Objective To investigate the antimicrobial resistance proifle of Shigella flexneri in Shanghai from 2010 to 2014 and examine the mechanism of fluoroquinolone resistance.Methods Kirby-Bauer method was used to determine the susceptibility of the S. flexneri strains to 14 antibiotics. The minimum inhibitory concentration (MIC) of ciprofloxacin was tested by E-test. Mutations within quinolone resistance determining regions (QRDR) of gyrA and parC and plasmid-mediated quinolone resistance (PMQR) genes,qnrA,qnrB,qnrS andaac(6')-Ib-crwere identified by polymerase chain reaction (PCR). All the products were subjected to sequencing analysis.Results More than 90 % of the 139S. flexneri isolates were resistant to ampicillin, streptomycin, tetracycline, and nalidixic acid, 40.3 % to ciprofloxacin and 30.2 % to cefepime, respectively. Genetic mutation of gyrA and parC was found in 98.6 % and 97.8 % of the strains, respectively. Three point mutations (Ser83, Asp87 and His211) were detected in gene gyrA and one point mutation (Ser80) was found in in gene parC. Plasmid-mediated resistant gene qnrS was found in 9 strains and aac(6')-Ib-cr in 6 strains.Conclusions The antibiotic resistance of S. flexneri is serious in Shanghai. The mutation rate within QRDR is high. Point mutation in Asp87 of gyrA is the main mechanism of quinolone resistance. The plasmid-mediated quinolone-resistant genes also play an important supplementary role.

Objective To establish a rapid staining method for facilitating initial identification of Legionella pneumophila in amoebal trophozoite. Methods Acanthamoeba polyphaga and Legionella pneumophila were co-cultured under laboratory condition. At consecutive time points during the culture, smears of the cultured products were made on glass slides for staining purposes. Different types of stainings including Gram′s staining, Gimenez staining, Giemsa staining and immunofluorescence were used to determine the best method for the identification of amoebal pathogens. Results Gimenez staining technique is simpler and yields better results as compared with the other three stainings. Gimenez stain gives the best color and contrast for amoeba and amoebal Legionella Amoeba trophozoites and/or cysts showed a distinct purplish blue with amoebal Legionella in red. Amoebal Legionella can be distinguished clearly at an earlier time of co-culture, providing a proper sensitivity. It takes only 10 minutes to finish the operation. The other techniques require the use of expensive reagents, are relatively time-consuming, and involve complex staining procedures. Conclusion Gimenez staining is of value for the initial identification of amoebal pathogens, and it is suitable for laboratory diagnosis.

Objective:To explore the rule of CD8+CD25+FoxP3+ regulator T cells(Treg) in the pathogenesis of pre-eclampsia (PE) in peripheral blood.Methods:This study included 24 gestational-age-matched healthy pregnant women and 46 pregnant women diagnosed with mild PE (MPE,n=24) or severe PE (SPE,n=22) during the third trimester of gestation.An 3 ml sample of peripheral blood was drawn from each subject and anti-coagulated with heparin sodiurm The percentage of CD8 + CD25 + FoxP3 + Treg cells was detected by flow cytometry.The cytokines IL-6,IL-17A,IL-10,IL-1,IL-33 and TGF-β31 were detected using Luminex200.Peripheral blood mononuclear cells (PBMCs) were isolated frum healthy controls and treated with IL-33,the percentages of CD8+ CD25+ FoxP3 + Treg cell were measured by flow cytometric detection.Results:Compared to that of healthy pregnant controls [0.48(0.21-0.96)％],MPE patients [0.32(0.19-0.63)％] and SPE patients [0.13(0.02-0.41)％] had lower percentages of CD8+CD25+FoxP3+Treg cells (P＜0.05).Compared to HP controls,higher levels of IL-6 and IL-17A were found in MPE and SPE patients(P＜0.05) and even higher in SPE patients.The levels of IL-10,IL-1[β and IL-33 were similar in all three groups (P＞0.05).Compared to HP contruls,the levels of TGF-[β1 was significantly increased in SPE and MPE patients(P＜0.05),but no significant differences were found between these two groups (P ＞ 0.05).The percentage of CD8+ CD25+ FoxP3+ Treg cells showed a negative correlation with the serum concentrations of IL-17A (r =-0.338,P =0.338),and a positive correlation with the serum concentrations of IL-33 (r =0.548,P =0.548).After PBMCs were treated with IL-33 for five days,the percentages of CD8+CD25+FoxP3+ Treg were significantly higher than those of the contruls (P＜0.05).Conclusion:These findings suggested that the reduced CD8+ CD25 + FoxP3 + Treg cells may play a role in the pathogenesis of ore-eclamosia.

