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Identification of Novel Small Molecule Growth Inhibitors Specific to Avian Pathogenic Escherichia coli
Kathayat, Dipak

2017, Master of Science, Ohio State University, Comparative and Veterinary Medicine.
Avian Pathogenic E. coli (APEC), an extraintestinal pathogenic E. coli (ExPEC), is one of the most common bacterial pathogens affecting chickens, turkeys, and other avian species. It causes multiple extra-intestinal infections which subsequently lead to high morbidity and mortality, production losses, and increased slaughter condemnation resulting severe economic loss to the global poultry industry. Antimicrobial medication is the major approach currently employed to reduce the incidence and mortality associated with this infection. However, multi-drug resistant (MDR) APEC strains are reported nowadays worldwide. Furthermore, vaccination which is used as a subsidiary approach to prevent infection frequency is not sufficient to provide protection against diverse heterologous APEC serotypes. Therefore, the objective of this study is to identify novel small molecule (SM) growth inhibitors of APEC and to evaluate toxicity and efficacy of identified SMs, in vitro and in vivo.
Here, using a Tecan Sunrise™ absorbance plate reader, a pre-selected enriched SM library containing 4,182 SMs was screened at 100 µM concentration against a predominant field APEC serotype, APEC O78, grown in minimal M63 media. Of the total 4,182 SMs, 41 SMs inhibited APEC O78 growth. The majority of growth inhibitory SMs were found belonging to chemical groups; quinolines, piperidines, pyrrolidinyls, and imidazoles. Among 41 SMs, 30 SMs exhibited bacteriostatic activity, while remaining 11 SMs displayed bactericidal activity and were selected for further studies. Dose-response analysis of these selected SMs revealed their dose-dependent activity with minimal inhibitory concentration (MIC) ranging 12.5 µM to 200 µM. These selected SMs were found broadly effective against various APEC serotypes such as O1, O2, O8, O15, O18, O35, O109, and O115. Six of these SMs exhibited narrow-spectral activity affecting 1-3 tested commensal bacteria. Except SM11, other SMs were least toxic to Caco-2 epithelial (<10%) and HD11 macrophage cells (<10%) at 200 µM concentration. Seven of these SMs were least hemolytic (<10%) to avian and sheep red blood cells, whereas remaining four SMs were more hemolytic (>20%) at 200 µM concentration. All of the selected SMs were found effective in significant (P<0.01) reduction of intracellular APEC O78, O1, and O2 survival in infected Caco-2, HD11 and THP-1 cells at varying concentrations ranging from 1X to 2X of MIC. No resistance was observed to any of these selected SMs when APEC O78 was treated at lethal (2X MBC) or sub-lethal (0.75X MIC) concentration of SMs. In vivo evaluation of these SMs using greater wax moth (Galleria mellonella) revealed their low toxicities (<10%), except for SM1. Furthermore, treatment with these SMs significantly (P<0.0001) extended the survival of infected larvae, except for SM8, and significantly (P<0.05) reduced the APEC load inside the larva, except for SM8 and SM9.
In summary, we have identified 11 novel anti-APEC SMs. Our future studies will focus on investigating; effects of these SMs on APEC preformed biofilms, interactions with antimicrobials that are currently being used, effects in infected chickens, and elucidation of their mechanism of actions. We expect that these studies will enable the development of novel narrow-spectrum SM antimicrobials for the control of APEC infections.
Gireesh Rajashekara (Advisor)
Chang-Won Lee (Committee Member)
Anastasia Vlasova (Committee Member)
122 p.

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Kathayat, D. (2017). Identification of Novel Small Molecule Growth Inhibitors Specific to Avian Pathogenic Escherichia coli . (Electronic Thesis or Dissertation). Retrieved from https://etd.ohiolink.edu/

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Kathayat, Dipak. "Identification of Novel Small Molecule Growth Inhibitors Specific to Avian Pathogenic Escherichia coli ." Electronic Thesis or Dissertation. Ohio State University, 2017. OhioLINK Electronic Theses and Dissertations Center. 10 Dec 2017.

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Kathayat, Dipak "Identification of Novel Small Molecule Growth Inhibitors Specific to Avian Pathogenic Escherichia coli ." Electronic Thesis or Dissertation. Ohio State University, 2017. https://etd.ohiolink.edu/

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