This study examined the efficacy of potassium lactate and lactic acid to control Campylobacter sp. and psychrotrophic bacteria on chicken. The objectives of the two studies conducted were to determine the optimal combination of potassium lactate and lactic acid to inhibit Campylobacter sp. in a challenge study and to inhibit naturally occurring Campylobacter sp. and psychrotrophic bacteria in a shelf life study. Boneless, skinless chicken breasts were injected with three levels of potassium lactate (0,1.5,2%), in conjunction with four levels of lactic acid. Lactic acid was injected (0, 0.1, 0.2, 0.3%) as well as applied directly to the surface (0.1% of weight of chicken breast). The chicken breasts were surface inoculated with a mixture of Campylobacter sp. and sampled over a period of 28 days at 11oC. The greatest inhibition was found using 2% potassium lactate in conjunction with any level of lactic acid (injected) or 0.1% lactic acid (surface application). Results of this study indicate that potassium lactate and lactic acid can be used to control the growth and/or survival of Campylobacter sp. on boneless chicken breasts. The second study eliminated the 1.5% potassium lactate and 0.2% and 0.3% lactic acid treatments and chicken breasts were not inoculated with Campylobacter sp.. This 4oC shelf life study occurred over 32 days, testing for Campylobacter species, psychrotrophic bacteria, as well as testing for sensory perceptions of color and odor changes in the chicken. The most effective treatment was the 2% potassium lactate-0.1% lactic acid surface treatment, demonstrating the most inhibition against both target populations. This treatment also had the greatest impact upon the odor of the chicken breasts. This treatment had the greatest difference from control samples, which was achieved by the inhibition of spoilage organisms on the chicken breasts. / Master of Science

Indiana University-Purdue University Indianapolis (IUPUI) / Mononuclear molybdenum enzymes catalyze a variety of reactions that are essential in the cycling of nitrogen, carbon, arsenic, and sulfur. For decades, the structure and function of these crucial enzymes have been investigated to develop a fundamental knowledge for this vast family of enzymes and the chemistries they catalyze. The dimethyl sulfoxide reductase (DMSOR) family is the most diverse family of molybdoenzymes and, the members of this family catalyze a myriad of reactions that are important in microbial life processes. Periplasmic nitrate reductase (Nap) is an important member of the DMSO reductase family that catalyzes the reduction of nitrate (NO3-) to nitrite (NO2-), and yet the physiological role of Nap is not completely clear. Enzymes in this family can transform multiple substrates; however, quantitative information about the substrate preference is sparse and more importantly, the reasons for the substrate selectivity are not clear. Substrate specificity is proposed to be tuned by the ligands coordinating the molybdenum atom in the active site. As such, periplasmic nitrate reductase is utilized as a vehicle to understand the substrate preference and delineate the mechanistic underpinning of these differences. To this end, NapA from Campylobacter jejuni has been heterologously overexpressed, and a series of variants, where the molybdenum-coordinating cysteine has been replaced with another amino acid, has been produced. The kinetic and biochemical properties of these variants will be discussed and compared with those of the native enzyme, providing quantitative information to understand the function.

