Objective:Based on the modified ultracentrifugation method, the outer membrane vesicles (OMV) secreted by Klebsiella pneumoniae were rapidly separated, identified and quantified. Methods:Standard strains of classic Klebsiella Pneumoniae (cKP) purchased from the Clinical Laboratory Center of the National Health Commission, and hypervirulent Klebsiella pneumoniae (hvKP) which was donated by Taiwan University were cultured in M9 basal media for 9 hours, and the OMV were extracted by modified ultracentrifugation. The shape and size of OMV were identified by transmission electron microscopy (TEM), relative quantification by Stewart phospholipids analysis method. Two groups were compared using independent samples t test. Results:It was observed under the TEM that most of the OMV secreted by cKP and hvKP showed spherical vesicle structure and a small part were irregular. The diameter of OMV ranged from 20 to 250 nm, multiple vesicles could be seen in clusters. Relative quantification found that the number of OMV secreted by hvKP were more than cKP ( P<0.05). Conclusions:This study successfully achieved the extraction, identification and quantification of OMV from Klebsiella pneumoniae through the modified ultracentrifugation method, which provided a foundation for further study about the function and mechanism of OMV, and also provided new ideas for the treatment of bacteria. Based on the ultracentrifugation method, the OMV secreted by Klebsiella pneumoniae were rapidly separated and extracted, then identified and quantified.

At present, the laser induced breakdown spectroscopy (LIBS) has already become an analysis method with great application value and bright prospects. It has been reported that the LIBS technology had been successfully applied in the field of clinical laboratory diagnostics such as the identification of pathogens, the diagnosis of malignant tumors, the identification of caries, the analysis of onychomycosis, the detection of toxic and harmful elements and other applications with positive results. The remote-LIBS technique provides feasibility for detecting pathogen which is with significance of the disease prevention and control. It is possible to apply LIBS technology to in vivo diagnostics in the future.

To dynamically investigate the distribution and antimicrobial resistance profiles of bacteremia pathogens isolated from different regions in China in 2011, 2013 and 2016. Non-repetitive isolates from nosocomial bloodstream infections were retrospectively collected and detected for antimicrobial susceptibility tests (AST) by agar dilution or microbroth dilution methods. Whonet 5.6 was used to analyze the AST data. Among 2 248 isolates, 1 657 (73.7%) were Gram-negative bacilli and 591 (26.3%) were Gram-positive cocci. The top five bacteremia pathogens were as follows, Escherichia coli (32.6%, 733/2 248), Klebsiella pneumoniae (14.5%, 327/2 248), Staphylococcus aureus (10.0%, 225/2 248), Acinetobacter baumannii (8.7%, 196/2 248) and Pseudomonas aeruginosa (6.2%, 140/2 248). Colistin (96.5%, 1 525/1 581, excluding innate resistant organisms), tigecycline (95.6%, 1 375/1 438, excluding innate resistant organisms), ceftazidine/clavulanate acid (89.2%, 1 112 /1 246), amikacin (86.4%, 1 382/1 599) and meropenem (85.7%, 1 376/1 605) showed relatively high susceptibility against Gram-negative bacilli. While tigecycline, teicoplanin and daptomycin (the susceptibility rates were 100.0%), vancomycin and linezolid (the susceptibility rates were 99.7%) demonstrated high susceptibility against Gram-positive cocci. The prevalence of extended-spectrum β-lactamases (ESBLs)-producing Enterobacteriaceae were 50.6% (206/407), 49.8% (136/273) and 38.9% (167/429) in 2011, 2013 and 2016 respectively; carbapenem-non-susceptible Enterobacteriaceae were 2.2% (9/408), 4.0% (16/402) and 3.9% (17/439) in 2011, 2013 and 2016 respectively; The prevalence of multidrug-resistant A. baumannii (MDRA) was 76.4% (55/72) in 2011, 82.7% (43/52) in 2013 and 87.5% (63/72) in 2016, respectively. The prevalence of multidrug-resistant P. aeruginosa (MDRP) was 9.8% (5/51) in 2011, 20.0% (7/35) in 2013 and 13.0% (7/54) in 2016, respectively. The prevalence of methicillin-resistant S. aureus (MRSA) was 51.9% (41/79) in 2011, 29.7% (19/64) in 2013 and 31.7% (26/82) in 2016, respectively. The prevalence of high level gentamicin resistance (HLGR) of Enterococcus faecium and Enterococcus faecalis were 43.2% (48/111) and 40.9% (27/66), respectively. The predominant organism of carbapenem-non-susceptible Enterobacteriaceae was K. pneumoniae with its proportion of 57.1% (24/42). Among 30 tigecycline-non-susceptible Enterobacteriaceae, K. pneumoniae was the most popular organism with 76.7% (23/30). Among 39 colistin-resistant Enterobacteriaceae, E. coli, Enterobacter cloacae and K. pneumoniae were constituted with the percent of 43.6 (17/39), 35.9 (14/39) and 15.4 (6/39), respectively. The Gram-negative bacilli (E. coli and K. pneumoniae were the major organisms) were the major pathogens of nosocomial bacteremia, to which tigecycline, colistin and carbapenems kept with highly in vitro susceptibility. Whereas, among the Gram-positive cocci, S. aureus was the top 1 isolated organism, followed by E. faecium, to which tigecycline, daptomycin, linezolid, vancomycin and teicoplanin kept with highly in vitro susceptibility. Isolation of colistin-resistant Enterobacteriaceae, tigecycline-non-susceptible Enterobacteriaceae, linezolid- or vancomycin-non-susceptible Gram-positive cocci suggests more attention should be paid to these resistant organisms and dynamic surveillance was essential.

