Antimicrobial resistance and genetic characterization of Shigella spp. in Shanxi Province, China, during 2006–2016

Background Shigella spp., facultative anaerobic bacilli of the family Enterobacteriaceae, are one of the most common causes of diarrheal diseases in human worldwide which have become a significant public health burden. So, we aimed to analyze the antimicrobial phenotypes and to elucidate the molecular mechanisms underlying resistance to cephalosporins and fluoroquinolones in Shigella isolates from patients with diarrhea in Shanxi Province. Results During 2006–2016, we isolated a total of 474 Shigella strains (including 337 S. flexneri and 137 S. sonnei). The isolates showed high rates of resistance to traditional antimicrobials, and 26, 18.1 and 3.0% of them exhibited resistance to cephalosporins, fluoroquinolones and co-resistance to cephalosporins and fluoroquinolones, respectively. Notably, 91.1% of these isolates, including 22 isolates that showed an ACTSuT profile, exhibited multidrug resistance (MDR). The resistance rates to cephalosporins in S. sonnei isolates were higher than those in S. flexneri. Conversely, the resistance rates to fluoroquinolones were considerably higher in S. flexneri isolates. Among the 123 cephalosporins-resistant isolates, the most common extended-spectrum beta-lactamase gene was blaTEM-1, followed by blaCTX-M, blaOXA-1, and blaSHV-12. Six subtypes of blaCTX-M were identified, blaCTX-M-14 (n = 36) and blaCTX-M-55 (n = 26) were found to be dominant. Of all the 86 isolates with resistance to fluoroquinolones and having at least one mutation (Ser83Leu, His211Tyr, or Asp87Gly) in the the quinolone resistance-determining regions of gyrA, 79 also had mutation of parC (Ser80Ile), whereas 7 contained plasmid-mediated quinolone resistance genes including qnrA, qnrB, qnrS, and aac(60)-Ib-cr. Furthermore, pulsed-field gel electrophoresis analysis (PFGE) showed a considerable genetic diversity in S. flexneri isolates. However, the S. sonnei isolates had a high genetic similarity. Conclusions Coexistence of diverse resistance genes causing the emergence and transmission of MDR might render the treatment of shigellosis difficult. Therefore, continuous surveillance might be needed to understand the actual disease burden and provide guidance for shigellosis. Electronic supplementary material The online version of this article (10.1186/s12866-019-1495-6) contains supplementary material, which is available to authorized users.


Background
Shigella spp., facultative anaerobic bacilli of the family Enterobacteriaceae, are one of the most common causes of diarrheal diseases in human worldwide and have become a significant public health burden [1]. Globally, nearly 167 million Shigella episodes per year are estimated, of which 99% are reported in developing countries. It is reported that almost 61% of all deaths attributed to shigellosis are in children under 5 years old [2]. In China, nearly half a million shigellosis cases are reported every year, which is situated at the top four notifiable infectious disease [2].
Researchers have classified the genus Shigella into 4 serogroups: S. dysenteriae, S. flexneri, S. boydii, and S. sonnei based on biochemical and serological properties. The S. flexneri is the predominant species in developing countries [1], where is in poor sanitation, such as in mainland China. Otherwise, the S. sonnei is mainly found in industrialized countries [2,3], and has been implicated in source outbreaks [4]. However, in some Asian countries and some developed regions of China, S. sonnei is gradually overtaking S. flexneri as the main pathogenic bacteria that cause shigellosis [5][6][7][8].
Infants, the elderly, and immunocompromised individuals with Shigella infection require antimicrobial treatment to shorten the clinical symptom duration and carriage and reduce the spread of infection [13]. The World Health Organization recommends fluoroquinolones and cephalosporins as the preferred drugs for the treatment of Shigella infections. With the extensive use of these antimicrobials, antimicrobial resistance is increasing remarkably in Shigella isolates. Since the first report of norfloxacin-resistant Shigella in 1949 in Japan [14], increasing number of Shigella isolates with multiple drug resistance (MDR, defined as resistance to three or more classes of antimicrobials) has been discovered in the world. It was reported that some factors could influence the antimicrobial susceptibility patterns of Shigella isolates, such as the geographic location, year, antimicrobial use, and antimicrobial agents [15]. However, few studies have investigated the antimicrobial resistance of Shigella in different cities of China, such as Shanghai and Beijing [5,6].
Selection of the most effective antimicrobial agents for shigellosis treatment requires the understanding of the antimicrobial susceptibility profiles of prevalent strains [16]. This study aimed to analyze the antimicrobial resistance profiles of Shigella isolates from Shanxi Province during 2006 to 2016, and to elucidate the molecular mechanisms underlying the emergence of MDR in these isolates.

