Minimum Inhibitory Concentration For Strains Research Articles

New insights: mupirocin resistance among methicillin-resistant Staphylococcus aureus (MRSA) infection in southern Indian region

: Methicillin-resistant (MRSA) remains to be the most concerned pathogen with enhanced multi-drug resistance in clinical set-ups. With an immediate need to control MRSA infection, there is a steep rise in the use of mupirocin as a decolonization regime.: A total of 65 MRSA samples were analysed in the present study. infection identified by standard conventional phenotypic methods. The antibiotic susceptibility pattern of mupirocin (5 μg/ml) was found using Kirby Bauer’s disc diffusion method and the minimum inhibitory concentration (MIC) determined using MIC E-test strips of mupirocin.: All the bacterial strains presented with MIC value less than 4 μg/ml, 23 isolates falls in the range of 0.05 μg/ml and 0.19 μg/ml, 12 strains MIC ranged between 0.125 and 0.640 μg/ml. However, five MRSA isolates showed no zone against mupirocin E-strip.: In present study, 92% of isolates were sensitive to mupirocin, this gives hope that mupirocin can be promising in the elimination of MRSA strains, a major concern in clinical practice. Also, the use of 5µg mupirocin discs is economical as compared to mupirocin E-strips, mupirocin discs can be used in a low resourceful clinical setups. However, our study revealed no difference in sensitivity for both the E-strip and disc method.

Combating bacterial resistance to Meropenem by infusion strategy applied to septic burn patients with vasopressor requirements or acute kidney injury to achieve the target

Introduction: Faced with the growing challenge to the use of antimicrobials for the adequate and effective therapy of nosocomial infections, international health agencies have reinforced that combating bacterial resistance and preventing the development of multidrug-resistant (MDR) strains are urgent, since a significant increase based on minimum inhibitory concentration (MIC) for therapeutic agents were reported by the committee of hospitals infection. Meropenem, a carbapenem agent, is widely prescribed for therapy of septic shock caused by susceptible Gram-negative bacteria. In general, the prolonged 3-hrs-infusion has been widely applied in these patients over the past 10 years providing coverage only against susceptible Gram-negative pathogens (MIC 2 mg/L), extended also to intermediate susceptible strains up to MIC 4 mg/L, according to Clinical Laboratory Standard Institute (CLSI database). However, new strategies have been recommended to combat the development of resistance to pathogens isolated from cultures to increase the coverage of this carbapenem agent up to MIC 8 mg/L, to avoid mutant selection with death in ICU. Subject: Clinical protocol was carried out to investigate the efficacy & safety of meropenem at the dose regimen recommended 1g q8h by prolonged infusion, based on serum levels and on cultures monitoring of isolates. Aim of protocol was to assess pharmacodynamics (PD) based on changes of pharmacokinetics (PK), which could affect the coverage of meropenem in septic burns patients with increased or decreased renal function. Pharmacokinetic-pharmacodynamics (PK/PD) tools were applied to investigate efficacy & safety. Methods-clinical protocol: Forty-eight major septic burn patients with high variability on renal function in ICU were included. Cultures were collected before meropenem therapy starts; all of them had nosocomial infection caused by Gram-negative pathogens isolated. Patients undergoing meropenem therapy at the initial stage of septic shock from day-0 to day-8 (D0-D8) and at the late stage of septic shock from day 8 to day 14 (D-8 to D-14) were investigated according to dose requirements based on creatinine clearance, drug serum levels (TDM), and coverage up to MIC 8 mg/L, dose dependent on renal function. Results: Coverage occurred for all patients of both groups after the extended infusion against susceptible Gram-negative strains up to MIC 2 mg/L (minimum inhibitory concentration), and up to MIC 4 mg/L, strains of intermediate susceptibility, according to Clinical Laboratory Standards Institute (CLSI, database) of our hospital. It was demonstrated in patients with renal function augmented by vasopressors, the superiority on coverage by trice that occurred in 24/27 patients (89%) after 4 hrs.-infusion at TDM3 against strains MIC 8 mg/L by comparison with coverage registered in 12/39 patients (30%) after 3 hrs.-infusion at TDM2. On the other hand, meropenem dose regimen must be adjusted to 1g q24h in patients with AKI to guarantee effectiveness & safety in those patients. In addition, after continuous venovenous haemodialysis-filtration (CVVHDF) installed in those patients, meropenem PK/PD target was attained up to MIC 8 mg/L in patients with the empirical dose regimen recommended of 1g q8h, 3hrs.-infusion. Conclusion: Precision medicine guarantees meropenem serum levels combined with cultures monitoring; consequently, must be applied routinely to guarantee coverage against Gram-negative nosocomial pathogens susceptible including strains of intermediate susceptibility (MIC 4-8 mg/L) to avoid mutant selection. Therefore, effective, and safe antimicrobial therapy for patients in septic shock, combined with a continuous monitoring of inflammatory biomarkers, should guide clinical management to ensure cure with early ICU discharge.

