Effect of Interleukin-10 on Gut-Derived Sepsis Caused by Pseudomonas aeruginosa in Mice

We evaluated the protective effect of interleukin-10 (IL-10) against murine gut-derived sepsis caused by Pseudomonas aeruginosa. Gut-derived sepsis was induced by administering cyclophosphamide and ampicillin while feeding P. aeruginosa to specific-pathogen-free mice. Treating mice with recombinant human IL-10 (rhIL-10) at 1.0 or 5.0 μg/mouse twice a day following the second cyclophosphamide administration significantly increased the survival rate compared to that of control mice treated with saline; however, treatment with rhIL-10 at 0.1 μg/mouse did not result in significant protection. Bacterial counts in the liver, spleen, and blood were all significantly lower in mice treated with rhIL-10 than in saline-treated control mice. Treatment with rhIL-10 significantly suppressed tumor necrosis factor alpha, interleukin-1β, interleukin-6, and gamma interferon levels in the serum of mice following induction of gut-derived sepsis. We also studied the effect of IL-10 on leukocyte recovery after cyclophosphamide treatment of mice. Administration of rhIL-10 intraperitoneally at 1.0 μg/mouse significantly accelerated the recovery of leukocytes in comparison with that of the group of saline-treated controls. These results indicate that IL-10 shows a protective effect against gut-derived P. aeruginosa sepsis. We suspect that the mechanism of this effect is that IL-10 regulates in vivo production of inflammatory cytokines. Furthermore, acceleration of leukocyte recovery by IL-10 after cyclophosphamide-induced depression may also play an important role in this protection.     

Microbial Growth Inhibition by Alternating Electric Fields in Mice with Pseudomonas aeruginosa Lung Infection

High-frequency, low-intensity electric fields generated by insulated electrodes have previously been shown to inhibit bacterial growth in vitro. In the present study, we tested the effect of these antimicrobial fields (AMFields) on the development of lung infection caused by Pseudomonas aeruginosa in mice. We demonstrate that AMFields (10 MHz) significantly inhibit bacterial growth in vivo, both as a stand-alone treatment and in combination with ceftazidime. In addition, we show that peripheral (skin) heating of about 2°C can contribute to bacterial growth inhibition in the lungs of mice. We suggest that the combination of alternating electric fields, together with the heat produced during their application, may serve as a novel antibacterial treatment modality.The 20th century was the golden era of the antibacterial agents, with millions of people owing their lives to the discovery of and treatment with the numerous antibiotic families used today. Surely, antibacterial agents will continue to play a major role in the battle against pathogenic bacteria in the 21st century; however, the extensive use of antibiotics holds a threat of a far less optimistic future due to the rapid rise of multidrug-resistant bacteria. Recently, the potential use of physical means as an aid to antibiotics in the battle against bacterial pathogens has been studied: photodynamic therapy (12, 21, 35), ultrasound wave therapy (7, 23, 25), thermotherapy (26), and weak electric currents (4, 6, 24, 32, 33) are all being tested as treatment modalities against pathogenic microorganisms. The major drawback of the methods mentioned above is their low therapeutic index due to the high levels of heating produced by ultrasonic waves, thermotherapy, and photodynamic therapy (36) and the activated oxygen generated by photodynamic therapy, both of which can damage the tissues in and around the target area (22). In addition, the use of conductive electrodes for the generation of electric currents is associated with the release of metal ions and free radicals at the electrode surface, all of which are toxic to living cells (18). Indeed, as of today, none of the above-mentioned means has matured into an approved treatment modality against bacterial pathogens.Recently, we demonstrated that low-intensity alternating electric fields of high frequencies (antimicrobial fields [AMFields]) have an in vitro inhibitory effect on the growth of pathogenic bacteria, including Staphylococcus aureus and Pseudomonas aeruginosa (8). AMFields were generated using electrically insulated electrodes and therefore were not associated with the production of free radicals, toxic metal ions, or electrolysis at the electrode surface. Fields of the relatively high frequency at which the AMFields effects were observed (with an optimum at 10 MHz) have no known effect on human cells (13, 14). Indeed, we found that AMFields have no effect on the growth of cell cultures in vitro (unpublished results). Furthermore, the high frequencies of the AMFields allow for the application of relatively high intensities without nerve and muscle stimulation, and their nonattenuated spread in tissues makes deep treatment realistic.In view of the above-described factors, the objective of the present study was to test the feasibility of using AMFields generated by insulated electrodes for the inhibition of P. aeruginosa proliferation in the infected lungs of mice.     

