The fungal pathogen Candida albicans and the opportunistic bacterial pathogens Enterobacter aerogenes and Enterobacter cloacae are common sources of human disease. The colonization of proximal anatomical locations by these pathogens suggests that interspecies polymicrobial interactions between Candida albicans and Enterobacter species occur. In order to understand mechanisms of diseases caused by these pathogens and to further the study of disease prevention, analyzation of their combined activities was conducted in this study. Changes in fungal morphology, cellular viability, and colony density were investigated using fungal and bacterial co-cultures. The effects of the Candida secreted quorum sensing molecule farnesol on Enterobacter aerogenes and Enterobacter cloacae was studied to observe changes in Enterobacter viability and colony density. The effects of the presence of Enterobacter species on Candida albicans was studied by observing changes in Candida morphology and colony density. The mutant strain of Candida albicans AlS6-/- was also cultured with Enterobacter to determine if the presence of the ALS6 surface glycoprotein gene affected Candida viability and colony density in the presence of Enterobacter species. Statistically significant decreases were observed in all studied metrics between experimental and control groups. This indicated that the interactions observed between Candida albicans and Enterobacter species represent reciprocal inhibitions of cellular functionalities. As Candida albicans is the primary cause of human fungal infections and Enterobacter species are common causes of opportunistic infections, the study of polymicrobial interactions between Candida and Enterobacter species as conducted in this study is important to furthering efforts of human disease inhibition.

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Human noroviruses (HuNoVs) are the leading cause of viral epidemic acute gastroenteritis and responsible for the deaths of over 200,000 children each year worldwide. HuNoV research has been hampered by the long absence of a readily reproducible cell culture system and a suitable small animal model, while gnotobiotic (Gn) pigs have been a unique animal model for understanding HuNoV pathogenesis and immunity, as well as evaluating vaccine and therapeutics. Recent reports of HuNoVs infection and replication in B cells supplemented with commensal bacteria Enterobacter cloacae and in Blab/c mice deficient in RAG/IL2RG have gained extensive attention, and my studies utilized the well-established Gn pig model to investigate the effects of these two interventions on HuNoV infection. Surprisingly, the colonization of E. cloacae inhibited HuNoV infectivity in Gn pigs, evidenced by the significantly reduced HuNoV shedding in feces and HuNoV titers in intestinal tissues and blood compared to control pigs. Moreover, HuNoV infection of enterocytes but not B cells was observed with or without E. cloacae colonization, indicating B cells were not a target cell type for HuNoV in Gn pigs. On the other hand, using RAG2/IL2RG deficient pigs generated by CRISPR/Cas9 system, with confirmed severe combined immunodeficiency, I evaluated the effects of host immune responses on HuNoV infection. Compared to wild-type Gn pigs, longer HuNoV shedding was observed in RAG2/IL2RG deficient pigs (16 versus 27 days), and higher HuNoV titers were detected in intestinal tissues and contents and in blood, indicating increased and prolonged HuNoV infection in RAG2/IL2RG deficient pigs. In addition, I evaluated dietary interventions including probiotics and rice bran using Gn pig model of HuNoV infection and diarrhea. While the colonization of probiotic bacteria Lactobacillus rhamnosus GG (LGG) and Escherichia coli Nissle 1917 (EcN) in Gn pigs completely inhibited HuNoV fecal shedding, the two cocktail regimens, in which rice bran feeding started either 7 days prior to or 1 day after viral inoculation in the LGG+EcN colonized Gn pigs, exhibited dramatic anti-HuNoV effects, including reduced incidence and shorter duration of diarrhea, as well as shorter duration of virus fecal shedding. The anti-HuNoV effects of the cocktail regimens were associated with the enhanced IFN-𝛾⁺ T cell responses, increased production of intestinal IgA and IgG, and longer villus length. Taken together, my dissertation work improves our understanding of HuNoV infection and immunity, and further supports for Gn pigs as a valuable model for future studies of human enteric virus infection, host immunity, and interventions. / Ph. D. / Human noroviruses (HuNoVs) are the leading cause of viral epidemic acute gastroenteritis. Using the gnotobiotic pig model of HuNoV infection and diarrhea, we found that (1) the colonization of a commensal bacterium E. cloacae inhibited HuNoV infectivity, and B cells were not a target cell type for HuNoV in gnotobiotic pigs. (2) Increased and prolonged HuNoV infection in RAG2/IL2RG deficient pigs, which had severe combined immunodeficiency. (3) The dietary supplementation of rice bran and colonization of two probiotic bacteria significantly reduced HuNoV infectivity and diarrhea, and the beneficial effects were associated with enhanced intestinal immunity and health. Taken together, the dissertation work improves our understanding of HuNoV infection and immunity, and further supports for gnotobiotic pigs as a valuable model for future studies of human enteric virus infection, host immunity, and interventions.

