Bactericidal properties of peracetic acid and hydrogen peroxide, alone and in combination, and chlorine and formaldehyde against bacterial water strains Laboratoire de bacteriologie, virologie et microbiologie industrielle, Faculte' des sciences pharmaceutiques, 31 alle'es Jules Guesde, 31400 Toulouse, France AND
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CORINNECABASSUDAND PHILIPPE APTEL Lyonnaise des Eaux, Laboratoire membranes, 20, avenue Didier Daurat, 31432 Toulouse, France Received August 13, 1991 Revision received December 13, 1991 Accepted January 27, 1992 ALASRI,A., ROQUES,C., MICHEL,G., CABASSUD,C., and APTEL,P. 1992. Bactericidal properties of peracetic acid and hydrogen peroxide, alone and in combination, and chlorine and formaldehyde against bacterial water strains. Can. J. Microbiol. 38: 635-642. The bactericidal properties of peracetic acid, hydrogen peroxide, chlorine, and formaldehyde were compared in vitro using a rapid micromethod. A combination of peracetic acid and hydrogen peroxide was also tested to assess interactions. The activities of these agents, which are widely used as disinfectants, were evaluated against water isolates and culture collection strains. Peracetic acid and chlorine exhibited an excellent antimicrobial activity, with a relatively rapid destruction of 10' bacteria/mL. The time-dependent bactericidal activities of hydrogen peroxide and formaldehyde were the lowest. The combination of peracetic acid and hydrogen peroxide, tested by a checkerboard micromethod, was found to be synergistic. The minimal bactericidal concentration was established in terms of time for a given mixture of peracetic acid and hydrogen peroxide. Determination of bactericidal concentrations showed that synergy was maintained with increasing contact time. Concentrations for minimal times of treatment by chemicals that provided interesting activities in vitro were tested for disinfection of ultrafiltration membranes. The bactericidal activities of peroxygen compounds were confirmed and synergism was maintained in working conditions. Chlorine showed a loss of efficacy when used on membranes. Key words: peracetic acid, hydrogen peroxide, chlorine, formaldehyde, minimal bactericidal concentration, ultrafiltration membranes. ALASRI,A., ROQUES,C., MICHEL,G., CABASSUD,C., et APTEL,P. 1992. Bactericidal properties of peracetic acid and hydrogen peroxide, alone and in combination, and chlorine and formaldehyde against bacterial water strains. Can. J . Microbiol. 38 : 635-642. Les proprietes bactericides de l'acide peracetique, du peroxyde d'hydrogene, du chlore et du formaldehyde ont ete comparees in vitro selon une micromethode rapide. Les interactions pouvant resulter de l'association de l'acide peracetique et du peroxyde d'hydrogene ont aussi ete evaluees. L'activite de ces composes largement utilises comme desinfectants a ete mesuree sur des souches isolees de l'eau et sur des souches de collection. L'acide peracetique et le chlore possedent une excellente activite antimicrobienne qui se traduit par une destruction relativement rapide de 10' bacteries/mL. En considerant le facteur temps, ce sont le peroxyde et la formaldehyde qui affichent les plus faibles activites bacteriennes. Selon les resultats obtenus par la methode de l'echiquier, l'association de l'acide peracetique et du peroxyde d'hydrogene est synergique. La concentration minimale bactericide a Cte etablie en fonction du temps pour un melange precis de peroxyde et d'acide peracetique. La mesure des concentrations bactericides a confirme que l'activite bactericide se maintenait avec l'augmentation du temps de contact. La concentration de ces agents chimiques qui en un temps minimum de contact presentent des activites interessantes in vitro a ete verifiee pour la desinfection des membranes d'ultrafiltration. L'activite bactericide des composes peroxygenes a ete confirmee et dans les conditions experimentales suivies, la synergie a ete maintenue. Le chlore perd de l'efficacite lorsqu'il est utilise sur des membranes. Mots cle's : acide peracetique, peroxyde d'hydrogene, chlore, concentration bactericide minimale, membranes d'ultrafiltration. [Traduit par la redaction]
Introduction Ultrafiltration membranes have begun to be widely used in today's water-treatment systems. Because of their adhesion and proliferation, bacteria are considered as a particular problem. Therefore sanitizing membranes by efficient disinfection is necessary for an optimal shelf life. Bacteria were isolated from stored membranes, and experiments were carried out to try to prevent microbial growth. The disinfection efficiency is strongly influenced by the biocide composition, it's concentration, the biocide-bacteria reaction time, temperature, and pH. Generally chemical disinfection is a compromise between the benefits of destroying microorganisms and the detriments of chemical toxicity and Printed in Canada / Imprime au Canada
undesirable by-product formation (Curver 1990). Modification of reaction time can reduce biocide concentrations to below the recommended limits. The aim of the present study was to determine the bactericidal activity of four biocides in terms of reaction time and to determine whether they could be used for membrane disinfection. The antimicrobial properties of peroxygen compounds have been recognized for many years, and a variety of applications have been developed. Peracetic acid is reported to be a most effective biocide and is used, both in aqueous solution and as an aerosol or vapor, for sterilization in gnotobiotic units (Doll et al. 1963; Greenspan et al. 1955).
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TABLE1. MBC (ppm) of the antimicrobial agents tested for water isolates Combination
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Time E. coli
5 min 30 min 1h 2h 3h 4h 5h 6h 7h
S. aureus
5 min 30 min 1h 2h 3h 4h 5h 6h 7h
P. aeruginosa
5 min 30 min 1h 2h 3h 4h 5h 6h 7h
PAA
PAA
It has also been proposed as an antiviral agent (Harakeh 1984; Kline and Hull 1960) and recently has been introduced as a disinfectant in water-treatment plants. Hydrogen peroxide is used as an antiseptic in dilute solutions (Baldry 1983) and has been advised for membrane regeneration in combination with peracetic acid (Byrne and Roesner 1990). Because of its broad spectrum of activity and relative ease of use, formaldehyde represents a choice agent for numerous decontamination applications and has done so since the 1890s (Fink et a/. 1988; Phillips 1973; Vesley and Lauer 1986). Chlorine is the most widely used water disinfectant. It is generally believed that chlorine inhibits essential cell enzyme systems through oxidation, but the exact mechanism of action is unltnown (Curver 1990).
