C8-oxo-HSl对P.aeruginosa在污水处理中生长特性影响

通过24孔板序批实验对群体效应(QS)信号分子C_8-oxo-HSL对铜绿假单胞菌(P.aeruginosa)在污水处理过程中生长特性影响作用进行分析探讨.研究发现C_8-oxo-HSL有效刺激P.aeruginosa大量生长,其对应的阙值浓度为10^(-10)g/L C_8-oxo-HSL.同时推知具有Lux I/R群体效应系统的革兰氏阴性菌可在较低的信号分子浓度作用下大量增殖.C_8-oxo-HSL还可有效刺激P.aeruginosa分泌大量蛋白质至紧密型胞外聚合物(TB-EPS)中,其对应的阙值浓度为10^(-7)g/LC_8-oxo-HSL.但C_8-oxo-HSL对P.aeruginosa的TB-EPS中多糖没有明显影响作用.C_8-oxo-HSL通过促使P.aeruginosa的增殖和团聚从而增强菌胶团稳定性.此外C_8-oxo-HSL可促进P.aeruginosa生物膜形成.但由于P.aeruginosa生物膜的形成同时和细菌菌量与胞外聚合物浓度直接相关.所以P.aeruginosa生物膜快速生长的C_8-oxo-HSL阙值浓度相对较高(约10^(-8)g/L).

EFFECTS OF RADIX ANGELICAE SINENSIS AND SHUANGHUANGLIAN ON A RAT MODEL OF CHRONIC PSEUDOMONAS AERUGINOSA PNEUMONIA

Objective. To study the effects of two kinds of Chinese herbal medicine, Radix angelicae sinensis(RAS)(当归)and Shuanghuanglian(SHL)(双黄连) on chronic Pseudomonas aeruginosa(PA)lung infection in a rat model mimicking cystic fibrosis(CF). Methods.Rats were divided into RAS, SHL and control groups. All rats were challenged intratracheally with alginate embedded PA and the treatments with herbal medicine started on the same day of challenge. The drugs were administered subcutaneously once a day for ten days and the control group was treated with sterile saline.The rats were sacrificed two weeks after challenge. Results. Significantly improved lung bacterial clearance(P<005, P<001) and milder macroscopic lung pathology (P<0005) were found in the two treated groups compared to the control group. In the SHL treated group, the neutrophil percent in the peripheral blood leukocytes(P<005), the antiPA IgG level in serum (P<005), the incidence of lung abscesses(P<0005) and the incidence of acute lung inflammation(P<005) were significantly lower than in the control group. The RAS treatment reduced fever(P<005), decreased the incidence of lung abscesses(P<0005) and lung mast cell number (P<005), and lowered antiPA IgG1 level in serum(P<005) when compared to the control group. The antiPA bacterial activity test in SHL was weakly positive whereas in RAS it was negative. Conclusion.The treatment with both herbal medicines could increase the resistance of the rats against PA lung infection and they therefore might be potential promising drugs for stimulation of the immune system in CF patients with chronic PA lung infection.

Pathogenic effects of biofilm with chronic pseudomonas aeruginosa lung infection in rats

Objective： To establish an animal model of P.aeruginosa biofilm associated with chronic pulmonary infection and investigate the pathogenic effects of biofilm. Methods： Experiments in vitro, measuring the MICS, MBCS of levofloxacin（LFX）, ceftazidime（CAZ） in PAO579 in alginate beads and planktonic PAO579. Rats were challenged with 0.1 ml of PAO579（109CFU/ml） in alginate beads or 0.1 ml of planktonic PAO579（109CFU/ml）, 3,7,14 days after challenging, bacteriological, pathological features were observed. Results： The MICS, MBCS of LFX, CAZ in PAO579 in alginate beads were higher than those in planktonic PAO579 in vitro. CFU/lung in alginate beads group was significantly higher than that in planktonic bacteria group（P = 0.002, P = 0.004, P = 0.002, respectively）; macroscopic lung pathology and the inflammation in alginate beads group were significantly more severe compared to those in planktonic bacteria group in vivo. Conclusion： P.aeruginosa biofilm protected bacterium from killing of antibiotics and might mediate the host immune damage in the lung tissue and made bacterium evade the host immune defense.

