Central metabolism is a key player in E. coli biofilm stimulation by sub-MIC antibiotics.

Exposure of Escherichia coli to sub-inhibitory antibiotics stimulates biofilm formation through poorly characterized mechanisms. Using a high-throughput Congo Red binding assay to report on biofilm matrix production, we screened ~4000 E. coli K12 deletion mutants for deficiencies in this biofilm stimulation response. We screened using three different antibiotics to identify core components of the biofilm stimulation response. Mutants lacking acnA, nuoE, or lpdA failed to respond to sub-MIC cefixime and novobiocin, implicating central metabolism and aerobic respiration in biofilm stimulation. These genes are members of the ArcA/B regulon-controlled by a respiration-sensitive two-component system. Mutants of arcA and arcB had a 'pre-activated' phenotype, where biofilm formation was already high relative to wild type in vehicle control conditions, and failed to increase further with the addition of sub-MIC cefixime. Using a tetrazolium dye and an in vivo NADH sensor, we showed spatial co-localization of increased metabolic activity with sub-lethal concentrations of the bactericidal antibiotics cefixime and novobiocin. Supporting a role for respiratory stress, the biofilm stimulation response to cefixime and novobiocin was inhibited when nitrate was provided as an alternative electron acceptor. Deletion of a gene encoding part of the machinery for respiring nitrate abolished its ameliorating effects, and nitrate respiration increased during growth with sub-MIC cefixime. Finally, in probing the generalizability of biofilm stimulation, we found that the stimulation response to translation inhibitors, unlike other antibiotic classes, was minimally affected by nitrate supplementation, suggesting that targeting the ribosome stimulates biofilm formation in distinct ways. By characterizing the biofilm stimulation response to sub-MIC antibiotics at a systems level, we identified multiple avenues for design of therapeutics that impair bacterial stress management.

Combinational Effect of Selected Medicinal Plants and Antibiotics Against Pathogenic Bacteria.

<b>Background and Objective:</b> The emergence of antibiotic resistance is a primary global health concern. As a result, there is an urgent need for new strategies to combat antibiotic-resistant bacteria. One of these essential strategies is the combination of medicinal plants and antibiotics as an alternative to using antibiotics alone which was the objective of this article. <b>Materials and Methods:</b> Nine plant materials were collected from different Egypt localities and then extracted by water. Water extracts were filtered and added with Mueller-Hinton agar during preparation. Nine test bacteria and 13 standard antibiotics were used in the disc diffusion sensitivity method. <b>Results:</b> The activity of Amikacin was increased when combined with most different plant extracts against <i>Escherichia coli</i> while antagonistic against <i>Pseudomonas aeruginosa</i>. Aztreonam, Ceftriaxone, Gentamicin and Nalidixic acid antibiotics showed antagonistic or indifferent effects when combined with most different plant extracts against <i>E. coli</i>, <i>Klebsiella pneumonia</i> and <i>P. aeruginosa</i>. The synergistic effect was achieved in Aztreonam when combined with all plant extracts, while Nalidixic acid showed antagonistic when combined with most plant extracts against <i>Proteus mirabilis</i>. The antagonistic effect was achieved in Aztreonam, Ceftriaxone and Nalidixic acid when combined with <i>Achillea fragrantissima</i>, <i>Artemisia monosperma</i> and <i>Leptadenia pyrotechnica</i>, also Aztreonam with <i>Lycium shawii</i> extract against <i>Salmonella typhimurium</i>. The <i>A. fragrantissima</i> and <i>A. monosperma</i> increase the activity of Novobiocin and Vancomycin against <i>Bacillus cereus</i> and Ampicillin and Cefazolin against <i>Staphylococcus aureus</i> but Novobiocin activity increased with most plant extracts against <i>S. aureus</i>. <b>Conclusion:</b> The combinations of antibiotics with the extracts of medicinal plants displayed varying degrees of effects, synergistic, antagonistic and indifferent according to antibiotic type, plant extract and test organism.