Objective To investigate the spectrum and antimicrobial resistance of major pathogens causing nosocomial infections in China, 2016. Methods Non-duplicated nosocomial cases as well as pathogens causing bloodstream infections ( BSI) , hospital-acquired pneumonia ( HAP) and intra-abdominal infections ( IAI ) from 12 teaching hospitals across China were collected. The minimum inhibitory concentrations (MICs) of important clinical common strains were determined by agar dilution method or broth microdilution method. The CLSI M100-S27 criteria was used for interpretation. Data were analyzed by using WHONET-5. 6 software. Results A total of 2060 cases were collected, including 894 cases from BSI, 630 cases from HAP and 536 cases from IAI. The MICs of 1896 important clinical common strains were determined. Escherichia coli and Klebsiella pneumoniae were the most prevalent pathogens causing BSI and IAI, while Acinetobacter baumanii and Pseudomonas aeruginosa were dominated in HAP. All Staphylococcus aureus were susceptible to tigecycline, linezolid, daptomycin and glycopeptides. Methicillin-resistant S. aureus accounted for 44. 4％ ( 75/169 ) of all the S. aureus. The rate of methicillin-resistant coagulase-negative staphylococci was 80. 9％ ( 72/89 ) . No Enterococcus strains were found resistant to tigecycline, linezolid or daptomycin. Vacomycin resistant enterococcus was found in Enterococcus faecium, accounting for 1. 8％ ( 2/111 ) of all E. faecium strains. Tigecycline, meropenem, amikacin, imipenem, and polymyxin B exhibited high potency against Enterobacteriaceae and the susceptibility rates were 96. 6％(865/895), 94. 3％ (859/911), 94. 2％ (858/911), 94. 1％ (857/911), and 91. 6％ (820/895), respectively. The prevalence of extended-spectrum β-lactamase was 58. 4％ ( 263/450 ) in E. coli and 28. 6％ ( 84/294 ) in K. pneumonia. The rate of carbapenem resistant K. pneumonia and E. coli was 15. 3％ ( 45/294 ) and 1. 8％ ( 8/450 ) , respectively. The percentage of polymyxin B resistant K. pneumonia and E. coli was 4. 1％ ( 12/294 ) and 4. 4％ ( 20/450 ) , respectively. The rate of tigecycline resistant K. pneumonia and E. coli was 2. 4％ ( 7/294 ) and 0. 2％ ( 1/450 ) , respectively. A. baumanii showed low susceptibility to the antimicrobial agents except tigecycline ( 91. 4％, 235/257 ) and polymyxin B (100％, 257/257). The rate of carbapenem resistant A. baumanii was 80. 5％ (207/257). The rate of carbapenem resistant P. aeruginosa was 31. 7％ ( 59/186 ) . Polymyxin B and amikacin demonstrated high antibacterial activity against P. aeruginosa with susceptility rate of 100％ ( 186/186 ) and 90. 9％ ( 169/186), respectively. Conclusions Nosocomial pathogens showed high susceptibilities against tigecycline and polymyxin B. Antimicrobial resistance in A. baumannii is a serious problem. The prevalence of carbapenem-resistant Enterobacteriaceae and polymyxin B resistant Enterobacteriaceae has increased, which should be monitored continuously in China.