Isolation, identification and characterization of cadmium-resistant Pseudomonas aeruginosa strain E_1

Strain E1 with resistance to 18 mmol/L cadmium (Cd), isolated from Cd-contaminated soil was identified by morphological observation, biochemical and physiological characterization and 16S rDNA sequence analysis. The resistance to heavy metals Cd, Cu, Co, Mn, Pb, Zn and 12 antibiotics was examined. The ability of removing Cd from solution was studied. The characterizations show that strain E1 is affiliated to Pseudomonas aeruginosa (P. aeruginosa). Strain E1 has high resistance to heavy metals and the order is found to be Cd>Mn>Zn>Cu>Pb>Co in solid media. Strain E1 also exhibits the resistance to 12 antibiotics. Both living and non-living cells of strain E1 can remove Cd from solution, and living cell has better biosorption than non-living cell.

A Modified Selective Medium Containing Benzalkonium Chloride (BKC) for the Isolation of <i>Pseudomonas aeruginosa</i>from Raw Milk

A modified selective medium (modified Cetrimide Agar, mCA) consisting of 200 μg/mL benzalkonium chloride (BKC) was developed for the isolation of Pseudomonas aeruginosa from raw milk. Initially, a total of 55 isolates were obtained from 14 raw milk samples collected from several dairy plants in Ankara, Turkey. Among these isolates, 19 were identified as Pseudomonas aeruginosa, 28 as Pseudomonas fluorescens, 4 as Acinetobacter baumannii, 2 as Enterobacter intermedium, 1 asEnterobacter agglomerans, and 1 as Escherichia coli using Microbact biochemical test kit. BKC was chosen as a selective agent to suppress growth of competitive flora because it is very effective against a wide range of Gram-negative bacteria while P. aeruginosa is resistant. MICs (minimum inhibitory concentration) for BKC were determined by agar dilution method. The concentration of 200 μg/mL BKC inhibited competitive flora, while 90% of P. aeruginosa strains were resistant. When the results of enumeration of P. aeruginosa and other Gram (-) bacteria in Cetrimide Agar (CA) and mCA were compared, it was observed that mCA was more selective than the standard CA in preventing the growth of competitive flora especially of P. fluorescens.

Prevalence and Resistance Pattern of <i>Pseudomonas aeruginosa</i>Isolated from Surface Water

Pseudomonas aeruginosa is one of the most common pathogenic bacteria, frequently found in different environmental samples. The prevalence of multidrug resistant isolates has become an alarming concern for both patients and their surroundings. The present study was carried out to record prevalence of P. aeruginosa in surface water of Dhaka city and to screen their antibiotic resistance pattern. The study was also extended to typing of resistant isolates according to extended spectrum beta lactamase production. Hereby, Kirby-Bauer method was applied to test antibiotic sensitivity according to Clinical and Laboratory Standards Institute. Then, the Ampicillin resistant isolates were screened for ESBL production by Double Disk Synergy Test (DDST). In these prospects, 52 water samples were tested, of which 32 were found positive for P. aeruginosa isolates. Hundred percent of the positive isolates were found to Ampicillin (AMP) resistant followed by 93.7% to both Tetracycline and Gentamycin and 71.8% to Co-triimoxazole. P. aeruginosa is completely susceptible to third generation antibiotics ciprofloxacin, Imipenem and Aztreonam followed by moderately susceptible to Polymyxin-B (78.2%) and Colistin (87.5%). According to DDST, all of the susceptible isolates were found positive for AMC type beta-lactamase production. It is evident from this study that the surface water is contaminated with antibiotic resistant P. aeruginosa and that through the water systems antibiotic resistance can be transferred to humans and animals. So, appropriate and rationale use of antibiotic should be applied to minimize the emergence of multidrug isolates to environment.

