Day 1 :
Keynote Forum
Palmer Taylor
Dean of the Skaggs School of Pharmacy & Pharmaceutical Sciences
Keynote: Development of orally bioavailable, blood-brain barrier penetrant antidotes to replace 2-PAM as an antidote in organophosphate exposure
Time : 09:50-10:40
Biography:
Palmer Taylor, Ph.D. holds the Sandra & Monroe Trout Chair of Pharmacology at the University of California, San Diego. He served as Founding Dean of the Skaggs School of Pharmacy & Pharmaceutical Sciences (2002-2014) and previously as Chair of the Department of Pharmacology in the School of School of Medicine (1987-2002). His research is directed to molecular pharmacology and neurotransmission, through study of the structures and functions of acetylcholinesterase (AChE) and nicotinic acetylcholine receptors (nAChRs). Of particular interest are molecular recognition and design of selective therapeutic agents, directed to the ï¡ï€·ï€nAChR subtype and to antidotes that reactivate of organophosphate-conjugated AChE. The soluble acetylcholine binding protein has been the template for design of agonists and antagonists of the ï¡ï€·ï€AChR subtype potentially useful in treatment of disorders of nervous system development and aging. His group has also been responsible for development of small zwitterion antidotes that serve as reactivators for organophosphate poisoning.
Abstract:
Current antidotes to organophosphate exposure from pesticides and the more insidious nerve agents of terrorism are limited by their inability to cross the blood-brain barrier, their rapid clearance and lack of oral bioavailability. To enhance antidote activity, we have developed a library of zwitterion antidotes with measured in vitro reactivation capabilities and capacity to cross the blood-brain barrier and reactivate cholinesterase in the central and peripheral nervous system. Lead compounds show good protection ratios in mice and have led to more detailed pharmacokinetic and tissue disposition studies in mice and macaques. Antidotes, in contrast to therapeutic agents in treatment of chronic disease, require immediate absorption and distribution to the target tissue of action. To this end, we have developed a small molecule intramuscular loading dose-oral maintenance dose scheme for the treatment of organophosphate poisoning from nerve agents used in terrorism and pesticides used in agriculture and home/gardens. By virtue of the ionization equilibria transitioning at physiological pH values between amine cation and oximate anion, both with pKa values between 8 and 9, a cationic or zwitterion species is available for reactivation of organophosphate conjugated AChE within the active center gorge in target tissues and a neutral species, that can readily cross the blood-brain barrier and exhibit oral bioavailability, treatment immediately after exposure by subcutaneous organophosphates or inhaled sarin vapor reverses toxicity in mice and macaques. The presentation will review the pharmacokinetics and tissue disposition required for successful antidote treatment of terrorism-inspired events.
Keynote Forum
Mewa Singh
Meda Biotech LLC, USA
Keynote: Hybrid-nano engineeringâ„¢ for Paclitaxel
Time : 11:00-11:50
Biography:
Dr. Mewa Singh heads research and product development Meda Biotech llc. With over 24 years of experience in the field of biopharmaceuticals. He provides expertise in Biotechnology, product development and the pharmaceuticals business market. Over the time period of 24 years. He has successfully launched products for diagnostics, vaccines, nutraceuticals and nanomedicines. He has applied five patents and is founder of two biotech start-ups (Meda Biotech llc and Nano Biotech (P) Ltd. He has performed at administrative and senior management positions which included Director of R&D, Chief Scientific Officer, and COO positions. He holds M.S. in Biochemistry, M Phil in Biochemistry, Microbiology and Immunology, and PhD in Microbiology and Immunology.. Developing over 160 Nanomedicines for Pharmaceuticals, Nutraceuticals and Agrochemicals, he has been working with the strength of conviction that a new concept of Hybrid drugs, is the solubility solution for insoluble drugs. His discovery of Hybrid-Nanoengineering™could have real significant therapeutic value for patients. This belief gave birth to a process of continual discovery and innovation, allowing him to develop many innovative medicines. Since 2002, his company Meda Biotech has been working to commercialize a technology that could make the treatment of cancer, pain and inflammation, more effective and less harmful to patients.
