J.G. Anderson

Inactivation of Bacterial Pathogens following Exposure to Light from a 405-Nanometer Light-Emitting Diode Array

ABSTRACT This study demonstrates the susceptibility of a variety of medically important bacteria to inactivation by 405-nm light from an array of light-emitting diodes (LEDs), without the application of exogenous photosensitizer molecules. Selected bacterial pathogens, all commonly associated with hospital-acquired infections, were exposed to the 405-nm LED array, and the results show that both gram-positive and gram-negative species were successfully inactivated, with the general trend showing gram-positive species to be more susceptible than gram-negative bacteria. Detailed investigation of the bactericidal effect of the blue-light treatment on Staphylococcus aureus suspensions, for a range of different population densities, demonstrated that 405-nm LED array illumination can cause complete inactivation at high population densities: inactivation levels corresponding to a 9-log10 reduction were achieved. The results, which show the inactivation of a wide range of medically important bacteria including methicillin-resistant Staphylococcus aureus, demonstrate that, with further development, narrow-spectrum 405-nm visible-light illumination from an LED source has the potential to provide a novel decontamination method with a wide range of potential applications.

Inactivation of food-borne enteropathogenic bacteria and spoilage fungi using pulsed-light

The lethality of high-intensity pulsed-light emissions from low and high ultraviolet (UV) light sources on predetermined microbial populations has been investigated. Prior to treatment, the bacterial enteropathogens Bacillus cereus, Escherichia coli, and Salmonella enteritidis and the food-spoilage fungi Aspergillus niger and Fusarium culmorum were seeded separately onto the surface of either tryptone soya yeast extract or malt extract agar plates. Prescribed microbial population densities were applied to the test media and these samples were exposed to one of two light sources. These were low-pressure, xenon filled, flash lamps that produced either high or low UV intensities. They were operated in pulsed mode, being driven by a stacked Blurnlein table generator. Microbial samples were treated by exposure to different numbers of light pulses. The treated bacterial populations were reduced by /spl sim/8 log orders after 1000 light-pulses of the higher UV intensity light and the fungal counts had a corresponding reduction of 4.5 log orders. The fungus, Aspergillus niger, was shown to be significantly more resistant in spore form to the intense UV light compared with Fusarium culmorum. This resistance has been attributed to the high level of UV absorbance associated with the dark pigment present in A. niger. The pulsed light source of lower UV intensity was shown to be significantly less effective in reducing microbial populations.

Light inactivation of food‐related pathogenic bacteria using a pulsed power source

The effects of high intensity light emissions, produced by a novel pulsed power energization technique (PPET), on the survival of bacterial populations of verocytotoxigenic Escherichia coli (serotype 0157:H7) and Listeria monocytogenes (serotype 4b) were investigated. Using this PPET approach, many megawatts (MW) of peak electrical power were dissipated in the light source in an extremely short energization time (about 1 μs). The light source was subjected to electric field levels greater than could be achieved under conventional continuous operation, which led to a greater production of the shorter bacteriocidal wavelengths of light. In the exposure experiments, pre‐determined bacterial populations were spread onto the surface of Trypone Soya Yeast Extract Agar and were then treated to a series of light pulses (spectral range of 200–530 nm) with an exposure time ranging from 1 to 512 μs. While results showed that as few as 64 light pulses of 1 μs duration were required to reduce E. coli 0157:H7 populations by 99·9% and Listeria populations by 99%, the greater the number of light pulses the larger the reduction in cell numbers (P < 0·01). Cell populations of E. coli 0157:H7 and Listeria were reduced by as much as 6 and 7 log10 orders at the upper exposure level of 512 μs, respectively. Survival data revealed that E. coli 0157:H7 was less resistant to the lethal effects of radiation (P < 0·01). These studies have shown that pulsed light emissions can significantly reduce populations of E. coli 0157:H7 and L. monocytogenes on exposed surfaces with exposure times which are 4–6 orders of magnitude lower than those required using continuous u.v. light sources.

