Aminoglycoside Family Research Articles

Fluorescent reporters give new insights into antibiotics-induced nonsense and frameshift mistranslation

We developed a reporter system based on simultaneous expression of two fluorescent proteins: GFP as a reporter of the capacity of protein synthesis and mutated mScarlet-I as a reporter of translational errors. Because of the unique stop codons or frameshift mutations introduced into the mScarlet-I gene, red fluorescence was produced only after a mistranslation event. These reporters allowed us to estimate mistranslation at a single cell level using either flow cytometry or fluorescence microscopy. We found that laboratory strains of Escherichia coli are more prone to mistranslation compared to the clinical isolates. As relevant for uropathogenic E. coli, growth in human urine elevated translational frameshifting compared to standard laboratory media, whereas different standard media had a small effect on translational fidelity. Antibiotic-induced mistranslation was studied by using amikacin (aminoglycoside family) and azithromycin (macrolide family). Bactericidal amikacin induced preferably stop-codon readthrough at a moderate level. Bacteriostatic azithromycin on the other hand induced both frameshifting and stop-codon readthrough at much higher level. Single cell analysis revealed that fluorescent reporter-protein signal can be lost due to leakage from a fraction of bacteria in the presence of antibiotics, demonstrating the complexity of the antimicrobial activity.

Lactiplantibacillus plantarum, lactiplantibacillus pentosus and inulin meal inclusion boost the metagenomic function of broiler chickens

BackgroundThe inclusion of alternative ingredients in poultry feed is foreseen to impact poultry gut microbiota. New feeding strategies (probiotics/prebiotics) must be adopted to allow sustainable productions. Therefore, the current study aimed to use metagenomics approaches to determine how dietary inclusion of prebiotic (inulin) plus a multi-strain probiotic mixture of Lactiplantibacillus plantarum and Lactiplantibacillus pentosus affected microbiota composition and functions of the gastro-intestinal tract of the broilers during production. Fecal samples were collected at the beginning of the trial and after 5, 11 and 32 days for metataxonomic analysis. At the end of the trial, broilers were submitted to anatomo-pathological investigations and caecal content was subjected to volatilome analysis and DNAseq.ResultsProbiotic plus prebiotic inclusion did not significantly influence bird performance and did not produce histopathological alterations or changes in blood measurements, which indicates that the probiotic did not impair the overall health status of the birds. The multi-strain probiotic plus inulin inclusion in broilers increased the abundance of Blautia, Faecalibacterium and Lachnospiraceae and as a consequence an increased level of butyric acid was observed. In addition, the administration of probiotics plus inulin modified the gut microbiota composition also at strain level since probiotics alone or in combination with inulin select specific Faecalibacterium prausnitzi strain populations. The metagenomic analysis showed in probiotic plus prebiotic fed broilers a higher number of genes required for branched-chain amino acid biosynthesis belonging to selected F. prausnitzi strains, which are crucial in increasing immune function resistance to pathogens. In the presence of the probiotic/prebiotic a reduction in the occurrence of antibiotic resistance genes belonging to aminoglycoside, beta-lactamase and lincosamide family was observed.ConclusionsThe positive microbiome modulation observed is particularly relevant, since the use of these alternative ingredients could promote a healthier status of the broiler’s gut.

Bacteriological study and its antibiotics susceptibility pattern of Otitis Media in Iraqi patients