Infectious diseases caused by pathogenic microorganisms are one of the largest socioeconomic burdens today. Although infectious diseases have been studied for decades, in numerous cases, the precise mechanisms involved in the multifaceted interaction between pathogen and host continue to be elusive. Thus, it still remains a challenge for researchers worldwide to develop novel strategies to investigate the molecular context of infectious diseases in order to devise preventive or at least anti-infective measures. One of the major drawbacks in trying to obtain in-depth knowledge of how bacterial pathogens elicit disease is the lack of suitable infection models to authentically mimic the disease progression in humans. Numerous studies rely on animal models to emulate the complex temporal interactions between host and pathogen occurring in humans. While they have greatly contributed to shed light on these interactions, they require high maintenance costs, are afflicted with ethical drawbacks, and are not always predictive for the infection outcome in human patients. Alternatively, in-vitro two-dimensional (2D) cell culture systems have served for decades as representatives of human host environments to study infectious diseases. These cell line-based models have been essential in uncovering virulence-determining factors of diverse pathogens as well as host defense mechanisms upon infection. However, they lack the morphological and cellular complexity of intact human tissues, limiting the insights than can be gained from studying host-pathogen interactions in these systems. The focus of this thesis was to establish and innovate intestinal human cell culture models to obtain in-vitro reconstructed three-dimensional (3D) tissue that can faithfully mimic pathogenesis-determining processes of the zoonotic bacterium Campylobacter jejuni (C. jejuni). Generally employed for reconstructive medicine, the field of tissue engineering provides excellent tools to generate organ-specific cell culture models in vitro, realistically recapitulating the distinctive architecture of human tissues. The models employed in this thesis are based on decellularized extracellular matrix (ECM) scaffolds of porcine intestinal origin. Reseeded with intestinal human cells, application of dynamic culture conditions promoted the formation of a highly polarized mucosal epithelium maintained by functional tight and adherens junctions. While most other in-vitro infection systems are limited to a flat monolayer, the tissue models developed in this thesis can display the characteristic 3D villi and crypt structure of human small intestine. First, experimental conditions were established for infection of a previously developed, statically cultivated intestinal tissue model with C. jejuni. This included successful isolation of bacterial colony forming units (CFUs), measurement of epithelial barrier function, as well as immunohistochemical and histological staining techniques. In this way, it became possible to follow the number of viable bacteria during the infection process as well as their translocation over the polarized epithelium of the tissue model. Upon infection with C. jejuni, disruption of tight and adherens junctions could be observed via confocal microscopy and permeability measurements of the epithelial barrier. Moreover, C. jejuni wildtype-specific colonization and barrier disruption became apparent in addition to niche-dependent bacterial localization within the 3D microarchitecture of the tissue model. Pathogenesis-related phenotypes of C. jejuni mutant strains in the 3D host environment deviated from those obtained with conventional in-vitro 2D monolayers but mimicked observations made in vivo. Furthermore, a genome-wide screen of a C. jejuni mutant library revealed significant differences for bacterial factors required or dispensable for interactions with unpolarized host cells or the highly prismatic epithelium provided by the intestinal tissue model. Elucidating the role of several previously uncharacterized factors specifically important for efficient colonization of a 3D human environment, promises to be an intriguing task for future research. At the frontline of the defense against invading pathogens is the protective, viscoelastic mucus layer overlying mucosal surfaces along the human gastrointestinal tract (GIT). The development of a mucus-producing 3D tissue model in this thesis was a vital step towards gaining a deeper understanding of the interdependency between bacterial pathogens and