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Characterizing The Role Of Pa5189 Of Pseudomonas Aeruginosa In Deletion And Overexpression Mutants, Seh Na Mellick

Theses/Capstones/Creative Projects

In the context of rising multidrug resistance in biofilm-forming pathogens like Pseudomonas aeruginosa, this study investigates the role of the understudied transcription factor PA5189 in antibiotic resistance and biofilm formation. PA5189 deletion and overexpression mutants were created in a parent P. aeruginosa strain using pEX18Tc-based recombinant suicide vectors, with genotypic verification of putative triparental conjugants achieved through restriction digestion and PCR. The study revealed that PA5189 overexpression significantly increases resistance to commonly used broad spectrum antibiotics such as ciprofloxacin and imipenem. Additionally, differential expression of PA5189 was found to notably affect biofilm formation, with variations contingent on the nutrient …

Turbidimetric Bioassays: A Solution To Antimicrobial Activity Detection In Asymptomatic Bacteriuria Isolates Against Uropathogenic Escherichia Coli, Ciara Kenneally, Craig P. Murphy, Roy D. Sleator, Eamonn P. Culligan

Department of Biological Sciences Publications

Traditional bacteriocin screening methods often face limitations due to diffusion-related challenges in agar matrices, which can prevent the peptides from reaching their target organism. Turbidimetric techniques offer a solution to these issues, eliminating diffusion-related problems and providing an initial quantification of bacteriocin efficacy in producer organisms. This study involved screening the cell-free supernatant (CFS) from eight uncharacterized asymptomatic bacteriuria (ABU) isolates and Escherichia coli 83972 for antimicrobial activity against clinical uropathogenic E. coli (UPEC) strains using turbidimetric growth methods. ABU isolates exhibiting activity against five or more UPEC strains were further characterized (PUTS 37, PUTS 58, PUTS 59, S-07-4, and …

Methionyl-Trna Synthetase Synthetic And Proofreading Activities Are Determinants Of Antibiotic Persistence, Whitney N. Wood, Miguel Angel Rubio, Lorenzo Eugenio Leiva, Gregory J. Phillips, Michael Ibba

Biology, Chemistry, and Environmental Sciences Faculty Articles and Research

Bacterial antibiotic persistence is a phenomenon where bacteria are exposed to an antibiotic and the majority of the population dies while a small subset enters a low metabolic, persistent, state and are able to survive. Once the antibiotic is removed the persistent population can resuscitate and continue growing. Several different molecular mechanisms and pathways have been implicated in this phenomenon. A common mechanism that may underly bacterial antibiotic persistence is perturbations in protein synthesis. To investigate this mechanism, we characterized four distinct metG mutants for their ability to increase antibiotic persistence. Two metG mutants encode changes near the catalytic site …

Diagnosis Of Urinary Tract Infections And Rapid Molecular Characterization Of Antibiotic Resistance, Mohammed Harris

All Dissertations

Urinary tract infections (UTIs) are one of the most common infectious clinical entities in both community and hospital settings. They have a broad range of clinical severity yet inflict large epidemiological burden of morbidity and mortality on patients and the healthcare system with billions of dollars in cost of treatment. Understanding what methods are optimal for diagnosing UTIs are critical to mitigate the marked impact and cost of these infections.

Chapter 1 and 2 in this work surveys the broad array of diagnostic modalities for UTIs and highlights their advantages and limitations in the context of the current standard of …

Effects Of Altering Physiologically Relevant Cholesterol Levels And Media Types On Porin Gene Expression In Klebsiella Pneumoniae And The Resulting Impact On Antibiotic Resistance, Megan R. Camden

UNF Graduate Theses and Dissertations

Klebsiella pneumoniae is a Gram-negative, non-motile bacterium belonging to the Enterobacteriaceae family. K. pneumoniae is a rising threat in the clinical setting, as there has been a large increase in the presence of antibiotic resistant isolates. While much research is conducted on laboratory and clinical strains of bacteria, not much is known regarding the impact that human physiology can have on bacterial gene expression, and in response, to antibiotic susceptibility. The goal of this study is to determine if physiologically relevant cholesterol levels and media types impact porin gene expression and antibiotic resistance in K. pneumoniae. To accomplish this, …

Effect Of Sub-Inhibitory Concentrations Of Nitrofurantoin, Ciprofloxacin And Trimethoprim On In-Vitro Biofilm Formation In Uropathogenic Escherichia Coli, Shane Whelan

ORBioM (Open Research BioSciences Meeting)

The purpose of the current study was to determine the effect of sublethal concentrations of nitrofurantoin, ciprofloxacin and trimethoprim on biofilm formation in 57 uropathogenic Escherichia coli strains (UPEC).

