The LD50 of Vibrio parahaemolyticus ATCC 17803 for 13- to 18-g male mice, strain ICR, as determined by the 50% endpoint method, was found to be an estimated 7 x 107 CFU, when administered intraperitoneally in 0.5 ml of 24-h broth culture, O. D. 0.5 at 650 nm, diluted to 10-0.72. The elicitation of protective antibody by this bacterium in male New Zealand White rabbits was demonstrated by testing control, baseline, and hyperimmune rabbit sera for their protective effect against 2 LD50 of the bacterium administered intraperitoneally in 13- to 18-g male mice, strain ICR. A comparison of these sera showed that there was a highly significant difference, at the 0.01 level, between the protection conferred by the hyperimmune sera and the protection conferred by the baseline or control area. Significant protection was demonstrated by hperimmune sera at dilutions as high as 1/32. Comparison of results of in vitro tests for preciptins and agglutinins in hyperimmune rabbit sera with results of in vivo tests for protective effect of hyperimmune rabbit sera led to a tentative conclusion that this passive protection afforded by the hyperimmune rabbit sera is probably conferred more by precipitins than by agglutinins.

Salmonellosis is a serious problem of public health that mainly infants, young children and people with immunodeficiencies face. Human direct contact with animals is one of the possible ways of transmission of the disease. An increasing trend of keeping exotic pets, including amphibians and reptiles, has been observed for recent years in the United States and developing European countries. Most of these animals are asymptomatic carriers of Salmonella. However, in this review we introduced new and the most dangerous outbreaks of salmonellosis caused by contact with amphibians and reptiles that appeared in all continents in last ten years. It was demonstrated that Salmonella strains isolated from cold-blooded animals such as animals and reptiles differ genetically from strains isolated from humans. It means that the reason of appearance of Salmonella outbreaks caused by transmission of pathogens from amphibians or reptiles to human are genetic changes including the activation of virulence factors that cause pathogenicity in humans. It is supposed that popularity of keeping amphibians and reptiles as pets has caused an increase in the demand for these animals and in the international trade of these species. The problem is also caused by breeder's unconsciousness of proper procedures of keeping exotic animals in a household. Therefore, the World Health Organization (WHO) has censored the most important issues to minimize the risk of salmonellosis, focusing mainly on RAS salmonellosis (reptile – associated salmonellosis), because this disease is the most common in the United States. It is estimated that about 74,000 of the United States citizens are getting infected by Salmonella strains transmitted from reptiles kept as pets every year. Educating people on this topic is a key preventive method of salmonellosis. This review can help future breeders how to keep reptiles and amphibians according to recommendation of World Health Organization.

Anthrax, a fatal bacterial infection is caused by Bacillus anthracis, a gram-positive, spore forming, capsulated, rod shaped organism. Centers for Disease Control (CDC) lists anthrax as Category A biological agent due to its severity of impact on human health, high mortality rate, acuteness of the disease and potential for delivery as a biological weapon. The currently available human vaccine in the United States (AVA anthrax vaccine adsorbed) is prepared from Alum adsorbed formalin treated supernatant culture of toxigenic, non-encapsulated strain of Bacillus anthracis with the principle component being protective antigen (PA83). Evaluation of anthrax vaccine given to nearly 400,000 US military personnel by Vaccine Adverse Event Reporting System (VAERS) showed adverse effects such as flu-like symptoms, local pain, large degree of inflammation, edema, malaise, rash, arthralgia, and headache following vaccination. All the adverse reactions are attributed to the composition of vaccine components. These vaccine preparations contain trace contaminants of lethal and edema factors that contribute to the adverse side effects. Also, the current method of vaccine manufacture has limited production capacity.The production of PA83, in plants through chloroplast genetic engineering might eliminate the concerns of adverse side effects and the levels of expression would ensure the availability of vaccine for the human population in an environmentally friendly approach. The primary concern is whether the PA83 purified from transgenic chloroplasts is as immunogenic as the PA83 in the AVA. For this, PA83 has been expressed in transgenic chloroplasts of Nicotiana tabacum var. petit Havana, by inserting the pag (2205 bp) with the N-terminal 6X histidine tag, into the chloroplast genome by homologous recombination. Chloroplast integration of the pag was confirmed by PCR and Southern analysis. The PA83 protein was detected in transgenic chloroplasts by immunoblot analysis using anti-PA83 antibodies. Maximum expression levels of PA83 (14.17% TSP) were observed in mature leaves upon continuous illumination, due to the presence psbA 5'UTR, a light and developmentally regulated translation enhancer sequence. The PA83 has been purified by affinity chromatography using Ni resin columns. Chloroplast derived PA83 was functional in vitro and was able to lyse the mouse macrophages when combined with the lethal factor. The in vitro assays showed that the crude extracts contained up to 20ug/ml of functional PA83.The immunization studies of PA83 on Balb/c mice, revealed highly immunogenic IgG titers. Subcutaneous immunization with purified chloroplast derived PA83 with adjuvant yielded IgG titers up to 1:320,000, similar to that of the group immunized with PA83 derived from Bacillus anthracis. Immunization of groups with PA83 combined with alhydrogel adjuvant showed four - eight times higher immune response than the groups without adjuvant. The higher expression levels of PA83 in transgenic chloroplasts might ensure the availability of anthrax vaccine to the general public and the high immune response observed in the mouse model would enable the replacement of the current AVA with a cleaner and safer vaccine.

