The Comparative Studies Of Drug Sensitivity Of Carbapenems Combine With Sulbactam Or Cefoperazone On Pseudomonas Aeruginosa

Objectives:To compare the drug sensitivity of carbapenems combine with sulbactam or cefoperazone on Pseudomonas Aeruginosa(PA) in vitro.Methods:1.Kirby-Bauer(K-B) assay:Kirby-Bauer(K-B) disc agar diffusion method was used to measure drug sensitivity in120clinical Pseudomonas Aeruginosa strains which were collected from Qilu hospital of Shandong University and the second affiliated hospital of Shandong University of Chinese medicine. Pseudomonas Aeruginosa strains were divided into sensitive strain, multi-drug-resistant strains(MDR), pan-resistant strains(PDR) and extensive Drug Resistant(XDR) according to the shapes of bacteriostasis circle. Sulbactam susceptibility paper(125μg) was made by ourselves. Both synergy and adding effect are calculated as Synergistie rate.2. Minimal inhibitory concentration(MIC) assay:2.1The preparation of antimicrobial stock solution.Pseudomonas Aeruginosa strains were measured by Minimal inhibitory concentration(MIC) assay and divided into sensitive group, multi-drug-resistant group(MDR), pan-resistant group(PDR) and extensive Drug Resistant(XDR) group, each group contains12strains. Biapenem, imipenem, fosfomycin, eeioperazone. sulbactam were diluted by sterile Phosphate buffer(pH7.2) to5120μg/mL, amikacin was diluted to1280ug/mL, ciprolloxacin was diluted to2560μg/mL 2.2The preparation of Single drug-well plates:Antimicrobial stock solutions of10μL of each above drug were separately added to90\μL MH broth and fold dilutions were made. All drugs were diluted as follows: Biapencm, imipenem, fosfomycin, Sulbactam and Cefopcrazone, cefoperazone, sulbactam were diluted from512μg/mL to0.016μg/mL, amikacin was diluted from128μg/mL to0.004μg/mL, ciprofloxacin was diluted from256μg/mL to0.008μg/mL.2.3Single drug MIC test:Bacterials suspension of0.5MCF were prepared and diluted with MH broth to l/200(the bacterial concentration was about5.0×105cfu/mL). The Single drug-well plates were added with50p.L diluted bacterials suspension, while negative and positive controls were established. Bacterials were cultured in37℃for18-24h and test each drug’s MIC.2.4The preparation of orifice plate for drug combinationAccording to23times to1/24times MIC of each drug, the concentration of drug in each holes were designed. Horizontally, from left to right, the concentration of A drug was gradually falling, vertically, from high to low, the concentration of B drug was gradually falling. The final concentration of drug was4times to the initial concentration. Every hole separately contained25μL A drug and25μL B drug.2.5Drug combination MIC test:The50μL MH broth with bacteria was added to every hole, while negative and positive controls were established. All bacteria were incubated for18-24hours and correspondingly got MIC values of each antimicrobial.2.6Fractional inhibitory concentration(FIC) of drug combination:FIC was indicated as following formula:A drug combined MIC/A drug used alone MIC+MIC B drug combined/B drug used alone MIC. Synergies:FIC index≤0.5, independce:0.5<FIC index<4, antagonism:FIC index>4. On account of natural resistance of Pseudomonas aeruginosa to sulbactam, sulbactam was not taking into account in the calculation of FIC.3. Drug combination killing curve:Respectively chose2strains of sensitive, MDR, XDR and PDR strains and respectively measured killing curve of imipenem and biapenem combined with sulbactam, cefoperazone, amikacin. On account of natural resistance of Pseudomonas aeruginosa to sulbactam, the concentration of sulbactam was same as which of imipcnem and biapenem. Due to our pre-test results, colonies were counted on when bacteria were incubated for Oh and8h.Compared with the most effective single drug group, synergy was defined when bacterial colonies reduced>2.0log10CFU/mL, independce was defined when bacterial colonies reduced the <1.0logl0CFU/mL, antagonism was defined when bacterial colonies increased>2.0loglOCFU/mL.Results1. Single drug susceptibility (K-B)In120Pseudomonas aeruginosa strains, the number of susceptible strains were86strains for amikacin,63strains for fosfomycin,61strains for ciprofloxacin,66strains for Ceftazidime,64strains for cefoperazone,65strains for cefoperazone/sulbactam,67strains for aztreonam,64strains for