Disparate properties of Burkholderia multivorans and Pseudomonas aeruginosa regarding outer membrane chemical permeabilization to the hydrophobic substances novobiocin and triclosan.

Burkholderia multivorans causes opportunistic pulmonary infections and is intrinsically resistant to many antibacterial compounds including the hydrophobic biocide triclosan. Chemical permeabilization of the Pseudomonas aeruginosa outer membrane affects sensitization to hydrophobic substances. The purpose of the present study was to determine if B. multivorans is similarly susceptive suggesting that outer membrane impermeability properties underlie triclosan resistance. Antibiograms and conventional macrobroth dilution bioassays were employed to establish baseline susceptibility levels to hydrophobic antibacterial compounds. Outer membrane permeabilizers compound 48/80, polymyxin B, polymyxin B-nonapeptide, and ethylenediaminetetraacetic acid were used in attempts to sensitize disparate B. multivorans isolates to the hydrophobic agents novobiocin and triclosan, and to potentiate partitioning of the hydrophobic fluorescent probe 1-N-phenylnapthylamine (NPN). The lipophilic agent resistance profiles for all B. multivorans strains were essentially the same as that of P. aeruginosa except that they were resistant to polymyxin B. Moreover, they resisted sensitization to hydrophobic compounds and remained inaccessible to NPN when treated with outer membrane permeabilizers. These data support the notion that while both phylogenetically-related organisms exhibit general intrinsic resistance properties to hydrophobic substances, the outer membrane of B. multivorans either resists permeabilization by chemical modification or sensitization is mitigated by a supplemental mechanism not present in P. aeruginosa.

CYP broth: a tool for Yersinia pestis isolation in ancient culture collections and field samples.

We developed a simple new selective LB-based medium, named CYP broth, suitable for recovering long-term stored Y. pestis subcultures and for isolation of Y. pestis strains from field-caught samples for the Plague surveillance. It aimed to inhibit the growth contaminating microorganisms and enrich Y. pestis growth through iron supplementation. The performance of CYP broth on microbial growth from different gram-negative and gram-positive strains from American Type Culture Collection (ATCC®) and other clinical isolates, field-caught rodent samples, and more importantly, on several vials of ancient Y. pestis subcultures was evaluated. Additionally, other pathogenic Yersinia species such as Y. pseudotuberculosis and Y. enterocolitica were also successfully isolated with CYP broth. Selectivity tests and bacterial growth performance on CYP broth (LB broth supplemented with Cefsulodine, Irgasan, Novobiocin, nystatin and ferrioxamine E) were evaluated in comparison with LB broth without additive; LB broth/CIN, LB broth/nystatin and with traditional agar media including LB agar without additive, and LB agar and Cefsulodin-Irgasan-Novobiocin Agar (CIN agar) supplemented with 50 µg/mL of nystatin. Of note, the CYP broth had a recovery twofold higher than those of the CIN supplemented media or other regular media. Additionally, selectivity tests and bacterial growth performance were also evaluated on CYP broth in the absence of ferrioxamine E. The cultures were incubated at 28 °C and visually inspected for microbiological growth analysis and O.D.625 nm measurement between 0 and 120 h. The presence and purity of Y. pestis growth were confirmed by bacteriophage and multiplex PCR tests. Altogether, CYP broth provides an enhanced growth of Y. pestis at 28 °C, while inhibiting contaminant microorganisms. The media is a simple, but powerful tool to improve the reactivation and decontamination of ancient Y. pestis culture collections and for the isolation of Y. pestis strains for the Plague surveillance from various backgrounds. KEY POINTS: • The newly described CYP broth improves the recuperation of ancient/contaminated Yersinia pestis culture collections • CYP broth was also efficient in reducing environmental contamination in field-capture samples, improving Y. pestis isolation • CYP broth can also be used for the isolation of Y. enterocolitica and Y. pseudotuberculosis.

Genetic and Chemical Screening Reveals Targets and Compounds to Potentiate Gram-Positive Antibiotics against Gram-Negative Bacteria.

