In vitro activity of carvacrol,cinnamaldehyde and thymol combined with antifungals against Malassezia pachydermatis

Malassezia pachydermatis is an important opportunistic agent of dermatitis and otitis in dogs. M. pachydermatis is generally treated with topical therapies using combinations of antifungal, antimicrobial and anti-inflammatory agents. We investigated the in vitro activities of carvacrol (CRV), cinnamaldehyde (CIN) and thymol (THY) alone and in combination with antifungal agents (fluconazole, itraconazole, ketoconazole, clotrimazole, miconazole, terbinafine and nystatin) against M. pachydermatis. The assays were performed according to the Clinical and Laboratory Standards Institute (CLSI), using Sabouraud dextrose broth and checkerboard microdilution. The mean fractional inhibitory concentration index (FICI) showed primary synergies for the combinations carvacrol + nystatin, thymol + nystatin, and carvacrol + miconazole (80%). In conclusion, the results obtained indicate that the phytochemicals tested showed relevant in vitro anti-M. pachydermatis activity. Future in vivo experiments are needed to elucidate the safety and therapeutic potential of these combinations.     

In vitro activity of antifungals against Zygomycetes

To date, no reference standard for therapy for zygomycosis has been established because there are insufficient clinical data with which to make such a judgement. Knowledge of the species responsible for the infection and its antifungal susceptibility profile has become increasingly important in the management of patients. Amphotericin B is the most active drug against all the species involved, followed by posaconazole, whereas voriconazole has no activity. Echinocandins are completely inactive in vitro , but may be an interesting option when used in combination with other drugs.     

In vitro antimicrobial effect of cefazolin and cefotaxime combined with minocycline against Vibrio cholerae non-O1 non-O139.

The most common clinical manifestation of Vibrio cholerae non-O1 non-O139 is gastroenteritis. This vibrion may also cause bacteremia, soft tissue infection, and other extraintestinal invasive disease, especially in immunocompromised patients. This study evaluated the current status of antimicrobial resistance in clinical isolates of V. cholerae non-O1 non-O139 in Taiwan as part of the SMART (Surveillance from Multicenter Antimicrobial Resistance in Taiwan) program. Minimal inhibitory concentrations (MICs) of 9 antimicrobial agents were determined by the agar dilution method. All of the isolates were susceptible to minocycline (MIC at which 90% of the isolates were inhibited [MIC(90)], 0.12 microg/mL), cefotaxime (MIC(90), 0.06 microg/mL), lomefloxacin (MIC(90), 0.12 microg/mL), levofloxacin (MIC(90), 0.03 microg/mL), ciprofloxacin (MIC(90), 0.03 microg/mL), moxifloxacin (MIC(90), 0.06 microg/mL), sparfloxacin (MIC(90), 0.06 microg/mL), gatifloxacin (MIC(90), 0.03 microg/mL), and cefazolin (MIC(90), 8 microg/mL). We conducted time-kill studies to evaluate the inhibitory activities of either cefazolin or minocycline alone or in combination against V. cholerae non-O1 non-O139 (Vc2). We also evaluated the inhibitory activity of cefazolin or cefotaxime combined with minocycline. The individual MICs of cefazolin, cefotaxime, and minocycline were 4 microg/mL, 0.0075 microg/mL, and 0.12 microg/mL, respectively, when approximately 5 x 105 colony-forming units/mL of V. cholerae non-O1 non-O139 was incubated. Bacterial growth was inhibited initially but resumed later when cefazolin, cefotaxime, or minocycline was used alone. When cefazoline or cefotaxime was combined with minocycline, V. cholerae non-O1 non-O139 was inhibited over 48 h and no regrowth was noted. We conclude that the combination of cefazolin or cefotaxime with minocycline has a synergistic inhibitory effect on V. cholerae non-O1 non-O139 in vitro.     

In vitro susceptibility of Cryptococcus neoformans isolates from patients with acquired immunodeficiency syndrome

Cryptococcus neoformans strains from 26 individual patients with acquired immunodeficiency syndrome (AIDS) and three isolates from patients without AIDS were tested for their susceptibility to amphotericin B, flucytosine, ketoconazole, and miconazole nitrate. Ninety percent of the C neoformans isolates from patients with AIDS were inhibited by drug concentrations within achievable serum levels. The minimum fungicidal concentration of the four tested antifungal agents, however, exceeded obtainable cerebrospinal fluid levels.     