Abstract:
Over the past decade, there is a common problem to number of drug candidates with solubility problems has steadily increased as a result of using combinatory chemistry and high-throughput screening in drug discovery. Presently, 70% of new entities (therapies and drugs) are poorly soluble and 40% of existing immediate-release oral drugs are considered practically insoluble in water. In the pursuit to achieve optimal concentration this often leads to dose escalation. Dose escalation is often undesirable for the following reasons. (1) Increases toxicity, (2) difficulty in designing formulation, (3) manufacturing and treatment costs increase dramatically, and (4) use of toxic carriers and solubilizing agents is increased.
Paclitaxel is one of them, even after so many years the delivery system is still compromised.
• $4.5 Billion market for Taxol in 2013
• Market is growing.
• Taxol patent has expired.
• Nano-technology has been touted as the “next new thing” for several years but has not been perfected for Taxol yet, until now.
• We are not using “me-too technology” like other Nano-medicine formulations.
• Patents were filed in 2012 and 2013.
• Abraxane is a very successful drug on the market currently (run rate > $1B) using nano technology, not really but micronized.
Meda biotech has developed a nano formulation for this molecule. Paclitaxel is attached with curcumin which is a safe natural molecule from turmeric natural anti-inflammatory molecule. Curcumin also known for controlling many type of cancer cells. The end formulation is nano formulation and has better cytotoxicity than Abraxane. It has better stability in acidic and alkaline environment.
Our double edge approach with improved solubility should be a winner
- Assessment of Bioequivalence | Bioavailability Studies
Location: HILL CREST HALL
Chair
Palmer Taylor
Dean of the Skaggs School of Pharmacy & Pharmaceutical Sciences
Session Introduction
Sudheendra K
Syngene Clinical Development, India
Title: Preparing for phase I unit regulatory inspections
Biography:
Sudheendra K has his expertise in managing the GCP Quality Management Systems at Syngene Clinical Development in the conduct of Phase I, Bioequivalence, Bioavailability studies. He has hosted many international regulatory inspections such as US FDA, EMA, ANVISA, Thailand GLP at Syngene Clinical Development. He is a trained biochemist and has been associated with GCP QMS management for the last 14+ years. He has also audited many vendors and service providers who provide support for the contract research organizations for the conduct of early phase studies. He has also audited many complex clinical trials Phase II and Phase III trials have been conducted at different hospital sites.
Email: sudheendra.kulkarni@syngeneintl.com
Abstract:
|
|
Speaker Photo:
Dimensions : 85 (W) x 119(H)
Speaker Logo:
Dimensions : 90 (W) x 90(H)
Yes Delete Logo
Speaker PDF:Speaker Interview PDF:Youtube Id:Vimeo Id:Plenary Title:Plenary Time:Plenary Name: Ex: Stuart Williams (Only First Name & Last Name)Plenary Affiliation:
Abstract:
Clinical Research Organizations and Industry have seen a paradigm shift towards the regulatory inspection scenario and data integrity standards that have taken the primary focus on the rights, safety and well-being of the trial subjects. The DI standards have become one of the key attention area for the regulatory inspections, not that the inspectors are looking for fraud and maleficence. The systems used in the research industry have become more complex in terms of metadata that is being generated across the companies. The metadata that is created in a clinical trial is enormous and the inspectors are very aware of the possible DI issues that occur from time-to-time. The functions such as User Access, Data Backup Policies, Audit Trail, etc. have become the focus of attention. ALCOA principle is applicable to both paper and electronic data, thereby to ensure that the DI standards are followed all the time by all the personnel involved who generate the clinical trial data. To ensure that the DI standards are maintained across the life cycle of the clinical trial, Quality Systems should be in place and the rigor of sustaining the checks be on a continual mode. This will ensure that the clinical trial data generated will meet all the applicable regulatory requirements. No doubt, in the coming days, the DI requirements shall be more demanding and key focus in clinical research.