多重耐药铜绿假单胞菌感染

铜绿假单胞菌广泛分布于土壤、水、植被中,是人体正常定植菌,一般不致病。当人体正常防御体系受损时,如气管插管、留置静脉导管、留置导尿管、中性粒细胞缺乏和免疫抑制状态等,铜绿假单胞菌侵入人体,成为条件致病菌。铜绿假单胞菌可引起包括皮肤软组织感染、肺炎、血流感染在内     

Elucidation of the Pharmacokinetic/Pharmacodynamic Determinant of Colistin Activity against Pseudomonas aeruginosa in Murine Thigh and Lung Infection Models

Colistin is increasingly used as last-line therapy against Gram-negative pathogens. The pharmacokinetic (PK)/pharmacodynamic (PD) index that best correlates with the efficacy of colistin remains undefined. The activity of colistin against three strains of Pseudomonas aeruginosa was studied in neutropenic mouse thigh and lung infection models. The PKs of unbound colistin were determined from single-dose PK studies together with extensive plasma protein binding analyses. Dose-fractionation studies were conducted over 24 h with a dose range of 5 to 160 mg/kg of body weight/day. The bacterial burden in the thigh or lung was measured at 24 h after the initiation of treatment. Relationships between antibacterial effect and measures of exposure to unbound (f) colistin (area under the concentration-time curve [fAUC/MIC], maximum concentration of drug in plasma [fC_max]/MIC, and the time that the concentration in plasma is greater than the MIC [fT > MIC]) were examined by using an inhibitory sigmoid maximum-effect model. Nonlinearity in the PKs of colistin, including its plasma protein binding, was observed. The PK/PD index that correlated best with its efficacy was fAUC/MIC in both the thigh infection model (R² = 87%) and the lung infection model (R² = 89%). The fAUC/MIC targets required to achieve 1-log and 2-log kill against the three strains were 15.6 to 22.8 and 27.6 to 36.1, respectively, in the thigh infection model, while the corresponding values were 12.2 to 16.7 and 36.9 to 45.9 in the lung infection model. The findings of this in vivo study indicate the importance of achieving adequate time-averaged exposure to colistin. The results will facilitate efforts to define the more rational design of dosage regimens for humans.Infections caused by multidrug-resistant Gram-negative bacteria, in particular, Pseudomonas aeruginosa, are presenting a critical challenge, and they are associated with a high mortality rate if they are not treated promptly and effectively (3). The Antimicrobial Availability Task Force of the Infectious Diseases Society of America, the FDA, and other organizations have highlighted the urgent need to develop new antibiotics with activity against Gram-negative organisms, including P. aeruginosa (4, 27). Unfortunately, novel agents with activity against P. aeruginosa may not be available in the next 9 to 11 years (24). In the meantime, colistin (polymyxin E) is often the only available active antibiotic and is increasingly used as the last line of therapy against Gram-negative “superbugs” (8, 13, 18).Colistin is a cationic lipopeptide antibiotic with concentration-dependent bactericidal activity against P. aeruginosa and other Gram-negative bacteria (18). It is administered parenterally in the form of its inactive prodrug, colistin methanesulfonate (CMS) (2). CMS/colistin entered clinical use in the late 1950s, but it fell out of favor in the 1970s due to concerns about the potential for nephrotoxicity and neurotoxicity (8, 18). Since CMS/colistin was never subjected to contemporary drug development procedures, there has been a substantial lack of preclinical and clinical pharmacokinetic (PK) and pharmacodynamic (PD) data that may be used to guide the selection of appropriate dosage regimens (18). As a result, the dosage regimens in use today were selected empirically and are not based upon solid PK/PD data. The urgency to establish optimized dosage regimens is highlighted by recent reports of colistin resistance (11, 27).To assist with the optimization of dosage regimens, the aims of the present study were to elucidate the PK/PD index of colistin, the antibacterially active form of CMS (2), that correlates best with efficacy against P. aeruginosa by using mouse thigh and lung infection models and to determine the target values of the index for various magnitudes of antibacterial effects.(This study was presented, in part, at the 47th Interscience Conference on Antimicrobial Agents and Chemotherapy, Chicago, IL, September 2007, abstr. A-5.)     