Investigation of antifungal activities of myrcene on Fusarium reference strains.

Antifungal effects of myrcene, the plant-based naturel compound, were investigated on Fusarium graminearum PH-1 and Fusarium culmorum FcUK99 references, for the first time. Minimum inhibitory concentration (MIC) and half of MIC (MIC50) of both Fusarium strains against myrcene were found as 25µg/µl and 12.5µg/µl, respectively. MIC50 application decreased the cell viabilities in the ratios of 34.90% and 33.91% in PH-1 and FcUK99, respectively (p < 0.01). The significantly increased catalase (CAT) activity was recorded in MIC50 treated strains (p < 0.01). Apoptosis-like process and cellular oxidative stress were also monitored with acridine orange/ethidium bromide (Ao/Eb) dual staining and 2',7'-dichlorodihydrofluorescein diacetate (DCF-DA) staining. The genomic template stability (GTS) percentages were calculated as 79% for PH-1 and 71% for FcUK99 via random amplified polymorphic DNA (RAPD). Methylation polymorphism values were calculated as 53.8% and 50.6% in PH-1 and 40.4% and 39% in FcUK99 for HapII and MspI, respectively by coupled restriction enzyme digestion-random amplification (CRED-RA). Methylation-sensitive amplified polymorphism (MSAP) revealed that myrcene caused both type I and type III epigenetic modifications in both genomes. MIC50 dose caused up to 13.86 ± 0.42-fold changes in the expressions of cat, mst20, and stuA, whereas downregulation in tri5 was recorded. Myrcene application did not change the retrotransposon movement in both species by the amplifying of idiomorphic retrotransposon patterns through inter-retrotransposon polymorphism-polymerase chain reaction (IRAP-PCR). This study demonstrated that myrcene is an effective compound in the management of phytopathogenic Fusarium species by causing morphological, genetic, epigenetic, and cellular alterations, and has a potential to utilize as an antifungal agent.

145. Harnessing the Inoculum Effect to Diagnose Molecular Mechanisms of Carbapenem Resistance

Abstract Background The global spread of carbapenem-resistant Enterobacterales (CRE) presents a major threat to public health. CRE employ two molecular mechanisms to evade carbapenems: 1) expression carbapenemases (CPases), which efficiently hydrolyze carbapenems or 2) disruption of porins, which reduces carbapenem influx to levels that can be hydrolyzed by certain beta-lactamases. Diagnosing mechanisms underlying carbapenem resistance is important for infection control and to administer appropriate treatments. Methods We measured Meropenem and Ertapenem minimum inhibitory concentrations (MICs) for 103 clinical Enterobacterales isolated from hospitals in Massachusetts and California using broth microdilution assays at 14 inocula spanning four orders of magnitude. They represented 30 isolates encoding CPases and intact porins, 46 isolates with disruptions in one or both of the porins responsible for carbapenem influx (OmpC and OmpF), 25 encoding CPases with disrupted porins, and two controls encoding intact porins and no CPases. Results We observed that the two mechanisms result in distinct profiles; first the carbapenem MICs of CPase-encoding strains show a strong inoculum dependence (Fig 1.A), whereas the MICs of porin deficient strains remain largely constant at all inocula (Fig 1.B). The synergistic action of these two mechanisms leads to high-level resistance that we termed “hyper-CRE” (Fig 1.C). Together these factors explain the level of resistance in nearly all our CRE isolates. To validate the hyper-CRE phenotype, we successfully employed CRISPR-based gene editing to show that knocking out the major porin in CPase-producing strains elevates their carbapenem resistance to hyper-CRE levels. We also determined 18% of our isolates changed susceptibility classification within the Clinical Laboratory and Standards Institute (CLSI) recommended inoculum range. This is worrisome for the treatment of infections with strains that are deemed susceptible via in vitro AST assays but are truly resistant in vivo. Conclusion Overall, our approach has demonstrated that measuring MICs at different inoculum can yield crucial diagnostic information about mechanisms of resistance which has important implications for patient care, infection control, and surveillance of emerging CPases. Disclosures All Authors: No reported disclosures.