新型隐球菌与肺泡上皮细胞的体外相互作用

目的研究新型隐球菌与肺泡上皮细胞的体外相互作用,探讨隐球菌肺部感染的发病机制。方法体外培养Ⅱ型肺泡上皮细胞A549(ATCC CCL-185),检测新型隐球菌2种变种对细胞的时间/浓度黏附率、通过率;检测新型隐球菌对细胞的损伤作用;透射电镜观察相互作用的超微结构。结果2种变种的新型隐球菌可以对A549细胞产生黏附与侵袭,黏附率与侵袭率呈现时间依赖性;同时还可以使A549细胞凋亡率升高,对其造成损伤,这与菌体的活力相关。超微结构可见隐球菌与肺泡上皮细胞的黏附与侵袭过程。2种变种之间在黏附率、通过率及对细胞的损伤作用方面差异无统计学意义。结论活的隐球菌黏附与侵袭肺泡上皮细胞是隐球菌感染肺部的重要条件,不同变种对肺部的易感性可能不存在差异。进一步明确二者的作用机制对隐球菌的发病机制研究具有重要意义。     

In vitro expression of vital virulent genes of clinical and environmental isolates of Cryptococcus neoformans/gattii in endothelial cells of human blood-brain barrier

BackgroundEvaluation of the pathogenesis of clinical and environmental cryptococcal isolates to the central nervous system is necessary for understanding the risk. This study was designed to determine the in vitro expression of six important virulent genes of Cryptococcus neoformans/gattii in Human Brain Microvascular Endothelial cells (hBMEC).MethodsThe hBMEC were infected with Cryptococcus to determine invasion and survival rate at 3, 12 and 24 hours by subsequent colony count of internalized yeasts. The whole RNA of the intracellular Cryptococcus was extracted to quantify the expression of CAP10, PLB1, ENA1, URE1, LAC1, and MATα genes by real-time quantitative PCR for 3 and 12 hours of infection.ResultsInvasion and survival rates were higher in clinical and standard strains of C. neoformans. A significant difference was observed among the clinical and environmental isolates for the expression of CAP10, ENA1, LAC1, MATα and URE1 at 3 hours, and ENA1, LAC1, MATα, PLB1 and URE1 at 12 hours. Clinical isolates showed significant upregulation of all the genes except PLB1, which was higher in environmental isolates. Relative expressions at the two time-points showed statistically significant (P = 0.043) changes for the clinical isolates and no significance (P = 0.063) for environmental isolates.ConclusionThe C. gattii (VGI) isolates showed significantly lower invasion and survival than C. neoformans (VNI, and VNII) irrespective of their sources. Clinical isolates exhibited higher expression for the majority of the virulent genes until 12 hours of infection, probably due to their better adaptation in the host system and enhanced pathogenicity than the environmental counterparts.     

Semi-mechanistic PK/PD modelling of combined polymyxin B and minocycline against a polymyxin-resistant strain of Acinetobacter baumannii

ObjectivesTo expand on previous reports of synergy between polymyxin B (PMB) and minocycline (MIN) against Acinetobacter baumannii; and to gain insight into the qualitative and quantitative determinants of their synergy.MethodsA semi-mechanistic pharmacokinetic/pharmacodynamic (PK/PD) model was developed on the basis of data from in vitro time-kill experiments with determination of resistant bacterial count to describe the effects of PMB and MIN alone and in combination. The model was enriched by complementary experiments providing information on the characteristics of the resistant subpopulation.ResultsThe model successfully described the data and made possible quantification of the strength of interaction between the two drugs and formulation of hypotheses about the mechanisms of the observed interaction. The effect of the combination was driven by MIN, with PMB acting as an helper drug; simulations at clinically achievable concentrations showed that 1.5 mg/L MIN +0.2 mg/L PMB is expected to produce sustained killing over 30 hours, while 0.3 mg/L MIN +1 mg/L PMB is met by bacterial regrowth. Interaction equations showed that maximal synergy is reached for PMB concentrations ≥0.1 mg/L and MIN concentrations ≥1 mg/L.ConclusionsSemi-mechanistic PK/PD modelling was used to investigate the quantitative determinants of synergy between PMB and MIN on a PMB-resistant A. baumannii strain. The developed model, improving on usual study techniques, showed asymmetry in the drug interaction, as PMB acted mostly as a helper to MIN, and provided simulations as a tool for future studies.     

