Serratia marcescens is an opportunistic pathogen involved in predominantly nosocomial infections, however, it is also implicated as a common cause of microbial keratitis. Since many S. marcecens strains are also resistant to multiple antibiotics, this organism represents a growing public health problem. S. marcescens MG1 utilises a regulatory system for regulation of swarming motility and exo-enzyme secretion that relies on the production of a diffusible signal identified as N-butanoyl-L-homoserine lactone (C4-HSL). The aim of this study was to determine the role of C4-HSL in surface colonisation (adhesion and biofilm formation). In this thesis, the development of a novel biofilm in S. marcescens MG1 is described. The biofilm comprises of an intricate and complex structure consisting of long filamentous cells, cell aggregates and cell chains. Two C4-HSL controlled genes (bsmA and bsmB) are shown to be crucial for biofilm formation. It is proposed that C4-HSL regulated bsmA and bsmB gene products are engaged in fine tuning aggregation at a specific time point in late biofilm development. Since adhesion is the first stage of colonisation, the role of C4-HSL in adhesion to a hydrophilic abiotic surface (HAS) and a human corneal epithelial (HCE) cell line was assessed. While adhesion to the HAS was found to be C4-HSL controlled, this was not the case for adhesion to the HCE cells. In adhesion to the HAS, mutations in the following C4-HSL regulated genes resulted in reduced adhesion; a sensor kinase gene (rssA), a type I transporter gene (lipB), bsmA and bsmB. These four genes were found to effect the expression of type I fimbriae which is proposed to be the adhesin affecting C4-HSL regulated adhesion. While C4-HSL is not involved in adhesion to the HCE cell line, the genes bsmA and bsmB are important. It is proposed that bsmA and bsmB dependent HCE adhesion is due to the requirement of these genes for type I fimbriae production. Furthermore, C4-HSL was found to regulate capsule polysaccharide and OmpX production and repress cytotoxic activity against HCE cells and erythrocytes. It is proposed that cytotoxicity is mediated by ShlA haemolysin.

Bacterial contamination of platelet concentrates (PCs) poses the major transfusion-associated infectious risk. Coagulase negative staphylococci (CoNS), the predominant platelet contaminants, are recognized as one of the leading causes of hospital-acquired infections due to their ability to form biofilms (surface-attached aggregates). In this study, 29 CoNS strains were characterized for their growth and biofilm formation abilities in media and PCs. Twenty-five strains were aerobic including Staphylococcus epidermidis, S. capitis, and S. chromogenes, while four were identified as the anaerobe S. saccharolyticus. Biofilm-associated icaA and icaD genes were amplified from eight strains. Interestingly, only six of those strains were biofilm-positive. Sequencing of S. capitis icaD revealed no mutations that could explain differences in biofilm phenotypes. Growth of CoNS in PCs varied significantly between strains. This study provides preliminary evidence that slow-growing biofilm-positive S. epidermidis are more likely to be missed during platelet culture, highlighting the need for improved screening methods.

Most Salmonella enterica serovars are believed to have a cyclical lifestyle involving both host-associated and environment-associated, persistent phases. Their ability to persist in the environment increases the probability that they will be transmitted. Our hypothesis is that the genetic factors required for cellular aggregation and biofilm formation are important for host-to-host transmission. A link between biofilm formation, environmental persistence and transmissibility has not been observed, due to the lack of an appropriate model. We developed a murine model of Salmonella transmission allowing us to study the genetic factors involved in the transmission process. To test the role of aggregation and biofilm formation we used the ∆csgD mutant, which is deficient in both processes. We also engineered luciferase reporter strains of Salmonella enterica serovar Typhimurium (Salmonella Typhimurium) to track infection within a mouse population before the onset of clinical signs using bioluminescent imaging. We determined that mice shed high levels of Salmonella Typhimurium in their feces when pre-treated with streptomycin. To observe the transmission efficiency of Salmonella, we tracked their spread from infected mice to naive mice, and determined that Salmonella could be transmitted only after pre-treatment with streptomycin. We compared the shedding potential and colonization levels of mice challenged with either wild-type Salmonella Typhimurium or the ∆csgD mutant and determined them to be statistically similar when challenged separately. We found that wild-type Salmonella Typhimurium persisted in fecal pellets at higher levels than the ∆csgD mutant. We compared both the short- and long- transmission potential of the ∆csgD mutant to wild type Salmonella Typhimurium, and found that the mutant did not have a defect in either process. Though not observed in our model, we believe that environmental persistence and biofilm formation are important for the transmission of Salmonella due to its cyclical lifestyle. The model we generated remains useful to test the role of other genes in transmission. It can be further refined to more accurately mimic environmental transmission of Salmonella. Further understanding of the transition of Salmonella from infected hosts to the environment and back into new hosts will aid in reducing its environmental persistence and transmission.

