The study explored the prevalence of biofilm formers and its association with multidrug resistance in Klebsiella Pneumonia, a gram-negative bacterium that has high propensity to form antibiotic resistant strains and forms biofilms. Biofilms are complex microbial community with attributes that vary from planktonic cells. Antibiotic resistance is a property that has shown evidence to be higher in biofilms as compared to planktonic cells. Multi-drug resistance, a higher form of antibiotic resistance, is defined as resistance to at least one agent in three or more antibiotic categories. A single-armed and a two-armed meta-analysis was done to assess prevalence of biofilm formers and to find association between biofilm formation capacity and multi drug resistance. The one-armed meta-analysis revealed 74% (95% CI: 64%-83%) prevalence of biofilm formers among clinical isolates of Klebsiella Pneumonia. The prevalence rate is comparable with that of prevalence rate attained by other bacterium by similar meta-analysis studies. This high prevalence of biofilm formers warrants for a paradigm shift in treatment strategies for treatment of infections. The two-armed meta-analysis showed that there was identical risk of multi drug resistance among the biofilm formers and non-biofilm formers. The result challenges the intrinsic capacity of planktonic cells to resist against antibiotics to achieve multi drug resistance. Further research to update the biofilm formation profiles and to understand the resistance mechanism in commonly occurring bacterial infections in of utmost importance.

The rise of antimicrobial resistance demands the development of more rapid screening methods for the detection of antimicrobial resistance in clinical samples to both give the patient the proper treatment and expedite the treatment of patients. Cerium oxide nanoparticles may serve a useful role in diagnostics due to their ability to exist in a mixed valence state and act as either oxidizing agents or reducing agents. Considering that cerium oxide nanoparticles have been shown to shift in absorbance upon oxidation, a useful method of antimicrobial resistance detection could be based on the oxidation of cerium oxide nanoparticles. Herein, an assay is described whereby cerium oxide nanoparticle oxidation is a function of glucose metabolism of bacterial samples in the presence of an antimicrobial agent. Cerium oxide nanoparticles were shown to have an absorbance in the range of 395nm upon oxidation by hydrogen peroxide whereas mixed valence cerium oxide nanoparticles lacked an absorbance around 395nm. In the presence the hydrogen peroxide-producing glucose oxidase and either increasing concentrations of glucose or bacterial medium supplemented with increasing concentrations of glucose, cerium oxide nanoparticles were shown to increase in absorbance at 395nm. This oxidation assay was capable of measuring differences in the absorbance of E. coli and S. aureus samples grown in the presence of inhibitory and non-inhibitory concentrations of ampicillin in as little as six hours. Therefore, this cerium oxide nanoparticle oxidation assay may be very useful for use in clinical laboratories for the detection of antimicrobial resistance due to the relatively low cost, no requirement for specialized equipment and, most importantly, the reduced incubation time of the assay to as little as six hours compared to current gold standard antimicrobial resistance detection methods that require 24 hours.; This assay may thus also help partially circumvent the issue of knowledge of antimicrobial resistance in infected patients before prescribing improper regimens.

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The objectives of this study were to evaluate the effect of number of stocker cattle in receiving pens (large: n=150 cattle; small: n=50 cattle) on 1) BRD morbidity/mortality, and performance, 2) antimicrobial use and prevalence of antimicrobial resistance in Mannheimia haemolytica, and 3) profitability of stocker operations. No differences were found for morbidity (p=0.5041). Mortality tended (p=0.0744) to be higher in large groups. BW increased (p LESS THAN 0.0001) over time. A treatment*day interaction (p=0.00592) was found for ADG, with largest gains for both groups from day 14-28. M. haemolytica recovery decreased (p=0.0002) over time. Antimicrobial resistance (p=0.0179) and MDR (p=0.0405) were higher in the small group. Treatment costs were higher in the small group ($1,093.53/hd) compared to large ($1,037.04/hd). Because of the nature of a pilot study, further research are needed to determine the effectiveness of reducing animals in a pen on health, growth, AMR, and profitability associated with stocker cattle.

The antimicrobial resistance (AMR) crisis, where new antibiotic discovery is not keeping pace with the emergence of resistant pathogens, is driven by mobile genetic elements (MGEs). MGEs can autonomously transfer between bacteria, along with AMR genes. The widespread use of antibiotics in the clinic, in agriculture, and animal husbandry, has accelerated the MGE-mediated transfer of AMR genes in the environment. However, despite playing such an important role in the AMR crisis, the dynamics and mechanisms behind the transmission of genes are poorly understood. Furthermore, which natural and man-made compounds inhibit or promote their movement in these environments is unknown. One method to combat the rise in AMR is to identify small molecules as probes to understand the molecular basis of transmission and apply this information to prevent MGE-mediated resistance dissemination. Since conjugation is the main mechanism for AMR gene transfer, targeting MGEs that use conjugation, such as conjugative plasmids (e.g. Tn1549) and conjugative transposons (e.g. F plasmid), has the potential to prevent the emergence of multi-drug resistant pathogens. In this work, a high throughput assay modeled after Tn1549 excision was screened against a library of known bioactive compounds to find modulators of the integrase and excisionase activity. Several fluoroquinolone antibiotics including ciprofloxacin were identified as dose-dependent inhibitors of excision, which acted by changing supercoiling levels in the cell. Ciprofloxacin enhanced conjugation frequency of Tn1549 at sub-MIC concentrations relative to an untreated control and inhibited conjugation frequency at higher concentrations. A second project was focused on a high throughput conjugation assay based on the separation of the lux operon between a donor and recipient cell, such that only transconjugants produce luminescence to reflect active gene transfer. This work furthers our understanding of the development of assays to target MGEs and screening for inhibitors of their movement. / Thesis / Master of Science (MSc) / Antibiotics are small molecules that cure bacterial infections. However, their efficacy is fading as a result of the ability of mobile genetic elements (MGEs) to spread antimicrobial resistance genes between bacteria. Conjugative plasmids (CPs) and conjugative transposons (CTns) are two of the major types of MGEs that contribute to the dissemination of antimicrobial resistance in pathogens. The goal of this research is to search for inhibitors of CTns and CPs in order to prevent the emergence of multi-drug resistant bacteria. High throughput assays were designed to model both a CTn (Tn1549) and a CP (F plasmid) to find small molecules targeting their movement. A screen of the Tn1549 excision assay identified fluoroquinolone antibiotics that inhibit excision in a dose-dependent manner and indirectly inhibit the integrase used to excise the CTn. Ciprofloxacin, a fluoroquinolone, inhibited the conjugation frequency of Tn1549. Future work will focus on identifying new inhibitors of these MGEs and their characterization.