HP
HP
Chlorine
Formaldehyde
identified by morphological and biochemical characteristics, the catalase test, oxidase activity, and the API system (Biomerieux Laboratories). Culture media Strains were maintained on trypticase soy agar (Biomerieux Laboratories). Minimal bactericidal concentrations (MBCs) were determined using trypticase soy broth (Biomerieux Laboratories). Chemical disactivation medium consisted of 1 egg yolk; 10 mL 5% sodium thiosulfate (Prolabo Laboratories); 5 mL Tween-80 (Aldrich-Chimie); and trypticase soy broth up to 100 mL. For disinfection of membranes deoxycholate lactose, pyocyanosel, and manitol salt agar media, respectively, were used for E. coli, P. aeruginosa, and S. aureus counts (Biomerieux Laboratories). Inoculated agar and microtiter plates were incubated at 37°C for 24 to 48 h for all strains.
Materials
Methods
Antimicrobial agents Disinfectant agents were obtained from the following sources: peracetic acid (PAA, 15% w/w), Interox Chemicals Ltd.; hydrogen peroxide (HP, 30/35%), Produits Chimiques du Ciron; sodium hypochlorite (12.5% active chlorine), S.A. Gede; and formaldehyde (37%), Aldrich-Chimie. Before use, biocides were added to sterile distilled water to obtain appropriate concentrations.
Activities of biocides were evaluated at first on microplates by MBC (5 log reduction in total number of bacteria for a contact time of 5 min). The more interesting biocides were then tested on hollow fibers.
Bacterial strains Strains tested were isolated from water (water samples were provided by the Lyonnaise des Eaux) or came from culture collections (Escherichia coli ATCC 8739; Pseudomonas aeruginosa ATCC 9027; and Staphylococcus aureus ATCC 6538 P). All strains were
Verification of disactivating medium Twofold serial dilution of biocides in distilled water (100 pL/well) were placed on a Nunclon (8 x 12) microtiter plate (96 cells). Disactivating medium (100 pL) was added to each well. After 10 min of disactivation, 10 pL of bacterial suspension was added to each well. After 5 min of contact, 10 pL from each well was transferred to a second microplate, which contained broth medium and which then was incubated. Three disactivation media
ALASRI ET
AL.
TABLE2. MBC (ppm) of the antimicrobial agents tested for culture collection strains Combination
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Time
E. coli
5 min 30 min 1h 2h 3h 4h 5h 6h 7h
S. aureus
5 min 30 min 1h 2h 3h 4h 5h 6h 7h
P. aeruginosa
5 min 30 min 1h 2h 3h 4h 5h 6h 7h
PAA
PAA
were tested. The disactivating medium described in the section Culture media gave the best bacterial growth for all the tested concentrations.
Determination of MBC with time, using a dilution-disactivation micromethod Twofold serial dilutions of biocides in distilled water (100 pL/well) were placed on a Nunclon (8 x 12) microtiter plate (96 wells). Bacterial suspension prepared in 0.9% NaCl solution (10 pL adjusted to lo9 organisms/ml) was added to each well. After different contact times (5 and 30 min and 1, 2, 3, 4, 5, 6, and 7 h), 10 pL from each well was transferred to a second microplate, containing 100 pL of disactivating medium per well. After 10 min of disactivation, 10 pL from each well was transferred to a third microplate with broth medium (100 pL/well), which was then incubated, and growth was observed by turbidity. Each MBC was determined four times for each strain. For each strain tested, the adequacy of growth conditions and the sterility of the medium were checked in columns 11 and 12 (control wells). Evaluation of interactions between PAA and HP Association tests were performed according to the checkerboard pattern (Amarouch 1984; Berenbaum et al. 1983; Beales and Sutherland 1983). Twofold serial dilutions of one biocide were tested in combination with concentrations of twofold dilutions of the other. The same method used for measuring the bactericidal effect, described above, was used for a contact time of 5 min. Each strain was tested at least twice. The fractional bactericidal concentration (FBC) index was determined as follows. The concentrations of PAA and H P that produced a bactericidal effect when used alone were called PAAa and HPa. The concentrations that produced the same effect when used in combination were called PAAc and HPc.
HP
HP
Chlorine
Formaldehyde
The expression reported by Berenbaum (1978), "PAAc/PAAa + HPc/HPa," gives the degree of interaction or FBC index. The following values are generally considered to define the degree of synergy: FBC I0.75, combination is synergistic; 0.75 2 FBC 5 1.1, additive; 1.1 > FBC 5 2, indifferent; FBC > 2. antagonistic.
Determination of MBC with time, using a combination of PAA and HP A combination of PAA and H P in a particular ratio (0.03% PAA and 3.91% HP), chosen on the basis of the MBC (biocides alone) and interaction degree, was tested to determine the FBC index in terms of contact time (5 and 30 min and 1, 2, 3, 4, 5, 6, and 7 h). Disinfection of hollow fibers The bactericidal efficacy of the chemicals studied for membrane disinfection was tested against water isolates. Bactericidal activities For their storage, hollow fiber filters are filled with ultrafiltrated water (0.01 pm) with adjunction of a disinfectant. Our bactericidal efficacy experiments were carried out on microprototypes (length, 25 cm; diameter, 1.5 cm) with 15 hollow fibers each (length, 24 cm). The microprototypes (MPs) were inoculated with a mixture of bactericidal suspensions of E. coli, P. aeruginosa, and S. aureus to obtain 10' organisms/mL of each species. After 1 min of shaking (Vortex 2, Shake 5, Scientific Industries) and 1 h of incubation (20°C), disinfectants were added to the hollow fibers. After each contact time (5 and 30 min 1, 2, 3, 4, and 5 h, and 1, 2,3, and 4 days) 0.5 mL of each M P test suspension was suspended in 4.5 mL disactivating medium. After 10 min, the suspension was diluted, and aerobic plate counts were made on specific media after 2 to 4 days incubation.
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-(3.
E. coli
V
5 30 l h 2 h 3 h 4 h 5 h 6 h 7 h min rnin Time
2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 ' 1 1 5 30 l h 2 h 3 h 4 h 5 h 6 h 7 h min rnin Time
FIG. 3. MBC of hydrogen peroxide against time for water strains.
FIG. 1. MBC of peracetic acid against time for water strains.
* *
E coli S. aureus -+ P. aeruginosa
A
5 30 min min
lh
2h
3h
4h
.
h
5h
.
-
b
6h
.
4
7h
Tune
FIG. 2. MBC of chlorine against time for water strains.
