Göttingen, Univ., Diss., 2001. / Computerdatei im Fernzugriff.

Göttingen, Univ., Diss., 2001. / Computerdatei im Fernzugriff.

Göttingen, Universiẗat, Diss., 2001.

Les cellules épithéliales jouent un rôle clé dans la mise en place et le maintien de l’homéostasie mais également dans les processus de défense contre les agents pathogènes, notamment via les récepteurs de l’immunité innée, comme le récepteur le Toll-like (TLR) 4. En effet, la stimulation de ce récepteur par son ligand, le lypopolysaccharide (LPS), le constituant majeur de la paroi des bactéries à Gram négatif, induit l’activation cellulaire et la production de médiateurs de l’inflammation, comme les cytokines et chimiokines pro-inflammatoires KC et MIP-2. Dans ce travail, j’ai étudié les mécanismes de régulation de la voie de signalisation du récepteur TLR4 au niveau des cellules épithéliales, notamment via, les microARN, de petits ARNs non codants qui peuvent réguler de manière extrêmement rapide et efficace l’expression de gènes cibles. Dans une première partie, en utilisant, une lignée de cellules du tubule collecteur rénal immortalisées, des cultures primaires de cellules de TC microdisséquées à partir de reins de souris transgéniques, ainsi qu’un modèle murin d’ITU ascendante, nous avons montré que la CsA induit l’expression du micro-ARN Let-7i, qui cible TLR4 et inhibe donc par conséquent l’activation cellulaire induite par le LPS. Dans une seconde partie, en utilisant une lignée de cellules épithéliales intestinales immortalisées ainsi qu’un modèle murin d’infection du tractus digestif par Citrobacter rodentium, nous avons pu montrer que l’IL-22 induit l’expression du miR-763 qui, en potentialisant la dégradation de la kinase IRAK1, une kinase clé de la voie de signalisation de TLR4, induit une inhibition de la voie de signalisation de TLR4 dans les cellules épithéliales intestinales. Mon travail de thèse a permis d’apporter un faisceau d’arguments montrant l’implication de microARN spécifiques dans la régulation de la voie de signalisation de TLR4 au niveau des épithéliums, suggérant que l’utilisation d’anti-miR synthétiques pourrait être efficace pour traiter certaines pathologies inflammatoires ou infectieuses. / Epithelial cells play a key role in the establishment and the maintenance of homeostasis, but also in the defense against pathogens, particulary via the receptors of innate immunity, such as the Toll-like receptor (TLR) 4. Indeed, the stimulation of this receptor by its ligand, the lypopolysaccharide (LPS), the major constituent of the wall of Gram-negative bacteria, induces cellular activation and production of inflammatory mediators, such as cytokines and pro-inflammatory chemokines KC and MIP-2. In this work, I studied the mechanisms of regulation of the signaling pathway of the TLR4 receptor in epithelial cells, specially via microRNAs, small non-coding RNAs which can regulate extremelys rapidly and efficiently the expression of genes. In a first part, using an immortalized renal collector tubule cell line, primary cultures of TC cells microdissected from transgenic mouse kidneys, as well as a mouse model of ascending UTI, CsA induces the expression of the Let-7i microRNA, which targets TLR4 and therefore inhibits the LPS induced cell activation. In a second part, using an immortalized intestinal epithelial cell line as well as a mouse model of Citrobacter rodentium digestive tract infection, we were able to show that IL-22 induces expression of miR-763 which, by potentiating the degradation of the kinase IRAK1, a key kinase of the TLR4 signaling pathway, induces an inhibition of the TLR4 signaling pathway in the intestinal epithelial cells. My thesis work has permitted to provide lines of evidence in the involvement of specific microRNAs in the regulation of the TLR4 signaling pathway in the epithelial cells, suggesting that the use of synthetic anti-miR could be effective to treat certain inflammatory or infectious diseases.