Gram-negative bacteria are intrinsically resistant to a plethora of antibiotics that effectively inhibit the growth of Gram-positive bacteria. The intrinsic resistance of Gram-negative bacteria to classes of antibiotics, including rifamycins, aminocoumarins, macrolides, glycopeptides, and oxazolidinones, has largely been attributed to their lack of accumulation within cells due to poor permeability across the outer membrane, susceptibility to efflux pumps, or a combination of these factors. Due to the difficulty in discovering antibiotics that can bypass these barriers, finding targets and compounds that increase the activity of these ineffective antibiotics against Gram-negative bacteria has the potential to expand the antibiotic spectrum. In this study, we investigated the genetic determinants for resistance to rifampicin, novobiocin, erythromycin, vancomycin, and linezolid to determine potential targets of antibiotic-potentiating compounds. We subsequently performed a high-throughput screen of ∼50,000 diverse, synthetic compounds to uncover molecules that potentiate the activity of at least one of the five Gram-positive-targeting antibiotics. This led to the discovery of two membrane active compounds capable of potentiating linezolid and an inhibitor of lipid A biosynthesis capable of potentiating rifampicin and vancomycin. Furthermore, we characterized the ability of known inhibitors of lipid A biosynthesis to potentiate the activity of rifampicin against Gram-negative pathogens.

Structural Basis of Peptide-Based Antimicrobial Inhibition of a Resistance-Nodulation-Cell Division Multidrug Efflux Pump.

Bacterial efflux pumps in the resistance-nodulation-cell division (RND) family of Gram-negative bacteria contribute significantly to the development of antimicrobial resistance by many pathogens. In this study, we selected the MtrD transporter protein of Neisseria gonorrhoeae as it is the sole RND pump possessed by this strictly human pathogen and can export multiple antimicrobials, including antibiotics, bile salts, detergents, dyes, and antimicrobial peptides. Using knowledge from our previously published structures of MtrD in the presence or absence of bound antibiotics as a model and the known ability of MtrCDE to export cationic antimicrobial peptides, we hypothesized that cationic peptides could be accommodated within MtrD binding sites. Furthermore, we thought that MtrD-bound peptides lacking antibacterial action could sensitize bacteria to an antibiotic normally exported by the MtrCDE efflux pump or other similar RND-type pumps possessed by different Gram-negative bacteria. We now report the identification of a novel nonantimicrobial cyclic cationic antimicrobial peptide, which we termed CASP (cationic antibiotic-sensitizing peptide). By single-particle cryo-electron microscopy, we found that CASP binds within the periplasmic cleft region of MtrD using overlapping and distinct amino acid contact sites that interact with another cyclic peptide (colistin) or a linear human cationic antimicrobial peptide derived from human LL-37. While CASP could not sensitize Neisseria gonorrhoeae to an antibiotic (novobiocin) that is a substrate for RND pumps, it could do so against multiple Gram-negative, rod-shaped bacteria. We propose that CASP (or future derivatives) could serve as an adjuvant for the antibiotic treatment of certain Gram-negative infections previously thwarted by RND transporters. IMPORTANCE RND efflux pumps can export numerous antimicrobials that enter Gram-negative bacteria, and their action can reduce the efficacy of antibiotics and provide decreased susceptibility to various host antimicrobials. Here, we identified a cationic antibiotic-sensitizing peptide (CASP) that binds within the periplasmic cleft of an RND transporter protein (MtrD) produced by Neisseria gonorrhoeae. Surprisingly, CASP was able to render rod-shaped Gram-negative bacteria, but not gonococci, susceptible to an antibiotic that is a substrate for the gonococcal MtrCDE efflux pump. CASP (or its future derivatives) could be used as an adjuvant to treat infections for which RND efflux contributes to multidrug resistance.

Genetic approaches to improve clorobiocin production in Streptomyces roseochromogenes NRRL 3504.

