Microbiology / Infectious Disease

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    Profiling Patient Exosomes as Key Regulators of Neurological Symptoms in Coronavirus Disease 2019 (COVID-19)
    (2022) Tate, Amanda; Phillips, Wendy; Borgmann, Kathleen
    The severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) pandemic, also known as coronavirus disease 2019 (COVID-19), has ignited the most significant world-wide health crisis of the 21st century. While acute COVID-19 infection has potentially lethal outcomes, there are also cases of post-acute sequelae of COVID-19 (PASC) infections, commonly known as 'long-haul' COVID-19. In these long-haul cases, individuals experience prolonged neurologic symptoms including loss of taste and smell, altered level of consciousness, weakness, and chronic fatigue, months after acute COVID-19 infection has subsided. Currently, more than half of COVID-19 survivors present with at least one long-haul COVID-19 symptom. Additionally, there are no effective treatments for these complex neurological symptoms due to lack of sufficient knowledge of the mechanisms in which COVID-19 affects brain function and contributes to long-haul COVID-19 symptoms. Exosomes are extracellular vesicles (EVs) containing genetic material, protein, metabolites and lipids, which mirror their cell of origin. The physiologic role of exosomes has yet to be clearly defined, but it is widely speculated that these EVs play a fundamental role in cell-cell communication. Virally infected cells are able to release EVs that interact with distant cells and alter their typical biological functions. Similar to other neurotropic viruses, EVs have been isolated and characterized in samples from patients with COVID-19. A recent respiratory virus mimetic experiment has demonstrated the ability of EVs to enter the brain and collect in microglial cells. Based on this current research, we suspect exosomes are contributing to the development of chronic neurological symptoms seen in individuals with long-haul COVID-19. We hypothesize that there are differences in exosomes isolated from individuals with and without neurological symptoms following COVID-19 infection. Exosomes were isolated from nasal swabs of COVID-19 patients using ultra-centrifugation, and separated from viral particles using density gradient purification with 20% sucrose buffer. Exosomal small RNAs were isolated, and then identified using RNA sequencing. Sequencing data was compared to both human and COVID-19 genomes using bioinformatics to determine expression levels and known functions of the small RNAs that are present. The exosomal RNAs characterized in this study will guide our future aims to identify the effects these EVs have on different neuronal cells. Differentially expressed cellular or viral RNAs could regulate long-haul COVID-19 neurological outcomes. We hope to gain an understanding of the role these EVs play in COVID-19 infections, as well as provide insight into potential therapeutic targets for individuals living with long-haul symptoms. Funding: This work was supported by US4 MD006882 from the National Institute on Minority Health and Health Disparities.
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    Variation of Best Fit Distributions in Single Cell Virus Dynamics Models
    (2022) Doty, Madison; Dobrovolny, Hana
    Purpose: Mathematical modeling of viral kinetics can be used to gain further insight into the viral replication cycle and virus-host interactions. However, many of the virus dynamics models do not incorporate the cell-to-cell heterogeneity of virus yield or the time-dependent factor of virus replication. A recent study of vesicular stomatitis virus (VSV) kinetics in single BHK cells determined that both virus production rate and yield of virus particles varies widely between individual cells of the same cell population. Methods: Here we use the results of the previously mentioned study to determine the distribution that best describes the time course of viral production within the single cells. We determined a list of eight potential distributions that are commonly used in viral kinetics models to fit to each data set by minimizing the sum of squared residuals. The model of best fit for each individual cell was determined using Akaike's Information Criterion (AICC ). Results: Results of this study show that the distribution that best describes viral production varies from cell to cell. Conclusion: This finding could have further reaching implications for incorporating time-dependent viral production into a standard model of virus kinetics in order to better reproduce the diversity of viral replication that occurs over time within a population of cells.