Cellulose, the most abundant biopolymer on earth, is composed of β – 1,4 – linked glucose units, which in turn form a highly ordered crystalline structure that is insoluble and recalcitrant to degradation. It is the world’s most attractive, abundant and renewable energy resource, representing the bioconversion of carbon dioxide into green plants. Cellulosic biomass, such as agricultural and forestry residues, waste paper and industrial waste can therefore be used as an inexpensive and abundantly available source of sugar for fermentation into fuel ethanol. The combustion of biofuels releases carbon dioxide which is thus recycled and hence the use of these fuels in transportation provides an alternative to fossil fuels, solving many environmental problems. The ability to degrade crystalline cellulose seems to be restricted to a specialized group of microorganisms which includes for example Clostridium, Cellulomonas, Cytophaga, Trichoderma etc. Hence the aim of this project was to create BioBricks using different cellulases from cellulose degraders and express them in different expression hosts like Escherichia coli, Bacillus subtilis, Citrobacter freundii etc., using two different promoters, spac and lac. It was observed that the expression of Cytophaga hutchinsonii cellulases (CHU_2103 and CHU_2802) and dehydrogenases (CHU_1944 and CHU_2315) was toxic to the E. coli host for some unknown reason. Therefore it was decided to use cellulases from Cellulomonas fimi, which are well characterized. BioBricks of cellulases (cenA and cex) from C. fimi were introduced into different expression hosts. It was observed that under our experimental conditions Citrobacter freundii SBS197 gave the best results. Both Pspac and Plac were functional in this organism with expression being higher when Pspac was used. When E. coli JM109 was used as an expression host, activity was only detected when the lac promoter was used to control the expression. Although the expression was higher when E. coli JM109 (containing Plac) was used as an expression host, almost all of this activity was residing within the cells, whereas when C. freundii SBS197 was used as an expression host, considerable activity was detected in the surrounding medium, which is essential for cellulose degradation. Growth curve studies were done to see if heterologous cellulases enable the host to use cellulosic substrates as a source of carbon. It was observed that C. freundii SBS197 expressing cenA and cex was able to use filter paper and Avicel as a source of carbon with maximum growth of up to 8.8×108 cfu/ml and 1.2×109 cfu/ml respectively. This was about 2 – 5 fold higher when compared to the control (vector and/or negative) strains. Filter paper completely disappeared within 3 – 4 days when C. freundii SBS197 was used. Slight degradation was observed when E. coli JM109 was used but there was no physical degradation seen when B. subtilis 168 was used as an expression host. Hence it was concluded that heterologous cellulases impart to C. freundii SBS197 with the ability to use cellulosic substrates as a source of carbon. The maximum growth obtained using these cultures is to our knowledge higher than what has been reported so far for recombinant organisms expressing heterologous cellulases using cellulosic substrates as a source of carbon.

Citrobacter freundii sind Gram-negative, bewegliche, stäbchenförmige Bakterien aus der Familie der Enterobacteriaceae. Als opportunistischer Erreger kann C. freundii beispielsweise Harnwegsinfektionen und Neugeborenen-Meningitis verursachen. Die Internalisierung von C. freundii 3009 in das Endosom von Harnblasenepithelzellen (T24) erfolgt in vitro über einen ausschließlich Mikrotubuli-abhängigen Mechanismus. Als genetische Grundlage der Invasionskompetenz von C. freundii 3009 wurde in Vorarbeiten eine im Chromosom lokalisierte Invasionsdeterminante identifiziert, die eine hohe Identität zum Typ 1 Fimbrien Gencluster (fim) von Salmonella enterica serovar Typhimurium aufweist. Diese in Plasmid pTO3 klonierte Invasionsdeterminante vermittelt nicht-invasiven E. coli K12 Stämmen Invasivität. Im Gegensatz dazu sind rekombinante E. coli K12 Klone, die das fim Operon aus S. enterica serovar Typhimurium bzw. E. coli oder andere E. coli Adhäsindeterminanten tragen, nicht invasiv. Im Rahmen dieser Arbeit wurden die für die Invasionsfähigkeit von