The MIC of nitrofurantoin, ciprofloxacin and trimethoprim was determined for 57 UPEC isolates. Biofilm formation for each isolate with and without sub-lethal concentrations of each antibiotic was then quantified, and the statistical significance of changes in biofilm formation was ascertained by way of a Dunnett's test.

The effects of sub-MIC antibiotics on biofilm formation of UPEC were variable. A total of 22.8% of strains were induced to form biofilm by nitrofurantoin, …

Resolving The Repression Pathway Of Virulence Gene Hila In Salmonella, Alexandra King, Lon Chubiz Phd, Brenda Pratte, Lauren Daugherty

Undergraduate Research Symposium

Salmonella is a relatively abundant, virulent species of bacteria that is most known for spreading gastrointestinal diseases through food. These illnesses result in approximately 1.35 million infections, including over 25,000 hospitalizations each year, in the U.S. alone (CDC.gov). As antibiotic resistance becomes an increasingly urgent public health problem, the importance of developing alternative treatment methods is only becoming more crucial. One of the genes responsible for this virulence is known as hilA. HilA is the main transcriptional regulator of Salmonella Pathogenicity Island-1 gene (UniProt). SPI-1 plays an important role in the invasion of Salmonella into epithelial cells. The proteins encoded …

Bacteriophages: Paving The Road For The Future Of Medicine, Luke Brinkerhoff

Honors Theses

Bacteriophages are a possible solution to antibiotic resistance, which is predicted to be detrimental world-wide by the year 2050. Personal field research was also conducted for a project studying the characteristics of two bacteriophages on a single bacterial host.

Diversity Of Bacteriophage In Burkholderia Species, Abigail Price

Honors Projects

Bacteriophages are viruses that infect and replicate within bacteria and offer the potential of a therapeutic alternative to chronic infections that do not respond to antibiotic-based therapies. B. vietnamiensis is one of a number of Burkholderia species involved with chronic drug resistant infections in the lungs of individuals with compromised respiratory systems, as found in those with chronic obstructive pulmonary disease and, most especially, are of particular significance in patients with cystic fibrosis. The diversity of the Burkholderia species is explored by using online databases and looking at bacteriophage or phage-encoding viruses found in B. vietnamiensis. The open reading frames …

The Role Of Coagulase-Negative Staphylococcal Secreted Products On Staphylococcus Aureus And Staphylococcus Lugdunensis Infections, Denny Chin

Electronic Thesis and Dissertation Repository

The Staphylococcus genus is comprised of over 40 bacterial species. The most well-studied species in this genus is the notorious human pathogen Staphylococcus aureus, a bacterium that produces coagulase among many other virulence factors. Since S. aureus is a major health burden and causes a plethora of diseases in humans, it has received significant attention and much research has been done to understand its biology to treat diseases caused by this pathogen. However, the coagulase-negative staphylococci (CoNS) make up most of the staphylococcal species and have received less attention since they are thought to have a lesser impact on …

Investigating Antimicrobial Properties Of Snake Venoms Against B. Cereus, B. Subtilis, E. Coli, And P. Vulgaris, Savannah Berger

Honors Program Theses and Research Projects

The increasing incidence of antibiotic resistant bacterial infections has provoked the attention of health officials and scientists as a major threat to global public health. Antibiotic resistance is the product of overprescription by doctors, ineffective or shortened dosing by the patient, and more. When a bacterial infection is ineffectively treated as such, persistent pathogenic cells are given the opportunity to proliferate and spread their resistance to other cells. With bacteria utilizing such tools to fight and ultimately resist our current treatment methods, investigation towards the next novel mechanism of inhibition is essential. Venom is of particular interest to many scientists …

Regulation, Mechanism Of Action, And Function Of A Small Toxin Protein In Ehec, Bikash Bogati

Doctoral Dissertations

The zor-orz locus identified in the chromosome of Escherichia coli O157:H7 EDL933 is a type I toxin-antitoxin system that consists of two homologous gene pairs zorO-orzO and zorP-orzP. The zor genes encode for small toxin proteins and the orz genes encode for small RNAs. Previously it was shown that overproduction of ZorO results in cell growth stasis whereas co-expression of orzO rescues cellular growth. Within, I demonstrate that in addition to growth inhibition, ZorO overproduction results in membrane depolarization and ATP depletion but does not impact the gross morphology of E. coli. In vivo translation and subsequent impacts …

