Drug repurposing: phosphate prodrugs of anticancer and antiviral FDA-approved nucleosides as novel antimicrobials.

OBJECTIVES: Following a drug repurposing approach, we aimed to investigate and compare the antibacterial and antibiofilm activities of different classes of phosphate prodrugs (HepDirect, cycloSal, SATE and mix SATE) of antiviral and anticancer FDA-approved nucleoside drugs [zidovudine (AZT), floxouridine (FUDR) and gemcitabine (GEM)] against a variety of pathogenic Gram-positive and -negative bacteria. METHODS: Ten prodrugs were synthesized and screened for antibacterial activity against seven Gram-negative and two Gram-positive isolates fully susceptible to traditional antibiotics, alongside six Gram-negative and five Gram-positive isolates with resistance mechanisms. Their ability to prevent and eradicate biofilms of different bacterial pathogens in relation to planktonic growth inhibition was also evaluated, together with their effect on proliferation, viability and apoptosis of different eukaryotic cells. RESULTS: The prodrugs showed decreased antibacterial activity compared with the parent nucleosides. cycloSal-GEM-monophosphate (MP) prodrugs 20a and 20b were the most active agents against Gram-positive bacteria (Enterococcus faecalis and Staphylococcus aureus) and retained their activity against antibiotic-resistant isolates. cycloSal-FUDR-MP 21a partially retained good activity against the Gram-positive bacteria E. faecalis, Enterococcus faecium and S. aureus. Most of the prodrugs tested displayed very potent preventive antibiofilm specific activity, but not curative. In terms of cytotoxicity, AZT prodrugs did not affect apoptosis or cell viability at the highest concentration tested, and only weak effects on apoptosis and/or cell viability were observed for GEM and FUDR prodrugs. CONCLUSIONS: Among the different prodrug approaches, the cycloSal prodrugs appeared the most effective. In particular, cycloSal (17a) and mix SATE (26) AZT prodrugs combine the lowest cytotoxicity with high and broad antibacterial and antibiofilm activity against Gram-negative bacteria.

Multiscale computing for science and engineering in the era of exascale performance.

In this position paper, we discuss two relevant topics: (i) generic multiscale computing on emerging exascale high-performing computing environments, and (ii) the scaling of such applications towards the exascale. We will introduce the different phases when developing a multiscale model and simulating it on available computing infrastructure, and argue that we could rely on it both on the conceptual modelling level and also when actually executing the multiscale simulation, and maybe should further develop generic frameworks and software tools to facilitate multiscale computing. Next, we focus on simulating multiscale models on high-end computing resources in the face of emerging exascale performance levels. We will argue that although applications could scale to exascale performance relying on weak scaling and maybe even on strong scaling, there are also clear arguments that such scaling may no longer apply for many applications on these emerging exascale machines and that we need to resort to what we would call multi-scaling. This article is part of the theme issue 'Multiscale modelling, simulation and computing: from the desktop to the exascale'.

FabR regulates Salmonella biofilm formation via its direct target FabB.

BACKGROUND: Biofilm formation is an important survival strategy of Salmonella in all environments. By mutant screening, we showed a knock-out mutant of fabR, encoding a repressor of unsaturated fatty acid biosynthesis (UFA), to have impaired biofilm formation. In order to unravel how this regulator impinges on Salmonella biofilm formation, we aimed at elucidating the S. Typhimurium FabR regulon. Hereto, we applied a combinatorial high-throughput approach, combining ChIP-chip with transcriptomics. RESULTS: All the previously identified E. coli FabR transcriptional target genes (fabA, fabB and yqfA) were shown to be direct S. Typhimurium FabR targets as well. As we found a fabB overexpressing strain to partly mimic the biofilm defect of the fabR mutant, the effect of FabR on biofilms can be attributed at least partly to FabB, which plays a key role in UFA biosynthesis. Additionally, ChIP-chip identified a number of novel direct FabR targets (the intergenic regions between hpaR/hpaG and ddg/ydfZ) and yet putative direct targets (i.a. genes involved in tRNA metabolism, ribosome synthesis and translation). Next to UFA biosynthesis, a number of these direct targets and other indirect targets identified by transcriptomics (e.g. ribosomal genes, ompA, ompC, ompX, osmB, osmC, sseI), could possibly contribute to the effect of FabR on biofilm formation. CONCLUSION: Overall, our results point at the importance of FabR and UFA biosynthesis in Salmonella biofilm formation and their role as potential targets for biofilm inhibitory strategies.