Colistin is effective in treatment of infections caused by multidrug-resistant Pseudomonas aeruginosa in cancer patients

The increasing incidence of infections caused by multidrug-resistant Pseudomonas aeruginosa is a worldwide health problem. Because no new antipseudomonal agents are expected to be available in the near future, we evaluated the safety and efficacy of colistin, an old drug with bactericidal activity against this organism. We collected clinical and demographic data on 95 cancer patients diagnosed with infections caused by multidrug-resistant P. aeruginosa between January 2001 and January 2004 and treated with either colistin (colistin group) or at least one active antipseudomonal agent (a beta-lactam antibiotic or a quinolone) (control group). We compared the results obtained for both groups. Thirty-one patients had been treated with colistin and 64 had been treated with an antipseudomonal non-colistin-containing regimen. Compared with the control group, patients in the colistin group had a lower median age (52 and 62 years, respectively; P = 0.012) but were more likely to have had nosocomial infections (87% and 64%, respectively; P = 0.02). Twenty-five patients (81%) in the colistin group and 40 patients (63%) in the control group had an APACHE II score of >15 (P = 0.074). The overall clinical response rates were 52% in the colistin group and 31% in the control group (P = 0.055). Multiple logistic regression analysis showed that those patients treated with colistin were 2.9 times (95% confidence interval, 1.1 to 7.6 times) more likely than those in the control group to experience a clinical response to therapy (P = 0.026). Colistin therapy was at least as effective and as safe a beta-lactam antibiotic or a quinolone in the treatment of infections caused by multidrug-resistant P. aeruginosa and, hence, may be a useful or preferred alternative therapy for this infection in cancer patients.     

Colistin methanesulfonate is an inactive prodrug of colistin against Pseudomonas aeruginosa

There is a dearth of information on the pharmacodynamics of "colistin," despite its increasing use as a last line of defense for treatment of infections caused by multidrug-resistant gram-negative organisms. The antimicrobial activities of colistin and colistin methanesulfonate (CMS) were investigated by studying the time-kill kinetics of each against a type culture of Pseudomonas aeruginosa in cation-adjusted Mueller-Hinton broth. The appearance of colistin from CMS spiked at 8.0 and 32 mg/liter was measured by high-performance liquid chromatography, which generated colistin concentration-time profiles. These concentration-time profiles were subsequently mimicked in other incubations, independent of CMS, by incrementally spiking colistin. When the cultures were spiked with CMS at either concentration, there was a substantial delay in the onset of the killing effect which was not evident until the concentrations of colistin generated from the hydrolysis of CMS had reached approximately 0.5 to 1 mg/liter (i.e., approximately 0.5 to 1 times the MIC for colistin). The time course of the killing effect was similar when colistin was added incrementally to achieve the same colistin concentration-time course observed from the hydrolysis of CMS. Given that the killing kinetics of CMS can be accounted for by the appearance of colistin, CMS is an inactive prodrug of colistin with activity against P. aeruginosa. This is the first study to demonstrate the formation of colistin in microbiological media containing CMS and to demonstrate that CMS is an inactive prodrug of colistin. These findings have important implications for susceptibility testing involving "colistin," in particular, for MIC measurement and for microbiological assays and pharmacokinetic and pharmacodynamic studies.     

Brain Penetration of Colistin in Mice Assessed by a Novel High-Performance Liquid Chromatographic Technique

