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.

Clostridioides difficile is a bacterial pathogen that causes pseudomembranous colitis and the majority of antibiotic-associated diarrheal cases. Broad-spectrum antibiotic usage disrupts the normal gut microbiota and thereby compromises colonization resistance, the main defense against C. difficile infection. Treatment options are limited to vancomycin, fidaxomicin, and the fecal microbiota transplant. Addressing the scarcity of these therapeutics, we documented two explorations in C. difficile drug discovery: (i) evaluation of antibacterial and toxin-suppressing activity of (+)-puupehenone and similar derivatives, and (ii) clarification of a discrepancy in the hypothesized mechanism of auranofin against C. difficile. A better understanding of how C. difficile colonizes and thrives in the gut can greatly benefit therapeutic development. Interestingly, C. difficile can scavenge nutrients such as amino acids and possibly even purines during infection. Amino acids including proline and glycine act as substrates for Stickland metabolism, a bioenergetics scheme that partially relies on enzymes containing selenium in the form of selenocysteine (e.g., D-proline reductase and glycine reductase). Purines such as xanthine and uric acid can be degraded by bacterial molybdenum hydroxylases harboring an uncharacterized form of selenium, though the role of these enzymes in C. difficile physiology is poorly understood. Selenium likely plays a key role in the scavenging of these nutrients during C. difficile infection. Our investigation of these selenium-dependent enzymes revealed two new findings in C. difficile biology: (i) a link between proline-dependent growth and D-proline reductase, characterized as an energy "addiction," and (ii) a previously uncharacterized selenium-dependent pathway involved in the catabolism of xanthine and uric acid. Overall, these physiological analyses of C. difficile provide promising candidates for therapeutics and key information regarding the organism's nutrient preferences.

Diss. (sammanfattning) Stockholm : Karol. inst., 2005. / Härtill 5 uppsatser.

<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.

Paracoccidioidomicose (PCM) é uma micose sistêmica causada pelo fungo Paracoccidioides brasiliensis e é considerada a 10º causa de morte dentre doenças infecciosas e parasitárias no Brasil. As células dendríticas (DCs) são as mais eficientes na apresentação de antígenos, e sendo de 100 a 1000 vezes mais eficientes que um adjuvante não específico. P10, motivo específico de 15 aminoácidos derivado da gp43 excretada pelo fungo, é reconhecido por linfócitos T, direciona para resposta Th1 e confere proteção no modelo experimental. Nesse sentido, analisamos a capacidade de DCs pulsadas com P10 em ativar uma resposta protetora. In vitro observamos que a proliferação de esplenócitos foi maior quando a reestimução era feira com P10 e DCs juntos. In vivo, camundongos infectados e tratados com DCs pulsadas com P10 apresentaram diminuição da carga fúngica e melhoria do tecido pulmonar, aumento de IFN-<font face=\"Symbol\">g/IL-12 e diminuição de IL-4/IL-10. Esses resultados evidenciam o potencial das DCs atuando como adjuvante para o P10 em uma vacina para o tratamento e cura da PCM. / Paracoccidiodomycosis (PCM) is a systemic mycosis caused by the fungus Paracoccidioides brasiliensis. In Brazil, it is ranked as the tenth cause of death among the chronic infectious and parasitic diseases. Dendritic cells (DCs) are the most efficient antigen presenting cells, and they are at least 100-1000 times more efficient than a non specific adjuvant. The P10, 15-amino acid peptide motif derived from gp43 secreted by the fungus, is recognized by T cells, induces Th1 response and protects mice in a murine model of the disease. In the present work we analyzed the ability of DCs pulsed with P10 to elicit a protective response. In vitro results showed that a stronger proliferation of the splenocytes was reached when induced by DCs with P10. In vivo, mice infected and treated with DCs pulsed with P10 showed reduce of the fungi burden with less inflamed areas, higher levels of IFN-<font face=\"Symbol\">g/IL-12 and lower levels of IL-10/IL-4. All that point out the importance of the adjuvant role of DCs when developing a vaccine using P10 for the treatment or cure of PCM.

A maioria dos tratamentos antifúngicos são longos e não eficazes, portanto, há uma necessidade de pesquisa de novas alternativas. A fim de prospectar novos epitopos para uma vacina para Paracoccidioides lutzii e Cryptococcus spp.. Foi desenvolvido um sistema com o objetivo de purificar epitopos de macrófagos no contexto de MHCII. Além disso, houve pesquisa de epítopos na parede celular e sobrenadante de Paracoccidioides spp. A atividade antifúngica da miltefosina foi avaliada contra Paracoccicioides spp.. A miltefosina demonstrou atividade inibitória similar à anfotericina B. A atividade fungicida ocorreu em baixas concentrações, se observou alterações ultraestruturais e formação de melanina. A atividade do C7a in vitro e in vivo contra Candida spp. foi analisada. Os resultados mostraram atividade fungicida em valores baixos. Além disso, foi visto inibição da formação de hifas / pseudohifas e alterações morfológicas. Os ensaios in vivo demonstraram uma diminuição significativa na infecção em um modelo de candidíase vaginal e sistêmica. / Most of antifungal treatments are not completely effective or long, thus there is a need to search new alternatives. In order to prospect new epitopes for a vaccine to Paracoccidioides lutzii and Cryptococcus spp.. A system was developed with the purpose of purify epitopes from macrophages in the context of MHCII. Also, a search for epitopes in the cell wall and supernatant of Paracoccidioides spp. was done. The antifungal activity of miltefosina was evaluated against Paracoccicioides spp.. Miltefosine demonstrated inhibitory activity similar to amphotericin B. Fungicidal activity occurred at low concentrations, ultrastructural alterations and melanin production were observed. The activity of C7a in vitro and in vivo against Candida spp. was analyzed. The results showed fungicidal activity at low values. Also, it was revealed that inhibition of formation of hyphae/pseudohyphae and morphological alterations. In vivo assays demonstrated a significant decrease of fungal border of intravaginal and intravenous infected mice.