JAC Advance Access originally published online on December 6, 2005
Journal of Antimicrobial Chemotherapy 2006 57(2):335-338; doi:10.1093/jac/dki432
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Use of a new alginate film test to study the bactericidal efficacy of the high-level disinfectant ortho-phthalaldehyde
1 School of Pharmacy and Biomolecular Sciences, University of Brighton, Brighton BN2 4GJ, UK; 2 Welsh School of Pharmacy, Cardiff University, Redwood Building, King Edward VII Avenue, Cardiff CF10 3XL, UK
* Corresponding author. Tel: +44(0)2920879088; Fax: +44(0)2920874149; E-mail: maillardj{at}cardiff.ac.uk
Received 4 March 2005; returned 19 April 2005; revised 22 June 2005; accepted 1 November 2005
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Abstract |
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Objectives: To evaluate the merit of a new alginate efficacy film test to determine the bactericidal activity of the high-level disinfectant ortho-phthalaldehyde (OPA).
Methods: The efficacy of OPA was investigated using a new sodium alginate surface film test against Mycobacterium chelonae NCIMB 1474 and Epping, and Pseudomonas aeruginosa NCIMB 10421 under different test conditions.
Results: OPA was highly bactericidal against P. aeruginosa but its mycobactericidal efficacy was seriously reduced and produced 
5 log reductions only at a concentration of 0.5% (w/v) within 30–60 min without organic load.
Conclusions: The sodium alginate film efficacy was reproducible between repeats. Inactivation results depended upon the concentration of OPA, contact time, the presence of an organic load and the bacterial genera.
Keywords: disinfection , time-kill assay , mycobacteria , Pseudomonas aeruginosa
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Introduction |
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ortho-Phthalaldehyde (OPA) is an aromatic dialdehyde and has been shown to have the potential to replace glutaraldehyde (GTA) for high-level disinfection (i.e. activity against vegetative bacteria, viruses but not necessarily spores).1,2 OPA has been found to be rapidly bactericidal, including against GTA-resistant mycobacteria,1–3 and sporicidal.4 Standard quantitative suspension and carrier tests are routinely used to assess the microbicidal activity of disinfectants. However, these methodologies suffer from some drawbacks, mainly with the preparation of the test inocula (e.g. use of bacterial suspension with high metabolic activity, loss of viability following a drying step on a carrier).1,2,5 Although the design of standard carrier tests has taken an appropriate step towards a better correlation of the efficacy of a disinfectant between in vitro and in situ situations, it falls short of testing the antimicrobial potential of a product against microorganisms grown as a biofilm. This is particularly pertinent since microorganisms associated with surfaces are often found as a biofilm and bacterial biofilms have been shown to be more resistant to disinfection. As for other practical tests, the standardization of the inoculum is of paramount importance.
This study reports the use of a sodium alginate film test for testing the efficacy of a high-level disinfectant.
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Materials and methods |
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Bacterial strains
Pseudomonas aeruginosa NCIMB 10421, Mycobacterium chelonae NCIMB 1474 and a GTA-resistant M. chelonae Epping (obtained from Dr P. A. Griffiths, City Hospital NHS Trust, Birmingham, UK) were used. The preparation of the mycobacterial inocula has been described elsewhere.2 P. aeruginosa was inoculated into 10 mL of tryptone soya broth (Oxoid Ltd, Basingstoke, UK) and grown for 18 h at 37°C using a mechanical shaker (Reciprocal Mixer, Denley, UK) on its lowest setting.
After the required incubation periods the bacterial suspensions were centrifuged three times at 3000 g for 10 min. The resulting pellets were then re-suspended in HEPES buffer (Fisher Scientific, Loughborough, UK) using a vortex for up to 10 min, without glass beads for P. aeruginosa or with ten 4 mm un-drilled glass beads (Fisher Scientific) for the mycobacteria, to break up aggregates formed during their growth.2
Chemicals and biocides
Solutions of 0.5, 0.2 and 0.0182% (w/v) OPA (pH 7.4) (Cidex, Advanced Sterilisation Products, Irvine, CA, USA) were freshly prepared for each experiment in sterile deionized water (note: hard water was not used to avoid adding another parameter at this time). The neutralizer sodium bisulphite (Aldrich, Gillingham, UK) was prepared using sterile deionized water and made up to 0.5% (w/v).