Aim To observe the improvement effects of parecoxib on ventricular remodeling after acute myocardial infarction in rats and its influence on PI3K/Akt signaling pathway.Methods Forty male Sprague-Dawley rats were randomly divided into five groups(n=8):sham operation group (group S), ventricular remodeling model group(group R), low dose of parecoxib group(group P1), middle dose of parecoxib group(group P2), and high dose of parecoxib group(group P3).A myocardial infarction model was established by ligating the left anterior descending branch(LAD) of coronary artery in group R, group P1, group P2 and group P3.One day after the operation,the rats were given intraperitoneal injection of 4,8,12 mg·kg-1 parecoxibin Group P1, group P2 and group P3,respectively, for two weeks.The same volume saline was given in group S and group R.Four weeks later, LVSP, LVEDP,+dp/dtmax,-dp/dtmax were monitored.The hearts were harvested to calculate left ventricular hypertrophy index.The pathological change of heart was examined with an optical microscope.The expressions of atrial natriuretic peptide(ANP) mRNA and brain natriuretic peptide(BNP) mRNA were detected by RT-PCR.The expression of PI3K,Akt,P-Akt and caspase-3 was detected by Western blot.Results Compared with group S, the cardiac function was decreased, the left ventricular hypertrophy index, the expression levels of ANP,BNP mRNA, caspase-3 were increased, and the expression levels of PI3K, P-Akt were reduced in group R(all P<0.05).Compared with group R, the cardiac function was ameliorated, the left ventricular hypertrophy index were reduced in group P2 and group P3(all P<0.05).The expression levels of ANP,BNP mRNA, Caspase-3 were decreased, and the expression levels of PI3K and P-Akt were increased in group P1,group P2 and group P3(all P<0.05).Conclusions Middle and high doses of parecoxib can mitigate the process of ventricular remodeling after myocardial infarctionand improve the myocardial function, and its underlying mechanism may be related to activating PI3K/Akt signaling pathway.

Aim To investigate the role of matrix metalloproteinase-9 down-regulation in the learning and memory dysfunction induced by propofol treatment in rats.Methods 7-day-old SD rats were randomly divided into three groups(n=18):control group(NS group) and repeated doses of propofol group(RP group) was intraperitoneally injected with normal saline and propofol respectively for consecutive seven days, single dose of propofol group(SP group) were intraperitoneally injected with normal saline first for consecutive six days, and then injected with propofol on 7th day.The blood gas and glucose levels were monitored of six rats randomly selected from each group.Morris water maze was conducted to test the learning and memory functions of the remaining rats.The expression of MMP-9, BDNF and caspase-3 was detected by Western blot, and the hippocampal neuron apoptosis was determinated by TUNEL staining.Results Compared with NS group and SP group, the escape latency in RP group was prolonged significantly, exploration time and the number of crossing the platform in RP group were markedly decreased(P<0.05).The expressions of MMP-9 and mBDNF in RP group declined, but the expression of proBDNF and the ratio of proBDNF/mBDNF in RP group were higher than those in NS group and SP group(P<0.05).Compared with NS group and SP group, the number of apoptotic neurons and the expression of cleaved-caspase-3 in RP group were increased significantly, but the expression of pro-Caspase3 in RP group was reduced(P<0.05).There was no difference between SP group and NS group regarding all the results(P>0.05).Conclusions Repeated exposure to propofol can lead to a decline in long-term learning and memory functions in neonatal rats, which may be related to the down-regulation of MMP-9 expression, proBDNF and mBDNF conversion disorder in hippocampus and the apoptosis of hippocampal neurons.However, single exposure to propofol has no significant effect.

AIM: To investigate the effects of propofol on the expression of tissue-type plasminogen activator (tPA) and matrix metalloproteinase 9 (MMP9) in the hippocampus and the cognitive function in neonatal rats.METHODS: The 7-day-old rats were randomly divided into 3 groups: the rats in control (CON) group were intraperitoneally injected with normal saline for 7 d;the rats in single dose of propofol anesthesia (SP) group were intraperitoneally injected with normal saline for 6 d and with propofol on the 7th day;the rats in repeated dose of propofol anesthesia (RP) group were intraperitoneally injected with propofol for 7 d.Blood glucose and blood gas analysis were tested in 6 rats of each group.The rats were randomly selected from each group to isolate the hippocampal tissues at 2 h, 24 h, 48 h, 72 h and 30 d after the last injection.The spatial learning and memory functions of the other rats aged 25 d were determined by Morris water maze.The morphological changes of the hippocampus were observed by HE staining and Nissl's staining.The expression of tPA and MMP9 at mRNA and protein levels was determined by RT-PCR and Western blot.RESULTS: Compared with group CON, the protein expression of tPA and MMP9 in RP group was significantly decreased at each time point, while no significant decrease was observed in SP group except at the time point of 24 h.Compared with CON group, the mRNA expression of tPA and MMP9 was down-regulated obviously in RP group, which was not significantly down-regulated in SP group.From the 3rd training day of Morris water maze beginning, the escape latency was prolonged, and the space exploration time and the number of crossing the original platform location were reduced in RP group compared with CON group and SP group, while no significant difference was observed between CON group and SP group.Compared with CON group, the number of nerve cells reduced and nerve cells arranged in disorder in the hippocampus in RP group.Moreover, the number of Nissl body decreased significantly and finally developed into neuronal degeneration and necrosis in RP group, and no significant difference between SP group and CON group was observed.CONCLUSION: Repeated dose of propofol anesthesia leads to long-term cognitive dysfunction in neonatal rats, which may be related to the down-regulation of tPA and MMP9 expression and destruction of normal morphology and function of neurons in hippocampus, whereas single dose of propofol anesthesia has no such effects.