Streptomyces roseochromogenes NRRL 3504 is best known as a producer of clorobiocin, a DNA replication inhibitor from the aminocoumarin family of antibiotics. This natural product currently draws attention as a promising adjuvant for co-application with other antibiotics against Gram-negative multidrug-resistant pathogens. Herein, we expand the genetic toolkit for NRRL 3504 by showing that a set of integrative and replicative vectors, not tested previously for this strain, could be conjugally transferred at high frequency from Escherichia coli to NRRL 3504. Using this approach, we leverage a cumate-inducible expression of cluster-situated regulatory gene novG to increase clorobiocin titers by 30-fold (up to approximately 200 mg/L). To our best knowledge, this is the highest level of clorobiocin production reported so far. Our findings set a working ground for further improvement of clorobiocin production as well as for the application of genetic methods to illuminate the cryptic secondary metabolome of NRRL 3504. Key Points • Efficient system for conjugative transfer of plasmids into NRRL 3504 was developed. • Expression of regulatory genes in NRRL 3504 led to increase in clorobiocin titer. • Secondary metabolome of NRRL 3504 becomes an accessible target for genetic manipulations using the expanded vector set and improved intergeneric conjugation protocol.

Proposal of a novel selective enrichment broth, NCT-mTSB, for isolation of Escherichia albertii from poultry samples.

AIMS: Escherichia albertii is an emerging diarrheagenic pathogen causing food- and water-borne infection in humans. However, no selective enrichment broths for E. albertii have ever been reported. In this study, we tested several basal media, selective supplements and culture conditions which enabled selective enrichment of E. albertii. METHODS AND RESULTS: We developed a selective enrichment broth, novobiocin-cefixime-tellurite supplemented modified tryptic soy broth (NCT-mTSB). NCT-mTSB supported the growth of 22 E. albertii strains, while inhibited growth of other Enterobacteriaceae at 37°C, except for Escherichia coli and Shigella spp. Enrichment of E. albertii was improved further by growth at 44°C, a temperature that suppresses growth of several strains of E. coli/Shigella. Combined use of NCT-mTSB with XR-DH-agar, xylose-rhamnose supplemented deoxycholate hydrogen sulphide agar, enabled isolation of E. albertii when at least 1 CFU of the bacterium was present per gram of chicken meat. This level of enrichment was superior to those obtained using buffered peptone water, modified-EC broth, or mTSB (with novobiocin). CONCLUSIONS: Novobiocin-cefixime-tellurite supplemented modified tryptic soy broth enabled effective enrichment of E. albertii from poultry samples and was helpful for isolation of this bacterium. SIGNIFICANCE AND IMPACT OF STUDY: To our knowledge, this is the first report of selective enrichment of E. albertii from poultry samples.

Multiplex PCR method for the detection of human norovirus, Salmonella spp., Shigella spp., and shiga toxin producing Escherichia coli in blackberry, coriander, lettuce and strawberry.

A multiplex PCR method was developed for the simultaneous detection of murine norovirus (MNV-1) as a surrogate for human norovirus (HuNoV) GI and GII, Salmonella spp., Shigella spp., and Shiga toxin producing Escherichia coli (STEC) in fresh produce. The toxicity of the glycine buffer on bacterial pathogens viability was evaluated. The growth of each of the three pathogens (previously stressed) was evaluated at 35 and 41.5 °C in modified buffered peptone water (mBPW) and trypticase soy broth (TSB), supplemented with vancomycin, novobiocin and brilliant green at two concentration levels. The selected conditions for simultaneous enrichment were: 41.5 °C/mBPW/supplemented with 8 ppm vancomycin, 0.6 ppm novobiocin and 0.2 ppm brilliant green. The pathogens and aerobic plate count (APC) growth was evaluated in the enrichment of lettuce, coriander, strawberry and blackberry under the best enrichment conditions. Starting from 1 to 10 CFU/mL, Salmonella reached from 7.63 to 8.91, Shigella 6.81 to 7.76 and STEC 7.43 to 9.27 log CFU/mL. The population reached for the APC was 5.11-6.56 log CFU/mL. Simultaneous detection by PCR was done using designed primers targeting invA, ipaH, stx1 and stx2 genes, and MNV-1. The detection sensitivity was 10-100 PFU for the MNV-1 and 1-10 CFU for each pathogenic bacteria. This protocol takes 6 h for MNV-1 and 24 h for Salmonella spp., Shigella spp., and STEC detection from the same food portion. In total, 200 samples were analyzed from retail markets from Queretaro, Mexico. Two strawberry samples were positive for HuNoV GI and one lettuce sample was positive for STEC. In conclusion, the method developed in this study is capable of detecting HuNoV GI and GII, Salmonella spp., Shigella spp and STEC from the same fresh produce sample.