Assesment Of Antibiotic Resistant Gene Expression In Clinical Isolates Of Pseudomonas Aeruginosa, Dustin Esmond

Biology Theses

Increasing prevalence of nosocomial infections by antimicrobial resistant pathogens resulting in higher mortality rates and financial burden is of great concern. Pseudomonas aeruginosa represents one of six highly virulent “ESKAPE” pathogens that exhibit considerable intrinsic drug resistance as well as mechanisms for acquiring further resistance. As many of these mechanisms are regulated through gene expression, we sought to identify regulatory strategies and patterns at play in 23 clinical isolates collected from Baku, Azerbaijan and Tyler, Texas, USA. Real-time quantitative polymerase chain reaction was performed on six gene targets implicated in resistance and contrasted with antibiotic phenotypes. We found AmpC cephalosporinase …

The Role Of The Msaabcr Operon In Cell Wall Integrity And Programmed Cell Death During Biofilm Development, Bibek G C

Dissertations

Staphylococcus aureus is an important human pathogen in both community and health care settings. Biggest challenges with S. aureus as a pathogen is its ability to acquire antibiotic resistance and produce robust biofilms. In this work, we investigated the nature of the cell wall defect in the msaABCR operon mutant in the Mu50 (VISA) and USA300 LAC methicillin-resistant Staphylococcus aureus (MRSA) strains. Results showed that msaABCR-mutant cells had decreased cell wall thickness and cell wall crosslinking in both strains. These defects are most likely due to increased murein hydrolase activity and/or nonspecific processing of murein hydrolases mediated by increased …

Characterizing The Diversity Of Cluster R Prophage In Mycobacterium Abscessus, Colin Welch

Honors College

Mycobacterium abscessus is a mycobacterial pathogen responsible for pulmonary and disseminated infections in susceptible individuals and often is resistant to all antibiotic therapies, with cure rates ranging from 25% to 58% (Degiacomi et al., 2019).Investigating the mechanisms of extensive resistance in M. abscessus lends opportunities to develop more effective treatments. Prophage, viral sequences integrated into bacterial genomes, contribute to virulence and fitness in many bacterial pathogens including Escherichia coli O157:H7 and Vibrio cholerae (Fortier & Sekulovic, 2013). Yet, prophage in pathogenic mycobacteria are not well understood or studied. Recent work has demonstrated that the Mycobacterium chelonae prophage, McProf can function …

Increasing Antibiotic Resistance In Shigella Bacteria In The United States, William J. Pharr

The Corinthian

Shigella bacteria cause half a million infections, 6,000 hospitalizations, and 70 deaths annually in the United States. These bacteria are of particular concern due to their high survivability, low infectious dose, and high adaptability. Cases of shigellosis from Shigella sonnei are becoming a more prevalent issue in the U.S. as the bacteria continues to develop higher resistance to today’s strongest antibiotics. Much of this resistance is connected to the exchange of genes between strains of Shigella due to insertion sequences (IS), intercontinental travel, and men who have sex with men (MSM). As a result of increased resistance, the use of …

Lmea, A Conserved Cell-Envelope Protein In Mycobacteria, Is Important For Antibiotic Resistance And Cell Envelope Permeability, Sarah Hassan Osman

Masters Theses

The cell envelope of mycobacteria is critical for the survival and virulence of pathogenic species during infection, and its biosynthesis has been a proven drug target. Therefore, finding new targets in the biosynthetic pathway of cell envelope components is of great interest. Mycobacterium smegmatis is a model organism for the study of the devastating pathogen Mycobacterium tuberculosis. Previously, lipomannan elongation factor A (LmeA) has been identified as a cell envelope protein that is critical for the control of mannan chain length of lipomannan (LM) and lipoarabinomannan (LAM), lipoglycan components of the cell envelope. The deletion mutant, ∆lmeA, accumulates abnormal LM/LAM …

Characterization Of The Broad-Spectrum Inhibitory Capability Of Alcaligenes Faecalis And A. Viscolactis Against Potential Pathogenic Microorganisms, Andrew Fuqua

Undergraduate Honors Theses

The recent rise of multidrug resistant microorganisms has grown from an isolated concern to a massive public health crisis. It has become imperative that scientists look for new ways to combat this issue. Due to the selective pressures of competition, bacteria and other microbes possess a host of defenses and weapons designed to exploit vulnerabilities in other microorganisms. Consequently, the study of these systems and microbial interactions has much to reveal in the search for novel antimicrobial treatments. Previous research from our laboratory has discovered that both Alcaligenes faecalis and Alcaligenes viscolactis, two rarely studied and generally non-virulent bacteria, …