A sensitive and reliable liquid chromatographic method was developed and validated for the determination of colistin concentrations in mouse brain homogenate. With a mobile phase consisting of acetonitrile-tetrahydrofuran-water (50:25:25 [vol/vol]) at a flow rate of 1 ml/min, a linear correlation between peak area and colistin concentration was observed over the concentration range of 93.8 to 3,000 ng/g in brain tissue (R² > 0.994). Intra- and interday coefficients of variation were 5.1 to 8.3% and 5.8 to 8.5%, respectively, and the recovery ranged from 85% to 94%. This assay was then utilized to determine the amount of colistin that permeated the blood-brain barrier over a 2-h period following bolus intravenous administration of colistin sulfate to mice. After a single dose of 5 mg/kg of body weight to mice, brain homogenate concentrations of colistin were very low, relative to plasma colistin concentrations, suggesting that colistin permeability across the healthy blood-brain barrier is negligible during this experimental period.Colistin (also known as polymyxin E) is a cationic polypeptide antibiotic and is bactericidal against multidrug-resistant gram-negative pathogens (8). Like other members of the polymyxin family, colistin consists of two major components (colistins A and B), with colistins A and B differing only in the fatty acid side chain. In studies conducted several decades ago, colistin was shown to result in adverse effects after intramuscular or intravenous administration to patients (6, 14); nephrotoxicity and neurotoxicity were the most commonly observed adverse effects. It is possible that the prevalence of such toxicity may have been exaggerated due to a lack of understanding of colistin pharmacology and the use of inappropriate doses (22). Over the last few years, however, resistance to many other antibiotics and limited development of new antibiotics have resulted in an increasing use of colistin, administered in the form of its inactive prodrug, colistin methanesulfonate (CMS) (1), for the treatment of infections caused by gram-negative pathogens, such as Pseudomonas aeruginosa and Acinetobacter baumannii (5). With its increased use, interest in the pharmacology of this old antibiotic has been rekindled and optimization of dosage regimens is therefore urgently required in order to maximize its efficacy while minimizing potential toxicity.Gram-negative bacterial species are increasingly reported to cause severe central nervous system (CNS) infections, such as meningitis (11, 24, 26) and ventriculitis (7, 30), which have a high mortality rate if untreated or treated inappropriately. There have been several clinical case reports of successful treatment of meningitis and ventriculitis by parenteral administration of CMS, either alone or in combination with other antibacterials (11, 12, 18), resulting in eradication of the gram-negative pathogens from the cerebrospinal fluid (CSF). These studies suggest that CMS, or colistin that is generated from CMS in vivo, has the potential to permeate the blood-CSF barrier (BCSFB). Indeed, using a microbiological assay, Jiménez-Mejías et al. were able to detect “colistin” in human CSF following intravenous administration of CMS (12). However, less is known about the ability of colistin to penetrate the blood-brain barrier (BBB), the endothelial lining of the cerebral blood vessels—a process which would be required in order to treat infections of the brain parenchyma, such as meningoencephalitis (27). A more thorough understanding of colistin penetration into the brain parenchyma, therefore, would be useful for optimizing treatment of such infections. In addition, the neurotoxicity associated with colistin (2) warrants a better understanding of colistin penetration across the BBB so that more rational dosing regimens can be designed with minimal potential for CNS-mediated toxicity. However, in order to accurately determine brain penetration of colistin, a robust and reliable analytical technique is required.Numerous methods based on microbiological (15, 16, 19), thin-layer chromatographic (TLC) (32), immunological (13), and liquid chromatography and mass spectrometry (10, 25) techniques have been developed to quantitate the polypeptide antibiotics in plasma and tissues. While liquid chromatography and mass spectrometry methods are quite sensitive, they are often not very accessible, and the other techniques listed above often lack sensitivity or selectivity required for accurate quantitation. With respect to colistin, several methods for assaying this polypeptide in rat or human plasma have been developed utilizing derivatizing reagents such as orthophthalaldehyde (4, 17) and 9-fluorenylmethyl chloroformate (FMOC-Cl) (20, 21). While these assays have been successfully used to measure concentrations of colistin in human and rodent plasma and some tissue samples (34), quantitative assays for measuring the concentration of this antibiotic in animal and human brain tissues are not available.The purpose of this study, therefore, was to develop a reliable and sensitive method to quantify colistin in mouse brain homogenate. Our method consists of protein precipitation with trichloroacetic acid (TCA), solid-phase extraction (SPE) of colistin from brain tissue, and derivatization with FMOC-Cl, followed by liquid chromatography with fluorescence detection. This method was then employed in our laboratory to determine the concentration of colistin in mouse brain homogenate following intravenous administration of colistin sulfate, to determine the potential of colistin to penetrate the BBB in healthy mice.     

Pharmacokinetic/Pharmacodynamic Investigation of Colistin against Pseudomonas aeruginosa Using an In Vitro Model

