3.1. Antibacterial Tests with Liquid CLO
A total of seven bacteria, known to be potentially pathogenic to humans and recognized as environmental contaminants, were selected for evaluation. It was found that most of the materials used routinely in the laboratory were appropriate for tests (i.e., they were resistant to CLO liquid and vapor), with the exception of polystyrene culture vessels (trays, tubes, flasks), which were degraded by direct contact with CLO. The resulting degradation product was toxic to bacteria and to cultured cells. Consequently polypropylene and glass vessels were used in the experiments. Other materials tested, such as metals, wood, hard plastic, glass and fabrics, appeared to be resistant to CLO.
Initial tests were carried out with
B. subtilis, using TTO, a well-known antibacterial oil [
13] as a positive control, and an equivalent number of unexposed bacteria as a negative control. The antibacterial efficacy was affected by time of exposure and concentration of CLO, as expected, but was not significantly influenced by the number of bacteria, as shown in
Figure 1. This latter point was important to establish since the number of viable organisms in the field are likely to vary considerably from a few bacteria to many thousands.
B. subtilis was readily killed by CLO after several minutes contact with the oil (
Figure 1). Concentrations of CLO down to 0.1% (in saline) completely killed the bacteria within one hour (
Figure 1 and
Figure 2). Further dilutions showed successively less efficacy.
Figure 1.
Antibacterial effect of CLO: Variation in bacterial number. Reaction mixtures (100 µL) contained either 1,500 cfu or 150,000 cfu of B. subtilis in saline with 1% CLO. At various times reactions were stopped by pelleting the bacteria in Eppendorf tubes, and the latter were washed in saline and assayed for cfu. Blue bars: low inoculum; red bars: high inoculum. % kill in comparison with unexposed controls. NC, untreated control.
Figure 1.
Antibacterial effect of CLO: Variation in bacterial number. Reaction mixtures (100 µL) contained either 1,500 cfu or 150,000 cfu of B. subtilis in saline with 1% CLO. At various times reactions were stopped by pelleting the bacteria in Eppendorf tubes, and the latter were washed in saline and assayed for cfu. Blue bars: low inoculum; red bars: high inoculum. % kill in comparison with unexposed controls. NC, untreated control.
Figure 2.
Effect of CLO against different bacteria. Reactions, containing 1,500 cfu of each bacterium in 100 µL of saline and the indicated concentration of CLO, were incubated at 22 °C for 60 min. Bacteria were then pelleted by centrifugation, washed and assayed for cfu, in comparison with unexposed control bacteria. BS = B. subtilis, HI = H. influenzae, SP = S. pyogenes, AB = A. baumannii, EF = E. fecalis.
Figure 2.
Effect of CLO against different bacteria. Reactions, containing 1,500 cfu of each bacterium in 100 µL of saline and the indicated concentration of CLO, were incubated at 22 °C for 60 min. Bacteria were then pelleted by centrifugation, washed and assayed for cfu, in comparison with unexposed control bacteria. BS = B. subtilis, HI = H. influenzae, SP = S. pyogenes, AB = A. baumannii, EF = E. fecalis.
Four of the other bacteria were tested by the same protocols, and all of them were readily killed by CLO down to high dilutions, as shown in
Figure 2, although the relative sensitivities varied.
H. influenzae (Gram –) and
S. pyogenes (Gram +) were as susceptible as
B. subtilis (Gram +), whereas
A. baumannii (Gram –) was slightly more resistant, and
E. fecalis (Gram +) was substantially more resistant. Nevertheless even the latter was completely killed by exposure to 1% CLO. In addition
S. enteritidis (Gram –) was very sensitive, but
E. coli (Gram –) was slightly more resistant (data not shown). To determine if these activities of CLO were bactericidal or bacteriostatic, representative treated culture plates were returned to the incubator for several days, but no growth or additional growth occurred. In addition when the treated “bacteria” were pelleted by centrifugation, washed in saline and resuspended in broth, no growth was observed. Thus the activities were bactericidal.
Since CLO is immiscible with aqueous solutions, incubations of CLO with bacterial suspensions required frequent agitation or mixing, although efficient bacterial killing was achieved. Attempts were made to improve the stability of the emulsions by incorporating small amounts of the neutral detergent Tween 80 into the oil, as recommended by studies on Taxandria oil [
20]. However the addition of either Tween 80 or Tween 20 did not improve the antibacterial efficiency of CLO any further and consequently they were omitted in subsequent tests.
