Quorum sensing (QS) is a process that allows bacteria to sense the population density of cells around them by communicating with each other via autoinducer molecules. This cross-communication is crucial in the regulation of bacterial processes such as bioluminescence, virulence, and biofilm formation. Previous research by Milburn and Makemson on Vibrio harveyi suggested that in addition of the known biosynthesis of three well-characterized autoinducers, dozens of unknown molecules are also produced and released to the environment by V. harveyi. This study was performed using electrospray tandem mass spectrometry with the purpose of detection and characterization of the extracellular molecules produced by V. harveyi, and assessment of their relationship to QS. A total of 11 molecules were characterized, from which three could be related to QS. These findings provide a glimpse of the nature of novel secondary metabolites produced by V. harveyi and provide the groundwork for further research.

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.

Typescript (photocopy). / Digitized by Kansas Correctional Industries

Digitized by Kansas State University Libraries

No description available.

No description available.

Typescript (photocopy). / Digitized by Kansas Correctional Industries

The role of microorganisms in the degradation of xenobiotics in the environment is well established. Bacteria from the genus Pseudomonas are particularly well adapted to the degradation of hydrocarbons, aromatics, and numerous other natural and introduced substrates. We have isolated a strain of Pseudomonas putida, designated PC2P15, that uses toluene, phenol, benzene, and a number of other substrates as its sole sources of carbon and energy.

Interest in the distribution of Clostridium botulinum type E was heightened by the sudden outbreak of human botulism from smoked whitefish chubs and canned tuna fish in 1963. The question arose as to how widely the organism is distributed among fish and shellfish in the Northwest and what potential hazard exists for the consumer of fish products. This sporeforming anaerobic orgnism is heat sensitive and had eluded detection in other surveys where heat shock had been used to eliminate non sporeforming contaminants. More recent study using other techniques than heat to facilitate recovery has shown this organism to be widespread, especially in the marine environment. This study was undertaken to find the incidence and distribution of C. botulinum in the marine organisms and environment of the Pacific Northwest and the food products derived therefrom. All species of fish were examined by incubating the gills and viscera individually in tryptone-peptone-glucose medium anaerobically at 28 C for four days and testing the culture filtrate for mouse toxicity by intraperitoneal injection. Toxic filtrates were typed by retesting them in mice protected by specific botulinal antitoxin of type A, B, E, or F. Among salmonid fish the proportions of specimens of each species yielding toxic filtrates were as follows. Sockeye salmon from the Columbia River, 14 of 59 (23.7%); Chinook salmon from the Columbia River, 19 of 106 (18.0%); Chinook salmon from the Pacific Ocean, 1 of 18 (5,6%); Coho salmon from the Columbia River, 10 of 19 (34.4%); Coho salmon from the Pacific Ocean, 13 of 186 (7.0%); Steelhead trout from the Alsea River, 7 of 37 (19.0%). About one-third of the toxic cultural filtrates were successfully typed and proven to contain botulinal toxin. Most of them proved to be type E toxin but 3 were type A, 3 were type B and one, a comparatively new type, type F, was isolated from a Sockeye salmon in the Columbia River. Pure cultures of Clostridium botulinum type E were isolated from 18 specimens and one specimen yielded a pure culture of type F from a sockeye salmon. This was the second time this type had been isolated. In all of the experimental groups the proportion of fish producing toxigenic cultures was significantly higher in those taken in the two rivers than those of the same species taken from the ocean waters. "Bottom fish" represented by Cod, Sole, Grouper and members of the Sebastodes group were also tested in the manner described above. The number of specimens yielding toxic filtrates were 28 of 157 (17.8%). When grouped according to location at which the fish were caught, those near the mouth of the Columbia River produced a greater percentage of toxic filtrates than did those caught off the open shore line. The results were as follows: Bottom fish from Astoria, 23 of 70 (32.8%), Botton fish from Coos Bay, 5 of 87 (5.6%). Sturgeon specimens produced 3 of 24 (12.5%) toxic filtrates. Most of the species contained type E; however, one type A and one type B were found on typing, with about one-third of the toxic filtrates being successfully typed. Environmental swab samples from the "deep sea" fillet processing plants produced 3 of 39 (7.7%) toxic filtrates. None of the 53 samples taken in the salmon processing plants produced toxic filtrates. Shellfish were collected along the ocean beach and in the estuaries. Three to five shellfish were combined into a single specimen and treated as described. All shellfish obtained from the estuaries demonstrated a higher percentage of toxic filtrates than those obtained from the ocean beach. The results were as follows: Razor clams, 11 of 75 (14.6%), Cockle clams, 12 of 15 (80.0%); Softshell clams, 8 of 12 (66.4%); Littleneck clams, 4 of 11(36.2%); Horseneck clams, 1 of 3 (33.3%); Oysters, 6 of 19 (31.6%); Dungeness crabs, 17 of 24 (71%). Only the razor clams were collected exclusively from the ocean beach. Loss of toxicity on holding mixed cultures at -15 C while awaiting typing was a continual problem. This accounts for only one-third of toxic filtrates being successfully typed. Electrophoretic analysis of the total bacterial proteins was carried out on cell sonicates and cell free culture filtrates by first growing cells for four days at 28 C anaerobically. The cells were separated, washed and disrupted with ultrasonic energy. The cell free culture filtrate was concentrated 10 fold by dialysis against polyethylene glycol 4000. Both the toxigenic organisms and the toxic filtrate demonstrated an extra common protein band in the upper third of the electrophoretic pattern not present in the nontoxic spectra. This band might represent the type E toxin. Differences could also be noted in the number of protein bands in the lower third of the patterns in different nontoxigenic strains and also when the toxigenic and nontoxigenic strains were compared. This could suggest an association with a phage in the toxigenic cultures. / Graduation date: 1970

Aerobic heterotrophic bacteria associated with oyster larvae feed algae Isochrysis galbana, Monochrysis lutheri and Pseudoisochrysis paradoxa were isolated and enumerated. The bacterial numbers ranged from 7.8x10³ to 3.9x10⁶ CFU per ml. The bacteria associated with Pseudoisochrysis sp. were identified and the majority of isolates belonged to genera Leucothrix (51%). Also present were members of Pseudomonas III sp. (19.1%), atypical Moraxella sp. (16.8%), Moraxella sp. (7.2%) and Flavobacterium sp. (5.9%). The growth of bacteria on marine agar was fastidious and took four days to form visible colonies. None of the bacterial isolates grew in buffered salt broth in which the algae had been grown. Marine broth supplemented with 0.1% beef extract best supported the growth of the isolates, while ferric citrate (3x10⁻⁴ M) supported their growth in buffered salt broth. The role of algae as a solid support for bacteria was investigated by studying the attachment of bacteria on glass slides suspended in the growth medium. The percent of Leucothrix sp. attached to the slide was 2.1-3.0%. This was four to six times greater than that of Staphylococcus (0.5%), an organism well known for its commensal growth on skin and mucus membranes of man and animal. Hydrophobic attraction, which is thought to play an important role in the orientation of bacteria to solid surfaces, was studied by measuring the adsorption of bacteria on hydrocarbons introduced to a bacterial suspension. Leucothrix sp. exhibited the strongest affinity, whereas Flavobacterium sp. and Pseudomonas sp. adsorbed the least. An attempt to obtain an axenic culture of algae by antibiotic treatment was unsuccessful due to the detrimental effect of antibiotics to both algae and bacteria. / Graduation date: 1982