| Shigellosis is a global problem that seriously jeopardizes public health.In developing countries,shigellosis has caused a large disease burden,with an estimated at least 80 million infections per year,mainly in children.The incidence of shigellosis is20.28/100 000 per year in China,and the number of the patients is more than 250,000each year.Due to the long-term use of antibiotics,antimicrobial resistance is becoming more and more serious,which has become a global problem to be solved urgently.In resent years,multidrug-resistant Shigella has become the major epidemic strain,which is widely prevalent and spread in different regions of the world,and even ceftriaxone and azithromycin resistant Shigella have appeared,which has brought great problems and challenges to clinical treatment and public health prevention and control.In 2015,there was even a water-borne outbreak caused by the ceftriaxone and azithromycin co-resistant Shigella sonnei in Guangxi of China,which aroused our great attention to this resistant phenotype.At present,the reports on the ceftriaxone and azithromycin co-resistant Shigella are extremely rare,and there is no study on the prevalence and distribution of the ceftriaxone and azithromycin co-resistant Shigella in China.Our study was the first to study the prevalence and distribution of Shigella resistant to ceftriaxone and azithromycin in China,providing a reference for the scientific prevention and control of shigellosis in the future.The purpose of this study was to clarify the prevalence and distribution of ceftriaxone and azithromycin co-resistant Shigella in China through the analysis of Shigella resistance and epidemiological information.Through antimicrobial resistance gene detection,plasmid profile,conjugation transfer,Southern hybridization and plasmid sequencing,the resistance mechanism of ceftriaxone and azithromycin co-resistant Shigella was revealed.Through the genome sequencing and phylogenetic analysis of representative strains of ceftriaxone and azithromycin co-resistant S.sonnei colected from different years and regions in China,the phylogenetic evolution of the strains was explored.In this study,antimicrobial susceptibility testing was performed on 4237 strains of Shigella isolated and identified in the laboratory over the years.A total of 1327?31.3%?strains of ceftriaxone-resistant Shigella were screened.Due to the lack of azithromycin resistance standard for Shigella at the beginning of the study,and the mph?A?gene is an important mechanism of mediating resistance to azithromycin for Shigella according to the literatures,mph?A?-positive was considered to be azithromycin resistance in our study,we screened the mph?A?gene of all strains,and 779 strains?18.4%?of mph?A?-positive Shigella were screened,and part of the mph?A?-positive Shigella strains were given to azithromycin susceptibility test,refering to the 2016 CLSI standard of resistance to azithromycin for Shigella,mph?A?-positive S.sonnei 100%?42/42?were resistant to azithromycin??32?g/mL?,and mph?A?-positive Shigella flexneri 73.9%?34/46?were resistant to azithromycin??16?g/mL?.Based on the above analysis,a total of 399 strains of ceftriaxone and azithromycin co-resistant Shigella were screened,with a total detection rate of 9.4%.Among them,211 strains were S.sonnei,and the detection rate was 11.5%;198 strains were S.flexneri with a detection rate of 7.8%.Ceftriaxone and azithromycin co-resistant Shigella have emerged in China since2003-2004,from 0.4%in 2003-2004 to 13.2%in 2013-2014,showing an increasing trend.Ceftriaxone and azithromycin-resistant Shigella was detected in 10 regions of Anhui?18.2%?,Henan?16.4%?,Shanghai?15.4%?,Gansu?8.4%?,Hubei?8.3%?,Guangxi?6.9%?,Shanxi?5.2%?,Beijing?4.0%?,Sichuan?3.8%?and Xinjiang?2.9%?in China,only four provinces-Liaoning,Yunnan,Guizhou and Guangdong-were not detected,possibly due to the low number of Shigella strains collected in these areas.Antimicrobial resistance analysis showed that the resistance rates of ceftriaxone and azithromycin co-resistant S.sonnei to ceftazidime,piperacillin,cefoperazone,cefazolin,tobramycin,gentamicin,ticarcillin,ticarcillin/clavuric acid,aztreonam,ampicillin and compound sulfamethoxazole were significantly higher than that of Shigella non-resistant to ceftriaxone and azithromycin?P<0.05?;but the resistance rates to tetracycline,chloramphenicol and norfloxacin were significantly lower than that of Shigella non-resistant to ceftriaxone and azithromycin?P<0.05?.The above results indicate that the ceftriaxone and azithromycin co-resistant Shigella is widely distributed in China,showing an increasingly epidemic trend,and the detection rate of co-resistance to ceftriaxone and azithromycin in S.sonnei is significantly higher than that of S.flexneri.In addition,the antibiotic resistance of ceftriaxone and azithromycin co-resistant Shigella was significantly higher than that of Shigella non-resistant