Otitis media is an acute upper respiratory tract infection-related inflammation of the middle ear and tympanic membrane, frequently affecting children. Typically, a subsequent bacterial infection complicates a viral infection, which ultimately causes the condition. The study aims to study the function of bacterial ear infections and its causes, as well as their resistance to medications, which was the focus of this investigation. The first axis of the research was the identification of bacterial isolates using recognized diagnostic tools, and the second axis was determining the antibiotic's resistance and sensitivity. Patients with otitis media were gathered from Al-Hakim General Hospital and Al-Sadr city hospital in Al-Najaf city between November 2020 and April 2021 for 100 clinical samples. More than 80 samples were found to be infected with bacteria. Bacterial strains found in this investigation are ( 30 ) isolates of Pseudomonas aeruginosa, (20) isolates of Klebsiella spp, (20) isolates of Proteus spp, ( 15 ) isolates of Staphylococcus aureus, (8) isolates Escherichia coli and (7) isolates Enterococcus fecalies. As part of this research, the disk diffusion method was used to assess how sensitive the test was. The results showed that Pseudomonas aeruginosa was resistant to most antibiotics, particularly the penicillin family, cephalosporin, and trimethoprim, with the existence of isolates resistant to meropenem. The investigation results varied for the quinolone, aminoglycoside, and macrolide families. Klebsiella spp. were tested for antibiotic sensitivity and found to be resistant to most antibiotics, particularly those in the penicillin family, cephalosporins, and trimethoprim. Some quinolones, aminoglycosides, and macrolides are also resistant. Proteus spp were resistant to most antibiotics, particularly the penicillin family (except for augmentin, which had some sensitive isolates) and cephalosporin (except for cefdinir and cefepime) had some susceptible isolates) and trimethoprim, in addition to the presence of isolates resistant to meropenem. There is a discrepancy in the examination results for the quinolone family. The aminoglycoside family is also highly resistant. S. aureus isolates were resistant to penicillin (except for augmentin, which some isolates were responsive to), trimethoprim, and quinolones, with the presence of isolates resistant to vancomycin. The macrolide class ( azithromycin) also has a significant resistance level. Escherichia coli is susceptible to meropenem, imipenem, and certain cephalosporin generations. Augmentin, cefepime, cephalothin, meropenem, imipenem, and azithromycin were ineffective against Enterococcus fecal. The conclusion is that Pseudomonas spp has a role in ear infections and the germs Klebsiella spp., Proteus spp., Staphylococcus aureus, Escherichia coli, and Enterococcus fecalies. Penicillin and cephalosporin resistance was seen in the majority of the identified isolates. The existence of isolates of Proteus and Pseudomonas species resistant to meropenem. Vancomycin-resistant strains of Staphylococcus aureus isolates are present. Keywords: Otitis media, Resistance antibiotic, S.aureus, P.aerginosa

Phenotypic Characterization and Prevalence of Carbapenemase-Producing Pseudomonas aeruginosa Isolates in Six Health Facilities in Cameroon

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen with a great ability to adapt to stress, in particular, to the selective pressure of antibiotics in the hospital environment. This pathogen constitutes a real public health concern, especially in low- and middle-income countries. In Cameroon, little is known about the drug resistance patterns of Pseudomonas aeruginosa. This study sought to determine the prevalence of Pseudomonas aeruginosa strains producing carbapenemases in six health facilities in the center, littoral, and west regions of Cameroon. An analytical cross-sectional study was conducted over a four-month period from July to October 2021. All Pseudomonas aeruginosa or suspected strains isolated from pathological products at the bacteriology laboratory of different health facilities were systematically collected and underwent a re-identification. After growing on cetrimide agar and successfully subculturing on nutrient agar, an oxidase test was performed on pure colonies, followed by biochemical identification (API 20NE system) of the bacterial suspension (0.5McFarland standard). Drug susceptibility testing for the detection of extended-spectrum beta-lactamases of overproduced inducible cephalosporinases and carbapenemases was performed according to adequate standard procedures. Of the 468 isolates collected, 347 (74.14%) were confirmed Pseudomonas aeruginosa after re-identification, of which 34.49% (120/347) produced inducible cephalosporinases (CAZR and C/TS) and 32.26% (112/347) extended-spectrum beta-lactamases. The prevalence of carbapenemase-producing P. aeruginosa (IMPR and C/TR) was 25.07% (87/347), with 17.24% (15/87) class A and 82.76% (72/87) class B. A high rate of resistance to penicillin (piperacillin: 70.58% and ticarcillin: 60.24%) was observed. We also noted a 34.49% resistance to ceftazidime, 30.22% to imipenem against 37.02% to meropenem, and 25.1% to ceftolozane/tazobactam (C/T). These strains also exhibited 79.57% resistance to quinolones and about 26% to aminoglycoside families. Multivariate analysis revealed that carbapenemase-producing Pseudomonas aeruginosa-related infections were significantly associated with hospitalization (p = 0.04), maternity (p = 0.03), surgery (p = 0.04), and intensive care wards (p = 0.04). This study highlighted a high burden of resistant strains of carbapenemase-producing Pseudomonas aeruginosa. Surveillance should be intensified to prevent the dissemination and spread of these strains.

The comparative effects of erythromycin and amikacin on acute respiratory Pseudomonas aeruginosa infection.

