Exploiting thiol-functionalized benzosiloxaboroles for achieving diverse substitution patterns - synthesis, characterization and biological evaluation of promising antibacterial agents.

Benzosiloxaboroles are an emerging class of medicinal agents possessing promising antimicrobial activity. Herein, the expedient synthesis of two novel thiol-functionalized benzosiloxaboroles 1e and 2e is reported. The presence of the SH group allowed for diverse structural modifications involving the thiol-Michael addition, oxidation, as well as nucleophilic substitution giving rise to a series of 27 new benzosiloxaboroles containing various polar functional groups, e.g., carbonyl, ester, amide, imide, nitrile, sulfonyl and sulfonamide, and pendant heterocyclic rings. The activity of the obtained compounds against selected bacterial and yeast strains, including multidrug-resistant clinical strains, was investigated. Compounds 6, 12, 20 and 22-24 show high activity against Staphylococcus aureus, including both methicillin-sensitive (MSSA) and methicillin-resistant (MRSA) strains, with MIC values in the range of 1.56-12.5 µg mL-1, while their cytotoxicity is relatively low. The in vitro assay performed with 2-(phenylsulfonyl)ethylthio derivative 20 revealed that, in contrast to the majority of known antibacterial oxaboroles, the plausible mechanism of antibacterial action, involving inhibition of the leucyl-tRNA synthetase enzyme, is not responsible for the antibacterial activity. Structural bioinformatic analysis involving molecular dynamics simulations provided a possible explanation for this finding.

Aromatic Diboronic Acids as Effective KPC/AmpC Inhibitors.

Over 30 compounds, including para-, meta-, and ortho-phenylenediboronic acids, ortho-substituted phenylboronic acids, benzenetriboronic acids, di- and triboronated thiophenes, and pyridine derivatives were investigated as potential ß-lactamase inhibitors. The highest activity against KPC-type carbapenemases was found for ortho-phenylenediboronic acid 3a, which at the concentration of 8/4 mg/L reduced carbapenems' MICs up to 16/8-fold, respectively. Checkerboard assays revealed strong synergy between carbapenems and 3a with the fractional inhibitory concentrations indices of 0.1-0.32. The nitrocefin hydrolysis test and the whole cell assay with E. coli DH5α transformant carrying blaKPC-3 proved KPC enzyme being its molecular target. para-Phenylenediboronic acids efficiently potentiated carbapenems against KPC-producers and ceftazidime against AmpC-producers, whereas meta-phenylenediboronic acids enhanced only ceftazidime activity against the latter ones. Finally, the statistical analysis confirmed that ortho-phenylenediboronic acids act synergistically with carbapenems significantly stronger than other groups. Since the obtained phenylenediboronic compounds are not toxic to MRC-5 human fibroblasts at the tested concentrations, they can be considered promising scaffolds for the future development of novel KPC/AmpC inhibitors. The complexation of KPC-2 with the most representative isomeric phenylenediboronic acids 1a, 2a, and 3a was modeled by quantum mechanics/molecular mechanics calculations. Compound 3a reached the most effective configuration enabling covalent binding to the catalytic Ser70 residue.

Efflux-Related Carbapenem Resistance in Acinetobacter baumannii Is Associated with Two-Component Regulatory Efflux Systems' Alteration and Insertion of ΔAbaR25-Type Island Fragment.