The effect of Some Boron Derivatives on Kanamycin Resistance and Survival of E.coli and P.aeruginosa in Lake Water

Objective To study MIC value of 7 boron derivatives namely [Boric acid （H3BO3）, Anhydrous Borax （Na2B407）, Sodium Borate （NaBO2）, Diammonium Tetraborate （NH4）2B4O7, Sodium Perborate （NaBO3）, Boron Trioxide （B203）, Potassium Tetraborate （K2B407）] on E. coil and P. aeruginosa and their effects on survival of bacteria in lake water and resistance against kanamycin antibiotic. Methods MIC values of Boron derivatives and antibiotic were studied by broth microdilution method. The effect of boron derivatives on survival of bacteria in lake water were also determined with plate count. Results Sodium perborate was determined as the substances. Effectiveness increased as temperature most effective substance among the studied increased. E. coil was more affected from P. aeruginosa in 8 mg/mL sodium perborate concentration in lake water. Moreover, it was determined that MIC value of kanamycin antibiotic decreased 200 times by especially treating P. aeruginosa with sodium perborate in lake water. However, it can be stated that this change in resistance did not arise from microorganisms. Conclusion Sodium perborate solution can be used supportedly in kanamycin antibiotic applications for P. aeruginosa. Future studies are necessary to explore the relation between sodium perborate and kanamycin which is effective on P. aeruginosa in lake water.

Analysis of Drug Resistance Spectra and Conjugative Plasmid Carrying Rates of P.aeruginosa and Acinetobacter

Antimicrobial susceptibility test was performed on 57 clinical isolates of P. aeruginosa and 36 clinical isolates of Acinetobacter with 11 antimicrobial agents including getamicin, amikacin, ciprofloxacin, ofloxacin, fleroxacin, piperacillin, cefotaxime, cefoperazone/sulbactam, ceftazidime, cefoperazone and doxycycline. Transferable drug resistance plasmid carrying rates of these clinical isolates were also studied. On the basis of the in vitro activities, 52.63%(30/57) of the isolated strains of P. aeruginosa were susceptible to antimicrobial agents selected (except doxycycline), 41.67%(15/36) of the isolated strains of Acinetobacter were susceptible to 11 antimicrobial agents. The sensitivity rate of P.aeruginosa and Acinetobacter to antimicrobial agents selected was 70% or greater to all except doxycycline. Furthermore, the sensitivity rate of P.aeruginosa to amikacin ciprofloxacin, ceftazidime, cefoperazone, cefoperazone/sulbactam, and that of Acinetobacter to cefoperazone/sulbactam, amikacin was more than 90%,among them amikacin, cefoperazone/sulbactam being the most effective. Plasmid analysis showed that 15.79%(9/57) P.aeruginosa strains and 13.89%(5/36) Acinetobacter strains carried plasmid. Conjugative plasmid carrying rates of P. aeruginosa strains and Acinetobacter strains were 7.02%(4/57), 13.89%(5/36), respectively. Conjugative plasmid didn′t play an important role in the formation and dissemination of drug resistance of P. aeruginosa and Acinetobacter.

Expression of IL-4 in a rat model of chronic pulmonary infection with biofilm formation induced by Pseudomonas aeruginosa

<正>The expression of IL-4 in a rat model of chronic pulmonary infection biofilm formation induced by Pseudomonas aeruginosa was investigated,in which SPF Wister rats were infected via trachea with 0.1 ml P.aeruginosa strain PAO579(10~9 CFU/ml)in alginate beads or the planktonic form of this bacterial strain(10~9 CFU/ml),and on 3,7 and 14 d after infection,the bacteriological and pathological changes were observed as well as the expression of the cytokine IL-4 was determined.It was demonstrated that the count of CFU per lung tissue in case of bacteria in alginate beads was significantly higher than that of bacteria in planktonic form,with more severe gross pathologic changes and inflammatory reactions in the alginate bead group in comparison with that of the planktonic forms(P=0.002,P=0.004 and P=0.002,respectively).In addition,the expression of IL-4 in the alginate bead group was also higher than that in the planktonic form(P=0.02,P=0.02 and P=0.022,respectively).A positive corre- lation between the level of IL-4 expression and the gross lung pathology in alginate bead group existed as demonstrated by simple regression analysis(r=0.78,P<0.02).It is concluded that the chronic pul- monary infection with biofilm formation induced by P.aeruginosa tends to have the priority to the Th2 immune response.