One of the most common causes of pneumonia is Pseudomonas aeruginosa (P. aeruginosa). As with other microbial pathogens, this bacterium tends to develop resistance to various antibiotics. Amikacin and erythromycin, which are from the aminoglycoside and macrolide antibiotic families, are used to treat respiratory infections caused by P. aeruginosa. This study explored whether amikacin, erythromycin or a combination of both works better against P. aeruginosa acute lung infection. For this study, 32 rats were used. The trachea of rats was exposed aseptically and their lung was infected with P. aeruginosa through trachea. Then, according to the group, they received amikacin, erythromycin or a combination of both for 1 week. Finally, they were euthanised on the 3rd and 7th days post-infection. The macroscopic and microscopic evaluations of the lungs, kidney and liver were performed. The right lung was collected for in vivo bacteriological analysis. The amikacin group (A group) had a statistically significantly lower macroscopic and microscopic scores than the other groups (p < 0.05). In vivo bacteriological test revealed that the A group had significantly lower lung bacterial load (p < 0.05). In summary, it was concluded that amikacin could help alleviate the respiratory infection caused by P. aeruginosa solely, and it was more effective than erythromycin.

Synthesis, Characterization, Antimicrobial Activity and Molecular Docking of a Dinuclear Nickel(II) Complex

Bis-(benzidinedioxime) nickel(II) acetate complex was synthesizedby reacting the metal ions with benzidinedioxime in 1:1 mole ratio.The nickel(II) complex was characterized by elemental analysis, NMR, and powder X-ray diffraction. The result obtained from CHN Elemental analysis and 1H nuclear magnetic resonance confirmed that the nickel(II) complex is synthesized by reacting benzidinedioxime with nickel(II) acetate in a 1:1 mole ratio. The powder XRD showed that two Ni(II) coordinated to two benzidinedioxime ligands via the nitrogen atoms of both oxime groups and the two azomethine nitrogen atoms. Powder X-ray diffraction (PXRD) indicated that the nickel(II) complex has little distorted tetrahedral geometry. Nickel(II) complex has a higher antimicrobial activist against K. pneumonia, B. subtilis, and Candida than kanamycin. In addition, the B. subtilis shows resistance against kanamycin (inhibition zone 0) while it shows high sensitivity against bis-benzidinedioxime nickel(II) acetate complex (inhibition zone 33.3 ± 0.82 mm, MIC 18.5 µg/mL). The average MICs of bis-benzidinedioxime nickel(II) acetate are found to be for inhibition of each of the five organisms in the range of 6.2–55.65 µg/mL. The molecular docking studies investigations confirmed that the bis-benzidinedioxime nickel(II) acetate is the most potent aminoglycoside enzymes bacterial inhibitors. The bis-benzidinedioxime nickel(II) acetate exhibited high binding energy with the receptor aminoglycoside-3-phosphotransferase enzyme than receptors of other aminoglycoside enzymes families.

The AMR-ARRAY: A modular bead array detecting β-lactam, (fluoro) quinolone, colistin, aminoglycoside and macrolide resistance determinants in Gram-negative bacteria

The aim of this study was to develop a highly multiplexed bead array to detect genes and/or mutations frequently associated with resistance to antimicrobials of the β-lactam, (fluoro)quinolone, colistin, macrolide and aminoglycoside families in Enterobacteriaceae such as Escherichia coli, Shigella spp. and Salmonella spp. Ligase Chain Reaction and the Luminex® technology were combined in a 53-plex assay designed to target selected genetic markers with 3 internal controls. The AMR-ARRAY consistently detected resistance determinants as compared to phenotypically expressed resistance for 94.7% (856/904) of the assessed resistances. When compared to resistance profiles inferred from whole genome sequencing results, the AMR-ARRAY showed a selectivity and specificity of 99.3% and 100%, respectively. The strong features of the AMR-ARRAY are (i) its competitive cost, currently 18€/sample (ii) its wide analytical scope, currently 50 markers covering 5 antimicrobial families, (iii) its robust and user-friendly design consisting in a single-tube assay conducted in 4 successive steps (iv) its relatively short turnaround time, less than 8 h (v) its ability to detect allelic variability at critical SNPs (vi) its open access and easily upgradable design, with probes sequences, procedure and software source code freely available. The use of the AMR-ARRAY as a screening method in official antimicrobial resistance monitoring could improve the granularity of the collected data and pinpoint remarkable isolates harbouring unusual resistance determinants thereby enabling fit-for-purpose selection of isolates for Whole Genome analysis.

