| Based on the structure of O antigen, Shigella includes about 40 serotypes, which can be divided into four species: Shigella.boydii, Shigella.dysenteriae, Shigella. flexneri, and Shigella. sonnei. Among them, S.flexneri is the most prevalent species in our country, cause 90% of infection. Serotypes F2a and F1a are the dominating ones for inducing infection. Recently several new subserotypes, such as F4c, F1c and F4x, have been identified in the world; and in our country, an additional new subserotype, SFxv has also been discovered in 2009.In this study, we isolated 62 atypical strains of S. ?exneri serotype 4 from the clinical samples, which belonged to two new subserotypes with distinct agglutination profiles from those reported previously. So they are provisionally named as F4cv and F4s. These strains were further analyzed with the methods of virulence gene, antimicrobial susceptibility test and gene variety. The followings are the findings in detail:Firstly, serological and biochemical identification were adopted on the atypical strains isolated from clinical surveillances. Monovalent antisera (Denka Seiken, Japanese) and monoclonal antibodies for Shigella flexneri (MASF, Reagensia AB, Sweden) were adopted to identify the serotypes. The reaction formula for monovalent antisera test was: F4cv: IV+, 7(8)+; F4s: IV+. For MASF, the reaction formula is: F4cv: B+, IV: I +, 7(8)+. F4s: B+, IV: I +. All these results revealed that the new strains had characters different with those reported previously and were temporarily named as F4cv and F4s.The biochemical test was conducted with API 20E biochemical assay kit (API Inc., France), and the results showed that both F4cv and F4s totally ferment glucose, glycol, melibiose and arabinose.They were identified as Shigella.spp. Besides the results mentioned above, the indole test for Fxv strain was positive.Bacterial susceptibility to antimicrobial agents was determined by Kirby-Bauer agarose method, and totally 14 kinds of antibiotics were tested. The results showed that all of F4cv was resistant to Nalidixic acid (NA), 96.72% to Tetracycline(TE), 93.44% to Ampicillin(AMP), 91.80% to Chloramphenicol (C), Amoxicillin-clavulanic acid (AMC) and Ampicillin-sulbactam(SAM). As for Trimethoprim -suifamethoxazol -e(SXT), the resistance is 86.89%. Among them, AMP and SXT were the first line antibiotics for treating shigellosis dysentery in our country. Nor?oxacin(NOR), one of the third generation of quinolone antibiotics, is the first choice for treating shigellosis. But the resistance of F4cv to NOR can be as high as 68.85%, which was higher than those reported in literatures. 19.67% of F4cv was resistant to Cipro?oxacin (CIP), and 18.03% to Levo?oxacin(LEV), one of the fourth generations of quinolone antibiotics. Furthermore, some strains were resistant to the third generation of cephalosporins, such as ceftazidime and cefotaxime. Simultaneously, it should be concerned that 3 strains of F4cv showed resistance to NOR and the third generation of cephalosporins. The drug resistant pattern of F4s was similar to F4cv. Compared with F4cv and F4s, strain Fxv was more susceptible to NOR, CIP and SXT. However, F4cv showed a higher resistance to the three antibiotics, with 68.85% resistance to NOR. Based on the results mentioned above, it was found that all of F4cv belonged to multidrug resistant strains, which showed an increased resistance to clinically first choice antibiotics, increased antibiotics resistance spectrum, increased complication in multidrug resistance, which posed many difficulties to the clinical treatment and disease control.All these strains were also subjected to the virulence genes testing with polymerase chain reaction (PCR) which included genes ipaH ial sen set1A set1B and virF. The results showed that there was lack of ial gene for F4s strains. All of 61 strains of F4cv carried set1A, set1B, and ipaH. And the percentage of carrying sen, ial, and virF were 83.61%, 83.62% and 75.41% respectively. The carrying of virulent genes for F4cv strains could be divided into 6 patterns. There are 37 strains of F4cv carried all the 6 genes, accounting for more than half of all strains (60.66%), which were dominant virulence gene patterns. All these results showed that these strains may have a stronger virulence, therefore, there were higher chances for them to induce the breakout of shigellosis, making them to be a potential risk for the outbreak of dysentery.The plasmids DNA were extracted by commercial kits and separated by horizontal electrophoresis in a 0.7% agarose; and these produced the plasmid profiles. 