroquinolones (FQs) are the most commonly used drugs for the treatment of shigellosis. However, the inexorable development of resistance by Shigella to the drugs has constrained the effective and adequate treatment of acute dysentery. Shigella resistance to FQ agents is produced by several mechanisms. Mutational alterations in DNA gyrase and/or topoisomerase IV genes, associated with high-level FQ resistance in clinical isolates, play a main role in FQ resistance. FQ resistance can also be acquired through quinolone resistance genes associated with plasmids including Qnr families, a variant of aminoglycoside acetyltransferase aac(6 ′)-Ib-cr and efflux pump qepA. Qnr proteins interfere with quinolone binding to DNA gyrase and topoisomerase IV. Plasmid-mediated qnrA was initially identified in clinical isolates from Klebsiella pneumoniae from the USA, in 1988 [Reference Martínez-Martínez, Pascual and Jacoby4], and since then, other plasmid-mediated quinolone resistance (PMQR) genes have been reported.

Recently, there have been numerous surveys on FQ resistance, mutations in quinolone resistance-determining regions (QRDRs) and the presence of PMQR in Shigella strains around the world. Studies describing the prevalence of PMQR determinants and mutations in QRDRs among S. sonnei in Jiangsu Province of China are limited. The objective of the present study was to examine the extent of FQ resistance and investigate the prevalence of PMQR and mutations in QRDRs of gyrA, gyrB, parC, and parE among a collection of S. sonnei clinical isolates between 2012 and 2015.

Sponsored by Jiangsu Province Center for Disease Control and Prevention (Nanjing, JS, China), 108 unduplicated clinical S. sonnei isolates were obtained from hospitalized patients in Jiangsu Province between January 2012 and December 2015, comprising 19, 25, 30, and 34 S. sonnei isolates during the period between 2012 and 2015 respectively, which indicated a slight annual increase in the number of isolates detected. All strains were confirmed by API system (bioMerieux, France) and slide agglutination test using Shigella antisera (Ningbo Tianrong Bio-pharmaceutical Co., Ltd., China). According to the guidelines of the CLSI (Clinical and Laboratory Standards Institute) [5], susceptibility testing to nalidixic acid and norfloxacin of all the isolates was performed by the disc diffusion (Kirby–Bauer) method. Of the 108 isolates, 92 (85·2%) were categorized as nalidixic acid resistant (NAL^R) and 8 (7·4%) displayed norfloxacin resistance (NOR^R). The proportion of NAL^R strains was higher than that during 2008 and 2010 in the same area while the number of NOR^R S. sonnei isolates was decreased in this study (χ ² test; P < 0·05) [Reference Shen6]. In addition, the number of NOR^R isolates collected during 2012 and 2013 was significantly greater than that collected during the subsequent 2 years (χ ² test; P < 0·05). Periodic monitoring and reporting of FQ resistance circulating in the area are of immense importance.

The gyrase and topoisomerase IV genes (gyrA, gyrB, parC, and parE) of all strains were amplified through polymerase chain reaction (PCR) using previously described primers [Reference Rahman7–Reference Dutta9]. The amplification products were then purified and sequenced, and sequence alignment was done using BLAST program. Of the 108 S. sonnei isolates, 94 contained mutations in the gyrA gene, and only three contained mutations in the parC gene, while no mutations in the gyrB or parE genes was detected. Of S. sonnei containing mutations, codon Ser83 was the most frequently affected (91/108), and these strains were resistant to nalidixic acid, indicating that the mutation at position 83 of gyrA was crucial for resistance to nalidixic acid, which is in agreement with a previous study reporting that 94·9% of the quinolone-resistant Shigella isolates had at least mutations at position 83 of gyrA [Reference Yang10]. Additionally, six isolates were observed with Asp87Asn/Gly/Tyr substitution, and replacement of Asp (GAC) to Tyr (TAC) was observed in three isolates without the occurrence of Ser83 mutation (Table 1). It is notable that two of the three Asp87Tyr strains showed susceptible to both nalidixic acid and norfloxacin. An earlier report pointed that mutation in codon 87 of gyrA was associated with nalidixic acid resistance [Reference Hirose11]. We previously reported that mutations in QRDRs also occurred to FQ susceptible Shigella flexneri isolates, separately or together [Reference Qin12]. Curiously, three S. sonnei strains in this study that exhibited NAL^R and possessed double mutations in gyrA (Ser83 and Asp87) and a single mutation in parC (Ser80), were not resistant to norfloxacin. This was not consistent with the previous report that multiple QRDR mutations are largely responsible for FQ resistance among Shigella isolates [Reference Folster13]. However, some studies have already reported similar phenomenon. In southeast China, high mutation rate of mutations both in gyrA83 and parC80 was detected among ciprofloxacin-susceptible S. flexneri (138/142) [Reference Pu14]. Moreover, in our previous report, 28 of 410 norfloxacin-susceptible S. flexneri isolates had mutations of Ser83, Asp87 in gyrA and Ser80 in parC [Reference Qin12].

Table 1.

FQ resistance, amino acid substitutions in gyrA and parC genes in terms of amino acid positions and PMQR determinants in Shigella sonnei isolates

NAL, nalidixic acid; NOR, norfloxacin; R, resistant; S, susceptible; I, intermediate resistant; −, negative.

Then, the S. sonnei isolates were also screened for PMQR genes including qnrA, qnrB, qnrC, qnrD, qnrS, aac(6′)-Ib-cr, and qepA by PCR [Reference Robicsek15–Reference Yamane19]. The qnrA gene was detected in nine of the collected isolates, achieving a prevalence rate of 8·3% (Table 1), and no qnrB, qnrC, qnrD, qnrS, aac(6 ′)-Ib-cr, and qepA genes were detected. All qnrA-positive isolates were resistant to nalidixic acid and only two of them were resistant to norfloxacin. It is worth noting that the qnrA-positive strains were observed with gyrA substitution at position Ser83 simultaneously. In China, qnrA1 was first reported in S. flexneri isolates and even coexisted with other PMQR determinants in 2010 [Reference Xiong20]. In China, the qnrS and aac(6′)-Ib-cr genes in S. flexneri were discovered in Zhejiang Province in 2009 [Reference Pu18]. Later in 2013, aac(6′)-Ib-cr appeared in S. sonnei and qepA gene was found [Reference Yang10]. Notably, all these reported PMQR-positive isolates were resistant to FQs except for the qnrA-positive isolates. Similarly, only two of the nine PMQR-positive isolates showed resistance to norfloxacin in this study. To the best of our knowledge, this is the first study reporting the existence of qnrA in S. sonnei isolates in China. Although PMQR genes provide a low level of quinolone resistance, they can promote mutations within the QRDR. In addition, considering the mobile characteristic of PMQR genes, it is essential to continue this type of surveillance and future research should focus on continual monitoring of the spread of PMQR determinants.

In conclusion, these new data demonstrate that there is a significant abundance of mutations in QRDR genes, which play a primary role in FQ resistance. In addition, we have reported the first incidence of qnrA gene in S. sonnei isolated from Jiangsu Province. Our findings emphasize on the need of continuing active surveillance of mechanisms of FQ resistance in S. sonnei, knowing that it is essential to safe and effective use of antimicrobial drugs for effective control of shigellosis.