Infections caused by the organism Staphylococcus aureus are one of the most common causes for community-associated and healthcare-acquired infections (HAI). Isolates of this bacterium found within the healthcare setting often demonstrate a higher prevalence of antibiotic resistance making these infections difficult to treat. Historically, considerable focus has been placed on methicillin-resistant S. aureus (MRSA), which are strains resistant to β-lactam antibiotics like penicillin, oxacillin and cephalosporins; however, methicillin-sensitive (MSSA) strains may also possess resistance to several first-line antibiotics. Resistance to antibiotics can be acquired through horizontal gene transfer (HGT) by means of mobile genetic elements or by random DNA mutations as product of DNA replication. Bacteria have elucidated these mechanisms to defend themselves from antibiotics and one cause that promotes resistance is the inappropriate use or prescription of antibiotics to treat infections, i.e., using antibiotics to treat COVID-19. Through the SARS-CoV-2 pandemic, the CDC reported an increased prescription for antibiotics, similarly, other previous studies reported that antibiotics were part of treatment plant in some patients with COVID-19. The aim of this thesis is to study the differences in antibiotic resistance profiles of Staphylococcus aureus strains collected from carriage and disease samples at Nemours Children's Hospital in Orlando, FL from 2019-2022. The focus will be on comparing the susceptibility of methicillin-sensitive and methicillin-resistant strains to various antibiotics. The results will provide clinicians with valuable information that will allow for better treatments and consideration for antibiotic use when creating a treatment plan for patients.

The objective of this project was to develop a DNA hybridization sensor that can detect the presence of E. coli and reveal its resistance to the drug gentamicin. This probe will enable rapid and user-friendly diagnostics of E. coli infections and analysis of bacterial gentamicin-susceptibility profile by interrogation of a fragment of E. coli 16S rRNA bearing a substitution in the gentamicin-resistant cells. The sensor is promising for the point-of-care use to provide a timely UTI diagnostic solution. A quick diagnosis of E. coli infection and antibiotic resistance is crucial for treatment. To design a hybridization probe, we proposed a split approach for target interrogation and catalytic activity of a peroxidase-like deoxyribozyme (PDz) as a signal reporter. PDz contains a series of guanine residues in a strand and has been shown to form a parallel guanine-quadruplex (G4). This G4, with the addition of a hemin cofactor, catalyzes the reaction similar to that of horseradish peroxidase. If a colorless organic indicator is added to the G4-PDz-hemin containing solution and mixed H2O2, a colored oxidation product is formed (e.g., a dark blue/green). The color change reports the presence of the catalytically active G4, which occurs only when the nucleotide sequence of the target is a perfect match. When the target is not a perfect match, for example, in the case of the drug-causing nucleotide substitution, the G4 does not form, and there is no color change. The probes tested in this paper show promising results of such a sensor by being able to catalyze the described colorimetric reaction to generate a strong signal in the presence of a "gentamicin-susceptible" target and show selectivity against the "gentamicin-resistant" target.

<p>Retractable surface appendages Type IV pili (T4P) are one of the major virulence determinants in the opportunistic pathogen <em>Pseudomonas aeruginosa </em>(Pa), that is the leading cause of mortality in CF patients. T4P are heteropolymers composed of the major-pilin subunit PilA and the less-abundant minor pilins (MPs), FimU/PilV/W/X/E. Pilins share high sequence and structural similarity with pseudopilins (XcpT/U/V/W/X), that are proposed to form a periplasmic-structure in the evolutionarily related Type II secretion system (T2SS). Similar to T4P system, the T2SS is a multi-subunit complex that spans the inner (IM) and the outer (OM) membranes. It involves a two-step process facilitating the secretion of toxins into the extracellular milieu from the periplasm.</p> <p>Using immunogold TEM analysis and Western blot we identified, under native conditions, the major pseudopilin of T2SS XcpT, is incorporated into the T4P appendage, thus appearing on the surface. This is in contrast to previous studies reporting, the otherwise periplasmic structure, the pseudopilus appears on the surface only upon over-expression of XcpT. Further, we identified this incorporation is strictly dependent on PilA expression, such that levels of surface-XcpT co-varied with the levels of surface-PilA. However, XcpT incorporation into the T4P fiber did not affect T4P-mediated twitching motility or T2SS-mediated elastase secretion. Based on these observations we proposed two explanations. Firstly, given the similarity between XcpT and type IV pilins, it is possible the pseudopilin is recognized by the T4P machinery and therefore is incorporated into the pilus. Secondly, since XcpT incorporation does not affect T4P-mediated motility, it may affect other properties of T4P, such adherence during biofilm formation, previously associated with surface-exposed pseudopilus. In addition, we also identified enhanced expression of <em>fimU</em> and <em>pilX</em> MPs drastically increased elastase secretion, through a yet to be discovered mechanism. Regardless, our results present an alternative role of both minor pilins and XcpT in their non-native systems suggesting there is more overlap between the T4P and T2S systems than previously appreciated. Further exploration of this overlap will aid in the study of the two systems in Pa, as well as in other pathogens.</p> / Master of Science (MSc)