Effect on bacterial adhesion to membranes The bacterial adhesion to membranes was observed every day after M P disinfection (4 days). Samples of hollow fibers (4 cm) taken from each test and cut (longitudinally and transversely) into four portions were dipped in 5 mL PBS (phosphate-buffered saline, Gibco Laboratories) and then shaken ultrasonically (1 min at 120 W, Bioblock Scientific). Agar plate counts were then determined. Experiments were performed at least twice for each biocide.
Results The in vitro activities of biocides tested on water and culture collection strains in terms of time (MBC in parts per million) are given in Tables 1 and 2.
V
5 30 l h 2 h 3 h 4 h 5 h 6 h 7 h rnin rnin Time FIG. 4. MBC of formaldehyde against time for water strains.
and PAA were observed to have a very low MBC, 6 and 12 PPm, respectively, after 5 rnin of reaction time (E. coli water strain). The action of PAA was poorly time dependent (Fig. 1): MBC values rapidly decreased from 12 to 3 ppm in 30 rnin and then remained stable for the 7 h of the test (E. coli water strain). Chlorine presented an intermediate action, requiring 3 h to obtain a straight line of MBC (Fig. 2). At less than 3 h, bactericidal activity was time dependent (MBC
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0 0
195 391
781 1563 3 125 6250 12500
HP ( P P ~ ) FIG. 5. Checkerboard determination of interactions for P. aeruginosa ATCC 9027. S, synergistic; A, additive; I, indifferent; T, antagonistic (on the basis of FBC index as described in Methods). Hatched area, visible growth.
value is 6 pprn at 5 min and decreased to 0.048 pprn after 3 h of contact time; E. coli water strain). Formaldehyde and H P were less bactericidal and more time dependent, showing a diminution of MBC as the reaction time increased to 5 h (Figs. 3 and 4): from 6240 and 3125 pprn (5 min) to 390 and 195 pprn (5 h), respectively (E. coli water strain). Three culture collection strains were tested by the checkerboard method to determine the type of interaction between PAA and HP. Differences in species responses to the checkerboard technique were nonsignificant. Thus a high degree of synergy was obtained for P. aeruginosa (Fig. 5), with 11 synergistic combinations (FBC index from 5 to 0.31). Echerichia coli (Fig. 6) showed 5 synergistic combinations (FBC index from 1 to 0.56) and S. aureus showed 4 (FBC index from 1 to 0.56; Fig. 7). A combination of PAA and H P selected on the basis of the MBC and the degree of synergy was tested in terms of time. The bactericidal activity presented an intermediate time dependence, with stabilization after 4 h of contact (Tables 1 and 2). In combination, the MBC of PAA can be reduced from 12 to 3 pprn with adjunction of 390 pprn of HP, and the MBC of H P can be reduced from 3125 to 390 pprn with adjunction of 3 pprn of PAA (E. coli water strain). The FBC index was calculated for each reaction time as described above to detect any change during the 7 h of bactericidal action (Figs. 8 and 9). Generally, synergy was maintained and the FBC index remained below 1, making the combination synergistic even in terms of time without any notable difference between species. Investigations on membranes showed that the bactericidal activities of PAA, alone and in combination with HP, were maintained. The concentration of 3.047 pprn PAA allows a reduction of S. aureus and P. aeruginosa by 5 log bacteria in 30 min. Total reduction for all three species was obtained after 1 h of contact time (Fig. 10).
FIG. 6. Checkerboard determination of interactions for E. coli ATCC 8739. S, synergistic; A, additive; I, indifferent; T, antagonistic (on the basis of FBC index as described in Methods). Hatched area, visible growth.
FIG. 7. Checkerboard determination of interactions for S. aureus ATCC 6538 P . S, synergistic; A, additive, I, indifferent, T, antagonistic (on the basis of FBC index as described in Methods). Hatched area, visible growth.
The combination of PAA and H P (0.75 pprn PAA + 97.65 pprn PH) gave similar results with bactericidal efficacy after 2 h (Fig. 11) and inhibition of bacterial adhesion (Table 3). Chlorine at 0.048 pprn reduced the number of bacteria by only 1 log (5 h contact time; Fig. 12); its effect on adhesion was not significant. However, a bactericidal effect was observed from 3 h of reaction time (Fig. 12), and total
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E. coli
-
+
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a
5min
30 min
Ih
2h
FIG. 8. FBC index of the association peracetic acid water strains.
3h
+
4h
5h
6h
S. aureus P.aeruginosa Synergistic Additive Indifferent
7h
hydrogen peroxide (0.03% PAA and 3.91% HP) in terms of time for various
E. coli
a
S. aureus
P.aeruginosa
+ Synergistic ,
FIG. 9. FBC index of the association peracetic acid collection strains.
+
*
Additive Indifferent
hydrogen peroxide (0.03% PAA and 3.91% HP) in terms of time for culture
inhibition of adhesion was also seen with 0.763 ppm chlorine.
Discussion The purpose of the present experiments, in which MBC was calculated in terms of time and in which the interaction between two disinfectants was tested, was to determine adequate times of application for optimal membrane disinfection.
No significant variability was observed in the bactericidal activity of biocides against the different species. However, slight MBC differences were observed and, in general, culture collection strains appeared to be more sensitive. PAA and chlorine present the highest bactericidal activities, as reported in the literature (Baldry 1983; Curver 1990; Pratt et al. 1988). PAA is not time dependent, making it a fast disinfectant, whereas the MBC of chlorine can be reduced at least fourfold after a contact time of 3 h with respect to
64 1
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* S. aureus
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4
minmin
minmin Time FIG. 10. Disinfection efficacy of hollow fibers as log viable bacteria per millilitre of storage suspension after treatment with 3.047 pprn of peracetic acid. T. bacteria, total bacteria.
T. bacteria
Time
FIG. 1 1 . Disinfection efficacy of hollow fibers as log of viable bacteria per millilitreof storage suspension after treatment with 0.75 pprn peracetic acid + 97.65 pprn hydrogen peroxide. T. bacteria, total bacteria.
TABLE3. Effect on bacterial adhesion to hollow fibers Time 5 1 2 3 4
h day days days days
Chlorine (0.048 ppm)
Chlorine (0.763 ppm)
PAA (3.047 ppm)
1.3 1.95 2.44 2.50 2.48
-
-
-
-
PAA and HP (0.75 + 97.65 ppm)
Control
-
NOTE:The preventive inhibition of adhesion is expressed as the log o f the number of viable bacteria per centimetre of hollow fiber after treatments. Aerobic plate counts were made after shaking membranes as described in Methods.