Structure-activity relationship of 2-hydroxy-2-aryl-2,3-dihydro-imidazo[1,2-a]pyrimidinium salts and 2N-substituted 4(5)-aryl-2-amino-1H-imidazoles as inhibitors of biofilm formation by Salmonella Typhimurium and Pseudomonas aeruginosa.

A library of 80 1-substituted 2-hydroxy-2-aryl-2,3-dihydro-imidazo[1,2-a]pyrimidinium salts and 54 2N-substituted 4(5)-aryl-2-amino-1H-imidazoles was synthesized and tested for the antagonistic effect against biofilm formation by Salmonella Typhimurium and Pseudomonas aeruginosa. The nature of the substituent at the 1-position of the salts was found to have a major effect on their biofilm inhibitory activity. Salts with an intermediate length n-alkyl or cyclo-alkyl chain (C7-C10) substituted at the 1-position in general prevented the biofilm formation of both species at low micromolar concentrations, while salts with a shorter n-alkyl or cyclo-alkyl chain (C1-C5) or longer n-alkyl chain (C11-C14) were much less potent. Salts with a long cyclo-alkyl chain however were found to be strong biofilm inhibitors. Furthermore, we demonstrated the biofilm inhibitory potential of salts with certain aromatic substituents at the 1-position, such as piperonyl or 3-methoxyphenetyl. The activity of the 2-aminomidazoles was found to be dependent on the nature of the 2N-substituent. Compounds with a n-butyl, iso-butyl, n-pentyl, cyclo-pentyl or n-hexyl chain at the 2N-position have an improved activity as compared to their unsubstituted counterparts, whereas compounds with shorter 2N-alkyl chains do have a reduced activity and compounds with longer 2N-alkyl chains do have an effect that is dependent on the nature of the substitution pattern of the 4(5)-phenyl ring. Finally, we demonstrated that introduction of a 3-methoxyphenethyl or piperonyl group at the 2N-position of the imidazoles could also result in an enhanced biofilm inhibition.

The small regulatory RNA molecule MicA is involved in Salmonella enterica serovar Typhimurium biofilm formation.

BACKGROUND: LuxS is the synthase enzyme of the quorum sensing signal AI-2. In Salmonella Typhimurium, it was previously shown that a luxS deletion mutant is impaired in biofilm formation. However, this phenotype could not be complemented by extracellular addition of quorum sensing signal molecules. RESULTS: Analysis of additional S. Typhimurium luxS mutants indicated that the LuxS enzyme itself is not a prerequisite for a wild type mature biofilm. However, in close proximity of the luxS coding sequence, a small RNA molecule, MicA, is encoded on the opposite DNA strand. Interference with the MicA expression level showed that a balanced MicA level is essential for mature Salmonella biofilm formation. Several MicA targets known to date have previously been reported to be implicated in biofilm formation in Salmonella or in other bacterial species. Additionally, we showed by RT-qPCR analysis that MicA levels are indeed altered in some luxS mutants, corresponding to their biofilm formation phenotype. CONCLUSIONS: We show that the S. Typhimurium biofilm formation phenotype of a luxS mutant in which the complete coding region is deleted, is dependent on the sRNA molecule MicA, encoded in the luxS adjacent genomic region, rather than on LuxS itself. Future studies are required to fully elucidate the role of MicA in Salmonella biofilm formation.

Biofilm Bacteria Use Stress Responses to Detect and Respond to Competitors.

