Objective To detect VP1 and 2A genes of Enterovirus type 71 (EV71) isolated from clinical specimens of patients with light or heavy symptoms and analyze the homogeneity and phylogenetic tree. Methods Fifty clinical specimens of children with hand-foot-and-mouth disease ( HFMD) were dealed with, which were tested by RT-PCR assay with specific primer pairs for EV71. EV71 isolates from patients with light or heavy clinical symptoms were tested by RT-PCR assay with two specific primer pairs for VP1 and 2A genes of EV71 respectively. All of the PCR products were sequenced and compared with that of previously isolated EV71 isolates available from GenBank by homogeneity and phylogenetic tree analyses. Results The RT-PCR results indicated that 30 isolates were EV71, 13 of 30 isolates were from clinical specimens of patients with light symptoms of hand-foot and mouth, the other were from clinical specimens of patients with heavy symptoms of complications. VP1 genes and 2A genes of 10 EV71 isolated strains including 5 light strains and 5 heavy strains were sequenced and compared with that of previously isolated 5 EV71 Chinese isolates available from GenBank (fuyangEU703814.1, xi_anHM003207. 1, shandongEU753418.1, shenzhenFJ607337.1, henanGU366191. 1) by homogeneity and phylogenetic tree analyses. The homogeneity of VP1 and 2A genes of the 10 EV71 isolated strains and 5 previously isolated strains were between 94.7% -99.4% and 93.6% -99.3% respectively, with the representative isolates of A and B genotypes was between 81.0%-84. 6% and 78. 4%-82. 2% respectively. The data suggested that all of the 10 Chinese isolates belong to EV71 genotype C. There were only 87.8% -90.2% homology among these 10 strains and the representative strains of C1, C2, C3 sub-genotypes of EV71 but 96. 8% -99.6% homology among these 10 strains and the representative strains of C4 sub-genotypes of EV71, this suggested that these 10 Chinese isolates composed the C4 sub-genotype, of the C genotype, that formed a single branch in the phylogenetic tree. Conclusion EV71 of sub-genotype C4 distributed in Mainland China, and VP1 genes have close genetic relationship between isolated strains. There is no obvious difference in 2A genes between clinical specimens of patients with light or heavy symptoms by homogeneity and phylogenetic tree analyses.

Objective To study the association of multi-drug resistance with mutations in mar gene in clinical isolates of Shigella.Methods Fifty-four clinical isolates of Shigella were collected.Susceptibility tests of tetracycline(TE),chloramphenieol(C),ampicillin(AM),cipr ofloxacin(CIP),and norfloxacin(NOR)were performed in a11 isolates.Hexane and cyclohexane were used to study the organic solvent tolerance of all clinical isolates.marOR genes of these strains were analyzed by potymerase chain reaction-single strand conformation polymorphism(PCR-SSCP)and were sequenced. Results Thirty-five multi-drug resistant strains were identified,and the rate of multi-drug resistance was 64.8%.Thirty-eight of 54 strains were tolerant to organic solvent,including 33 multi-drug resistance strains,3 drug resistant strains and 2 sensitive strains. marOR gene was found in all strains by PCR, and 5 multi-drug resistant strains,(14.29%)carrying marOR gene mutations in multi-drug resistant strains were found by SSCP analysis.DNA sequence analysis of marOR gene revealed that there was a 4 base deletion from base 1376 to 1379 in marO gene that resulted in frame-shift mutation.However,this deletion mutation didn;t exist in the standard strain S51250 and sensitive strains.There were 10 point mutations of marR gene in multi-drug resistant strains,2 of them resulting in amino acid changes:1752(G→A)Gly→ser,1854(T→c)Tyr→His.The rest mutations were all nonsense,and some of them occurred in sensitive strains or in many other strains.Conclusion The marO gene mutation may play an important role in the regulation of multi-drug resistance in Shigella spp.

Objective To detect the mutations of the marOR gene and study the relations with the expressing level of the acrAB-tolC efflux pump in Shigella. Methods marOR genes were amplified by PCR for 100 clinical isolates and 5 reference strains of Shigella. The PCR products were digested by restriction endonuclease Taq Ⅰ , then analyzed by single strand conformation polymorphism(SSCP). The marOR genes of the mutated strains and sensitive strain were sequenced and the expressing leveLs of acrA, acrB and talC were determined by RT-PCR. Susceptibility tests of tetracycline (TE), chloramphenicol (C), ampicillin (Am) , gentamycin (GM), norfloxacin (NOR) and selectrin (SMZ-TMP) were performed in sequenced strains. Results marOR genes were found in all strains detected. SSCP analysis found the rate of mutations in marOR genes was 23%. Among 11 marOR gene-mutated strains which were sequenced, there were 9 strains having a four-base absence and three single-base mutations in different loci. The expressing levels of the acrAB-tolC efflux pump in the 11 strains were higher than those in sensitive strains and reference strain. Furthermore the 11 strains were multi-drug resistance. Conclusion The mutation rate of marOR gene in Shigella was high and the acrAB-tolC efflux pump genes were over-expressive in marOR gene-mutated strains which were multi-antibiotic resistance in the study.