5 min exposure. H P and formaldehyde show weaker bactericidal activities. These two biocides are time dependent, and their MBC can be highly decreased after 5 h of contact time. The same situation has been observed in biological safety cabinets, where sterilization with formaldehyde is insufficient below 4 h of contact time (Fink et al. 1988). A direct link between disinfection rapidity and formaldehyde concentration was also cited by Druilles (1984) and Lejeune et al. (1986). The bactericidal activity of H P occurs as a result of multiple cellular injuries, which can to a certain extent be repaired (Campbell and Dimmick 1966). In .the present work, the experimental conditions allow the survival of a few cells after short exposures to HP; longer contact times are necessary for total elimination. The checkerboard method shows that combination of PAA and H P is synergistic and that the degree of synergy varies little between the species. This synergism may be used to reduce the levels of active substances. The combination of PAA and H P chosen from the MBC and FBC indices shows that synergism is maintained over time, in keeping with the values defined for weak or moderate synergy, usually within the FBC range 0.25-0.75 (Kerry et al. 1975; Neu 1977; Salem et al. 1975). This is probably due to a complementary bactericidal effect and also to a reaction of H P
with residual acetic acid present in the PAA formulation to produce additional PAA, which is more effective. The investigations were made on hollow fibers in the same conditions as in industrial disinfection. The concentrations tested were those determined from the minimal bactericidal time (the lowest contact time from which MBC cannot be reduced) of chemicals providing interesting activities in vitro: PAA, alone and with adjunction of hydrogen peroxide, and chlorine. The bactericidal efficacy of peroxygen compounds was confirmed with the membrane experiments. In general, more time dependence was observed and P. aeruginosa was more sensitive than in in vitro tests. A total reduction of bacteria by 5 log and inhibition of adhesion were obtained. The absence of adhesion was due to the rapid killing of bacteria before the production of extracellular polymeric compounds, glycocalyx formation, and specific adhesion, which could make them more resistant to biocides (Baldry 1983; Exner et al. 1987; Marshall et al. 1971). The concentration of 0.048 pprn chlorine is ineffective, as chlorine is known for its susceptibility to organic and inorganic matter (Curver 1990). In the present case the consumption of chlorine by oxidation of organic matters contained in filter membranes can induce a loss of it's bacteri-
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4
* 0
S. aureus, C1
T. bacteria, C1 T. bacteria, C2
0 5 30 l h 2 h 3 h 4 h 5 h I d 2 d 3 d 4 d min min Time '
FIG. 12. Disinfection efficacy of hollow fibers as log of viable bacteria per rnillilitre of storage suspension after treatment by 0.763 (Cl) or 0.048 (C2) ppm of chlorine. P. aerug., Pseudomonas aeruginosa; T. bacteria, total bacteria.
cidal activity. Increasing its concentration by a factor of 15 gives successful results. Currently, the application of these biocides to hollow fiber sterilization is limited because high concentrations could induce changes in the physical properties of the membranes. However, because disinfection efficiency is relative to contact time, determination of the lowest MBC values using the minimal bactericidal time is fundamental for the application of these biocides in disinfection of membranes. Synergism allows a diminution in biocide concentrations and will be of a great use in this field. Therefore the present study provides a good approach to determining the optimal conditions of disinfectant compounds in terms of contact time and possible interesting combinations. Acknowledgments This work was supported by the Lyonnaise des Eaux, Laboratoire membranes. We are grateful to Ms. Sandrine Sarrat and Aline Lafonta for their help. Amarouch, H. 1984. Micromethode semi-automatiske et informatisee pour la determination de la CMI, de la CMB, du TMB, du type d'interaction entre 2 et 3 antimicrobiens et de la cytotoxicitk des antibactkriens. Thkse de 3' cycle, Institut National polytechnique de Toulouse, France. pp. 35-46. Baldry, M.G.C. 1983. The bactericidal, fungicidal and sporocidal properties of hydrogen peroxide and peracetic acid. J . Appl. Bacteriol. 54: 4 17-423. Beales, A.S., and Sutherland, R. 1983. Antibiotic: assessment of antimicrobial activity and resistance. Edited by A. Denver-Russel, A., and L.B. Quesnel. Measurement of combined antibiotic action. Soc. Appl. Bacteriol. Tech. Ser. 18: 299-315.