C. freundii 3009 essentiellen Gene der klonierten Invasionsdeterminante ermittelt. Dies geschah zum einen durch Subklonierung von Teilen des Plasmids pTO3. In anschließenden Invasionsassays wurden die entsprechenden E. coli K12 Klone hinsichtlich ihrer Fähigkeit zur Invasion untersucht. Die Internalisierung des Wildtyps C. freundii 3009 sowie der invasiven rekombinanten E. coli K12 Stämme konnte nicht nur, wie erwartet, mittels Mannose, sondern auch mittels Chitinhydrolysat [(GlcNAc)n] inhibiert werden. Zum anderen wurden C. freundii Wildtypmutanten konstruiert, in denen der zentrale Teil der Invasionsdeterminante deletiert ist. Diese chromosomalen Deletionsmutanten weisen im Vergleich zum Wildtyp 3009 eine deutlich reduzierte Invasionsrate in die humane Harnblasenepithelzellinie T24 auf. Für die Komplementante wurde die Wiederherstellung des invasiven Phänotyps demonstriert. Darüber hinaus ist bekannt, dass C. freundii 3009 in humane mikrovaskuläre Endothelzellen aus dem Gehirn (HBMEC) eindringen und dort replizieren kann. In der vorliegenden Arbeit wurde gezeigt, dass sowohl C. freundii 3009 als auch der, die fimCf Determinante tragende, rekombinante E. coli Stamm HB101pPH1 in der Lage sind, im Tiermodell mit neugeborenen Ratten die Blut-Hirn-Schranke zu überwinden. Neben der Analyse der Funktion der klonierten C. freundii 3009 fim Determinante in Invasionsassays und mittels Mannose-sensitiver Hefeagglutination, wurden die Genprodukte selbst ebenfalls nachgewiesen. Durch radioaktive Markierung der für die Invasivität essentiellen Proteine nach induzierter Transkription in einem T7 Phagenexpressionssystem konnten die Molekulargewichte von FimCfA, FimCfD, FimCfF und FimCfH ermittelt werden. Diese stimmen mit den von der DNA Sequenz abgeleiteten Molekulargewichten gut überein. Sowohl mit C. freundii 3009 als auch mit entsprechenden rekombinanten E. coli K12 Stämmen gelang es in Westernblots, das Adhäsin FimCfH an der Bakterienoberfläche nachzuweisen. Dies konnte auch für FimCfF in denselben rekombinanten E. coli K12 Stämmen gezeigt werden. Allerdings scheinen die FimCf Proteine nicht zu einem Pilus assembliert zu werden, da in elektronenmikroskopischen Untersuchungen von C. freundii 3009 oder die fimCf Determinante tragenden E. coli K12 Stämmen niemals Fimbrien beobachtet werden konnten. Da auch bestimmte Typ 1 Fimbrien Varianten aus E. coli über das Adhäsin FimEcH die Internalisierung der Bakterien in Blasenepithelzellen zu induzieren vermögen, wurden die fimEc Gencluster aus den beiden human pathogenen E. coli Stämmen 536 (uropathogenes Isolat) und IHE3034 (Neugeborenen-Meningitis Isolat) kloniert. Zudem wurde die Invasionsfähigkeit des E. coli K1 Stamms IHE3034 und der fim negativen Insertionsmutante IHE3034-2 charakterisiert. Typ 1 Fimbrien werden als wichtige Virulenzfaktoren von uropathogenen E. coli (UPEC) angesehen. Trotzdem konnte eine Reihe von klinischen UPEC Isolaten identifiziert werden, die diesen Adhäsintyp nicht mehr exprimieren. Genetische Untersuchungen im Rahmen dieser Arbeit ergaben, dass in 11 Isolaten das fimEc Operon vollständig aus dem Chromosom der Bakterien deletiert ist. In den übrigen 6 Isolaten ist noch ein Teil des 3´-Endes des Gens fimECH vorhanden. Außerdem ist in die Deletionsstelle dieser 6 Isolate ein IS1 Element von E. coli inseriert. In den übrigen uropathogenen Isolaten sind auch angrenzende DNA Bereiche von der Deletion betroffen. Abschließend kann festgestellt werden, dass eine fim ähnliche Determinante in C. freundii 3009 als Invasionssystem fungiert. Die Typ 1 Fimbrien der E. coli Stämme 536 und IHE3034 sind zwar notwendig, aber nicht hinreichend für die Invasivität. Obwohl Typ 1 Fimbrien als wichtige Virulenzfaktoren von uropathogenen E. coli gelten, ist dennoch in einer Reihe von klinischen UPEC Isolaten das fimEc Operon deletiert. / Citrobacter freundii, which belong to the family of Enterobacteriaceae, are Gram-negative, motile and rod shaped bacteria. As an opportunistic pathogen they are known to be involved in urinary tract infections as well as in newborn meningitis. In vitro, the internalization of C. freundii 3009 into the endosomes of the urinary bladder epithelial cell line T24 exclusively follows a microtubule dependent mechanism. Previously, a chromosomal invasion determinant of C. freundii 3009 has been identified and cloned in plasmid pTO3. This invasion determinant