Background
Shigellosis continues to be a major public health problem in non-industrialized countries. It was reported that about 91 million cases of shigellosis occurred, with 414,000 people dying in Asia annually [
1]. In China, the morbility of
Shigella infections each year is unusually high. Wang et al. [
2] have reported that in 2000 0.8-1.7 million episodes of shigellosis occurred in seven different geographical regions, China.
For shigellosis, antibiotic therapy can reduce the duration and severity of the illness [
3]. However, with the long-term overuse of antimicrobials, the resistant
Shigella has prevailed all over the word, and production of extended-spectrum β-lactamases (ESBLs) or AmpC enzymes is one of the most important resistance mechanisms. In China extended-spectrum cephalosporin (ESC)-resistant
Shigella spp. has also been described in recent years [
3‐
7]. However, as China has a vast territory area, the spectrum of antimicrobial resistance and the mechanisms of resistance may vary in different regions. Therefore, it is necessary to explore the variations of ESC resistance in clinical isolates of
Shigella spp. from different geographical areas.
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, such as the invasion-associated locus (
ial), the invasion plasmid antigen H gene (
ipaH),
Shigella enterotoxin 1 (ShET-1) gene (
set1A and
set1B),
Shigella enterotoxin 2 (ShET-2) gene (
sen) and
virA gene [
8,
9]. Estimating the existence of virulence determinants in
Shigella would help us better understand its pathogenicity. However, investigations into clinical
Shigella spp. virulence factors are still rare in the world.
The purpose of this study was to investigate the resistance, the cephalosporin resistance mechanisms and the prevalence of putative virulence genes in ESC-resistant Shigella from patients with dysentery in Xiaoshan District, suburban of Hangzhou city, Zhejiang province, China. Finally we further ascertained the genotypes of these strains by ERIC-PCR (enterobacterial repetitive intergenic consensus sequence PCR) typing.
Discussion
This investigation reported the prevalence and resistance of ESC-resistant
Shigella, and the molecular analysis of cephalosporin resistance genes and virulence determinants in clinical isolates from Xiaoshan District, Hangzhou, China collected over a period of 5 years. In the economic undeveloped regions,
S. flexneri is the most frequently isolated
Shigella species. A similar situation also exists in China, according to previous data [
2,
4,
6,
16‐
18]. However, in our study
S. sonnei was the most common cause of bacterial dysentery, which was consistent with the findings in industrialized countries. In recent years, the data from Kaengkhoi District of Thialand, Ho Chi Minh City of Vietnam, South Korea, Taiwan and the East, North and Northeast regions of China, which are the newly industrialized regions, also displayed a striking species shift from
S. flexneri to
S. sonnei[
18‐
22]. Therefore, the species transition of shigellosis in the present study may be related to the economic growth in Xiaoshan District, suburban of Hangzhou city with higher economic indicators. Of course, other factors may also play a role and need further researches.
Through the resistance data analysis, we found that more than half of the
S. flexneri and
S. sonnei isolates were resistant to ESC (cefotaxime). Data from 8 Asian countries showed a high prevalence of resistance to the frontline antibiotics AMP (53.0%) and SXT (81.0%) among
Shigella isolates [
21]. However, the resistance rates to the both drugs among all our
Shigella isolates were more higher (AMP, 97.8%; SXT, 85.4%), and were consistent with the data from other investigations, mainland China [
6,
16,
17]. The data from Table
2 showed that SAM was not appropriate for the treatment of diarrhea caused by
S. flexneri, regardless of the strains with ESC-resistance (96.6%) or susceptibility (85.2%); On the contrary, it can be used to prescribe for
S. sonnei infections, especially for the cefotaxime susceptible strains. Although the resistance rate of
Shigella to PIP was high (83.7%) in this study, TZP was of very high anti-
Shigella activity (Table 2).