Colistin plays a key role in treatment of serious infections by Pseudomonas aeruginosa. The aims of this study were to (i) identify the pharmacokinetic/pharmacodynamic (PK/PD) index (i.e., the area under the unbound concentration-time curve to MIC ratio [ƒAUC/MIC], the unbound maximal concentration to MIC ratio [ƒC_max/MIC], or the cumulative percentage of a 24-h period that unbound concentrations exceed the MIC [ƒT_>MIC]) that best predicts colistin efficacy and (ii) determine the values for the predictive PK/PD index required to achieve various magnitudes of killing effect. Studies were conducted in a one-compartment in vitro PK/PD model for 24 h using P. aeruginosa ATCC 27853, PAO1, and the multidrug-resistant mucoid clinical isolate 19056 muc. Six intermittent dosing intervals, with a range of ƒC_max colistin concentrations, and two continuous infusion regimens were examined. PK/PD indices varied from 0.06 to 18 for targeted ƒC_max/MIC, 0.36 to 312 for ƒAUC/MIC, and 0 to 100% for ƒT_>MIC. A Hill-type model was fit to killing effect data, which were expressed as the log₁₀ ratio of the area under the CFU/ml curve for treated regimens versus control. With ƒC_max values equal to or above the MIC, rapid killing was observed following the first dose; substantial regrowth occurred by 24 h with most regimens. The overall killing effect was best correlated with ƒAUC/MIC (R² = 0.931) compared to ƒC_max/MIC (R² = 0.868) and ƒT_>MIC (R² = 0.785). The magnitudes of ƒAUC/MIC required for 1- and 2-log₁₀ reductions in the area under the CFU/ml curve relative to growth control were 22.6 and 30.4, 27.1 and 35.7, and 5.04 and 6.81 for ATCC 27853, PAO1, and 19056 muc, respectively. The PK/PD targets identified will assist in designing optimal dosing strategies for colistin.Globally there is a growing threat from the emergence of multidrug-resistant (MDR) microorganisms (38), especially among a number of important Gram-negative bacterial pathogens (16, 29, 38). Colistin (polymyxin E) still retains significant activity against many of these MDR Gram-negative pathogens, including Pseudomonas aeruginosa, Acinetobacter baumannii, and Klebsiella pneumoniae, which often leaves it as the only therapeutic option available (19, 26). With very few new chemical entities against Gram-negative infections in the drug development pipeline (29, 30, 38), particularly against P. aeruginosa (38), the use of colistin, a once-neglected antibiotic, has increased dramatically over the last 5 years (11, 26).Colistin is available commercially as colistin sulfate (hereafter referred to as colistin) and sodium colistin methanesulfonate (CMS), which is administered parenterally. CMS is an inactive prodrug of colistin (3) and, after parenteral administration, colistin is formed in vivo (21, 27, 33). Despite its newfound importance in therapy, there is a dearth of information on the pharmacokinetic (PK) and pharmacodynamic (PD) properties of colistin, a situation of significant concern given that resistance to colistin is beginning to emerge (1, 15, 18, 26, 28). Thus, the aims of the present study were to utilize an in vitro PK/PD model to (i) identify the PK/PD index (i.e., the area under the unbound concentration-time curve to MIC ratio [ƒAUC/MIC], the unbound maximal concentration to MIC ratio [ƒC_max/MIC], or the cumulative percentage of a 24-h period that unbound concentrations exceed the MIC [ƒT_>MIC]) that best predicts colistin efficacy and (ii) determine the magnitude of the predictive PK/PD index required to achieve various magnitudes of killing effect.(Parts of the present study were presented at the 48th Annual Interscience Conference on Antimicrobial Agents and Chemotherapy [ICAAC], Washington, DC, 25 to 28 October 2008, and at the Second American Conference on Pharmacometrics, Mashantucket, CT, 4 to 7 October 2009.)     

Effect of clarithromycin on Pseudomonas aeruginosa biofilms

Using an experimental in vitro culture system, we investigated the effect of clarithromycin on biofilm formation by a leucine-requiring Pseudomonas aeruginosa mutant strain (HU1). Biofilm formation on celldesks in a chemically defined medium was assessed by viable cell count as well as by measurement of glycocalyx production and scanning electron-microscopic observation. Cells proliferated exponentially until day 3 and remained stationary afterwards. The amount of glycocalyx, simultaneously semiquantified, showed a linear increase from day 1 to day 12. Scanning electron microscopy revealed firm biofilms on day 5. Three different concentrations of Clarithromycin (CAM) (minimum inhibitory concentration MIC 64 microg/ml) were added continuously at the early and late phases of biofilm formation, and the antibiofilm effect of CAM was evaluated by the changes in cell count and glycocalyx production. CAM was effective on biofilms at 100 microg/ml but neither at 1 nor at 10 microg/ml. It is suggested that glycocalyx production started following bacterial multiplication and continued even after the cells had entered the stationary phase to form mature biofilms. No antibiofilm effect of CAM was observed at sub-MIC.     

Silver Sulfadiazine: Effect on the Ultrastructure of Pseudomonas aeruginosa

Pseudomonas aeruginosa exposed to silver sulfadiazine (AgSu) were examined in an electron microscope. The treated cells were distorted in shape, and structures (blebs) protruded from the cell surface. These "blebs" appeared to arise from the cell wall. A strain of P. aeruginosa resistant to AgSu did not display these changes. Upon exposure of P. aeruginosa to silver nitrate, none of these changes was seen; rather, such cells are characterized by large, central aggregations of nuclear material. The results are consistent with previous findings which suggested that AgSu acted at the cell surface.     