The efflux pumps, beside the class D carbapenem-hydrolysing enzymes (CHLDs), are being increasingly investigated as a mechanism of carbapenem resistance in Acinetobacter baumannii. This study investigates the contribution of efflux mechanism to carbapenem resistance in 61 acquired blaCHDL-genes-carrying A. baumannii clinical strains isolated in Warsaw, Poland. Studies were conducted using phenotypic (susceptibility testing to carbapenems ± efflux pump inhibitors (EPIs)) and molecular (determining expression levels of efflux operon with regulatory-gene and whole genome sequencing (WGS)) methods. EPIs reduced carbapenem resistance of 14/61 isolates. Upregulation (5-67-fold) of adeB was observed together with mutations in the sequences of AdeRS local and of BaeS global regulators in all 15 selected isolates. Long-read WGS of isolate no. AB96 revealed the presence of AbaR25 resistance island and its two disrupted elements: the first contained a duplicate ISAba1-blaOXA-23, and the second was located between adeR and adeA in the efflux operon. This insert was flanked by two copies of ISAba1, and one of them provides a strong promoter for adeABC, elevating the adeB expression levels. Our study for the first time reports the involvement of the insertion of the ΔAbaR25-type resistance island fragment with ISAba1 element upstream the efflux operon in the carbapenem resistance of A. baumannii.

Mutant Prevention Concentration, Frequency of Spontaneous Mutant Selection, and Mutant Selection Window-a New Approach to the In Vitro Determination of the Antimicrobial Potency of Compounds.

The analysis of antimicrobial activity is usually MIC- and minimal bactericidal concentration (MBC)-focused, though also crucial are resistance-related parameters, e.g., the frequency of spontaneous mutant selection (FSMS), the mutant prevention concentration (MPC), and the mutant selection window (MSW). In vitro-determined MPCs, however, are sometimes variable, poorly repeatable, and not always reproducible in vivo. We propose a new approach to the in vitro determination of MSWs, along with novel parameters: MPC-D, MSW-D (for dominant mutants, i.e., selected with a high frequency, without a fitness loss), and MPC-F, MSW-F (for inferior mutants, i.e., with an impaired fitness). We also propose a new method for preparing the high-density inoculum (>1011 CFU/mL). In this study, the MPC and MPC-D (limited by FSMS of <10-10) of ciprofloxacin, linezolid, and novel benzosiloxaborole (No37) were determined for Staphylococcus aureus ATCC 29213 using the standard agar method, while the MPC-D and MPC-F were determined by the novel broth method. Regardless of the method, MSWs1010 of linezolid and No37 were the same. However, MSWs1010 of ciprofloxacin in the broth method was narrower than in the agar method. In the broth method, the 24-h incubation of ~1010 CFU in a drug-containing broth differentiates the mutants that can dominate the cell population from those that can only be selected under exposure. We consider MPC-Ds in the agar method to be less variable and more repeatable than MPCs. Meanwhile, the broth method may decrease discrepancies between in vitro and in vivo MSWs. The proposed approaches may help establish MPC-D-related resistance-restricting therapies.

The Contribution of Efflux Systems to Levofloxacin Resistance in Stenotrophomonas maltophilia Clinical Strains Isolated in Warsaw, Poland.

Levofloxacin is considered an alternative treatment option of Stenotrophomonas maltophilia infections to trimethoprim/sulfamethoxazole. The fluoroquinolone resistance in S. maltophilia is usually caused by an overproduction of efflux pumps. In this study, the contribution of efflux systems to levofloxacin resistance in S. maltophilia clinical isolates was demonstrated using phenotypic (minimal inhibitory concentrations, MICs, of antibiotics determination ± efflux pump inhibitors, EPIs) and molecular (real-time polymerase-chain-reaction and sequencing) methods. Previously, the occurrence of genes encoding ten efflux pumps was shown in 94 studied isolates. Additionally, 44/94 isolates demonstrated reduction in susceptibility to levofloxacin. Only 5 of 13 isolates (with ≥4-fold reduction in levofloxacin MIC) in the presence of EPIs showed an increased susceptibility to levofloxacin and other antibiotics. The overexpression of smeD and smeV genes (in five and one isolate, respectively) of 5 tested efflux pump operons was demonstrated. Sequencing analysis revealed 20-35 nucleotide mutations in local regulatory genes such as smeT and smeRv. However, mutations leading to an amino acid change were shown only in smeT (Arg123Lys, Asp182Glu, Asp204Glu) for one isolate and in smeRv (Gly266Ser) for the other isolate. Our data indicate that the overproduction of the SmeVWX efflux system, unlike SmeDEF, plays a significant role in the levofloxacin resistance.