Development of a Duplex Real-Time PCR Method for the Pharmaceutical Rapid Microbial Detection of <i>Staphylococcus aureus</i>and <i>Pseudomonas aeruginosa</i>

Objective: To develop a duplex real-time PCR assay for pharmaceutical rapid microbial detection of Staphylococcus aureus and Pseudomonas aeruginosa. Methods: The specific primers and probes were designed to amplify the femB gene of S. aureus and the DNA gyrase subunit B gene of P. aeruginosa. The sensitivity of the system was detected by a multiple proportional dilution method. In order to examine the specificity of the system, other twenty-one bacteria strains were assayed simultaneously. Results: A highly sensitive and specific duplex real-time PCR assay for the detection of S. aureus and P. aeruginosa was established. The sensitivity was 50 copies/μL. The specificity was 100%. The whole detection procedure can be finished within 2.5 h. Conclusion: The duplex real-time PCR method is efficient in detecting with good sensitivity and specificity. There is a good prospect of this method applying in disease prevention and pharmaceutical industry due to the simultaneous detection of two pathogens.

Resistance Trends among <i>Pseudomonas aeruginosa</i>Isolates in a Tertiary Care Centre in South Gujarat

It is necessary to determine the susceptibility pattern of clinical isolates especially nosocomial one in the clinical settings for making strategy for effective empirical treatment & to reduce incidence of multidrug resistant bugs. Aim of this study was to detect the antimicrobial susceptibility pattern of P. aeruginosa isolates from clinical samples between January 2014 to December 2015, received at department of Microbiology, GMC, Surat. Clinical isolates were confirmed as P. aeruginosa by phenotypic methods/Vitek2 compact system as per availability. Genetic sequencing could not be performed due to unavailability. Antimicrobial susceptibility tests were performed by Kirby-Bauer disc diffusion method/Vitek2 compact system & Interpretation was done according Clinical and Laboratory Standards Institute (CLSI) of that year [1] [2]. Seven hundred fifty seven P. aeruginosa strains were studied during the study period. Most of the isolates were from surgery ward (62%), followed by orthopaedic ward (15%). 65% of the total isolates were from swab samples followed by urine (7%), pus, fluid (5%) & devices (4%). 60% isolates were resistant to Ceftazidime & for other drugs resistance pattern was as follows: Cefepime (52%), Levofloxacin (49%), Ticarcillin/clavulanic acid (49%), Meropenem & Gentamycin (44%), Ciprofloxacin (43%), Amikacin (41%), Tobramycin (39%), Netlimycin (36%), Piperacillin (32%), Aztreonam (31%), Piperacillin/tazobactam (26%), Imipenem (23%) , Doripenem (12%) & Gatifloxacin (10%). As there is predominance of isolates from surgical ward in present study & resistance to carbapenem group of drugs was also found, indicating that most of the infection caused by Pseudomonas aeruginosa may be nosocomial.

Serotypes, Antibiogram and Genetic Relatedness of <i>Pseudomonas aeruginosa</i>Isolates from Urinary Tract Infections at Urology and Nephrology Center, Mansoura, Egypt

Background: Pseudomonas aeruginosa (P. aeruginosa) is an opportunistic pathogen that represents a major problem in many hospitals because of its increased resistance to antibiotics and the ability to cause nosocomial infections. The present study aimed to phenotype and genotype isolates of P. aeruginosa from inpatients with UTIs at Urology and Nephrology center, Mansoura, Egypt to study their relatedness. Methods: Thirty nine isolates of P. aeruginosa were phenotypically typed by determination of O-serotypes by slide agglutination technique and antimicrobial resistance patterns by disk-diffusion method. The genetic diversity of isolates was illustrated by performing RAPD-PCR using M13 primer. Results: Serotypes O11, O6 and O10 were the most prevalent. Isolates showed high resistance rates to antipseudmonal antibiotics with high incidence (51.3%) of multidrug resistance (MDR). Amikacin was the most effective. A significant correlation was found between O6, O10 and MDR. A relatively high polymorphism was demonstrated among P. aeruginosa isolates by using RAPD-M13 fingerprinting. Cross transmission was suggested by phenotypically and clonally identical isolates. Conclusion: The study demonstrates the role of combining both classical and molecular typing as a valuable mean to study the origin and cross transmission of P. aeruginosa in UTIs for better assessment of treatment and infection control.