Industrial Composting of Sewage Sludge: Study of the Bacteriome, Sanitation, and Antibiotic-Resistant Strains.

Wastewater treatment generates a huge amount of sewage sludge, which is a source of environmental pollution. Among the alternatives for the management of this waste, industrial composting stands out as one of the most relevant. The objective of this study was to analyze the bacterial population linked to this process and to determine its effectiveness for the reduction, and even elimination, of microorganisms and pathogens present in these organic wastes. For this purpose, the bacteriome and the fecal bacteria contamination of samples from different sewage sludge industrial composting facilities were evaluated. In addition, fecal bacteria indicators and pathogens, such as Salmonella, were isolated from samples collected at key stages of the process and characterized for antibiotic resistance to macrolide, β-lactam, quinolone, and aminoglycoside families. 16S rRNA phylogeny data revealed that the process clearly evolved toward a prevalence of Firmicutes and Actinobacteria phyla, removing the fecal load. Moreover, antibiotic-resistant microorganisms present in the raw materials were reduced, since these were isolated only in the bio-oxidative phase. Therefore, industrial composting of sewage sludge results in a bio-safe final product suitable for use in a variety of applications.

Involvement of oxidative stress and toll-like receptor-4 signaling pathways in gentamicin-induced nephrotoxicity in male Sprague Dawley rats

Gentamicin (GM) is an antibiotic belonging to an aminoglycoside family that might induce nephrotoxicity in human and animal models via oxidative stress. Toll-like receptors (TLRs) are part of innate immune systems that participate in inflammatory responses. In this regard, we investigated the effect of GM on kidney functional and structural parameters, enzymatic antioxidant levels, and mRNA expression of TLR4 and IL6 in the rat kidney. Adult male Sprague Dawley rats were randomly divided into two groups (n = 10): Control and Gentamicin (100 mg/kg, i.p.). After ten days of GM administration, a blood sample was taken, and the kidneys were removed. The serum levels of creatinine (Cr) and blood urea nitrogen (BUN) were measured. Furthermore, the right kidney was preserved in formalin 10% for hematoxylin and eosin (H&E) staining, and the left kidney was kept at −80 °C for molecular and oxidative indexes analysis. Administration of GM caused tubular damages and functional disturbance. So that, Cr and BUN values in the GM group were higher than Control group. Furthermore, molecular findings showed upregulation of TLR4 and IL-6 mRNA expression in renal tissue of the GM-received group. In this study, superoxide dismutase (SOD) activity was slightly increased as a compensatory mechanism in response to elevated malondialdehyde (MDA) levels in the GM-treated group. On the other hand, the activity of catalase (CAT) and glutathione peroxidase (GPx) were significantly declined. Our results demonstrated that oxidative stress and subsequent TLR4 upregulation signaling pathways are involved in GM-induced nephrotoxicity.

&lt;p&gt;&lt;em&gt;Acinetobacter baumannii&lt;/em&gt; Sequence Types Harboring Genes Encoding Aminoglycoside Modifying Enzymes and 16SrRNA Methylase; a Multicenter Study from Pakistan&lt;/p&gt;

IntroductionThe aminoglycosides are widely used for the therapeutic management of infections caused by gram-negative bacteria, including the Acinetobacter baumannii strains. However, the resistance to the members of the aminoglycoside family, such as amikacin, gentamicin, and tobramycin, is increasingly being common among the clinical isolates.PurposeThis study aimed to investigate the presence of 16SrRNA methylases and aminoglycoside modifying enzymes (AMEs) genes among aminoglycoside resistant A. baumannii isolates and to study the genetic diversity of the clinical population of A. baumannii in local hospitals.Material and MethodsThe 143 A. baumannii clinical strains were analyzed for antimicrobial susceptibility, genetic screening for enzymes conferring aminoglycosides resistance followed by the multilocus sequence typing.ResultsThe 133/143 (93%) isolates were non-susceptible to at least one of the tested aminoglycosides, including amikacin, gentamicin, and tobramycin. The MIC distribution has shown that 87.486.7% strains were resistant to amikacin and gentamicin, respectively. The aphA6, aadB, aacC1, and aphA1 were found in 74.1%, 59.4%, 16.1%, and 11.2% isolates, respectively, whereas the armA was found in 28% of the strains having a higher MIC value (MIC; ≥256µg/mL). The MLST data have shown that the ST589 and ST2 were the most common STs and corresponded to 51 (35.7%) and 38 (26.6%) isolates, respectively, and few of the isolates corresponding to these STs were found to harbor the armA gene with a variable genotypic profile for AMEs.DiscussionThe study has reported the incidence of various enzymes conferring aminoglycoside resistance among the A. baumannii clones for the first time from Pakistan. The findings suggest the possibility of transmission of aminoglycoside resistance determinants through the lateral gene transfer as well as clonal dissemination.

