[Effect of Phenylalanine-arginine-beta-naphthylamide to Ciprofloxacin Minimum Inhibitory Concentration Values and Expression of Efflux Pump System Genes in Acinetobacter baumannii Isolates]. / Acinetobacter baumannii Izolatlarinda Fenilalanin-arjinin-beta-naftilamidin Siprofloksasinin Minimal Inhibitör Konsantrasyon Degerleri ve Disa Atim Pompasi Genlerinin Ekspresyonu Üzerine Etkisi.

The most realistic approach in recent years is researching the resistance inhibition rather than synthesizing new compounds. In this study, we aimed to determine i) the effect of phenylalanine-argininebeta-naphthylamide (PAßN), on minimum inhibition concentrations (MICs) of ciprofloxacin (CIP), ii) to obtain the CIP+PAßN concentration that inhibits CIP resistance and iii) to show that this inhibition is caused by the effect of PAßN on the expression of efflux pump (EF) system genes. Acinetobacter baumannii isolates were collected from Trakya University Hospital. In 67 isolates determined to be resistant to CIP, CIP susceptibility was investigated in presence of PAßN once again. Isolates determined to have four or more fold decrease in ciprofloxacin MIC values were included in checkerboard assay and quantitative real-time reverse transcriptase polymerase chain reaction (qrRT-PCR). Fractional inhibition concentrations (FICs) were calculated through the PAßN concentrations that inhibit ciprofloxacin resistance, by the checkerboard assay results. With the combination of CIP+PAßN, the effect of the concentrations at which inhibition occurs, to the expression levels of EF system genes (adeA, adeB, adeR, adeS, adeF, adeG, adeH, adeL) was investigated by qrRT-PCR. By the checker board assay, a synergistic effect was determined between PAßN and CIP in 11 isolates, while in other isolates the effect was determined to be additive. In some isolates resistant to CIP, CIP + PAßN combination inhibited the resistance and increased CIP susceptibility. In the presence of 25 mg/L and 100 mg/L PAßN, 22 (32.83%) and 27 (40.3%) of 67 isolates became sensitive to CIP, respectively. In seven isolates, 12.5 µg/ml PAßN concentration eliminated CIP resistance by decreasing CIP MIC value to 1 µg/ml. Also, in one isolate the MIC value was 0.5 µg/ml in the presence of 25 µg/ml PAßN and 1 µg/ml in the presence of 1.5625 µg/ml PAßN. After analyzing the expression levels of EF genes (adeA, adeB, adeC, adeF, adeG, adeH, adeL, adeR and adeS) by the qRt-PCR method, it was determined that with the addition of PAßN to media containing CIP, the expression levels of the genes decreased (p<0.05). The aim of the study has been achieved with the results obtained. These results highlighted the importance of research on the inhibition of resistance, as well as the synthesis of new antimicrobial compounds. Combined use of inhibitors and antibiotics should be considered as an alternative treatment method. Thus, existing antibiotics can be included in the treatment again, saving time and money. It will be possible to use these findings in further studies to elucidate the mechanism of action of new inhibitor candidate compounds and associate them with the expression of DAP genes, also by investigating mutations in the regulatory gene regions in isolates with over-expression levels.

[Effect of N-acetylcystein on Antibiofilm Efficiency of Antibiotics in Staphylococci Isolates]. / Stafilokok Izolatlarinda Antibiyotiklerin Antibiyofilm Etkinligi Üzerine N-asetilsisteinin Etkisi.