shows high identity to the type 1 fimbrial gene cluster (fim) of Salmonella enterica serovar Typhimurium. Interestingly, non-invasive E. coli K12 strains become invasive, when they are transformed with plasmid pTO3. In contrast, recombinant E. coli K12 strains harbouring the fim operon of either S. enterica serovar Typhimurium and E. coli, respectively, or other E. coli adhesin determinants were not invasive. In this work the genes of this chromosomal determinant, which are essential for invasiveness, were identified. This was achieved on one hand by subcloning parts of the invasion determinant of plasmid pTO3. Subsequently, the ability of the resulting plasmids to confer invasiveness to E. coli K12 strains was examined by gentamicin protection assays. Interestingly, the internalization of those recombinant E. coli K12 strains is not only inhibited by an analogue of the natural receptor of the adhesin FimCfH, namely mannose, but also by chitin hydrolysate [(GlcNAc)n]. On the other hand, mutants of wild type C. freundii 3009 were constructed by deleting the central part of this particular invasion determinant. These chromosomal deletion mutants showed a considerable lower invasion rate of the human epithelial cell line T24 in comparison to the wild type. Complementation of one of these mutants fully restored the wild type phenotype. Furthermore, it is known that C. freundii 3009 is capable to invade and replicate in human brain microvascular endothelial cells (HBMEC). The results presented here demonstrate that not only C. freundii 3009 is capable to breach the blood-brain-barrier in a newborn rat animal model, but also recombinant E. coli strain HB101pPH1 which carries the chromosomal fimCf determinant. In addition to the functional analysis of the C. freundii 3009 fim determinant by performing gentamicin protection assays and mannose sensitive yeast agglutination, the expression of the corresponding gene products was shown. Proteins essential for invasion, namely FimCfA, FimCfD, FimCfF and FimCfH, were specifically radio labelled using a T7 phage expression system and their molecular weight was determined. The observed molecular weights are in agreement with the calculated ones from deduced amino acid sequences. Furthermore, the presence of the fimbrial adhesin minor subunit FimCfH on the outer surface of C. freundii 3009 as well as appropriate recombinant E. coli K12 strains was shown using Westernblot technique. Expression of the minor subunit FimCfF on the outer surface was also shown for the recombinant E. coli K12 strains using the same technique. However, electron microscopic studies of C. freundii 3009 or E. coli K12 strains harbouring the fimCf determinant indicate that FimCf protein subunits are not assembled in hair like appendages called fimbriae. Since certain type 1 fimbrial variants of E. coli are able to induce bacterial internalization by bladder epithelial cells via the adhesin FimEcH, the fimEc gene cluster from human pathogenic E. coli strains 536 (uropathogenic isolate) and IHE3034 (newborn meningitis isolate) were cloned in order to examine the role of type 1 fimbriae of these strains in invasiveness. In addition, E. coli K1 strain IHE3034 and the isogenic fim negative insertion mutant IHE3034-2 were characterized for their invasion capability. Although type 1 fimbriae are known to be an indispensable virulence factor of uropathogenic E. coli (UPEC), several clinical UPEC strains, which are incapable of expressing this particular adhesin, were isolated and identified. By performing genetic analysis, it could be demonstrated that the fimEc operon is completely deleted from the chromosome in 11 of those isolates. In another 6 isolates the presence of only the 3´-end of the gene fimECH could be found. Furthermore, an IS1 element inserted in the fim deletion was identified in these 6 strains. In the remaining 11 isolates also the DNA region adjacent to the fimEc cluster was affected by this deletion process. In conclusion, it was ascertained that in C. freundii 3009 a fim like determinant functions as an invasion system. Type 1 fimbriae of E. coli strains 536 and IHE3034 are necessary but not sufficient to confer invasiveness. Although type 1 fimbriae of uropathogenic E. coli are known to be an important virulence factor, in several clinical uropathogenic E. coli strains the fimEc operon was deleted.