Sudheendra K
Managing the GCP Quality Management Systems at Syngene Clinical Development in the conduct of Phase I
Title: Preparing for phase I unit regulatory inspections
Time : 11:50-12:20
Biography:
Sudheendra K has his expertise in managing the GCP Quality Management Systems at Syngene Clinical Development in the conduct of Phase I, Bioequivalence, Bioavailability studies. He has hosted many international regulatory inspections such as US FDA, EMA, ANVISA, Thailand GLP at Syngene Clinical Development. He is a trained biochemist and has been associated with GCP QMS management for the last 14+ years. He has also audited many vendors and service providers who provide support for the contract research organizations for the conduct of early phase studies. He has also audited many complex clinical trials Phase II and Phase III trials have been conducted at different hospital sites.
Abstract:
Clinical Research Organizations and Industry have seen a paradigm shift towards the regulatory inspection scenario and data integrity standards that have taken the primary focus on the rights, safety and well-being of the trial subjects. The DI standards have become one of the key attention area for the regulatory inspections, not that the inspectors are looking for fraud and maleficence. The systems used in the research industry have become more complex in terms of metadata that is being generated across the companies. The metadata that is created in a clinical trial is enormous and the inspectors are very aware of the possible DI issues that occur from time-to-time. The functions such as User Access, Data Backup Policies, Audit Trail, etc. have become the focus of attention. ALCOA principle is applicable to both paper and electronic data, thereby to ensure that the DI standards are followed all the time by all the personnel involved who generate the clinical trial data. To ensure that the DI standards are maintained across the life cycle of the clinical trial, Quality Systems should be in place and the rigor of sustaining the checks be on a continual mode. This will ensure that the clinical trial data generated will meet all the applicable regulatory requirements. No doubt, in the coming days, the DI requirements shall be more demanding and key focus in clinical research.
Ashish A Mungantiwar
President Medical Services in Macleod’s Pharmaceuticals Limited
Title: Bioequivalence inspection
Time : 12:20-12:50
Biography:
Ashish Mungantiwar a PhD pharmacologist by profession. He is working as President Medical Services in Macleod’s Pharmaceuticals Limited. He heads various departments like Bioequivalence, Clinical trial and Pharmacovigilance. As Head of Bioequivalence he is served as Study Director for more than 2000 Bioequivalence studies. He has successfully handled more than 25 inspections from USFDA, WHO, EU and ANVISA. He has attended meeting with regulators of WHO and EU during approval process of the product. In order to keep his academic temperament alive he works as PhD guide in industry and has already guided 5 PhD students. He is invited speaker in various international conferences.
Abstract:
Statement of the Problem: Bioequivalence study is conducted on healthy volunteer/patient to compare pharmacokinetic parameter of test product and reference product. Generic company conducting/sponsoring the study should be aware of risks of rejection and non-compliance.
Methodology & Theoretical Orientation: Current topic will cover list of inspection findings from various regulatory agencies like US, EU and WHO and How to be ready for unannounced inspection, Presentation will discuss current inspection trends and queries asked during dossier assessment.
Conclusion & Significance: This presentation will critically review inspector and assessors expectation on Bioequivalence studies. Understanding such expectation will help for early and successful approval of the product.
Muneesh Garg
Sitec Labs. Pvt. Ltd, India
Title: Assessment of bioequivalence between two formulations of Salmeterol xinafoate/Fluticasone propionate HFA pMDI 25/250 mcg per actuation in healthy volunteers
Time : 13:50-14:20
Biography:
Dr. Muneesh Garg has completed his MD (Physician) from Dagestan State Medical Academy, Russia and MD (Pharmacology) from Goverment Medical College, Patiala, Punjab, India. He has more than 18 years of experience in academia, and clinical research. He is the principal investigator of Sitec Labs Pvt Ltd., Navi Mumbai, India, for more than 11 years and has completed about 1000 BA/BE studies. He has published many research papers in reputed journals.
