Objective To knock down hemolysin hlyA gene and insert green fluorescence protein gene of vibrio cholerae vaccine candidate NFYY101. Methods Amplified fragments of hlyA gene upstream (hlyAup) and downstream (hlyAdown),lacz-GFPuv,and hlyAup-laczGFPuv-hlyAdown, and plasmids treated with specific enzymes were utilized to construct recombinant plasmids pUC18-hlyAup-laczGFPuv-hlyAdown and pCVD442-hlyAup-laczGFPuv-hlyAdown. Following the construction of the recombinant suicide plasmids ,a vaccine candidate was constructed by homologous recombination ,while SM10λpir carrying pCVD442- hlyAup-laczGFPuv-hlyAdown was utilized as the donor strain to transfect NFYY101. Results Construction of recombinant suicide plasmids pCVD442- hlyAup-laczGFPuv-hlyAdown was satisfactory that hemolysin hlyA gene was knocked out and green fluorescence protein gene was successfully inserted of vibrio cholerae vaccine candidate NFYY101. Conclusion Construction of the recombinant suicide plasmid pCVD442-hlyAup-laczGFPuv-hlyAdown successfully can be used for knocked out the hlyA gene and added green fluorescence protein gene as genetic marker into the chromosomal DNA of vibrio cholerae vaccine candidate.

Objective To probe into the epidemic feature, pa tho genic spectrum and main risk factors of diarrhea during the field training in ar my. Methods Detection of morbidity,investigation of risk factor s and detachment of pathogen were performed. Results The diarrh eal incidence rate of army of field training was 4.36%(106/2 433), which was higher than that of hold garrison(0.98%, 2/204). The incidence rate of officers was lower than that of soldiers. Two incidence peaks concerned with the army motion course were foun d. The detection rate of lapactic bacteria from fecal of patients was 63.1%(65/1 03) and most of them were ETEC, followed by the plesiomonas shigelloides. Se ven ind ividual factors concerned with diarrhea disease were found with logistic analysi s, including dr inking unboiled water, going out to eating and so on. Conclusions Incidence of diarrhea in the army increased obviously during the field training and the chief pathogen are ETEC and plesiomonas shigelloides, at the same time, t he occurrence of diarrhea is related with many individual factors.

Objective To establish an infection model using Caenorhabditis elegans (C.elegans)-extensively drug-resistant Klebsiella pneumoniae (XDRKP)system.Methods Clinically isolated XDRKP strains were used to infect C.elegans in the liquid killing assay,the nematode survival and the number of bacteria in C.elegans digestive tract was observed.Results C.elegans was significantly retarded after being infected by XDRKP,different concentra-tions of XDRKP led to different patterns of the worm death.Log-rank test showed that survival curves of C. elegans infected with 1 .5×106 CFU/mL of XDRKP and E.coli OP50 (control)were not significantly different (χ2 =0.08,P >0.05);survival curves of C.elegans infected with 1 .5 ×107 CFU/mL,1 .5 ×108 CFU/mL of XDRKP and E.coli OP50 were significantly different(χ2 =229.37,275.98,respectively,both P <0.001).The survival rates of 1 .5×108 and 1 .5 ×107 CFU/mL XDRKP groups were both lower than that of the control group.Supernatant suspension obtained from test was performed bacterial culture,identification and antimicrobial susceptibility testing, XDRKP was determined.After being infected with XDRKP 4,6,12,and 24 hours,the total number of bacteria in C.elegans were(0.28±0.02)×105 CFU/mL,(0.50 ±0.38)×105 CFU/mL,(1 .73 ±0.56)×105 CFU/mL,and (2.62±0.53)×105 CFU/mL,respectively,the number of bacteria in C.elegans digestive tract was significantly different at different time points (F =1 363.39,P <0.001).Conclusion The infection model of C.elegans-XDRKP is established successfully.

Objective To investigate the current situation on the infection of Acinetobacter baumannii in ICU and evaluate the efficiency of resistance control project which was performed in Xiaolan hospital,so as to provide scientific basis for clinical rational use of antibiotics.Methods Used VITEK-2 microbiology analysis system for the pathogen identification and drug sensitivity.Compared drug resistance of Acinetobacter baumannii isolated from the samples of ICU patients in 2012 (before the project executed)with that in 2013 (after the project executed).Results 71 strains and 57strains Acinetobacter bauman-nii were isolated from the ICU in 2012 (before the project executed)and in 2013 (after the project executed)respectively, which majorly isolated from the samples of respiratory system,and occupied 94.4% and 84.2% respectively.The infection rate of multi-resistant Acinetobacter baumannii was decreased from 77.5% to 66.7%,and the resistance rate of Acinetobact-er baumannii to cefepime,ceftazidime,cefatriaxone,tobramycin and cotrimoxazole decreased distinctly after the implementa-tion of drug resistance control management (χ2 > 3.84,P <0.05).Conclusion The drug resistance rates of Acinetobacter baumannii to 14 kinds of routine antibacterial drugs in 2013 were lower than that in 2012 in ICU.Among them,the drug re-sistance rates of Acinetobacter baumannii to 5 kinds of antibacterial drugs decreased distinctly.