Efflux Inhibition in Clinical Isolates of Pseudomonas Aeruginosa Using Sertraline for Ciprofloxacin Resistance Breakdown

Background and Aim The efflux pump in Pseudomonas aeruginosa inhibits the effect of ciprofloxacin by releasing quinolones out of the cell. It is important to find compounds to inactivate or inhibit its activity to continue using the antibiotics. The present study was done to investigate using sertraline as an efflux pump inhibitor in P. aeruginosa to reduce antibiotic resistance. Methods &amp; Materials P. aeruginosa strains were isolated from clinical sources and identified by routine microbiological methods. Resistance of the isolates to ciprofloxacin was evaluated by Kirby–Bauer test. Resistance breakdown was investigated by adding sertraline to the Moller Hinton agar medium and determining the zone of inhibition of ciprofloxacin. Minimum inhibitory concentration (MIC) by microplate dilution method and Minimum bactericidal concentration (MBC) by culture and MTT method were done for the isolates and ATCC 27853. The presence of the efflux pump was evaluated by the phenotypic method using sertraline and serial dilution method of the liquid medium in a microplate, on ciprofloxacin-resistant strains. The presence of the producing gene of this pump was determined by the genotyping method in resistant strains by performing PCR. The standard PAO1 strain of P. aeruginosa was used as a positive control. Ethical Considerations This study was approved by the Ethics Committee of the Faculty of Medical Sciences of Islamic Azad University, Brojerd Branch (Code: IR.IAU.B.REC.1401.011). Results Based on Kirby–Bauer test results, three strains were considered resistant to ciprofloxacin. MIC of drug-resistant strains was between 32 and 64 mg/ml and MBC was between 16 and 32 mg/ml. By performing electrophoresis on the PCR products, it was determined that the tested strains contained the mexA gene encoding the efflux pump. In the agar medium without sertraline, the zone of inhibition around the ciprofloxacin disc was zero, but after adding sertraline, the diameter of the halo increased to 25 mm. The minimum inhibitory concentration of ciprofloxacin in the isolates before adding 25 µg of sertraline was 128 µg/ml and after adding sertraline, it was 4 µg/ml. Conclusion It was concluded that sertraline inhibited the efficiency of the efflux pump in resistant P. aeruginosa isolates and reduced ciprofloxacin resistance.

Comparison of the Anti-bacterial Efficacy of Saussurea costus and Melaleuca alternifolia Against Porphyromonas gingivalis, Streptococcus mutans, and Enterococcus faecalis: An in-vitro Study.

The aim was to compare the in-vitro antibacterial effectiveness of two herbal extracts (a) Saussurea-costus (S. costus) and (b) Melaleuca-alternifolia (M. alternifolia) against Porphyromonas gingivalis (P. gingivalis), Streptococcus mutans (S. mutans) and Enterococcus faecalis (E. faecalis). Aqueous extracts from M. alternifolia were prepared by adding 2 grams of S. costus and M. alternifolia, respectively to 100 ml distilled water. Bacterial strains of P. gingivalis, E. faecalis and S. mutans were treated into 3 groups. In groups 1 and 2, bacterial strains were treated with aqueous extracts of S. costus and M. alternifolia, respectively. In the control-group, bacterial strains were exposed to distilled water. Antibacterial activity of the samples and nanoparticles was determined. The minimum-inhibitory-concentration (MIC) values were determined using the microdilution method. P < 0.01 was considered statistically significant. The MIC for all bacterial strains treated with S. costus was significantly higher than that of M. alternifolia (P < 0.001). There was no significant difference in MIC for strains of P. gingivalis, E. faecalis and S. mutans treated with S. costus. For bacterial strains treated with M. alternifolia, the MIC was significantly higher for P. gingivalis compared with E. faecalis and S. mutans strains (P < 0.01). There was no difference in MIC for E. faecalis and S. mutans strains treated with M. alternifolia. The in-vitro antibacterial efficacy of M. alternifolia is higher than S. costus against P. gingivalis, E. faecalis and S. mutans.

Effects of four efflux pump inhibitors on the activities of clarithromycin against Mycobacterium abscessus

