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Objective To explore the application potential of forced expiratory volume in three second/forced vital capacity (FEV3/FVC) in early lung diseases,such as early airway obstruction and mild gas trap.Methods A total of 288 patients (excluding those with restrictive ventilation dysfunction) who underwent pulmonary function examination in the pulmonary function room of our hospital from January 2014 to October 2017 were collected.288 patients were divided into three groups.Group A:FEV3/FVC and forced expiratory volume in one second/forced vital capacity (FEV1/FVC) were normal;Group B:FEV3/FVC decreased alone;Group C:FEV1/FVC decreased.The general data and pulmonary function indexes of the three groups were compared.Results Compared with group A,group B had lower FEV1 ％ and diffusion capacity for carbon monoxide of the lung (DLCO％),but higher total lung capacity (TLC％),residual volume (RV％) and RV/TLC.Compared with group B,group C had higher TLC ％,RV％,RV/TLC％,while FEV1％,DLCO％ reduce more remarkably.There were significant differences in the three groups of small airway function (P ≤ 0.01).FEV3/FVC was positively correlated with max expiratory at 50％ FVC (MEF50％),max expiratory at 75％ FVC (MEF25％) and maximal mid expiratory flow (MMEF％).The correlation coefficients were respectively 0.613,0.610,0.608 (P ≤0.01).When FEV3/FVC serves as an indicator to determine airway obstruction,the specificity of it is 45.7％,sensitivity 98.5％,and negative predictive value 99％,positive predictive value 35.5％.Conclusions FEV3/FVC individual decline is the indication of early lung diseases such as mild airway obstruction,mild gas trap and diffuse disorder.
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Objective To detect biofilm formation and biofilm-associated genes of methicillin-resistant Staphylococcus aureus (MRSA) in clinical isolates. Methods The biofilm were determined by microtiter plate assay (MPA) and congo red agar (CRA) and the biofilm-associated genes icaA,sarA,fnbA,fnbB were detected by PCR in 33 strains of MRSA in clinical isolates. Results Of the 33 MRSA isolates, 29(87.9%) were MPA positive, 16(48.5%) were CRA positive; The icaA gene was present in 39.4% of isolated strains. Furthermore, 69.7% of strains harboured the sarA gene, 39.4% were fnbA positive and 75.8% were fnbB positive. As many as 87.9% strains had the ability to form biofilm in vitro. 44.8% of MRSA formed biofilm in ica-dependent mechanism and 55.2% of MRSA isolates formed biofilm in ica-independent mechanism. Of the biofilm positive MRSA, 75.9% were sarA positive, 37.9% were fnbA positive and 79.3% were fnbB positive. Conclusion Most of the MRSA strains formed biofilm in ica-independent mechanism. fnbB and sarA gene shows higher frequency among the biofilm-associated genes of MRSA, it may infer that most of the MRSA strains biofilm formation are fnbB-mediated. Meanwhile, sarA may be a positive regulator of fnbB, and thus drives the biofilm formation.
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Objective To observe the role of preoperative lung function test in predicting the risk of postoperation pulmonary complications in patients with chronic obstructive pulmonary disease (COPD) accepting non chest operations. Methods 80 patients accepting non-invasive chest operations during Oct 2006 to May 2013 in the third affiliated hospital of SYSU were studied retrospectively. All the patients accepted lung function test 1 week before operation. Based on the lung function records, patients were divided into 2 groups. 40 of them in COPD group, 40 in control group. The incidence rate of postoperation pulmonary complications in different group and the relationship between the severity of lung function decreasing and the rate of postoperation pulmonary complications were investigated. The differences of the American Society of Anesthesiologists (ASA) Physical Status Classification, body mass index, smoking index, length of stay, hospitalization costs between the 2 groups were also studied. Results The incidence rate of postoperation pulmonary disease in COPD group was 30% (12/40) while the rate in control group was 12.5% (5/40), the statistic difference was significant (P = 0.046). There was remarkable relationship between the severity of lung function decreasing and the rate of postoperation pulmonary complications(P=0.005), patients with mild to moderate lung function decreasing would be safer in operation, but patients with severe lung function decreasing would be in high risk(r=-0.451). Patients in COPD group were older than the control group, but there were no significant difference on body mass index, smoking index, length of stay, hospitalization costs between the 2 groups (P > 0.05). There was no relationship between ASA physical status classification and postoperation pulmonary complications. Conclusion Incidence of postoperation pulmonary complications in patients with COPD is high, which mainly manifests as pneumonia. It was important to test the lung function before non-invasive chest operations, especially in patients with COPD(P>0.05).