Biofilms are often responsible for the difficulties in the treatment of infectious diseases due to their properties that facilitate escape from antibiotic effect and their antiphagocytic effects. At least 65% of all infectious diseases are associated with biofilm-forming bacteria. As Staphylococcus aureus and Staphylococcus epidermidis are among the most common agents of hospital infections and the infections are mostly biofilm-related, they pose an important problem. In infectious isolates, the minimum biofilm eradication concentration (MBEK) values of biofilm forms are much higher than the minimum inhibition concentration (MIC) values of planktonic forms. This situation requires the use of much higher doses of antibiotics in the treatment of infections and causes an increase in antibiotic resistance. The N-acetylcysteine (NAC) molecule is known to be effective against biofilm by disrupting mature biofilms and reducing the adhesion of bacteria to surfaces. In this study, it was aimed to demonstrate i) the biofilm-forming abilities ii) the change in ampicillin and vancomycin MIC values in the presence of NAC molecules, iii) the change in the MBEK values of these antibiotics in the presence of NAC molecule and iv) the change in the expression levels of genes thought to be related to biofilm formation in the presence of the NAC molecule among S.aureus (n= 38) and S.epidermidis (n= 12) isolates isolated from various clinical specimens in Trakya University Health Research and Application Center. In this study, microplate crystal violet method was used to demonstrate the biofilm formation in staphylococci. Broth microdilution and checkerboard method were used to demonstrate the change in the presence of NAC molecule of the MIC and MBEC values of ampicillin and vancomycin. The effect of NAC on the expression of intercellular binding proteins A and D (icaA, icaD) and Staphylococcus regulatory protein A (sarA) genes, which are the genes involved in biofilm formation in staphylococci, was determined by quantitative real-time Polymerase Chain Reaction (qRt-PCR) method. The Student-t test was used to compare the control and experimental groups (concentrations detected with synergy and additive effect); p˂ 0.05 was accepted as the limit value of significance. In this study, when the NAC molecule was used together with ampicillin and vancomycin, it was determined that this combination lowers the MIC values of staphylococcus isolates and staphylococcal biofilm MBEK values; and also the expression levels of icaA, icaD and sarA which were effective in biofilm formation in staphylococci have not changed and decreased. As a result, in this study, it has been determined that the NAC molecule can be a new alternative for combined drug therapy and is promising in terms of bringing a new approach to treatment. In addition, it is thought that it is possible to use the NAC molecule together with different microorganisms and antimicrobial agents, and the results obtained in this study are considered to be guiding for further studies on this subject.

Development of effective AmB/AmB-αCD complex double loaded liposomes using a factorial design for systemic fungal infection treatment.

Amphotericin B (AmB) is a very potent antibiotic which still remains as the gold standard for the treatment of systemic fungal infections. AmB is a member of Biopharmaceutical Classification System Class IV, mainly characterized by its poor solubility and low permeability. In this study, AmB/AmB-α cyclodextrin complex double loaded liposomes (DLLs) were developed using the design of experiments (DoE®) approach to optimize/determine the effects of lipid composition and other parameters on final product properties such as encapsulation efficacy, particle size, polydispersity index, and zeta potential. Experimental design 24 was used for optimization of these properties in which four factors were studied in two levels. DLLs showed much higher physical stability than liposomes loaded only with free AmB by the means of particle size, zeta potential and encapsulation efficiency, in addition exhibited sustained release of AmB over 72 h (26.7%) with faster onset time. On the other hand, fourfold improved antimicrobial efficiency, minimum inhibitory concentration (0.125 µg/ml), and minimum fungicidal concentration (0.5 µg/ml) was determined by DLLs against C. albicans compared to Ambisome®. Dose dependent effects of the DLLs were investigated by cytotoxicity studies on Vero and L-929 cells. No significant cytotoxicity observed for AmB/AmB-αCD complex DLLs and Ambisome at tested concentrations while free AmB caused severe cytotoxicity. Lastly the developed DLLs did not cause an increase in NGAL (an early biomarker for acute kidney toxicity) levels for both Vero and HK-2 cell lines compared to free AmB.

Design, synthesis, molecular docking, density functional theory and antimicrobial studies of some novel benzoxazole derivatives as structural bioisosteres of nucleotides.

