Abstract: Objective To observe the phenotypic characteristics of 3 wild-type plague phages under different experimental environments, providing scientific evidence for the identification of phage biological characteristics and the study of their interaction with host bacteria in the future. Methods The sensitivity of 3 wild-type plague phages were detected by using liquid culture method, emisolid medium method and micro-liquid culture method based on OmniLog TM microbial identification system. Results The growth result based on LB liquid medium showed that the growth of plague phage 476 for 20-24 hours at both 28 ℃ and 37 ℃was better than that of plague phages 087 and 072204 at 37 ℃, and the growth of plague phages 087 was better than that of plague phages 072204 at 37 ℃. With the attenuated plague bacterium EV76 as the host bacterium, phage 476 was able to form visible plaque on double-layer agar medium for 20-20 hours at both 28 ℃ and 37 ℃, phages 087 and 072204 were only able to form opaque plaque on double-layer agar medium for 20-24 hours at 37 ℃. The growth results based on OmniLogTM system showed that when plague phage was lysed in EV76 strain at 33 ℃, the first row appeared as a straight line with a peak of no more than 100 in the 96-well microplate curve chart. As the phage quantity decreased, the dilution plate appeared with growth curve similar to EV76 strain in turn, and the color of tetrazolium dyes in the experimental wells gradually deepened as the phage number decreased and the host bacteria number increased. Therefore, it indicates that phage 476 was sensitively at both 28 ℃ and 37 ℃, while phage 087 and 072204 were temperature-dependent only at 37 ℃ to attenuated plague bacterium EV76. Conclusions The lysing ability of 3 wild-type plague phages are temperature-dependent, and the growth results are consistent under the three experimental conditions.

Objective To construct a technical platform for scarless gene modification of Yersinia pestis and to study the functions of its specific genes.Methods The resistance fragment, including upstream and downstream homologous arms of targeted regions, was reamplified by asymmetric PCR.The amplicons were introduced into Y.pestis harboring plasmid pKD46.With the induction of L-arabinose,the recombinant related enzymes: Exo, Beta and Gam, were expressed to guide the homologous recombination.A donor plasmid, pKSI-1, which carried the desired modification fragment flanking by I-SceⅠ recognition sites, was introduced into Y.pestis as the second step of λ-Red recombination with the help of pREDTKI.Results and Conclusion Two mutant strains:△waaA and waaA(△9nt), were successfully constructed for Y.pestis strain 201.Scarless modification introduces no extra modification to the genome, and it is ideal for comprehensive functional genomic studies.

Objective To investigate the biological characteristics and epidemiological significance of Yersinia pestis strains in Qilian County,Qinghai Province,in order to provide a scientific basis for plague prevention and control.Method Totally 67 strains were separated from kinds of host in Qilian County,Qinghai Province from 1958 to 2011,to do biochemical test,toxicity test,virulence factors evaluation,plasmid analysis and different region (DFR) genotyping.Results According to biochemical typing,48 of the 50 strains tested were Qing-Tibet Plateau ecotype,15 were Qilian Mountain ecotype,and the remaining 4 were different ecotypes from the plague foci in Qinghai plateau.The strains had 8 genomovars,and were given priority to genomovar8 (42 strains),secondly,genomovar44 (15 strains),genomovar5 (4 strains),genomovar7 (2 strains),genomovar19 (1 strain),genomovar30 (1 strain),genomovar32 (1 strain),and genomovar34 (1 strain).A proportion of 95.52％ (64/67) of the strains contained 3 kinds of plasmid-6 × 106,45 × 106,and 52 × 106;85.07％ (57/67) contained all the four virulence factors,and 96.00％ (48/50) were velogenic strains.Conclusion The strains separated in Qilian County,Qinghai Province have the characteristics of Qinghai-Tibet Plateau plague's pathogen and have strong toxicity,so we should enhance the plague monitoring and give more publicity to plague prevention to prevent animal plague spreading to human.