Abstract:
Salmeterol xinafoate and fluticasone propionate has been shown to be effective and well tolerated in the treatment of asthma. Our research aimed to determine the bioequivalence between two formulations (test and reference) of salmeterol xinafoate/fluticasone propionate HFA pMDI 25/250 mcg per actuation with and without charcoal blockade. Study-1 was a single dose, randomized, 4-period, 2-sequence, laboratory-blinded, crossover replicate design conducted in 42 healthy volunteers under fasting conditions with concurrent oral charcoal administration (to block gastrointestinal absorption). Study-2 was a single dose, randomized, 2-period, 2-sequence, laboratory-blinded, crossover design conducted in 74 healthy volunteers under fasting conditions without concurrent oral charcoal administration. Both the studies had a minimum washout period of 14 days. Blood samples were collected up to 18 hours post-dose and 36 hours post-dose for pharmacokinetic profiling for study-1 and 2 respectively. Safety evaluations included monitoring adverse events and vital signs as well as clinical laboratory assessments. Plasma concentrations of salmeterol xinafoate and fluticasone propionate were determined using a validated LC-MS/MS method. The 90% CI of the difference between the test and reference for salmeterol xinafoate was 94.10-113.20, and 96.44-116.69 for Cmax, and AUC0-t respectively in study-1. The 90% CI of the difference between the test and reference for salmeterol xinafoate was 83.44-100.29 and 104.08-120.08 and for fluticasone propionate was 91.08-105.07, and 99.86-115.61 for Cmax and AUC0-t respectively in study-2. Since the 90% CI for Cmax and AUC0-t was within the 80–125% interval in both the studies, it was concluded that test and reference formulations of salmeterol xinafoate/fluticasone propionate HFA pMDI 25/250 mcg per actuation are bioequivalent in their rate and extent of absorption with and without charcoal blockade.
Nadin Almosnid
Middle Tennessee State University, USA
Title: In vitro cytotoxicity and initial safety evaluation of extracts from 16 plants used in Yao ethno medicine
Time : 14:20-14:55
Biography:
Dr. Nadin Almosnid is working as Associate professor. She has been published 12 International Journals, 22 National Journals and attended 23 conferences. She belongs to Pharmaceutics and Pharmaceutical Technology specialization.
Abstract:
Cancer is the second cause of death due to the limited treatment options. Because of the side effects and drug resistance of the current chemotherapy, there is an urgent need to develop new chemotherapeutic agents. Based on an ethno pharmacological survey, sixteen plant species widely used in the Medicines of Yao ethnic group in China, which is a special branch of traditional Chinese medicine (TCM), were collected and 64 plant extracts were prepared from these plants. An in vitro cytotoxicity screening was conducted with a panel of four human cancer cell lines namely, lung cancer A549, breast cancer BT20 and MCF-7, bone cancer U2OS. Besides, their possible toxicity was evaluated using two normal human cell lines, lung HPL1A and breast HMEC. A potent activity (IC50 < 5 µg/mL) was observed for ethanol extract from Melaleuca leucadendra Linnagainst against U2OS cells. The most potent extract was acetate extract from Elephantopus scaber against all tested cancer cells (BT20, A549, U2S, and MCF7) with IC50 range between 3.1-24 µg/mL. However, most of the antitumor behaviours were against U2OS cell with IC50 49.3-4 µg/ml. The selectivity index of the active samples varied from 0 to > 100. Additionally, extracts that showed the anti-proliferation activity exhibited the ability of inducing apoptosis in U2OS cells. This study provides in vitro evidence for the traditional use of these ethno medicinal plants, and validates the safety of these plant extracts.