Objective To construct a prokaryotic expression system of ahpC gene of Helicobacter pylori. Methods The ahpC gene was amplified from Hp chromosomal DNA by PCR technique and cloned into the expression vector pET-30a. The recombinant vector pET30a-ahpC was identified by DNA sequencing and transformed to E.coli BL21 (DE3) for expression under induction by IPTG. The expression product was analyzed by SDS-PAGE. Results PCR product showed that ahpC gene consisted of 594bp. The gene fragment that was inserted into the recombinant vector was identified to GenBank for 99%. SDS-PAGE showed that the induced protein was expressed highly in the host bacterium. Conclusion A prokaryotic high-expression system for ahpC gene has been successfully constructed. It can highly express r-AhpC protein in E.coli.

Objective To detect the influence of marOR mutations on antibiotic resistance in Shigella spp. Methods The marOR gene with four-base deletion was amplified by overlap PCR, then inserted in a T-vector and transformed into DH5α. The clone of marOR gene with four-base deletion and three point mutations was prepared from the strain having these mutations. Electrophoresis and sequencing were preformed to certify the correction of the cloned genes. Drug susceptibility tests were preformed for the strains harbouring the different clones [DH5α, DH5α (T), DH5α (marOR), DH5α (marOR-CATT), DH5α(marOR-CATT + 3m)]. Results Compared with the control strain (DH5α-T), the antibiotic resistances of marOR with four-base deletion [DH5α (marOR-CATT)] were higher to streptomycin, tobramycin, cefazolin and cefalexin, and the antibiotic resistances of marOR with four-base deletion and three point mutations [DH5α (marOR-CATT + 3m)] were higher to streptomycin and to tetracycline. The antibiotic resistances of DH5α (marOR-CATT) and DH5α (marOR-CATT +3m) to streptomycin, tetracycline, chloramphenicol, cefazolin, levofloxacin, ciprofloxacin and norfloxacin were higher than DH5α (marOR). The diameters of the antibiotics except the trimethoprim between DH5α (marOR-CATT) and DH5α (marORCATT +3m) had not significant disparity. Conclusion The four-base deletion in 1376-1379 sites of the marOR gene increased the resistance of Shigella spp to some antibiotics. The point mutations in 1411, 1417,1435 sites of the marOR gene have little influence on the antibiotic resistance of Shigella spp.

This study was aimed to explore the features of clustered regularly interspaced short palindromic repeats (CRISPR) structures in Shigella by using bioinformatics. We used bioinformatics methods, including BLAST, alignment and RNA structure prediction, to analyze the CRISPR structures of Shigella genomes. The results showed that the CRISPRs existed in the four groups of Shigella, and the flanking sequences of upstream CRISPRs could be classified into the same group with those of the downstream. We also found some relatively conserved palindromic motifs in the leader sequences. Repeat sequences had the same group with corresponding flanking sequences, and could be classified into two different types by their RNA secondary structures, which contain "stem" and "ring". Some spacers were found to homologize with part sequences of plasmids or phages. The study indicated that there were correlations between repeat sequences and flanking sequences, and the repeats might act as a kind of recognition mechanism to mediate the interaction between foreign genetic elements and Cas proteins.
Base Sequence ; Clustered Regularly Interspaced Short Palindromic Repeats ; Computational Biology ; Genome, Bacterial ; Plasmids ; Shigella ; genetics

OBJECTIVETo investigate the characteristic of integrons and the relationship between integrons and antimicrobial resistance in Shigella spp.

METHODSNinety Shigella strains (83 S. flexneri and 7 S. sonnei) were isolated from the stools of patients in China. Susceptibility to 8 antimicrobials was tested for all isolated strains. PCR, RFLP and sequencing analysis of integrons were applied to all of them.