No description available.

Bacterial biofilms are microbial communities that adhere to abiotic or biotic surfaces. Biofilm formation (BF) studies in E. coli have primarily concentrated on uropathogenic E. coli, commensal K-12 and enterohemorrhagic E. coli O157:H7. This does not include the vast diversity of environmental strains. Quorum sensing (QS) is a means by which bacteria can communication with one another through the production of signalling molecules. The autoinducer 2 (AI-2) QS system is utilized by E. coli and several other bacterial species for controlling gene expression. The role of AI-2 in E. coli BF varies among different strains. For example in the K-12 strain, AI-2 regulates motility, and thus can affect BF; whereas in O157:H7, AI-2 has a more metabolic role. Interestingly, in strain O157:H7, motility is controlled by a newly discovered QS system regulated by the autoinducer 3 (AI-3) molecule plus the mammalian hormones epinephrine (Epi) and norepinephrine (Ne). The purpose of this study was to investigate the ability of a panel of environmental E. coli strains to form biofilms and to determine whether QS is involved in the process. A new pathotype of E. coli, adherent invasive E. coli (AIEC) which is associated with Crohn’s disease was included in the investigation. Study 1 sought to determine whether BF under different media conditions correlated with the presence of genes involved in the AI-2 QS system or adhesin factors. Media conditions were the principal variable affecting the BF. Study 2 examined the role of the AI-2 and AI-3/Epi/Ne QS systems in motility and BF by the AIEC strain. It was discovered that the AI-3 system is involved in motility; whereas the AI-2 system had no effect on BF or motility. In Study 3, microarray gene expression analysis and invasion assays were performed using qseB or qseC mutants. These genes encode the two-component regulatory system recognizing AI-3 or its cognate, epinephrine. Our findings indicate that alternative pathways likely account for the BF observed for the qseB and qseC mutants. It was concluded that the AI-3/Epi/Ne QS system partially controls AIEC motility and the invasion of epithelial cells.

Bacterial biofilms are microbial communities that adhere to abiotic or biotic surfaces. Biofilm formation (BF) studies in E. coli have primarily concentrated on uropathogenic E. coli, commensal K-12 and enterohemorrhagic E. coli O157:H7. This does not include the vast diversity of environmental strains. Quorum sensing (QS) is a means by which bacteria can communication with one another through the production of signalling molecules. The autoinducer 2 (AI-2) QS system is utilized by E. coli and several other bacterial species for controlling gene expression. The role of AI-2 in E. coli BF varies among different strains. For example in the K-12 strain, AI-2 regulates motility, and thus can affect BF; whereas in O157:H7, AI-2 has a more metabolic role. Interestingly, in strain O157:H7, motility is controlled by a newly discovered QS system regulated by the autoinducer 3 (AI-3) molecule plus the mammalian hormones epinephrine (Epi) and norepinephrine (Ne). The purpose of this study was to investigate the ability of a panel of environmental E. coli strains to form biofilms and to determine whether QS is involved in the process. A new pathotype of E. coli, adherent invasive E. coli (AIEC) which is associated with Crohn’s disease was included in the investigation. Study 1 sought to determine whether BF under different media conditions correlated with the presence of genes involved in the AI-2 QS system or adhesin factors. Media conditions were the principal variable affecting the BF. Study 2 examined the role of the AI-2 and AI-3/Epi/Ne QS systems in motility and BF by the AIEC strain. It was discovered that the AI-3 system is involved in motility; whereas the AI-2 system had no effect on BF or motility. In Study 3, microarray gene expression analysis and invasion assays were performed using qseB or qseC mutants. These genes encode the two-component regulatory system recognizing AI-3 or its cognate, epinephrine. Our findings indicate that alternative pathways likely account for the BF observed for the qseB and qseC mutants. It was concluded that the AI-3/Epi/Ne QS system partially controls AIEC motility and the invasion of epithelial cells.

Bacterial contamination of platelet concentrates (PCs) poses the major transfusion-associated infectious risk. Coagulase negative staphylococci (CoNS), the predominant platelet contaminants, are recognized as one of the leading causes of hospital-acquired infections due to their ability to form biofilms (surface-attached aggregates). In this study, 29 CoNS strains were characterized for their growth and biofilm formation abilities in media and PCs. Twenty-five strains were aerobic including Staphylococcus epidermidis, S. capitis, and S. chromogenes, while four were identified as the anaerobe S. saccharolyticus. Biofilm-associated icaA and icaD genes were amplified from eight strains. Interestingly, only six of those strains were biofilm-positive. Sequencing of S. capitis icaD revealed no mutations that could explain differences in biofilm phenotypes. Growth of CoNS in PCs varied significantly between strains. This study provides preliminary evidence that slow-growing biofilm-positive S. epidermidis are more likely to be missed during platelet culture, highlighting the need for improved screening methods.