Oxazoline scaffold in synthesis of benzosiloxaboroles and related ring-expanded heterocycles: diverse reactivity, structural peculiarities and antimicrobial activity.

Two isomeric benzosiloxaborole derivatives 3a and 5a bearing fluorine and 4,4-dimethyl-2-oxazolin-2-yl substituents attached to the aromatic rings were obtained. Both compounds were prone to hydrolytic cleavage of the oxazoline ring after initial protonation or methylation of the nitrogen atom. The derivative 3c featuring N-methylammoniumalkyl ester functionality was successfully subjected to N-sulfonylation and N-acylation reactions to give respective derivatives which demonstrates its potential for modular synthesis of structurally extended benzosiloxaboroles. Compound 5c bearing N-ammoniumalkyl ester underwent conversion to a unique macrocyclic dimer due to siloxaborole ring opening. Furthermore, an unexpected 4-electron reduction of the oxazoline ring occurred during an attempted synthesis of 5a. The reaction gave rise to an unprecedented 7-membered heterocyclic system 4a comprising a relatively stable B-O-B-O-Si linkage and stabilized by an intramolecular N-B coordination. It could be cleaved to derivative 4c bearing BOH and SiMe2OH groups which acts as a pseudo-diol as demonstrated by formation of an adduct with Tavaborole. Apart from the multinuclear NMR spectroscopy characterization, crystal structures of the obtained products were determined in many cases by X-ray diffraction. Investigation of biological activity of the obtained compounds revealed that derivatives 3e and 3f with pendant N-methyl arylsulfonamide groups exhibit high activity against Gram-positive cocci such as methicillin-sensitive Staphylococcus aureus ATCC 6538P, methicillin-resistant S. aureus (MRSA) ATCC 43300 as well as the MRSA clinical strains, with MIC values in the range of 3.12-6.25 mg L-1. These two compounds also showed activity against Enterococcus faecalis ATCC 29212 and Enterococcus faecium ATCC 6057 (with MICs of 25-50 mg L-1). The results of the antimicrobial activity and cytotoxicity studies indicate that 3e and 3f can be considered as potential antibacterial agents, especially against S. aureus MRSA.

Phenotypic and Molecular Characteristics of the MDR Efflux Pump Gene-Carrying Stenotrophomonas maltophilia Strains Isolated in Warsaw, Poland.

An increase of nosocomial infections caused by Stenotrophomonas maltophilia strains has recently been observed all over the world. The isolation of these bacteria from the blood is of particular concern. In this study we performed the phenotypic and genotypic characterization of 94 S. maltophilia isolates, including isolates from patients hospitalized in a tertiary Warsaw hospital (n = 79) and from outpatients (n = 15). All isolates were found to be susceptible to trimethoprim-sulfamethoxazole and minocycline, while 44/94 isolates demonstrated a reduction in susceptibility to levofloxacin. A large genetic variation was observed among the isolates tested by pulsed-field gel electrophoresis. A clonal relationship with 100% similarity was observed between isolates within two sub-pulsotypes: the first included nine bloodstream isolates and the second involved six. Multilocus sequence typing showed two new sequence types (ST498 and ST499) deposited in public databases for molecular typing. Moreover, the presence of genes encoding ten different efflux pumps from the resistance-nodulation-division family and the ATP-binding cassette family was shown in the majority of the 94 isolates. The obtained knowledge about the prevalence of efflux pump genes in clinical S. maltophilia strains makes it possible to predict the scale of the risk of resistance emergence in strains as a result of gene overexpression.

Analysis of blaCHDL Genes and Insertion Sequences Related to Carbapenem Resistance in Acinetobacter baumannii Clinical Strains Isolated in Warsaw, Poland.