host-site specific mucins. The presence of a mucus layer conferred C. jejuni wildtype-specific protection against epithelial barrier disruption by the pathogen and prevented a high bacterial burden during the course of infection. Moreover, results obtained in this thesis provide evidence in vitro that the characteristic corkscrew morphology of C. jejuni indeed grants a distinct advantage in colonizing mucous surfaces. Overall, the results obtained within this thesis highlight the strength of the tissue models to combine crucial features of native human intestine into accessible in-vitro infection models. Translation of these systems into infection research demonstrated their ability to expose in-vivo like infection outcomes. While displaying complex organotypic architecture and highly prismatic cellular morphology, these tissue models still represent an imperfect reflection of human tissue. Future advancements towards inclusion of human primary and immune cells will strive for even more comprehensive model systems exhibiting intricate multicellular networks of in-vivo tissue. Nevertheless, the work presented in this thesis emphasizes the necessity to investigate host-pathogen interactions in infection models authentically mimicking the natural host environment, as they remain among the most vital parts in understanding and counteracting infectious diseases. / In der heutigen Zeit tragen insbesondere durch pathogene Mikroorganismen ausgelöste Infektionskrankheiten zur sozioökonomischen Belastung bei. Obwohl bereits jahrzehntelang an der Entstehung von Infektionskrankheiten geforscht wird, bleiben in zahlreichen Fällen die genauen Mechanismen, welche an den vielfältigen Interaktionen zwischen Pathogen und Wirt beteiligt sind, unbeschrieben. Gerade deshalb bleibt es für Wissenschaftler weltweit eine Herausforderung, neue Strategien zur Untersuchung des molekularen Kontexts von Infektionskrankheiten zu entwickeln, um präventive oder zumindest anti-infektive Maßnahmen ergreifen zu können. In den meisten Fällen ist jedoch das Fehlen geeigneter Infektionsmodelle, mit denen der Krankheitsverlauf im Menschen authentisch nachgestellt werden kann, eines der größten Hindernisse um detailliertes Wissen darüber gewinnen zu können wie bakterielle Pathogene die Krankheit auslösen. Zahlreiche Studien sind dabei auf Tiermodelle angewiesen, um die komplexen zeitlichen Abläufe zwischen Wirt und Pathogen im menschlichen Körper nachzuahmen. Während diese Modelle in hohem Maß dazu beigetragen haben, Aufschluss über diese Abläufe zu geben, sind sie doch sehr kostenintensiv, mit ethischen Bedenken behaftet und können nicht immer die Folgen einer Infektion im menschlichen Patienten vorhersagen. Seit Jahrzehnten werden daher alternativ in-vitro 2D Zellkultursysteme eingesetzt, um den Verlauf von Infektionskrankheiten zu erforschen, welche die Bedingungen im menschlichen Wirt wiederspiegeln sollen. Diese auf Zelllinien basierenden Modelle sind essentiell in der Entdeckung von Virulenzfaktoren diverser Pathogene, aber auch in der Aufklärung von wirtsspezifischen Abwehrmechanismen. Dennoch fehlt ihnen die morphologische und zelluläre Komplexität von intaktem menschlichen Gewebe. Dadurch sind die Erkenntnisse, die mit diesen Systemen über Infektionsverläufe gewonnen werden können, limitiert. Die vorgelegte Arbeit konzentriert sich auf die Etablierung und Weiterentwicklung intestinaler, humaner Zellkulturmodelle, um dreidimensionales Gewebe in vitro zu rekonstruieren mit dem Ziel, Pathogenese-beeinflussende Prozesse des zoonotischen Bakteriums C. jejuni nachzustellen. Das Fachgebiet der Gewebezüchtung wird üblicherweise für rekonstruktive Medizin eingesetzt und bietet exzellente Mittel zur in-vitro Herstellung organspezifischer Zellkulturmodelle, welche die unverkennbare Mikroarchitektur humanen Gewebes realistisch nachempfinden können. Die in dieser Arbeit verwendeten Modelle basieren auf einem extrazellulären Matrixgerüst, das aus der Dezellularisierung von Schweinedarm gewonnen wurde. Durch die Wiederbesiedelung mit human Kolonzellen und der Kultivierung unter dynamischen Bedingungen entwickelte sich ein hochpolarisiertes mucosales Epithel, das durch funktionale Zell-Zell-Kontakte (tight und adherens junctions) aufrechterhalten wird. Während andere in-vitro Infektionssysteme meist durch die Präsenz einer flachen Zellschicht limitiert werden, entwickelt das in dieser Arbeit eingeführte Gewebemodell die für den menschlichen Dünndarm charakteristische Architektur aus Villi und Krypten. Zunächst wurden