Results
Bacterial isolates, serotyping, and biochemical characterization During our 11-year routine surveillance (from 2006 and 2016) of shigellosis, a total of 474 Shigella isolates, including 337 S. flexneri (71.1%) strains and 137 S. sonnei (28.9%) isolates, but no S. dysenteriae and S. boydii were identified from patients with diarrhea in Shanxi Province. The age of the patients ranged from 2 months to 87 years (Fig. 1). The patients aged 15-59 years accounted for the highest proportion of 36.5% of all age groups (n = 173), whereas patients over 60 years old were the least susceptible, with a proportion of 6.3% (n = 30). Among all age groups, the proportion of males was higher than that of females (Fig. 1b). The male to female ratio for the patients was 1.42:1. Among the Shigella isolates, the constituent ratio of S. flexneri was higher than that of S. sonnei isolates every year, except in 2011 and 2016 (Fig. 2). Several S. flexneri serotypes were found in the 337 S. flexneri isolates, including serotypes 1a, 1b, 2a, 2b, 4c, and 5b. Notably, serotypes 4c and 1a were the main serotypes, accounting for 43 and 30%, respectively (Fig. 3). These results suggested that S. sonnei and S. flexneri are the prevalent species in Shanxi Province of China, especially the S. flexneri serotypes 4c and 1a.
Moreover, notable differences were noted in antibiotic resistance profiles especially those changed significantly  (Table 2).

Molecular analysis of antibiotic-resistant determinants and integrons
A total of 195 Shigella isolates (including 109 cephalosporin-resistant isolates, 72 quinolone-resistant isolates, and 14 co-resistance isolates) were tested for the presence of antimicrobial resistance determinants and integrons. PCR results showed that all 195 tested isolates were negative for bla VIM and bla NDM , but positive for bla SHV, bla TEM , bla OXA , bla CTX-M , intI1 and intI2 gene regions (Table 4)     1 CLSI class: means resistance to one third-generation cephalosporin or one quinolone 2 CLSI classes: means resistance to two third-generation cephalosporin or two quinolones, or one third-generation cephalosporin and one quinolone 3 CLSI classes: means resistance to one third-generation cephalosporin and two quinolones, or one third-generation cephalosporin and two quinolones 4 CLSI classes: means resistance to two third-generation cephalosporins and two quinolones 5 CLSI classes: means resistance to three third-generation cephalosporins and two quinolones We have added these under the Table 3 in our manuscript AT/S: resistance to ampicillin, trimethoprim-sulfamethoxazole ACT/S: resistance to ampicillin, chloramphenicol, trimethoprim-sulfamethoxazole ACTSuT: resistance to ampicillin, chloramphenicol, tobramycin, Trimethoprim/ sulfamethoxazole and tetracycline 120 S. flexneri strains. All class 1 integrons harbored bla OXA-1 , which is present on the Tn2603 transposons [17] and aadA1 gene cassettes, whereas class 2 integrons are included in dfrA1, sat1, and aadA1 gene cassettes. Among the 86 quinolone-resistant isolates, no point mutations were noted in the QRDRs of gyrB and parE, but point mutations were noted in gyrA and parC in the most resistant isolates. All the 86 quinolone-resistant isolates had the gyrA mutation of Ser83Leu and His211-Tyr; 7 isolates had the gyrA mutation of Asp87Gly; and 79 had the parC mutation of Ser80Ile. Seven isolates were positive for qnrA, qnrB and acc(6′)-Ib-cr. Forty-five (52.3%) isolates were positive for qnrS.

