The results of the previous experiments showed that using a light dose of 0.88 J of HeNe laser light and 1.18 J of GaAIAs laser light lethal photosensitisation of L. casei was only possible at a neutral pH, and not under acidic conditions, when the bacteria were sensitised by both TBO and AIPCS2. Similarly A.
naeslundii could only be sensitised by AIPCS2 to killing by 1.18 J of GaAIAs
laser light when at a neutral pH. This set of experiments was carried out to determine if a bactericidal effect could be achieved at a low pH by increasing the light dose. Bacterial suspensions at pH 4.5 were sensitised with either TBO or AIPCS2 and exposed to 0.88 - 4.40 J of HeNe laser light or 1.18 - 5.90 J of
GaAIAs laser light respectively. The numbers of surviving bacteria were determined by viable counting as detailed in 2.6. As the previous studies
bacterial viability these controls were omitted from this part of the study. Also as the sensitiser alone had little or no effect on the bacterial viability these controls were also omitted.
4.5.2 - RESULTS v ia b le c o u n t (cfu) 1 .0 0 0 E + 10 1 .OOOE + 0 9 - 1 .0 0 0 E + 0 8 1 .0 0 0 E + 07 1,0 0 0 .0 0 0 1 0 0 ,0 0 0 1 0 ,0 0 0 1 ,0 0 0 100 0 .8 8 1 .3 2 1 .7 6 2 .6 4 3 .5 2 4 .4 0 energy dose (J) L-S - Ü L + S +
Figure 4.9 - Effect of increasing light doses on the lethal photosensitisation of
L casei with TBO and HeNe laser light at pH 4.5.
L-S- : exposure to neither the laser light nor the sensitiser L+S+ : exposure to both the laser light and the sensitiser
viable count (cfu) 1.000E + 10 1.000E +09 - 1.000E + 08 1.000E + 07 1,000,000 100,000 10,000 1,000 1.18 1.77 2.36 3.54 4.72 5.90 energy dose (J) L -S - ■ l+ S +
Figure 4.10 - Effect of increasing light doses on the lethal photosensitisation of L. case/ with AIPCS2 and GaAIAs laser light at pH 4.5.
L-S- : exposure to neither the laser light nor the sensitiser L+S+ : exposure to both the laser light and the sensitiser
viable count (cfu) 1.000E + 08 1.0 00E +07 1 ,000,000 100,000 10,000 1,000 100 10 0.1 m 1.18 1.77 2. 36 3. 54 4. 72 5.90 e n e r g y d o s e (J) L-S - i l L + S +
Figure 4.11 - Effect of increasing light doses on the lethal photosensitisation of
A. naeslundii with AIPCS2 and GaAIAs laser light at pH 4.5.
L-S- ; exposure to neither the laser light nor the sensitiser L+S+ : exposure to both the laser light and the sensitiser
No bacterial kill was observed on exposure of TBO sensitised L case/ at pH 4.5 to 0.88 J of HeNe laser light. However, exposure to 1.32 J of HeNe laser light resulted in a kill of 1.5 x 10® cfu (26 %). On exposure of TBO sensitised L.
case/ at pH 4.5 to 1.76 J of HeNe laser light a reduction of 5.7 x 10® cfu (99.8 %) was observed, a kill similar to that achieved at a neutral pH (see Figure 5.3). When the bacteria were exposed to energy doses higher than 1.76 J (2.64 - 4.44 J) no survivng bacteria were detected.
When AlPcSa sensititised L case/were exposed to 1.18 J of GaAIAs laser light at pH 4.5 a very small and insignificant kill of 8 % was observed. Increasing the energy dose to 1.77 J resulted in an increased kill of 1.3 x 10® cfu (14 %). This however was statistically insignificant and only exposure to 2.36 J of GaAIAs laser light resulted in a significant kill (9.1 x 10® cfu (99.9 %) similar to that observed at a neutral pH (see Figure 5.7). Increasing energy doses resulted in an increase in bacterial kill and on exposure of the sensitised bacteria to 5.9 J no surviving bacteria were detected.
A similar pattern of results was observed on exposure of AIPCS2 sensitised A.
naeslundii at pH 4.5 to increasing doses of GaAIAs laser light. On exposure of
the bacteria to 1.18 and 1.77 J of GaAIAs laser light all the bacteria survived. However, on exposure to 2.36 J of laser light a kill of 6.4 x 10^ cfu (98 %) was observed and increasing light doses caused increasing bacterial kill.