Objective: To investigate the CRISPR genotypes (clusters) and regional distribution of Yersinia pestis in Qinghai-plateau. Methods: One hundred and two isolates of Y. pestis isolated from human plague patients, host animal and insect vectors from Qinghai-plateau were selected. The DNAs were extracted using the traditional sodium dodecyl sulfate decomposition and phenol-chloroform method. Three CRISPR loci YPa, YPb and YPc of 102 isolates of Y. pesits were amplified and sequenced, and then the CRISPR sequence analysis was carried out by comparing the latest published CRISPR spacer dictionary and the NCBI database to identify the spacer and spacer array. CRISPR genotyping of isolates of Y. pesits were finally conducted according to the polymorphism of the spacer arrays and the regional distribution pattern of isolates of Y. pesits in Qinghai-plateau was described. Results: Forty spacers including 22 of YPa, 13 of YPb and 5 of YPc were observed among 102 isolates of Y. pestis in Qinghai-plateau, of which 5 spacers (a1', a103, a104, b4'' and b4''') were firstly identified. Meanwhile, 16, 10, and 5 different spacer arrays were obtained in YPa, YPb and YPc respectively, including 11 new spacer arrays detected in this study. One hundred and two isolates were divided into 24 CRISPR genotypes and classified into 9 CRISPR clusters (Cb4, Cb4', Cb2, Ca37, Ca7, Ca7', CaΔ5', Ca35' and Cc3'). Each dominant cluster presented significant aggregation geographically: Ca7 were found in Yushu, Nangqian, Chenduo, Zaduo, Zhiduo and Qumalai countries. Ca7' were found in Xunhua, Tongren, Zeku, Tongde, Maqin and Guinan countries. CaΔ5' were restricted to Qilian, Gangcha, Menyuan and Datong countries. CaΔ35' were found in Huangyuan, Haiyan, Gangcha, Tianjun, Delingha, Wulan, Doulan, Gonghe, Xinghai, Guide and Tongde countries. Conclusion CRISPR-based genotyping analyses showed complicated population of Y. pestis in Qinghai-plateau. Four clusters, Ca7, Ca7', CaΔ5' and Ca35' were the most epidemic dominant four clusters and presented obvious regional distribution patterns, which instructed us to strengthen the surveillance and prevention and control by CRISPR-genotyping technique.

Bacteriophages are abundantly distributed viruses , which are able to infect bacteria .Bacteriophages are becoming a focus of attention for microbiologists , as they can cause pollution to the fermentation industry and might serve as alternative therapies for antibiotic-resistant pathogenic bacteria .Effective bacteriophage infection normally involves adsorp-tion, injection, replication, assembly and release , against which bacteria have developed various resistance strategies .The research progress in the bacteriophage resistance mechanisms is briefly reviewed in this paper .

Objective By the method of multiple polymerase chain reaction (PCR),we intend to amplify different regions (DFR) of Yersinia pestis DNA,and to establish a multiple DFR genotyping technique for detection of Yersinia pestis.Methods According to the product size of 23 DFRs and pMT plasmid,24 primers were optimized and combined,then multiple primers in one PCR reaction system were added,and positive template DNA was amplified.Meanwhile,200 wild strain DNAs were amplified by multiple PCR and normal PCR,to verify the coincidence rate of the two methods.Results Totally 24 target segments were amplified through the positive DNA template.Through different permutation and combination,24 primers were optimized and combined into 9 groups.Totally 200 wild strain DNAs were used for verification,the coincidence rate of multiple PCR and normal PCR was 100％.Conclusions Multiple PCR is applicable and feasible for DFR genotyping of Yersinia pestis.It is an efficient,economic and high accuracy experimental method for large quantities of Yersinia pestis DFR genotyping.