Objective: To detect the effects of four efflux pump inhibitors on the minimum inhibitory concentration of clarithromycin (CLA) against Mycobacterium abscessus (M. abscessus) in vitro, and to explore the role of efflux pump in CLA resistance of M. abscessus. Methods: Four frequently-used efflux pump inhibitors (Carbonyl Cyanide 3-chlorophenylhydrazone, CCCP, N, N'-dicyclohexylcarbodiimide, DCC, Verapamil, VP, Reserpine, RSP) were evaluated in this study. The minimum inhibitory concentration (MIC) values of clarithromycin against M. abscessus reference strain and 60 clinical strains with or without efflux pump inhibitors were detected by Alamar Blue method. Sequence analysis of erm(41) and rrl genes known to be associated with CLA resistance in M. abscessus was performed to analyze the correlation between the effect of efflux pump inhibitors on MIC and mutation of resistance-related genes. Results: CCCP, DCC, VP and RSP could reduce the MIC of M. abscessus to CLA, and the effect of RSP was weaker than the other three efflux pump inhibitors. Among the sixty M. abscessus clinical strains, ten strains were resistant to clarithromycin, seven of which had rrl gene mutation. The CLA resistance rate of smooth phenotype isolates was higher than that of rough phenotype isolates. At 3 day of clarithromycin incubation, the MICs of resistant strains were all reduced by efflux pump inhibitors. Compared with the strains with rrl gene mutation, efflux pump inhibitors had a greater effect on the strains without rrl gene mutation. At 14 day of clarithromycin incubation, 83% of M. abscessus subsp. abscessus, were induced to be resistant, and all of them were T28 sequence type of erm(41). With the occurrence of induced drug resistance, the effect of efflux pump inhibitor on CLA MIC decreased. Efflux pump inhibitors had no statistically significant diffence in the effect of effcux pump inhibitors on CLA MIC levels in different phenotypes of isolates. Conclusions: Efflux pump is involved in the resistance process of M. abscessus to CLA. Efflux pump inhibitors reduce the drug resistance to clarithromycin against M. abscessus in different degrees. The use of efflux pump inhibitors may provide a new way to alleviate the drug resistance of M. abscessus.

Stochastic establishment of β-lactam-resistant Escherichia coli mutants reveals conditions for collective resistance.

For antibiotic resistance to arise, new resistant mutants must establish in a bacterial population before they can spread via natural selection. Comprehending the stochastic factors that influence mutant establishment is crucial for a quantitative understanding of antibiotic resistance emergence. Here, we quantify the single-cell establishment probability of four Escherichia coli strains expressing β-lactamase alleles with different activity against the antibiotic cefotaxime, as a function of antibiotic concentration in both unstructured (liquid) and structured (agar) environments. We show that concentrations well below the minimum inhibitory concentration (MIC) can substantially hamper establishment, particularly for highly resistant mutants. While the pattern of establishment suppression is comparable in both tested environments, we find greater variability in establishment probability on agar. Using a simple branching model, we investigate possible sources of this stochasticity, including environment-dependent lineage variability, but cannot reject other possible causes. Lastly, we use the single-cell establishment probability to predict each strain's MIC in the absence of social interactions. We observe substantially higher measured than predicted MIC values, particularly for highly resistant strains, which indicates cooperative effects among resistant cells at large cell numbers, such as in standard MIC assays.

Cytotoxicity, early safety screening, and antimicrobial potential of minor oxime constituents of essential oils and aromatic extracts

Due to market and legislative expectations, there is a constant need to explore new potential antimicrobial agents for functional perfumery. In this study, we evaluated the antimicrobial activity of 53 low molecular oximes and the corresponding carbonyl compounds against Escherichia coli, Enterococcus hirae, Pseudomonas aeruginosa, Bacillus cereus, Staphylococcus aureus, Aspergillus brasiliensis, Legionella pneumophila and Candida albicans. The most potent compound was α-isomethylionone oxime, which exhibited a minimum inhibitory concentration (MIC) of 18.75 µg/mL against E. hirae. The evaluation of the MICs for bacterial and fungal strains was performed for selected compounds, for example, the MIC of 2-phenylpropionaldehyde, cis-jasmone oxime, and trans-cinnamaldehyde measured against A. brasiliensis was 37.50 µg/mL. ADME-Tox (Absorption, Distribution, Metabolism, Excretion, and Toxicity) and 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) cell viability assays were performed to assess the cytotoxicity of tested compounds. ADME-Tox indicated the safety and promising properties of selected compounds, which enables their usage as nontoxic supporting antibacterial agents. The results of the in vitro MTS assay were consistent with the ADME-Tox results. None of the compounds tested was toxic to Human Embryonic Kidney 293T (HEK293T) cells, with all cell viabilities exceeding 85%.

Origanum vulgare L. essential oil inhibits virulence patterns of Candida spp. and potentiates the effects of fluconazole and nystatin in vitro

