Development of novel oral formulations prepared via hot melt extrusion for targeted delivery of photosensitizer to the colon.

Colon-residing bacteria, such as vancomycin-resistant Enterococcus faecalis and Bacteroides fragilis, can cause a range of serious clinical infections. Photodynamic antimicrobial chemotherapy (PACT) may be a novel treatment option for these multidrug resistant organisms. The aim of this study was to formulate a Eudragit®-based drug delivery system, via hot melt extrusion (HME), for targeting colonic release of photosensitizer. The susceptibility of E. faecalis and B. fragilis to PACT mediated by methylene blue (MB), meso-tetra(N-methyl-4-pyridyl)porphine tetra-tosylate (TMP), or 5-aminolevulinic acid hexyl-ester (h-ALA) was determined, with tetrachlorodecaoxide (TCDO), an oxygen-releasing compound, added in some studies. Results show that, for MB, an average of 30% of the total drug load was released over a 6-h period. For TMP and h-ALA, these values were 50% and 16% respectively. No drug was released in the acidic media. Levels of E. faecalis and B. fragilis were reduced by up to 4.67 and 7.73 logs, respectively, on PACT exposure under anaerobic conditions, with increased kill associated with TCDO. With these formulations, photosensitizer release could potentially be targeted to the colon, and colon-residing pathogens killed by PACT. TCDO could be used in vivo to generate oxygen, which could significantly impact on the success of PACT in the clinic.

Induction and repair of DNA strand breaks in Bacteroides fragilis.

Alkaline sucrose gradient sedimentation was used to establish whether strand breakage and repair take place in the DNA of UV-irradiated Bacteroides fragilis during the removal of pyrimidine dimers. A B. fragilis wild-type strain and two of its repair mutants, a mitomycin C sensitive mutant (MTC25) having wild-type levels of UV survival, and a UV-sensitive, mitomycin C sensitive mutant (UVS9), were investigated. Under anaerobic conditions, far-UV irradiation induced metabolically regulated strand breakage and resynthesis in the wild-type strain, but this was markedly reduced in both the MTC25 and UVS9 mutants. Approximately half of the strand breaks generated by the various strains were rejoined during further holding in buffer. Under replicating conditions, complete repair of strand breaks in the wild type was observed. Caffeine treatment under anaerobic conditions caused direct DNA strand breakage in B. fragilis cells but did not inhibit UV-induced breakage or repair.

Evaluation of a fluorescent antibody technique for the rapid enumeration of Bacteroides fragilis group of organisms in water.

The Bacteroides fragilis group has been evaluated as a prospective rapid indicator of faecal contamination of water. Fluorescent antibody (FA) stained B. fragilis group bacteria were enumerated microscopically and compared with faecal coliform or Escherichia coli counts as indicators of faecal contamination. Environmental samples included surface waters (raw drinking water and known contaminated water). Laboratory disinfection experiments with ozone, chlorine and u.v. radiation were also performed. Bacteroides FA counts specifically detected recent human faecal contamination in field samples in 2-3 h. Samples with a high content of particulates or debris limited the sensitivity to about 10 FA counts/ml. Viable counts showed that the sensitivity to all three disinfection agents was essentially the same for Bacteroides and E. coli. Fluorescent antibody counts of Bacteroides, conversely, were not altered by any of the agents. Therefore, the Bacteroides FA method is not recommended for routine monitoring but may be useful for cases where extensive human faecal contamination is suspected (e.g. pipeline rupture or pollution of recreational water) and where rapid remedial action must be taken to protect the public health.

Isolation and physiological characterization of mitomycin C-sensitive/UV-sensitive mutants in Bacteroides fragilis.

Mutants of Bacteroides fragilis sensitive to mitomycin C were isolated after mutagenesis with ethyl methane sulphonate. One mutant (MTC25) was markedly sensitive to mitomycin C but was unaffected as regards UV sensitivity; another mutant (UVS9) was sensitive to UV radiation but was only moderately sensitive to mitomycin C. Caffeine decreased the survival after UV-irradiation of the wild-type, MTC25 and UVS9 strains by the same relative amount. Aerobic liquid holding recovery occurred in each of the three strains. The MTC25 and UVS9 mutants showed reduced host cell phage reactivation. The wild-type, MTC25 and UVS9 strains all showed UV- and H2O2-induced phage reactivation. The physiological characterization of the MTC25 and UVS9 mutants indicates that it is possible to differentiate between mechanisms for the repair of mitomycin C- and UV-induced DNA damage in B. fragilis.