Objective To explore the primary teeth caries status of preschoolers in Pudong community and its relevant factors of parental knowledge-belief-practice (KBP).Methods Using stratified random cluster sampling,a total of 1 179 children from 9 kindergartens in Hudong community in Pudong new area were examined.Oral cavity check-ups and parental questionnaire survey were conduced to understand the primary teeth caries rate of preschoolers and its related influencing factors.Results The prevalence rate of primary teeth caries was 53.2％ and DMFT 2.79.The prevalence rate of primary teeth caries had statistical significance in different age groups (P ＜ 0.05).Multi-factor analysis showed that the influencing factors of primary teeth caries were age (P ＜ 0.05),parental education level (P ＜ 0.05),parental assistance of teeth brushing (P ＜ 0.05),children eating sweets before bedtime (P ＜ 0.05) and parental awareness for treating primary teeth (P ＜ 0.05).Conclusions The prevalence rate of primary teeth caries is high among the preschoolers of Pudong.And age,parental education level,parental preventive knowledge of teeth caries and poor eating habits are significant factors correlated with the incidence of dental caries in children.

Objective To explore the correlation between social support and the self-efficacy among community patients with chronic obstructive pulmonary diseases ( COPD ) .Methods From October 2008 to March 2009 ,320 community patients with COPD were recruited from a community health service center by using a convenience sampling.The research data were taken by questionnaires and scale .The Spearman’s correlation analysis was done to analyze the correlation of social support and the self-efficacy.Results The total score of social support of the patients with COPD was ( 37 .64 ±5 .96 ) , which was lower than the national norm (40.80 ±7.64), and the total score of self-efficacy was (74.24 ±9.50).The number of patients with medium-level self-efficacy was 286 (89.4%).The overall social support was positively correlated with self-efficacy (r=0.553,P<0.01), and it was also positively correlated with the various dimensions of self-efficacy (r=0.190-0.515,P <0.05).Conclusions Social support and the self-efficacy are closely related.It is important to promote patients ’ self-efficacy and mobilize the social and family support by various approaches .

Objective To explore the relationship between self-efficacy and self-management behaviors of patients with chronic obstructive pulmonary disease (COPD).Methods A total of 350 COPD patients form 5 residents' committees in shanghai were recruited by using a convenient sampling method and were scored using Chinese Self-efficacy Scale (CSES)and the self-management behaviors scale.Spearman's correlation analysis was performed to detect the relation of self-efficacy with self-management behaviors.Results Three hundred and twenty adults were included in this investigation,and their average FEV1/FVC was (57.86 ± 7.06)％,average score of self-efficacy was 74.2 ± 9.5.In self-management behaviors,time spent on physical exercise was (16.2 ± 33.9) minutes per week,and endurance exercises accounted for (109.0±49.0) minutes per week.The score of cognitive symptom management practice was 0.9 ± 1.0 and communication with physicians was 0.7 ± 0.8.Total score of self-efficacy was positively correlated with self-management behavior in each dimension (r values were 0.522,0.407,0.330 and 0.044,respectively ; all P ＜ 0.01).Conclusions Self-efficacy and self-management behaviors of COPD patients need to be improved,and self-efficacy may be related to self-management behaviors.

Objective To investigate the self-efficacy levels and its influencing factors of community-based patients with chronic obstructive pulmonary diseases (COPD).Methods From October 2008 to March 2009,320 community COPD patients were recruited from a Shanghai community.They undertook questionnaires,scale survey and pulmonary function testing so as to investigate the influencing factors of self-efficacy.Results The total scale of self-efficacy was 74.24 ± 9.50 and the level of selfefficacy in 286 cases( 89.4％ )was intermediate.The knowledge of COPD,social supports,forced expiratory volume at 1 second (FEV1)/forced vital capacity (FVC) and self-management level were entered into regression equation and could explain 57.1％ of the total variance of independent variables.Conclusions The knowledge of COPD,social supports,FEV1/FVC and self-management level are the major influencing factors of self-efficacy in the COPD patients.We should improve the knowledge of disease and strengthen the psychological care and social supports so as to improve their quality of life.