Genetics and physiology of Salmonella houtenae isolated from a river in Mexico provides insight into the aquatic habitat influence on its adaptation and pathogenesis

Salmonella enterica subsp. houtenae (IV) is a non-enteric subspecies of the genus Salmonella and has recently been implicated in extraintestinal diseases in humans. In Mexico, its reported that rivers are a reservoir of Salmonella houtenae, however, detailed information about the virulence and infective capacity of this bacterium are limited. Here, we present the high-quality genome draft of Salmonella houtenae str. CFSAN039533 isolated from a river in Culiacan, Mexico. In addition, its virulence, antimicrobial resistance profile and the use of carbon sources for its primary metabolism are analyzed. The genome sequence of CFSAN03953 strain comprises 4.74-Mb which contains 4266 protein coding genes and 77 tRNA genes. The strain belongs to the Marine serotype (48: g, z51: -), and showed several virulence genes related to the SPI-1, SPI-2, SPI-3, SPI-5 and fimbriae genes. The strain exhibited a susceptible phenotype, which contrasted with the resistance genes of aminoglycosides family identified into the genome. Carbon source degradation evaluation along with the genomic analysis, indicated that the strain can use a relatively broad spectrum of compounds related to the central metabolic pathways. This study provides information on the genetics and physiology of Salmonella houtenae that confirms its ability to survive and adapt to the environment. In addition, its pathogenic potential and infective capacity towards warm-blooded hosts are demonstrated.

Analysis of behavioral changes in zebrafish (Danio rerio) larvae caused by aminoglycoside-induced damage to the lateral line and muscles

Zebrafish behavior is influenced by the lateral line hair cells and muscles. Drug-induced behavioral changes can serve as indicators in the evaluation of drug toxicity. The aminoglycoside family of antibiotics comprise a number of agents, including neomycin (NM) and gentamicin (GM). We hypothesized that NM and GM exert different effects on zebrafish larvae through their action on the lateral line and muscle fibers, inducing different swimming behavioral patterns such as locomotor behavior and the startle response. In this study, 125 μM NM and 5, 10, 20 μM GM induced hair cell damage in the anterior and posterior lateral lines of zebrafish larvae. However, unlike GM, 125 μM NM also caused muscle damage. Locomotor behavior was decreased in the 125 μM NM-exposed group compared to the group exposed to GM. Furthermore, 125 μM NM exposure induced significantly different patterns of various indices of startle behavior compared with the GM exposure groups. Additionally, the larvae exhibited different startle responses depending on the concentration of GM. These results suggest that GM may be the drug-of-choice for analyzing behavioral changes in zebrafish caused by damage to the lateral line alone. Our study highlights the importance of confirming muscle damage in behavioral analyses using zebrafish.

Antibiotic Susceptibility and Molecular Detection of Pseudomonas aeruginosa Isolated from Bovine Mastitis

This study aimed to isolate Pseudomonas aeruginosa from cattle (bovine) milk with mastitis to characterize its antimicrobial susceptibility against some antibiotics, and to identify aminoglycoside acetyltransferase (aac-3-Ib) gene.A total of 100 bovine milk samples were collected randomly from different local cow farms at districts of Wasit governorate, Iraq. Six P. aeruginosa isolates were obtained using bacterial culture method and further identified by Analytical Profile Index (API-20E). The antibiotic sensitivity test was performed by disc diffusion methods. Among the 5 antibiotics used, the highest resistance (100%) was found with Nalidixic acid andtetracycline, follow by gentamicin (50%) and the lowest resistance rate (16.6%, and 33.3%) was to the ciprofloxacin and cephalothin respectively. PCR was performed for all the gentamicin resistant isolates that be among the aminoglycoside family. The where the frequency of aac(3)-Ib gene have product (530bp) was to 3 of P. aeruginosa isolates. From the finding of present study we concluded that P.aeruginosa thatisolated from mastitic bovinehave been developed resistance against aminoglycosides through presence of aac(3)-Ibgene and the ciprofloxacin and cephalothin can be taken as good choice of treatment