Andras Fodor
University of Pannonia, Hungary
Title: Agrobacterium: a Suitable Genetic System for Analysis Resistance to Xenorhabdus Antimicrobial Peptide Complexes Extremely Active in Food Spoilage (Zoonic) Bacteria
Time : 16:20-16:40
Biography:
Present position: Retired from Senior Research Associate position from the University of Pannonia, Georgikon Faculty, Department of the Animal Sciences & Animal Breeding,
Diploma: Geneticist – biologist & Teacher Certification for High School, 234/1964
PhD: Committee for Doctoral Awards of the Hungarian Academy of Sciences, October 7, 1974, Budapest, Hungary; PhD: Candidate of Biological Sciences; (No. 6.162).
Biological Doctor’s Degree: D-1674/1974, Eötvös Loránd University, Budapest, Hungary, December 3, 1974.
Habilitation: Habilitation Committee of the Eötvös Loránd University, Budapest, Hungary, December 13, 2000, Budapest, Hungary.
Széchenyi Professorship Award: March 19, 1999, Hungarian Ministry of Education
Fulbright Research Grant, a Fulbright Grant Biological Science Grant (1214102) and funds from Valent Biosciences, both awarded to András Fodor to conduct research in the lab of Heidi Goodrich-Blair at the University of Wisconsin-Madison, USA, 2015.
Abstract:
The emergence of antibiotic resistance proves an increasing challenge n prevention and control in microbial (zoonic) pathogens in foods. The aim of this work is to establish a suitable experimental system for genetic analysis of antimicrobial peptide-resistance in a genetically well-characterized Gram-negative target, and found Agrobacterium tumefaciens. Intentionally, not a single but a multiple antimicrobial peptide-complex (EMA_PF2, isolated from Xenorhabdus budapestensis, with a reproducible MALDI profile), was chosen as a model to be bio-assayed in liquid cultures. Antimicrobial activity of EMA_PF2 is demonstrated on t Gram-positive Rhodococcus equi, Erysipelothrix rhusiopathiae, Staphylococcus aureus, Streptococcus equi, Corynebacterium pseudotuberculosis, and Listeria monocytogenes) and Gram negative Salmonella Gallinarum, Salmonella Derby, Bordetella bronchiseptica, Escherichia coli, Pasteurella multocida, Aeromonas hydrophila strains including multi-resistant ones. A. tumefaciens A281 strain with C58 chromosome (originated from a nopaline-catabolizing strain) and intact (transfer-DNA, T-DNA carrying) pTiBo542 plasmid (with agropine (L, L, -succinamopine) catabolizing genes) proved fully resistant to EMA_PF2; while the studied disarmed (transfer-DNA-deleted, del-T-DNA) plasmid-harboring derivatives (AGL1; EHA105 and A4T) were fully sensitive. By contrast, all 5 examined pTi58-plasmid-cured derivatives of C58 nopaline-catabolizing strains proved resistant to EMA_PF2. Two octopine-catabolizing strains behaved differently. Agrobacterium strains sensitive to EMA_PF2, harboring disarmed pTiBo542 provide a system for genetic complementation analysis of resistance to antimicrobial peptides using complementary sequences from resistant strains.
Alejandro Saúl Padrón Yaquis
Center for Drug Research and Development, Cuba
Title: Bioequivalence of two prolonged release diclofenac sodium formulations in healthy volunteers
Time : 15:50-16:20
Biography:
Alejandro Saul Padron Yaquis has completed his PhD from Habana University. He has graduated from Dmitry Mendeleev University of Chemical Technology of Russia. He is the General Director of the Center for Pharmaceuticals Research and Development at BioCubaFarma Organization. He has published more than 25 papers in reputed journals and has been serving as the Director of a Bioequivalence Unit. He has provided training and support for the conduction of BE study in other countries.
Abstract:
Introduction: The implementation of generic drug development programs constitutes a basic component of global health policy. The aim of this work is to determine the existence of bioequivalence between two prolonged release diclofenac sodium formulations in healthy volunteers.