RESULTSHigh prevalence of multi-drug resistance (95.6%) was identified. Of the isolates 79 (87.8%) carried integrase genes of class 1 integron (3.3%), class 2 integron (10.0%) or both (74.4%). No intI3 was detected in the tested isolates. The prevalence of intI2 was significantly higher in isolates with multi-drug resistance to at least 3 antibiotics than that in isolates with resistance to 2 and less antibiotics (P<0.05). Gene cassettes dfrA17-aadA5, dfrA12-orfF-aadA2 of class 1 integron and dfrA1-sat1-aadA1 of class 2 integron were identified.

CONCLUSIONThe class 2 integron may play a role in the emergence of multi-drug resistance in Shigella spp.

Base Sequence ; China ; DNA Primers ; Drug Resistance, Microbial ; Drug Resistance, Multiple ; Integrons ; Polymerase Chain Reaction ; Polymorphism, Restriction Fragment Length ; Shigella ; drug effects ; genetics

Objective To detect the distribution of clustered regularly interspaced short palindromic repeat(CRISPR)associated protein genes cas1 and cas2 in Shigella and to understand the characteristics of CRISPR with relationship between CRISPR and related characteristics on drug resistance. Methods CRISPR associated protein genes cas1 and cas2 in Shigella were detected by PCR,with its products sequenced and compared.Results The CRISPR-associated protein genes cas1 and cas2 were found in all the 196 Shigella isolates which were isolated at different times and locations in China. Consistencies showed through related sequencing appepared as follows:cas2,cas1 (a) and cas1(b)were 96.44%,97.61%and 96.97%,respectively. There were two mutations including 3177129 site(C→G)and 3177126 site(G→C)of cas1(b)gene in 2003135 strain which were not found in the corresponding sites of Z23 and 2008113. Results showed that in terms of both susceptibility and antibiotic-resistance,strain 2003135 was stronger than Z23 and 2008113. Conclusion CRISPR system widely existed in Shigella,with the level of drug resistance in cas1(b) gene mutant strains higher than in wild strains. Cas1(b)gene mutation might be one of the reasons causing the different levels of resistance.

Objective To explore the stability of resistant phenotypes and changes of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) gene system on four Shigella strains in the absence of antibiotics.Methods Four clinical isolated Shigella strains that resistant to different antibiotics were consecutive passaged for 90 times without antibiotics.Agar dilution method was used to determine the minimum inhibitory concentration of Shigella strains.After sequence analysis with PCR,CRISPR Finder and Clustal X 2.1 were applied to identify the changes of CRISPR loci in the Shigella strains.Results After the consecutive transfer of 90 generations,sensitivity to certain antibiotics of four Shigella strains with different drug resistant spectrums increased.Mel-sf1998024/zz resistance to ampicillin,cephalexin,cefotaxime,chloramphenicol decreased,mel-s2014026/sx resistance to norfloxacin,trimethoprim decreased,mel-sf2004004/sx drug resistance to ampicillin,cefuroxime,cefotaxime,chloramphenicol,trimethoprim decreased and mel-sf2013004/bj resistance to chloramphenicol decreased.The spacer of which matched gene codes Cas and its upstream repeat in 3'end of CRISPR3 got lost in mel-sf1998024/zz and mel-sf2013004/bj.Conclusions Shigella strains could reduce or lose their resistance to some antibiotics after consecutive transfers,without the interference of antibiotics.CRISPR3 locus had dynamic spacers in Shigella strains while CRISPR3 locus and cas genes might have been co-evolved.

Objective To investigate the association between phage-mediated shiga toxin and molecular distribution of CRISPR in Escherichia (E.) coli O26:H11 or NM.Methods A total of 135 E.coli O26:H11 or NM strains were collected from NCBI database.Software CRT and CRISPR Finder were used to extract CRISPR and Excel was used to assign the spacer of unique number and type CRISPR.And the relationship between CRISPR and stx phage was analyzed.Results All the 135 E.coli O26:H11 or NM strains had the CRISPR.For CRISPRI,CRISPR2.1,CRISPR2.2 and CRISPR3-4,19,22,1 and 1 subtypes were found,respectively.According to the four CRISPR sites,the strains could be divided into 40 subtypes.Stx-phage was only observed in the group C of CRISPR.Compared with E.coli of stx-phage negative,E.coli with stx-phage harbored more spacers.Conclusions CRISPR loci was extensively existed in E.coli O26:H11 or NM,and many subtypes were found in these strains.The presence of stx-phage was related to the molecular distribution of CRISPR in E.coli O26:H11 or NM.CRISPR might be a valuable biomarker to identify strains with high virulent potential.