pipcracillin/tazobactam,83strains for imipenem,82strains for biapenem,85strains for meropenem,104strains for polymyxin.120Pseudomonas aeruginosa strains were divided into56strains Un-susceptible strains,36strains for MDR,13strains for XDR,15strains for PDR.2. Drug combination susceptibility test (K-B)The most effective drugs combined with biapenem were fosfomycin(82strains), sulbactam(51strains), amikacin(41strains), cefoperazone/sulbactam(36strains). The most effective drugs combined with imipenem were fosfomycin(82strains), sulbactam(48strains), amikacin(44strains). However, antagonism emerged when imipenem combined with cefoperazone/sulbactam(44strains) and cefoperazone (68strains). There was no difference in Synergistic rate when carbapenem drugs combined with sulbactam to different susceptibility Pseudomonas aeruginosa strains.3. Drug combination MIC test:The results of the drug combination MIC test werebasically the same with that of the drug combination susceptibility test (K-B). The most effective drugs combined with imipenem or biapenem were fosfomycin(27strains,42strains), amikacin(33strains,31strains), sulbactam(21strains,22strains). However, antagonism emerged when imipenem combined with cefoperazone (24strains) and cefoperazone/sulbactam(6strains). There was antagonism to Pseudomonas aeruginosa strains when Imipenem combined with cefoperazone, because Imipenem combined with sulbactam have no antagonism.4.Drug combination killing curve There were8Pscudonionas aeruginosa strains in synergism when biapencm combined with sulbactam,8strains with amikacin,4strains with cefoperazone/sulbactam,4strains with cefoperazone. Similarly, there were8Pseudomonas aeruginosa strains in synergism when imipenem combined with sulbactam,4strains with amikacin,4strains with cefoperazone/sulbactam,4strains with cefoperazone. There were respectively4Pseudomonas aeruginosa strains in independence when biapenem or imipenem combined with cefoperazone/sulbactam or cefoperazone.Conclusion1. The most effective drugs combined with biapenem were fosfomycin, sulbactam, amikacin, cefoperazone/sulbactam2. The most effective drugs combined with imipenem were fosfomycin, sulbactam, amikacin3. The combination of carbapenems and sulbactam or fosfomycin could be used in the treatment of resistant PA infections, but imipenem combined with cefoperazone was not recommended. Objectives:To complare the invasiveness of conidia of Aspergillus fumigatus on A549cells are pretreated by different concentrations dexamethasone, and to explore the relationships among dexamethasone, fibronectin of A549cell and invasiveness of conidia.Methods:(1) After the pretreatment of antibody of fibronectin or/and dexamethasone, the A549cells were attacked by conidia, after4h, the extracellular free conidia were cleaned away, the rest immobilized conidia were released by disintegrating cells with Triton-X100and quantified by counting the colony-forming units. The invasiveness of conidia was measured by the invasion rate (%).(2) The relative transcripts of fibronectin gene of cells pretreated by different concentrations dexamethasone for24h were tested by fluorogenic quantitative PCR(RT-PCR) and the expression of fibronectin of cells were pretreated for48h were tested by Westen Blot and immunofluorescence.Results:(1) Significant decreasement of invasiveness of conidia was seen in anti-fibronectin group (14.42±1.68vsl9.17%±2.53%, P<0.05) but no difference exist in anti-fibronectin&25mg/L dexamethasone group to the control. There was no significant relevance between dexamethasone’ concentration and the invasiveness of conidia in4h group, but in24h, the invasiveness in25mg/L(24.66%±2.41%) is higher than that in the control (19.17%±2.53%) and1mg/L (19.93%±3.06%), and that in5mg/L (22.47%±2.46%) group is higher than the control, P<0.05(2) The relative transcripts of fibronectin gene of25mg/L (9.19×10-3±1.2×103) is higher than the control (4.61×10-3±1.54×10-3) and mg/L (6.20×10-3±.93×10-3),5mg//L(7.94×103±2.24±10-3) higher than the control, P<0.05. High concentration dexamethasone promotes fibronectin production after48h interaction.Conclusion:Dexamethasone can enhance invasiveness of conidia of aspergillus fumigatus by promoting cell fibronectin expression, it can partly explain the patients are taken large dose and long-term glucocorticoid therapy are susceptible by aspergillus fumigatus.