The role of oxygen in the visible-light inactivation of Staphylococcus aureus

Exposure to visible-light causes the photoinactivation of certain bacteria by a process that is believed to involve the photo-stimulation of endogenous intracellular porphyrins. Studies with some bacterial species have reported that this process is oxygen-dependent. This study examines the role of oxygen in the visible-light inactivation of Staphylococcus aureus. Suspensions of S. aureus were exposed to broadband visible-light under both oxygen depletion and oxygen enhancement conditions to determine whether these environmental modifications had any effect on the staphylococcal inactivation rate. Oxygen enhancement was achieved by flowing oxygen over the surface of the bacterial sample during light inactivation and results demonstrated an increased rate of staphylococcal inactivation, with approximately 3.5 times less specific dose being required for inactivation compared to that for a non-enhanced control. Oxygen depletion, achieved through the addition of oxygen scavengers to the S. aureus suspension, further demonstrated the essential role of oxygen in the light inactivation process, with significantly reduced staphylococcal inactivation being observed in the presence of oxygen scavengers. The results of the present study demonstrate that the presence of oxygen is important for the visible-light inactivation of S. aureus, thus providing supporting evidence that the nature of the mechanism occurring within the visible-light-exposed staphylococci is photodynamic inactivation through the photo-excitation of intracellular porphyrins.

Putative virulence factor expression by clinical and food isolates of Bacillus spp. after growth in reconstituted infant milk formulae

ABSTRACT Forty-seven strains representing 14 differentBacillus species isolated from clinical and food samples were grown in reconstituted infant milk formulae (IMF) and subsequently assessed for adherence to, invasion of, and cytotoxicity toward HEp-2 and Caco-2 cells. Cell-free supernatant fluids from 38 strains (81%) were shown to be cytotoxic, 43 strains (91%) adhered to the test cell lines, and 23 strains (49%) demonstrated various levels of invasion. Of the 21 Bacillus cereus strains examined, 5 (24%) were invasive. A larger percentage of clinically derivedBacillus species (20%) than of similar species tested from the food environment were invasive. Increased invasion occurred after growth of selected Bacillus species in reconstituted IMF containing glucose. While PCR primer studies revealed that many different Bacillus species contained DNA sequences encoding the hemolysin BL (HBL) enterotoxin complex andB. cereus enterotoxin T, not all of these isolates expressed these diarrheagenic genes after growth in reconstituted IMF. Of the 47 Bacillus isolates examined, 3 isolates of B. cereus and 1 isolate ofB. subtilis produced the HBL enterotoxin after 18 h of growth in brain heart infusion broth. However, eight isolates belonging to the species B. cereus,B. licheniformis, B. circulans, and B. megaterium were found to produce this enterotoxin after growth in reconstituted IMF when assessed with theB. cereus enterotoxin (diarrheal type) reversed passive latex agglutination (RPLA) kit. It is concluded that several Bacillus species occurring occasionally in clinical specimens and food samples are of potential medical significance due to the expression of putative virulence factors.

The Production of Conidiophores and Conidia by Newly Germinated Conidia of Aspergillus niger (Microcycle Conidiation)

SUMMARY: The ability of Aspergillus niger conidia to produce conidiophores after germination in shaken culture at 30° was stimulated by the inclusion of glutamate in the medium. Incubation of the conidia at 35° to 41° increased swelling of the conidia and also the proportion which produced conidiophores. Although conidiophore initiation was stimulated at temperatures between 35° and 41°, maturation was poor and optimum conidiation was obtained by incubation at these temperatures followed by 30°. Conidiophore formation from conidia required a prior period of spore metabolism and at temperatures between 30° and 41° did not occur until several hours after germination. Direct conidiophore production from conidia in the complete absence of vegetative growth was achieved by incubation of the conidia at 44° (which allows only swelling) for a prolonged period (48 h.) followed by 30°. Although vegetative growth was absent the conidiophores were similar to, but smaller than, normal subaerial conidiophores and viable conidia were produced. These conidia differed from subaerial spores in lacking the dark pigmented spore coat.

