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Efficacy of a quaternary ammonium compound against planktonic and sessile populations of differentLegionella pneumophila strains

Efficacy of a quaternary ammonium compound against planktonic and sessile populations of... Annals of Microbiology, 57 (1) 121-125 (2007) Efficacy of a quaternary ammonium compound against planktonic and sessile populations of different Legionella pneumophila strains Ayten KIMIRAN-ERDEM*, Nazmiye Ozlem SANLI-YURUDU, Aysin COTUK Istanbul University, Faculty of Science, Department of Biology, 34418, Istanbul, Turkey Received 1 November 2006 / Accepted 21 December 2006 TM Abstract - Efficacy of Gemacide PN-50 (a quaternary ammonium compound) as a commercial formulation recommended for disin- fecting heat exchangers was determined for both planktonic and sessile populations of various Legionella pneumophila strains. The qua- ternary ammonium compound (QAC) was preferred as an alternative due to the emerging resistance of potentially pathogenic bacteria against different biocides. Planktonic L. pneumophila strains were suspended in tap water while sessile ones were grown on stainless steel that is used in construction of the cooling towers, then both group of strains were exposed to the biocide. The sensitivity of both planktonic and sessile populations of L. pneumophila strains to the biocide was different. The biocide was found effective below recom- mended dosages (1000-2000 mg/L) against planktonic populations of L. pneumophila, whereas it was determined that higher than the recommended dosages were required for sessile populations. The environmental isolates were more resistant to the biocide than the ATCC isolate was. The results indicated that studying only the planktonic populations of L. pneumophila for biocide tests might not be sufficient to provide the optimum dosage and contact time information for field trials. Therefore, biocidal activity of a water treatment chemical must be evaluated in terms of dosage and contact times on both planktonic and sessile bacteria. TM Key words: Legionella pneumophila, biocide, quaternary ammonium compounds (QACs), Gemacide PN-50 , biocidal activity, biofilm. INTRODUCTION nate legionellae. Therefore, in order to prevent legionellosis outbreaks, contaminated water systems must be disinfected Legionella pneumophila, the causative agent of and the most effective and practical biocide need to be eval- Legionnaires’ disease and Pontiac fever, can be isolated uated (Elsmore, 1986). Various biocides have been exam- from a variety of potential human contact areas, such as ined for their efficacies in both laboratory simulations and heat rejection systems (cooling tower systems, evaporative field treatment of Legionella contaminations on cooling tow- condensers) and potable water sources (Wright et al., 1991; ers and in potable water systems (Skaliy et al., 1980; Grace Yamamoto et al., 1991; Fields et al., 2002). It has been et al., 1981; Elsmore, 1986; Yamamoto et al., 1991; suggested that the primary mode of dispersion of the etio- Elsmore, 1993; Green and Pirrie, 1993; Surman et al., logical agent is an aerosol; inhalation of contaminated 1993; Lin et al., 1998; Viera et al., 1999; Walker et al., aerosols may result in outbreaks (Wright et al., 1991; 1999; Gao et al., 2000; Donlan et al., 2002; Fields et al., Mcnally et al., 2000). In addition to aerosol forms, in man- 2002; Kim et al., 2002; Kool, 2002). made water systems, legionellae, like most other aquatic In this study, we preferred a quaternary ammonium TM bacteria often exist as attached complex consortia (Green compound (Gemacide PN-50 ), which is a commercial for- and Pirrie, 1993; Murga et al., 2001; Petti et al., 2004). mulation, recommended by manufacturer for decontamina- Biofilms and sediments might create significant problems tion of heat exchangers. QACs have been actively deployed such as fouling (Johansen et al., 1997), heat transfer resist- since the 1930s with no apparent reduction in their effec- ance (Wright et al., 1991), filter plugging (Laopaiboon et al., tiveness (McBain, 2004). These are a widely used group of 2002) and metal corrosion (Momba and Binda, 2002), as non-oxidizing biocides for control of microbial growth in a well as increased resistance to antimicrobial compounds variety of applications, such as cooling tower systems, dis- (Green and Pirrie, 1993; Surman et al., 1993; Viera et al., infection and sterilisation, corrosion inhibition, wood preser- 1999; Kool, 2002) in water systems. vation, dispersion agents and in food production (Lutey, Control measures need to be considered for all water 1995; Laopaiboon et al., 2002; Majtàn and Majtànovà, TM systems, because they can harbour, amplify and dissemi- 2003; Korukoglu et al., 2006). Gemacide PN-50 is an alternative compound to oxidizing agents, safe for humans, stable in solution even at elevated temperatures and a wide TM range of pH. Gemacide PN-50 has no corrosive effect on common industrial materials such as stainless steel or alu- * Corresponding author. Phone: +90 212 455 57 00; TM minium (Gemsan, Undated). Gemacide PN-50 does not Fax: +90 212 528 05 27; E-mail: aytenkimiran@yahoo.com, kimiran@istanbul.edu.tr need to get inside the microbial cells like many other antimi- 122 A. Kimiran-Erdem et al. crobial agents do. It is semi-soluble in membranes and it Determination of biocidal activity with qualitative test exhibits its effect by disrupting the membrane integrity. method. TM Although Gemacide PN-50 biocide is already recommend- Planktonic bacteria. In the current study, the qualitative ed for disinfecting heat exchangers, there is a lack of pub- suspension test method by Skaliy et al. (1980) was modi- lished data about the efficacy of this compound against both fied based on ASTM (1991) E 645–91 standard test method. planktonic and sessile populations of legionellae. In various This method was used for determination of the efficacy of countries, alternative biocides have been searched due to microbicides used in cooling systems. the emerging resistance of the microorganisms against anti- The bactericidal activity of different biocide concentra- bacterial compounds. Therefore, in the current study, tions was assessed by adding 5 mL of bacterial suspension inhibitory characteristics of this biocide were investigated into 500 mL water containing different dosages of biocide, against both suspended and sessile bacteria. in a 1-litre glass Erlenmeyer flask. After shaking this mix- th rd th th ture gently, 1 mL aliquots were drawn at 0 , 3 , 6 , 24 th and 168 hours of incubation, and transferred into 9 mL of MATERIALS AND METHODS appropriate neutralizer (0.4 % sodium dodecyl sulphate in Buffered Yeast Extract Broth (BYEB). A 10-mL aliquot of the Test organisms. All tests were performed with three dif- neutralized sample was serially diluted (1:10) with sterile ferent L. pneumophila serogroup 1 strains confirmed by tap water. From each of three suitable dilutions, 0.1 mL was monoclonal antibody tests. C1 and C2 strains were isolated drawn and inoculated on BCYEA. After inoculation, plates from a cooling tower in the vicinity of Istanbul and potable were sealed in polyethylene bags to prevent dehydration water, respectively. A standard strain (SG 1 ATCC 33152) (Surman et al., 1993). After a 7-day incubation at 37 ºC, was obtained from Hertfordshire University Biodeterioration the number of survivors was determined by counting the Center. Since repeated subculturing on artificial media may colonies (CFU/mL) using a Colony Counter Device (åCOLyte affect the resistance of organisms to biocidal effect and may Super Colony Counter, Synbiosis). Plates on which colonies cause mutations, strains were maintained in phosphate did not develop were incubated for an additional 14 days buffer and glycerin suspension and stored at –70 ºC in cry- before discarding. At the time points given above, biocide- otubes. Freeze-dried cultures were not passaged more than bacteria suspensions were incubated at 25 ºC. At each time 3 times, as suggested (Skaliy et al. 1980; Elsmore, 1993). interval, 1 mL aliquots were processed as described above. 