Materials and methods: A phase I, randomized, crossover, double-blind clinical trial was conducted where Voltaren Retard® (Novartis, Switzerland) and a prolonged release Cuban diclofenac sodium formulation (CIDEM, Havana, Cuba) were compared. The sampling period was 40 hours, with a washout time of 15 days between each period. Thirty-six healthy volunteers of both sexes, aged between 18 and 50, were included. All subjects received orally a single dose of 100 mg (one tablet) of the corresponding formulation in each period.
Results: The quantification of diclofenac sodium in plasma by HPLC demonstrated that both formulations could be considered as bioequivalent. Mean values of the key pharmacokinetic parameters were: AUC (4924.25 vs. 4928.32 μg/mL), Cmax (1046.97 vs. 1042.19 μg/mL); median Tmax was 2 hours for both formulations. The confidence intervals for AUC and Cmax were 98.3-101.7 and 98.75-101.2, respectively. The most frequent adverse events, with both formulations, were headache (11.1%), increase in transaminases values (11.1%), increase in urea (11.1%) and hypertension (8.3%), all of them mild.
Conclusions: Cuban prolonged release diclofenac sodium formulation was bioequivalent with the international leader formulation Voltaren Retard®.
Barkat Ali Khan
Islamic International University, Pakistan
Title: Fabrication of Optimized Diclofenac Potassium Micro-particles Using Response Surface Methodology
Time : 14:55-15:30
Biography:
Dr. Barkat Ali Khan: Was born on 20 September 1982 in, Bannu, Khyber Pakhtunkhwa, Pakistan. He completed his B. Pharm. in 2005 from Gomal University D.I Khan, M. Phil. And PhD (Pharmaceutics) in 2010 and 2013 respectively from the Islamia University of Bahawalpur under the supervision of renowned professor Dr. Navid Akhtar. He is serving as a Lecturer in the department of Pharmaceutics at faculty of Pharmacy, Gomal University D.I Khan, Pakistan, since January 2012. He also served the school of pharmacy, Kampala international university Uganda; east Africa for one year. He has more than 100 publications in national and international journals. His research interest is in the area of cosmetics, skin preparations, novel drug delivery systems such as nanoparticles, microparticles and liposomes and their design, preparation optimization and evaluation. He is the chief editor of journal of pharmaceutical and cosmetics sciences. He is serving as Editorial Board Member and reviewer for many reputed journals. He received scholarship for his master and PhD studies from the higher education commission (HEC) of Pakistan. He was granted travel expenses to present his research papers at Thailand and Turkey by HEC.
Abstract:
Aims: This study was conducted to prepare modified release Diclofenac potassium loaded ethyl cellulose micro particles.
Methods: 13 trail formulations were studied to form an optimized formulation. Non-solvent addition coacervation method was used for the preparation. Response Surface Methodology (RSM) was applied for modified release formulation optimization. Solid state study was conducted for optimized formulation both qualitatively and quantitatively.
Results: As the polymer concentration (X1) and stirring speed (X2) increases Entrapment efficiency (Y3) also increases. Stirring speed (X2) has great effect over particles size (Y4). As polymer concentration (X1) and stirring speed (X2) increases compressibility index (CI:Y5) also increases. Optimized formulation was selected from 30 predicted values. Polymer concentration (X1) for optimized formulation was 1.96gm and stirring speed was 602rpm. Characterization study of modified formulation of Diclofenac potassium were given as; %DR after 1st hour (Y1) was 29.793%, % DR after 7th hr (Y2), E.E(Y3) was 94.511%, particle size was 343.70µm and CI (Y5) was 13.46%. In vitro dissolution study showed sustained release for 12 hrs. Kinetic study showed high R2 values for zero order (0.9318) and Higuchi model (0.9962). Both qualitative and quantitative analysis proved the stability of optimized micro particles.
Conclusion: SEM showed that particles of optimized formulation were nearly spherical, light yellowish in colour, and having porous and rough surface entrapping drug crystals. Fourier transform spectrophotometry showed that drug is stable in polymer and having no interactions, X-rays powder diffraction (XRD) showed decrease in crystallinity of Diclofenac Potassium in optimized formulation and differential scanning calorimetry (DSC) proved the thermal stability of formulation.