Organic load
Bovine albumin (Oxoid Ltd) was added to the biocide solutions (at 0.3 or 3 g/L) in order to determine whether the presence of organic material would interfere with the antimicrobial activity of OPA. In addition, the efficacy of OPA was investigated with no bovine albumin.
Preparation of the bacterial ‘biofilm’ inocula
A 1 mL sample of bacterial suspension was added to a cooled 3% (w/v) sodium alginate solution (BDH Chemicals Ltd, Poole, UK). The final concentration in the bacteria/alginate mixture was 
1.5–5 x 108 cfu/mL. Custom-made sterile polypropylene (a material used in endoscopes) discs (Advanced Sterilisation Products) were added aseptically to specially designed sterile perspex moulds and 0.4 mL aliquots of alginate/bacteria were pipetted onto the surface of the discs. The moulds were lowered into sterile beakers containing 100 mL of 2% (w/v) calcium chloride (BDH chemicals, Poole, UK) and left for 5 min. The discs were then removed and placed into 90 mm Petri dishes containing 40 mL of 2% calcium chloride solution. After 1 h residence time the discs were rinsed with five changes of 10 mL of sterile distilled water to remove unbound organisms. The washed surfaces were then placed into sterile beakers containing 30 mL of nutrient broth (Oxoid Ltd) with 0.2% (w/v) calcium chloride and incubated for 18–24 h for P. aeruginosa or 4 days for the mycobacteria. Following incubation, remaining unbound organisms were removed by rinsing with five changes of 10 mL of sterile distilled water.
Sodium alginate surface film efficacy test
Sodium alginate/bacterial ‘biofilms’ were exposed to 20 mL of OPA at the appropriate concentration with or without organic load. In the control, OPA was replaced with sterile deionized water. After the required contact time, polypropylene discs were aseptically transferred to a 5 mm Petri dish containing 20 mL of neutralizer. Neutralization was allowed to occur for at least 20 min. The films were then rinsed in five changes of 10 mL of sterile distilled water and dissolved in 10 mL of McIlvaine's buffer (0.1 M citric acid and 0.2 M disodium phosphate at pH 7.4; Fisher Scientific). Survivors were determined using a Miles–Misra drop counting method (mycobacteria) or the spread plate method (P. aeruginosa). The microbicidal effect (ME) was calculated as follows: ME = log Nc – log Nb where Nc and Nb represent the numbers of cfu/mL in the control and biocide solutions, respectively.
Controls
Before testing the bactericidal activity of OPA, neutralization tests were carried out to check the efficacy of the neutralizer (0.5% w/v sodium bisulphite) to quench the activity of the aldehyde. Two alginate films were produced for each test; the first film was added to 20 mL of neutralizer and 0.2 mL of OPA at the required concentration. The second film was added to 20 mL of neutralizer and 0.2 mL of sterile distilled water. Both films were left for at least 15 min at room temperature. The films were then rinsed five times using sterile distilled water, dissolved in McIlvaine's buffer and plated out onto TSA for P. aeruginosa or 7H11 for the mycobacteria.
Statistical analysis
Results presented here represent the mean of at least five repeats. A two-way ANOVA was carried out to determine the efficacy of different OPA concentrations against all three test organisms under different conditions.
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Results |
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The use of sodium bisulphite 0.5% (w/v) was found to be a satisfactory neutralizer to quench the activity of OPA at the different concentrations investigated (data not shown). In addition, sodium bisulphite 0.5% (w/v) also proved to be non-toxic to the test organisms used in this study (data not shown).
The bacterial concentration recovered from untreated alginate films was highly reproducible: 8.680 ± 0.161 (n = 45), 8.960 ± 0.380 (n = 45) and 8.350 ± 0.280 (n = 45) log cfu/mL for P. aeruginosa, M. chelonae NCIMB 1474 and M. Chelonae Epping, respectively.