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Berenbaum, M.C. 1978. A method for testing for synergy with any number of agents. J. Infect. Dis. 137: 123-127. Berenbaum, M.C., Yu, L.V., and Terrance, P . 1983. Synergy with double and triple antibiotic combinations compared. J . Antimicrob. Chemother. 12: 555-563. Byrne, W., and Roesner, M. 1990. Membrane regeneration program t o restore performance of severely fouled RO membranes. Membrane & Separation Technology News. National Meeting of the North American Membrane Society, May 23-31. Campbell, J.E., and Dimmick, R.L. 1966. Effect of 3% hydrogen peroxide on the viability of Serratia marcescens. J. Bacteriol. 91: 925-929. Curver, J.E. 1990. Water disinfection-a comparison of chlorine, ozone, UV light, and membrane filtration. Proceedings of the 1990, membrane conference and high technology, separation symposium, Boston, Mass. pp. 5 19-528. Doll, J.P., Trexler, P.C., Reynolds, L. I., and Bernard, G.R. 1963. The use of peracetic acid t o obtain germfree invertebrate eggs for gnotobiotic studies. Am. Midl. Nat. 69: 231-239. Druilles, J. 1984. Mkthode de contrale d'efficacitk. Application a diffkrents prockdks (form01 et aerosol). These de 3' cycle Pharmacie, Universitk de Montpelier I, France. Exner, M., Tuschewitzki, G. J., and Scharnagel, J. 1987. Influence on biofilm by chemical disinfectants and mechanical cleaning. Zentralbl. Bakteriol. Parasitenkd. Infektionskr. Hyg. Abt. 1 . Orig. Reike B, 183: 549-563. Fink, R., Liberman, D.F., et al. 1988. Biological safety cabinets, decontamination or sterilization with paraformaldehyde. Am. Ind. Hyg. Assoc. J. 49: 277-279. Greenspan, F.R., Johnson, M.A., and Trexler, P.C. 1955. Peracetic acid aerosols. Proceedings of the 42nd Annual Meeting of the Chemical Specialities Manufacturers' Association. pp. 59-63. Harakeh, M.S. 1984. Inactivation of enterovirus, rotavirus and bacteriophages by peracetic acid in a municipal sewage effluent. FEMS Microbiol. Lett. 23: 27-30. Kerry, D. W., Hamilton-Miller, J .M.T., and Brumfitt, W. 1975. Trimethoprim and rifampicin: in vitro activities separately and in combination. J. Antimicrob. Chemother. 1: 417-427. Kline, L.B., and Hull, R.N. 1960. The virucidal properties of peracetic acid. Am. J. Clin. Pathol. 33: 30-33. Lejeune, B., Lesaout, J., and Lebras, M.P. 1986. ~ t u d ede l'efficacitk de la dksinfection dite terminale. Mkdecine et Maladies Infectieuses, pp. 291-295. Marshall, K.C., Stout, R., and Mitchell, R. 1971. Mechanism of the initial events in the sorption of marine bacteria t o surfaces. J. Gen. Microbiol. 68: 337-387. Neu, H.C. 1977. Micellinam-an amidino penicillin which acts synergistically with other P-lactam compounds. J. Antimicrob. Chemother. 3: 43-52. Phillips, C.R. 1973. Lectures on sterilization. Gaseous sterilization. Edited by J.H. Brewer and N.C. Durham. Duke University Press, Durham, N.C. pp. 35-50. Pratt, J.R., Bowers, N.J., Niederlehner, B.R., and Cairns, J. 1988. Effect of chlorine on microbial communities in naturally derived microcosms. Environ. Toxicol. Chem. 7: 679-687. Salem, A.R., Jackson, D.D., and McFadzean, J.A. 1975. An investigation of interactions between metronidazole ('Flagyl') and other antibacterial agents. J . Antimicrob. Chemother. 1: 378-391. Vesley, D., and Lauer, J. 1986. Decontamination, sterilization, disinfection, and antisepsis in the microbiology laboratory. Edited by B.M. Miller. American Society for Microbiology, Washington, D.C. pp. 194-196.
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CORINNECABASSUDAND PHILIPPE APTEL Lyonnaise des Eaux, Laboratoire membranes, 20, avenue Didier Daurat, 31432 Toulouse, France Received August 13, 1991 Revision received December 13, 1991 Accepted January 27, 1992 ALASRI,A., ROQUES,C., MICHEL,G., CABASSUD,C., and APTEL,P. 1992. Bactericidal properties of peracetic acid and hydrogen peroxide, alone and in combination, and chlorine and formaldehyde against bacterial water strains. Can. J. Microbiol. 38: 635-642. The bactericidal properties of peracetic acid, hydrogen peroxide, chlorine, and formaldehyde were compared in vitro using a rapid micromethod. A combination of peracetic acid and hydrogen peroxide was also tested to assess interactions. The activities of these agents, which are widely used as disinfectants, were evaluated against water isolates and culture collection strains. Peracetic acid and chlorine exhibited an excellent antimicrobial activity, with a relatively rapid destruction of 10' bacteria/mL. The time-dependent bactericidal activities of hydrogen peroxide and formaldehyde were the lowest. The combination of peracetic acid and hydrogen peroxide, tested by a checkerboard micromethod, was found to be synergistic. The minimal bactericidal concentration was established in terms of time for a given mixture of peracetic acid and hydrogen peroxide. Determination of bactericidal concentrations showed that synergy was maintained with increasing contact time. Concentrations for minimal times of treatment by chemicals that provided interesting activities in vitro were tested for disinfection of ultrafiltration membranes. The bactericidal activities of peroxygen compounds were confirmed and synergism was maintained in working conditions. Chlorine showed a loss of efficacy when used on membranes. Key words: peracetic acid, hydrogen peroxide, chlorine, formaldehyde, minimal bactericidal concentration, ultrafiltration membranes. ALASRI,A., ROQUES,C., MICHEL,G., CABASSUD,C., et APTEL,P. 1992. Bactericidal properties of peracetic acid and hydrogen peroxide, alone and in combination, and chlorine and formaldehyde against bacterial water strains. Can. J . Microbiol. 38 : 635-642. Les proprietes bactericides de l'acide peracetique, du peroxyde d'hydrogene, du chlore et du formaldehyde ont ete comparees in vitro selon une micromethode rapide. Les interactions pouvant resulter de l'association de l'acide peracetique et du peroxyde d'hydrogene ont aussi ete evaluees. L'activite de ces composes largement utilises comme desinfectants a ete mesuree sur des souches isolees de l'eau et sur des souches de collection. L'acide peracetique et le chlore possedent une excellente activite antimicrobienne qui se traduit par une destruction relativement rapide de 10' bacteries/mL. En considerant le facteur temps, ce sont le peroxyde et la formaldehyde qui affichent les plus faibles activites bacteriennes. Selon les resultats obtenus par la methode de l'echiquier, l'association de l'acide peracetique et du peroxyde d'hydrogene est synergique. La concentration minimale bactericide a Cte etablie en fonction du temps pour un melange precis de peroxyde et d'acide peracetique. La mesure des concentrations bactericides a confirme que l'activite bactericide se maintenait avec l'augmentation du temps de contact. La concentration de ces agents chimiques qui en un temps minimum de contact presentent des activites interessantes in vitro a ete verifiee pour la desinfection des membranes d'ultrafiltration. L'activite bactericide des composes peroxygenes a ete confirmee et dans les conditions experimentales suivies, la synergie a ete maintenue. Le chlore perd de l'efficacite lorsqu'il est utilise sur des membranes. Mots cle's : acide peracetique, peroxyde d'hydrogene, chlore, concentration bactericide minimale, membranes d'ultrafiltration. [Traduit par la redaction]
Introduction Ultrafiltration membranes have begun to be widely used in today's water-treatment systems. Because of their adhesion and proliferation, bacteria are considered as a particular problem. Therefore sanitizing membranes by efficient disinfection is necessary for an optimal shelf life. Bacteria were isolated from stored membranes, and experiments were carried out to try to prevent microbial growth. The disinfection efficiency is strongly influenced by the biocide composition, it's concentration, the biocide-bacteria reaction time, temperature, and pH. Generally chemical disinfection is a compromise between the benefits of destroying microorganisms and the detriments of chemical toxicity and Printed in Canada / Imprime au Canada
undesirable by-product formation (Curver 1990). Modification of reaction time can reduce biocide concentrations to below the recommended limits. The aim of the present study was to determine the bactericidal activity of four biocides in terms of reaction time and to determine whether they could be used for membrane disinfection. The antimicrobial properties of peroxygen compounds have been recognized for many years, and a variety of applications have been developed. Peracetic acid is reported to be a most effective biocide and is used, both in aqueous solution and as an aerosol or vapor, for sterilization in gnotobiotic units (Doll et al. 1963; Greenspan et al. 1955).
