Plasmid transfer dynamics in Salmonella and related species and factors that influence the transfer efficiency

The presentation shows my final project after passed the Candidacy examination of the CEMB program and presented by me to all committee members. I am honored to work in the Food & Drug Administration (FDA), Microbiology Division, Jefferson, AR as a visitor (doctoral candidate for the project illustrative purpose only) under the supervision of Dr. Steven Foley.

Transcript

1. Plasmid transfer dynamics in Salmonella and related species and factors that influence the transfer efficiency SUAD ALGARNI PhD Candidate Cell & Molecular Biology Research Advisor: Dr. Steven L. Foley Deputy Director, Division of Microbiology FDA, National Center for Toxicological Research Administrative Advisor: Dr. Douglas D. Rhoads Director, Cell & Molecular Biology Graduate Program University of Arkansas Committee Chair: Dr. Steven C. Ricke Chair, Donald “Buddy” Wray in Food Safety University of Arkansas
2. Graduate Committee: Dr. Douglas D. Rhoads Dr. Steven L. Foley Dr. Young Min Kwon Dr. Steven C. Ricke Thank you for agreeing to serve on my Graduate Committee!
3. EDUCATION: • Doctoral of Philosophy in Cell & Molecular Biology 2014 - Present University of Arkansas, Fayetteville, Arkansas, United States. • Master of Health sciences in Medical Laboratory Sciences 2011 - 2013 Quinnipiac University, Hamden, Connecticut, United States. • Bachelor of science in Zoology 2000 - 2004 King Abdelaziz University, Jeddah, kingdom of Saudi Arabia. Academic History and Background
4. Previous experience in the CEMB Program SPECIAL SKILLS LABORATORY TECHNIQUES: • Preparation of Total DNA & RNA Extraction • RT-qPCR using real-time analysis • Prepare a next-gen cDNA library • Sequence the library on the Next-gen Illumina MiSeq DNA Sequencer • Bioinformatics to analyze millions of DNA sequence reads • Spectroscopy skills: NanoDrop 2000 UV-Vis Spectrophotometer, Qubit 3 Fluorometer.
5. Previous experience in the CEMB Program • Program languages (Python, Jupyternotebook, QIIME pipeline, Galaxy) • Other (R Studio, Excel, Unix os, Mac OS, Windows OS)
6. Table of my current coursework (completed) Courses Terms Description Grade BIOL 5153 Spring 2018 PROGRAMMING FOR BIOLOGISTS B BIOL 5343 Spring 2017 ADVANCED IMMUNOLOGY A BIOL 580V Fall 2017 SPECIAL TOPICS IN BIOL SCIENCE (SCIENTIFIC WRITING) B BIOL 5873 Fall 2016 MICROBIAL GENETICS B CHEG 5113 Fall 2015 TRANSPORT PROCESSES I A CHEM 5813 Fall 2014 BIOCHEMISTRY I A CHEM 5843 Spring 2015 BIOCHEMISTRY II B POSC 5873 Fall 2017 FOODBORNE PATHOGENS A POSC 5932 Fall 2015 CARDIOVASCULAR PHYSIOLOGY A STAT 4101L Fall 2016 INTRODUCTION TO R A STAT 4373 Spring 2016 EXPERIMENTAL DESIGN B CNEX 799V Fall 2017 CANDIDACY EXAM PASSED PASSED
7. Regulation of bacterial conjugation 1. The bacterial conjugation system 2. The potential human health risk due to the horizontal gene transfer of virulence factors to Salmonella I. Controlling and reducing of the spread of virulence plasmids in Salmonella 3. Bacterial conjugation mechanisms I. Bacterial Transfer Genetics (Conjugative Plasmids) 4. Copy number control and plasmid replication control I. Theta Replication II. Rolling-Circle Replication III. Strand Displacement Replication Chapter 1: Literature review and introduction
8. 5. Role of histone-like nucleoid-structuring (H-NS) protein and other DNA binding proteins in the regulation of plasmids. 6. The effect of exogenous agents on conjugation efficiency I. Quorum-Sensing Molecules (AI-2) II. Antimicrobial Agents A. Tetracycline B. Chloramphenicol C. Ampicillin 7. SOS response mediators Chapter 1: Literature review and introduction
9. Evaluation of mobile genetics elements and dynamics 1. Definition of bacterial antimicrobial resistance mobilome 2. Evaluation of resistance plasmid mobilome 3. Evaluation of gut and food mobilome 4. Mobilome clinical microbial safety 5. Mobilome evaluation in environmental Chapter 1: Literature review and introduction
10. Chapter 1: Literature review and introduction Bacterial Antimicrobial Resistance 1. Antimicrobial agents or antibiotics 2. Bacterial antibiotic resistances and acquired antibiotic resistance 3. The plasmids dynamics of antimicrobial resistance genes 4. The role of the evolutionary history of mobile genetic elements and antimicrobial resistance 5. The global impact of the change in antibiotics resistance on human health and food product safety 6. The genetics mechanisms between antibiotics resistance genes to different classes of antimicrobial agents 7. The epidemic of multidrug resistant of Salmonella enterica strains in human health
11. Chapter 2: Approach and Methods Antimicrobial selective pressure experiment: To determine the number of transconjugants produced under the different selection conditions and the relative impact on antimicrobial exposure to plasmid transfer and identify the plasmids that transfer Donor Recipient Conjugation (plasmid transfer) + Chromosome Plasmid Salmonella strains E.coli J53
12. Chapter 2: Materials and Methods 16 μg/m l 8 μg/m l 4 μg/m l 2 μg/m l 1 μg/m l No Abx Combine and add to pre- selective pressure media Na AzideR Recipient Incubate for 2 hours, then remove antibiotic media Add enriched media and incubate to facilitate cell recovery Perform dilutions, streak onto selective media, incubate and enumerate bacterial growth Antim icrobial Exposure
13. Chapter 2: Initial results of selective pressure experiments Figure 2: Average relative number of transconjugants formed among nine different Salmonella isolates (listed on the below of the graph) following exposure to different concentration of Tetracycline.