3.2. Possible Effect of Light
Some plant-based antimicrobials are influenced by ambient light. In theory the antibacterial activity could be enhanced by light (due to the presence of photosensitizers, [
18]), or reduced as a result of photo-degradation. Since the CLO applications in buildings could take place in either the presence or absence of ambient light, it was important to establish that the efficacy of the CLO would not be affected significantly. This was tested with
B. subtilis, but the efficacy of killing by CLO was essentially the same in light or dark, as shown in
Figure 3.
Figure 3.
Effect of light exposure on antibacterial property of CLO. Reactions contained 1,500 cfu of B. subtilis and the indicated concentration of CLO. Half the reactions were exposed to fluorescent light in the biosafety cabinet, and the others were completely covered in aluminum foil to exclude light. Reactions were processed and assayed for cfu as usual, in comparison to the untreated control (NC).
Figure 3.
Effect of light exposure on antibacterial property of CLO. Reactions contained 1,500 cfu of B. subtilis and the indicated concentration of CLO. Half the reactions were exposed to fluorescent light in the biosafety cabinet, and the others were completely covered in aluminum foil to exclude light. Reactions were processed and assayed for cfu as usual, in comparison to the untreated control (NC).
3.3. Sporicidal Activity of CLO
Conventional methods of producing spores for study often contain mixtures of spores and vegetative cells. In order to ensure that prospective antimicrobials are really effective against spores, it is desirable to experiment with preparations that are relatively free of dead and vegetative forms [
10]. Recently a method of purifying spores was reported for
Clostridium difficile (an anaerobic intestinal pathogen), which involved centrifugation of partly purified spores through gradients of Histodenz [
19]. The spores of
C. difficile sedimented through 50% Histodenz, leaving them free from the vegetative cells and dead organisms, which were found in the layers of less dense Histodenz.
In our experiments with different concentrations of Histodenz, it was shown that purification of
B. subtilis spores worked best by sedimentation through 45% Histodenz (they did not sediment through 50%). This purified spore preparation was tested in a time course experiment, and the results are summarised in
Table 1. Increasing exposure times and concentrations resulted in greater killing of the spores, although the rate and level of killing achieved was not as great as that observed with the vegetative
B. subtilis.
Table 1.
Sporicidal activity of CLO; % kill (cfu).
Table 1.
Sporicidal activity of CLO; % kill (cfu).
Exposure time hours | 1% CLO | 5% CLO | 10% CLO |
---|
24 | 30.5 | 48.0 | 63.6 |
48 | 41.2 | 50.0 | 88.0 |
72 | 64.5 | 70.2 | 93.4 |
3.4. Antibacterial Effects of CLO Vapor on Dried Films
In subsequent experiments the bacteria were used as dried films on glass slides, to represent normal conditions of surface contamination. The treated films were reconstituted in saline solution after exposure, followed by serial dilution and measurement of CFUs on agar plates. The films, containing different numbers of viable bacteria, were found to be just as vulnerable to liquid CLO as bacterial suspensions (data not shown).
The next series of experiments consisted of exposing similar dried films of bacteria to the vapor of CLO. This treatment was also very effective, although the time of exposure required for efficient killing was significantly longer than with the liquid oil. Results are shown in
Figure 4 for the three bacteria,
B. subtilis,
H. influenzae, and
S. pyogenes. In separate tests dried films of
A. baumannii showed a similar level of susceptibility.
Figure 4.
Effect of CLO-vapor on dried bacteria. 100 µL each bacterium were air dried on glass slides, and exposed to the vapor of CLO in a glass vessel, at 22 ºC. The bacteria were then reconstituted in saline and assayed for CFU, in comparison with unexposed bacteria. BS = B. subtilis, HI = H. influenzae, SP = S. pyogenes.
Figure 4.
Effect of CLO-vapor on dried bacteria. 100 µL each bacterium were air dried on glass slides, and exposed to the vapor of CLO in a glass vessel, at 22 ºC. The bacteria were then reconstituted in saline and assayed for CFU, in comparison with unexposed bacteria. BS = B. subtilis, HI = H. influenzae, SP = S. pyogenes.