OBJECTIVE: To investigate the phylogenetics and prevalence of bloodstream infections with ST131, the antimicrobial resistance profiles of the pathogens, and the clinical features. METHODS: Non-duplicate isolates were collected from 144 patients with bloodstream infections in our hospital between January and December, 2016.The phylogenetic groups of the isolates were analyzed using multiplex PCR, and O serotyping of ST131 strains was performed by allele-specific PCR.The clinical characteristics of the 144 patients were analyzed to define the differences in the clinical features between patients with ST131 infection and those with non-ST131 infection.Antibiotic susceptibility of the isolates was determined using the Vitek 2 compact system. RESULTS: The phylogenetic group analysis showed a domination by group B2 (41.0%[59/144]), followed by group F, group B1 and group E, which accounted for 16.7%(24/144), 13.9%(20/144), and 13.2% (19/144), respectively.Nine strains (6.3%) of were identified to be ST131 strains, among which 8 were O25b-B2-ST131 strains and 1 was O16-B2-ST131 strain.Of the 9 cases of ST131 infection, 7(77.8%) were found to occur in a nosocomial setting.The demographic characteristics and clinical features of the ST131-infected patients were similar to those of non-ST131-infected patients.ST131 strains were sensitive to piperacillin/tazobactam, imipenem, ertapenem, and amikacin, but showed high resistance rates to cefazolin, ceftriaxone, ciprofloxacin, levofloxacin, gentamicin, and trimethoprim/ sulfamethoxazole (all over 50%).The positivity rate of ESBLs in the ST131 strains was 77.8%, and the multidrug resistance rate reached 88.9%, which was higher than that of non-ST131 isolates, but the difference was not statistically significant. CONCLUSIONS The most common phylogenetic groups of isolates from patients with bloodstream infections are group B2 and F, and the positivity rate of ST131 is low.We for the first time detected O16-ST131 in patients with blood-borne infections in China.The clinical features of ST131-infected patients are similar to those of non-ST131-infected patients.The positivity rate of ESBLs and the multidrug resistance rate are high in ST131 strains, which may raise concerns in the future.
Anti-Bacterial Agents ; therapeutic use ; Bacteremia ; drug therapy ; epidemiology ; microbiology ; China ; Drug Resistance, Bacterial ; Escherichia coli ; classification ; drug effects ; genetics ; Escherichia coli Infections ; drug therapy ; epidemiology ; microbiology ; Genotype ; Humans ; Microbial Sensitivity Tests ; Molecular Epidemiology ; Phylogeny ; Species Specificity