Acinetobacter baumannii is an important cause of nosocomial infections worldwide. The elucidation of the carbapenem resistance mechanisms of hospital strains is necessary for the effective treatment and prevention of resistance gene transmission. The main mechanism of carbapenem resistance in A. baumannii is carbapenemases, whose expressions are affected by the presence of insertion sequences (ISs) upstream of blaCHDL genes. In this study, 61 imipenem-nonsusceptible A. baumannii isolates were characterized using phenotypic (drug-susceptibility profile using CarbaAcineto NP) and molecular methods. Pulsed field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST) methods were utilized for the genotyping. The majority of isolates (59/61) carried one of the following acquired blaCHDL genes: blaOXA-24-like (39/59), ISAba1-blaOXA-23-like (14/59) or ISAba3-blaOXA-58-like (6/59). Whole genome sequence analysis of 15 selected isolates identified the following intrinsic blaOXA-66 (OXA-51-like; n = 15) and acquired class D ß-lactamases (CHDLs): ISAba1-blaOXA-23 (OXA-23-like; n = 7), ISAba3-blaOXA-58-ISAba3 (OXA-58-like; n = 2) and blaOXA-72 (OXA-24-like; n = 6). The isolates were classified into 21 pulsotypes using PFGE, and the representative 15 isolates were found to belong to sequence type ST2 of the Pasteur MLST scheme from the global IC2 clone. The Oxford MLST scheme revealed the diversity among these studied isolates, and identified five sequence types (ST195, ST208, ST208/ST1806, ST348 and ST425). CHDL-type carbapenemases and insertion elements upstream of the blaCHDL genes were found to be widespread among Polish A. baumannii clinical isolates, and this contributed to their carbapenem resistance.

Antimicrobial and KPC/AmpC inhibitory activity of functionalized benzosiloxaboroles.

A series of 22 benzosiloxaboroles, silicon analogues of strong antimicrobial agents - benzoxaboroles, have been synthesized and tested against ß-lactamases KPC- and pAmpC-producing strains of Gram-negative rods. Comprehensive structural-property relationship studies supported by molecular modelling as well as biological studies reveal that 6-B(OH)2-substituted derivative 27 strongly inhibits the activity of cephalosporinases (chromosomally encoded AmpC and plasmid encoded CMY-2) and KPC carbapenemases. It also shows strong ability to inhibit growth of the strains producing KPC-3 when combined with meropenem. In addition, halogen-substituted (mono-, di- or tetra-) benzosiloxaboroles demonstrate high antifungal activity (MIC 1.56-6.25 mg/L) against C. tropicalis, C. guilliermondii and S. cerevisiae. The highest activity against pathogenic yeasts (C. albicans, C. krusei and C. parapsilosis - MICs 12.5 mg/L) and against Gram-positive cocci (S. aureus and E. faecalis - 6.25 mg/L and 25 mg/L respectively) was displayed by 6,7-dichloro-substituted benzosiloxaborole. The studied systems exhibit low cytotoxity toward human lung fibroblasts.

Non-antibiotics, Efflux Pumps and Drug Resistance of Gram-negative Rods.

Non-antibiotic medicinal products consist of drugs with diverse activity against bacteria. Many non-antibiotics demonstrate direct anti-bacterial activity against Gram-positive cocci. The activity observed against Gram-negative rods is much lower and non-antibiotics primarily from the following groups: non-steroidal anti-inflammatory drugs, cardiovascular and antidepressant medicinal products demonstrate this activity. It has been shown that the low activity of some non-antibiotics or the absence of activity against Gram-negative rods is related, among other things, to the extrusion of these compounds from bacterial cells by multi-drug resistance efflux pumps. Substrates for the resistance-nodulation-division efflux systems include the following non-antibiotics: salicylate, diclofenac, ibuprofen, mefenamic acid, naproxen, amitriptyline, alendronate sodium, nicergoline, and ticlopidine. In addition, interactions between non-antibiotics and multi-drug resistance efflux pumps have been observed. It has also been revealed that depending on the concentration, salicylate induces expression of multi-drug resistance efflux pumps in Escherichia coli, Salmonella enterica subsp. enterica serotype Typhimurium, and Burkholderia cenocepacia. However, salicylate does not affect the expression of the resistance-nodulation-division efflux systems in Stenotrophomonas maltophilia and Acinetobacter baumannii. Most importantly, there were no effects of medicinal products containing some non-antibiotic active substances, except salicylate, as substrates of multi-drug resistance efflux pumps, on the induction of Gram-negative rod resistance to quinolones.