A series of some novel 2-(p-tert-butylphenyl)-5-(3-substituted-propionamido)benzoxazole derivatives have been designed, synthesized, evaluated for antimicrobial activity and have performed molecular docking studies against penicillin-binding protein 4 (PBP4) and active and allosteric site of PBP2a; were calculated some theoretical quantum parameters and absorption, distribution, metabolism and excretion (ADME) descriptors. B9 acted at minimum inhibitory concentration (MIC) = 8 µg/mL against S. aureus, E. faecalis and their drug-resistant isolates and also formed with GLU145 (1.74 Å) and ILE144 (1.89 Å) two hydrogen bonds at allosteric site of PBP2a with Glide emodel score: -42.168. ΔE of compound B9 had moderate value of all compounds with 0.14742.[Formula: see text]Communicated by Ramaswamy H. Sarma.

Synthesis and molecular docking studies of some novel antimicrobial benzamides.

Common use of classical antibiotics has caused to the growing emergence of many resistant strains of pathogenic bacteria. Therefore, we aimed to synthesize a number of N-(2-hydroxy-(4 or 5)-nitrophenyl)benzamide derivatives as a new class of antimicrobial compounds. Moreover, our second goal is to predict the interaction between active structures and enzymes (DNA -gyrase and FtsA) in the binding mode. In this study, thirteen N-(2-hydroxy-(4 or 5-nitrophenyl)-substituted-benzamides were synthesized and determined for their antimicrobial activity using the microdilution method. According to this work, none of the compounds showed any activity against Candida albicans and its clinical isolate. Some of the benzamides (4N1, 5N1, 5N2) displayed very significant activity against Staphylococcus aureus and MSSA with <4 µg/ml MIC value, even they were found to be more potent than ceftazidime. 4N1 was also found to be more effective than gentamicin against Enterococcus faecalis clinical isolate. Molecular docking studies revealed that 4N1, 5N1, and 5N2 showed a good interactions with DNA-gyrase. Moreover, 5N1 has interacted with FtsA enzyme in the binding mode, as well. Only compound 5N4 displayed very good activity against Escherichia coli ATCC 25922. These findings showed us that 4N1, 5N1, 5N2, and 5N4 could be lead compounds to discover new antibacterial candidates against multidrug-resistant strains.

Synthesis and antimicrobial evaluation of some novel sulfonylamido-benzoxazoles.

A series of 2-(p-substituted phenyl)-5-[(4-substituted phenyl) sulfonylamido]-benzoxazoles were synthesized and tested for their antimicrobial activities. The structures of the new derivatives were elucidated by spectral techniques. The minimum inhibitory concentrations (MIC) of the new benzoxazoles were determined against standard bacterial and fungal strains and drug-resistant isolates and compared to those of several reference drugs.

[Investigation of antimicrobial and antibiofilm effects of some preservatives used in drugs, cosmetics and food products]. / Ilaç, kozmetik ve gida ürünlerinde kullanilan bazi koruyucularin antimikrobiyal ve antibiyofilm etkisinin arastirilmasi