Faculty of Science School of Molecular and Cell Biology 9904041r lthompson@csir.co.za / The production of H2 was monitored using an automated, semi-continuously fed anaerobic fluidised bed bioreactor containing 2 facultatively anaerobic bacteria, Enterobacter cloacae (E. cloacae Ecl) and Citrobacter freundii (C. freundii Cf1). Shake flask tests using Endo formulation with modified C:N:P ratios, showed that a 334:28:5.6 ratio gave the highest attached counts of E. cloacae Ecl and C. freundii Cf1 in both single and binary species biofilms grown on granular activated carbon. Once the reactor had achieved steady state after 30 days using the modified C:N:P ratio, pH, redox potential, temperature, volatile fatty acids and the H2 production rate were monitored. The H2 production rate of 95 mmol H2 / (l x h) compared favourably with previous studies. Bacterial biofilms counts for both E. cloacae Ecl and C. freundii Cf1 remained high around 9.0 log cfu/g granular activated carbon, although biomass overgrowth could not be controlled in the reactor. The efficiency of converting sucrose into H2 was calculated at 20.5%. Therefore use of this technology to power a 5.0kW proton exchange fuel cell for a single rural household is currently not feasible due to the high organic load required. Pooling of wastewater generation capacity, improvement of bacterial strain selection and feed formulation, pH control, gas removal and purification are factors that need to be considered for future improvement of conversion efficiencies. Use of this technology would be most suited for industrial processes generating large volumes of wastewater high in carbohydrates. Alternatively, municipal wastewater treatment facilities could be converted into electricity generating facilities through the combination of this technology and proton exchange membrane fuel cells.

Microorganisms, particularly bacteria, can associate together to form complex communities called biofilms. These communities are embedded in extracellular polymeric substances and can form on numerous surfaces such as implanted devices (catheters, central lines, joint replacement) in patients. These biofilms cause bloodstream and systemic infections that are difficult to treat and increase the chances of sepsis. Previously, our laboratory has identified a protein secreted by Klebsiella that has inhibitory effects on other members of the Enterobacteriacea bacterial family, namely Citrobacter and Enterobacter. Our current interest lies in the ability of the protein to potentially inhibit this bacterial family from establishing biofilms. In the present study, we wanted to explore: (1) if it is possible to form Citrobacter and Enterobacter biofilms in 6-well plates and on microscope coverslips; (2) if treating these biofilms with the secreted protein shows inhibition similar to previous planktonic cultures; (3) if these biofilms and inhibition could be visualized by a variety of staining techniques. To determine if it is possible to create Citrobacter and Enterobacter biofilms, bacteria were inoculated into 6-well plates, grown under static conditions in a 37°C incubator for 24 hours, and stained with crystal violet. Images showed that robust biofilms grew in the 6-well control plates while wells treated with the Klebsiella protein displayed reduced biofilms. To determine if it was possible to see Citrobacter and Enterobacter biofilms at a microscopic level, microscope slides were placed into 6-well plates, treated as above, and the slides were Gram stained. Images show thick biofilms consisting of Gram negative rods on control slides, while slides treated with the Klebsiella molecule become sparse and poorly grown. To determine if Citrobacter and Enterobacter biofilms could be fluorescently labeled for visualization, the same process was employed as above, but stained with a LIVE/DEAD cell viability kit where live cells fluoresce green and dead cells fluoresce red. Control slides showed bright thick green fluorescing biofilms while slides treated with the Klebsiella molecule had fewer green fluorescing cells and some red cells. From these observations, it is our conclusion that: (1) it is possible to grow Citrobacter and Enterobacter biofilms on both 6-well plates and microscope slides; (2) Citrobacter and Enterobacter biofilms can be visualized both by simple staining and fluorescent staining; (3) Citrobacter and Enterobacter biofilms are inhibited by Klebsiella secreted proteins. Currently, the identity of this protein is unknown. However, it is possible that this unknown protein could be of future use in the treatment of bacterial biofilms one identified.