Detection Of Streptomycin Residues In Sheep Carcasses In Duhok Province, Kurdistan Region, Iraq

In general, the Iraqi farmers of breeding and fattening of sheep using a broad-spectrum antibiotics like tetracycline and aminoglycosides family for treatment and prophylaxis without considering to the withdrawal periods of these drugs before slaughtering the animals. The aim of the present study was to determine the residues of streptomycin (member of aminoglycosides group) in sheep meat and edible tissues using enzyme linked immunosorbent assay (ELISA) method. A total of 88 samples including longissimus dorsi, diaphragmatic muscles, liver and kidney were collected from 22 sheep carcasses slaughtered in Duhok abattoir, Kurdistan, Iraq. The result revealed that streptomycin residues were found in 12 samples (54.5%) of the sheep carcasses distributed as following 36.36 %, 45.45 %, 22.72 % and 50 % of Longissimus dorsi, diaphragm muscle, liver and kidney samples., respectively. The minimal streptomycin concentration in ovine meat samples was 3.001 ppb and the maximum concentration was 690.307 ppb. The results of this study show that sheep meat, which is sold in Dohuk governorate, often contains the residues of antibiotics due to slaughtered animals might have been treated with veterinary drugs and the proper withdrawal period was not respected before slaughtering.

Fluorescent Trimethylated Naphthyridine Derivative with an Aminoalkyl Side Chain as the Tightest Non-aminoglycoside Ligand for the Bacterial A-site RNA.

The bacterial ribosomal decoding region of the aminoacyl-tRNA site (A-site) is one of the most validated target RNAs for antibiotic agents. Although natural aminoglycosides are well-characterized A-site binding ligands, high off-target effects and the growing emergence of bacterial resistance against aminoglycosides limit their clinical use. To circumvent these concerns with the aminoglycoside family, non-aminoglycoside A-site binding ligands have great potential as novel antibiotics against bacterial infections. This work describes a new class of small heterocyclic ligands based on the 2-amino-5,6,7-trimethyl-1,8-naphthyridine (ATMND) structure for the bacterial (Escherichia coli) A-site. ATMND possessing an aminoethyl side chain is found to strongly and selectively bind to the internal loop of the A-site (Kd =0.44 μm; pH 7.0, I=0.06 m, 5 °C). Significantly, this ligand shows the tightest binding reported to date among non-aminoglycoside ligands. The binding study based on the thermodynamics and molecular modelling reveals key molecular interactions of ATMND-C2 -NH2 for high affinity to the A-site. This ligand is also demonstrated to be applicable to the fluorescence indicator displacement assay for assessing ligand/A-site interactions.

Effects of feeding pasteurized waste milk to dairy calves on phenotypes and genotypes of antimicrobial resistance in fecal Escherichia coli isolates before and after weaning

The aim of this study was to evaluate the effects of feeding pasteurized waste milk (pWM) to calves on antimicrobial resistance of fecal Escherichia coli at both phenotypic and genotypic levels. Fifty-two Holstein female calves (3 ± 1.3 d of age) were fed 1 of the 2 different types of milk: milk replacer (MR) without antimicrobials or pWM with β-lactam residues until weaning at 49 d of age. Fecal swabs of all calves were obtained on d 0, 35, and 56 of the study and 3 E. coli isolates per sample were studied. Phenotypic resistance was tested by the disk diffusion method against a panel of 12 antimicrobials. A total of 13 resistance genes consisting of β-lactam, sulfonamide, tetracycline, and aminoglycoside families were examined by PCR. Feeding pWM to calves increased the presence of phenotypic resistance to ampicillin, cephalotin, ceftiofur, and florfenicol in fecal E. coli compared with MR-fed calves. However, the presence of resistance to sulfonamides, tetracyclines, and aminoglycosides was common in dairy calves independent of their milk-feeding source, suggesting other factors apart from the feeding source are involved in the emergence of antimicrobial resistance.