BackgroundRecurrence and resistance of Candida spp. infections is associated with the ability of these microorganisms to present several virulence patterns such as morphogenesis, adhesion, and biofilm formation. In the search for agents with antivirulence activity, essential oils could represent a strategy to act against biofilms and to potentiate antifungal drugs.ObjectiveTo evaluate the antivirulence effect of Origanum vulgare L. essential oil (O-EO) against Candida spp. and to potentiate the effect of fluconazole and nystatin.MethodsThe effect of O-EO was evaluated on ATCC reference strains of C. albicans and non-albicans Candida species. Minimum inhibitory concentration (MIC) was determined through broth microdilution assay. Adhesion to microplates was determined by crystal violet (CV) assay. An adapted scratch assay in 24-well was used to determine the effect of essential oil on biofilms proliferation. Viability of biofilms was evaluated by MTT reduction assay and through a checkerboard assay we determined if O-EO could act synergistically with fluconazole and nystatin.ResultsMIC for C. albicans ATCC-90029 and ATCC-10231 was 0.01 mg/L and 0.97 mg/L, respectively. For non-albicans Candida strains MIC values were 2.6 mg/L for C. dubliniensis ATCC-CD36 and 5.3 mg/L for C. krusei ATCC-6258. By using these concentrations, O-EO inhibited morphogenesis, adhesion, and proliferation at least by 50% for the strains assayed. In formed biofilms O-EO decreased viability in ATCC 90029 and ATCC 10231 strains (IC50 7.4 and 2.8 mg/L respectively). Finally, we show that O-EO interacted synergistically with fluconazole and nystatin.ConclusionsThis study demonstrate that O-EO could be considered to improve the antifungal treatment against Candida spp.

Genotypic Resistance of Pyrazinamide but Not Minimum Inhibitory Concentration Is Associated With Longer Time to Sputum Culture Conversion in Patients With Multidrug-resistant Tuberculosis.

Pyrazinamide (PZA) resistance in multidrug-resistant tuberculosis (MDR-TB) is common; yet, it is not clear how it affects interim and treatment outcomes. Although rarely performed, phenotypic drug susceptibility testing (pDST) is used to define PZA resistance, but genotypic DST (gDST) and minimum inhibitory concentration (MIC) could be beneficial. We aimed to assess the impact of PZA gDST and MIC on time to sputum culture conversion (SCC) and treatment outcome in patients with MDR-TB. Clinical, microbiological, and treatment data were collected in this cohort study for all patients diagnosed with MDR-TB in Sweden from 1992-2014. MIC, pDST, and whole-genome sequencing of the pncA, rpsA, and panD genes were used to define PZA resistance. A Cox regression model was used for statistical analyses. Of 157 patients with MDR-TB, 56.1% (n = 88) had PZA-resistant strains and 49.7% (n = 78) were treated with PZA. In crude and adjusted analysis (hazard ratio [HR], 0.49; 95% conficence interval [CI], .29-.82; P = .007), PZA gDST resistance was associated with a 29-day longer time to SCC. A 2-fold decrease in dilutions of PZA MIC for PZA-susceptible strains showed no association with SCC in crude or adjusted analyses (HR, 0.98; 95% CI, .73-1.31; P = .89). MIC and gDST for PZA were not associated with treatment outcome. In patients with MDR-TB, gDST PZA resistance was associated with a longer time to SCC. Rapid PZA gDST is important to identify patients who may benefit from PZA treatment.

Stochastic bacterial population dynamics restrict the establishment of antibiotic resistance from single cells

A better understanding of how antibiotic exposure impacts the evolution of resistance in bacterial populations is crucial for designing more sustainable treatment strategies. The conventional approach to this question is to measure the range of concentrations over which resistant strain(s) are selectively favored over a sensitive strain. Here, we instead investigate how antibiotic concentration impacts the initial establishment of resistance from single cells, mimicking the clonal expansion of a resistant lineage following mutation or horizontal gene transfer. Using two Pseudomonas aeruginosa strains carrying resistance plasmids, we show that single resistant cells have <5% probability of detectable outgrowth at antibiotic concentrations as low as one-eighth of the resistant strain's minimum inhibitory concentration (MIC). This low probability of establishment is due to detrimental effects of antibiotics on resistant cells, coupled with the inherently stochastic nature of cell division and death on the single-cell level, which leads to loss of many nascent resistant lineages. Our findings suggest that moderate doses of antibiotics, well below the MIC of resistant strains, may effectively restrict de novo emergence of resistance even though they cannot clear already-large resistant populations.

Lung Collapse during Mini-Thoracotomy Reduces Penetration of Cefuroxime to the Tissue: Interstitial Microdialysis Study in Animal Models.