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    In vitro and in vivo development of rifampin resistance with Staphylococcus aureus clinical isolates
    (2022) Madunezim, Quentin
    Purpose: Rifampin is an older antibiotic used to treat several types of bacterial infections including tuberculosis. It is highly active against Staphylococcus aureus and effective against infections with this pathogen. Rifampin is a unique antibiotic that has the capacity to penetrate through thick bacterial biofilms and reach bacteria that may be harder to treat using other antibiotics. Rifampin's mechanism of action involves inhibiting bacterial RNA Polymerase by forming a stable drug-enzyme complex, however mutations in the rpoB gene that encodes the beta subunit of RNA polymerase can cause resistance in S. aureus. Resistant S.Aureus can then form biofilms on implanted medical devices such as prosthetic hips or indwelling catheters, making it even harder to treat and difficult on the lives of these individuals. The focus of our research was to determine if mutations in the rpoB gene of S. aureus induced in vitro leads to a decrease in pathogenicity of the bacteria, can be duplicated in an animal model during treatment and if rifampin can still be effective against these resistant mutants. Methods: The minimum inhibitory concentration (MIC) of rifampin was determined for several isolates of S. aureus using a broth microdilution method. These susceptible strains of S. aureus were then serially passaged on agar plates containing rifampin at multiples of the MIC. Colonies were selected from plates with the higher rifampin concentration, saved and then passaged again at increasing concentrations of rifampin. The change in rifampin MIC was confirmed for each isolate and passage by broth microdilution. The same serial passage was then conducted in broth. To confirm the purity, isolates were cultured on MSA plates to determine if contamination was present. Results: MICs for rifampin against the S. aureus isolates tested increased from 0.008 µg/mL for the parent strain to 16 µg/mL for selected strain after 3 passages on agar. Similar results were obtained following serial passaging in broth with rifampin MICs going from 0.002 µg/mL to 0.008 µg/mL. The observed increase in rifampin MIC for each isolate was found to be stable. Conclusion: Rifampin-resistant mutants were generated both in S. aureus 134-3 and 005-4 isolate strains following serial passages on agar plates and broth containing sub-inhibitory concentrations of rifampin. Rifampin MICs increased from parent to the resistant generated mutants (0.002 ug/mL to 16 ug/mL). Future studies will include looking at the same parent strains in an animal model of biofilm infection and demonstrating development of resistance to rifampin during therapy.
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    Effects of Fosfomycin-Resistant Mutants on Bacterial Growth and Efficacy
    (2022) Sule, Olagoke; Weiss, William
    Introduction: Fosfomycin (FOS) is an antibiotic used in treating urinary tract infections (UTI) and cystitis (bladder infection) in women. The antibiotic, which is bactericidal, works by inhibiting the bacterial cell wall biogenesis by inactivating the enzyme UDP-N-acetylglucosamine-3-enolpyruvyltransferase, also known as MurA. However, several mechanisms of FOS resistance have been reported. The resistance mechanisms involve reduced uptake of FOS due to a defect in one of the two transporters caused by mutations in the structural gene. Objective: This study looked at the differences in virulence and efficacy in the murine UTI model with FOS-resistant mutants. Method: The study started with two FOS-susceptible strains (E. coli 045 and 167). Isolated colonies from each parent strain were inoculated into Tryptic Soy Broth (TSB) then incubated for 5 hours. Next, the broth culture was swabbed onto a TSA plate. A 200 ug FOS disc was placed on the agar surface and incubated. The following day, colonies were selected from inside or around the edge of the zone of inhibition surrounding the FOS disc. These colonies were inoculated into TSB and incubated. This process was repeated for three passages (E. coli 045{A, B, C}and 167{A, B, C}). Next, Mueller-Hinton agar was prepped and used to determine the FOS minimum inhibitory concentration (MIC) for each of the passages of the two strains. Finally, a growth curve in Mueller-Hinton broth was performed to determine the effect the passages had on the growth patterns over time at 0, 1, 2, 4, and 6 hrs. Results: Fosfomycin MICs increased from 8 ug/mL to 512ug/mL from the parent to the resistant generated mutants on the Mueller-Hinton agar. It showed consistency in FOS resistance from the parent to the resistant generated mutants. Growth curves exhibited comparable patterns for parent and resistant mutants. Conclusion: Fosfomycin-resistant mutants were generated both an E. coli 045 and 167 strain following serial passages on plates with a FOS disc. Using the Mueller-Hinton agar method, Fosfomycin MICs increased from parent to the resistant generated mutants (8 ug/mL to 512ug/mL). These results indicate that strains with elevated MICs may still be successfully treatable with Fosfomycin. Future studies will include looking at the same strains in an animal model of UTI and determining if the mutation is stable and can be treated with FOS.