Objective To investigate the antimicrobial resistance profile of the clinical isolates collected from selected hospitals across China. Methods Twenty-nine general hospitals and five children's hospitals were involved in this program. Antimicrobial susceptibility testing was carried out according to a unified protocol using Kirby-Bauer method or automated systems. Results were interpreted according to CLSI 2017 breakpoints. Results A total of 190 610 clinical isolates were collected from January to December 2017, of which gram negative organisms accounted for 70.8％ (134 951/190 610) and gram positive cocci 29.2％ (55 649/190 610). The prevalence of methicillin-resistant strains was 35.3％ in S. aureus (MRSA) and 80.3％ in coagulase negative Staphylococcus (MRCNS) on average. MR strains showed much higher resistance rates to most of the other antimicrobial agents than MS strains. However, 91.6％ of MRSA strains were still susceptible to trimethoprim-sulfamethoxazole, while 86.2％ of MRCNS strains were susceptible to rifampin. No staphylococcal strains were found resistant to vancomycin. E. faecalis strains showed much lower resistance rates to most of the drugs tested (except chloramphenicol) than E. faecium. Vancomycin-resistant Enterococcus (VRE) was identified in both E. faecalis and E. faecium. The identified VRE strains were mainly vanA, vanB or vanM type based on phenotype or genotype. The proportion of PSSP or PRSP strains in the non-meningitis S.pneumoniae strains isolated from children decreased but the proportion of PISP strains increased when compared to the data of 2016. Enterobacteriaceae strains were still highly susceptible to carbapenems. Overall, less than 10％ of these strains (excluding Klebsiella spp.) were resistant to carbapenems. The prevalence of imipenem-resistant K. pneumoniae increased from 3.0％ in 2005 to 20.9％ in 2017, and meropenem-resistant K. pneumoniae increased from 2.9％ in 2005 to 24.0％ in 2017, more than 8-fold increase. About 66.7％ and 69.3％ of Acinetobacter (A. baumannii accounts for 91.5％) strains were resistant to imipenem and meropenem, respectively. Compared with the data of year 2016, P. aeruginosa strains showed decreasing resistance rate to carbapenems. Conclusions Bacterial resistance is still on the rise. It is necessary to strengthen hospital infection control and stewardship of antimicrobial agents. The communication between laboratorians and clinicians should be further improved in addition to surveillance of bacterial resistance.

Objective To explore the clinical application value of the prealbumin (PA),total bile acid (TBA),and red blood cell volume distribution width (RDW) in chronic liver disease.Methods Totally 393 cases of patients with chronic liver disease admitted by Xiangya Hospital of Central South University from March 2015 to March 2016 were selected as group observation,and were divided into chronic hepatitis,compensated liver cirrhosis,decompensated liver cirrhosis and primary liver cancer.At the same time,200 cases of healthy volunteers were collected as normal control.Serum prealbumin and total bile acids were tested as well as the RDW of all cases.SPSS 17.0 software was used for data statistics processing.The receiver-operating characteristic curve (ROC) was drawn to evaluate the diagnosis value of the indexes in chronic liver disease severity.Results PA in the observation group was significantly lower than normal control,while its TBA and RDW were significantly higher than normal control.All of three parameters in patients,especially with liver cirrhosis and decompensated liver cirrhosis,had higher positive rate.When the clinical diagnosis was taken as gold standard,the best level of PA to diagnose primary liver cancer,chronic hepatitis and decornpensated liver cirrhosis was 244.7 mg/L,238.5 mg/L and 132.8 mg/L,the AUC was 0.973,0.909 and 0.879,the sensitivity was 92.3％,95.1％ and 85.6％,and the specificity was 95.8％,72.8％ and 79.7％;the best level of RDW to diagnose primary liver cancer,chronic hepatitis and decompensated liver cirrhosis was 13.2％,13.8％ and 14.3％,the AUC was 0.816,0.827 and 0.818,the sensitivity was 66.7％,77.4％ and 72.2％,and the specificity was 79.5％,73.8％ and 77.3％.When combined detection of PA and RDW,the diagnostic performance had improved significantly.Conclusions Serum prealbumin and total bile acid,as well as the whole blood RDW may objectively reflect the injury of liver metabolism and synthesis function,and for the early diagnosis and prognosis of patients with chronic liver disease has a important clinical significance.