Effect of UV irradiation on macromolecular synthesis and colony formation in Bacteroides fragilis.

Irradiation of Bacteroides fragilis cells with far-UV light resulted in the immediate, rapid and extensive degradation of DNA which continued for 40 to 60 min after irradiation. During the degradation phase, DNA synthesis was decreased but was never totally inhibited. DNA degradation after irradiation was inhibited by chloramphenicol and caffeine. DNA synthesis in irradiated cells was reduced by chloramphenicol but resumed after 100 min at the same exponential rate as in irradiated cells without chloramphenicol. Irradiated cells continued to synthesize DNA for 40 min in the presence of caffeine but after this time DNA synthesis was completely inhibited and never recovered. RNA and protein synthesis were decreased by UV irradiation and the degree of inhibition was proportional to the UV dose. Colony formation was not affected immediately by UV irradiation and continued for a dose-dependent period before inhibition. There was an inverse relationship between UV dose and inhibition of colony formation which occurred sooner in cells irradiated with lower doses of UV light. The characteristics of DNA synthesis in B. fragilis cells after UV irradiation differ from those in wild-type Escherichia coli cells, where DNA synthesis is stopped immediately by UV irradiation, but resemble those in E. coli recA mutant cells where extensive degradation occurs following UV irradiation.

Effect of oxygen and UV irradiation on nucleic acid and protein syntheses in Bacteroides fragilis.

Macromolecular synthesis in Bacteroides fragilis was decreased by oxygen. DNA degradation and synthesis were inhibited by UV irradiation and oxygen, but RNA and protein syntheses were relatively unaffected.

UV light induction of proteins in Bacteroides fragilis under anaerobic conditions.

Far-UV irradiation of Bacteroides fragilis cells under anaerobic conditions resulted in the induction of a new 95,000-molecular-weight protein and the increased synthesis of two proteins with molecular weights of 90,000 and 70,000. The latter two proteins were synthesized in small amounts in unirradiated cells. The induction of a 37,000- to 40,000-molecular-weight protein was not observed in irradiated B. fragilis cells. Caffeine, which affected the survival of irradiated B. fragilis cells and reduced host cell-mediated UV reactivation, specifically inhibited the induction of the 95,000-, 90,000-, and 70,000-molecular-weight proteins. Sodium arsenite did not affect the induction of the three inducible proteins or the survival of irradiated B. fragilis cells.

Effect of oxygen radicals and peroxide on survival after ultraviolet irradiation and liquid holding recovery of Bacteroides fragilis.

The survival of Bacteroides fragilis cells after far-ultraviolet irradiation under anaerobic and aerobic conditions and the liquid holding recovery under aerobic conditions were not affected by peroxide or quenchers of toxic oxygen derivatives.

Effect of oxygen on liquid holding recovery of Bacteroides fragilis.

Liquid holding recovery (LHR) in ultraviolet-irradiated Bacteroides fragilis cells occurred under aerobic conditions but was inhibited by anaerobic conditions. The increase in survival after aerobic LHR resulted in an increase in the shoulder regions of the ultraviolet survival curves. Maximum LHR was obtained after holding the cells for 2 to 3 h. LHR was temperature dependent, and in stationary-phase cells LHR was independent of nutrients. Higher levels of LHR occurred in exponential-phase cells than in stationary-phase cells, and LHR was affected by nutrients in exponential-phase cells. Sublethal concentrations of caffeine and acriflavine inhibited LHR. In addition to LHR, minimal medium recovery also occurred in the concentration of [3H]thymine-containing dimers in the acid-insoluble fraction of the cells. A corresponding increase in [3H]thymine-containing dimers was observed in the acid-soluble fraction after LHR. Although a small proportion of irradiated cells produced filaments, this phenomenon was not directly related to LHR in B. fragilis.

Effect of oxygen on Bacteroides fragilis survival after far-ultraviolet irradiation.

Bacteroides fragilis cells were more sensitive to far-ultraviolet radiation under aerobic conditions than under anaerobic conditions. The percentage of pyrimidine dimers assayed after irradiation under both conditions was similar.