PFGE analysis
PFGE was performed to determine the genetic relatedness among the 75 randomly selected Shigella isolates from different years and regions in Shanxi Province. The results of PFGE suggested that the 38 S. flexneri isolates generated 36 PFGE patterns (Fig. 5 a). All isolates could be categorized into four distinct groups (A-D) with a similarity of approximately 82%, including F1a, F2a, F2b, F4c, and F5b serotypes. This suggests considerable genetic diversity among the 38 S. flexneri isolates between different regions and years in Shanxi Province. Notably,    (Fig. 5 b), but formed a single cluster except one isolate (LJ-11-005) with a similarity of 82%. This result suggested that the S. sonnei strains had high genetically similarity in Shanxi Province.

Discussion
The emergence of novel and atypical bacterial serotypes in nature is attributed to serotype conversion, which often occurs in response to the protective host immune response [18]. Since the 1990s, several new S. flexneri serotypes (e.g., 1c and SFxv) have emerged and become the most prevalent ones in some countries [19].  [20]. Data on the prevalence of S. flexneri serotypes causing shigellosis in mainland China from 2001 to 2010 suggest that SFxv is the second most predominant serotype after 2a [21]. However, our results showed that the top three common Shigella serotypes in Shanxi Province were S. sonnei, S. flexneri serotypes 4c and 1a, which differed from those reported previously [18][19][20][21]. Interestingly, our data indicated that S. sonnei has replaced S. flexneri as the predominant species causing shigellosis in Shanxi Province, which was consistent with the findings of previous studies [5,6]. The increasing of proportion of S. sonnei is related to regional economic development and sanitary conditions. Shanxi is a developing and mountainous province with poor sanitary, which could promote the increasing of S. sonnei strains. Furthermore, it could also be conducive to the prevalence and dissemination of S. sonnei strains with MDR. In our study, S. flexneri tended to gradually increase and reach a peak in 2011, and then slowly decline again, whereas S. sonnei showed an opposite tendency. These trends and patterns were similar with those noted in developed countries [22]. The increasing antimicrobial resistance of Shigella species is a major problem in the treatment of Shigella gastroenteritis, especially of the MDR Shigella strains. Approximately 91.1% of the strains in our study showed MDR profiles, which is significantly higher than the rate of 41.6% (1762/4234) from the NARMS report (2005~2014) [23]. All the MDR strains were highly resistant to the traditional antimicrobials such as ampicillin, ticarcillin, trimethoprim/sulfamethoxazole, and tetracycline. One of the reasons for the rapid accumulation of resistance has been reported to be the excessive or inappropriate use of antibiotics in outpatients in China [24,25].
Fluoroquinolones and third-generation cephalosporins are the recommended first-line and alternatives drugs by the World Health Organization for empiric shigellosis treatment [26]. Our study further indicated that the current resistance patterns have changed, and empirical therapy should be modified in accordance with these changes. Thus, the treatment should be based on the susceptibility patterns and antimicrobials with current resistance might become effective in the future.
Moreover, in our study, 26.2% of cephalosporinsresistant Shigella isolates were found, which was considerably higher than the rate indicated in the NARMS report (lower than 1% from 2005 to 2014). The S. sonnei isolates showed higher resistance rates to cephalosporins, whereas the S. flexneri isolates had higher level resistance to fluoroquinolones. More importantly, we found 14 MDR isolates with co-resistance to fluoroquinolones and cephalosporins. If these MDR strains are prevalent worldwide, it might become a remarkable global public health concern. Our findings indicated that continuing monitor the antimicrobial resistance of Shigella isolates is necessary to help determine the appropriate antimicrobial therapy for patients with Shigella infection. More importantly, determining the mechanisms of antimicrobial resistance is necessary to assist in developing measures to prevent antibiotic resistance.
The increasing antibiotic resistance and rate led us to investigate the genetics and mechanisms of antibiotic resistance. Under the influence of various antibiotics, bacteria have a strong ability to obtain resistance genes for survival. Class 1 and class 2 integrons, which contain resistance genes and can be coordinately excised or integrated [2], might account for the horizontal transfer of resistance genes. In our study, 71.3% (n = 139) and 85.6% (n = 167) of isolates harbored class 1 and class 2 integrons, followed by the bla OXA-1 + aadA1 and dfrA1 + sat1 + aadA1 gene cassettes, conferring resistance to trimethoprim and streptomycin [27].