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    Disseminated Cryptococcus Neoformans Infection in a Renal Transplant Patient
    (2022) Tangirala, Praharsha; Mathew, Trina; Lee, Jack; Arispe, Ryan; Yalamanchili, Harika; Sengodan, Mohan; Machaiah, Madhrira; Balamuthusamy, Saravanan; Balamuthusamy, Saravanan
    Background: Cryptoccocus Neoformans is a fungus mainly found in the environment that infects humans via inhalation and usually affects the lungs and central nervous system. Most people remain asymptomatic; however, immunocompromised patients are most susceptible to this pathogen, particularly HIV/AIDs patients. We present a case of a renal transplant patient with disseminated Cryptococcus Neoformans infection. Case Presentation: A 71 y/o female presents to the emergency department for further evaluation of a fever of unknown origin that has been going on for 6 days. Outpatient workup was initiated by the transplant service; however, due to persistently high fevers she was admitted for further workup and management. Patient reports that her fevers mainly occur at night and reach a maximum of 104-105 F. In the morning the fever decreases to 102 F with Tylenol. Denies any other significant symptoms, recent travel, sick contacts, alcohol, tobacco, or drug use. Past history is significant for CKD due to IgA nephropathy with renal transplant 2.5 years ago. Patient is currently on a chronic immunosuppressive regimen of Mycophenolate and Tacrolimus and infection prophylaxis with TMP-SMX and Valganciclovir. Upon admission, vitals were within normal limits and physical exam was unremarkable. Labs showed negative urinalysis, influenza, COVID-19, and rapid strep antigen test. Lactic acid, magnesium, coagulation studies, TSH, and troponin were all within normal limits. BUN was elevated at 32 and Creatinine at 1.8. White blood cell count was decreased at 2.8k, hemoglobin decreased at 10.9, and hematocrit decreased at 34.2. Chest X-ray showed nodular opacifications involving the right mid to upper lung, possibly masses or mass like infiltrates. Malignancy at this point was high on the differential. CT of the chest was then obtained, which revealed a right upper lobe mass and bilateral pulmonary lymph node involvement that was concerning for metastatic disease. On admission day 2, a CT guided lung biopsy was done. Preliminary reads were suggestive of fungal etiology and no malignancy with the final report of histoplasmosis, but clinical correlation was recommended. On day 5, Cryptococcus Antigen (Ag) titer was obtained and was elevated. On day 6, bronchoscopy with cultures using MALDI-TOF revealed Cryptococcus neoformans and no malignant cells. Given concomitant cytopenia, patient was started on treatment for disseminated Cryptococcal disease with ambisome and flucytosine. However, patient developed an AKI likely from ambisone, and the regimen was switched to PO Voriconazole BID. On day 10, patient was switched to high dose fluconazole. On day 12, repeat Cryptococcal Ag titers showed an increase from 1:40 to 1:320, which was concerning for a high fungal burden. Ambisone was added back on with the fluconazole; however, due to worsening renal function ambisome was held again. Ultimately, the patient wanted to go home and she was discharged on high dose fluconazole per infectious disease recommendation with PCP and transplant service outpatient follow up. Discussion: This case demonstrates that although lobar masses in the elderly can be highly suspicious for malignancy, rarer causes such as Cryptococcus Neoformans should be considered on the differential.