Abstract 3214: Systematic drug repurposing for faster cures of pediatric cancer identifies that Digoxin prolongs survival in a PDOX model of group 4 medulloblastoma

Abstract Background: Medulloblastoma (MB) is the most common malignant brain tumor of childhood. While the current standard care for MB leads to long term survival in approximately 75 % of patients; recurrent and refractory MB continues to have dismal outcomes. In addition, long term survivors face significant treatment-related sequelae especially poor neurocognitive outcomes. Drug repositioning for known drugs is a promising potential strategy not only for drug discovery but also for accelerated translation into the clinical setting. To systematically explore thousands of known drugs available, we integrated computational biology and empirical biology methods to find old drugs for new indications in MB. Results: A non-parametric, bootstrapping based simulated annealing (NPBSA) algorithm was employed to identify driver signaling pathways for over 1,800 patients with Group 3 and Group 4 MB through an integrative analysis on their mRNA expression, DNA-copy number, DNA-methylation and DNA-seq profiles. Then, drug functional networks were constructed based on gene expression profiles under drug treatment as well as chemical structures and were clustered into drug modules with potential mechanisms of action. By evaluating targeted effects of 1,309 drugs from connectivity map database within each drug module in driver signaling pathways, we identified a group of known cardiac glycosides that top ranked among the total drug candidates for the Group 3 and 4 MB subtypes. In addition to traditional chemotherapeutic agents, members of the cardiac aminoglycoside family were repeatedly identified as potential therapeutic agents for MB. These findings were validated in multiple MB-derived cell lines which showed high rates of growth inhibition by cardiac aminoglycosides compared to controls. To evaluate if this growth inhibition in vitro correlated to prolonged survival in vivo, an extensively characterized patient-derived orthotopic-xenograft (PDOX) model of Group 4 MB (ICb-1078MB) was treated with digoxin (2 mg/kg i.p.) for 2 cycles of 14 days. Digoxin treatment significantly prolonged survival to 113 days from a median of 92 days in untreated controls (p=0.001). Histological evaluation of recurrent tumors following digoxin treatment demonstrated changes in the pattern of tumor spread, vascularity and necrosis compared to untreated controls. Conclusions: Leveraging big data in the domains of pharmacogenomics and the notion of drug functional networks and driver signaling pathways represents a powerful tool to repurpose known drugs for new indications in pediatric cancers. Using this integrative biology approach, we identified the cardiac aminoglycosides family generally and Digoxin, specifically, as potential novel agents in the treatment of pediatric medulloblastoma Citation Format: Lei Huang, Sarah G. Injac, Hong Zhao, Qi Lin, Mari Kogiso, Chris T. Man, Xiao-Nan Li, Ching C. Lau, Stephen T. Wong. Systematic drug repurposing for faster cures of pediatric cancer identifies that Digoxin prolongs survival in a PDOX model of group 4 medulloblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3214. doi:10.1158/1538-7445.AM2017-3214

P11.08 Systematic drug repurposing for faster cures of pediatric cancer identifies that Digoxin prolongs survival in a PDOX model of group 4 medulloblastoma

Abstract Background: Medulloblastoma (MB) is the most common malignant brain tumor of childhood. While the current standard care for MB leads to long term survival in approximately 75 % of patients; recurrent and refractory MB continues to have dismal outcomes. In addition, long term survivors face significant treatment-related sequelae especially poor neurocognitive outcomes. Drug repositioning for known drugs is a promising potential strategy not only for drug discovery but also for accelerated translation into the clinical setting. To systematically explore thousands of known drugs available, we integrated computational biology and empirical biology methods to find old drugs for new indications in MB. Results: A non-parametric, bootstrapping based simulated annealing (NPBSA) algorithm was employed to identify driver signaling pathways for over 1,800 patients with Group 3 and Group 4 MB through an integrative analysis on their mRNA expression, DNA-copy number, DNA-methylation and DNA-seq profiles. Then, drug functional networks were constructed based on gene expression profiles under drug treatment as well as chemical structures and were clustered into drug modules with potential mechanisms of action. By evaluating targeted effects of 1,309 drugs from connectivity map database within each drug module in driver signaling pathways, we identified a group of known cardiac glycosides that top ranked among the total drug candidates for the Group 3 and 4 MB subtypes. In addition to traditional chemotherapeutic agents, members of the cardiac aminoglycoside family were repeatedly identified as potential therapeutic agents for MB. These findings were validated in multiple MB-derived cell lines which showed high rates of growth inhibition by cardiac aminoglycosides compared to controls. To evaluate if this growth inhibition in vitro correlated to prolonged survival in vivo, an extensively characterized patient-derived orthotopic-xenograft (PDOX) model of Group 4 MB (ICb-1078MB) was treated with digoxin (2 mg/kg i.p.) for 2 cycles of 14 days. Digoxin treatment significantly prolonged survival to 113 days from a median of 92 days in untreated controls (p=0.001). Histological evaluation of recurrent tumors following digoxin treatment demonstrated changes in the pattern of tumor spread, vascularity and necrosis compared to untreated controls. Conclusions: Leveraging big data in the domains of pharmacogenomics and the notion of drug functional networks and driver signaling pathways represents a powerful tool to repurpose known drugs for new indications in pediatric cancers. Using this integrative biology approach, we identified the cardiac aminoglycosides family generally and Digoxin, specifically, as potential novel agents in the treatment of pediatric medulloblastoma.