Background: Single-lung ventilation facilitates surgical exposure during minimally invasive cardiac surgery. However, a deeper knowledge of antibiotic distribution within a collapsed lung is necessary for effective antibiotic prophylaxis of pneumonia. Patients and Methods: The pharmacokinetics/pharmacodynamics (PK/PD) of cefuroxime were compared between the plasma and interstitial fluid (ISF) of collapsed and ventilated lungs in 10 anesthetized pigs, which were ventilated through a double-lumen endotracheal cannula. Cefuroxime (20 mg/kg) was administered in single 30-minute intravenous infusion. Samples of blood and lung microdialysate were collected until six hours post-dose. Ultrafiltration, in vivo retrodialysis, and high-performance liquid chromatography-tandem mass spectrometry were used to determine plasma and ISF concentrations of free drug. The concentrations were examined with non-compartmental analysis and compartmental modeling. Results: The concentration of free cefuroxime in ISF was lower in the non-ventilated lung than the ventilated one, evidenced by a lung penetration factor of 47% versus 63% (p < 0.05), the ratio between maximum concentrations (65%, p < 0.05), and the ratio between the areas under the concentration-time curve (78%, p = 0.12). The time needed to reach a minimum inhibitory concentration (MIC) was 30%-40% longer for a collapsed lung than for a ventilated one. In addition, a delay of 10-40 minutes was observed for lung ISF compared with plasma. The mean residence time values (ISF collapsed lung > ISF ventilated lung > plasma) could explain the absence of practically important differences in the time interval with the concentration of cefuroxime exceeding the MICs of sensitive strains (≤4 mg/L). Conclusion: The concentration of cefuroxime in the ISF of a collapsed porcine lung is lower than in a ventilated one; furthermore, its equilibration with plasma is delayed. Administration of the first cefuroxime dose earlier or at a higher rate may be warranted, as well as dose intensification of the perioperative prophylaxis of pneumonia caused by pathogens with higher MICs.

Inhibitory effects of aptamer targeted teicoplanin encapsulated PLGA nanoparticles for Staphylococcus aureus strains.

Emergence of resistance to traditional antibiotic treatments necessitates alternative delivery systems. Teicoplanin is a glycopeptide antibiotic used in the treatments of serious infections caused by Gram-positive bacteria, including Methicillin Resistant Staphylococcus aureus (MRSA). One strategy to keep up with antibiotic resistance development is to limit dose and amount during treatments. Targeted delivery systems of antibiotics have been suggested as a mechanism to slow-down the evolution of resistance and to increase efficiency of the antimicrobials on already resistant pathogens. In this study, we report teicoplanin delivery nanoparticles of Poly Lactic-co-Glycolic Acid (PLGA), which are functionalized with S. aureus specific aptamers. A 32-fold decrease in minimum inhibitory concentration (MIC) values of teicoplanin for S. aureus was demonstrated for susceptible strains and about 64-fold decline in MIC value was achieved for moderately resistant clinical isolates of MRSA upon teicoplanin treatment with aptamer-PLGA nanoparticles. Although teicoplanin delivery in PLGA nanoparticles without targeting demonstrated eightfold decrease in MIC of susceptible strains of S. aureus and S. epidermidis and twofold in MIC of resistant strains, the aptamer targeting specifically decreased MIC for S. aureus, but not for S. epidermidis. Therefore, aptamer-targeted PLGA delivery of antibiotic can be an attractive alternative to combat with some of the multi-drug resistant bacterial pathogens.

Przeciwgrzybicza aktywność olejku melisowego (Oleum Melissae)

Introduction. A lot of plant produced essential oils, which are applied in prophylaxis and therapy of medicine. Melissa was known and used in I century BC. It produced the oil, which possess following properties: antiarteriosclerotic, anticancer, sedative, antidepression, antimigraine, antiasthmatic, antirheumatic and antioxidant. Its contain: geraniol, β-caryophyllene, geranial, thymol, neral, geranyl acetate, linalol, cytronellol, citronellal and α-humulen. It exhibited antibacterial and antifungal activity. Aim. The aim of this study was to indicate of susceptibility of yeastlike fungi to melissae essential oil. Material and methods. The strains of fungi were isolated from oral cavity from patients with candidosis. A total 23 strains of yeastlike fungi from genus of Candida albicans (22 strains), C. glabrata (5), C. guilliermondii (2), C. humicola (2), C. kefyr (3), C. krusei (5), C. lusitaniae (2), C. parapsilosis (5), C. tropicalis (6), C. utilis (1) and 9 reference strains were tested. Investigated was carried out using plate dilution technique in Sabouraud’s agar. The melissae oil (Semifarm) was dissolved in DMSO and then in aseptic distilled water. Inoculum contain 105 CFU per spot was transferred with Steers replicator upon the agar with and without essential oil (strains growth control). The concentrations of melissae oil were: 2.0, 1.0, 0.5, 0.25 and 0.12 mg/ml. Incubation was performed in aerobic conditions in temp. 37°C. Incubation of agar plates were performed in aerobic condition at temp. 37°C, at 24-48 hours. Minimum inhibitory concentration (MIC) was interpreted as the lowest concentrations of melissa oil which inhibited the growth of yeastlike fungi. Results. The dates indicated that the strains of fungi was susceptible to oil in concentrations 0.25-2.0 mg/ml. The 19 (86%) of strains from genus Candida albicans was inhibited in concentrations 0.25-0.5 mg/ml. On the same values of MIC’s were susceptible the strains of Candida albicans, Candida glabrata and Candida humicola (MIC 0.5 mg/ml). The fungi from genus of Candida kefyr, Candida krusei, Candida lusitaniae and Candida tropicalis were less sensitive. The growth of this yeastlike fungi was inhibited by concentrations of melissae oil in range 0.5-2.0 mg/ml. The oil was the lowest active towards genus Candida lusitaniae and Candida utilis. The MIC for these strains was from 1.0 to 2.0 mg/ml. From all tested genus Candida strains 11 (21%) of them was susceptible to melissa oil in range 2.0 mg/ml. Conclusions. Melissa oil was the most active towards strains of Candida albicans, Candida glabrata and Candida humicola. The lowest sensitive to oil were the strains from genus Candida lusitaniae and Candida utilis. The melissa oil characterized a high activity towards all tested strains of yeastlike fungi from genus of Candida.

