Molecular characterization and analysis of high-level multidrug-resistance of Shigella flexneri serotype 4s strains from China

To conduct the first comprehensive analysis of Shigella flexneri serotype 4s, a novel serotype found in 2010, we identified 24 serotype 4s isolates from 1973 shigellosis cases in China (2002–2014). The isolates were characterized by single nucleotide polymorphism (SNP) phylogenetic analysis, pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) to determine their genetic relatedness, and analysed further for their antimicrobial susceptibilities and antimicrobial resistance determinants. The PFGE and SNP phylogenetic analyses suggest that S. flexneri serotype 4s strains are derived from multiple serotypes, including two predominant serotypes in China: serotype X variant and serotype II. Three new sequence types were identified by MLST. All isolates were resistant to ticarcillin, ampicillin and tetracycline, with high-level resistance to third-generation cephalosporins. Notably, all the isolates were multidrug resistant (MDR), with the highest levels of resistance observed for eight antimicrobials classes. Most isolates contain various antimicrobial resistance determinants. In conclusion, we found that serotype 4s isolates have multiple evolutionary sources, diverse biochemical characteristics and genomes, and highly prevalent multidrug resistance and antimicrobial-resistant determinants. With few clinical treatment options, continuous monitoring and timely intervention against this emerging MDR serotype is essential. The possibility that serotype 4s will become the next predominant serotype exists.


Results
Bacterial isolation and biochemical characterization. Twenty-four S. flexneri serotype 4s isolates were identified amongst the 1973 S. flexneri isolates collected from the eastern, western, southern, northern and central regions of China during our 13-year routine surveillance (from 2002 to 2014) of shigellosis. This serotype was relatively dispersed across China according to our surveillance results, suggesting that S. flexneri serotype 4s is still relatively rare in China, and that there is not currently an outbreak of this serotype. Biochemical characterization showed that the 4s isolates displayed inconsistent biochemical features (Table 1). Four isolates from Shanghai and three isolates from Guangxi produced indole, one isolate (SH4s01) could not ferment melibiose but could ferment sorbitol, and SH4s04 also could not ferment melibiose. These features differ from those of the first-reported 4s isolate 17 , which could ferment melibiose, but could not produce indole and did not use sorbitol.
Pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) analyses. S. flexneri serotype 4s was shown previously to have evolved from the S. flexneri serotype X variant (SFxv) 17  compare the genetic similarity and verify the evolutionary relationship between these two serotypes, PFGE was performed (Fig. 1). The banding profiles of several of the serotype 4s isolates shared more than 90% similarity with that of SFxv. We also compared the profiles of S. flexneri serotype 4s isolates with those of other S. flexneri serotypes (1a, 1b, 1c, 2a, 2b, 2c, 4a, 4b, X, Y and 6). Surprisingly, there were two clusters containing S. flexneri serotype 4s and S. flexneri serotype II (2a, 2b and 2c) isolates each with a similarity score of 100%. This result suggested that S. flexneri serotype 4s may have evolved from S. flexneri serotype II, indicating multiple evolutionary sources. Several S. flexneri serotype 4s isolates (such as SH4s02 and SH4s07) showed distinct banding profiles or low similarities with other strains, suggesting a high level of genetic diversity among the S. flexneri serotype 4s strains. There was no significant association between the geographical areas, years of isolation or resistance profiles of isolates that shared similar PFGE patterns.