Biochemical indexes and blood composition in early lactation health,ketosis and hypocalcemia dairy cows were analyzed to make sure the milk composition characteristics with related diseases,the correlation analysis between early lactating dairy milk composition and blood biochemical were also make in order to provide support for the cattle health assessment.According to theblood index,72 Holstein cows 7-21 d postpartum,were divided into group subclinical hypocalcemia,ketosis test group and control group,24 heads each group.The blood and milk of cows were collected and used to analyze the correlation between blood biochemical indexes and milk composition.The results showed that ketosis and hypocalcemia induced the level of milk protein and non fat milk solids decreased,while the content of citric acid in milk increased.The correlation equation between citric acid in milk and serum NEFA,BHBA and GLU was y=3.192x-0.802,(R2 =0.363),y=4.594x-0.793,(R2 =0.320),y=1.228x+0.775,(R2 =0.261),in which x was the content of citric acid in milk.The results showed that the content of citric acid in milk had positively related to blood NEFA,which could be used as an early marker for the diagnosis of negative energy balance.The levels of BUN and ALB in blood can be used to evaluate the levels of milk protein and urea.

Objective: To understand the epidemiological characteristics and antibiotic resistance profiles of Campylobacter spp. in Shanghai from 2013 to 2016. Methods: Stool samples collected from diarrhea outpatients were cultured for Campylobacter spp., using the membrane filter method in 23 hospitals under the sentinel programs, from 2013 to 2016. All the strains were identified by biochemical tests and PCR. Broth microdilution method was used to investigate the antibiotic resistance of 179 Campylobacter spp. strains that including azithromycin, ciprofloxacin, erythromycin, gentamicin, tetracycline, nalidixic acid, telycin, klinthromycin and flurbenicol. Results: A total of 179 Campylobacter spp. strains were isolated from 10 444 stool samples (1.7%). Campylobacter jejuni and Campylobacter coli appeared as the predominant ones (94.4% and 5.6%). The incidence rate was higher in children than that in adults, with peaks of infections mainly from April to June and October to December. Campylobacter jejuni strains seemed highly resistant to ciprofloxacin (96.4%), tetracycline (83.4%) and nalidixic acid (81.7%). The resistant rates appeared higher on Campylobacter coli strains that isolated from patients. Some strains were resistant to multi-drugs. Conclusions Campylobacter spp. seemed one of the important pathogens that isolated from outpatients with diarrhea, in Shanghai. Both age and season related characteristics of Campylobacter spp. were seen. Campylobacter spp. isolated from patients was highly resistant to ciprofloxacin, tetracycline and nalidixic acid.

Objective To investigate the antimicrobial characteristics of shigella flexneri and to analyze the correlation between acquired resistance genes and mobile genetic elements.Methods 139 strains of shigella flexneri collected from each district of Centers for Disease Control and Prevention in Shanghai from 2010 to 2014 were recovered.The K-B method was used to determine the susceptibility of the strains to 13 antibiotics.And then 17 kinds of acquired resistance genes to β-lactams, sulfonamides, aminoglycosides and 7 kinds of genetic markers of mobile genetic elements were analyzed by PCR.Index cluster analysis was performed to explore the correlation between them.Results Among 139 strains of Shigella flexneri,3 kinds of genetic markers of mobile genetic elements,ISEcp1,intI1 and trbC,3 kinds of acquired β-lactam-resistance genes,CTX-M,OXA and TEM,2 kinds of acquired aminoglycoside-resistance genes,ant(3")-I and aac(6′)-Ib, 1 kind of overlapping gene of quaternary ammonium disinfectant and sulfonamides, qacEΔ1-sull and 1 kind of sulfamethoxazole/trimethoprim-resistant gene, dfrA1 were detected.The resistence genes,OXA,ant(3")-I and drfA1 were highly related with each other,which were mediated by Class 1 integron.TEM,qacEΔl-sull and aac(6′)-Ib were highly related with each other,which were mediated by trbC.Conclusion Acquired multidrug resistance gene transfer mediated by a variety of mobile genetic elements may have largely contributed to the spreading of resistant strains of Shigella.