Preservatives are added to food, drugs and other pharmaceutical products to avoid microbial contamination. For antimicrobial activity testing and preservative efficacy testing, vegetative forms of the standard test organisms are used. However, microbial biofilm formation may occur on living tissues, medical implants, industrial or drinking water pipes, natural aquatic systems, glass and plastic surfaces. In our study, it was aimed to determine the antimicrobial and antibiofilm effects of some preservatives used in drug, cosmetics and food products and to compare the minimum biofilm inhibitory concentration (MBIC) of microbial biofilm formed on glass surfaces which are commonly used in those areas and the minimum inhibitory concentration (MIC) values of the planktonic forms. In the study Pseudomonas aeruginosa ATCC 27853, Salmonella Thyphimurium SL1344, Staphylococcus aureus ATCC 6538, Staphylococcus epidermidis NCTC 11047, Enterococcus faecalis ATCC 29212 and Candida albicans ATCC 10231 were used as the standard strains; sodium nitrate, methylparaben, prophylparaben, potassium sorbate and sodium benzoate as the preservatives; ampicillin, vancomycin, gentamicin, ciprofloxacin, amphotericin B and itraconazole as the antimicrobial agents. MIC values were determined through the guidelines of CLSI M100-S18 and M27-A3 protocols. BioTimer method was used to determine the MBIC values. The value of "colony forming unit (CFU)/glass beads" was calculated using the graphics drawn by plotting the time of color change for phenol red or resazurin against log10CFU. All experiments were done with four media at different pH values namely pH: 7, pH: 6.5, pH: 6 and pH: 5.5. According to the results of tests on planktonic forms of the microorganisms, sodium benzoate was determined to be the most effective preservative against all the microorganisms tested except S.aureus and E.faecalis. The most effective preservative against S.aureus and E.faecalis was prophylparaben. Prophylparaben was also effective against S.epidermidis. However, in our study it was determined that preservatives were not effective against biofilm forms even if the inoculum was lower, equal to or higher than the inocula of the planktonic forms. The data obtained from this study indicated that preservatives used to prevent microbial contamination in pharmaceutical, cosmetic and food products, are not effective against biofilm forms of the microorganisms. This study is thought to be a guide for further studies to be held in the investigation of antimicrobial and antibiofilm effects of preservatives used in drugs, cosmetics and food industry.

Synthesis and antimicrobial evaluation of 2-(p-substituted phenyl)-5-[(4-substituted piperazin-l-yl)acetamido]-benzoxazoles.

A series of 2-(p-substituted phenyl)-5-(2-{4-[(p-chloro-fluorophenyl)/phenyl] piperazin-1-yl}acetamido)-benzoxazoles were synthesized and tested for their antimicrobial activities. The structures of the new derivatives were elucidated by spectral techniques. The minimum inhibitory concentrations (MIC) of the new benzoxazoles, along with those of previously synthesized analogues, were determined against standard bacterial and fungal strains and drug-resistant isolates, and compared with those of several reference drugs. The new benzoxazole derivatives were found to possess a broad spectrum of antimicrobial activity with MIC values of 32-1024 µg/ml. Although the standard drugs were more active against the tested pathogens, the activities of the new benzoxazoles and the reference drugs were largely similar against the drug-resistant isolates.

Synthesis and different biological activities of novel benzoxazoles.

A series of 2-[4-(4-substitutedbenzamido/phenylacetamido/butanamido)phenyl]-5-ethylsulphonyl-benzoxazole derivatives were synthesized and biologically evaluated as possible antimicrobial agents and inhibitors of tyrosinase, acetylcholinesterase (AChE), and butyrylcholinesterase (BChE). The results demonstrated that the synthesized compounds exhibited a broad spectrum of activity with minimum inhibitory concentration (MIC) values of 128-16 µg/ml against some Gram-positive, Gram-negative bacteria as well as Candida albicans and C. krusei. The compound 10 displayed higher activity in this series against methicilline resistant Staphylococcus aureus (MRSA) with a MIC value of 16 µg/ml than the compared control drugs ampicillin and ceftriaxone. Compound 14 showed moderate tyrosinase inhibition, however, none of the compounds showed effect as inhibitor of AChE and BChE.

Novel benzoxazoles: synthesis and antibacterial, antifungal, and antitubercular activity against antibiotic-resistant and -sensitive microbes.

A new series of 5-(p-substituted benzamido/phenylacetamido)-2-(p-tert-butylphenyl)benzoxazole derivatives were synthesized and evaluated for their antibacterial, antifungal, and antimycobacterial activities against antibiotic-resistant and -sensitive Staphylococcus aureus, Enterococcus faecalis, Escherichia coli, Klebsiella pneumoniae, and Mycobacterium tuberculosis as well as against Candida albicans and Candida krusei. The compounds possessed broad-spectrum activity against all of the tested Gram-positive and Gram-negative bacteria and yeasts, their minimum inhibitory concentrations (MICs) ranging between 16-128 microg/ml. One compound exhibited significant antibacterial activity (16 microg/ml) against an antibiotic-resistant Enterococcus faecalis isolate, having twice the potency of the compared standard drugs vancomycin and gentamycin sulfate. The compounds also showed moderate antitubercular activity with MIC values between 8-128 microg/ml against Mycobacterium tuberculosis and its clinical isolate.