Serratia marcescens is an opportunistic pathogen involved in predominantly nosocomial infections, however, it is also implicated as a common cause of microbial keratitis. Since many S. marcecens strains are also resistant to multiple antibiotics, this organism represents a growing public health problem. S. marcescens MG1 utilises a regulatory system for regulation of swarming motility and exo-enzyme secretion that relies on the production of a diffusible signal identified as N-butanoyl-L-homoserine lactone (C4-HSL). The aim of this study was to determine the role of C4-HSL in surface colonisation (adhesion and biofilm formation). In this thesis, the development of a novel biofilm in S. marcescens MG1 is described. The biofilm comprises of an intricate and complex structure consisting of long filamentous cells, cell aggregates and cell chains. Two C4-HSL controlled genes (bsmA and bsmB) are shown to be crucial for biofilm formation. It is proposed that C4-HSL regulated bsmA and bsmB gene products are engaged in fine tuning aggregation at a specific time point in late biofilm development. Since adhesion is the first stage of colonisation, the role of C4-HSL in adhesion to a hydrophilic abiotic surface (HAS) and a human corneal epithelial (HCE) cell line was assessed. While adhesion to the HAS was found to be C4-HSL controlled, this was not the case for adhesion to the HCE cells. In adhesion to the HAS, mutations in the following C4-HSL regulated genes resulted in reduced adhesion; a sensor kinase gene (rssA), a type I transporter gene (lipB), bsmA and bsmB. These four genes were found to effect the expression of type I fimbriae which is proposed to be the adhesin affecting C4-HSL regulated adhesion. While C4-HSL is not involved in adhesion to the HCE cell line, the genes bsmA and bsmB are important. It is proposed that bsmA and bsmB dependent HCE adhesion is due to the requirement of these genes for type I fimbriae production. Furthermore, C4-HSL was found to regulate capsule polysaccharide and OmpX production and repress cytotoxic activity against HCE cells and erythrocytes. It is proposed that cytotoxicity is mediated by ShlA haemolysin.

Serratia marcescens is an opportunistic pathogen involved in predominantly nosocomial infections, however, it is also implicated as a common cause of microbial keratitis. Since many S. marcecens strains are also resistant to multiple antibiotics, this organism represents a growing public health problem. S. marcescens MG1 utilises a regulatory system for regulation of swarming motility and exo-enzyme secretion that relies on the production of a diffusible signal identified as N-butanoyl-L-homoserine lactone (C4-HSL). The aim of this study was to determine the role of C4-HSL in surface colonisation (adhesion and biofilm formation). In this thesis, the development of a novel biofilm in S. marcescens MG1 is described. The biofilm comprises of an intricate and complex structure consisting of long filamentous cells, cell aggregates and cell chains. Two C4-HSL controlled genes (bsmA and bsmB) are shown to be crucial for biofilm formation. It is proposed that C4-HSL regulated bsmA and bsmB gene products are engaged in fine tuning aggregation at a specific time point in late biofilm development. Since adhesion is the first stage of colonisation, the role of C4-HSL in adhesion to a hydrophilic abiotic surface (HAS) and a human corneal epithelial (HCE) cell line was assessed. While adhesion to the HAS was found to be C4-HSL controlled, this was not the case for adhesion to the HCE cells. In adhesion to the HAS, mutations in the following C4-HSL regulated genes resulted in reduced adhesion; a sensor kinase gene (rssA), a type I transporter gene (lipB), bsmA and bsmB. These four genes were found to effect the expression of type I fimbriae which is proposed to be the adhesin affecting C4-HSL regulated adhesion. While C4-HSL is not involved in adhesion to the HCE cell line, the genes bsmA and bsmB are important. It is proposed that bsmA and bsmB dependent HCE adhesion is due to the requirement of these genes for type I fimbriae production. Furthermore, C4-HSL was found to regulate capsule polysaccharide and OmpX production and repress cytotoxic activity against HCE cells and erythrocytes. It is proposed that cytotoxicity is mediated by ShlA haemolysin.

Zugl.: Hamburg-Harburg, Techn. Universiẗat, Diss., 1999. / Zugl.: Hamburg-Harburg, Techn. Univ., Diss., 1999.