OBJECTIVETo study the in vitro antibacterial activity of meropenem combined with doxycycline, ciprofloxacin, sulbactam or cefoperazone/sulbactam against clinically isolated extensively drug-resistant Acinetobacter baumannii (XDRAB).

METHODSUsing a checker board synergy design, the minimal inhibitory concentration (MIC) of antibiotics against 50 isolates of XDRAB was determined by broth microdilution antifungal susceptibility test. The fractional inhibitory concentration (FIC) index was calculated to determine the combined effect of the antibiotics.

RESULTSMeropenem showed significantly reduced MIC50 and enhanced antimicrobial activities when combined with doxycycline, sulbactam or cefoperazone/sulbactam. The FIC results suggested that the main actions of doxycycline, sulbactam, and cefoperazone/sulbactam were synergistic (38%, 26%, and 10%, respectively) and addictive (62%, 74%, and 90%, respectively) without indifferent or antagonistic effects. The main actions of meropenem combined with ciprofloxacin were additive (56%) and indifference (44%) with synergistic and antagonistic effects.

CONCLUSIONMeropenem combined with doxycycline, sulbactam or cefoperazone/sulbactam shows excellent activity against clinical isolates of XDRAB.

Objective To study the in vitro antibacterial activity of meropenem combined with doxycycline, ciprofloxacin, sulbactam or cefoperazone/sulbactam against clinically isolated extensively drug-resistant Acinetobacter baumannii (XDRAB). Methods Using a checker board synergy design, the minimal inhibitory concentration (MIC) of antibiotics against 50 isolates of XDRAB was determined by broth microdilution antifungal susceptibility test. The fractional inhibitory concentration (FIC) index was calculated to determine the combined effect of the antibiotics. Results Meropenem showed significantly reduced MIC50 and enhanced antimicrobial activities when combined with doxycycline, sulbactam or cefoperazone/sulbactam. The FIC results suggested that the main actions of doxycycline, sulbactam, and cefoperazone/sulbactam were synergistic (38%, 26%, and 10%, respectively) and addictive (62%, 74%, and 90%, respectively) without indifferent or antagonistic effects. The main actions of meropenem combined with ciprofloxacin were additive (56%) and indifference (44%) with synergistic and antagonistic effects. Conclusion Meropenem combined with doxycycline, sulbactam or cefoperazone/sulbactam shows excellent activity against clinical isolates of XDRAB.

Objective To study the in vitro antibacterial activity of meropenem combined with doxycycline, ciprofloxacin, sulbactam or cefoperazone/sulbactam against clinically isolated extensively drug-resistant Acinetobacter baumannii (XDRAB). Methods Using a checker board synergy design, the minimal inhibitory concentration (MIC) of antibiotics against 50 isolates of XDRAB was determined by broth microdilution antifungal susceptibility test. The fractional inhibitory concentration (FIC) index was calculated to determine the combined effect of the antibiotics. Results Meropenem showed significantly reduced MIC50 and enhanced antimicrobial activities when combined with doxycycline, sulbactam or cefoperazone/sulbactam. The FIC results suggested that the main actions of doxycycline, sulbactam, and cefoperazone/sulbactam were synergistic (38%, 26%, and 10%, respectively) and addictive (62%, 74%, and 90%, respectively) without indifferent or antagonistic effects. The main actions of meropenem combined with ciprofloxacin were additive (56%) and indifference (44%) with synergistic and antagonistic effects. Conclusion Meropenem combined with doxycycline, sulbactam or cefoperazone/sulbactam shows excellent activity against clinical isolates of XDRAB.