Einführung: Enterobacter (E.) cloacae subsp. cloacae sind wichtige Humanpathogene, insbesondere bei stationär untergebrachten Patienten. Sie sind in der Lage, Medizinprodukte zu kontaminieren, und es wurde über nosokomiale Krankheitsausbrüche in Verbindung mit der Kolonisation von chirurgischen Utensilien berichtet. Es ist daher wichtig, die Wirksamkeit und Effizienz von Desinfektionsmitteln gegenüber dieser Bakterienart zu bestimmen. Ziele: Die aktuelle Studie wurde durchgeführt, um nachzuweisen, ob Peressigsäure, Ethanol, Benzalkoniumchlorid und Natriumhypochlorit, welche weit verbreitet in kommerziellen Desinfektionsmitteln enthalten sind, eine ausreichende Wirksamkeit gegen multiresistente, von Patienten im Krankenhaus isolierte, E. cloacae aufweisen. Material und Methoden: Sechs multiresistente E. cloacae Isolate, die von Patienten in einem klinischen Umfeld gewonnen wurden, wurden getestet und mit dem E. cloacae Typstamm verglichen. Die Studien wurden in vitro mit Peressigsäure, Ethanol, Benzalkoniumchlorid und Natriumhypochlorit nach den Richtlinien der Desinfektionsmittel-Kommission des Verbundes für Angewandte Hygiene e.V. durchgeführt. Die Tests umfassten qualitative und quantitative Suspensionstests zur Bestimmung der bakteriziden Wirkung, den sogenannten Keimträgertest und die Bestimmung der minimalen Hemmkonzentrationen. Ergebnisse: Die Studienergebnisse zeigten, dass multiresistente E. cloacae Stämme genauso empfindlich gegenüber Desinfektionsmitteln waren wie der Typstamm. Organische Belastung interagierte stark mit Natriumhypochlorit und minderte dadurch seine Wirksamkeit, während Peressigsäure und Ethanol nicht durch organische Verunreinigung beeinflusst wurden. Die Kontaktzeit hatte nur einen geringen Einfluss auf die bakterizide Wirkung. Im Gegensatz dazu spielten bei Benzalkoniumchlorid organische Verunreinigung und die Kontaktzeit eine wichtige Rolle. Insgesamt waren die minimalen Hemmkonzentrationen und die bakterizid wirksamen Konzentrationen niedriger als die für kommerzielle Produkte gebräuchlichen Konzentrationen. In den Keimträgertests hatte das Trocknen auf einer glatten Oberfläche einen Einfluss auf das Überleben eines Stammes von E. cloacae. Die Ergebnisse zeigten auch, dass sich die Wirksamkeit der Desinfektionsmittel in den verschiedenen verwendeten Tests deutlich unterscheiden kann. Die Ergebnisse waren schwer mit anderen Studien zu vergleichen, da eine internationale Durchführungsrichtlinie für die Prüfung der Wirksamkeit von Desinfektionsmitteln gegen multiresistente Bakterien fehlt. Fazit: Peressigsäure, Ethanol, Benzalkoniumchlorid und Natriumhypochlorit eignen sich zur Desinfektion von multiresistenten E. cloacae. Die Wirksamkeit von Natriumhypochlorit und Benzalkoniumchlorid wird jedoch stark durch organische Stoffe beeinflusst. Dies unterstreicht die Bedeutung geeigneter Reinigungsmaßnahmen vor der Desinfektion. Wenn dies erfolgt ist, erweisen sich die getesteten Desinfektionsmittel gegen multiresistente E. cloacae genauso effektiv wie gegen den Typstamm.:1. Introduction ............................................................ 1 2. Literature Review ....................................................... 3 2.1 Enterobacteriaceae ..................................................... 4 2.1.1 General properties ................................................... 4 2.1.2 Enterobacter cloacae complex ......................................... 4 2.2 Multidrug-resistant bacteria and disinfectant “resistance” ............. 6 2.3 Disinfectant testing ................................................... 7 2.4 Active substances investigated in this study ........................... 8 2.4.1 Peracetic acid (PAA) ................................................. 8 2.4.2 Ethanol (ETH) ........................................................ 9 2.4.3 Benzalkonium chloride (BKC) .......................................... 9 2.4.4 Sodium hypochlorite (NaOCl) .......................................... 10 3. Materials and Methods ................................................... 11 3.1 Materials .............................................................. 11 3.2 Methods ................................................................ 14 3.2.1 Culture and storage of bacteria ...................................... 14 3.2.2 Preparation of disinfectants and the neutralizing agent .............. 14 3.2.3 Minimum inhibitory concentration (MIC) ............................... 15 3.2.4 Qualitative suspension test .......................................... 15 3.2.5 Quantitative suspension test.......................................... 16 3.2.6 Surface disinfection without mechanical action (germ carrier test) ... 16 3.2.7 Statistical analysis ................................................. 17 4. Results ................................................................. 18 4.1 Minimum inhibitory concentrations ...................................... 18 4.2 Qualitative suspension tests ........................................... 18 4.3 Quantitative suspension tests .......................................... 21 4.4 Surface disinfection without mechanical action (germ carrier test) ..... 24 5. Discussion .............................................................. 28 6. Summary ................................................................. 31 7. Zusammenfassung ......................................................... 33 8. References .............................................................. 35 9. Appendix ................................................................ 