In addition, of the 123 cephalosporin-resistant isolates, 73.2% harbored the bla TEM-1 resistance gene, 57.7% harbored bla CTX-M , most of which were bla CTX-M-14 , followed by bla CTX-M-55 , bla CTX-M-15 , bla CTX-M-28, and bla CTX-M-64 . Further, 39.8% harbored bla OXA , and 14.6% harbored bla SHV . The bla TEM-1 gene exists at high frequencies in antibiotic-resistance bacteria and often confers resistance to penicillin and other β-lactamic antibiotics [28]. whereas the OXA-type β-lactamic, with high hydrolytic activity against oxacillin and cloxacillin often confer resistance to ampicillin and cephalothin [29]. Sequencing analysis showed that all the bla OXA genes were bla OXA-1 , which is consistent with the findings of a previous study on Shigella strains [30]. Plasmid-mediated transfer of different bla CTX-M genes was thought to be the reason for introduction of these genes into the isolates at different times [25], indicating that bla CTX-M-14 and bla CTX-M-55 genes might have long been circulating among Shigella isolates in Shanxi Province.
PMQR was initially identified in Klebsiella pneumoniae in 1998 [31]; since then, various types of PMQR genes have been detected worldwide. Quinolone levels and/or fluoroquinolone resistance have been mostly attributed to mutations in the target enzymes gyrase (gyrA and gyrB) and topoisomerase IV (parC and parE), and the presence of plasmid-borne mechanisms owing to the proteins encoded by qnrA, qnrB, qnrS and aac(6′)-Ib-cr [32,33]. The mutations of gyrA and parC at positions 67-106 are known to be the predominant mutations that can lead to fluoroquinolone resistance [34]. The gyrA Ser83Leu is the most frequently observed in Shigella species, and usually results in high-level resistance to the first-generation quinolone nalidixic acid [35]. The presence of additional mutations of gyrA (Asp87Gly/Asn and His211Tyr) and parC (Ser80Ile) results in resistance to fluoroquinolones [36]. Moreover, the mutation of His211Tyr in gyrA is very common in fluoroquinoloneresistant Shigella [30]. In our study, 100% of the quinolone-resistant Shigella isolates had point mutations in gyrA (Ser83Leu, Asp87Gly/Asn, and His211Tyr) and parC (Ser80Ile). S. flexneri serotypes (such as 1a, 2a, 2b, and 4c) carrying the qnrS gene have been globally reported with low incidence [37,38]. In our study, 45 (52.3%) of the strains contained qnrS, of which 11 showed high resistance to levofloxacin and norfloxacin. The aac(6′)-Ib-cr is reported to be responsible for lowlevel resistance to fluoroquinolones [39] and was first isolated from Shigella strains in 1998 [38]. Moreover, seven of the quinolone-resistant isolates were aac(6′)-Ibcr-positive, suggesting that the qnrS and aac(6′)-Ib-cr genes had long been present in Shanxi Province. The qnrA and qnrB were reported to be located on plasmids carrying bla genes (such as bla SHV and bla CTX ) [40]. In this study, seven strains also contained qnrA and qnrB each, and the qnrB-positive isolate coharbored bla CTX-M-55, bla OXA , and bla TEM . Our results are consistent with those of previous studies and the theory suggesting quinolone resistance determinants alone might have a weak effect on resistance levels; however, when combined with other determinants, resistance can be obtained [41]. The various resistance genes facilitate the dissemination of resistance determinants and the survival of bacteria under the selective pressure of various antibiotics.
Besides, in our study, the PFGE dendrogram showed that the S. sonnei isolates are closely related (82% similarity), indicating that they are possibly derived from a common parental strain. In contrast, the S. flexneri isolates (including F1a, F2a, F2b, F4c, and F5b serotypes) showed lower degrees of similarity, suggesting that they are likely derived from diverse sources, such as from different years, sources or origins. And the group B PFGE pattern was the major PFGE type of S. flexneri in Shanxi Province. Although PFGE has high concordance with epidemiological and genetical relatedness, and is considered as the "gold standard" fingerprinting method used for the discrimination and identification within PulseNet, it might not be effective in some Shigella or Salmonella species, which warrants further investigation with complementary molecular tools as multilocus sequence typing (MLST) [42].