Identification of proteins, drying oils, waxes and resins in the works of art micro-samples by chromatographic and mass spectrometric techniques.

Simplified method for simultaneous identification of proteins, drying oils, waxes, and resins in the works-of-art samples was developed. Liquid chromatography with mass spectrometry and gas chromatography with mass spectrometry were used to identify natural materials most frequently encountered in historical paintings. Protein binders were extracted with ammonia and purified using miniaturized solid-phase microextraction (Omix tips) to efficiently suppress matrix interferences. Zwitterionic stationary phase was used for separation of 16 underivatized amino acids analysis with hydrophilic interaction liquid chromatography that was subsequently quantified with liquid chromatography with mass spectrometry. Gas chromatography with mass spectrometry was used to analyze drying oils, waxes, and resins after one-step saponification/transmethylation with (m-trifluoromethylphenyl)trimethylammonium hydroxide (Meth-Prep II). While the drawback of this reagent is low reactivity towards hydroxyl groups, sample pretreatment was much simpler as compared to the other methods. Fatty acids derivatization with the Meth-Prep II reagent was compared with their silylation using N,O-bis(trimethylsilyl) trifluoroacetamide/trimethylchlorosilane mixture. It was concluded that fatty acids analysis as their methyl esters instead of trimethylsilyl esters had a minor impact on the method sensitivity. The developed method was used to analyze samples from 16th and 17th century historical paintings.

Prevalence of ESBL-producing Pseudomonas aeruginosa isolates in Warsaw, Poland, detected by various phenotypic and genotypic methods.

Knowledge of the prevalence of ESBL enzymes among P. aeruginosa strains compared to the Enterobacteraiceae family is limited. The phenotypic tests recommended by EUCAST for the detection of ESBL-producing Enterobacteriaceae are not always suited for P. aeruginosa strains. This is mainly due to the presence of other families of ESBLs in P. aeruginosa isolates more often than in Enterobacteriaceae, production of natural AmpC cephalosporinase and its overexpression, and co-production of metallo-ß-lactamases. The aim of this study was to determine the occurrence of ESBLs in P. aeruginosa isolated from patients from hospitals in Warsaw, to evaluate the ESBL production of these isolates using currently available phenotypic tests, their modifications, multiplex PCR and molecular typing of ESBL-positive isolates by PFGE. Clinical isolates of P. aeruginosa were collected in 2000-2014 from four Warsaw hospitals. Based on the data obtained in this study, we suggest using three DDST methods with inhibitors, such as clavulanic acid, sulbactam and imipenem, to detect ESBL-producing P. aeruginosa strains. Depending on the appearance of the plates, we suggest a reduction in the distance between discs with antibiotics to 15 mm and the addition of boronic acid at 0.4 mg per disc. The analysed isolates carried genes encoding ESBL from the families VEB (69 isolates with VEB-9), GES (6 with GES-1, 1 GES-5, 5 GES-13 and 2 with GES-15), OXA-2 (12 with OXA-15, 1 OXA-141, 1 OXA-210, 1 OXA-543 and 1 with OXA-544) and OXA-10 (5 isolates with OXA-74 and one with OXA-142). The most important result of this study was the discovery of three new genes, blaGES-15, blaOXA-141 and blaOXA-142; their nucleotide sequences have been submitted to the NCBI GenBank. It is also very important to note that this is the first report on the epidemiological problem of VEB-9-producing bacterial strains, not only in Poland but also worldwide.