14. Chapter 3: Objective and outline The objective of the research study: Evaluate differences in the expression of genes which are important for the regulation of plasmid transfer in Salmonella strains. Second research study outline: 1) Abstract 2) Introduction 3) Materials and Methods 4) Results 5) Discussion
15. Chapter 3: Gene expression analysis of bacterial conjugation under selective pressure Salmonella strains (donor) and E.coli (recipient ) strains rRNA depletion and cDNA library preparation RNA will be collected from donor and recipient using the RNeasy extraction kit Cluster generation and sequence Illumina mRNA data analysis software in the Illumina BaseSpace environment and/or CLC Genomics Workbench Mix and expose to antimicrobial selective pressure The resultant expression data will incorporate into a pathway analysis program (Ingenuity pathways Analysis (Qaigen)
16. Chapter 4: Objectives and outline The objectives of the research study: Development of tools and approaches to study the diversity and dissemination potential of plasmids associated with antimicrobial resistance and virulence. 1) Abstract 2) Introduction 3) Materials and Methods 4) Results 5) Discussion
17. Chapter 4: Plasmid gene database development and downstream applications of the information
18. Search GenBank using Microbial Nucleotide BLAST: searched the reference sequences for the different Inc Groups against the “Complete plasmids” database Develop a comprehensive list of plasmids and their corresponding metadata, including accession numbers hyperlinked to the GenBank record Generate multi-record GenBank files for uploaded into the FeatureExtract 1.2 program and submit the query The output, which contains the individual CDS information from the different genomes will be formatted (cleaned up) for inclusion into the plasmid gene database for further analyses Chapter 4: Data extraction and database development
19. Chapter 4: Plasmid gene function characterization Product IncA/C IncFIB IncI1 aminoglycoside (2'') adenyltransferase (AadA2) X X X aminoglycoside 3'-adenyltransferase X X aminoglycoside 3'-phosphotransferase (APH(3')-I) X X X aminoglycoside 6'-N-acetyltransferase AAC(6')-Ib X X aminoglycoside 6'-N-acetyltransferase AacA34 X aminoglycoside 6'-N-acetyltransferaseAAC(6')-33 X aminoglycoside 6'-phosphotransferase, StrB X aminoglycoside acetyltransferase (AacC) X X X aminoglycoside adenyltransferase (AadA1) X X X aminoglycoside N(3')-acetyltransferase III(ACC(3)-III) X aminoglycoside N-acetyltransferase AAC(3)-II X X X aminoglycoside N-acetyltransferase AAC(3)-IV X aminoglycoside N-acetyltransferase AAC(3)-VIa X aminoglycoside N-acetyltransferase AAC(6')-29 X aminoglycoside N-acetyltransferase AAC(6')-Ian X aminoglycoside N-acetyltransferase AAC(6')-Ib3 X aminoglycoside N-acetyltransferase AAC(6')-IIc X aminoglycoside nucleotidyltransferase X X aminoglycoside O-phosphotransferase APH(3')-II X X aminoglycoside O-phosphotransferase APH(3')-VIa X aminoglycoside O-phosphotransferase APH(4)-Ia X Product IncA/C IncFIB IncI1 arsenate reductase ArsC X X X arsenical efflux pump membrane protein ArsB X X X arsenical pump-driving ATPase X X X arsenical resistance operon transcriptionalrepressor ArsD X X X arsenical resistance protein ArsH X ArsR family transcriptional regulator X X X arylsulfatase X cobalamin biosynthesis protein CobW X CopG family protein repressor X X copper ABC transporter substrate-bindingprotein X copper resistance protein X copper-sensitivity suppressor C X copper-translocating P-type ATPase X CusA/CzcA family heavy metal efflux RNDtransporter X ethidium bromide resistance protein X Ethidium bromide-methyl