OPA 0.5% (w/v) produced a 5 log reduction within 1 min against P. aeruginosa without organic load and within 5 min with bovine albumin (Table 1). As the concentration of OPA decreased, the time to produce 5 log reductions increased. Thus it took 30 min to achieve such a bactericidal effect with OPA 0.0182% (w/v) with or without a low concentration of organic load. It was noted that generally an increase in organic load concentration decreased the efficacy of OPA. Notably, the lowest concentration of OPA failed to achieve 5 log reductions with 3 g/L bovine albumin (Table 1).
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The overall efficacy of OPA under all test conditions was low against the two mycobacterial strains. Indeed a 5 log reduction in number was only achieved at the highest concentration, without organic load within 30 and 60 min, for M. chelonae NCIMB 1474 and Epping, respectively (Table 1). The presence of organic load severely hindered (P < 0.098) the activity of the dialdehyde. The lowest OPA concentration (0.0182% w/v) failed to achieve any kill. The GTA-resistant M. chelonae Epping was slightly less susceptible than its standard counterpart. This was particularly noticeable at an OPA concentration of 0.2% (w/v) (Table 1).
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Discussion |
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In this study, OPA (0.5% w/v) showed a good bactericidal activity against P. aeruginosa but a limited activity against the two mycobacterial strains. When evaluated with the standard suspension2 and carrier test,3 OPA (0.5% w/v) showed a high bactericidal and mycobactericidal activity. European standard suspension and carrier tests require that a biocide must produce a 5 log reduction in bacterial numbers within 5 min. It was interesting to note that at an OPA concentration of 0.5% (w/v), the presence of an organic load had some effect on the aldehyde efficacy. The effect of 3 g/L of bovine albumin on OPA (0.5% w/v) efficacy tested in suspension and carrier tests did not seem to have such a quenching effect (data not shown). These results justify the use of organic load when the efficacy of a surface disinfectant is investigated. The decrease in activity of low concentrations of the aldehyde in the presence of bovine albumin is not unexpected since, by nature, the aldehyde is a cross-linking agent6 and as such its activity should be quenched by a high level of proteins. The effect of sodium alginate on the aldehyde penetration or availability is unclear and is subject to current investigation. The assumed ‘biofilm’ nature of the microorganisms in the film test would also account for the reduced efficacy of the biocide. In nature a biofilm is made up of a number of cell aggregates, which are interspersed throughout an extensive exopolymer matrix.7 Sodium alginate should simulate this matrix. Although, the assumed ‘biofilm’ phenotype of the bacteria embedded in the alginate was not confirmed by chemical or genetic tests, the results obtained in this study showed that this is most probably the case, notably when compared with results from tests using planktonic cultures.2,3
This study highlighted that the alginate film test generated reproducible inocula and reproducible results between different repeats. The efficacy of OPA clearly depended upon the OPA concentration, contact time, the presence of an organic load and the bacterial genera. Mycobacteria are usually considered to have some intrinsic insusceptibility to biocides as a result of high levels of mycolic acid,8 which increase impermeability of the cell wall limiting biocide uptake.9 The reason for the reduced mycobactericidal effect of OPA against the GTA-resistant M. chelonae Epping using this methodology is at present unclear, although a combination of a higher hydrophobicity10 together with a reduced OPA availability might partly explain such observations. A similar protocol using poloxamer hydrogels has been described, although in that case the dissolution of the film was temperature dependent and required 5 min at <15°C.11,12 The advantage of the sodium alginate film test is that the dissolution of the film is not temperature dependent and hence would allow this technology to be used at a low temperature.
These results and the fact that this method adds more stringency to the efficacy testing of a biocide warrant the alginate film test to be investigated further.
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Acknowledgements |
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We are grateful for the financial support from the University of Brighton and to Advanced Sterilisation Products (Irvine, CA, USA) for providing a research studentship (to J. C. N. S.).
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References |
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