5 6 For the experiments, freeze-dried cultures of strains were The controls consisted of 10 -10 cells per mL in 500 mL grown on Buffered Charcoal Yeast Extract Agar (BCYEA, Cl -free sterile tap water containing no biocide. The rest of Oxoid) at 37 ºC in an atmosphere containing 2.5% CO . the experimental conditions were the same as above. Cultures were grown to late log phase, about 5-7 days of incubation, then cells were harvested and a suspension was Sessile bacteria. Bacterial suspensions (1 mL) were added prepared in Cl -free sterile tap water to a concentration of into 9 mL of BYEB in screw-capped bottles and then incu- Mc Farland 1 standard. Sterile tap water was preferred for bated at 37 ºC for 48 h in an atmosphere containing 2.5% dilutions because of the toxicity of sodium chloride against CO . Legionella inocula were transferred to beakers con- legionellae (Weiss and Westfall, 1984). The suspension was taining sterile stainless steel coupons to generate biofilm diluted in 1/10 ratio and used in the experiments. bacteria and the biofilm was allowed to develop on both sides of the coupons for 7 days at 37 ºC with slow stirring TM Biocide. Different dosages of Gemacide PN–50 , were (200 rpm, F 1.9 cm). After the incubation period, at 0, 3, 6 prepared in sterile demineralised water. Concentrations of and 24 h contact times, 6 coupons were aseptically 10000, 5000, 2000, 1000, 100, 10, 1 mg/L were used for removed, 3 of them were tested for biocidal activity while suspended bacteria and of 10000, 5000, 2000 and 1000 the other 3 were used as controls to determine the number mg/L were used for attached bacteria. Dosages for sus- of bacteria. After the incubation with biocide, test coupons pended bacteria were determined by MIC (minimum were transferred aseptically to 10 mL sterile neutralizer and inhibitory concentration) tests. were vortexed to detach bacteria. To determine the number of cells in biofilms, the content of tubes was diluted in 1/100 Toxicity test for neutralizer. Recommended neutralizer ratio and 0.1 mL of this dilution was cultivated in triplicate TM for Gemacide PN-50 by the manufacturer is a combination on BCYEA. The Petri plates were incubated for 7 days at 37 of Tween 80 (3%) and lecithin (0.3%). Instead of this com- ºC and following the incubation, colonies were counted. bination, sodium dodecyl sulphate (0.4%), which was Plates on which colonies did not develop were incubated for shown to have no toxicity effect on the organisms, was used an additional 14 days before discarding (Skaliy et al., 1980). as the quenching agent (Skaliy et al., 1980; Elsmore, 1993; Control coupons were transferred aseptically to tubes CEN, 1997; European Pharmacopeia, 2001; Johnston et al., containing 10 mL Cl -free sterile tap water and the tubes 2002). were vortexed to detach attached bacteria. The content of the tubes were diluted in 1/100 ratio and 0.1 mL of this Preparation of coupons. Stainless steel coupons (1 cm , solution was cultivated in BCYEA. 0.8 mm in thickness) were cleaned using a neutral deter- At the end of the qualitative suspension and biofilm gent (Triton) and then rinsed with water before autoclaving tests, the effect of biocide against C1 and C2 strains as well (120 °C, 20 min). After sterilisation and testing for the pres- as the standard strain were evaluated in respect to time and ence of any residual detergent, the coupons were placed biocide concentration. onto bacterial lawns on BCYEA plates using sterile forceps Statistical Analysis. Results were analysed statistically by and the plates were incubated at 37 ºC for 7 days. Plates