Evaluation of Azadirachta indica cultivated in Egypt against biofilm formation by Pseudomonas aeruginosa: Insights from molecular docking studies targeting LasR

Background:Azadirachta indica(A.indica),commonly known as neem,is a widely distributed medicinal plant in Asia and Africa and is well known to have a wide spectrum of biological activity.A.indica is considered a skin food that was traditionally used in different cultures to treat a wide range of skin disorders.A.indica was reported to possess antibacterial activity against Pseudomonas aeruginosa(P.aeruginosa)which is considered the most common biofilm model organism.This study aims to investigate the ability of A.indica cultivated in Egypt to inhibit/reduce the biofilm formation by P.aeruginosa.Methods:The microtiter plate assay was used to evaluate the anti-biofilm activity of neem,cultivated in Egypt,leaves against P.aeruginosa as well as the ability to reduce the activity of P.aeruginosa.To investigate the phytocompounds responsible for their bioactivity and to explore potential interactions between their bioactive components and one of the quorum-sensing regulatory proteins of P.aeruginosa involved in biofilm formation,liquid chromatography-mass spectrometric and molecular docking studies were done.Results:Results showed that methanol extract of leaves can reduce the formation of P.aeruginosa biofilm at lower concentrations than those reported in other regions with 1.25 mg/mL as the optimum concentration.The two-way analysis of variance revealed the significance of the extract effect and its concentration on the reduction of biofilm formation(P<0.05).Liquid chromatography-mass spectrometric study revealed the presence of fourteen compounds that belong to limonoids and flavonoids.Molecular docking analysis against LasR,the quorum-sensing regulatory protein,of P.aeruginosa supported these findings.Nimbolinin,a limonoid,has achieved the highest Libdock score of 138.769.Conclusion:It was concluded that A.indica,cultivated in Egypt,leaves can target LasR as a new mechanism of action for biofilm control by A.indica and therefore could be a good source of leads for anti-biofilm medicine.

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.

Phenolic compounds affect production of pyocyanin, swarming motility and biofilm formation of Pseudomonas aeruginosa

Objective: To investigate the effects of plant-derived phenolic compounds(i.e. caffeic acid, cinnamic acid, ferulic acid and vanillic acid) on the production of quorum sensing regulated virulence factors such as pyocyanin, biofilm formation and swarming motility of Pseudomonas aeruginosa(P. aeruginosa) isolates.Methods: Fourteen clinical P. aeruginosa isolates obtained from urine samples and P. aeruginosa PA01 strain were included in the study. The antibacterial effects of phenolic compounds were screened by well diffusion assay. Pyocyanin and biofilm activity were measured from culture supernatants and the absorbance values were measured using a spectrophotometer. Swarming plates supplemented with phenolic acids were point inoculated with P. aeruginosa strains and the ability to swarm was determined by measuring the distance of swarming from the central inoculation site.Results: Tested phenolic compounds reduced the production of pyocyanin and biofilm formation without affecting growth compared to untreated cultures. Moreover, these compounds blocked about 50% of biofilm production and swarming motility in P. aeruginosa isolates.Conclusions: We may suggest that if swarming and consecutive biofilm formation could be inhibited by the natural products as shown in our study, the bacteria could not attach to the surfaces and produce chronic infections. Antimicrobials and natural products could be combined and the dosage of antimicrobials could be reduced to overcome antimicrobial resistance and drug side effects.

Effects of Combined Treatment with Sansanmycin and Macrolides on Pseudomonas aeruginosa and Formation of Biofilm

Objective To observe the effects of combined treatment with sansanmycin and macrolides on Pseudomonas aeruginosa and formation of biofilm. Methods Micro-dilution method was used to determine the minimal inhibitory concentrations （MICs） of sansanmycin, gentamycin, carbenicillin, polymyxin B, roxithromycin, piperacillin, and tazobactam. PAl and PA27853 biofilms were observed under optical microscope after staining and under SEM after treatment with sansanmycin at different dosages and combined treatment with sansanmycin and roxithromycin. Viable bacteria in PAl and PA27853 biofilms were counted after treatment with sansanmycin at different dosages or combined treatment with sansanmycin and roxithromycin. Results The MIC of sansanmycin was lower than that of gentamycin and polymyxin B, but was higher than that of carbenicillin. Roxithromycin enhanced the penetration of sansanmycin to PAl and PA27853 strains through biofilms. PAl and PA27853 biofilms were gradually cleared with the increased dosages of sansanmycin or with the combined sansanmycin and roxithromycin. Conclusion Sub-MIC levels of roxithromycin and sansanmycin substantially inhibit the generation of biofilms and proliferation of bacteria. Therefore, combined antibiotics can be used in treatment of intractable bacterial infection.