Yinzhihuang Oral Liquid Ameliorates Hyperbilirubinemia Induced by δ-Aminolevulinic Acid and Novobiocin in Neonatal Rats.

Yinzhihuang oral liquid (YZH) is a traditional Chinese medicine that has been widely used in Asia to prevent and treat neonatal hyperbilirubinemia, but the published preclinical studies on its anti-hyperbilirubinemia effect are conducted in adult animals, partly due to the lack of preclinical neonatal hyperbilirubinemia animal models. In the present study, we tested six reagents to induce hyperbilirubinemia in neonatal rats, and established two appropriate neonatal hyperbilirubinemia rat models by subcutaneous injection of δ-Aminolevulinic acid (ALA, 200 mg/kg) or novobiocin (NOVO, 200 mg/kg). Oral treatment of YZH (80, 160 and 320 mg/kg) significantly decreased serum conjugated bilirubin levels in ALA-treated neonatal rats and serum unconjugated bilirubin levels in NOVO-treated neonatal rats, respectively. Additionally, pre-treatment of YZH also prevented the increase of serum bilirubin levels in both ALA- and NOVO-treated rats. Mechanistically, YZH significantly up-regulated the mRNA expression of genes involved in hepatic bilirubin disposition (organic anion-transporting polypeptide 1b2, Oatp1b2; multidrug resistance-associated protein 2, Mrp2) and bilirubin conjugation (UDP-glucuronosyltransferase 1a1, Ugt1a1). Additionally, YZH up-regulated the mRNA expression of cytochrome P450 1A1 (Cyp1a1), the target gene of aryl hydrocarbon receptor (AhR), and increased the nuclear protein levels of AhR in livers of neonatal rats. YZH and its two active ingredients, namely baicalin (BCL) and 4'-hydroxyacetophenone (4-HT), up-regulated the mRNA expression of AhR target genes (CYP1A1 and UGT1A1) and increased nuclear protein levels of AhR in HepG2 cells. In conclusion, the present study provides two neonatal hyperbilirubinemia animal models and evaluates the anti-hyperbilirubinemia effect and mechanisms of YZH in neonatal animals.

Customized optical mapping by CRISPR-Cas9 mediated DNA labeling with multiple sgRNAs.

Whole-genome mapping technologies have been developed as a complementary tool to provide scaffolds for genome assembly and structural variation analysis (1,2). We recently introduced a novel DNA labeling strategy based on a CRISPR-Cas9 genome editing system, which can target any 20bp sequences. The labeling strategy is specifically useful in targeting repetitive sequences, and sequences not accessible to other labeling methods. In this report, we present customized mapping strategies that extend the applications of CRISPR-Cas9 DNA labeling. We first design a CRISPR-Cas9 labeling strategy to interrogate and differentiate the single allele differences in NGG protospacer adjacent motifs (PAM sequence). Combined with sequence motif labeling, we can pinpoint the single-base differences in highly conserved sequences. In the second strategy, we design mapping patterns across a genome by selecting sets of specific single-guide RNAs (sgRNAs) for labeling multiple loci of a genomic region or a whole genome. By developing and optimizing a single tube synthesis of multiple sgRNAs, we demonstrate the utility of CRISPR-Cas9 mapping with 162 sgRNAs targeting the 2Mb Haemophilus influenzae chromosome. These CRISPR-Cas9 mapping approaches could be particularly useful for applications in defining long-distance haplotypes and pinpointing the breakpoints in large structural variants in complex genomes and microbial mixtures.

Evaluation of novobiocin and telmisartan for anti-CHIKV activity.