4 .6 -DISCUSSION
These results have shown that all four target bacteria could be killed by lethal photosensitisation at acidic pHs. However, L .case/ and A. naeslundii appeared
required exposure to a higher energy dose for a significant bactericidal effect to be achieved. As in other parts of the study, the laser light in the absence of the sensitiser was found to have no effect on bacterial viability.
Other studies have also shown that the kills achieved by lethal photosensitisation are pH dependent. Ito et a! (1977) demonstrated that the inactivation of yeast cells by toluidine blue increased in the neutral pH region. The effect of pH on the killing of yeasts cells was shown to be similar to the pH dependent quantum yield of sensitised singlet oxygen production. Nitzan at al
(1987) have shown that at pH 6.5 photodynamic killing of S. aureus was at a maximum compared to that achieved at pH 7.6. They have also shown in the same study that binding of the sensitiser (haematoporphyrin derivative) was also at a maximum at pH 6.5 with only minor binding at pH 7.6. They proposed several reasons for the pH dependence of bacterial killing. The first was that singlet oxygen production could be affected by pH, secondly that the haematoporphyrin derivative may be in a monomeric or dimereric form at pH 6.5 rather than in an oligomeric form which could occur at a pH below 6. They also suggested that as bacterial membrane fluidity changes as a function of pH, perhaps at pH 6.5 membrane fluidity is appropriate for good penetration of reactive species. Lastly they suggest that the bacterial cells bind more porphyrin at pH 6.5. Pottier et al (1975) have also demonstrated singlet oxygen generation in an aqueous system to be largely pH dependant when toluidine blue was used as a sensitiser and this has been attributed to the protonation of the triplet state sensitiser in acidic conditions. The efficiency of the triplet state in its reaction with oxygen is affected which in turn affects singlet oxygen production. The lifetime of triplet state methylene blue, a commonly used
sensitiser, has also been shown to be pH dependent (Kelly and Tuite (1993). As an analogue of methylene blue, TBO may also be affected in the same manner. This could be a possible explanation for the reduced susceptibility of
L. case/ to photosensitised killing with TBO under acidic conditions. pH affects the triplet state of AIPCS2 in that as the pH is lowered the lifetime of the triplet
state sensitiser is shortened. However, effects of pH caused by changes in the triplet state and effects caused by aggregation of the sensitiser are difficult to differentiate as AIPCS2 is prone to aggregation at low pHs. However, it would
be expected that any effect on the singlet oxygen generation by pH would be observed with all four target bacteria yet the results of this study have shown that killing of only two species was affected by pH. This therefore suggests that some other factor may also contribute to the pH effect observed in this study. Nitzan also suggested that pH may cause the formation of an oligomeric haematoporphyrin derivative. In aqueous solution the spectrum of phenothiazinium dyes such as TBO is markedly affected by dimérisation and oligomerisation (Lai et al (1984). Therefore if the spectrum becomes altered so too does the maximum absorbtion of the sensitiser shifting it away from the wavelength of the laser light used. Phthalocyanines are analogues of porphyrins and therefore may be affected in a similar manner by pH, resulting in the formation of an oligomeric sensitiser which would be less effective. The suggestion that membrane fluidity is affected by pH may account for the differences in susceptibility of the four target bacteria to killing by low power laser light. If under acidic conditions the membranes of L easel and A.
dependent on the location of the sensitiser in the cell and whether the dominant mechanism is mediated by radicals or by singlet oxygen. With a radical mechanism the sensitiser must be in close contact with the substrate (site of action) whereas a unique feature of singlet oxygen involvement is that reactive species are capable of acting at sites other than those at which they are produced. Binding of the sensitiser to the target cell is also suggested as a possible reason for pH affecting the killing of bacteria. However in this study it appears from the spectrophotometric measurements that pH does not affect binding of the sensitiser to the target cells. The fact that L case/ and
A.naeslundii appear slightly less susceptible to lethal photosensitisation under
acidic conditions does not therefore appear to be related to binding of the sensitiser. The more likely explanation may be that changes in membrane permeability as a result of pH could have affected the penetration of reactive species.
CHAPTER FIVE - EFFECT OF THE PHYSIOLOGICAL STATE OF THE BACTERIA ON LETHAL PHOTOSENSITISATION