Synthesis and antimicrobial activity of novel benzoxazoles.

A series of 2-(p-substituted-benzyl)-5-[[4-(p-chloro/fluoro-phenyl)piperazin-1-yl]acetamido]-benzoxazoles were synthesized in need of new compounds for the fight against microbial pathogens. Their structures were elucidated by spectral techniques. These new derivatives, along with previously synthesized 2-(p-substituted-benzyl)-5-substituted-benzoxazoles, were evaluated for their antibacterial and antifungal activities against standard strains and drug-resistant isolates in comparison with ampicillin, gentamicin sulfate, ofloxacin, vancomycin, fluconazole, and amphotericin B trihydrate. The minimum inhibitory concentration (MIC) of each compound was determined by a two-fold serial dilution technique. The compounds were found to possess a broad spectrum of antimicrobial activities with MIC values of 32-256 microg/ml. Although standard drugs were more active against the pathogenes employed in this study, the activities of the new benzoxazoles and reference drugs against drug-resistant isolates of the microorganisms were largely similar.

[Comparison of two different methods for the investigation of in vitro susceptibilities of planktonic and biofilm forming Candida species to antifungal agents]. / Candida türlerinin biyofilm olusturan ve planktonik formlarinin antifungal ajanlara karsi in vitro duyarliliklarinin arastirilmasinda iki farkli yöntemin karsilastirilmasi

Microdilution method that determines the minimum inhibitory concentrations (MIC) of antifungal agents against Candida spp. is still the only method used in laboratories for both biofilm and planktonic forms. However, it was determined in several studies that there were susceptibility differences between the biofilm and planktonic forms of the same microorganism. The aims of this study were the determination of in vitro susceptibilities of planktonic and biofilm forms of Candida strains against antifungal agents, the comparison of the data obtained from planktonic and biofilm forms and the evaluation of two different methods used for the detection of susceptibilities of biofilm forms. Candida albicans ATCC 10231, Candida parapsilosis ATCC 90028 and Candida krusei ATCC 6258 were used as reference strains together with clinical isolates of one of each C.albicans, C.parapsilosis and Candida tropicalis. Microdilution method was used to determine the susceptibilities of planktonic forms of the strains according to CLSI M27-A3 standards, and MIC values of fluconazole, itraconazole, flucytosine, amphotericin B and nystatin were determined. For the detection of antifungal susceptibilities of Candida spp. biofilm forms, Calgary biofilm method (CBM) and BioTimer assay (BTA) were used, and minimum biofilm eradication concentration (MBEC) and minimum biofilm inhibition concentration (MBIC) values of the same antifungals were determined. The difference between MIC and CBM-MBEC, CBM-MBEC and BTA-MBEC, CBM-MBEC and BTAMBIC values were found statistically significant (p < 0.05). In general CBM-MBEC values were found to be higher than MIC values. However, MBEC values were not always very reliable since the exact number of the microorganisms in biofilm can not be determined. BTA-MBIC values were also generally lower than the MBEC values and higher than the MIC values. Statistically significant difference between two methods was determined only for the MBEC values of flucytosine (p= 0.002) and itraconazole (p = 0.025). For flucytosine (p = 0.001) and itraconazole (p = 0.001), there was also a significant difference between CBM-MBEC and BTA-MBIC values, however, the difference was not significant (p > 0.05) for the other antifungal agents. These findings supported that antifungal susceptibilities of biofilm forming Candida strains should also be investigated. However, MBEC and MBIC of the antifungal agents should not always be expected to be higher than the MIC values since the mechanism of action of the specific antifungal agents and the first inoculum concentration of the microorganisms might differ.