In Vitro Antibiofilm Activity of Fosfomycin Alone and in Combination with Other Antibiotics against Multidrug-Resistant and Extensively Drug-Resistant Pseudomonas aeruginosa.

BACKGROUND: Due to its rapid resistance development and ability to form biofilms, treatment of Pseudomonas aeruginosa infections is becoming more complicated by the day. Drug combinations may help reduce both resistance and biofilm formation. METHODS: Using the microtiter plate assay, we investigated the in vitro inhibition of biofilm formation and the disruption of preformed biofilms in multidrug-resistant and extensively drug-resistant clinical isolates of P. aeruginosa in the presence of peak plasma levels of eight antipseudomonal antibiotics alone and in combination with fosfomycin: ceftazidime, piperacillin/tazobactam, cefepime, imipenem, gentamicin, amikacin, ciprofloxacin and colistin. RESULTS: Combination therapy was significantly superior to monotherapy in its inhibition of biofilm formation. The highest inhibition rates were observed for combinations with colistin, cefepime and ceftazidime. CONCLUSION: Our results support fosfomycin combination therapy as an enhanced prophylactic option. Moreover, combinations with ß-lactam antibiotics and colistin demonstrated a more potent inhibition effect on biofilm formation than protein synthesis inhibitors.

Synthesis and Biological Evaluation of New Quinoline and Anthranilic Acid Derivatives as Potential Quorum Sensing Inhibitors.

Inhibiting quorum sensing (QS), a central communication system, is a promising strategy to combat bacterial pathogens without antibiotics. Here, we designed novel hybrid compounds targeting the PQS (Pseudomonas quinolone signal)-dependent quorum sensing (QS) of Pseudomonas aeruginosa that is one of the multidrug-resistant and highly virulent pathogens with urgent need of new antibacterial strategies. We synthesized 12 compounds using standard procedures to combine halogen-substituted anthranilic acids with 4-(2-aminoethyl/4-aminobuthyl)amino-7-chloroquinoline, linked via 1,3,4-oxadiazole. Their antibiofilm activities were first pre-screened using Gram-negative Chromobacterium violaceum-based reporter, which identified compounds 15-19 and 23 with the highest anti-QS and minimal bactericidal effects in a single experiment. These five compounds were then evaluated against P. aeruginosa PAO1 to assess their ability to prevent biofilm formation, eradicate pre-formed biofilms, and inhibit virulence using pyocyanin as a representative marker. Compound 15 displayed the most potent antibiofilm effect, reducing biofilm formation by nearly 50% and pre-formed biofilm masses by 25%. On the other hand, compound 23 exhibited the most significant antivirulence effect, reducing pyocyanin synthesis by over 70%. Thus, our study highlights the potential of 1,3,4-oxadiazoles 15 and 23 as promising scaffolds to combat P. aeruginosa. Additionally, interactive QS systems should be considered to achieve maximal anti-QS activity against this clinically relevant species.

In vitro killing of multidrug/extensively drug-resistant Pseudomonas aeruginosa by fosfomycin alone or in combination with antipseudomonal antibiotics.

Pseudomonas aeruginosa is a leading cause of nosocomial infections. Given the constant rise in resistance, adequate therapy is increasingly demanding. Fosfomycin recently became an appealing treatment option of bacterial infections due to multidrug-resistant bacteria (MDR). So far, fosfomycin synergy with other antibiotics has been assessed in studies, but only a limited number focused on MDR P. aeruginosa and on the effect of these combinations on the duration of the postantibiotic effect (PAE). We investigated synergy of fosfomycin with an array of antipseudomonal antibiotics using gradient diffusion strip cross method and time-kill method, and their effect on the duration of PAE against 51 variously resistant P. aeruginosa isolates. The highest rate of synergy was observed for combination with ceftazidime (23.4%) and gentamicin (19.1%). The PAE of antibiotic combinations was superior to that of the drugs alone. Our findings indicate that fosfomycin combination therapy may be a valuable treatment alternative.