The Impact of Efflux Pump Inhibitors on the Activity of Selected Non-Antibiotic Medicinal Products against Gram-Negative Bacteria.

The potential role of non-antibiotic medicinal products in the treatment of multidrug-resistant Gram-negative bacteria has recently been investigated. It is highly likely that the presence of efflux pumps may be one of the reasons for the weak activity of non-antibiotics, as in the case of some non-steroidal anti-inflammatory drugs (NSAIDs), against Gram-negative rods. The activity of eight drugs of potential non-antibiotic activity, active substance standards, and relevant medicinal products were analysed with and without of efflux pump inhibitors against 180 strains of five Gram-negative rod species by minimum inhibitory concentration (MIC) value determination in the presence of 1 mM MgSO4. Furthermore, the influence of non-antibiotics on the susceptibility of clinical strains to quinolones with or without PAßN (Phe-Arg-ß-naphthylamide) was investigated. The impacts of PAßN on the susceptibility of bacteria to non-antibiotics suggests that amitriptyline, alendronate, nicergoline, and ticlopidine are substrates of efflux pumps in Gram-negative rods. Amitriptyline/Amitriptylinum showed the highest direct antibacterial activity, with MICs ranging 100-800 mg/L against all studied species. Significant decreases in the MIC values of other active substances (acyclovir, atorvastatin, and famotidine) tested with pump inhibitors were not observed. The investigated non-antibiotic medicinal products did not alter the MICs of quinolones in the absence and in the presence of PAßN to the studied clinical strains of five groups of species.

The Influence of Efflux Pump Inhibitors on the Activity of Non-Antibiotic NSAIDS against Gram-Negative Rods.

BACKGROUND: Most patients with bacterial infections suffer from fever and various pains that require complex treatments with antibiotics, antipyretics, and analgaesics. The most common drugs used to relieve these symptoms are non-steroidal anti-inflammatory drugs (NSAIDs), which are not typically considered antibiotics. Here, we investigate the effects of NSAIDs on bacterial susceptibility to antibiotics and the modulation of bacterial efflux pumps. METHODOLOGY: The activity of 12 NSAID active substances, paracetamol (acetaminophen), and eight relevant medicinal products was analyzed with or without pump inhibitors against 89 strains of Gram-negative rods by determining the MICs. Furthermore, the effects of NSAIDs on the susceptibility of clinical strains to antimicrobial agents with or without PAßN (Phe-Arg-ß-naphtylamide) were measured. RESULTS: The MICs of diclofenac, mefenamic acid, ibuprofen, and naproxen, in the presence of PAßN, were significantly (≥4-fold) reduced, decreasing to 25-1600 mg/L, against the majority of the studied strains. In the case of acetylsalicylic acid only for 5 and 7 out of 12 strains of P. mirabilis and E. coli, respectively, a 4-fold increase in susceptibility in the presence of PAßN was observed. The presence of Aspirin resulted in a 4-fold increase in the MIC of ofloxacin against only two strains of E. coli among 48 tested clinical strains, which included species such as E. coli, K. pneumoniae, P. aeruginosa, and S. maltophilia. Besides, the medicinal products containing the following NSAIDs, diclofenac, mefenamic acid, ibuprofen, and naproxen, did not cause the decrease of clinical strains' susceptibility to antibiotics. CONCLUSIONS: The effects of PAßN on the susceptibility of bacteria to NSAIDs indicate that some NSAIDs are substrates for efflux pumps in Gram-negative rods. Morever, Aspirin probably induced efflux-mediated resistance to fluoroquinolones in a few E. coli strains.

CHEMICAL COMPOSITION AND ANTIBACTERIAL ACTIVITY OF SOME MEDICINAL PLANTS FROM LAMIACEAE FAMILY.