Activity of Lividomycin Against Pseudomonas aeruginosa: Its Inactivation by Phosphorylation Induced by Resistant Strains

The antibacterial activity and enzymatic inactivation of lividomycin, a new aminoglycosidic antibiotic, were studied with 13 strains of Pseudomonas aeruginosa. The minimal inhibitory concentration of lividomycin was 12.5 to 25 mug/ml, and three strains were resistant to high concentrations of lividomycin (more than 200 mug/ml). It was found that P. aeruginosa TI-13 and K-11, highly lividomycin-resistant strains of clinical origin, strongly inactivated the drug. The third resistant strain, Km-41/R, was developed in vitro. Unlike the other resistant strains, Km-41/R, was developed in vitro. Unlike the other resistant strains, Km-41/R did not inactivate the drug, indicating that different mechanisms were involved in lividomycin resistance. By use of a cell-free extract from P. aeruginosa TI-13, the inactivation of lividomycin was found to be caused by the formation of a monophosphorylated product of the drug.     

Effect of Varidase (streptodornase) on biofilm formed by Pseudomonas aeruginosa

The biofilm of Pseudomonas aeruginosa could be removed by Varidase (streptodornase) that was used as defibrinating drug. After cultivating the biofilm on a silicone chip in a modified alginate-producing medium in vitro, it was treated with Varidase or DNase I. In both treatments, the biofilm was removed depending on the concentration of the reagents. Varidase was apparently effective under the condition that the biofilm was exposed to more than 625 U/ml (a quarter of the concentration of the medical use) for 3 h, twice, at 37 degrees C. The result of this experiment indicates that (a) the DNases, DNase I and Varidase, were effective in destroying the biofilm of P. aeruginosa in vitro, and (b) in a clinical field, Varidase could be useful for P. aeruginosa focal infection, such as urinary tract infection, by removing the biofilm.     

猴头菌提取物对小鼠D半-乳糖衰老模型脑组织的抗氧化作用

目的探讨猴头菌提取物对D-半乳糖所致小鼠衰老模型脑组织的抗氧化作用。方法选择7~8周龄雄性小鼠60只,随机分为阴性对照组、阳性对照组及猴头菌提取物高、中、低剂量治疗组,每组12只。阳性对照组及3个治疗组按100 mg/(kg.d)皮下注射D-半乳糖造模,阴性对照组以相同方式予同剂量0.9%氯化钠注射液。造模后,猴头菌提取物低、中、高治疗组分别按1.5、3.0、4.5 g/(kg.d)予猴头菌提取物,阴性对照组和阳性对照组予同剂量蒸馏水,连续实验4周。检测小鼠脑组织中超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)和脑、血清中丙二醛(MDA)含量。结果经3个剂量猴头菌提取物治疗后,衰老小鼠脑组织中SOD、GSH-Px的表达显著升高,MDA含量显著下降,与阳性对照组比较差异有统计学意义(P<0.01,P<0.05)。结论猴头菌提取物对D-半乳糖所致衰老小鼠脑组织具有明显抗氧化作用。     

Antiviral Effect of Pyran Against Systemic Infection of Mice with Herpes Simplex Virus Type 2

The immunomodulator pyran markedly protected 5-week-old mice from lethal intravenous infection with herpes simplex virus type 2. The 50% lethal dose was increased almost 100-fold in pyran-treated mice as compared with controls. Although the protection was not as marked in older mice (10 and 16 weeks old), there was a significant increase in mean survival time. When the pathogenesis of herpesvirus disease was monitored in control and drug-treated mice, the effect of pyran was most evident in the spinal cord, where virus was recovered from 20 of 25 control mice and from only 6 of 25 pyran-treated mice. There was also a significant reduction in the titer of virus present, and virus appeared later in the spinal cord of pyran-treated mice than in control mice. The protective effect of pyran was observed only when the drug was administered 24 h before viral challenge, was seen after both intraperitoneal and intravenous injection, and was not due to direct inactivation of the virus.     

Susceptibility of 1,500 Isolates of Pseudomonas aeruginosa to Gentamicin, Carbenicillin, Colistin, and Polymyxin B

A series of 1,500 strains of Pseudomonas aeruginosa was collected from a variety of sources to provide a group of strains which would truly represent the species. All of them were pyocine typed, and a wide range of types was included among the isolates from each source. The gentamicin, carbenicillin, colistin, and polymyxin minimal inhibitory concentration of each strain was measured by the agar dilution method employing the Steers inocula replicator. Over 99% of strains were inhibited by 8 mug of gentamicin per ml, by 256 mug of carbenicillin per ml, and by 4 mug of colistin per ml. The small number of strains more resistant to each antibiotic comprised a variety of different pyocine types. Few strains were found to be susceptible to tetracycline, chloramphenicol, kanamycin, or streptomycin at the single concentration tested.     