Impact of Sulfidation of Silver Nanoparticles on Established<i>P. aeruginosa Biofilm</i>

Silver nanoparticles (Ag-NPs), one of the most common types of nanomaterials in medical fields and consumer products, are known to have antimicrobial effects;these materials also undergo a series of chemical and biological transformations in the environment. Although the pristine form of silver nanoparticles has been studied, less is known about the impacts of the transformed Ag-NPs on biological systems. This knowledge gap hinders the progress of effectively assessing the impacts of Ag-NPs on the environment and human health. In this study, we demonstrate that the most common form of transformed Ag-NPs, sulfidized silver nano-particles (Ag2S-NPs), show less damage on established Pseudomonas aeruginosa GFP (ATCC? 10145 GFP?) biofilm than the pristine form of the nanoparticle. At a dosage of 0.625 mg/L, the total biomass in the biofilm decreased 64% after being exposed to Ag-NPs and 44% after exposure to Ag2S-NPs. Live biofilms were also interrogated. We observed high reduction in live population for biofilm exposed to Ag-NPs and relatively low reduction by Ag2S-NPs at exposure concentrations higher than 0.625 mg/L. Compared with Ag-NPs, the lower solubility of Ag2S-NPs results in less Ag+ diffusion into established biofilms. Our results suggest that the sulfidation of Ag-NPs reduces their impacts on established biofilms, indicating that the transformed Ag-NPs may have less environmental or human health risks.

射干提取液对铜绿假单胞菌PA11株R质粒体内外消除作用

目的 :以射干提取液为质粒消除剂 ,对铜绿脓假单胞菌 PA1 1株 R质粒进行体内外消除试验 ,使消除子恢复对抗生素的敏感性。方法 :取射干水煎剂对 PA1 1株进行最小抑菌浓度 ( MIC)试验 ,统计 MIC50 和 MIC90 。结果 :射干水煎剂对 PA1 1株 MIC为 31 .2 5 g· L-1,MIC50 为7.81 g·L-1,MIC90 为 1 5 .62 g· L-1。结论

不同磷浓度和N/P对铜绿微囊藻生长及水环境因子的影响

以铜绿微囊藻为实验用藻,在反应器中设置总磷浓度分别为0.02、0.05、0.1、0.2、0.4、0.5、1和2 mg/L等8个浓度,同时针对每个总磷浓度,配置N/P为1∶1、2∶1、4∶1、8∶1、16∶1和40∶1等6个比例,对反应器中藻的繁殖情况进行连续检测,同时检测水中DO、p H值的相应变化情况。结果表明,不同总磷浓度对铜绿微囊藻的繁殖的作用具有差异性,浓度在0.02～0.05 mg/L范围时,藻的繁殖缓慢,在0.1 mg/L以上时,藻的繁殖速度明显加快,当达到0.5 mg/L以上时,不同总磷浓度条件下藻的繁殖速度基本相近;当总磷浓度在0.1mg/L以上时,N/P对藻繁殖的影响才体现出来,N/P在8∶1、16∶1和40∶1时对藻繁殖的影响远大于其他比例,8∶1和16∶1是藻繁殖的最佳N/P比例。与此同时,铜绿微囊藻细胞密度的变化也明显导致了水中DO、p H值的变化,在藻细胞快速增长阶段,DO、p H值呈现上升趋势,在藻细胞稳定阶段,DO、p H值呈现下降趋势。

BPIFB1在铜绿假单胞菌引起的炎症反应中的调节作用

目的研究杀菌性/通透性增强蛋白折叠结构1(BPIFB1)在铜绿假单胞菌引起的炎症反应中的作用机制和调节途径。方法应用RNA干涉、特异性蛋白激酶抑制剂阻断法,通过ELISA、Western blot法测定BPIFB1与脂多糖(LPS)共同作用于RAW264.7细胞后膜表面CD14、TLR受体以及胞内相关信号转导通路分子表达水平的变化情况。结果 BPIFB1与LPS作用后RAW264.7细胞表面CD14、TLR4和MyD88的表达水平明显下降;当细胞中MyD88、TRAF6、NF-κB等蛋白分子的表达被各自特异性siRNA所阻断后,BPIFB1与LPS抑制细胞因子TNF-α过表达的能力也被显著抑制;当用BPIFB1与LPS处理过的细胞用相应蛋白激酶抑制剂作用后,细胞内相关通路蛋白p38、pERK1/2、Akt1磷酸化水平被明显抑制。结论 BPIFB1与LPS结合通过阻抑相关胞内细胞因子的表达,降低了铜绿假单胞菌所产生的细胞炎性反应程度。