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TABLE1. MBC (ppm) of the antimicrobial agents tested for water isolates Combination
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Time E. coli
5 min 30 min 1h 2h 3h 4h 5h 6h 7h
S. aureus
5 min 30 min 1h 2h 3h 4h 5h 6h 7h
P. aeruginosa
5 min 30 min 1h 2h 3h 4h 5h 6h 7h
PAA
PAA
It has also been proposed as an antiviral agent (Harakeh 1984; Kline and Hull 1960) and recently has been introduced as a disinfectant in water-treatment plants. Hydrogen peroxide is used as an antiseptic in dilute solutions (Baldry 1983) and has been advised for membrane regeneration in combination with peracetic acid (Byrne and Roesner 1990). Because of its broad spectrum of activity and relative ease of use, formaldehyde represents a choice agent for numerous decontamination applications and has done so since the 1890s (Fink et a/. 1988; Phillips 1973; Vesley and Lauer 1986). Chlorine is the most widely used water disinfectant. It is generally believed that chlorine inhibits essential cell enzyme systems through oxidation, but the exact mechanism of action is unltnown (Curver 1990).
HP
HP
Chlorine
Formaldehyde
identified by morphological and biochemical characteristics, the catalase test, oxidase activity, and the API system (Biomerieux Laboratories). Culture media Strains were maintained on trypticase soy agar (Biomerieux Laboratories). Minimal bactericidal concentrations (MBCs) were determined using trypticase soy broth (Biomerieux Laboratories). Chemical disactivation medium consisted of 1 egg yolk; 10 mL 5% sodium thiosulfate (Prolabo Laboratories); 5 mL Tween-80 (Aldrich-Chimie); and trypticase soy broth up to 100 mL. For disinfection of membranes deoxycholate lactose, pyocyanosel, and manitol salt agar media, respectively, were used for E. coli, P. aeruginosa, and S. aureus counts (Biomerieux Laboratories). Inoculated agar and microtiter plates were incubated at 37°C for 24 to 48 h for all strains.
Materials
Methods
Antimicrobial agents Disinfectant agents were obtained from the following sources: peracetic acid (PAA, 15% w/w), Interox Chemicals Ltd.; hydrogen peroxide (HP, 30/35%), Produits Chimiques du Ciron; sodium hypochlorite (12.5% active chlorine), S.A. Gede; and formaldehyde (37%), Aldrich-Chimie. Before use, biocides were added to sterile distilled water to obtain appropriate concentrations.
Activities of biocides were evaluated at first on microplates by MBC (5 log reduction in total number of bacteria for a contact time of 5 min). The more interesting biocides were then tested on hollow fibers.
Bacterial strains Strains tested were isolated from water (water samples were provided by the Lyonnaise des Eaux) or came from culture collections (Escherichia coli ATCC 8739; Pseudomonas aeruginosa ATCC 9027; and Staphylococcus aureus ATCC 6538 P). All strains were
Verification of disactivating medium Twofold serial dilution of biocides in distilled water (100 pL/well) were placed on a Nunclon (8 x 12) microtiter plate (96 cells). Disactivating medium (100 pL) was added to each well. After 10 min of disactivation, 10 pL of bacterial suspension was added to each well. After 5 min of contact, 10 pL from each well was transferred to a second microplate, which contained broth medium and which then was incubated. Three disactivation media
ALASRI ET
AL.
TABLE2. MBC (ppm) of the antimicrobial agents tested for culture collection strains Combination
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Time
E. coli
5 min 30 min 1h 2h 3h 4h 5h 6h 7h
S. aureus
5 min 30 min 1h 2h 3h 4h 5h 6h 7h
P. aeruginosa
5 min 30 min 1h 2h 3h 4h 5h 6h 7h
PAA
PAA
were tested. The disactivating medium described in the section Culture media gave the best bacterial growth for all the tested concentrations.
Determination of MBC with time, using a dilution-disactivation micromethod Twofold serial dilutions of biocides in distilled water (100 pL/well) were placed on a Nunclon (8 x 12) microtiter plate (96 wells). Bacterial suspension prepared in 0.9% NaCl solution (10 pL adjusted to lo9 organisms/ml) was added to each well. After different contact times (5 and 30 min and 1, 2, 3, 4, 5, 6, and 7 h), 10 pL from each well was transferred to a second microplate, containing 100 pL of disactivating medium per well. After 10 min of disactivation, 10 pL from each well was transferred to a third microplate with broth medium (100 pL/well), which was then incubated, and growth was observed by turbidity. Each MBC was determined four times for each strain. For each strain tested, the adequacy of growth conditions and the sterility of the medium were checked in columns 11 and 12 (control wells). Evaluation of interactions between PAA and HP Association tests were performed according to the checkerboard pattern (Amarouch 1984; Berenbaum et al. 1983; Beales and Sutherland 1983). Twofold serial dilutions of one biocide were tested in combination with concentrations of twofold dilutions of the other. The same method used for measuring the bactericidal effect, described above, was used for a contact time of 5 min. Each strain was tested at least twice. The fractional bactericidal concentration (FBC) index was determined as follows. The concentrations of PAA and H P that produced a bactericidal effect when used alone were called PAAa and HPa. The concentrations that produced the same effect when used in combination were called PAAc and HPc.
HP
HP
Chlorine
Formaldehyde
The expression reported by Berenbaum (1978), "PAAc/PAAa + HPc/HPa," gives the degree of interaction or FBC index. The following values are generally considered to define the degree of synergy: FBC I0.75, combination is synergistic; 0.75 2 FBC 5 1.1, additive; 1.1 > FBC 5 2, indifferent; FBC > 2. antagonistic.