Synthesis, antibacterial and cytotoxic activity evaluation of hydroxyurea derivatives.

5 Synthesis and biological evaluation of a series (N = 16) of cyclic and acyclic hydroxyurea derivatives, including benzotriazole-, isocyanuric acid- and biuret-containing compounds, are disclosed. 1-N-(benzyloxycarbamoyl)benzotriazole was used as a benzyloxyisocyanate donor, a useful intermediate in the preparation of substituted hydroxyurea. Antibacterial activities of synthesized hydroxyurea derivatives were tested on three E. coli strains, i.e., a strain susceptible to antibiotics, a strain resistant to macrolide antibiotics and a strain resistant to aminoglycoside antibiotics. Six compounds (three acyclic and three cyclic hydroxyureas) showed growth inhibition of the tested E. coli strains, with different specificity toward each strain. Results of the cytotoxic activity evaluation revealed that twelve out of sixteen test compounds were cytotoxic to human acute monocytic leukemia THP-1 and/or human acute T cell leukemia Jurkat cell line. 1-(N-hydroxycarbamoyl) benzotriazole () increased the metabolic activity of both cell lines. Two compounds, 1-(N-hydroxycarbamoyl) benzotriazole (5) and N,N',N''-trihydroxybiuret (15), were identified as potential NO donors.

Improvement of pBBR1MCS plasmids, a very useful series of broad-host-range cloning vectors.

pBBR1MCS vectors are small in size, contain unique cloning sites within the lacZα gene, and are mobilizable and compatible with various plasmid incompatibility groups. We cloned four genes for aminoglycoside resistance methyltransferases from the Arm and Kam families into pBBR1MCS-3 and expressed them in Escherichia coli. The activity of two of these enzymes was impaired because of the fusion with the first 20 amino acids of the ß-galactosidase α-peptide derived from the pBBR1MCS-3 vector. In order to overcome this problem, we introduced by site-directed mutagenesis a new NdeI restriction site into pBBR1MCS-3 to generate a start codon directly at the beginning of lacZα gene. We modified the pBBR1MCS-2, 4 and 5 plasmids in the same manner and obtained the enhanced pBBR1MCS_START vector series that retains all the useful features of the previous vectors, but eliminates the unknown effect of the fusion with the ß-galactosidase α-peptide.

Structural basis for the methylation of A1408 in 16S rRNA by a panaminoglycoside resistance methyltransferase NpmA from a clinical isolate and analysis of the NpmA interactions with the 30S ribosomal subunit.

NpmA, a methyltransferase that confers resistance to aminoglycosides was identified in an Escherichia coli clinical isolate. It belongs to the kanamycin-apramycin methyltransferase (Kam) family and specifically methylates the 16S rRNA at the N1 position of A1408. We determined the structures of apo-NpmA and its complexes with S-adenosylmethionine (AdoMet) and S-adenosylhomocysteine (AdoHcy) at 2.4, 2.7 and 1.68 Å, respectively. We generated a number of NpmA variants with alanine substitutions and studied their ability to bind the cofactor, to methylate A1408 in the 30S subunit, and to confer resistance to kanamycin in vivo. Residues D30, W107 and W197 were found to be essential. We have also analyzed the interactions between NpmA and the 30S subunit by footprinting experiments and computational docking. Helices 24, 42 and 44 were found to be the main NpmA-binding site. Both experimental and theoretical analyses suggest that NpmA flips out the target nucleotide A1408 to carry out the methylation. NpmA is plasmid-encoded and can be transferred between pathogenic bacteria; therefore it poses a threat to the successful use of aminoglycosides in clinical practice. The results presented here will assist in the development of specific NpmA inhibitors that could restore the potential of aminoglycoside antibiotics.

Tobramycin at subinhibitory concentration inhibits the RhlI/R quorum sensing system in a Pseudomonas aeruginosa environmental isolate.