Chemical composition and antibacterial activity of aqueous (ethanolic and methanolic) extracts from herbs often used in Polish cuisine and traditional herbal medicine including thyme (Thymus vulgaris L.), rosemary (Rosmarinus officinalis L.), oregano (Origanum vulgare L.), peppermint (Mentha piperita L.) and sage (Salvia officinalis L.) were compared. The aqueous ethanolic extracts contained slightly higher levels of phenolics compared to the aqueous methanolic extracts. In turn, GC-MS analysis showed that the aqueous methanolic extracts of thyme, rosemary and sage contained several additional compounds such as eugenol or ledol. The present studies also indicated that the bacterial species applied in the experiment exhibited different sensitivities towards tested extracts. Staphylococcus aureus strains were found to be the most sensitive bacteria to aqueous (ethanolic and methanolic) rosemary and sage extracts and aqueous methanolic thyme extract. Klebsiella pneumoniae ATCC 13883 and Proteus vulgaris NCTC 4635 were more susceptible to the aqueous methanolic thyme extract. However, Listeria monocytogenes 1043S was the most sensitive to the aqueous ethanolic rosemary extract. Gram-positive bacteria were generally more sensitive to the tested extracts than Gram-negative ones.

Modification of the susceptibility of gram-negative rods producing ESßLS to ß-lactams by the efflux phenomenon.

The production of ß-lactamases is the most important mechanism of Gram-negative rod resistance to ß-lactams. Resistance to ceftazidime and cefepime in clinical isolates of Enterobacteriaceae (especially ESßL-positive E. coli and K. pneumoniae) and P. aeruginosa is life-threatening. However, all strains of the above mentioned species possess chromosomally encoded RND efflux pump systems in addition to ß-lactamase production. The main goal of this study was to assess the role of efflux pump systems in cefepime and/or ceftazidime resistant phenotypes of ESßL-positive clinical strains of Enterobacteriaceae and P. aeruginosa. The influence of the efflux pump inhibitor PAßN on the minimum inhibitory concentration (MIC) values of tested cephalosporins was species-dependent. Generally, a significant reduction (at least four-fold) of ß-lactam MICs was observed in the presence of PAßN only in the case of P. aeruginosa clinical isolates as well as the ESßL-producing transformant PAO1161 ΔampC. The usage of this agent resulted in the restoration of susceptibility to cefepime and/or ceftazidime in the majority of the P. aeruginosa ESßL-positive strains with low and moderate resistance to the above cephalosporins. Moreover, an outer membrane permeabilizing effect in the presence of PAßN was identified. Strain-dependent ß-lactamase leakage upon PAßN or ß-lactam treatment was demonstrated. The most important observation was the restoration of susceptibility of P. aeruginosa WUM226 to cefepime (MIC decrease from 32 to 4 mg/L) and ceftazidime (MIC decrease from 128 to 4 mg/L) in the presence of PAßN, which occurred despite an almost complete lack of ß-lactamase leakage from bacterial cells. In conclusion, these data indicate that RND efflux pumps can modify the susceptibility to ß-lactams in Gram-negative rods producing ESßLs. However, this phenomenon occurs only in P. aeruginosa strains and was not observed among E. coli and K. pneumoniae strains, representing the Enterobacteriaceae family.

Defect in dolichol-dependent glycosylation increases sensitivity of Saccharomyces cerevisiae towards anti-fungal drugs.