HPLC法同时测定不同配伍比例三棱-莪术药对中P-香豆酸、阿魏酸的含量

目的:建立同时测定三棱-莪术药对中P-香豆酸、阿魏酸含量的方法,探讨不同比例药对中二者含量的变化。方法:采用高效液相色谱法。色谱柱为Agilent 20RBAX XDB-C_(18),流动相为乙腈-0.1%冰醋酸溶液(梯度洗脱),流速为1.0 m L/min,检测波长为266 nm,柱温为30℃,进样量为10μL。结果:P-香豆酸、阿魏酸检测质量浓度线性范围分别为4.218 6~21.093μg/m L(r=0.999 8)、1.836 0~9.180μg/m L(r=0.999 9);精密度、稳定性、重复性试验的RSD<2.0%;加样回收率分别为98.72%~100.30%(RSD=0.18%,n=9)、99.11%~100.45%(RSD=0.46%,n=9)。三棱-莪术药对4个配伍比例(1∶1、2∶1、1∶2、1∶0,m/m)中,三棱-莪术比例(m/m)为2∶1时P-香豆酸、阿魏酸含量最高。结论:该方法操作简便,精密度、稳定性、重复性好,可用于三棱-莪术药对中P-香豆酸、阿魏酸含量的同时测定;该药对中三棱-莪术比例(m/m)为2∶1时主要有效成分含量最高。

光限制胁迫协同pH与氮磷比对铜绿微囊藻生长的影响

以铜绿微囊藻为试验材料,应用正交试验法,在培养温度(25℃)及接种量相同的情况下,研究光限制胁迫协同pH与氮磷比对铜绿微囊藻生长的影响.影响铜绿微囊藻生长的因素顺序为:pH>光限制胁迫天数>氮磷比,pH为铜绿微囊藻生长的显著性影响因素(F=63.111 5),且在pH 10.5、氮磷比15∶1及光限制胁迫5 d的条件下正常光照培养4 d后藻细胞数量最大.

铜绿假单胞菌PA0057基因的克隆、表达及其重组蛋白活性的初步研究

铜绿假单胞菌PA0057基因为Ⅳ类未知基因,通过NCBI比对,发现PA0057基因编码的蛋白与金属β-内酰胺酶同源性为96%,因此推测PA0057蛋白可能具有金属β内酰胺酶活性.提取铜绿假单胞菌PAOI基因组DNA,利用设计的引物通过PCR方法扩增得到PA0057基因,将其克隆至克隆载体pGEM-T,并转化人大肠杆菌DH5α;而后将其克隆至表达载体pET28a中,转化人大肠杆菌BL21(DE3)中,IPTG诱导表达,并通过Ni-NTA亲和柱进行纯化,纯化后蛋白超滤浓缩得高浓度蛋白;通过药敏纸片检测PA0057蛋白活性.

新型尿苷肽类化合物Sansanmycin P的提取分离、结构鉴定及活性研究

目的从放线菌Streptomyces sp SS发酵液中分离新型活性尿苷肽类似物。方法发酵液经D4006大孔吸附树脂、DEAE-葡聚糖凝胶及制备液相分离纯化,获得单一化合物;根据UV、MS、MS/MS、1D和2D NMR确定化合物结构,并进行抗菌活性研究。结果分离纯化得到1个N-末端含有1-甲基-6-羟基四氢异喹啉的尿苷肽类化合物Sansanmycin P。初步体外抗菌活性,抗铜绿假单胞菌MIC>32μg/ml,抗结核杆菌MIC>32μg/ml。结论 Sansanmycin P为全新结构Sansanmycins类似物,具有一定抗结核杆菌和铜绿假单胞菌活性。