The MLST results showed that sequence type (ST) 100 was predominant among the S. flexneri isolates (Fig. 1). Three STs were found in the 4s isolates (Supplementary Text S1). GX4s03 was designated as a new type of isolate because its rpoS gene, which is most similar to rpoS15, contains a new point mutation. In the ropS gene of GX4s01, a 19-bp sequence was inserted, making this gene contain a new allele sequence. Without the inserted sequence, the GX4s01 ropS sequence is identical to allele rpoS15, indicating that this new allele is also a mutation Figure 1. PFGE profiles, antimicrobial susceptibility profiles and sequence types (STs) of Shigella flexneri serotype 4s, 2 and xv isolates. The serotype, origin, strain number, year of isolation and ST are listed after each PFGE profile. AMK, amikacin; AMP, ampicillin; ATM, aztreonam; CHL, chloramphenicol; CAZ, ceftazidime; CFP, cefoperazone; CFZ, cefazolin; CRO, ceftriaxone; FEP, cefepime; FOX, cefoxitin; GEN, gentamicin; IPM, imipenem; LVX, levofloxacin; NIT, nitrofurantoin; NOR, norfloxacin; PIP, piperacillin; SXT, trimethoprim/sulfamethoxazole; TET, tetracycline; TIC, ticarcillin; TIM, ticarcillin/clavulanic acid constant 2; TOB, tobramycin. Black indicates "resistant", grey indicates "intermediate susceptibility" and blank indicates "sensitive". Stars represent 4s strains whose PFGE patterns are identical. Four cornered stars represent 4s strains whose PFGE patterns are very different from the others. Diamonds represent 4s strains with new MLST types. Triangles represent 4s strains with resistance to at least 7 antibiotic classes. based on rpoS15. We attributed one new ST, SH4s05, to the new allele combination of 15 housekeeping genes. The SH4s05 allele number for rpoS is 64, while the other allele number is consistent with the ST100 number and this new allele combination is not recorded in the MLST database. All of the new DNA sequences and new allele combinations have been submitted to the MLST database (http://www.shigatox.net/ecmlst/cgi-bin/index). Phylogenetic analysis. We sequenced the whole genomes of the 24 S. flexneri serotype 4s strains and performed a SNP phylogenetic analysis based on the sequences. To evaluate the similarity between serotype 4s and other serotypes, we selected and downloaded the whole genome sequences of 35 strains, including most of the S. flexneri serotypes in the Shigella Genome Sequencing Consortium collection 14 previously analysed and found to be distributed in seven phylogenetic groups (PG1-PG7) 14 .
The phylogenetic tree showed that most of the serotype 4s strains fell within one group mixed with PG3 strains such as the S. flexneri serotypes 1a, 2a, 2b, X, Xv, Y and Yv (Fig. 2), suggesting a high level of genome similarity among them 14 . This result is consistent with our PFGE results in that the patterns of serotype 4s show similarity with those of serotype II and Xv. Only three isolates (SH4s02, HN4s04 and SH4s04) fell outside the main clusters, showing independent origins. Although SH4s02 and SH4s07 had distinct PFGE typing patterns with others, the phylogenetic tree revealed that SH4s07 is embedded in the main cluster. This inconsistence indicated that SH4s07 shared similar core regions but different restriction enzyme sites with other serotype 4s strains.
To get more information of the subtle differences among serotype 4s strains, we also performed a SNP phylogenetic analysis only based on the whole genome sequences of the 4s serotype (Supplementary Figure S1). More shared core SNPs were analysed compared with Fig. 2 because of the close relationships among these strains. So although HN4s04 and SH4s04 are close in Fig. 2, they are distinguishable in Supplementary Figure S1. Overall, the phylogenetic tree become more diverse, and the phylogenetic tree showed that these 24 strains were grouped into 3 distinct clusters, suggesting possible multiple origins of 4s serotype.

Molecular analysis of antimicrobial resistance genes and integrons. All 24 isolates were tested for
the presence of AMR (antimicrobial resistance) determinants and integrons, including bla VIM , bla NDM , bla SHV , bla TEM , bla OXA , bla CTX-M , qnrS, aac(6′ )-Ib-cr, intI1 and intI2. Point mutations within gyrA, gyrB, parC and parE were also examined ( Table 3). All isolates were negative for bla SHV , bla VIM and bla NDM ; however, all 24 isolates contained both bla TEM-1 and bla OXA-1 . Seven isolates contained bla CTX-M-14 or bla CTX-M-79 , and these isolates showed a high degree of resistance to the β -lactamase antibiotics ceftriaxone, cefoperazone and cefazolin. Three isolates carried the following plasmid-mediated quinolone resistance (PMQR) genes: BJ4s03 and GX4s01 carried qnrS, and HN4s06 carried both qnrS and aac(6′ )-Ib-cr. Only two isolates showed resistance to levofloxacin and norfloxacin (quinolone antibiotics) based on the antimicrobial susceptibility testing results, although none of the isolates contained qnrA, qnrB or qnrD. Analysis of point mutations in the quinolone resistance-determining regions (QRDRs) showed that all isolates contained the gyrA Ser83Leu and His211Tyr substitutions, along with the parC Ser80Ile mutation. Isolate SH4s09 also contained a Ser83Asn substitution in gyrA (Table 3). No point mutations were found in gyrB or parE. All isolates harboured class 1 integrons with bla OXA-1 + aadA12 gene cassettes, and two isolates from Henan harboured dfrA17 + aadA5 or aacA4 + cmlA1 gene cassettes. All isolates also contained class 2 integrons with dfrA1 + sat1 + aadA1 gene cassettes.