Two temperature-sensitive Saccharomyces cerevisiae mutants, sec59-1 and dpm1-6, impaired, respectively, in dolichol kinase (Sec59p) and dolichyl phosphate mannose (DolPMan) synthase (Dpm1p), have an aberrant cell wall structure and composition. We tested their sensitivity to four classes of antifungal drugs used in clinical practice: 5-fluorocytosine, amphotericin B, caspofungin and itraconasole. The strains were resistant to itraconazole and sensitive to the other drugs used. The minimal inhibitory concentration (MIC) of caspofungin and amphotericin B was two-fold lower for sec59-1 and dpm1-6 than for the respective wild-type strains. The sensitivity of both mutants could be brought back to the wild-type level by a multicopy suppressor of the thermosensitive phenotype, the RER2 gene, encoding cis-prenyltransferase involved in dolichol biosynthesis. Biochemical analysis revealed slight changes of the cell wall composition, different in the mutants as compared to the wild-type in response to the drugs. Our data strongly support a relationship between dolichol phosphate level, protein glycosylation and antifungal sensitivity.

Synthesis and antimicrobial and nitric oxide synthase inhibitory activities of novel isothiourea derivatives.

The reaction of substituted benzylhalides, or of halomethyl derivatives of thiophene or furane, with thiourea or its derivatives yielded the respective isothioureas as hydrohalide salts. The products (a total of 17, including 16 novel compounds) were tested for activity against five Gram-positive and nine Gram-negative bacterial strains, six yeast species and two protozoan species. The most active against Gram-positive bacteria were S-(2,4-dinitrobenzyl)isothiourea hydrochloride (MIC range for four out of five strains tested: 12.5-25 microg/mL) and S-(2,3,4,5,6-pentabromobenzyl)isothiourea hydrobromide (MIC range: 12.5-50 microg/mL). The lowest MICs of novel isothioureas for yeast and Gram-negative bacteria ranged between 50 and 100 microg/mL. Nine novel isothioureas showed appreciable genotoxicity in the Bacillus subtilis 'rec-assay' test, the most potent being S-2-(5-nitrofuran-2-ylmethyl)isothiourea and S-(2-nitrobenzyl) isothiourea. At 10 muM concentration, S-(3,4-dichlorobenzyl)isothiourea hydrochloride and S-(2,3,4,5,6-pentabromobenzyl)isothiourea hydrobromide inhibited Ca(2+)/calmodulin-dependent (non-inducible) nitric oxide synthase activity in normal rat brain homogenates stronger (p < 0.05) than the reference drug 7-nitroindazole (by 78, 76 and 60%, respectively); ten other new isothiourea derivatives significantly inhibited the activity to a lower extent (by 28-60%). These results extend the list of promising isothioureas with substantial activity in vitro and suggest that an in-depth study of toxicity, antimicrobial properties in vivo and nitric oxide synthase isoform selectivity of selected novel compounds is warranted.

A novel isoquinoline alkaloid, DD-carboxypeptidase inhibitor, with antibacterial activity isolated from Streptomyces sp. 8812. Part I: Taxonomy, fermentation, isolation and biological activities.

A novel isoquinoline alkaloid of molecular formula C10H9NO4, labeled JS-1, was isolated from the culture broth of Streptomyces sp. 8812. It was purified by acetone protein precipitation from the culture supernatant, followed by anion exchange and C18 RP HPLC columns. JS-1 is an inhibitor of exocellular DD-carboxypeptidases/transpeptidases (DD-peptidases) 64-575 II from Saccharopolyspora erythraea 64-575 II, and R39 from Actinomadura R39. JS-1 exhibits activity against Gram-negative bacteria, such as Bordetella bronchiseptica, Stenotrophomonas maltophilia, Proteus vulgaris, P. mirabilis, Burkholderia cepacia and Acinetobacter baumanii, with MIC values 10-160 microg ml(-1), and against Gram-positive bacteria, such as Staphylococcus aureus, with MIC values 40-206 microg ml(-1).

Synthesis of 1-{4-[4-(adamant-1-yl)phenoxymethyl]-2-(4-bromophenyl)- 1,3-dioxolan-2-ylmethyl}imidazole with expected antifungal and antibacterial activity.

A multistep synthesis of a new analogue of ketoconazole, which was expected to show antifungal and antibacterial activity, has been described.