Bacteria use complex regulatory networks to cope with stress, but the function of these networks in natural habitats is poorly understood. The competition sensing hypothesis states that bacterial stress response systems can serve to detect ecological competition, but studying regulatory responses in diverse communities is challenging. Here, we solve this problem by using differential fluorescence induction to screen the Salmonella Typhimurium genome for loci that respond, at the single-cell level, to life in biofilms with competing strains of S. Typhimurium and Escherichia coli. This screening reveals the presence of competing strains drives up the expression of genes associated with biofilm matrix production (CsgD pathway), epithelial invasion (SPI1 invasion system), and, finally, chemical efflux and antibiotic tolerance (TolC efflux pump and AadA aminoglycoside 3-adenyltransferase). We validate that these regulatory changes result in the predicted phenotypic changes in biofilm, mammalian cell invasion, and antibiotic tolerance. We further show that these responses arise via activation of major stress responses, providing direct support for the competition sensing hypothesis. Moreover, inactivation of the type VI secretion system (T6SS) of a competitor annuls the responses to competition, indicating that T6SS-derived cell damage activates these stress response systems. Our work shows that bacteria use stress responses to detect and respond to competition in a manner important for major phenotypes, including biofilm formation, virulence, and antibiotic tolerance.

2D proteome analysis initiates new insights on the Salmonella Typhimurium LuxS protein.

BACKGROUND: Quorum sensing is a term describing a bacterial communication system mediated by the production and recognition of small signaling molecules. The LuxS enzyme, catalyzing the synthesis of AI-2, is conserved in a wide diversity of bacteria. AI-2 has therefore been suggested as an interspecies quorum sensing signal. To investigate the role of endogenous AI-2 in protein expression of the Gram-negative pathogen Salmonella enterica serovar Typhimurium (S. Typhimurium), we performed a 2D-DIGE proteomics experiment comparing total protein extract of wildtype S. Typhimurium with that of a luxS mutant, unable to produce AI-2. RESULTS: Differential proteome analysis of wildtype S. Typhimurium versus a luxS mutant revealed relatively few changes beyond the known effect on phase 2 flagellin. However, two highly differentially expressed protein spots with similar molecular weight but differing isoelectric point, were identified as LuxS whereas the S. Typhimurium genome contains only one luxS gene. This observation was further explored and we show that the S. Typhimurium LuxS protein can undergo posttranslational modification at a catalytic cysteine residue. Additionally, by constructing LuxS-betala and LuxS-PhoA fusion proteins, we demonstrate that S. Typhimurium LuxS can substitute the cognate signal peptide sequences of beta-lactamase and alkaline phosphatase for translocation across the cytoplasmic membrane in S. Typhimurium. This was further confirmed by fractionation of S. Typhimurium protein extracts, followed by Western blot analysis. CONCLUSION: 2D-DIGE analysis of a luxS mutant vs. wildtype Salmonella Typhimurium did not reveal new insights into the role of AI-2/LuxS in Salmonella as only a small amount of proteins were differentially expressed. However, subsequent in depth analysis of the LuxS protein itself revealed two interesting features: posttranslational modification and potential translocation across the cytoplasmic membrane. As the S. Typhimurium LuxS protein does not contain obvious signal motifs, it is speculated that LuxS is a new member of so called moonlighting proteins. These observations might have consequences in future studies on AI-2 quorum signaling in S. Typhimurium.

Brominated furanones inhibit biofilm formation by Salmonella enterica serovar Typhimurium.

Salmonella enterica serovar Typhimurium is a main cause of bacterial food-borne diseases. As Salmonella can form biofilms in which it is better protected against antimicrobial agents on a wide diversity of surfaces, it is of interest to explore ways to inhibit biofilm formation. Brominated furanones, originally extracted from the marine alga Delisea pulchra, are known to interfere with biofilm formation in several pathogens. In this study, we have synthesized a small focused library of brominated furanones and tested their activity against S. enterica serovar Typhimurium biofilm formation. We show that several furanones inhibit Salmonella biofilm formation at non-growth-inhibiting concentrations. The most interesting compounds are (Z)-4-bromo-5-(bromomethylene)-3-alkyl-2(5H)-furanones with chain lengths of two to six carbon atoms. A microarray study was performed to analyze the gene expression profiles of Salmonella in the presence of (Z)-4-bromo-5-(bromomethylene)-3-ethyl-2(5H)-furanone. The induced genes include genes that are involved in metabolism, stress response, and drug sensitivity. Most of the repressed genes are involved in metabolism, the type III secretion system, and flagellar biosynthesis. Follow-up experiments confirmed that this furanone interferes with the synthesis of flagella by Salmonella. No evidence was found that furanones act on the currently known quorum-sensing systems in Salmonella. Interestingly, pretreatment with furanones rendered Salmonella biofilms more susceptible to antibiotic treatment. Conclusively, this work demonstrates that particular brominated furanones have potential in the prevention of biofilm formation by Salmonella serovar Typhimurium.