Objective:To analyze the identification and antibiotics susceptibility of Herbaspirillum in catheter-related bloodstream infection, and improve the awareness and attention of the rare pathogenic microorganisms for clinicians and microbiologists. Methods:The bacterium was isolated from a positive blood culture of a hemodialysis patient with chronic renal failure. The smear of isolate was prepared, stained and observed by microscope. The single colonies were identified by mass spectrometry and VITEK 2 Compact identification and antibiotics sensitivity analysis system, respectively. Then, 16S ribosomal DNA (rDNA) was amplified and sequenced, and bacterial genome was sequenced.Results:The gram-negative bacilli was found in the positive blood culture bottle. After incubated on blood agar for 16 hours, milky white, bulging and non-haemolytic colonies were observed. The identification result was Burkholderia cepacian by VITEK 2_Compact and antimicrobial susceptibility testing showed resistance to aztreonam and polymyxin but sensitive to other drugs in N335 card. The isolate could not be identified by VITEK MS with clinical database. However, it was identified as Herbaspirillum huttiense/Herbaspirillum aquaticum with research database. The 16S rDNA of the strain was consistent with Herbaspirillum huttiense and Herbaspirillum aquaticum (more than 99%). High-throughput bacterial genome sequencing revealed that the isolate in this case shared 100% homology with Herbaspirillum huttiense subsp putei IAM 15032 in Genbank database, which confirmed that the isolate was Herbaspirillum huttiense. Conclusions:There are more and more environmental microorganisms evolved into human pathogenic bacteria. Herbaspirillum species are easy to be misidentified because its biochemical characteristics are similar to other strains.

Objective:To assess the effect of serum heat inactivation on the detection of 2019 novel coronavirus (2019-nCoV) specific IgM and IgG antibodies by colloidal gold method.Methods:The serum specimens were collected from a total of 106 Coronavirus Disease 2019 (COVID-19) patients and 52 control subjects. Both the fresh serum and the heat inactivated serum samples from the same patient were detected simultaneously with the 2019-nCoV IgM and IgG antibodies detection kit (colloidal gold method). According to the patient′s onset time, the positive rates of antibodies production profile were calculated. The influence of heat inactivation on the detection rates of antibodies at different stages of disease after onset was analyzed.Results:The test results of the specimens of the healthy control group before and after inactivation were all negative. For the 106 specimens of COVID-19 patients, the detection rates of 2019-nCoV specific IgM and IgG antibodies were reduced after heating at 56 ℃ for 30 min. The positive rates of IgM antibodies significantly decreased from 66.04% (70/106) to 43.40% (46/106) ( χ2=22.042, P=0.000), while the positive rates of IgG antibodies slightly decreased from 81.13% (86/106) to 76.42% (81/106) ( χ2=0.800, P=0.063). Further analysis revealed that there was a significant difference in the positive rates of IgM antibodies before and after heat inactivation in the 3rd, 5th and 6th week after onset. However, there was no statistically significant difference in the detection rates of IgG antibodies before and after serum heat inactivation in different periods of onset. Conclusions:Heat inactivation significantly decreased the detection rates of 2019-nCoV specific IgM antibodies, which may lead to serological false negative results.

Objective To investigate the expression level of Kruppel-like transcription factor family member KLF11 in intestinal mucosal tissues of Crohn's disease (CD) and its regulatory effect on intestinal inflammation in CD-like colitis. Methods We examined KLF11 expression levels in diseased and normal colon mucosal tissues from 12 CD patients and 12 patients with colorectal cancer using immunofluorescence staining. KLF11 expression was also detected in the colon mucosal tissues of a mouse model of 2,4,6-trinitrobenesulfonic acid (TNBS)-induced colitis. A recombinant adenoviral vector was used to upregulate KLF11 expression in the mouse models and the changes in intestinal inflammation was observed. A Caco-2 cell model with stable KLF11 overexpression was constructed by lentiviral infection. The effect of KLF11 overexpression on expressions of JAK2/STAT3 signaling pathway proteins was investigated using immunoblotting in both the mouse and cell models. The mouse models were treated with coumermycin A1, a JAK2/STAT3 signaling pathway agonist, and the changes in intestinal inflammatory responses were observed. Results The expression level of KLF11 was significantly lowered in both the clinical specimens of diseased colon mucosal tissues and the colon tissues of mice with TNBS-induced colitis (P<0.05). Adenovirus-mediated upregulation of KLF11 significantly improved intestinal inflammation and reduced the expression levels of inflammatory factors in the intestinal mucosa of the colitis mouse models (P<0.05). Overexpression of KLF11 significantly inhibited the expression levels of p-JAK2 and p-STAT3 in intestinal mucosal tissues of the mouse models and in Caco-2 cells (P<0.05). Treatment with coumermycin A1 obviously inhibited the effect of KLF11 upregulation for improving colitis and significantly increased the expression levels of inflammatory factors in the intestinal mucosa of the mouse models (P<0.05). Conclusion KLF11 is downregulated in the intestinal mucosa in CD, and upregulation of KLF11 can improve intestinal inflammation and reduce the production of inflammatory factors probably by inhibiting the JAK2/STAT3 signaling pathway.