experimentelle Bedingungen für die Infektion eines zuvor entwickelten, statisch kultivierten Dünndarmmodells mit C. jejuni etabliert. Dies beinhaltete die erfolgreiche Isolierung koloniebildender Einheiten, die Messung der epithelialen Barrierefunktion, sowie immunhistochemische und histologische Färbetechniken. Dadurch konnte die Anzahl der Bakterien sowie deren Translokalisierung über das polarisierte Epithel während des Infektionsprozesses nachvollzogen werden. Außerdem konnte die Beeinträchtigung von Zell-Zell-Kontakten durch konfokale Mikroskopie und Permeabilitätsmessungen der epithelialen Barriere beobachtet werden. Neben der Bestimmung der Kolonisierungsrate von C. jejuni Isolaten und der dadurch hervorgerufenen spezifischen Zerstörung der epithelialen Barriere konnten die Bakterien auch innerhalb der 3D Mikroarchitektur des Gewebemodells lokalisiert werden. Außerdem konnte im Rahmen der 3D Gewebeumgebung beobachtet werden, dass Pathogenese-relevante Phänotypen von C. jejuni Mutantenstämmen im Vergleich zu konventionellen in-vitro 2D Zellschichten abwichen, diese aber dafür mit den in-vivo gemachten Beobachtungen übereinstimmten. Darüber hinaus wies die genomweite Suche einer C. jejuni Mutantenbibliothek signifikante Unterschiede zwischen bakteriellen Faktoren, die für die Interaktion mit nicht polarisierten Wirtszellen oder dem hochprismatischen Epithel des Gewebemodells bedeutsam oder entbehrlich waren, auf. Die Aufklärung der Funktion einiger bisher nicht charakterisierter Faktoren, die zu einer effizienten Kolonisierung menschlichen Gewebes beitragen, verspricht eine faszinierende Aufgabe für die zukünftige Forschung zu werden. Die vorderste Verteidigungslinie gegen eindringende Pathogene bildet die schützende, viskoelastische Mukusschicht, die mukosale Oberflächen entlang des menschlichen Gastrointestinaltrakts überzieht. Mit der Entwicklung eines mukusproduzierenden Gewebemodells in der hier vorgelegten Arbeit gelang ein entscheidender Schritt zur Erforschung der Wechselbeziehungen zwischen bakteriellen Pathogenen und wirtsspezifischen Muzinen. Während des Infektionsverlaufs wurde das unterliegende Epithel durch die Anwesenheit der Mukusschicht vor der Zerstörung durch die Mikroben geschützt und eine erhöhte bakterielle Belastung verhindert. Darüber hinaus liefern die Resultate dieser Arbeit einen in-vitro Nachweis für den bakteriellen Vorteil einer spiralförmigen Morphologie, um muköse Oberflächen zu besiedeln. Zusammenfassend unterstreicht diese Arbeit das Potential der hier entwickelten Gewebemodelle, entscheidende Eigenschaften des menschlichen Darms in einem leicht zugänglichen in-vitro Infektionsmodell zu vereinigen. Der Einsatz dieser Modelle im Rahmen der Infektionsforschung bewies deren Fähigkeit in-vivo beobachtete Infektionsverläufe widerzuspiegeln. Während diese Infektionsmodelle bereits organotypische Architektur und hochprismatische Zellmorphologie aufweisen, ist ihre Darstellung von menschlichem Gewebe noch nicht perfekt. Durch den Einsatz von humanen Primär- und Immunzellen wird es in Zukunft möglich sein, noch umfassendere Modellsysteme zu entwickeln, die komplexe multizelluläre Netzwerke von in-vivo Geweben aufweisen. Nichtsdestotrotz verdeutlicht die hier vorgelegte Arbeit wie wichtig es ist, die Interaktionen zwischen Wirt und Pathogen innerhalb von Infektionsmodellen zu erforschen, welche die natürliche Wirtsumgebung wiedergeben. Dies spielt eine entscheidende Rolle, um die Entstehung von Infektionskrankheiten nachvollziehen und ihnen entgegenwirken zu können.

The antimicrobial efficacy of carvacrol, trans-cinnamaldehyde, eugenol and thyme oil (0.5, 1, 2 and 5% v/v) was evaluated against Salmonella on turkey breast cutlets as 2 min dip treatments at 4°C. Carvacrol revealed maximum efficacy against Salmonella on turkey cutlets and was selected for 30s and 60s dip treatments at 4°C. Only 5% carvacrol was effective and exhibited 1.0 and 1.8 log CFU/g reduction of Salmonella with 30s and 60s dip treatments, respectively. However, carvacrol showed synergistic activity with carbon dioxide in modified atmosphere packaging (95% CO2/5% O2) against Salmonella and Campylobacter and caused 1.0-2.0 log CFU/g reductions with lower concentrations (0.25, 0.5 and 1.0%). This combination reduced the growth of lactic acid bacteria. In the third experiment, efficacy of lauric arginate (200 and 400ppm) against C. jejuni was tested on chicken breast fillets. Both these concentrations significantly reduced C. jejuni on chicken fillets (1.0-1.5 log CFU/g) at 4°C.