Chikungunya has re-emerged as an epidemic with global distribution and high morbidity, necessitating the need for effective therapeutics. We utilized already approved drugs with a good safety profile used in other diseases for their new property of anti-chikungunya activity. It provides a base for a fast and efficient approach to bring a novel therapy from bench to bedside by the process of drug-repositioning. We utilized an in-silico drug screening with FDA approved molecule library to identify inhibitors of the chikungunya nsP2 protease, a multifunctional and essential non-structural protein required for virus replication. Telmisartan, an anti-hypertension drug, and the antibiotic novobiocin emerged among top hits on the screen. Further, SPR experiments revealed strong in-vitro binding of telmisartan and novobiocin to nsP2 protein. Additionally, small angle x-ray scattering suggested binding of molecules to nsP2 and post-binding compaction and retention of monomeric state in the protein-inhibitor complex. Protease activity measurement revealed that both compounds inhibited nsP2 protease activity with IC50 values in the low micromolar range. More importantly, plaque formation assays could show the effectiveness of these drugs in suppressing virus propagation in host cells. We propose novobiocin and telmisartan as potential inhibitors of chikungunya replication. Further research is required to establish the molecules as antivirals of clinical relevance against chikungunya.

Dilipid Ultrashort Tetrabasic Peptidomimetics Potentiate Novobiocin and Rifampicin Against Multidrug-Resistant Gram-Negative Bacteria.

The development of new antibacterial agents and therapeutic approaches is of high importance to address the global problem of antibiotic resistance. Although antimicrobial peptides are known to synergize with certain antibiotics, their clinical application is limited by their systemic toxicity, protease instability, and high production cost. To overcome these problems, nine dilipid ultrashort tetrabasic peptidomimetics (dUSTBPs) were prepared consisting of three basic amino acids separated by a molecular scaffold, bis(3-aminopropyl)glycine, and were ligated to two fatty acids. Several nonhemolytic dUSTBPs were shown to enhance the activity of several antibiotics against pathogenic Gram-negative bacteria. More importantly, dUSTBP 8, consisting of three l-arginine units and a dilipid of 8 carbons long, potentiated novobiocin and rifampicin consistently against multidrug-resistant (MDR) clinical isolates of Pseudomonas aeruginosa, Acinetobacter baumannii, and Enterobacteriaceae. Preliminary studies suggested that dUSTBPs were likely to potentiate antibiotics through outer membrane permeabilization and/or disruption of active efflux and that dUSTBP 8 exhibited enhanced resistance to trypsin in comparison to the previously described di-C9-KKKK-NH2 antibiotic potentiator. The antibacterial activity of rifampicin and novobiocin was enhanced by dUSTBP 8 comparable to other known outer membrane permeabilizing potentiators including the gold standard polymyxin B nonapeptide. Our results indicate that ultrashort tetrabasic peptidomimetics are potent adjuvants that repurpose novobiocin and rifampicin as potent agents against priority MDR Gram-negative pathogens.

Repurposing of Streptomyces antibiotics as adenosine deaminase inhibitors by pharmacophore modeling, docking, molecular dynamics, and in vitro studies.

Adenosine deaminase (ADA) is an enzyme present in purine metabolic pathway. Its inhibitors are considered to be potent drug lead compounds against inflammatory and malignant diseases. This study aimed to test ADA inhibitory activity of some Streptomyces secondary metabolites by using computational and in vitro methods. The in silico screening of the inhibitory properties has been carried out using pharmacophore modeling, docking, and molecular dynamics studies. The in vitro validation of the selected antibiotics has been carried out by enzyme kinetics and fluorescent spectroscopic studies. The results indicated that novobiocin, an aminocoumarin antibiotic from Streptomyces niveus, has significant inhibition on ADA activity. Hence, the antibiotic can be used as a lead compound for the development of potential ADA inhibitors.

Real-life effect of a microcrystalline tyrosine adjuvanted mite immunotherapy in patients with allergic rhinitis.