Objective To study the relationship between biofilm-forming ability,distribution of quorum sensing related genes and antibiotic resistance in clinical isolates of Pseudomonas aeruginosa.Methods The biofilm-forming ability of 94 clinical isolates was analyzed semi-quantitatively by crystal violet staining.The antibiotic resistance of the isolates was determined by K-B method.Quorum sensing related genes,lasI,lasR,rhlR and rhlI,were detected by PCR.The diffe,rences of drug resistance of Pseudomonas aeruginosa with different biofilm-forming ability and the effects of quorum sensing related genes on biofilm-forming ability were analyzed.Results Of the 94 isolates,89(94.7％) showed biofilm-forming ability.The 89 isolates consisted of 22(23.4％) isolates with weakly positive biofilm-forming ability,44 (46.8 ％) with positive biofilm-forming ability and 23 (24.5 ％) with strongly positive biofilm-forming ability.The strains of Pseudomonas aeruginosa with different biofilm-forming ability showed different drug resistance rates to amikacin,tobramycin and gentamicin (P ＜ 0.05).The drug resistance rate of the strains with strong positive biofilm-forming ability to amikacin was higher than that of the strains with positive and weakly positive biofilm-forming ability(P ＜ 0.05),and the drug resistance rates to tobramycin and gentamicin were higher than those of the strains with weakly positive biofilm-forming ability(P ＜ 0.05).Of the 94 isolates,91 strains carried lasI,lasR,rhlI and rhlR gene and 2 strains only lost lasR gene,and 1 strain lost all the 4 genes.The strains with only lasR gene deficiency or all the lasI,lasR,rhlI and rhlR gene deficiencies showed negative biofilm-forming ability,and were sensitive to conventional antimicrobial agents.Conclusion Most of the clinical isolates of Pseudomonas aeruginosa in this study showed strong ability of biofilm-forming ability which may correlate positively to partial antibiotic resistance.The quorum sensing related genes may affect biofilm formation of Pseudomonas aeruginosa.

Objective To investigate the diagnostic value of seven tumour-associated autoantibodies,including p53,PGP9.5,SOX2,GAGE7,GBU4-5,MAGE A1 and CAGE autoantibodies,in the newly diagnosed patients with lung cancer.Methods A total of 108 patients with newly diagnosed lung cancer,120 with benign lung diseases,227 with other cancers and 96 healthy controls were enrolled in the study.Their serum levels of seven autoantibodies were detected by enzyme linked immunosorbent assay (ELISA).The ROC curve was drawn and used to analyze their diagnostic efficiency for lung cancer.The diagnostic value of the combination of seven autoantibodies in different groups was also compared.Results The serum levels of seven autoantibodies in lung cancer patients were significantly higher than those in the patients with benign lung diseases or other cancers and healthy controls (P ＜ 0.05).The sensitivity,specificity and AUCROc of the combination of seven autoantibodies in the preliminary diagnosis of lung cancer were 62.00％,89.80％ and 0.769,respectively,and its sensitivity and AUCROC were higher than those of single autoantibody.The positive rate of the combination of seven autoantibodies in lung cancer patients was significantly higher than those in healthy controls (x2 =50.885,P ＜ 0.01) and the patients with benign lung diseases (x2 =56.341,P ＜ 0.01) or other cancers (x2 =46.812,P ＜ 0.01).Conclusion The combination detection of seven autoantibodies,including p53,PGP9.5,SOX2,GAGE7,GBU4-5,MAGE A1 and CAGE,may serve as potential markers for the diagnosis of lung cancer.

Objective To investigate the drug resistant mechanism and homology of three strains of carbapenem-resistant Klebsiella pneumoniae (K.pneumoniae) isolated from different sites of one patient.Methods Three strains of carbapenem-resistant K.pneumoniae were isolated from femoral vein catheter tip,wound secretions and sputum of a patient with severe burns,respectively.Their carbapenemase,metallo-β-lactamase (MBL) and drug resistance genes were detected by modified Hodge test,double-disk synergy test and combination disk diffusion and PCR,respectively,and homology and biological typing were analyzed by enterobacterial repetitive intergenic consensus-PCR (ERIC-PCR) assay and multilocus sequence typing (MLST) technology,respectively.Results The carbapenemase and MBL of three strains of carbapenem-resistant K.pneumoniae were negative and positive,respectively.The blaNDM-1 gene was identified from the three strains,but other drug resistance genes such as blanC,blaGES,blaIMP,blaSPM,blaVIM,blaGIM and blaOXA-48 were not detected.ERIC-PCR showed that three isolates belonged to the same genotype,and MLST showed that they were type ST17.Conclusion Carring blaNDM-1 gene is the main cause leading to the drug resistance of three strains of carbapenem-resistant K.pneumoniae,and they belong to the same genotype.