Conversion of <i>Campylobacter upsaliensis</i> to the nonculturable but viable coccoid form was characterized. Chloramphenicol did not prevent the conversion. Severe decreases in isocitrate dehydrogenase activity and oxygen uptake and extensive degradation of ribosomal RNA suggest that the coccoid form is a degenerative form rather than part of a life cycle. The autolysins of spiral and coccoid forms of <i>C. upsaliensis</i> were also studied. Autolytic activity in the soluble and sediment fractions of sonicates of the spiral and the coccoid form of <i>C. upsaliensis</i> could not be demonstrated by native (nondenaturing) PAGE. Autolysins were detected, however, by using denaturing SDS-PAGE gels containing either purified <i>E. coli </i> peptidoglycan or whole cells of <i>Micrococcus luteus</i> as the turbid substrate, with subsequent renaturation by treatment with Triton X-100 buffer. In renaturing gels that contained <i>E. coli</i> peptidoglycan, 14 autolytic bands were detected ranging from 200 kDa to 12 kDa. In similar gels containing whole cells of <i>M. luteus</i> , only a single band appeared having a molecular weight of 34 kDa. This band corresponded to one of the bands present in the gels containing <i>E. coli </i> peptidoglycan. This common autolysin was isolated by adsorbing it from <i>C. upsaliensis</i> lysates onto <i>M. luteus</i> cells and then subjecting these cells to renaturing SDS-PAGE in gels containing <i>E. coli</i> peptidoglycan. The 34 kDa autolysin differed from a single 51 kDa autolysin unique to the <i>M. luteus cells</i>. The 34 kDa autolysin was isolated from an SDS-PAGE gel and was pure when tested by isoelectric focusing. The N-terminal amino acid sequence analysis showed the first 15 amino acids of the 34 kDa autolysin to have 67% identity with a part of antigenic protein PEB4 of <i>Campylobacter jejuni</i>. The purified autolysin was used to immunize rabbits and the antibodies produced precipitated autolytic activity from cell lysates. The specificity of the antibodies was shown by Western blotting: only a single specific band occurred, with a molecular weight of 34 kDa, and thus it seems unlikely that the 34 kDa autolysin was derived from any of the other autolysins that were detected. / Ph. D.

A comparative study of the microaerophilic <u>Campylobacter jejuni</u> strain H840 with an aerotolerant mutant, MC711-01, revealed that catalase and, to a lesser extent, SOD activity is correlated with the aerotolerance and enhanced resistance of MC7ll-0l to H₂O₂. When cells were cultured under 6% oxygen, the specific activity of catalase was significantly higher in crude extracts of MC711-01 than of H840. The catalase activity of MC711-01 more than doubled when cells were cultured under 21% oxygen, and this activity was 2.6 times greater than that of H84O; no corresponding increase was observed in strain H840. There was no significant difference in the mean SOD activity of the two strains when cultured under 6% oxygen or in H840 cells cultured under either 6% or 21% oxygen; however, the SOD activity of MC711-01 increased 1.5 times when cells were cultured under 21% oxygen. Survival studies revealed that MC711-01 was significantly more E resistant to H₂O₂ when cultured under either 6% or 21% oxygen. However, both MC711-01 and H84O were more susceptible to H₂O₂ when grown under 21% oxygen, indicating that both strains might be more highly stressed when the cells are grown at this oxygen tension. The present study suggests that in <u>C. jejuni</u>, the level of catalase activity may influence the degree of susceptibility to H₂O₂ and consequently, the degree of aerotolerance. / Master of Science

Pyruvate:ferredoxin oxidoreductase activity was demonstrated in microaerophilic members of Campylobacter, Wolinella, and Helicobacter. Arcobacter cryaerophila (sic) and Arcobacter nitrofigilis, two aerobic species, lacked any detectable activity. Under anaerobic conditions crude extracts of Campylobacter, Helicobacter and Wolinella were capable of reducing the electron carriers benzyl viologen and metronidazole in the presence of pyruvate. Addition of Clostridium pasteurianum ferredoxin to the metronidazole-linked reaction enhanced metronidazole-reducing activity, suggesting that electron transport to artificial electron carriers is facilitated by ferredoxin. All species exhibited varying degrees of sensitivity to metronidazole (MIC = <0.8 to 25 µg/ml) except Arcobacter cryaerophila, which was resistant to > 100 µg/ml. This further supports the theory that these organisms possess ferredoxin-linked reactions. The presence of the oxygen-labile enzyme pyruvate:ferredoxin oxidoreductase may be related to the inability of these microaerophilic bacteria to grow in normal atmospheric levels of oxygen. Under aerobic conditions crude extracts of the organisms were also capable of reducing NAD in the presence of pyruvate. This might be accounted for by an NAD-linked pyruvate dehydrogenase; alternatively, it might be due to an enzymatic reduction of NAD by electrons from the reduced ferredoxin generated during the ferredoxin-linked pyruvate oxidoreductase reaction. / Master of Science