Small bowel obstruction due to antegrade and retrograde intussusception after gastric bypass: three case reports in two patients, literature review, and recommendations for diagnosis and treatment.

Intestinal obstruction due to intussusception, although rare, is a complication that can cause small bowel obstruction after Roux-en-Y gastric bypass. Possible causes include disordered intestinal motility, the presence of staple lines and other lead points in the intestinal wall, and the anatomic peculiarities of the enteroenterostomy. In the case of antegrade intussusception, the lead point is usually identifiable and can involve either limb. Retrograde intussusception is more common but usually has a featureless entry point beginning a few inches below the intestinal anastomosis, with the intussusceptum traversing the enteroenterostomy into either the biliary or Roux limb. Abdominal computed tomography scan is necessary for diagnosis, and early exploration is recommended to avoid ischemia and bowel resection.

Gene expression analysis of monospecies Salmonella typhimurium biofilms using differential fluorescence induction.

Bacterial biofilm formation is an important cause of environmental persistence of food-borne pathogens, such as Salmonella Typhimurium. As the ensemble of bacterial cells within a biofilm represents different physiological states, even for monospecies biofilms, gene expression patterns in these multicellular assemblages show a high degree of heterogeneity. This heterogeneity might mask differential gene expression that occurs only in subpopulations of the entire biofilm population when using methods that average expression output. In an attempt to address this problem and to refine expression analysis in biofilm studies, we used the Differential Fluorescence Induction (DFI) technique to gain more insight in S. Typhimurium biofilm gene expression. Using this single cell approach, we were able to identify 26 genetic loci showing biofilm specific increased expression. For a selected number of identified genes, we confirmed the DFI results by the construction of defined promoter fusions, measurement of relative gene expression levels and construction of mutants. Overall, we have shown for the first time that the DFI technique can be used in biofilm research. The fact that this analysis revealed genes that have not been linked with Salmonella biofilm formation in previous studies using different approaches illustrates that no single technique, in casu biofilm formation, is able to identify all genes related to a given phenotype.

Structure-activity relationship of 4(5)-aryl-2-amino-1H-imidazoles, N1-substituted 2-aminoimidazoles and imidazo[1,2-a]pyrimidinium salts as inhibitors of biofilm formation by Salmonella typhimurium and Pseudomonas aeruginosa.

A library of 112 4(5)-aryl-2-amino-1H-imidazoles, 4,5-diphenyl-2-amino-1H-imidazoles, and N1-substituted 4(5)-phenyl-2-aminoimidazoles was synthesized and tested for the antagonistic effect against biofilm formation by Salmonella Typhimurium and Pseudomonas aeruginosa. The substitution pattern of the 4(5)-phenyl group and the nature of the N1-substituent were found to have a major effect on the biofilm inhibitory activity. The most active compounds of this series were shown to inhibit the biofilm formation at low micromolar concentrations. Furthermore, the influence of 6 imidazo[1,2-a]pyrimidines and 18 imidazo[1,2-a]pyrimidinium salts on the biofilm formation was tested. These compounds are the chemical precursors of the 2-aminoimidazoles in our synthesis pathway. A good correlation was found between the activity of the imidazo[1,2-a]pyrimidinium salts and their corresponding 2-aminoimidazoles, supporting the hypothesis that the imidazo[1,2-a]pyrimidinium salts are possibly cleaved by cellular nucleophiles to form the active 2-aminoimidazoles. However, the imidazo[1,2-a]pyrimidines did not show any biofilm inhibitory activity, indicating that these molecules are not susceptible to in situ degradation to 2-aminoimidazoles. Finally, we demonstrated the lack of biofilm inhibitory activity of an array of 37 2N-substituted 2-aminopyrimidines, which are the chemical precursors of the imidazo[1,2-a]pyrimidinium salts in our synthesis pathway.