Molecular typing and in vitro resistance of Cryptococcus neoformans clinical isolates obtained in Germany between 2011 and 2017

Cryptococcosis is a fungal infection of the central nervous system predominantly caused by Cryptococcus neoformans in immunocompromised patients. In several countries worldwide, up to 50% of isolates show in vitro resistance to clinically used antifungals including fluconazole. No prospective data on susceptibility to antifungal drugs are available for Germany. In this study, we characterised all C. neoformans isolates collected from individual patients’ samples at the German reference laboratory for cryptococcosis 2011 and 2017 (n = 133) by multi-locus sequence typing and phenotypic drug susceptibility testing. We identified serotype A/genotype VNI isolates belonging to clonal complexes previously described from Europe, Africa, Asia and South America as the most prevalent agents of cryptococcosis in Germany. Overall, we observed minimal inhibitory concentrations (MICs) above the epidemiological cut-offs (ECVs) in 1.6% of isolates regarding fluconazole and 2.3% of isolates regarding 5-flucytosine. Here, two C. neoformans var. grubii isolates displayed decreased drug susceptibility to fluconazole, one of them additionally to 5-flucytosine. We also found 5-flucytosine MICs above the ECV for two C. neoformans var. neoformans isolates. We identified a novel mutation in the ERG11 gene which might be associated with the elevated fluconazole MIC in one of the isolates. The clinical importance of the detected in vitro resistance is documented by patient histories showing relapsed infection or primary fatal disease. Of note, sertraline demonstrated antifungal activity comparable to previous reports. Systematic collection of susceptibility data in combination with molecular typing of C. neoformans is important to comprehensively assess the spread of isolates and to understand their drug resistance patterns.     

In vitro activity of ceftriaxone combined with tazobactam against anaerobic bacteria

The in vitro activity of ceftriaxone combined with tazobactam against 190 strains of anaerobic bacteria was compared with that of amoxicillin with clavulanic acid, ampicillin with sulbactam, piperacillin alone and with tazobactam, cefoxitin, and imipenem, i.e. beta-lactam antibiotics established in the treatment of anaerobic infections. All anaerobes tested were susceptible to 32 mg/l ceftriaxone when tazobactam was added at fixed ratios (ceftriaxone to tazobactam) of 2:1 and 8:1 and at constant concentrations of 2,4 and 8 mg/l, respectively. When 4 mg/l tazobactam was added, the MICs of ceftriaxone for 83 of 94 strains of theBacteroides fragilis group were reduced by a factor of 8 to 512; for eight strains, this reduction was two to fourfold. Only the MICs of ceftriaxone for threeBacteroides fragilis strains were not influenced.     

In vitro synergistic effect of colistin and ampicillin/sulbactam with several antibiotics against clinical strains of multi-drug resistant Acinetobacter baumannii

PurposeNowadays, Acinetobacter baumannii is resistant to almost all available antibiotics. The evaluation of synergistic effects between the antibiotics against this pathogen is among the efforts to counteract its antimicrobial resistance. This study aimed to evaluate possible synergistic effect of colistin and ampicillin/sulbactam (separately) with several antibiotics against clinical isolates of multi-drug resistant (MDR) A. baumannii.MethodsAcinetobacter baumannii strains were isolated from biological samples of hospitalized patients with any type of nosocomial infection related to this pathogen. Only MDR strains (resistance to at least three classes of antibiotics including cephalosporins, fluoroquinolones, and aminoglycosides) were included in the study. After determining the minimum inhibitory concentration (MIC) of antibiotics against the isolates by broth microdilution test, the checkerboard method was used for evaluation of any possible synergistic effect of both colistin and ampicillin/sulbactam with several other antibiotics.ResultsTwenty isolates underwent synergy test for colistin and 20 isolates for ampicillin/sulbacatam. Doxycycline (55%), azithromycin (35%), and co-trimoxazole (35%) had the most frequency of synergistic effect with colistin. On the other hand, amikacin and gentamicin (55%), doxycycline (50%), co-trimoxazole (45%), azithromycin (40%), and cefepime (40%) had the most frequency of synergistic effect with ampicillin/sulbactam. No antagonistic effect was observed for both antibiotics.ConclusionColistin and ampicillin/sulbactam have substantial synergistic effect with several antibiotics especially doxycycline, co-trimoxazole, azithromycin, and amikacin (with ampicillin/sulbactam) against MDR strains of Acinetobacter baumannii.     

Effect of granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor on polymorphonuclear neutrophils, monocytes or monocyte-derived macrophages combined with voriconazole against Cryptococcus neoformans.