Aim: Evaluate the effectiveness and safety of immunotherapy with Acarovac Plus® in a 1-year prospective multicentered real-life study. Methods: A total of 118 adults with allergic rhinitis sensitized to Dermatophagoides received subcutaneous immunotherapy with Acarovac Plus. Treatment outcomes were evaluated at baseline, 6 months and 1 year after treatment initiation. Primary end point was the evolution of the combined symptom and medication score. Secondary end points included other effectiveness outcomes and measurement of product tolerability. Results: Acarovac Plus induced significant improvements in primary and secondary end points after 6 months compared with baseline. These differences persisted after 1 year of treatment (p < 0.001; baseline vs 1 year): combined symptom and medication score (1.60 vs 0.79). No serious adverse events were recorded. Conclusion: Acarovac Plus for 1 year was effective and well tolerated in a real-life setting.

Homodimeric Tobramycin Adjuvant Repurposes Novobiocin as an Effective Antibacterial Agent against Gram-Negative Bacteria.

Low permeability across the outer membrane is a major reason why most antibiotics are ineffective against Gram-negative bacteria. Agents that permeabilize the outer membrane are typically toxic at their effective concentrations. Here, we report the development of a broad-spectrum homodimeric tobramycin adjuvant that is nontoxic and more potent than the gold standard permeabilizing agent, polymyxin B nonapeptide. In pilot studies, the adjuvant confers potent bactericidal activity on novobiocin against Gram-negative bacteria, including carbapenem-resistant and colistin-resistant strains bearing plasmid-borne mcr-1 genes. Resistance development to the combination was significantly reduced, relative to novobiocin alone, and there was no induction of cross-resistance to other antibiotics, including the gyrase-acting fluoroquinolones. Tobramycin homodimer may allow the use of lower doses of novobiocin, overcoming its twin problem of efficacy and toxicity.

Clinical efficiency and safety of Hsp90 inhibitor Novobiocin in avian tibial dyschondroplasia.

Tibial dyschondroplasia (TD) is a bone defect of broilers and other poultry birds that disturbs growth plate and it causes lameness. Previously we evaluated differential expression of multiple genes involved in growth plate angiogenesis and reported the safety and efficacious of medicinal plant root extracted for controlling TD. In this study, clinical and protective effect of an antibiotic Novobiocin (Hsp90 inhibitor) and expression of Hsp90 and proteoglycan aggrecan was examined. The chicks were divided into three groups; Control, thiram-induced TD, and Novobiocin injected TD. After the induction of TD, the Novobiocin was administered through intraperitoneal route to TD-affected birds until the end of the experiment. The expressions and localization of Hsp90 were evaluated by qRT-PCR, immunohistochemistry (IHC) and western blot, respectively. Morphological, histological examinations, and serum biomarker levels were evaluated to assess specificity and protective effects of Novobiocin. The results showed that TD causing retarded growth, enlarged growth plate, distended chondrocytes, irregular columns of cells, decreased antioxidant capacity, reduced protein levels of proteoglycan aggrecan, and upregulated in Hsp90 expression (p < 0.05) in dyschondroplastic birds as compared with control. Novobiocin treatment restored growth plate morphology, reducing width, stimulated chondrocyte differentiation, sprouting blood vessels, corrected oxidative imbalance, decreased Hsp90 expressions and increased aggrecan level. Novobiocin treatment controlled lameness and improved growth in broiler chicken induced by thiram. In conclusion, the accumulation of the cartilage and up-regulated Hsp90 are associated with TD pathogenesis and irregular chondrocyte morphology in TD is along with reduced aggrecan levels in the growth plate. Our results indicate that Novobiocin treatment has potential to reduce TD by controlling the expression of Hsp90 in addition to improve growth and hepatic toxicity in broiler chicken.

Combinatorial drug discovery in nanoliter droplets.