The Effects Of Farnesol, A Quorum Sensing Molecule From Candida Albicans, On Alcaligenes Faecalis, Savannah Hutson

Undergraduate Honors Theses

Quorum sensing molecules have become a recent focus of study to learn if and how they can be used, both on their own and in conjecture with current antimicrobial methods, as a means of bacterial control. One such quorum sensing molecule is the sesquiterpene alcohol, Farnesol, which is synthesized and released by the fungus, Candida albicans. In most in-vivo cases, our laboratory has shown that Alcaligenes faecalis overtakes C. albicans, preventing its growth. However, as a way to counteract this inhibitory effect, Farnesol may be one way that Candida has found to fight back. In this study, we …

Antibiotic Resistant Bacteria, Antibiotic Resistance Genes And Potential Drivers In The Aquatic Environments, Shuo Shen

Dissertations

As antibiotic resistance genes in aquatic environment have been increasing across the world, affecting water quality and public health, many studies documented concentrations of antibiotic resistance genes and some studies discussed their potential drivers. However, systematic and quantitative reviews that link antibiotic resistance genes (ARGs) to anthropogenic and environmental factors are limited. Nevertheless, this information will be important for developing regulation policy on controlling antibiotic use and therefore reducing potential risks to antibiotic resistance. I conducted meta-analysis of ARGs concentration at a global scale using Bayesian inference to explore climatic and socio-economic factors as drivers. I found local-scale climatic variables …

Identification Of Antibiotic Producing Soil Bacteria Against Bacillus Subtilis, Morgan Brockhouse, Dr. Lori Scott

Identifying and Characterizing Novel Antibiotic Producing Microbes From the Soil

This project is a collaboration with the Tiny Earth Project Initiative (TEPI), which is a global network of educators and students focused on student-sourcing antibiotic discovery from soil. Individual strains of soil bacteria were isolated and produced antibiotic against Bacillus subtilis. Two of these samples were sequenced using the 16S rRNA gene to reveal they are very closely related to the genus Pseudomonas.

Assessing The Prevalence Of Multidrug Resistant Salmonella Enterica From Stream Sediment And Poultry Litter In The Shenandoah Valley Of Virginia, Noah Greenman

Masters Theses, 2010-2019

Multidrug resistant Salmonella enterica present in stream sediment and poultry litter represent a critical health concern. A small number of S. enterica serotypes are responsible for most lab-confirmed infections in the US each year. To assess the prevalence of these significant strains, we isolated 88 S. enterica from stream sediment and poultry litter. Sequence data for all isolates were generated using an Illumina® sequencing platform, with long-reads for some isolates from the Oxford Nanopore MinION™ used in a hybrid genome assembly approach. Isolates were typed according to their serotype and multi-locus sequence type using SeqSero/SISTR and Enterobase respectively. Antibiotic resistance …

Biofilm Producing Clinical Staphylococcus Aureus Isolates Augmented Prevalence Of Antibiotic Resistant Cases In Tertiary Care Hospitals Of Nepal, Sarita Manandhar, Anjana Singh, Ajit Varma, Shanti Pandey, Neeraj Shrivastava

Student Publications

Staphylococcus aureus, a notorious human pathogen, is a major cause of the community as well as healthcare associated infections. It can cause a diversity of recalcitrant infections mainly due to the acquisition of resistance to multiple drugs, its diverse range of virulence factors, and the ability to produce biofilm in indwelling medical devices. Such biofilm associated chronic infections often lead to increase in morbidity and mortality posing a high socio-economic burden, especially in developing countries. Since biofilm formation and antibiotic resistance function dependent on each other, detection of biofilm expression in clinical isolates would be advantageous in treatment decision. …

Comparison Of Beta-Lactamase Genes In Environmental Rahnella Isolates, Katherine D'Angelo, Ray A. Larsen

Honors Projects

Antibiotic resistance is a growing problem in the field of healthcare. Antibiotics are becoming less effective as species of bacteria adapt and share resistance mechanisms. If transmission of mechanisms can be better understood at the molecular level, inhibitors could be developed to lessen the likelihood of antibiotic resistance. In this study, Rahnella spp were isolated from environmental sources on MacConkey plates containing 100 µg ml^-1 ampicillin, and confirmed by 16S rRNA gene sequencing. Whole genomic DNA was extracted from isolates and initial amplifications were performed by polymerase chain reaction (PCR) using primers specific for 16S amplification. New primers were …