If the inhibition zone diameter of CAZ is ≥21 mm or that of FEP ≥18 mm, the two antibiotics can be reported susceptibility for enterobacteriaceae, irrespective of the isolates producing ESBLs or not, according to the CLSI [
10]. In all
Shigella isolates studied, a similar results of resistance to CAZ and FEP (16.6% and 18.5%) were observed, which were higher than that reported by Yang et al. (5.2% and 6.5%) [
16]. However, the resistance rates in Table
2 indicated that the two antibiotics were more suitable for the empiric therapy of the infection of ESC-resistant
S. sonnei than that of ESC-resistant
S. flexneri infection.
Fluoroquinolones are the popular antibiotics for the treatment of serious shigellosis in both adults and children. Research results from Gu et al. [
23] showed the resistance rate to CIP was 29.1% between 2007 and 2009 in the Asia-Africa area. Data from Henan Province, China displayed that 21% and 79% of
S. flexneri strains showed high- or low-level resistance to CIP, respectively [
6]. Yang et al. [
16] reported that 27.9% and 9.7%
Shigella were resistance to CIP and LEV, respectively, in Anhui province, China. In our study, a similar resistance rate to CIP (24.2%) and a higher resistance rate to LEV (16.0%) were exhibited (Table
2). Among the fluoroquinolone resistant isolates, 95.3% (82/86, resistance to CIP) and 89.5% (51/57, resistance to LEV) strains belonged to
S. flexneri. The possible cause was that
S. flexneri isolates often possessed plasmid-mediated quinolone resistance (PMQR) determinants or mutations in quinolone resistance-determining regions (QRDR) of gyrase and topoisomerase genes. This situation had been described in isolates from other regions of China by Zhang et al. [
4], Zhu et al. [
24] and Pu et al. [
25].
So far, at least 109 variants of CTX-M enzymes (CTX-M-1 to 124) have been described. Of these CTX-Ms, 19 variants (CTX-M-15, 16, 19, 23, 25, 27, 32, 35, 37, 40, 42, 53, 54, 55, 57, 58, 62, 64, 82, 93) exhibit the increased hydrolysis activity against ceftazidime, and the others display a much higher rate of hydrolysis of cefotaxime than ceftazidime [
26]. CTX-M-15 is the most usually detected CTX-M variant that hydrolyze ceftazidime at high level in enterobacteriaceae [
26]. In this study, 28
bla
CTX-M-15
positive ESC-resistant
Shigella isolates were all resistant to ceftazidime (data not shown). No other CTX-M variant genes mediating high level ceftazidime resistance were found (Table
3). In the
bla
CTX-M
genes with higher catalytic efficiencies against cefotaxime than ceftazidime,
bla
CTX-M-14
was the most prevalent one (53.0%), and coincides with the data published worldwide in clinically important pathogens [
26]. OXA-30 belongs to the class D oxacillinase group III, and mediates resistance to cefepime but not ceftazidime [
27]. Unfortunately, 52 (26.3%) of our ESC-resistant
Shigella isolates hosted
bla
OXA-30
, and 12 of them carried
bla
CTX-M-15
concomitantly and conferred resistance to cefotaxime, ceftazidime and cefepime (Table
3). In the past decade, an emergence of ESBL-producing
Shigella spp. carrying different types of ESBL genes has been described in different countries and regions [
28]. However, only a few studies have reported in the world the existence of AmpC β-lactamases encoded by
bla
CMY-2
or
bla
DHA-1
in
Shigella spp. [
4,
25,
28‐
31]. In this study, we also found 2 AmpC β-lactamase producers with
bla
DHA-1
and
bla
CMY-2
in the three-dimensional extract test positive
Shigella strains. The
bla
CMY-2
and the
bla
DHA-1
existed in 1
S. flexneri with
bla
CTX-M-14
and 1
S. sonnei with
bla
CTX-M-15
and
bla
OXA-30
, respectively (Table
3).