Determination of MBC with time, using a combination of PAA and HP A combination of PAA and H P in a particular ratio (0.03% PAA and 3.91% HP), chosen on the basis of the MBC (biocides alone) and interaction degree, was tested to determine the FBC index in terms of contact time (5 and 30 min and 1, 2, 3, 4, 5, 6, and 7 h). Disinfection of hollow fibers The bactericidal efficacy of the chemicals studied for membrane disinfection was tested against water isolates. Bactericidal activities For their storage, hollow fiber filters are filled with ultrafiltrated water (0.01 pm) with adjunction of a disinfectant. Our bactericidal efficacy experiments were carried out on microprototypes (length, 25 cm; diameter, 1.5 cm) with 15 hollow fibers each (length, 24 cm). The microprototypes (MPs) were inoculated with a mixture of bactericidal suspensions of E. coli, P. aeruginosa, and S. aureus to obtain 10' organisms/mL of each species. After 1 min of shaking (Vortex 2, Shake 5, Scientific Industries) and 1 h of incubation (20°C), disinfectants were added to the hollow fibers. After each contact time (5 and 30 min 1, 2, 3, 4, and 5 h, and 1, 2,3, and 4 days) 0.5 mL of each M P test suspension was suspended in 4.5 mL disactivating medium. After 10 min, the suspension was diluted, and aerobic plate counts were made on specific media after 2 to 4 days incubation.
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-(3.
E. coli
V
5 30 l h 2 h 3 h 4 h 5 h 6 h 7 h min rnin Time
2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 ' 1 1 5 30 l h 2 h 3 h 4 h 5 h 6 h 7 h min rnin Time
FIG. 3. MBC of hydrogen peroxide against time for water strains.
FIG. 1. MBC of peracetic acid against time for water strains.
* *
E coli S. aureus -+ P. aeruginosa
A
5 30 min min
lh
2h
3h
4h
.
h
5h
.
-
b
6h
.
4
7h
Tune
FIG. 2. MBC of chlorine against time for water strains.
Effect on bacterial adhesion to membranes The bacterial adhesion to membranes was observed every day after M P disinfection (4 days). Samples of hollow fibers (4 cm) taken from each test and cut (longitudinally and transversely) into four portions were dipped in 5 mL PBS (phosphate-buffered saline, Gibco Laboratories) and then shaken ultrasonically (1 min at 120 W, Bioblock Scientific). Agar plate counts were then determined. Experiments were performed at least twice for each biocide.
Results The in vitro activities of biocides tested on water and culture collection strains in terms of time (MBC in parts per million) are given in Tables 1 and 2.
V
5 30 l h 2 h 3 h 4 h 5 h 6 h 7 h rnin rnin Time FIG. 4. MBC of formaldehyde against time for water strains.
and PAA were observed to have a very low MBC, 6 and 12 PPm, respectively, after 5 rnin of reaction time (E. coli water strain). The action of PAA was poorly time dependent (Fig. 1): MBC values rapidly decreased from 12 to 3 ppm in 30 rnin and then remained stable for the 7 h of the test (E. coli water strain). Chlorine presented an intermediate action, requiring 3 h to obtain a straight line of MBC (Fig. 2). At less than 3 h, bactericidal activity was time dependent (MBC
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0 0
195 391
781 1563 3 125 6250 12500
HP ( P P ~ ) FIG. 5. Checkerboard determination of interactions for P. aeruginosa ATCC 9027. S, synergistic; A, additive; I, indifferent; T, antagonistic (on the basis of FBC index as described in Methods). Hatched area, visible growth.
value is 6 pprn at 5 min and decreased to 0.048 pprn after 3 h of contact time; E. coli water strain). Formaldehyde and H P were less bactericidal and more time dependent, showing a diminution of MBC as the reaction time increased to 5 h (Figs. 3 and 4): from 6240 and 3125 pprn (5 min) to 390 and 195 pprn (5 h), respectively (E. coli water strain). Three culture collection strains were tested by the checkerboard method to determine the type of interaction between PAA and HP. Differences in species responses to the checkerboard technique were nonsignificant. Thus a high degree of synergy was obtained for P. aeruginosa (Fig. 5), with 11 synergistic combinations (FBC index from 5 to 0.31). Echerichia coli (Fig. 6) showed 5 synergistic combinations (FBC index from 1 to 0.56) and S. aureus showed 4 (FBC index from 1 to 0.56; Fig. 7). A combination of PAA and H P selected on the basis of the MBC and the degree of synergy was tested in terms of time. The bactericidal activity presented an intermediate time dependence, with stabilization after 4 h of contact (Tables 1 and 2). In combination, the MBC of PAA can be reduced from 12 to 3 pprn with adjunction of 390 pprn of HP, and the MBC of H P can be reduced from 3125 to 390 pprn with adjunction of 3 pprn of PAA (E. coli water strain). The FBC index was calculated for each reaction time as described above to detect any change during the 7 h of bactericidal action (Figs. 8 and 9). Generally, synergy was maintained and the FBC index remained below 1, making the combination synergistic even in terms of time without any notable difference between species. Investigations on membranes showed that the bactericidal activities of PAA, alone and in combination with HP, were maintained. The concentration of 3.047 pprn PAA allows a reduction of S. aureus and P. aeruginosa by 5 log bacteria in 30 min. Total reduction for all three species was obtained after 1 h of contact time (Fig. 10).
FIG. 6. Checkerboard determination of interactions for E. coli ATCC 8739. S, synergistic; A, additive; I, indifferent; T, antagonistic (on the basis of FBC index as described in Methods). Hatched area, visible growth.
FIG. 7. Checkerboard determination of interactions for S. aureus ATCC 6538 P . S, synergistic; A, additive, I, indifferent, T, antagonistic (on the basis of FBC index as described in Methods). Hatched area, visible growth.
The combination of PAA and H P (0.75 pprn PAA + 97.65 pprn PH) gave similar results with bactericidal efficacy after 2 h (Fig. 11) and inhibition of bacterial adhesion (Table 3). Chlorine at 0.048 pprn reduced the number of bacteria by only 1 log (5 h contact time; Fig. 12); its effect on adhesion was not significant. However, a bactericidal effect was observed from 3 h of reaction time (Fig. 12), and total
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E. coli
-
+
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a
5min
30 min
Ih
2h
FIG. 8. FBC index of the association peracetic acid water strains.