The Making Of A Pathogen: Implications Of Phage Domestication In Acinetobacter Baumannii, Allison Welp

Undergraduate Theses

Mutations and horizontal gene transfer have allowed for rapid evolution of many species of bacteria, allowing them to become more virulent and resistant to antibiotics. As a result of these changes, Acinetobacter baumannii has become one of the most prominent drug-resistant bacteria in hospitals. This nosocomial pathogen is capable of causing a range of infections—from pneumonia to sepsis—and is extremely difficult to eradicate from hospital settings. Despite its current status, this species has not always been apparent in healthcare. The emergence of this organism has been extremely rapid; once an innocuous environmental organism, A. baumannii is now resistant to all …

Whole Genome Sequencing As A Tool For Identifying Phenotypic Properties And Underlying Genetic Mechanisms In Staphylococcus Pseudintermedius, Matthew C. Riley

Doctoral Dissertations

Staphylococcus pseudintermedius is a Gram-positive bacterial opportunistic pathogen commonly associated with dermal infections in canines, but capable of causing serious disease in other species. Reports of human infections caused by S. pseudintermedius along with an increase in resistance to multiple antibiotics highlights the importance of this organism. Whole genome sequencing can allow large scale investigation of genetic mechanisms underlying phenotypic properties that contribute to the expansion of successful S. pseudintermedius clonal lineages.

The increase in multidrug and methicillin-resistant S. pseudintermedius (MRSP) may result from horizontal transfer of genetic material between bacterial isolates, yet is thought to be rare in Staphylococci …

Transduction As The Method Of Horizontal Gene Transfer Of The Staphylococcal Chromosomal Cassette Mec (Sccmec), Amber B. Sauder

Senior Honors Projects, 2010-2019

Methicillin-resistant Staphylococcus aureus (MRSA) gains resistance to β-lactam antibiotics through a mutated penicillin binding protein (PBP2a) encoded on the SCCmec element. In combination with the recombinase encoded by ccr, these two genes are used as markers of the mobile genetic element (SCCmec). Due to recent increases in community acquired MRSA infections, the mechanisms of antibiotic resistance gene transfer have gained attention. Transduction, a method of horizontal gene transfer mediated by bacteriophage, is believed to be responsible for the movement of the SCCmec element. Recent studies have shown the transduction of the SCCmec element in clinical isolates; however, this study is …

Pseudomonas Aeruginosa Ampr Transcriptional Regulatory Network, Deepak Balasubramanian

FIU Electronic Theses and Dissertations

In Enterobacteriaceae, the transcriptional regulator AmpR, a member of the LysR family, regulates the expression of a chromosomal β-lactamase AmpC. The regulatory repertoire of AmpR is broader in Pseudomonas aeruginosa, an opportunistic pathogen responsible for numerous acute and chronic infections including cystic fibrosis. Previous studies showed that in addition to regulating ampC, P. aeruginosa AmpR regulates the sigma factor AlgT/U and production of some quorum sensing (QS)-regulated virulence factors. In order to better understand the ampR regulon, the transcriptional profiles generated using DNA microarrays and RNA-Seq of the prototypic P. aeruginosa PAO1 strain with its isogenic ampR deletion …

Eluding Antibiotic Resistance: Capitalizing On Antimicrobial Peptides Interaction With The Lipid Bilayer, Danielle M. Mcgrath

Dissertations & Theses (Open Access)

It is widely accepted that the emergence of drug-resistant pathogens is the result of the overuse and misuse of antibiotics. Infectious Disease Society of America, Center for Disease Control and World Health Organization continue to view, with concern, the lack of antibiotics in development, especially those against Gram-negative bacteria.

Antimicrobial peptides (AMPs) have been proposed as an alternative to antibiotics due to their selective activity against microbes and minor ability to induce resistance. For example, the Food and Drug Administration approved Daptomycin (DAP) in 2003 for treatment of severe skin infections caused by susceptible Gram-positive organisms. Currently, there are 12 …

Escherichia Coli And Antibiotic Resistance To Tetracycline Antibiotics, Taylor L. Dodgen

Senior Honors Theses

Escherichia coli cells growing under ideal conditions are able to complete one reproduction cycle in as little as every twenty minutes. Since so many generations are able to be observed, one should theoretically be able to observe thousands of generations and determine evolution’s effects over a short period of time. In this experiment, E. coli K12 cells were cultured under ideal growth conditions but in the presence of antibiotics as a selective environmental stress in order to select for resistance. This was accomplished by serially passing colonies that were in close contact with two different, but similar antibiotics over a …