Background: Infections caused by carbapenem-resistant Enterobacteriaceae such as Escherichia coli and Klebsiella pneumoniae are among the most urgent threats of the infectious disease realm. The incidence of these infections has only been increasing over the years and due to very limited treatment options, mortality is estimated at about 50%. Methods: To evaluate the in vitro activity of meropenem and polymyxin B against carbapenem-resistant Enterobacteriaceae, antimicrobial susceptibility testing and time-kill studies were performed on K. pneumoniae clinical isolates representing a wide range of meropenem resistance (MICs 4 – 128 mg/L). Results: Regrowth was observed at clinically relevant concentrations of meropenem alone (4, 16, and 64 mg/L) or polymyxin B alone (0.25 and 1 mg/L) within 24 hours. However, meropenem and polymyxin B in combination were consistently bactericidal, achieving synergistic activity in strains with lower meropenem resistance (MICs ≤32 mg/L). Conclusions: Our findings are in agreement with the limited available literature, but we add that the synergistic interaction between meropenem and polymyxin B is dependent on the degree of meropenem resistance in KPC-producing K. pneumoniae. This data suggests that lower level resistance to carbapenems may be amenable to antimicrobial combinations involving a carbapenem and a polymyxin.

Antibiotic resistance is an ever-growing topic of concern within the medical field causing researchers to examine the mechanisms of resistance to develop new antimicrobials. Bacteria’s ability to form biofilms is one mechanism which aids in antimicrobial resistance. Staphylococcus aureus is of special interest as it is one of the most frequent biofilm-forming bacteria found on medical devices causing infections and posing dangerous threats in a clinical setting. A recently developed antimicrobial gel has been shown to have profound effects on treating bacterial infections and wound healing. This research is centered upon examining the antimicrobial effects of this gel on the three different stages of biofilm formation in clinical and laboratory strains of S. aureus. Through a series of experiments examining the effects this gel has on S. aureus at the stages of biofilm attachment, maturation, and dispersion, the gel has shown significant levels of inhibition. These findings indicate that the novel gel disrupts biofilm forming processes of S. aureus, which provides useful information for fighting infections in the medical field. Further research on the uses and effects of this new gel could lead possibility using the antimicrobial compound for a variety of clinical purposes.

Bacteria secrete and sense extracellular signals from neighboring members of a colony in a phenomenon called quorum sensing. These signals vary from species to species but allow for changes in the behavior of a colony based on changes to cell density, environment, or nutrient supply. Of particular interest to human health is the quorum sensing system of Streptococcus pneumoniae as this pathogen accounts for around one million infection-related deaths per year and is difficult to combat largely due to its ability to form biofilms. These polysaccharide coverings protect entire bacterial colonies from antimicrobial agents as well as allow them to adhere well to the nasopharynx passages of organisms, making them hard to remove. To gain a better understanding of quorum sensing in S. pneumoniae, we propose experiments to study its biofilm formation and its interactions with black carbon, a biochar shown previously to interact with the quorum sensing systems of related bacteria species. We hypothesize that inhalation of black carbon will aggravate a S. pneumoniae infection by promoting biofilm-forming quorum sensing systems making it easier for this bacteria to adhere to and remain on mammal lungs. We propose to first explore the competency and biofilm quorum sensing systems in S. pneumoniae to identify any shared signals between the two using RT-PCR and FITC-Dextran experiments. Further experiments will analyze black carbon particles’ effects on bacterial colonies grown on plates and present on the lung linings of mammals.

Lyme disease is one of the most common infectious diseases present in the United States today and it is clear that the changing climate will affect the geographical range of it across the country. Climate change may impact the range of the Lyme vector species, ticks, which will in turn expand the range of human risk. Although I could not successfully map the possible spread of tick populations or Lyme disease incidence as a result of climate change, my research shows a direct connection between infected tick geographic distribution and key climatic variables, such as temperature, humidity, and precipitation. It is expected that as the climate changes, particularly as it warms, the range of suitable habitat for ticks will expand into high latitudes and altitudes. The expansion of tick populations will put previously unaffected human populations at greater risk of Lyme disease. It is essential that further research be done to confirm the possible consequences of climate change on Lyme disease in the United States and to gain a more precise understanding of how and where effects will be seen. Health officials and policymakers must be informed so they can properly educate and prepare people preemptively for potential Lyme disease outbreaks.