A technique for the prediction of the conditions leading to mould growth in buildings

Epidemiological evidence suggests that the presence of mould growth in buildings can have a detrimental effect on the well-being of occupants. Based on an extensive literature review, growth limits for six generic mould categories have been formulated in terms of the minimum combination of temperature and relative humidity for which growth will occur on building materials. These limits were incorporated within the ESP-r system for building energy and environmental simulation in order to provide a design tool which can predict the likelihood and extent of mould infestation. The systems new predictive capability has been tested against monitored data and mycological samples taken from a mould infested house.

High-intensity narrow-spectrum light inactivation and wavelength sensitivity of Staphylococcus aureus

This study was conducted to investigate the bactericidal effects of visible light on methicillin-sensitive and methicillin-resistant Staphylococcus aureus (MRSA), and subsequently identify the wavelength sensitivity of S. aureus, in order to establish the wavelengths inducing maximum inactivation. Staphylococcus aureus, including MRSA strains, were shown to be inactivated by exposure to high-intensity visible light, and, more specifically, through a series of studies using a xenon broadband white-light source in conjunction with a selection of optical filters, it was found that inactivation of S. aureus occurs upon exposure to blue light of wavelengths between 400 and 420 nm, with maximum inactivation occurring at 405+/-5 nm. This visible-light inactivation was achieved without the addition of exogenous photosensitisers. The significant safety benefit of these blue-light wavelengths over UV light, in addition to their ability to inactivate medically important microorganisms such as MRSA, emphasises the potential of exploiting these non-UV wavelengths for disinfection applications.

Bactericidal Effects of 405 nm Light Exposure Demonstrated by Inactivation of Escherichia, Salmonella, Shigella, Listeria, and Mycobacterium Species in Liquid Suspensions and on Exposed Surfaces

The bactericidal effect of 405 nm light was investigated on taxonomically diverse bacterial pathogens from the genera Salmonella, Shigella, Escherichia, Listeria, and Mycobacterium. High-intensity 405 nm light, generated from an array of 405-nm light-emitting diodes (LEDs), was used to inactivate bacteria in liquid suspension and on exposed surfaces. L. monocytogenes was most readily inactivated in suspension, whereas S. enterica was most resistant. In surface exposure tests, L. monocytogenes was more susceptible than Gram-negative enteric bacteria to 405 nm light when exposed on an agar surface but interestingly less susceptible than S. enterica after drying onto PVC and acrylic surfaces. The study findings, that 405 nm light inactivates diverse types of bacteria in liquids and on surfaces, in addition to the safety advantages of this visible (non-UV wavelength) light, indicate the potential of this technology for a range of decontamination applications.

Inactivation of mycobacterium paratuberculosis by pulsed electric fields

ABSTRACT The influence of treatment temperature and pulsed electric fields (PEF) on the viability of Mycobacterium paratuberculosiscells suspended in 0.1% (wt/vol) peptone water and in sterilized cows milk was assessed by direct viable counts and by transmission electron microscopy (TEM). PEF treatment at 50°C (2,500 pulses at 30 kV/cm) reduced the level of viable M. paratuberculosis cells by approximately 5.3 and 5.9 log10 CFU/ml in 0.1% peptone water and in cows milk, respectively, while PEF treatment of M. paratuberculosisat lower temperatures resulted in less lethality. Heating alone at 50°C for 25 min or at 72°C for 25 s (extended high-temperature, short-time pasteurization) resulted in reductions ofM. paratuberculosis of approximately 0.01 and 2.4 log10 CFU/ml, respectively. TEM studies revealed that exposure to PEF treatment resulted in substantial damage at the cellular level to M. paratuberculosis.