viologen resistanceprotein EmrE X Product IncA/C IncFIB IncI1 Aerobactin siderophore ferric receptor protein (IutA) X aerobactin synthase IucA X antitoxin X X X antitoxin CcdA X X antitoxin RelB X bacteriocin immunity protein X bacteriocin microcin B17 X colicin 1B X X X colicin 1B immunity protein (Cbi) X X X colicin Ia immunity protein X X colicin Ia protein X X colicin M activity protein Cma X X colicin M immunity protein (Cmi) X X Product IncA/C IncFIB IncI1 class 1 integron integrase IntI1 X X X insertion element IS1 protein InsA X X X insertion element IS1 protein InsB X X X insertion element IS1 protein InsD X X insertion element IS1 protein InsE X insertion element IS1 protein InsF X insertion element IS1 protein OrfB X insertion element IS629 protein X insertion element ISKpn14 X Antimicrobial Resistance Genes Metal/ Disinfectant Res. Genes Virulence Genes Insertion Sequences, Transposons and Integrons
20. Chapter 4: Development of predictive database tools for plasmid transfer IncA/C traA traB traC traD traE traF traG traH traI traJ traK traL traM traN traO traP traQ traR traS traT traU traV traW traX traY IncFIA traA traB traC traD traE traF traG traH traI traJ traK traL traM traN traO traP traQ traR traS traT traU traV traW traX traY IncFIB traA traB traC traD traE traF traG traH traI traJ traK traL traM traN traO traP traQ traR traS traT traU traV traW traX traY IncI1 trbA trbB trbC pilI pilJ pilJ pilK pilL pilM pilN pilO pilP pilQ pilR pilS pilT pilU pilV traA traB traC traD traE traF traG traH traI traJ traK traL traM traN traO traP traQ traR traS traT traU traV traW traX traY IncHI1 thrA thrB thrC thrD thrE thrF thrG thrH thrI thrJ thrK thrL thrM thrN thrO thrP thrQ thrR thrS thrT thrU thrV thrW IncHI2 thrA thrB thrC thrD thrE thrF thrG thrH thrI thrJ thrK thrL thrM thrN thrO thrP thrQ thrR thrS thrT thrU thrV thrW Toxin Genes col1A(cia) col1B (cib) colA colD colE1 colM colN cvpA cvaA cvaB cvaC cka cba cua ccl imm IncA/C traA traB traC traD traE traF traG traH traI traJ traK traL traM traN traO traP traQ traR traS traT traU traV traW traX traY IncFIA traA traB traC traD traE traF traG traH traI traJ traK traL traM traN traO traP traQ traR traS traT traU traV traW traX traY IncFIB traA traB traC traD traE traF traG traH traI traJ traK traL traM traN traO traP traQ traR traS traT traU traV traW traX traY IncI1 trbA trbB trbC pilI pilJ pilJ pilK pilL pilM pilN pilO pilP pilQ pilR pilS pilT pilU pilV traA traB traC traD traE traF traG traH traI traJ traK traL traM traN traO traP traQ traR traS traT traU traV traW traX traY IncHI1 thrA thrB thrC thrD thrE thrF thrG thrH thrI thrJ thrK thrL thrM thrN thrO thrP thrQ thrR thrS thrT thrU thrV thrW IncHI2 thrA thrB thrC thrD thrE thrF thrG thrH thrI thrJ thrK thrL thrM thrN thrO thrP thrQ thrR thrS thrT thrU thrV thrW Toxin Genes col1A(cia) col1B (cib) colA colD colE1 colM colN cvpA cvaA cvaB cvaC cka cba cua ccl imm WGS Data Carries IncA/C and IncFIB plasmids that are conjugative in Salmonella, but not all E. coli Prediction
21. IncA/C traA traB traC traD traE traF traG traH traI traJ traK traL traM traN traO traP traQ traR traS traT traU traV traW traX traY IncFIA traA traB traC traD traE traF traG traH traI traJ traK traL traM traN traO traP traQ traR traS traT traU traV traW traX traY IncFIB traA traB traC traD traE traF traG traH traI traJ traK traL traM traN traO traP traQ traR traS traT traU traV traW traX traY IncI1 trbA trbB trbC pilI pilJ pilJ pilK pilL pilM pilN pilO pilP pilQ pilR pilS pilT pilU pilV traA traB traC traD traE traF traG traH traI traJ traK traL traM traN traO traP traQ traR traS traT traU traV traW traX traY IncHI1 thrA thrB