The antifungal activity of voriconazole (VCZ) was tested against Cryptococcus neoformans (Cn) with and without the addition of polymorphonuclear neutrophils (PMN), monocytes or monocyte-derived macrophages (MDM) in vitro. Human effector cells with and without the addition of VCZ were incubated with Cn for 24 h. PMN, mono and MDM alone resulted in 61%, 34% and 23% inhibition of Cn, respectively (n = 3, P<0.01). VCZ at 0.01 and 0.05 microg ml(-1) alone resulted in 48% inhibition and 19% killing (n = 6). The addition of VCZ at 0.01 and 0.05 microg ml(-1) to human effector cells enhanced killing of Cn by 51% and 71% for the PMN, 41% and 58% for the mono, and 14% and 34% for the MDM, respectively. The addition of either granulocyte colony-stimulating factor (G-CSF) or granulocyte-macrophage colony stimulating factor (GM-CSF) significantly enhanced the ability of human effector cells to kill Cn. G-CSF and GM-CSF plus PMN resulted in 47% and 46% killing, respectively; GM-CSF plus monocytes or MDM resulted in 31% or 22% killing, respectively. G-CSF and GM-CSF further enhanced the collaborative killing effect of human effector cells and VCZ. At 0.01 and 0.05 microg ml(-1) of VCZ, G-CSF or GM-CSF enhanced PMN killing to 92% and 93% or 87% and 94%, respectively. GM-CSF enhanced both mono and MDM with VCZ at 0.01 and 0.05 microg ml(-1) in killing Cn to 62% and 86%, and 61% and 84%, respectively. These results suggest that VCZ would have good efficacy in the treatment of Cn infection in humans. Furthermore, VCZ would have enhanced efficacy in clinical settings where either G-CSF or GM-CSF was being used.     

美满霉素联合栀子苷对大鼠局灶性脑缺血再灌注损伤的保护作用

目的:观察美满霉素联合栀子苷对大鼠缺血性脑损伤神经元凋亡的拮抗作用和对脑组织的保护作用。方法:SD大鼠随机分为假手术组、脑缺血再灌注组、美满霉素组、栀子苷组和美满霉素联合栀子苷组。用线栓法建立大鼠右侧局灶性脑缺血再灌注模型,在体分别观察美满霉素和栀子苷单一和两者联合干预缺血性脑损伤。TTC染色观察脑梗死面积,H-E染色观察海马神经元形态学的变化,TUNEL染色观察凋亡情况。结果:美满霉素和栀子苷均能有效促进脑损伤后海马组织的恢复并能拮抗神经元的凋亡,与脑缺血再灌注组相比差异具有统计学意义;两者联合应用较单一使用具有较好的效果。结论:美满霉素联合栀子苷对缺血性脑组织具有保护作用并能有效拮抗神经元的凋亡。     

In vitro activity of antimicrobial agents against oxacillin resistant staphylococci with special reference to Staphylococcus haemolyticus

One hundred and sixty seven isolates of staphylococci isolated from the inpatients of a tertiary care referral hospital in South India were speciated and activity of oxacillin, glycopeptides, linezolid and quinupristin/dalfopristin against these isolates was tested by broth microdilution method. Of the 114 coagulase negative staphylococci (CoNS), 49.1 % were S. haemolyticus, isolated predominantly from urine (64.6%), while the rest belonged to 11 other species. More than half the isolates of S. aureus (52.8%) and 68.4% of the CoNS were oxacillin resistant. All the strains were uniformly susceptible to vancomycin, linezolid and quinupristin/dalfopristin; but 25.6% isolates of S. haemolyticus showed reduced susceptibility to teicoplanin (MIC: 8-16 mg/L). Our study demonstrates the high prevalence of oxacillin resistance among hospital isolates of S. aureus and CoNS in India. Vancomycin, along with the newer agents like linezolid and quinupristin/dalfopristin remains the drug of choice for treating multi drug resistant staphylococcal infections.     

DC-CIK细胞协同索拉菲尼对肝癌细胞的杀伤作用

目的:观察DC-CIK共培养细胞(DC-CIK)联合索拉菲尼( sorafenib)对肝癌细胞BEL-7402的体外杀伤效应.方法:取健康人外周血单个核细胞,加入不同细胞因子促进DC及CIK细胞成熟后混合共培养.MTT法检测用药后BEL-7402细胞增殖的情况CCK8试剂盒检测DC-CIK共培养细胞联合索拉菲尼对BEL...