BACKGROUND: Antibiotics are not only small molecules with therapeutic activity in killing or inhibiting microbial growth, but can also act as signaling molecules affecting gene expression in bacterial communities. A few studies have demonstrated the effect of tobramycin as a signal molecule on gene expression at the transcriptional level and its effect on bacterial physiology and virulence. These have shown that subinhibitory concentrations (SICs) of tobramycin induce biofilm formation and enhance the capabilities of P. aeruginosa to colonize specific environments. METHODS: Environmental P. aeruginosa strain PUPa3 was grown in the presence of different concentrations of tobramycin and it was determined at which highest concentration SIC, growth, total protein levels and translation efficiency were not affected. At SIC it was then established if phenotypes related to cell-cell signaling known as quorum sensing were altered. RESULTS: In this study it was determined whether tobramycin sensing/response at SICs was affecting the two independent AHL QS systems in an environmental P. aeruginosa strain. It is reasonable to assume that P. aeruginosa encounters tobramycin in nature since it is produced by niche mate Streptomyces tenebrarius. It was established that SICs of tobramycin inhibited the RhlI/R system by reducing levels of C4-HSL production. This effect was not due to a decrease of rhlI transcription and required tobramycin-ribosome interaction. CONCLUSIONS: Tobramycin signaling in P. aeruginosa occurs and different strains can have a different response. Understanding the tobramycin response by an environmental P. aeruginosa will highlight possible inter-species signalling taking place in nature and can possible also have important implications in the mode of utilization for human use of this very important antibiotic.

Structural basis for the methylation of G1405 in 16S rRNA by aminoglycoside resistance methyltransferase Sgm from an antibiotic producer: a diversity of active sites in m7G methyltransferases.

Sgm (Sisomicin-gentamicin methyltransferase) from antibiotic-producing bacterium Micromonospora zionensis is an enzyme that confers resistance to aminoglycosides like gentamicin and sisomicin by specifically methylating G1405 in bacterial 16S rRNA. Sgm belongs to the aminoglycoside resistance methyltransferase (Arm) family of enzymes that have been recently found to spread by horizontal gene transfer among disease-causing bacteria. Structural characterization of Arm enzymes is the key to understand their mechanism of action and to develop inhibitors that would block their activity. Here we report the structure of Sgm in complex with cofactors S-adenosylmethionine (AdoMet) and S-adenosylhomocysteine (AdoHcy) at 2.0 and 2.1 A resolution, respectively, and results of mutagenesis and rRNA footprinting, and protein-substrate docking. We propose the mechanism of methylation of G1405 by Sgm and compare it with other m(7)G methyltransferases, revealing a surprising diversity of active sites and binding modes for the same basic reaction of RNA modification. This analysis can serve as a stepping stone towards developing drugs that would specifically block the activity of Arm methyltransferases and thereby re-sensitize pathogenic bacteria to aminoglycoside antibiotics.

The aminoglycoside resistance methyltransferase Sgm impedes RsmF methylation at an adjacent rRNA nucleotide in the ribosomal A site.

Ribosome-targeting antibiotics block protein synthesis by binding at functionally important regions of the bacterial rRNA. Resistance is often conferred by addition of a methyl group at the antibiotic binding site within an rRNA region that is already highly modified with several nucleotide methylations. In bacterial rRNA, each methylation requires its own specific methyltransferase enzyme, and this raises the question as to how an extra methyltransferase conferring antibiotic resistance can be accommodated and how it can gain access to its nucleotide target within a short and functionally crowded stretch of the rRNA sequence. Here, we show that the Sgm methyltransferase confers resistance to 4,6-disubstituted deoxystreptamine aminoglycosides by introducing the 16S rRNA modification m(7)G1405 within the ribosomal A site. This region of Escherichia coli 16S rRNA already contains several methylated nucleotides including m(4)Cm1402 and m(5)C1407. Modification at m(5)C1407 by the methyltransferase RsmF is impeded as Sgm gains access to its adjacent G1405 target on the 30S ribosomal subunit. An Sgm mutant (G135A), which is impaired in S-adenosylmethionine binding and confers lower resistance, is less able to interfere with RsmF methylation on the 30S subunit. The two methylations at 16S rRNA nucleotide m(4)Cm1402 are unaffected by both the wild-type and the mutant versions of Sgm. The data indicate that interplay between resistance methyltransferases and the cell's own indigenous methyltransferases can play an important role in determining resistance levels.