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

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 （109 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 correlation 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 pulmonary infection with biofilm formation induced by P. aeruginosa tends to have the priority to the Th2 immune response.

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.

Antifouling Properties of Electrospun Polymeric Coatings Induced by Controlled Surface Morphology

Nosocomial infections affect implanted medical devices and greatly challenge their functional outcomes,becoming sometimes life threatening for the patients.Therefore,aggressive antibiotic therapies are administered,which often require the use of last-resort drugs,if the infection is caused by multi-drug-resistant bacteria.Reducing the risk of bacterial contamination of medical devices in the hospitals has thus become an emerging issue.Promising routes to control these infections are based on materials provided with intrinsic bactericidal properties(i.e.,chemical action)and on the design of surface coatings able to limit bacteria adhesion and fouling phenomena(i.e.,physical action),thus preventing bacterial biofilm formation.Here,we report the development and validation of coatings made of layer-by-layer deposition of electrospun poly(vinylidene fluoride-co-trifluoro ethylene)P(VDF-TrFE)fibers with controlled orientations,which ultimately gave rise to antifouling surfaces.The obtained 10-layer surface morphology with 90°orientation fibers was able to efficiently prevent the adhesion of bacteria,by establishing a superhydrophobic-like behavior compatible with the Cassie-Baxter regimen.Moreover,the results highlighted that surface wettability and bacteria adhesion could be controlled using fibers with diameter comparable to bacteria size(i.e.,achievable via electrospinning process),by tuning the intra-fiber spacing,with relevant implications in the future design of biomedical surface coatings.

Comparison of inhibitory effect between DL-tryptophan and lactoferrin on Pseudomonas aeruginosa biofilm formation in wound dressing

Objective: To compare the inhibitory effect between DL-tryptophan and bovine lactoferrin on biofilm formed by isolated Pseudomonas aeruginosa strains. Methods: The study was carried out on 40 patients suffering from surgical site infection. Wound pus was collected using sterile swabs after isolation, and identified by common bacteriological methods. Isolated Pseudomonas aeruginosa strains were grown on biofilm enhancing materials, and then the inhibitory effects of different concentrations of DL-tryptophan and lactoferrin were tested using scanning electron microscopy and microtitre plate methods. Results: There was no significant difference in the inhibitory effect between DL-tryptophan and lactoferrin at 0.5 mg/mL. While in concentration of 1 mg/mL and 2 mg/mL, tryptophan showed more significant inhibitory effect than lactoferrin. Conclusions: Both DL-tryptophan and bovine lactoferrin have inhibitory effect on Pseudomonas aeruginosa biofilm formation in a dose dependent manner, and the inhibitory effect of DL-tryptophan is stronger.

Spions Increase Biofilm Formation by <i>Pseudomonas aeruginosa</i>

Limited research has suggested iron oxide nanoparticles (FeNP) have an inhibitory effect against several different genera of bacteria: Staphylococcus, Bacillus and Pseudomonas spp. In this study we looked at the effect of three different sets of Fe3O4 nanoparticles (FeNPs) on the development of Pseudomonas aeruginosa PAO1 biofilms. Two of the tested NPs were SPIONs (Superparamagnetic Iron Oxide Nanoparticles). Exposure of cells to the SPIONs at concentrations up to 200 μg/ml resulted in an increase in biofilm biomass by 16 h under static conditions and a corresponding increase in cell density in the bulk liquid. In contrast, these biofilms had decreased levels of extracellular DNA (eDNA). Fe(II) levels in the supernatants of biofilms formed in the presence of FeNPs exceeded 100 μM compared with 20 μM in control media without cells. Spent cell supernatants had little effect on Fe(II) levels. Cells also had an effect on the aggregation behavior of these nanoparticles. SPIONs incubated with cells exhibited a decrease in the number and size of FeNP aggregates visible using light microscopy. SPIONs resuspended in fresh media or spent culture supernatants formed large aggregates visible in the light microscope upon exposure to a supermagnet;and could be pelleted magnetically in microtitre plate wells. In contrast, SPION FeNPs incubated with cells were unaffected by exposure to the supermagnet and could not be pelleted. The results of this study indicate a need to reconsider the effects of FeNPs on bacterial growth and biofilm formation and the effect the bacterial cells may have on the use and recovery of SPIONs.