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Objective To assess antifungal activity of aspirin and fluconazole administered alone or in combination against Candida albicans biofilms in vitro,and to evaluate the combined effect of these two drugs.Methods The MIC50 of aspirin and fluconzole against biofilm-associated adherent cells were determined respectively,then the efficacy of combinations of aspirin and fluconazole were evaluated by calculating the fractional inhibitory concentration (FIC) index.The influence of aspirin on the mRNA expression of gene ALS3,HWP1 in biofilm cells were analyzed by fluorescent quantitative PCR assay.Results The MIC50 of aspirin for ATCC64550,clinical strains 14215,15346,15538,16335 were ＞ 1440 mg/L,＞ 1440 mg/L,1440 mg/L,720 mg/L and 1440 mg/L respectively,the MIC50 of fluconazole for biofilms cells of all the strains were ＞ 64 mg/L.Aspirin did not enhance the antifungal effect of fluconazole against biofilm formed by ATCC64550,but synergistic and additive effects were found for the combination of aspirin and fluconazole to block the biofilm formation by clinical isolates (FIC index =0.75,0.5,0.75,0.75).Quantitative real-time PCR analysis showed aspirin could reduce the transcript level of ALS3 and HWP1.Conclusion Aspirin could inhibit C.albicans biofilm formation; it may increase the sensitivity of biofilm cells of C.albicans to fluconazole.
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Objective To investigate the lipoteichoic acid(LTA) induced apoptosis and the expression of inflammatory cytokines in human alveolar macrophage (AM) and the anti-apoptotic and anti-inflamatory effect of moxifloxacin (MXF).Methods Obtained human AM from bronchoalveolar lavage and used MTT assay to observe the effects of LTA and MXF on cell activity,optical microscope to investigate the change of the cell morphology,flow cytometry to assess cell apoptosis,RT-PCR to detect the mRNA levels of TLR2,IL-1 β,IL-8 and TNF-α,ELISA for the production of IL-8 to exam RT-PCR.Results LTA showed cytotoxicity on AM in a dose-dependent manner ( P＜0.05 ) ; MXF inhibited the effect of LTA without cytotoxicicy ( P＜0.05 ).LTA promoted apoptosis ( P＜0.05 ) and the mRNA expressions of TRL2,IL-1 β,IL-8 and TNF-α significantly in AM (P＜0.05),the peaks and peak time ofthe above factors were (3.56±0.03) at 12 h,(46.63±7.06) at 6 h,(28.07±1.24) at 12 h and (2.34 ±0.50) at 3 h respectively and increased the release of IL-8 protein level at 24 h (P＜0.05).MXF inhibited the cell apoptosis and the above mRNA expression at 12h ( P＜0.05 ),and inhibited the IL-8 protein level at 24 h( P＜0.05 ).Conclusion LTA showed cytotoxicity on AM,induced AM apoptosis and increased the expression of TLR2,IL-I β,IL-8 and TNF-α of AM ; MXF could protect AM through inhibiting of the above effects and may play a key role beside bactericidal effect in gram-positive bacteria pneumonia.