Nm23-H1 promotes adhesion of CAL 27 cells in vitro.

nm23-H1 was found to diminish metastatic potential of carcinoma cell lines and therefore was placed in the group of metastatic suppressor genes. Its protein product has a function of a nucleoside diphosphate kinase (NDPK) as well as protein kinase and nuclease. Though it was found that Nm23-H1 is involved in many cellular processes, it is still not known how it promotes metastatic suppressor activity. Since the process of metastasis is dependent on adhesion properties of cells, the goal of our work was to describe the adhesion properties of CAL 27 cells (oral squamous cell carcinoma of the tongue) overexpressing FLAG/nm23-H1. In our experiments, cells overexpressing nm23-H1 show reduced migratory and invasive potential. Additionally, cells overexpressing nm23-H1 adhere stronger on substrates (collagen IV and fibronectin) and show more spread morphology than the control cells. Results obtained by EGF induction of migration revealed that the adhesion strength predetermined cell response to chemoattractant and that Nm23-H1, in this cell type, does not interfere with, EGF induced, Ras signaling pathway. These data contribute to the overall knowledge about nm23-H1 and its role in cell adhesion, migration, and invasion, especially in oral squamous cell carcinoma.

Modeling and experimental analyses reveal a two-domain structure and amino acids important for the activity of aminoglycoside resistance methyltransferase Sgm.

Methyltransferases that carry out posttranscriptional N7-methylation of G1405 in 16S rRNA confer bacterial resistance to aminoglycoside antibiotics, including kanamycin and gentamicin. Genes encoding enzymes from this family (hereafter referred to as Arm, for aminoglycoside resistance methyltransferases) have been recently found to spread by horizontal gene transfer between various human pathogens. The knowledge of the Arm protein structure would lay the groundwork for the development of potential resistance inhibitors, which could be used to restore the potential of aminoglycosides to act against the resistant pathogens. We analyzed the sequence-function relationships of Sgm MTase, a member of the Arm family, by limited proteolysis and site-directed and random mutagenesis. We also modeled the structure of Sgm using bioinformatics techniques and used the model to provide a structural context for experimental results. We found that Sgm comprises two domains and we characterized a number of functionally compromised point mutants with substitutions of invariant or conserved residues. Our study provides a low-resolution (residue-level) model of sequence-structure-function relationships in the Arm family of enzymes and reveals the cofactor-binding and substrate-binding sites. These functional regions will be prime targets for further experimental and theoretical studies aimed at defining the reaction mechanism of m7 G1405 methylation, increasing the resolution of the model and developing Arm-specific inhibitors.

Virtual screening and experimental verification to identify potential inhibitors of the ErmC methyltransferase responsible for bacterial resistance against macrolide antibiotics.

Methyltransferases from the Erm family catalyze S-adenosyl-L-methionine-dependent modification of a specific adenine residue in bacterial 23S rRNA, thereby conferring resistance to clinically important macrolide, lincosamide, and streptogramin B antibiotics. Thus far, no inhibitors of these enzymes have been identified or designed that would effectively abolish the resistance in vivo. We used the crystal structure of ErmC' methyltransferase as a target for structure-based virtual screening of a database composed of 58,679 lead-like compounds. Among 77 compounds selected for experimental validation (63 predicted to bind to the catalytic pocket and 14 compounds predicted to bind to the putative RNA binding site), we found several novel inhibitors that decrease the minimal inhibitory concentration of a macrolide antibiotic erythromycin toward an Escherichia coli strain that constitutively expresses ErmC'. Eight of them have IC(50) values in the micromolar range. Analysis of docking models of the identified inhibitors suggests a novel strategy to develop potent and clinically useful inhibitors.