Made available in DSpace on 2016-03-15T14:15:03Z (GMT). No. of bitstreams: 2 222.pdf: 7280517 bytes, checksum: 5c1c665cf2ba85668951a0c1daaafd4e (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2014 / O Parque Nacional Serra da Capivara (PNSC) é um ambiente propício para o desenvolvimento e conservação de grande número de espécies endêmicas da caatinga, além de ser uma região de importância ímpar, pois é o maior sítio arqueológico do Brasil. A água nesta região é escassa e o contato das pessoas e seus animais domésticos e de criação com os animais silvestres faz com que aumente a possibilidade de circulação de possíveis patógenos, possibilitando a emergência de doenças tanto no homem quanto em animais. Este trabalho teve como objetivo analisar a presença e circulação de bactérias potencialmente enteropatogênicas e zoonóticas na região do Parque Nacional Serra da Capivara, localizado no sudeste do Estado do Piauí Brasil. Coletas de amostras de água, fezes humanas e de animais silvestres e de criação foram realizadas em diferentes regiões no interior e no seu entorno,durante as idas ao campo, computando um montante de 170 amostras coletadas. (...) Não foi possível o isolamento de amostras do gênero Campylobacter, importante enteropatógeno de impacto para a Saúde Pública. O percentual de resistência das amostras a pelo menos uma das drogas testadas foi de 71 por cento sendo mais representativos para os fármacos: penicilina, cefoxitina e tetraciclina. A vigilância dos sorovares de Salmonella em ambientes aquáticos constitui um importante elemento para o monitoramento de infecções humanas e animais. / Neste estudo foram identificados 11sorovares de Salmonella: Worthington, Panama, Brooklyn, Glasgow, Braenderup, Isangi, Grumpensis, Rubislaw, Miami, Cerro, Thompson e duas amostras identificadas como Salmonella enterica subsp. houtenae. Quando analisadas quanto ao seu perfil filogenético, observou-se 14 clones distintos. Isolados com mesma origem clonal circulam em diferentes pontos de coletas no interior e entorno do Parque Nacional Serra da Capivara. Os dados obtidos neste estudo são relevantes para a Saúde Pública, pois contribuem para o monitoramento ambiental a partir do conhecimento da epidemiologia dos enteropatógenos circulantes no ambiente, impactando animais e a população que habita na região do PNSC. / The National Park Serra da Capivara (PNSC) is a proper environment for the developmentand conservation of a great number of species of the caatinga and endemic species, it is also aregion of singular importance for being the largest archaeological site in Brazil. The water inthis region is scant and the contact of people and of their animal whether they are pets or forbreeding, with the wild animals incresases the probability of circulation of pathogens, makingit possible for the appearance of deseases in men and in other animals. This study had theobjective of analisyng the presence and circulation of bacterias potencially enteropathenogenic and zoonotic in the region of the National Park Serra da Capivara ,located in the southeast of the state of Piaui Brazil. Water samples, feces of humans and ofbreeding and wild animals were collected in different regions inside and in the surroundings of the PNSC, during trips to the field mounting up to 170 samples collected. Five Hundredand twenty two isolated were obtained to the following genus/species of the family Enterobacteriaceae: Escherichia coli (32.6 percent); Enterobacter spp. (22.8 percent); Klebsiella spp (11.8 percent); Salmonella spp (10 percent), Citrobacter spp. (9.2 percent) amongst others. The isolation of thesample of the genus Campylobacter, important enteropathogen of public health impact, wasnot possible. The percentage of resistence of the samples to, at least, one of the drugs testedwas from 71 percent and the percentages of resistence were more representative for the drugs: penicilin, cefoxitin and tetracyclin. The surveillance of Salmonella serovars in aquatic environment constitutes an important element for the monitoring of animal and humaninfections. (AU)^ien / In this study 11 different Salmonella serovars were identified: Worthington, Panama, Brooklyn, Glasgow, Braenderup, Isangi, Grumpensis, Rubislaw, Miami, Cerro, Thompson and 2 samples identified as S. enterica subsp. houtenae. When analysed accordingto their filogenetic profile, they produced 14 different clones. Isolate samples with sameclonal origen circulate in distinct points of collect inside and in the surroundings of the PNSC. The data obtained in this study are relevant for public health, because they contribute to the environmental monitoring based on epidemiology knowledge of the enteropathogens circuling in the environment, causing impact on the animals and in the population living in the area of the PNSC. (AU)^ien

Due to the increasing prevalence of multi-drug resistant (MDR) bacteria, it is now important to begin the search for novel means of defending against such resistant infections. Enterobacteriaceae is a clinically relevant family of bacteria that has shown extensive resistance to many antibiotics, especially after biofilm formation. Inhibitory poly-microbial interactions within this family have been observed. It is known that Citrobacter freundii (CF) growth is significantly inhibited by Klebsiella pneumoniae (KP) through a secreted protein. In this study, the potential KP bacteriocin was screened for its inhibitory effects on CF at various phases of biofilm development. The suspected KP bacteriocin was also tested for its ability to decrease the dosage of antibiotics necessary to inhibit CF growth. Using spectrophotometric analysis, it was shown that the combined treatment of streptomycin and the KP protein allowed a decrease in the minimum inhibitory concentration of streptomycin needed from 50 μM to 32 μM. The combined treatment also yielded increased inhibition at the initial attachment phase of CF infection, as well as after biofilm development. The study uses the secreted KP protein to show the use of poly-microbial interactions within clinical applications. Future projects concerning this KP molecule can pursue the use of a C. elegans model to determine its efficacy in vitro.