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<p><b>OBJECTIVE</b>To explore the molecular mechanism of the differences in biofilm formation abilities of Candida albicans isolated from the respiratory tract.</p><p><b>METHODS</b>The biofilms formed by Candida albicans isolates from the respiratory tract and the standard strain ATCC90028 were examined for bacterial proliferation using XTT reduction assay. The mRNA expression of CPH1, EFG1, ALS3 and HWP1 in the isolates were measured with fluorescent quantitative RT-PCR.</p><p><b>RESULTS</b>XTT reduction assay demonstrated a strong ability of biofilm formation in 8 clinical isolates, and a relatively low biofilm formation ability in 7 clinical isolates and ATCC90028 strain. The strong and weak biofilm formers showed significant differences in ALS3 and HWP1 mRNA expressions (P<0.05) but not in EFG1 or CPH1 mRNA expressions (P>0.05).</p><p><b>CONCLUSION</b>The clinical isolates from the respiratory tract have different biofilm formation abilities under regulation by genes other than the transcription factors CPH1 and EFG1.</p>
Subject(s)
Humans , Biofilms , Candida albicans , Classification , Genetics , Physiology , DNA-Binding Proteins , Metabolism , Exons , Fungal Proteins , Metabolism , Membrane Glycoproteins , Metabolism , Phenotype , Respiratory System , Microbiology , Transcription Factors , MetabolismABSTRACT
AIM:Panton-Valentine leukocidin(PVL)is a pore-forming toxin secreted by Staphylococcus aureus epidemiologieally associated with the often-lethal necrotizing pneumonia.Until now,the mechanisms of pathogene-sis of PVL leading to the fatal pulmonia remains undefined and also acquired plenty of the toxins is difficult.In the present study，we obtain recombinant staphylococcal F and S components of the Panton-Valentine leukocidin by gene engineering and evaluate its biological activity in vitro,which provides an experimental basis for the further studies of its biological func-tion and its toxicity in pneumonia.METHODS:The full-length of F and S components of PVL gene amplified from the strain of Staphylococcus aureus DNA by hiSh-fidelity PCR was cloned into prokaryotic expression vector pET22b(+),and the vector was transformed into BL21(DE3)plysS to construct a prokaryotie expression system.The integrity of the opening-reading frame of each construct was verified by DNA sequencing.The recombinant PVL(rPVL)was induced by1.0 mmol/L IPTG.The expressed products were identified by SDS-PAGE and the fusion proteins(6His-LukS-PV and 6His-LukF-PV)were purified from lysates of transfeeted E.coli cells by affinity chromatography on nitrilotriacetic acid columns.The eytolytie activity was tested by incubation of rPVL with human polymorphonuclear neutrophils(PMNs)in vitro.RESULTS:The nueleotide sequence of the cloned PVL gene was the same as that of reported in GenBank.E coli BL21(DE3)plysS containing recombinant vectors grow at 37℃causes some proteins to accumulate as inclusion bodies.while incubation at 30℃led to a significant amount of soluble active proteins which accounted for about 31.7％ of the total bacterial protein.The relative molecular weight showed on SDS-PAGE profile was consistent with the expected value which the LukS-PV protein was about 34 kD.and the LukF-PV protein was about 35 kD.The purified rPVL was obtained and its cytolytic activity to PMNs was demonstrated.CONCLUSION:The genes of 1ukS-PV and lukF-PV are successfully cloned into plasmid pET22b(+)and expressed in E coli respectively.which provide a basis for analyzing the toxicity related to the diseases and further studies about the pathogenesis of PVL.
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Objective To elucidate the molecular basis for induced resistance of N. gonorrhoeae to ceftriaxone in vitro. Methods The reference strain ATCC49226 and clinical isolate ZSSY00205 of N. gon-orrhoeae were exposed to subinhibitory concentration of ceftriaxone for the induction of resistance. Then,suppression subtractive hybridization was performed with the pre-induction parent strains as drivers and post-induction mutant strains as testers to create a subtractive cDNA library. Following that, a total of 192 clones were randomly selected from the library, and arrayed by spotting onto nylon membranes. Finally, dif-ferentially expressed genes were screened by hybridization with labeled-RsaI restriction fragments from the sensitive and resistant N.gonorrhoeae strains respectively, and analyzed by sequencing and homology research using Blast program. Results A subtractive library for these resistant N.gonorrhoeae strains was generated by SSH technique. Microarray analysis and homology research confirmed 5 genes related to ceftriaxone resistance, i.e. mtrR, mtrC, gyrB, rpsJ and PJD1. Conclusions The induced resistance of N. gonorrhoeae to ceftriaxone may be associated with mtrR, mtrC, gyrB, rpsJ and PJD1 genes which probably mediate the resistance by enhancing the activity of efflux pump system.