Conclusions
In summary, we reported the distribution of Shigella serotypes and analyzed the common occurrence of MDR and resistance mechanisms in Shigella isolates in Shanxi Province during 2006 and 2016, China. The diverse antimicrobial resistance patterns and multi-types resistance genes were observed. Future studies should be focused on identifying ways to prevent the dissemination of these antimicrobial-resistance genes. Our data might provide a strategy for the treatment of infections caused by Shigella strains in Shanxi Province, China. Therefore, continuous surveillance might be imperative to determine the distribution and resistance development of Shigella, and to understand the actual disease burden and provide guidance for the clinical treatment of shigellosis. Furthermore, without treatment of shigellosis, especially caused the MDR Shigella, it might become a dominant strain and be prevalent in Shanxi Province, and spread worldwide, leading to the outbreaks of Shigella and causing significant public health and disease burden.

Materials
Bacterial isolates, serotyping, and biochemical characterization All the Shigella strains were isolated from fresh fecal samples, which were collected from outpatients with diarrhea or dysentery in four sentinel hospitals and two regional Centers for Disease Control and Prevention (one in Taiyuan City and the other in Yicheng County) in Shanxi Province based on a provincial pathogen monitoring system. Basic epidemiological data (name, age, gender, date, and region of isolation of patients) were recorded for each isolate. We screened for Shigella species by using the methods as reported previously [12,30]. Resultant colonies on the Salmonella-Shigella (SS) agar were transferred to our Microbiology Laboratory of Shanxi CDC for further confirmation. API 20E test strips (bioMerieux Vitek; Marcy-1′Etoile, France) and two specific serotyping kits were used to identify all the types and groups of S. flexneri. The slide agglutination test was used for serological reactions as reported previously [43].

Antimicrobial susceptibility testing
The antimicrobial susceptibility of all the Shigella isolates (474 Shigella strains, including 137 S. sonnei and 337 S. flexneri) was determined by analyzing the minimum inhibitory concentrations (MICs) of 21 antimicrobial agents, which were tested using the Sensititre semiautomated antimicrobial susceptibility system (TREK Diagnostics, Inc., Westlake, OH, USA) and the Sensititre 96-well plate PRCM2F (Thermo Fisher Scientific Ine, West Sussex, UK) according to the recommendations of the Clinical and Laboratory Standards Institute (CLSI, 2019) [44], as described previously [12,30]. The 21 antimicrobial agents included CAZ, CRO, FEP, CFP, CFZ, FOX, IPM, NIT, PIP, AMP, TIC, TE, TO, GEN, AK, ATM, C, TIM, LEV, NOR, and SXT. The Escherichia coli ATCC 25922 was used as quality control.

Pulsed-field gel electrophoresis (PFGE)
The genetic relationship among the Shigella species isolated from Shanxi Province was determined by analyzing 37 S. sonnei strains and 38 S. flexneri strains by using pulsed-field gel electrophoresis (PFGE) according to the standard protocol for Shigella outlined by PulseNet [50]. Macrorestriction patterns and dendrograms were analyzed and constructed using the methods as described previously [12,30], but with a different position tolerance of 1.5%.

Statistical analysis
Statistical analysis was performed using Chi-square test by using SPSS statistical package v. 19.0 (SPSS Inc., Chicago, IL). We compared the antibiotic resistance rates between the ages, gender, serotypes, and locations of the patients. A P value of < 0.05 was considered statistically significant.