thrC thrD thrE thrF thrG thrH thrI thrJ thrK thrL thrM thrN thrO thrP thrQ thrR thrS thrT thrU thrV thrW IncHI2 thrA thrB thrC thrD thrE thrF thrG thrH thrI thrJ thrK thrL thrM thrN thrO thrP thrQ thrR thrS thrT thrU thrV thrW Toxin Genes col1A(cia) col1B (cib) colA colD colE1 colM colN cvpA cvaA cvaB cvaC cka cba cua ccl imm WGS Data Carries IncA/C plasmid that is NOT conjugative in Salmonella or E. coli Prediction IncA/C traA traB traC traD traE traF traG traH traI traJ traK traL traM traN traO traP traQ traR traS traT traU traV traW traX traY IncFIA traA traB traC traD traE traF traG traH traI traJ traK traL traM traN traO traP traQ traR traS traT traU traV traW traX traY IncFIB traA traB traC traD traE traF traG traH traI traJ traK traL traM traN traO traP traQ traR traS traT traU traV traW traX traY IncI1 trbA trbB trbC pilI pilJ pilJ pilK pilL pilM pilN pilO pilP pilQ pilR pilS pilT pilU pilV traA traB traC traD traE traF traG traH traI traJ traK traL traM traN traO traP traQ traR traS traT traU traV traW traX traY IncHI1 thrA thrB thrC thrD thrE thrF thrG thrH thrI thrJ thrK thrL thrM thrN thrO thrP thrQ thrR thrS thrT thrU thrV thrW IncHI2 thrA thrB thrC thrD thrE thrF thrG thrH thrI thrJ thrK thrL thrM thrN thrO thrP thrQ thrR thrS thrT thrU thrV thrW Toxin Genes col1A(cia) col1B (cib) colA colD colE1 colM colN cvpA cvaA cvaB cvaC cka cba cua ccl imm IncA/C traA traB traC traD traE traF traG traH traI traJ traK traL traM traN traO traP traQ traR traS traT traU traV traW traX traY IncFIA traA traB traC traD traE traF traG traH traI traJ traK traL traM traN traO traP traQ traR traS traT traU traV traW traX traY IncFIB traA traB traC traD traE traF traG traH traI traJ traK traL traM traN traO traP traQ traR traS traT traU traV traW traX traY IncI1 trbA trbB trbC pilI pilJ pilJ pilK pilL pilM pilN pilO pilP pilQ pilR pilS pilT pilU pilV traA traB traC traD traE traF traG traH traI traJ traK traL traM traN traO traP traQ traR traS traT traU traV traW traX traY IncHI1 thrA thrB thrC thrD thrE thrF thrG thrH thrI thrJ thrK thrL thrM thrN thrO thrP thrQ thrR thrS thrT thrU thrV thrW IncHI2 thrA thrB thrC thrD thrE thrF thrG thrH thrI thrJ thrK thrL thrM thrN thrO thrP thrQ thrR thrS thrT thrU thrV thrW Toxin Genes col1A(cia) col1B (cib) colA colD colE1 colM colN cvpA cvaA cvaB cvaC cka cba cua ccl imm IncA/C traA traB traC traD traE traF traG traH traI traJ traK traL traM traN traO traP traQ traR traS traT traU traV traW traX traY IncFIA traA traB traC traD traE traF traG traH traI traJ traK traL traM traN traO traP traQ traR traS traT traU traV traW traX traY IncFIB traA traB traC traD traE traF traG traH traI traJ traK traL traM traN traO traP traQ traR traS traT traU traV traW traX traY IncI1 trbA trbB trbC pilI pilJ pilJ pilK pilL pilM pilN pilO pilP pilQ pilR pilS pilT pilU pilV traA traB traC traD traE traF traG traH traI traJ traK traL traM traN traO traP traQ traR traS traT traU traV traW traX traY IncHI1 thrA thrB thrC thrD thrE thrF thrG thrH thrI thrJ thrK thrL thrM thrN thrO thrP thrQ thrR thrS thrT thrU thrV thrW IncHI2 thrA thrB thrC thrD thrE thrF thrG thrH thrI thrJ thrK thrL thrM thrN thrO thrP thrQ thrR thrS thrT thrU thrV thrW Toxin Genes col1A(cia) col1B (cib) colA colD colE1 colM colN cvpA cvaA cvaB cvaC cka cba cua ccl imm WGS Data Carries IncFIB and IncI1 plasmids that are conjugative in Salmonella and most E. coli Prediction Chapter 4: Development of predictive database tools for plasmid transfer
22. Chapter 5: Summary and Overall Conclusions • Conclusion and wrap-up (potentially could be enough new/different material that could lead a separate publication from those mentioned above).