49 Acknowledgments ............................................................ 50 / Introduction: Enterobacter (E.) cloacae subsp. cloacae are important human pathogens, particularly in hospitalized patients. They tend to contaminate various medical devices and nosocomial outbreaks have been reported to be associated with the colonization of surgical equipment. Therefore, it is critical to determine the efficacy and effectiveness of disinfectants against this bacterial species. Objectives: The current study was undertaken to prove whether single active ingredients (i.e. peracetic acid, ethanol, benzalkonium chloride, and sodium hypochlorite) of widely used commercial disinfectants provide proper efficacy against multidrug-resistant human isolates of E. cloacae. Material and Methods: Six multidrug-resistant E. cloacae isolates obtained from patients in a clinical setting were tested and compared to the E. cloacae type strain. The studies were performed in vitro using peracetic acid, ethanol, benzalkonium chloride and sodium hypochlorite following the guidelines specified by the Disinfectants Commission within the Association of Applied Hygiene. Tests included determination of minimum inhibitory concentrations, bactericidal values by qualitative and quantitative suspension tests, and so-called germ carrier tests. The influence of exposure time and organic load on bacteriostatic and bactericidal concentrations was evaluated for each disinfectant using the two-tailed Mann-Whitney U-test. Results: Study results showed that multidrug-resistant E. cloacae strains were equally susceptible to disinfectants as the type strain. Organic matter highly interfered with sodium hypochlorite thereby decreasing its efficacy whereas peracetic acid and ethanol were not influenced by organic soiling. Contact time had only a minor effect on bactericidal values. This was in contrast to benzalkonium chloride where organic soiling and contact time played an important role. On the whole, minimum inhibitory concentrations and bactericidal concentrations were lower than in-use concentrations of commercial products. Drying on smooth surfaces in the carrier tests had an effect on the survival of one E. cloacae strain. Results also showed that efficacious values determined by the different tests used may differ distinctly. Results were difficult to compare with other studies because an international practical standard for testing disinfectant efficacy against multidrug-resistant bacteria is missing. Conclusion: Peracetic acid, ethanol, benzalkonium chloride and sodium hypochlorite are suitable to disinfect multidrug-resistant E. cloacae but the effectiveness of sodium hypochlorite and benzalkonium chloride is strongly influenced by organic matter. This underlines the importance of proper cleaning measures before disinfection. When this is done, the tested disinfectants proved to be as efficient against multidrug-resistant E. cloacae as against the type strain.:1. Introduction ............................................................ 1 2. Literature Review ....................................................... 3 2.1 Enterobacteriaceae ..................................................... 4 2.1.1 General properties ................................................... 4 2.1.2 Enterobacter cloacae complex ......................................... 4 2.2 Multidrug-resistant bacteria and disinfectant “resistance” ............. 6 2.3 Disinfectant testing ................................................... 7 2.4 Active substances investigated in this study ........................... 8 2.4.1 Peracetic acid (PAA) ................................................. 8 2.4.2 Ethanol (ETH) ........................................................ 9 2.4.3 Benzalkonium chloride (BKC) .......................................... 9 2.4.4 Sodium hypochlorite (NaOCl) .......................................... 10 3. Materials and Methods ................................................... 11 3.1 Materials .............................................................. 11 3.2 Methods ................................................................ 14 3.2.1 Culture and storage of bacteria ...................................... 14 3.2.2 Preparation of disinfectants and the neutralizing agent .............. 14 3.2.3 Minimum inhibitory concentration (MIC) ............................... 15 3.2.4 Qualitative suspension test .......................................... 15 3.2.5 Quantitative suspension test.......................................... 16 3.2.6 Surface disinfection without mechanical action (germ carrier test) ... 16 3.2.7 Statistical analysis ................................................. 17 4. Results ................................................................. 18 4.1 Minimum inhibitory concentrations ...................................... 18 4.2 Qualitative suspension tests ........................................... 18 4.3 Quantitative suspension tests .......................................... 21 4.4 Surface disinfection without mechanical action (germ carrier test) ..... 24 5. Discussion .............................................................. 28 6. Summary ................................................................. 31 7. Zusammenfassung ......................................................... 33 8. References .............................................................. 35 9. Appendix ................................................................ 49 Acknowledgments ............................................................ 50