Effect of Triethylenetetramine Dihydrochloride on the Antibiotic Susceptibility of Pseudomonas aeruginosa

A chelating agent, triethylenetetramine dihydrochloride, interacted synergistically in vitro with both gentamicin and carbenicillin against a clinical isolate of Pseudomonas aeruginosa designated Ps 15. The minimal inhibitory concentrations of carbenicillin and gentamicin for Ps 15 in a 50% serum-Trypticase soy broth were 250 and 72.9 mug/ml, respectively. However, addition of triethylenetetramine dihydrochloride to the 50% serum-Trypticase soy broth reduced the minimal inhibitory concentration of both antibiotics approximately 10-fold. A comparison of the growth of Ps 15 in 50% serum-Trypticase soy broth containing either of the antibiotics showed that a rapid decrease in the percentage of survivors only occurred when the chelating agent was present.     

Quorum-Quenching Acylase Reduces the Virulence of Pseudomonas aeruginosa in a Caenorhabditis elegans Infection Model

The Pseudomonas aeruginosa PAO1 gene pvdQ encodes an acyl-homoserine lactone (AHL) acylase capable of degrading N-(3-oxododecanoyl)-l-homoserine lactone by cleaving the AHL amide. PvdQ has been proven to function as a quorum quencher in vitro in a number of phenotypic assays. To address the question of whether PvdQ also shows quorum-quenching properties in vivo, an infection model based on the nematode Caenorhabditis elegans was explored. In a fast-acting paralysis assay, strain PAO1(pMEpvdQ), which overproduces PvdQ, was shown to be less virulent than the wild-type strain. More than 75% of the nematodes exposed to PAO1(pMEpvdQ) survived and continued to grow when using this strain as a food source. Interestingly, in a slow-killing assay monitoring the survival of the nematodes throughout a 4-day course, strain PAO1-ΔpvdQ was shown to be more virulent than the wild-type strain, confirming the role of PvdQ as a virulence-reducing agent. It was observed that larval stage 1 (L1) to L3-stage larvae benefit much more from protection by PvdQ than L4 worms. Finally, purified PvdQ protein was added to C. elegans worms infected with wild-type PAO1, and this resulted in reduced pathogenicity and increased the life span of the nematodes. From our observations we can conclude that PvdQ might be a strong candidate for antibacterial therapy against Pseudomonas infections.Pseudomonas aeruginosa is an opportunistic gram-negative pathogen of vertebrates and a primary pathogen of insects (17). It mainly infects individuals who are immunocompromised, such as human immunodeficiency virus-infected patients, as well as those who have cystic fibrosis. In addition, those having disruptions in normal barriers caused by severe burns or indwelling medical devices are at risk. Hospital-acquired P. aeruginosa pneumonias and septicemias are frequently lethal (2, 3). To facilitate the establishment of infection, P. aeruginosa produces an impressive array of both cell-associated and extracellular virulence factors, such as proteases and phospholipases, and also small molecules, including rhamnolipid, phenazines, and cyanide (17). Expression of many of the extracellular factors is cell density controlled, does not occur until the late logarithmic phase of growth, and is mediated through specific quorum-sensing signal molecules (23). Two of these molecules, N-butanoyl-l-homoserine lactone (C4-HSL) and N-(3-oxododecanoyl)-l-homoserine lactone (3-oxo-C12-HSL), have been studied in great detail. In our laboratory, we previously demonstrated that PA2385(pvdQ) from P. aeruginosa PAO1 encodes an acyl-homoserine lactone (AHL) acylase. Analysis of the gene product showed that the posttranslational processing of the acylase as well as the hydrolysis reaction type are similar to those of the beta-lactam acylases, strongly suggesting that the PvdQ protein is a member of the N-terminal nucleophile hydrolase superfamily. The main AHL signaling molecule of P. aeruginosa PAO1, 3-oxo-C12-HSL, is degraded by PvdQ (16). Addition of the purified protein to PAO1 cultures completely inhibited accumulation of 3-oxo-C12-HSL and production of the signal molecule 2-heptyl-3-hydroxy-4(1H)-quinolone and reduced production of the virulence factors elastase and pyocyanin. Similar results were obtained when pvdQ was overexpressed in P. aeruginosa (16). These results demonstrate that this protein has in situ quorum-quenching activity. This AHL acylase may enable P. aeruginosa PAO1 to modulate its own quorum-sensing-dependent pathogenic potential and, moreover, offers possibilities for novel antipseudomonal therapies.To test our hypothesis that PvdQ can exert its beneficial functions also in vivo, we chose to study its effect on the infection of the nematode Caenorhabditis elegans. This model has been used before in multiple pathogenicity studies of Cryptococcus neoformans (13) and gram-positive (6) and gram-negative (9, 10, 11) bacteria. Infection with P. aeruginosa strain PA14 was found to result in fast (hours) or slow (days) killing, depending on the growth medium used (19, 20). When Darby and colleagues (2) used the system to study P. aeruginosa PAO1, a lethal paralysis of the worms was observed, indicating another mechanism by which P. aeruginosa can kill C. elegans. It was shown that quorum-sensing-dependent hydrogen cyanide production on rich medium by P. aeruginosa PAO1 is the causative agent for the fast paralysis (5). Under the same conditions, an attenuation of paralysis by an AHL acylase from Ralstonia sp. strain XJ12B upon expression in P. aeruginosa PAO1 was observed (12). Those authors performed the assays with a mixed population of worms only and did not test for slow killing. In this study we show that P. aeruginosa PAO1 can also elicit a slow-killing response when grown in low-nutrient medium. Moreover, we report that not only overexpression of the gene from its host but also external addition of the purified PvdQ renders P. aeruginosa PAO1 less pathogenic to C. elegans and increases the life span of the infected animals in both slow- and fast-killing assays. As the correlation between the bacterial virulence factors required for pathogenesis in mammals and in C. elegans has been shown to be very high (20), we propose that the external addition of purified AHL acylases may be developed into a novel quorum-quenching therapy.     