61 strains of F4cv could be categorized into multiple plasmid profiles. Among them, 33 strains (54.10%) carried the plasmid with the size of 20kb, and 57 strains (93.44%) with 10.5kb, 32 strains (52.46%) with 8.7kb, 56 strains (91.80%) with the 4.8kb, 59 strains (96.72%) with 3.9kb, 54 strains (88.52%) with 3.5 kb, 60 strains (98.36%) with both 2.2kb and 1.8 kb. The F4s strain carried the plasmids with the size of 10.5kb, 4.8kb, 3.9kb, 2.2kb and 1.8kb, while 20kb, 8.7kb and 3.5kb were missing. This pattern was the same with two strains of F4cv. The plasmids with the size of 8.7kb and 3.5kb were absent in Fxv strain. BioNumerics was adopted to analyze the results which showed that F4s was one distinct type, which also had the same profiles with the two strains of F4cv, implying that they carried the same plasmid, and had close relationship. When the similarity coefficient was set to 90%, F4cv and Fxv belonged to the same category. There were differences among the plasmid profiles of F4s, F4cv, Fxv and Fx. But the categorized analysis showed that they had comparatively nearer relationship.The genotypes were analyzed with the PFGE standard methods recommended by PulseNet. XbaI and NotI restriction enzymes were used for PFGE, and finally NotI restriction enzyme was applied. The electrophoresis profiles of F4s, F4cv and Fxv showed the difference of 1-5 bands, indicating a closer genetic relationship of them. The electrophoresis profiles were categorized by BioNumerics software. When the similarity coefficient was set as 92%, 3 serotypes of shigella could be divided into 27 band patterns and 7 clusters, which were named from type A to G. 61 strains of F4cv could be divided into 25 PFGE band patterns, 7 clusters, showing the genetic varieties of the strains. The analysis showed that F4s was a distinct PFGE band type. When the similarity coefficient was 96%, both F4s and F4cv belong to the same cluster of B, which showed the close relation implying that F4s may evolve from F4cv. On the other hand, both Fxv and F4cv belonged to one cluster of type A, showing F4cv may evolve from Fxv.Fifteen housekeeper genes provided by EcMLST database were amplified with PCR method, followed by sequence testing of the PCR product. After the results were uploaded to the EcMLST database and compared with the known sequences, the sequence types of each strain could be acquired. Based on this, we identified that all 61 strains of F4cv and F4s belonged to a new sequence type named ST100. Comparing with the other STs in the database, it showed there was only one locus difference with ST18, ST86, implying a near relationship. Bionumerics software was adopted to build up phylogenetic chart, and the results showed clonal group 10 containing ST100 evolved from the clonal group 14, which contains EHEC. The cluster analysis on the MLST and PFGE results showed the categorizing ability of MLST was much lower than that of PFGE.A panel of 5 VNTRs (Variable Number Tandem Repeats) with good repeated varieties, including SF3 SF6 SF11 SF12 SF34 were selected with PCR method. Based on the PCR product sequence, electrophoresis profiles, BioNumerics software was adopted to analyze and identify the variability. It showed that MLVA could divide 67 strains of shigella into 45 types; among which, 61 strains of F4cv could be divided into 43 types. F4s was categorized into one distinct type, and Fxv was another type. The minimum spanning tree showed that F4s and Fxv were located on different branches of the two groups are derived from the F4cv branch, shows they are different with strain F4cv in genotypes, but they also have genetic evolutionary relationships with F4cv. Different strains of F4cv were not the same in the relationship, stating they had different kinds of variety.Based on the analyzing methods mentioned above, the F4cv and F4s serotypes were identified as new subserotypes, with the same biochemical characteristics and similar situations for antibiotics resistance. Compared with the Fxv, F4cv had an increased antibiotics resistance, and there was the emergence of strong drug-resistant strains. The genotype analysis based on PFGE, MLST, MLVA methods showed that F4s, F4cv, Fxv might have close genetic relationship to each other, and both F4s and F4cv might evolve from Fxv. The fact that the Shigella bacteria can trigger the emergence of new epidemic outbreaks, along with its increased resistance to the ?rst-line antibiotics for treating shigellosis, pose challenges to the prevention and control of bacillary dysentery. By analyzing phenotype and genotypes of new strains, our current study addressed the issues mentioned above, which is meaningful by providing theoretic support for treating and controlling shigellosis. |
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