Antimicrobial Susceptibility,Biofilm Production and Adhesion to HEp-2 Cells of Pseudomonas aeruginosa Strains Isolated from Clinical Samples

A hundred Pseudomonas aeruginosa strains from several clinical specimens from five hospitals in Sao Luís-MA were evaluated for biofilm production, prevalence of the gene algD, adhesion to HEp-2 cells and antimicrobial susceptibility. The most affected clinical specimens and hospital sectors were also evaluated. Most isolates were obtained from the tracheal aspirate (21.0%) and the most affected hospital sector was the ICU (43.0%). The antibiotics with the highest sensitivity rate were amikacin, piperacillin/tazobactam, fluoroquinolones, gentamicin and meropenem and the ones with the highest resistance rate were aztreonam, ceftazidime and cefepime. All samples were sensitive to polymyxin B. In relation to the expression of the gene for ESBL, 50.0% (17/34) of the multiresistant strains showed the enzyme TEM. Most strains showed high hydrophobicity and 96% of the isolates produced biofilm on a polystyrene microplate, 52% were capsule producers, 19% showed mannose-sensitive fimbriae and 39% expressed the gene algD. We observed adhesion to HEp-2 cells and to the coverslip. These factors may be reported in the pathogenesis of this bacterium, what represents a potential risk for colonization of medical devices which favor the establishment of chronic nosocomial infections.

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.

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 El 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.

Adjuvant role of Pseudomonas flagellin for Acinetobacter baumannii biofilm associated protein

AIM To study immunogenicity of Pseudomonas N terminal flagellin as an adjuvant for Acinetobacter baumannii(A. baumanni) biofilm associated protein(Bap).METHODS The N terminal flagellin gene was amplified. The p ET28a(+) and polymerase chain reaction products weredigested with HindⅢ and Eco R Ⅰ. The ligation of N terminal flagellin into p ET28a(?+) was performed using T4 DNA ligase and was then transformed into Escherichia coli BL21(DE3) as a suitable expression host. p ET28a(?+) vector harboring a conserved region of Bap from our previous work was used. The recombinant proteins were expressed, analyzed by SDS-PAGE method and was purified by affinity chromatography with His-Tag residues followed by confirmation with western blotting. Mice were immunized with recombinant N terminal flagellin and Bap subunits. The immunized animals were intranasally(i.n) challenged with A. baumannii and Pseudomonas aeruginosa(P. aeruginosa).RESULTS The flagellin enhanced the immunogenicity of Bap causing an increase in specific Ig G titers in serum(P < 0.001). Internal organs, i.e., liver, lung and spleen of the BapFlagellin immunized group challenged with A. baumannii showed significantly lower bacterial load compared to the control group. The bacterial loads were studied in internal organs. A. baumannii infected immunized animals with Bap-Flagellin exhibited internal organs with minor bacterial load while P. aeruginosa PAO1 infected group showed heavy bacterial load of(4.3 ± 0.12) × 106,(1.1 ± 0.01) × 106 and(2.2 ± 0.22) × 106 per gram of lungs, liver and spleen respectively. Bacterial loads were detected per gram of lungs, liver and spleen of the mice group immunized with Bap were(1.2 ± 0.06) × 107,(11.1 ± 0.041) × 105 and(3.6 ± 0.42) × 106 respectively. In vivo neutralization assay indicated that all experimental mice groups, except for Flagellin administered group was significantly(P < 0.05) protected against A. baumannii. CONCLUSION These results demonstrate that P. aeruginosa Flagellin as an adjuvant for Bap A. baumannii could be a useful model to evaluate new vaccine against A. baumannii.

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.

绿脓杆菌Pseudomonas aeruginosa BTE-1直接电催化特征

研究产电绿脓杆菌P.aeruginosa BTE-1的电化学催化特征.结果表明,在厌氧条件下,P.aeruginosa BTE-1菌株不能分泌可充当电子介体的绿脓菌素,但可依靠在电极表面形成生物膜而呈现直接电催化性能.P.aeruginosaBTE-1在电极表面形成生物膜与其在特定电极电位下向电极传递电子的过程直接相关,适宜的电位为0.2 V(vs.SCE),电位过高可能会损害P.aeruginosa BTE-1细胞.室温范围内升高温度可增强P.aeruginosa BTE-1生物膜的电催化活性,但过高的温度(>60℃)会抑制生物膜电催化活性.循环伏安曲线显示,在厌氧条件下形成的P.aerugi-nosa BTE-1生物膜,具有与典型产电菌株G.sulfurreducens相近的氧化还原电位(-0.4 V～-0.2 V,vs.SCE).P.aeruginosa BTE-1生物膜可电催化酵母抽取物和葡萄糖,但不能电催化醋酸盐.

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.

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.