Anti-Biofilm Activity of Grapefruit Seed Extract against Staphylococcus aureus and Escherichia coli.

Grapefruit seed extract (GSE) is a safe and effective preservative that is used widely in the food industry. However, there are few studies addressing the anti-biofilm effect of GSE. In this study, the anti-biofilm effect of GSE was investigated against biofilm-forming strains of Staphylococcus aureus and Escherichia coli. The GSE minimum inhibitory concentration (MIC) for S. aureus and E. coli were 25 μg/ml and 250 μg/ml, respectively. To investigate biofilm inhibition and degradation effect, crystal violet assay and stainless steel were used. Biofilm formation rates of four strains (S. aureus 7, S. aureus 8, E. coli ATCC 25922, and E. coli O157:H4 FRIK 125) were 55.8%, 70.2%, 55.4%, and 20.6% at 1/2 × MIC of GSE, respectively. The degradation effect of GSE on biofilms attached to stainless steel coupons was observed (≥ 1 log CFU/coupon) after exposure to concentrations above the MIC for all strains and 1/2 × MIC for S. aureus 7. In addition, the specific mechanisms of this anti-biofilm effect were investigated by evaluating hydrophobicity, auto-aggregation, exopolysaccharide (EPS) production rate, and motility. Significant changes in EPS production rate and motility were observed in both S. aureus and E. coli in the presence of GSE, while changes in hydrophobicity were observed only in E. coli. No relationship was seen between auto-aggregation and biofilm formation. Therefore, our results suggest that GSE might be used as an anti-biofilm agent that is effective against S. aureus and E. coli.

Antioxidant activity and evidence for synergism of Cannabis sativa (L.) essential oil with antimicrobial standards

The present work was conducted to study the antioxidant activity and to determine for the first time the antimicrobial potential of Moroccan Cannabis sativa essential oil (EO) singly or in combination with two conventional antibiotics (fluconazol and ciprofloxacin) against some resistant and pathogenic microorganisms. The chemical composition analyzed by means of GC/MS showed that C. sativa EO was characterized by the dominance of (E)-caryophyllene (35.0%), α-humulene (12.8%) and caryophyllene oxide (10.6%). Results from antioxidant tests showed that C. sativa EO exhibit a moderate potency (IC50 = 1.6 ± 0.1 mg/m L for 2,2- diphenyl-1-picrylhydrazyl (DPPH) assay, IC50 = 1.8 ± 0.2 mg/mL for β-carotene/linoleic acid assay, and IC50 = 0.9 ± 0.1 mg/m L for ferric reducing power assay). Regarding antimicrobial assays, the results demonstrated that C. sativa EO displayed interesting antimicrobial activities with minimum inhibitory concentration (MIC) values in the range from (1.2 to 37.8) mg/m L for tested microorganisms. The combination of the studied EO with fluconazol and ciprofloxacin showed a significant decrease in their individual MICs. In fact, at sub-inhibitory concentrations, C. sativa EO contributed to the decrease of ciprofloxacin MICs of bacterial strains by 2- to 64-fold and by16-fold regarding fluconazol MICs against Candida spp. These findings suggest that C. sativa EO can be considered as a potential source of natural antioxidant and antimicrobials, and constitute in combinational treatments a promising strategy to overcome the intense use of antibiotics against some infectious diseases.

In vitro effect of clindamycin against Bacteroides and Parabacteroides isolates in Poland.