Aerotolerance of Campylobacter jejuni ATCC 29428 and one of its aerotolerant mutants (strain MC711-01) was measured at 37°C and 42°C. The aerotolerance of C. jejuni was higher at 42°C than at 37°C. Three different lots of Gibco dehydrated Brucella broth were used to prepare Brucella agar. The agar media were then tested to see if they differed in their ability to support growth of C. jejuni. However, only slight differences in viable counts of C. jejuni were obtained between lots. Ageing of dehydrated Brucella medium for 2½ months and hydrated Brucella medium for 1½ months greatly affected the growth of C. jejuni and decreased its aerotolerance. This is probably due to the deterioration of the sodium bisulfite in Brucella medium during storage, because addition of 0.01% sodium bisulfite (the same amount as contained in the Brucella medium) to the aged medium (dehydrated or hydrated form) restored the ability of the medium to support growth of C. jejuni under various O₂ levels equivalent to or even better than that obtained with fresh Brucella medium. Moreover, when Brucella agar was prepared from the individual chemical and peptone components, only the medium containing the 0.01% bisulfite yielded colony counts of C. jejuni similar to that obtained on fresh commercial Brucella medium. When sodium bisulfite was omitted, viable counts and aerotolerance were decreased. / Master of Science

Campylobacter is a foodborne pathogen commonly found in live poultry and raw poultry products. Identifying areas of contamination or modes of transmission during commercial processing can lead to strategies to reduce the level of Campylobacter on finished products. Monitoring levels of airborne Campylobacter may be useful for identifying the presence or relative concentration of the pathogen in a processing plant environment. In this study, air sampling was used to detect and quantify Campylobacter in a commercial chicken processing plant by location within the plant and collection time during the day. Air was sampled from evisceration and post-chill areas in a poultry processing plant on four days and at 4 hour intervals onto Campy-Cefex agar plates or gelatin filters that were subsequently transferred to Campy-Cefex agar plates. Additionally, pre-evisceration and post-chill carcass rinses were analyzed quantitatively for Campylobacter. The mean level of airborne Campylobacter was 5 CFU/1000L of air sampled (10% samples positive) in comparison with 413 CFU/mL from carcass rinses (70% samples positive). Higher concentrations were found in carcass rinse samples from pre-evisceration. Airborne Campylobacter was detected from the evisceration area more frequently than from the post-chill carcass area of the plant (P < 0.05). This study shows that airborne Campylobacter can be quantified with a selective agar and with gelatin filter collection. Further research is needed to prove the utility of airborne detection of Campylobacter for estimating the relative contamination level of live poultry flocks and the processing plant environment and the potential for cross-contamination. / Master of Science in Life Sciences

Campylobacter and Salmonella are foodborne pathogens commonly associated with raw poultry. Although there has been much research done on isolating these pathogens from poultry production environments using cloacal swabs, fecal samples, intestinal tract contents and dissection, research involving environmental sampling has been limited. New and/or improved environmental sampling methods may provide an easy, convenient, and less time-consuming way to collect samples. Coupling these sampling methods with PCR may provide a relatively simple, rapid, and robust means of testing for foodborne pathogens in a chicken house or flock prior to slaughter. Air, boot and sponge samples were collected from three commercial chicken grow-out houses located in southwestern Virginia when flocks were three, four, and five weeks old. Air samples were collected onto gelatin filters. Fecal/litter samples were collected from disposable booties worn over investigator's protective shoe coverings. Pre-moistened sponges were used to sample house feed pans and water dispensers on drink lines. A PCR method was used to qualitatively detect Campylobacter jejuni and Salmonella spp. Campylobacter jejuni was detected at each farm (house), across all three ages (3, 4, and 5 weeks), and from each sample type. Salmonella was not detected in any of the environmental samples. For all 270 samples, 41% (110/270) were positive for Campylobacter. Collectively, 28% (25/90) of air, 44% (40/90) of sponge, and 50% (45/90) of bootie samples were positive for Campylobacter. The methods used in this study are non-invasive to live animals, relatively rapid and specific, and could enable poultry processing facilities to coordinate scheduled processing of flocks with lower pathogen incidence, as a way to reduce post-slaughter pathogen transmission. / Master of Science