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 serotypes in some countries 6,19 . SFxv first appeared in Henan Province, China, in 2001, and was one of the predominant serotypes in Shanxi, Gansu, and Anhui Provinces from 2002 to 2006 6,20 . 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 serotype 2a 21 . S. flexneri serotype 4s, a novel serotype identified in 2010, is believed to have evolved from serotype SFxv 17 . However, our results based on the SNP phylogenetic and PFGE analyses showed that this novel serotype is not only derived from serotype SFxv but also from S. flexneri serotype II (including serotypes 2a, 2b and 2c). A previous study reported that Yv, another new S. flexneri serotype, is derived from serotypes Y, SFxv and 2a 22 , suggesting that novel serotypes may be more likely to evolve from predominant serotypes. In addition, the SNP phylogenetic and PFGE analyses showed that S. flexneri serotype 4s strains have high genome diversity, suggesting that this serotype may also be derived from other serotypes such as 1a, X, Y or Yv.
The small number of S. flexneri serotype 4s isolates among the 1973 S. flexneri shigellosis isolates suggested there was no widespread epidemic of this serotype in China. MDR acquisition might explain this finding because drug resistance is often associated with a fitness cost, which is usually related to the replication and maintenance of AMR determinants in bacteria 23 . This fitness cost may result in infections of longer duration in a host and decreased transmission rates in a population. Although this factor may limit a widespread epidemic of S. flexneri serotype 4s, the extent to which S. flexneri serotype 4s is prevalent in the human population is probably influenced by many factors. Consequently, continuous surveillance of S. flexneri serotype 4s is very necessary. First, the present data indicate that serotype 4s evolved from S. flexneri serotype II and SFxv strains, which are the predominant serotypes in China. Therefore, S. flexneri serotype 4s strains still have the potential to cause an epidemic because of limited host immunity to their new surface antigens. Second, all of the isolates examined to date are MDR, with some isolates displaying resistance to eight antibiotic classes. These isolates have high levels of resistance to the antibiotics classed as "critically important" by the World Health Organization 24 (i.e., ampicillin, ceftriaxone and ticarcillin). Hence, once an epidemic or outbreak occurs, few effective antimicrobials would be available to clinicians, making infections more difficult to treat.
Analysis of antibiotic resistance genes can help us to understand the fundamental factors leading to bacterial resistance, and assist in developing measures to prevent resistance. In our research, all of the S. flexneri serotype 4s isolates contained both bla TEM-1 and bla OXA-1 . The OXA-1 β -lactamase has a high level of hydrolytic activity against oxacillin and cloxacillin, and confers resistance to ampicillin and cephalothin 25 , while TEM-1 confers resistance to penicillins and the early cephalosporins. bla TEM-1 is present in almost all extended-spectrum β -lactamase (ESBL)-producing isolates. It is constantly evolving and has generated a number of descendent alleles that confer resistance to most β -lactamase antibiotics 26 . In recent years, these two resistance genes have been found to exist at high frequencies in Shigella and other Enterobacteriaceae. They are clinically important because they confer resistance to the first generation cephalosporin antibiotics such as ampicillin, which was the principal antibiotic used to treat shigellosis decades ago 27 . Ampicillin resistance in Shigella has forced clinicians to seek new effective agents for the treatment of shigellosis. According to the recent recommendations of IDSA (Infectious Diseases Society of America), ceftriaxone (a third generation cephalosporin) and quinolones such as ciprofloxacin and norfloxacin are the treatment options for shigellosis 28 . In addition to bla TEM-1 and bla SHV , bla CTX-M has been associated with ESBL-producing strains since 1995 29 . It shares less than 40% identity with bla TEM and bla SHV , and comprises a relatively heterogeneous family of 40 members, which can be divided into five different groups 30,31 . The β -lactamase enzymes encoded by bacteria have a wide substrate range, hydrolysing penicillins as well as first-, second-and third-generation cephalosporins 32    types belong to the CTX-M-1 group and are plasmid encoded 35 . The S. flexneri serotype 4s isolates that contained these two genes showed a high degree of resistance to third-generation cephalosporins compared with the other isolates ( Fig. 1 and Table 3). Quinolone levels and/or fluoroquinolone resistance correlate with mutations in the target enzymes gyrase (gyrA and gyrB) and topoisomerase IV (parC and parE), and the presence of plasmid-borne mechanisms encoded by qnrA, qnrB, qnrS and aac(6′ )-Ib-cr 36,37 . The predominant mutations that give rise to fluoroquinolone resistance usually occur in the QRDR, from positions 67-106 of GyrA and ParC 38 . The mutation at position 83 of gyrA is the most frequently observed in Shigella species, and usually results in high-level resistance to the first-generation quinolone nalidixic acid 39 . The presence of additional mutation(s) in gyrA and/or another target gene such as parC will increase the level of