泛耐药铜绿假单胞菌的感染现状及危险因素分析

目的探讨医院内泛耐药铜绿假单胞菌(PDRPA)的感染现状及其危险因素,为临床控制PDRPA感染提供依据。方法对该院2012年3月至2014年2月期间的铜绿假单胞菌进行院内感染监测,回顾性分析相应病例的临床资料,分析PDRPA感染的危险因素。结果 PDRPA主要分布于重症医学科和神经外科,以痰液为主,分别占泛耐药菌株标本来源的59.5%和31.0%;年龄、住院时间、使用抗菌药物时间、使用碳青霉烯类、使用头孢菌素类、使用喹诺酮类、糖尿病、冠心病、深静脉插管、气管插管、使用呼吸机、鼻饲胃管与PDRPA感染明显相关(P0.05),而与肿瘤、使用氨基糖苷类、混合感染无明显相关性(P0.05)。结论年龄、住院时间、使用抗菌药物时间、使用碳青霉烯类、使用头孢菌素类、使用喹诺酮类、糖尿病、冠心病、深静脉插管、气管插管、使用呼吸机、鼻饲胃管是PDRPA感染的危险因素,因此,控制糖尿病、冠心病,合理使用抗菌药物,减少侵袭性操作是预防和控制PDRPA院内感染的关键。     

Blue Light Rescues Mice from Potentially Fatal Pseudomonas aeruginosa Burn Infection: Efficacy,Safety, and Mechanism of Action

Blue light has attracted increasing attention due to its intrinsic antimicrobial effect without the addition of exogenous photosensitizers. However, the use of blue light for wound infections has not been established yet. In this study, we demonstrated the efficacy of blue light at 415 nm for the treatment of acute, potentially lethal Pseudomonas aeruginosa burn infections in mice. Our in vitro studies demonstrated that the inactivation rate of P. aeruginosa cells by blue light was approximately 35-fold higher than that of keratinocytes (P = 0.0014). Transmission electron microscopy revealed blue light-mediated intracellular damage to P. aeruginosa cells. Fluorescence spectroscopy suggested that coproporphyrin III and/or uroporphyrin III are possibly the intracellular photosensitive chromophores associated with the blue light inactivation of P. aeruginosa. In vivo studies using an in vivo bioluminescence imaging technique and an area-under-the-bioluminescence-time-curve (AUBC) analysis showed that a single exposure of blue light at 55.8 J/cm², applied 30 min after bacterial inoculation to the infected mouse burns, reduced the AUBC by approximately 100-fold in comparison with untreated and infected mouse burns (P < 0.0001). Histological analyses and terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) assays indicated no significant damage in the mouse skin exposed to blue light at the effective antimicrobial dose. Survival analyses revealed that blue light increased the survival rate of the infected mice from 18.2% to 100% (P < 0.0001). In conclusion, blue light therapy might offer an effective and safe alternative to conventional antimicrobial therapy for P. aeruginosa burn infections.