Alejandro Saúl Padrón Yaquis
Center for Drug Research and Development, Cuba
Title: Bioequivalence of two prolonged release diclofenac sodium formulations in healthy volunteers
Time : 15:50-16:20
Biography:
Alejandro Saul Padron Yaquis has completed his PhD at the age of 33 years from Habana University. He graduated from Russian University of Chemistry D. I. Mendeleiev. He is the Director of the Center for Pharmaceuticals Research and Development, one of the enterprises of BioCubaFarma Organization. He has published more than 25 papers in reputed journals and has been serving as Director of a Bioequivalence Unit. He has provided training and support for the conduction of BE study in other countries.
Abstract:
Andras Fodor
University of Pannonia, Hungary
Title: Agrobacterium: a Suitable Genetic System for Analysis Resistance to Xenorhabdus Antimicrobial Peptide Complexes Extremely Active in Food Spoilage (Zoonic) Bacteria
Time : 16:20-16:40
Biography:
Present position: Retired from Senior Research Associate position from the University of Pannonia, Georgikon Faculty, Department of the Animal Sciences & Animal Breeding,
Diploma: Geneticist – biologist & Teacher Certification for High School, 234/1964
PhD: Committee for Doctoral Awards of the Hungarian Academy of Sciences, October 7, 1974, Budapest, Hungary; PhD: Candidate of Biological Sciences; (No. 6.162).
Biological Doctor’s Degree: D-1674/1974, Eötvös Loránd University, Budapest, Hungary, December 3, 1974.
Habilitation: Habilitation Committee of the Eötvös Loránd University, Budapest, Hungary, December 13, 2000, Budapest, Hungary.
Széchenyi Professorship Award: March 19, 1999, Hungarian Ministry of Education.
Fulbright Research Grant, a Fulbright Grant Biological Science Grant (1214102) and funds from Valent Biosciences, both awarded to András Fodor to conduct research in the lab of Heidi Goodrich-Blair at the University of Wisconsin-Madison, USA, 2015.
Abstract:
The emergence of antibiotic resistance proves an increasing challenge n prevention and control in microbial (zoonic) pathogens in foods. The aim of this work is to establish a suitable experimental system for genetic analysis of antimicrobial peptide-resistance in a genetically well-characterized Gram-negative target, and found Agrobacterium tumefaciens. Intentionally, not a single but a multiple antimicrobial peptide-complex (EMA_PF2, isolated from Xenorhabdus budapestensis, with a reproducible MALDI profile), was chosen as a model to be bio-assayed in liquid cultures. Antimicrobial activity of EMA_PF2 is demonstrated on t Gram-positive Rhodococcus equi, Erysipelothrix rhusiopathiae, Staphylococcus aureus, Streptococcus equi, Corynebacterium pseudotuberculosis, and Listeria monocytogenes) and Gram negative Salmonella Gallinarum, Salmonella Derby, Bordetella bronchiseptica, Escherichia coli, Pasteurella multocida, Aeromonas hydrophila strains including multi-resistant ones. A. tumefaciens A281 strain with C58 chromosome (originated from a nopaline-catabolizing strain) and intact (transfer-DNA, T-DNA carrying) pTiBo542 plasmid (with agropine (L, L, -succinamopine) catabolizing genes) proved fully resistant to EMA_PF2; while the studied disarmed (transfer-DNA-deleted, del-T-DNA) plasmid-harboring derivatives (AGL1; EHA105 and A4T) were fully sensitive. By contrast, all 5 examined pTi58-plasmid-cured derivatives of C58 nopaline-catabolizing strains proved resistant to EMA_PF2. Two octopine-catabolizing strains behaved differently. Agrobacterium strains sensitive to EMA_PF2, harboring disarmed pTiBo542 provide a system for genetic complementation analysis of resistance to antimicrobial peptides using complementary sequences from resistant strains.