resistance to fluoroquinolones 40 . In this study, all isolates had gyrA Ser83Leu and parC Ser80Ile mutations, and one strain had an additional Asp87Asn mutation in gyrA. Three of the strains contained qnrS, with two of these strains showing high resistance to levofloxacin and norfloxacin. One strain also contained aac(6′ )-Ib-cr. Our results are consistent with the previous theory that quinolone resistance determinants alone may have a weak effect on resistance levels, but can augment resistance when combined with other determinants 41 . Moreover, the point mutation found outside of the QRDR, His211Tyr in gyrA, is very common in fluoroquinolone-resistant Shigella 42 . In the present research, all isolates contained the His211Tyr mutation, regardless of their fluoroquinolone sensitivities. Therefore, the way in which this mutation contributes to drug resistance should be the subject of further research.
Antibiotic resistance and the way it disseminates in Shigella species is related to the presence of resistance gene cassettes in class 1 and class 2 integrons 43 . In our research, we identified nine types of gene cassette distributed amongst the class 1 and class 2 integrons. The bla OXA-1 and aadA2 cassettes in class 1 integrons are usually co-ordinately integrated, and encode resistance to ampicillin (oxa-1) and streptomycin (aadA) 44,45 . Strains harbouring dfrA17 and aadA5 cassettes were resistant to both trimethoprim and streptomycin 46 , while the aacA4 and cmlA1 cassettes usually confer resistance to aminoglycosides and chloramphenicol, respectively 47 . The dfrA1, sat1, and aadA1 cassettes usually band together in class 2 integrons, and confer resistance to trimethoprim, streptothricin, and streptomycin, respectively 45,47 . Recent reports show that these two integrons have high prevalence rates in Shigella species 42,48 . The current findings regarding integrons in the serotype 4s isolates are consistent with these previous results, which suggest the pervasive existence of integrons, and the importance of antibiotic resistance disseminated by integrons.
It has been shown previously that the provision of clean water and good sanitation reduces the incidence of S. flexneri infections in people 49 . S. flexneri strains can survive for several months in contaminated water 50 and foodstuffs 51 . In China, although the water and food hygiene problems are gradually improving, unsafe water and poor sanitation still exists in many areas and this is the main factor driving epidemics and outbreaks of S. flexneri 52,53 . BJ4s01 HN4s01 HN4s02 HN4s03 HN4s04 HN4s05 HN4s06 Of concern is that most of the infections caused by S. flexneri 4s strains in our research resulted from the intake of contaminated water or food according to our basic epidemiological data. The persistence of S. flexneri in the contaminated environment also increases the chance of transmission of AMR determinants between different subtypes. We tested for the presence of multiple AMR determinants in the 4s serotype and a high prevalence rate was observed. It has been shown that most of these determinants can be obtained on multiple occasions and maintained in multiple lineages for protracted periods of time 14 . We also observed that the serotype II and Xv strains whose PFGE patterns show similarity with that of serotype 4s also exhibited high antibiotic resistance levels, while the other serotype II and Xv strains did not show such high resistance levels. Hence, we are inclined to believe that serotype 4s is horizontally transferred to serotype II and Xv strains with high antibiotic resistance, thereby transforming them into serotype 4s, rather than them being transferred along with AMR determinants into the sensitive serotype II and Xv strains. In summary, comprehensive control of the contaminated environment is important for decreasing the rate of antibiotic resistance in S. flexneri and for reducing its prevalence. S. flexneri serotype 4s strains have shown a high level of multidrug resistance since they were first reported in 2010. In the present research, the antibiotic resistance and molecular characteristics of the 4s isolates collected from 2003 to 2013 were analysed. All of the isolates were MDR. They also had a high prevalence of antimicrobial-resistant genes and high genome diversity. The increasing drug resistance in novel S. flexneri serotypes has made prevention and treatment of shigellosis more difficult, and drug resistance poses a public health threat in areas where shigellosis is endemic. Because there are no reports of serotype 4s isolates outside of China, there is little concern, and therefore research, on this serotype. But the possibility of serotype 4s existing elsewhere in the world cannot be ruled out. In some parts of the world, the identification of new S. flexneri serotypes may be hindered by the high price of commercial antisera or the limited types of antisera available. Considering that novel serotype Xv has developed into a predominant serotype, second only to serotype 2a, in a short time in China 6 , it is important to monitor the prevalence and antibiotic resistance pattern of serotype 4s strains to enable timely intervention in the case of an epidemic. In addition, judicious use of antibiotics by physicians is needed to minimize the selection pressure of antibiotics on Shigella isolates. Further research should now be conducted to develop an effective vaccine against shigellosis.