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Objective To study influencing factor of mechanical ventilation (MV) time in patients withchronic obstructive pulmonary disease (COPD). Method Totally 128 patients with COPD and respiratory failurerequiting ventilation support were retrespectively investigated. The clinical characteristics of patients before and af-ter intuhation during the respiratory intensive care unit (RICU) stay were recorded and analyzed, t- test, X2 testand stepwise logistic regression analysis were used for statistical analysis. Results In the 128 patients, 61%,20%, and 9% of the patients required MV longer than 7, 14 and 21 days, respectively. There were no significantdifferences in the history of COPD, smoking, lung function, and complications between patients with MV>7 d, 14d, 21 d and patients with MV<7 d, 14 d, 21 d. Multiple regression analysis showed APACHE Ⅱ score (OR:2.3; 95% Ca: 1.2-5.7, P = 0.02) was an independent factor for MV > 7 days, while shock (OR: 0.7;95% CI: 1.0-1.9, P = 0.04) and decreased serum albumin (OR: 0.4, 95% CI: 0.2-0.8, P = 0.003)were an independent factors for MV > 21 days. The ventilator-associated pneumonia (VAP) was the most impor-rant risk factor for the time of MV. Conclusions APACHE Ⅱ score, serum albumin levels, hock and VAP arethe main factors affecting MV time in patients with COPD.
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OBJECTIVE To analyze homology,antimicrobial susceptibility and metallo-?-lactamase gene type of metallo-?lactamase producing Pseudomonas aeruginosa in burn wards.METHODS Antimicrobial susceptibility tests were performed with E-tests.Using 2-mercaptoethanol disc synergy test to screen metallo-?-lactamase(positive) strains from imipenem-resistant P.aeruginosa in burn wards.Metallo-?-lactamase gene and integrase gene were amplified and sequenced.Resistance plasmid transfer and curing experiments were implemented to study the transfer of imipenem resistance and plasmid DNA was extracted and purified with Qiagen Plasmid Mini Kit.Random amplified polymorphic DNA(RAPD) typing was carried out to analyze the homology of the isolates.(RESULTS) Six strains of P.aeruginosa were suggested to produce metallo-?-(lactamase) by 2(-mercaptoethanol) disc synergy test.Using primers described for bla(VIM),the amplifications were observed among all 6 isolates and a VIM-2 metallo-?-(lactamase) gene was identified by sequencing.All isolates which producing VIM-2 metallo-?-(lactamase) had class 1 integrase gene and derived from a same clonal origin.CONCLUSIONS(VIM-2) metallo-?-(lactamase) producing P.aeruginosa is prevalent in burn wards,all isolates which producing VIM-2 metallo-?-(lactamase) have class 1 integrase gene.
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Objective To study the mutations of Pseudomonas aeruginosa oprD gene in Imipenem-resistant clinical isolates. Methods Random amplified polymorphic DNA typing(RAPD) was carried out to analyse the homology in 10 Imipenem-resistant clinical isolates. Pseudomonas aeruginosa oprD gene in 10 Imipenem-resistant clinical isolates were amplified by polymerase chain reaction and sequenced. Results Ten Imipenem-resistant clinical isolates were divided into four different clones which can not produce metallo ?-lactamase. As compared with sequence X63152, oprD gene of Pseudomonas aeruginosa varied greatly. The aberration rates exceed 50%. There were multiple point mutations within 276～387 bp coding region of 30, 11, 9, 20, 31 strains. Due to the mutations of 308 bp G→C and 344 bp C→A, threonine and diaminocaproic acid were replaced by serine and threonine respectively. The DNA deletion of 393～412 bp in oprD gene contributes to the frame shift mutation in the following nucleotide sequence. The deletion of 264～273 bp in the coding region of oprD gene in 13 and 21 clone strains leads to frame shift mutations forming terminal codon TAA(319～321 bp).Base substitutions and multiple point mutations were obvious in the coding regions of 1 and 22 clone strains. Their aberration rates were 54.03% and 74.89% respectively. Conclusions The mutations of Pseudomonas aeruginosa oprD gene in Imipenem-resistant clinical isolates are various.
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OBJECTIVE To study the antimicrobial susceptibility and molecular epidemiology of imipenem-resistant Pseudomonas aeruginosa isolated from patients in burn wards in a hospital of Guangzhou. METHODS A total of 48 imipenem-resistant isolates were collected,antimicrobial susceptibility was tested by KirbyBauer disk diffusion method.Random amplified polymorphic DNA(RAPD) typing was carried out to analyze the homology. RESULTS All isolates were multiresistant,the orders of sensitivity rates of antibiotics were CIP,TOB,AMK,GEN,and FEP.Forty eight strains were classified into types A,B,C,and D based on RAPD pattern,types A and B were the most epidemic clones. CONCLUSIONS The prevalence of imipenemresistant P.aeruginosa in burn wards of the hospital is due to nosocomial infection.The prevalent strains are multiresistant.