In this study, we detected several pathogenic genes (
ial,
ipaH,
set1,
sen and
virA) for 198 ESC- resistant
Shigella isolates (Table
4). It has been shown that
ial takes responsibility for penetration of epithelial cell by
Shigella and
ipaH also for spread from cell to cell [
32,
33]. All
Shigella species studied were positive for the
ipaH as expected because this gene exists in multiple copies on both the chromosome and the plasmid of
Shigella. Conversely, the
ial gene is exclusively located on the plasmid and was only detected in some
Shigella isolates [
8]. Indeed, less frequent examination of
ial gene had been described by Luscher and Altwegg [
34], Kingombe et al. [
35] and Thong et al. [
36]. However, this gene was found in all our ESC-resistant
Shigella strains. Another virulence factor VirA is involved in the uptake, motility, and cell to cell transmission of
Shigella within the human host. It is an essential virulence factor in
Shigella disease pathogenesis [
9]. The positive rate of
virA implied all the isolates of our collection might have the ability (Table
4). The
set1 chromosomal gene encodes
Shigella enterotoxin 1 (ShET-1, composed of one A and five B subunits), which is generated by
S. flexneri (mainly in type 2a) and not found in other
Shigella spp. [
8,
12,
36‐
41]. The
sen gene encoding
Shigella enterotoxin 2 (ShET-2) is carried on a 140 MDa virulence plasmid. And the
sen is present in all
Shigella species. The both toxins are deemed to play a part in the clinical manifestation of shigellosis [
8]. In our study, 79.3% of ESC-resistant
S. flexneri strains were found to be
set1A and
set1B positive (62.1% isolates were serotype f2a, Table
4), and this is agreement with the previous results; however, 17.1% (24/140) ESC-resistant
S. sonnei isolates also carried
set1A and/or
set1B genes (Table
4). The
set1A and
set1B genes are located on the
she pathogenicity island (PAI), a chromosomal, laterally acquired, integrative element of
S. flexneri[
37]. Integrase-mediated excision can occur for the
she PAI, and result in the formation of a circular excision product, which is a substrate for lateral transfer processes e.g. conjugation, packaging into phage particles and recombinase-mediated integration into the chromosome [
42]. This may be the cause that the two determinants can be found in our ESC-resistant
S. sonnei isolates studied. And the deficiency of
set1A or
set1B, or the existence of point mutations in the primer binding sites may be the possible explanation for the both genes not coexisting in some ESC-resistant
S. sonnei isolates (Table
4). Furthermore, we found that the
S. flexneri isolates with
set1 were more resistance to CIP, LEV (p < 0.001, each) and FEP (p = 0.019) than those without
set1; for
S. sonnei, the
set1 positive isolates were more likely resistance to SAM (p < 0.001), CIP, LEV (p < 0 .001, each) and FEP (p = 0.002), and more likely sensitivity to CAZ (p = 0.005) than
set1 negative ones (data not shown). Nevertheless, we do not think there was correlation between
set1 and the antibiotics resistance, because none of reports have described currently that the invasion genetic elements carrying virulence genes known contain resistance determinants simultaneously in
Shigella spp.. These resistance differences may be only due to the spread of resistant plasmids among the different strains.
The results of ERIC-PCR typing showed that most of the cases of ESC-resistant S. flexneri and S. sonnei infections were caused by several identical strains, respectively [for S. flexneri isolates, the percentage of type A and type B accounting for 63.8% (37/58); for S. sonnei, type A, type B and type C encompassing 69.3% (97/140) of the isolates]. This indicates that clonal dissemination was likely to contribute most to the spread of ESC-resistant S. flexneri and S. sonnei within the region studied. Of 46 ESC-resistant S. flexneri isolates with the second largest number of the virulence gene composition (ia1 + ipaH + virA + setlA + setlB + sen) being resistant to 4 to 8 antibiotics, 60.3% belonged to type A (43.1%) and type B (17.2%). Of 114 ESC-resistant S. sonnei isolates with the first largest number of the virulence gene composition (ia1 + ipaH + vir + sen) being resistant to 3 to 6 antibiotics, 71.1% belonged to type A (57.9%) and type B (13.2%), but isolates that hosted the set1 gene were more heterogeneous in ERIC-PCR pattern.
In the present study all the Shigella strains were isolated from intestinal clinic patients. No patients had been hospitalized or died following episodes of shigellosis. According to the clinical reports, early stage of shigellosis the patients infected by ESBL genes positive isolates were not more severe than those of the patients infected by ESBL genes negative isolates. However, most of these patients had longer course of treatment, because physicians used to treat diarrhea by prescribing cefotaxime or ceftriaxone (particularly for children) in the region studied. When treatment failed, other drugs (such as fluoroquinolone or β-lactamase inhibitors) would be used as a substitute for continuing treatment. In addition, all the patients with Shigella infection in the study were treated with antibiotics, therefore, we had no relevant data to compare the course of disease treated using antibiotics with that of disease treated without antibiotics.
Authors’ contributions
CLZ performed the experiments; QZL designed the study, analyzed the clinical data and wrote this manuscript; JW, XC, LMS and YYG collected the clinical samples. All authors read and approved the final manuscript.