Combinatorial drug treatment strategies perturb biological networks synergistically to achieve therapeutic effects and represent major opportunities to develop advanced treatments across a variety of human disease areas. However, the discovery of new combinatorial treatments is challenged by the sheer scale of combinatorial chemical space. Here, we report a high-throughput system for nanoliter-scale phenotypic screening that formulates a chemical library in nanoliter droplet emulsions and automates the construction of chemical combinations en masse using parallel droplet processing. We applied this system to predict synergy between more than 4,000 investigational and approved drugs and a panel of 10 antibiotics against Escherichia coli, a model gram-negative pathogen. We found a range of drugs not previously indicated for infectious disease that synergize with antibiotics. Our validated hits include drugs that synergize with the antibiotics vancomycin, erythromycin, and novobiocin, which are used against gram-positive bacteria but are not effective by themselves to resolve gram-negative infections.

Reversal of resistance in bacteria underlies synergistic effect of essential oils with conventional antibiotics.

The pervasive of bacterial resistance earnestly threaten the prevention and the treatment of infectious diseases. Therefore, scientific communities take precedence over development of new antimicrobial agents. The aim of the study was to determine antimicrobial potency of three North-African essential oils Pituranthos chloranthus, Teucruim ramosissimum and Pistacia lentiscus individually, and in combination with antibiotics, to inhibit the growth of highly resistant clinical pathogen. Bacteria clinically isolated from patients, subsequently, challenged to a panel of drugs to determine the antibiotic-resistance profiles. Drugs displaying clinically irrelevant CMI were subjected to further studies in order to rescue antibiotic actions. Singular activity of essential oils and activity when combined with an antibiotic was hence elucidated. The results obtained highlighted the occurrence of strong antibacterial potential of essential oils when administrated alone. In the interactive experiment essential oils were found highly effective in reducing the resistance of Methicillin-resistant Staphylococcus aureus to amoxicillin, tetracycline, piperacillin, ofloxacin and oxacillin and resistance of Acinetobacter baumannii to amoxicillin and to ofloxacin in interactive manner. Furthermore, the results proved synergism among essential oils and both antibiotics ofloxacin and novobiocin against the Extended-Spectrum Beta-Lactamase producing E. coli (ESBL). Time kill kinetics was performed with a combination of sub-inhibitory concentrations to confirm the efficiency and killing rate of the combination over time. Further, the hypothetical toxicity of essential oils against human keratinocytes HaCat and murine spleenocytes were examined. The chemical composition of essential oils was assessed by GC/MS analysis and the major constituents found were sabinene, limonene, terpinen-4-ol, and ß-eudesmol.

Evaluation of Chromogenic Medium for Selective Isolation of Yersinia.

Cefsulodin-irgasan-novobiocin agar (CIN) has been used as a selective agar to detect Yersinia in food or human patients; however, its components can inhibit the growth of some strains of Yersinia enterocolitica serovar O3 and Y. pseudotuberculosis. Recently, a new Yersinia selective agar, CHROMagar Yersinia enterocolitica (CAYe), was developed and evaluated as a novel selective agar for pathogenic Y. enterocolitica. In this research, a total of 251Yersinia strains (176 pathogenic Y. enterocolitica, 59 Y. pseudotuberculosis, and 16 non-pathogenic Yersinia) were cultured on both CIN and CAYe for comparison. Except for 10 of 104 pathogenic Y. enterocolitica O3 strains and 59 Y. pseudotuberculosis strains, 198 Yersinia isolates grew on both media after 48 hr of incubation at 32℃. Of the 10 pathogenic Y. enterocolitica O3 which could not grow on CIN or CAYe, 9 strains could not grow on CIN with supplements and 1 strain could not grow CAYe with supplements. Of 9 strains which did not grow on CIN with supplements, 3 strains could not grow on CIN without supplements. However, 1 strain which did not grow on CAYe with supplements could grow on CAYe without supplements. All of the Y. pseudotuberculosis strains could grow on CIN with/without supplements and on CAYe without supplements. The results indicate that the inhibition of the growth of Y. enterocolitica O3 on CIN is related to the components of CIN; however, the inhibition on CAYe appears to be related to the supplements in CAYe. Therefore, CAYe may be a more useful selective medium than CIN for pathogenic Y. enterocolitica .