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The Enterobacteria constitute a group of bacteria that are small rods and can be mobile or immobile. This group is characterized by the ability to conduct facultative anaerobiosis and include Escherichia coli, Klebsiella, Salmonella, Shigella, Pasteurella, Yersinia, Enterobacter, Serratia, Proteus, Vibrio, Aeromonas, and Photobacterium. Many bacterial genera from this group colonize the intestines of animals, and also include the plant pathogen Erwinia. Escherichia coli and Klebsiella are the major pathogens associated with human urinary tract infection (1, 2, 3).
Extended Spectrum Beta-Lactamases (ESBLs) are found in both gram-positive and gram-negative bacteria and are even present in some species of algae. The gene encoding the antibiotic resistance is found on plasmids, transposons, and inserted in genes in bacteria all over the world and in multiple species. Although much research has been conducted to characterize the microbial enzyme biochemically, PCR analysis remains the "gold standard (4, 5, 6)."
E. coli isolates from the environment are generally succeptible to beta-lactam antibiotics because of the absence of a strong promoter sequence for the expression of the gene for Beta-Lactamase. However, extensive use of antibiotics and the spread of genetic elements among the bacteria have resulted in development of Beta-lactam resistant strains. Consequently, Beta-lactam resistant strains of E. coli have been isolated from hospitals and clinics around the world (7, 8, 9).
Klebsiella is a member of the Enterobacteria that is a small, non-motile rod, and the bacterium is often associated with urinary tract infections. Klebsiella is capable of nonaerobic metabolism and is found in water and soil, in addition to the mammalian intestinal and urinary tracts. Klebsiella possesses the enzyme nitrogenase, and is capable of nitrogen fixation under nonaerobic conditions, but the bacterium loses this capability under the aerobic environment in mammalian intestinal and urinary tracts (1, 10, 11, 12).
Aims and Objectives
The binding of E.coli to urothelial cells is mediated by pili via the FIMH protein. The receptor for the binding has been elucidated to be uroplakin 1a in mouse cells (13). Mutation of key residues in FIMH attenuates binging of E.coli to the FIMH urinary receptor and consequently reduces colonization of the bacteria in the bladder. The binding is mediated by mannose residues and relies on a key pocket in the FIMH protein (14, 15).
Isolates of E. coli from a septic lamb were utilized to characterize the cytotoxic necrotizing factor type 2, and it was discovered that this protein enters effected epithelial cells and targets Rho protein, which subsequently reorganizes actin filaments into stress fibers in the effected host cells (16, 17, 18).
Photodynamic therapy (PDT) has been utilized to treat many epidermal conditions in humans. These include skin cancer, acne, skin rejuvination, hidradenitis suppurativa, psoriasis, cutaneous T-cell lymphoma, disseminated actinic porokeratosis, localized scleroderma, and vulval lichen sclerosis. Other applications include anal and vulvar carcinoma, palliation of metastatic breast cancer to skin, Barrett's esophagus, and retinal macular degeneration (19, 20, 21).
PDT is characterized by the application of moderate light (50mW/s) for a moderate amount of time (15 minutes) to the infected area which creates a photosensitization period followed by a destruction period. PDT application results in a 5 to 6 log decrease in infectious, antiobiotic resistant bacteria by destruction of amino acids and polylysine tracts present in bacterial proteins, as well as the creation of destructive reactive oxygen species (22, 23, 24). It is proposed in this work that PDT be utilized to irradiate E. coli and Klebsiella beta-lactamase positive bacteria from cultures of human urothelial cells.
Materials and Methods
Human urothelial cells will be cultured according to standard techniques. Required solutions include Trypsin Versene/Trypsin-EDTA, 0.1% EDTA in PBS, Trypsin inhibitor, and freezing medium (growth medium with 10% DMSO). Passage of cells into T25 flasks will be as follows: aspirate off medium, add 5 ml 0.1% EDTA and incubate 3-5 min at 37 C, aspirate off EDTA and add 0.5 ml Trypsin Versene and rock 3-5 min at 37 C, dilute 5 uL Trypsin inhibitor (TI) into 5 ml medium, tap flask to separate cells and add diluted TI, tap flask and pipette up and down to ensure single suspension and count cells, transfer cells to 15 ml centrifuge tube, centrifuge cells at 350 g (1000 rpm) for 4 min in a Labfuge, aspirate supernatant and resuspend cells in warm growth medium, transfer cells to flasks at one to three or one to six. Cells usually take 1 week to reach confluence and medium should be changed 3 times per week. One T-25 flask yields approximately 1x106 cells and one T-75 flask yields approximately 3x106 cells. Do not allow cells to become over confluent and do not use cells after passage 10 as cells change genetically. Seeding density of cells is 1x105 in 6 cm dishes and 1x104 in 6 wells dishes. Three cryovials of cells can be frozen down from one T-75 flask and stored in liquid nitrogen.
ESBL positive strains of E. coli and Klebsiellia will be added to T-25 flasks of urothelial cells and incubated at 37 C for 24 hours to facilitate binding of the bacteria to the human cells. T-25 flasks of urothelial cells and bacteria will be subjected to photodynamic light to irradicate bacteria. Irradiated and non-irradated flasks will be processed for cell lysate and proteins will be subjected to western blots and probed with anti-FIMH monoclonal antibody (25) and visualized with chemiluminescence (26). Blots will be exposed to photographic film and signal will be quantified with scanning and quantifying software (27). Signal will be compared to that obtained from non-irradiated lysates to determine effectiveness of irradication by photodynamic light of ESBL-positive E coli and Klebsiellia.
Western blot studies of FIMH should be complimented with fluorescence microscopy studies of bacterial attachment to urothelial cells by treating cells and bacteria grown on glass slides with fluorescently-labeled ESBL positive E. coli and Klebsiella. Bacteria can be labeled with a DNA oligonucleotide probe specific for the FIMH gene linked to the fluorophore Texas Red (28). Human urothelial cells can be labeled with Hoechst blue fluorphore (29). Photodynamic irradiated samples can be compared to non-irradiated controls to determine the decrease in bacterial binding. Fluorescence signals can be quantitated with computer software to obtain quantitative data (30).