3h
+
4h
5h
6h
S. aureus P.aeruginosa Synergistic Additive Indifferent
7h
hydrogen peroxide (0.03% PAA and 3.91% HP) in terms of time for various
E. coli
a
S. aureus
P.aeruginosa
+ Synergistic ,
FIG. 9. FBC index of the association peracetic acid collection strains.
+
*
Additive Indifferent
hydrogen peroxide (0.03% PAA and 3.91% HP) in terms of time for culture
inhibition of adhesion was also seen with 0.763 ppm chlorine.
Discussion The purpose of the present experiments, in which MBC was calculated in terms of time and in which the interaction between two disinfectants was tested, was to determine adequate times of application for optimal membrane disinfection.
No significant variability was observed in the bactericidal activity of biocides against the different species. However, slight MBC differences were observed and, in general, culture collection strains appeared to be more sensitive. PAA and chlorine present the highest bactericidal activities, as reported in the literature (Baldry 1983; Curver 1990; Pratt et al. 1988). PAA is not time dependent, making it a fast disinfectant, whereas the MBC of chlorine can be reduced at least fourfold after a contact time of 3 h with respect to
64 1
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* S. aureus
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4
minmin
minmin Time FIG. 10. Disinfection efficacy of hollow fibers as log viable bacteria per millilitre of storage suspension after treatment with 3.047 pprn of peracetic acid. T. bacteria, total bacteria.
T. bacteria
Time
FIG. 1 1 . Disinfection efficacy of hollow fibers as log of viable bacteria per millilitreof storage suspension after treatment with 0.75 pprn peracetic acid + 97.65 pprn hydrogen peroxide. T. bacteria, total bacteria.
TABLE3. Effect on bacterial adhesion to hollow fibers Time 5 1 2 3 4
h day days days days
Chlorine (0.048 ppm)
Chlorine (0.763 ppm)
PAA (3.047 ppm)
1.3 1.95 2.44 2.50 2.48
-
-
-
-
PAA and HP (0.75 + 97.65 ppm)
Control
-
NOTE:The preventive inhibition of adhesion is expressed as the log o f the number of viable bacteria per centimetre of hollow fiber after treatments. Aerobic plate counts were made after shaking membranes as described in Methods.
5 min exposure. H P and formaldehyde show weaker bactericidal activities. These two biocides are time dependent, and their MBC can be highly decreased after 5 h of contact time. The same situation has been observed in biological safety cabinets, where sterilization with formaldehyde is insufficient below 4 h of contact time (Fink et al. 1988). A direct link between disinfection rapidity and formaldehyde concentration was also cited by Druilles (1984) and Lejeune et al. (1986). The bactericidal activity of H P occurs as a result of multiple cellular injuries, which can to a certain extent be repaired (Campbell and Dimmick 1966). In .the present work, the experimental conditions allow the survival of a few cells after short exposures to HP; longer contact times are necessary for total elimination. The checkerboard method shows that combination of PAA and H P is synergistic and that the degree of synergy varies little between the species. This synergism may be used to reduce the levels of active substances. The combination of PAA and H P chosen from the MBC and FBC indices shows that synergism is maintained over time, in keeping with the values defined for weak or moderate synergy, usually within the FBC range 0.25-0.75 (Kerry et al. 1975; Neu 1977; Salem et al. 1975). This is probably due to a complementary bactericidal effect and also to a reaction of H P
with residual acetic acid present in the PAA formulation to produce additional PAA, which is more effective. The investigations were made on hollow fibers in the same conditions as in industrial disinfection. The concentrations tested were those determined from the minimal bactericidal time (the lowest contact time from which MBC cannot be reduced) of chemicals providing interesting activities in vitro: PAA, alone and with adjunction of hydrogen peroxide, and chlorine. The bactericidal efficacy of peroxygen compounds was confirmed with the membrane experiments. In general, more time dependence was observed and P. aeruginosa was more sensitive than in in vitro tests. A total reduction of bacteria by 5 log and inhibition of adhesion were obtained. The absence of adhesion was due to the rapid killing of bacteria before the production of extracellular polymeric compounds, glycocalyx formation, and specific adhesion, which could make them more resistant to biocides (Baldry 1983; Exner et al. 1987; Marshall et al. 1971). The concentration of 0.048 pprn chlorine is ineffective, as chlorine is known for its susceptibility to organic and inorganic matter (Curver 1990). In the present case the consumption of chlorine by oxidation of organic matters contained in filter membranes can induce a loss of it's bacteri-
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4
* 0
S. aureus, C1
T. bacteria, C1 T. bacteria, C2
0 5 30 l h 2 h 3 h 4 h 5 h I d 2 d 3 d 4 d min min Time '
FIG. 12. Disinfection efficacy of hollow fibers as log of viable bacteria per rnillilitre of storage suspension after treatment by 0.763 (Cl) or 0.048 (C2) ppm of chlorine. P. aerug., Pseudomonas aeruginosa; T. bacteria, total bacteria.
cidal activity. Increasing its concentration by a factor of 15 gives successful results. Currently, the application of these biocides to hollow fiber sterilization is limited because high concentrations could induce changes in the physical properties of the membranes. However, because disinfection efficiency is relative to contact time, determination of the lowest MBC values using the minimal bactericidal time is fundamental for the application of these biocides in disinfection of membranes. Synergism allows a diminution in biocide concentrations and will be of a great use in this field. Therefore the present study provides a good approach to determining the optimal conditions of disinfectant compounds in terms of contact time and possible interesting combinations. Acknowledgments This work was supported by the Lyonnaise des Eaux, Laboratoire membranes. We are grateful to Ms. Sandrine Sarrat and Aline Lafonta for their help. Amarouch, H. 1984. Micromethode semi-automatiske et informatisee pour la determination de la CMI, de la CMB, du TMB, du type d'interaction entre 2 et 3 antimicrobiens et de la cytotoxicitk des antibactkriens. Thkse de 3' cycle, Institut National polytechnique de Toulouse, France. pp. 35-46. Baldry, M.G.C. 1983. The bactericidal, fungicidal and sporocidal properties of hydrogen peroxide and peracetic acid. J . Appl. Bacteriol. 54: 4 17-423. Beales, A.S., and Sutherland, R. 1983. Antibiotic: assessment of antimicrobial activity and resistance. Edited by A. Denver-Russel, A., and L.B. Quesnel. Measurement of combined antibiotic action. Soc. Appl. Bacteriol. Tech. Ser. 18: 299-315.
1992
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