The aims of this study were (i) to analyse strains of the genera Bacteroides and Parabacteroides isolated from clinical specimens for phenotypic resistance to clindamycin, (ii) to detect erm genes in the isolates and (iii) to determine any correlation between in vitro resistance and the presence of erm genes. The Bacteroides and Parabacteroides isolates analysed were obtained from patients hospitalised at teaching hospitals in Poland. Antimicrobial susceptibility testing was performed by Etest and the results were interpreted according to European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines. All isolates were analysed by PCR for the presence of the resistance genes ermF, ermB and ermG. Resistance to clindamycin was detected in 31.0% (62/200) of all evaluated isolates, with the ermF and ermB genes detected in 31.0% (62/200) and 0.5% (1/200) of isolates, respectively. No isolates with ermG were detected among the evaluated strains. Pearson's test showed an almost perfect correlation between clindamycin minimum inhibitory concentrations (MICs) and the presence of ermF in Bacteroides spp. and Parabacteroides distasonis isolates, although the ermF gene was also present in 10 clindamycin-susceptible isolates of Bacteroides spp. This study demonstrated a substantial proportion of Bacteroides (22.5-100% depending on the species) and 50.0% of Parabacteroides strains exhibiting resistance to clindamycin. The clindamycin MIC for resistant strains in each case was ≥256mg/L. Resistance to clindamycin in Bacteroides and Parabacteroides species is correlated mainly with the presence of the ermF gene.

In Vitro Activity of Two Nanoparticles on Clinical Isolates of Candida parapsilosis, Showing Resistance Against Antifungal Agents in Children

Background: Resistance of pathogenic fungi to conventionally available antifungal agents has been increasing and has become a serious problem. On the other hand, nanoscience has emerged as a powerful tool capable of developing and designing new antimicrobial drugs. Objectives: The aim of this study was to test minimum inhibitory concentrations (MICs) of silver (Ag-NPs) and selenium (Se-NPs) nanoparticles, both alone and in combination with antifungal agents fluconazole (FLU), itraconazole (ITR), and amphotericin B (AMB) on resistant C. parapsilosis strains. Methods: Eight C. parapsilosis strains resistant to antifungal agents (FLU, ITR, AMB) and the standard strain of C. parapsilosis (ATCC 22019) as a control were used in this study. The standard protocol of antifungal susceptibility testing for yeast was performed by the broth micro-dilution method described in the clinical and laboratory standards institute (CLSI) guidelines, document M 27-S4. Results and Conclusions: MICs of standard and resistant strains were different against nano compounds, both alone and in combination with antifungal drugs. The growth of yeast was inhibited by Ag-NPs at concentrations as low as 2 µg /mL (against FLCR1 and FLCR3) and 0.25 µg /mL (against ITRR2 and AMBR2). Antifungal activity testing of Se-NPs revealed that it can inhibit the growth of yeasts at concentrations as low as 4 µg /mL (against FLCR1 and FLCR3) and 0.5 mg/mL (against ITRR2, ITRR3 and AMBR1). The results demonstrated that Se-NPs had lower activity than Ag-NPs, but both (Se-NPs and Ag-NPs) had higher activity than common antifungal agents. Further in vitro and in vivo studies are required for this.

Antimicrobial susceptibilities of Chlamydia trachomatis isolated from the urethra and pharynx of Japanese males.

Sexually transmitted infections due to Chlamydia trachomatis (C.trachomatis) are a worldwide public health problem. The aim of this study was to investigate the drug susceptibilities of C.trachomatis strains isolated from the urethra and pharynx of Japanese males. Urethral and pharyngeal swabs were collected between 2013 and 2014 from Japanese males with urethritis. Using a McCoy cell line, 18 chlamydial strains were isolated from urethra in 18 patients and 7 from the pharynx in 7 of the 18 patients. The minimum inhibitory concentrations (MICs) of levofloxacin (LVFX) and azithromycin (AZM) were measured using the standard method of the Japanese Society of Chemotherapy. The MICs of LVFX and AZM against urethral chlamydial strains were 0.125-0.5μg/mL and 0.125-0.25μg/mL, respectively. In pharyngeal strains, the MICs of LVFX and AZM were 0.125-0.25μg/mL and 0.125-0.25μg/mL, respectively. In 7 patients with chlamydial strains isolated from both the urethra and pharynx, the MICs of LVFX between these strains were identical in 3 of 6 patients (no growth was observed for one pharyngeal strain), while the MICs of AZM between these strains were identical in all 6 patients (not performed for one patient). Our data suggest that C.trachomatis strains isolated from the urethra and pharynx of Japanese males are susceptible to LVFX and AZM. Although measuring the MICs of chlamydial strains is labor intensive, it is a significant surveillance tool for treating chlamydial infections and preventing the spread of STIs.