to ceftriaxone and azithromycin.Therefore,we should strengthen the epidemiological surveillance of the ceftriaxone and azithromycin co-resistant Shigella,so as to provide reference for the scientific prevention and control of shigellosis.In this study,the resistance genes of 399 strains of ceftriaxone and azithromycin co-resistant Shigella were detected by conventional PCR.The results showed that blaCTX-M?88.7%?was the main resistance gene in ESBLs,followed by bla TEM?65.4%?and blaOXA?41.6%?,blaVIM was not detected?In blaCTX-M,sequencing analysis showed that blaCTX-M-14?59.9%?was the most prevalent,followed by bla CTX-M-55?18.1%?and blaCTX-M-15?12.1%?.Among the integron genes,the positive rate of IntI2 was as high as93.2%,the positive rate of IntI1 was 67.7%,and the positive rate of the class 2 integron variable region was up to 70.7%.S1-PFGE and Southern Blot hybridization analysis were performed on 78 strains of blaCTX-M-and mph?A?-positive Shigella,except that one blaCTX-M-15 gene was not on the plasmid and one bla CTX-M-55 gene was on different plasmids with mph?A?gene,the remaining strains of blaCTX-M-14,blaCTX-M-55 and blaCTX-M-15 genes are simultaneously located on the same size plasmid with mph?A?gene,respectively.Moreover,plasmid conjugation transfer experiments confirmed that these plasmids were able to transfer horizontally in a natural state.Ten strains of Plasmids sequencing analysis showed that the types of these plasmids included IncF?8/10?,IncI?1/10?and IncB/O/K/Z?1/10?,with IncF as the dominant type.On the upstream of blaCTX-M gene,the insertion sequence ISEcp1 is mainly present,and IS26 and IS6100are mainly flanked the mph?A?gene.The presence of these insertion sequences may be related to the expression,activation or transfer of the bla CTX-M and mph?A?genes.Our study demonstrates that the plasmid co-carrying the blaCTX-M and mph?A?genes is an important mechanism mediating the co-resistance of Shigella to ceftriaxone and azithromycin,and may serve as the important mechanism for intraspecific,interspecies and inter-genus genes transfer,leading to the rapid spread of this genotype,which poses a serious challenges to the prevention and treatment of shigellosis in the future.Base on the high detection rate and growth trend of the ceftriaxone and azithromycin co-resistance in S.sonnei,our study mainly studied the phylogenetic evolution of S.sonnei,especially for ceftriaxone and azithromycin co-resistant S.sonnei,and explored the significance of the resistance genes transfer in evolution.A total of408 genome-wide sequencing of S.sonnei were completed,including 133 strains of cefdinella and azithromycin co-resistant S.sonnei and 275 other S.sonnei.In this study,the reddog process established by Holt Laboratories was used for SNP identification.Based on this,the phylogenetic tree was constructed by using the maximum likelihood method.BEAST was used to predict the SNP substitution rate and the most recent common ancestor,and SRST2 was used for resistance genes analysis.The results showed that S.sonnei had formed four unique genetic lineages among the global evolution process,and the lineages in China mainly originated in Lineage III,and gradually formed an independent lineage in the late propagation and evolution process,and the strains in Guangxi,especially the ceftriaxone and azithromycin co-resistant S.sonnei,appeared obviously clustering phenomenon in the phylogenetic tree,forming a relatively independent small branch.Phylogeny and divergence date by Bayesian speculates that S.sonnei was introduced into China in the early 1980s,and it has been popular in the late 1980s,and began to form a relatively independent lineage in China in the 1990s.From the distribution of resistance genes on the phylogenetic tree,there are significant differences between foreign lineages and Chinese lineage,the types and quantities of resistance genes of Chinese lineage are significantly more than those of foreign lineages,widespread with blaTEM,blaCTX-M and mph?A?genes,and strains carrying both bla CTX-M and mph?A?genes even formed a relatively independent small lineage in China,indicating that these resistance genes transfer played an important role in the spread and evolution of Shigella in China.In summary,our study carried out the study on the epidemic distribution and resistance mechanism of the ceftriaxone and azithromycin co-resistant Shigella in China,and the evolution of S.sonnei in China by whole genome sequencing,clarifying the importance of resistance genes transfer in the evolution of S.sonnei.These findings play an important guiding role for the monitoring of shigellosis in the future,and provide a scientific reference for effective prevention of public health caused by ceftriaxone and azithromycin co-resistant Shigella and the development of scientific and rational prevention and control strategies. |
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