Methods
Bacterial strains, serotyping and biochemical characterization. All of the Shigella strains were isolated from faecal samples from patients with diarrhoea or dysentery. The faecal samples, collected by sentinel hospitals in 13 provinces or municipalities in the eastern, western, southern, northern and central regions of China, were screened for Shigella species as follows. Samples were streaked onto Salmonella-Shigella (SS) agar and then incubated overnight at 37 °C. Resultant suspected Shigella colonies were picked and streaked directly onto SS agar and incubated overnight at 37 °C. The resultant colonies were subcultured on Luria-Bertani agar plates and grown in a 37 °C incubator. Next, the strains were submitted to our laboratory and identified to species and serotype levels. Specific serotypes were identified using monovalent antisera (Denka Seiken, Tokyo, Japan) and monoclonal antibodies (MASF IV-1 and MASF IV-2, Reagensia AB, Stockholm, Sweden). During our 13-year routine surveillance of shigellosis (from 2002 to 2014), a total of 1973 S. flexneri strains were isolated, and these included 24 S. flexneri serotype 4s isolates. API 20E test strips (bioMerieux Vitek, Marcy-L'Etoile, France) were used for biochemical characterization of the isolates following the manufacturer's recommendations. All experiments were performed in accordance with relevant guidelines and the experimental protocols were approved by Institute of Disease Control and Prevention, Academy of Military Medical Sciences. The informed consent was obtained from all subjects.

PFGE.
The genetic relatedness of the S. flexneri serotype 4s isolates was analysed using NotI macrorestriction of genomic DNA and PFGE according to the standard protocol for S. flexneri, as described previously 54 . Pattern analysis was carried out using BioNumerics software version 6.0 (Applied-Maths, Sint-Martens-Latem, Belgium). The Dice coefficient of similarity was calculated with a position tolerance of 0.8%, and the dendrogram was constructed based on the unweighted-pair group method of averages.
MLST. All isolates were assigned to multilocus sequence types using the protocols described at http://www.  56 with default parameters and a k-mer size of 13 bp. Concatenated core SNPs were then aligned for maximum likelihood phylogenetic tree construction with MEGA 57 , using the GTR model and 1000 bootstrap replicates. Phylogenetic tree of only 24 S. flexneri serotype 4s strains were also constructed as described above. The Whole Genome Shotgun project of S. flexneri serotype 4s has been deposited at GenBank under the accession LVIF00000000-LVJC00000000.
Antimicrobial susceptibility testing. The MICs of 21 antimicrobials against the S. flexneri serotype 4s isolates were determined next. We used the Sensititre semi-automated antimicrobial susceptibility system (TREK Diagnostics, Inc., Westlake, OH, USA) and the Sensititre Gram-negative custom plate PRCM2F for the analysis (the antimicrobials included ceftazidime, ceftriaxone, cefepime, cefoperazone, cefazolin, cefoxitin, nitrofurantoin, imipenem, piperacillin, ampicillin, tetracycline, gentamicin, tobramycin, amikacin, aztreonam, chloramphenicol, ticarcillin, ticarcillin/clavulanic acid constant 2, levofloxacin, norfloxacin, and trimethoprim/sulfamethoxazole) according to the manufacturer's instructions. The MICs were interpreted according to the recommendations of the Clinical and Laboratory Standards Institute. Escherichia coli strain ATCC 25922 was used as the quality control strain.