Antimicrobial Resistance Characteristics and Phylogenetic Groups of Escherichia coli Isolated From Diarrheic Calves in Southeast of Iran

Background: Antimicrobial resistance is one of the main challenges in diarrheal diseases in human and animals. Regardless to the main reason of the disease, approximately all antimicrobial actions including treatment, control and prevention are mostly centralized against Escherichia coli (E. coli) strains. Objectives: This work purposed to antimicrobial resistance (AR) and determinate virulence genes and phylogenetic groups in E. coli isolates (n=170) obtained from calves with diarrhea. Materials and methods: Isolates were molecular characterized for 17 AR genes and 3 phylogenetic sequences. AR phenotyping were performed on all strains for 12 antimicrobial agents by using disc diffusion method. Results: All AR genes but qnrS were identified with different prevalence in E. coli isolates that the most common genes were aadA (20%), blaTEM (11.7%) and sulII (11.2 %) belonging to aminoglycoside, β-lactamase and sulphonamide families, respectively. Resistance to the penicillin and sulphamethoxazole drugs was found in 100% of isolates and followed by tetracycline (73.5%), streptomycin (60%), trimethoprim sulphamethoxazole (56.5%) and kanamycin (53.5%). The phylogenetic groups A and B1 considerably surrounded the majority of isolates with the frequency of 65.8% and 30.6%, respectively. Conclusions: In Iran, diarrheic calves have an important role as reservoir of resistant E. coli strains against the some drugs which are registered for treatment of calf diarrhea.

Analysis of Amikacin in Human Serum By UHPLC With Fluorescence Detector Using Chloro-Formate Reagent With Glycine

Background: Amikacin belongs to aminoglycosides family, commonly administered in the treatment of systemic infections due to gram negative bacteria. Its narrow therapeutic index results in adverse effects like nephrotoxicity and ototoxicity. Objective: Optimize an ultra-high performance liquid chromatography (UHPLC) based analytical method for the determination of amikacin sulfate in human serum using derivatizaon with FMOCCl and glycine. Methods: Pre-column derivatization reaction of amikacin performed using fluorescence reagent 9-fluorenylmethyl chloroformate (FMOC-Cl) at ambient temperature in the presence of borate buffer (0.2 M). Stabilizing reagent glycine (0.1 M) added into the reaction mixture solution after completion of the derivatization reaction for stabilization of fluorescent complex product. Fluorimetric detection of amikacin was performed at excitation and emission wavelength of 265 nm and 315 nm respectively, using C18 UHPLC column. The reported method was validated by performing linearity, precision, recovery and ruggedness. Results: The optimum mobile phase composition was found to be Acetonitrile:water in the ratio of 70:30 (v/v) at flow rate of 0.4 ml/min. A linear response of amikacin in serum samples ranging from 0.5-10 μg/ml was obtained, with correlation co-efficient of 1.00. The limit of detection (LOD) was found to be 50 ng/ml. Both inter- and intra-day analysis co-efficient values were found to be less than 10%. Conclusion: The developed UHPLC method will be useful for pre-clinical and pharmacokinetic study of amikacin in human serum. Key words: Amikacin Sulphate, Ultra High Performance Liquid Chromatography (UHPLC), 9-Fluorenylmethyl chloroformate (FMOC-Cl) reagent, Borate buffer, Glycine, Fluorescence detector.