were then examined for the presence of growth inhibition student’s t-test. Differences were considered significant when P <0.05. All the experiments described in this study zones around the coupons (Johansen et al., 1997). Ann. Microbiol., 57 (1), 121-125 (2007) 123 were done in triplicates. meric quaternary ammonium was found to be ineffective by Thomas et al. (1999) against Legionella bacteria. McCall et RESULTS AND DISCUSSION al. (1999) reported that poly[oxyethylene (dimethyliminio) ethylene-(dimethyliminio) ethylene dichloride] was not In the current study, inhibitory characteristics of Gemacide effective against Legionella bacteria (both planktonic and TM PN-50 , a QAC, were investigated against both suspended biofilm) when a single dosage of ~10 mg/L (maximum and biofilm bacteria. dosage recommended by a manufacturer) was applied in a It was found that the biocide of 10000, 5000, 2000, model plumbing system. 1000 or 100 mg/L concentrations killed all strains of bacte- It was observed in previous reports that QACs were also rial suspensions immediately (Table 1). At 10 mg/L concen- used in combination with different compounds such as iso- tration of biocide achieved > 5 log reduction for standard propanol (Skaliy et al., 1980) or organo-tin compounds strain and strain C1 in 168-hours contact time. Gemacide (Grace et al., 1981). Skaliy et al. (1980) showed that the TM PN-50 at 1 mg/L had no lethal effect on C1 and C2 strains mixture of didecyl dimethyl ammonium chloride and iso- in 168-hours contact time, while it killed the standard strain propanol at 72 mg/l and 144 mg/l respectively, achieved > in 3-hours contact time. The differences between the 5 log decrease after 3-hour contact time. Yet, it is not clear TM Gemacide PN-50 treated (10 mg/L, 168 h) and untreated whether the ammonium or the tin or isopropanol compound samples were statistically significant (P < 0.05) for C1 is the primary active ingredient. In the current study, it was strain. found that when the QAC was used alone, the effect Manufacturer’s recommended dosages for Gemacide obtained was not significantly different then that reported TM PN-50 was 0.1% (1000 mg/L) or 0.2% (2000 mg/L), by Skaliy et al. (1980). however we found that 100 mg/L dosage for all strains and Elsmore (1993) has stated that there is no standard 10 mg/L for standard strain achieved > 5 log reduction at method for the evaluation of biocides against legionellae, zero time. There are a few studies about the individual but some criteria must be noted; the contact time of the usage of QACs (Elsmore, 1986; McCall et al., 1999; Thomas biocide, the amount of bacteria in the initial inocula, and the et al., 1999). Elsmore (1986) determined that 800 mg/L validity and expression of the results. Elsmore (1993) has cationic poliquaternary ammonium compound was the min- also stated that a desirable water treatment biocide must imum inhibiting concentration for L. pneumophila. A poly- provide a 4-log reduction in bacteria, following a minimum -1 TM TABLE 1 - Survival of strains of Legionella pneumophila (log CFU mL ) exposed to Gemacide PN-50 at different contact times and concentrations Planktonic strains Gemacide Survivors (Log of no. ± S.E.) (mg/L) 0 h 3 h 6 h 24 h 168 h C1 Control 5.6 ± 0.03 5.5 ± 0.04 5.5 ± 0.02 5.7 ± 0.03 5.5 ± 0.01 10000 0.0 0.0 0.0 0.0 0.0 5000 0.0 0.0 0.0 0.0 0.0 2000 0.0 0.0 0.0 0.0 0.0 1000 0.0 0.0 0.0 0.0 0.0 100 0.0 0.0 0.0 0.0 0.0 10 3.7 ± 0.01 2.5 ± 0.03 2.5 ± 0.06 1 0.0 1 5.2 ± 0.03 4.7 ± 0.05 5.0 ± 0.04 4.8 ± 0.04 4.2 ± 0.02 C2 Control 5.8 ± 0.01 5.9 ± 0.03 5.8 5.8 ± 0.01 5.8 ± 0.01 10000 0.0 0.0 0.0 0.0 0.0 5000 0.0 0.0 0.0 0.0 0.0 2000 0.0 0.0 0.0 0.0 0.0 1000 0.0 0.0 0.0 0.0 0.0 100 0.0 0.0 0.0 0.0 0.0 10 5.6 ± 0.09 6.4 ± 0.01 5.0 ± 0.02 5.9 ± 0.02 5.9 ± 0.02 1 6.0 ± 0.03 6.4 ± 0.01 5.9 ± 0.04 5.5 ± 0.02 5.9 ± 0.04 Standard strain Control 5.7 ± 0.05 5.7 ± 0.01 5.7 ± 0.02 5.7 ± 0.03 5.7 ± 0.01 10000 0.0 0.0 0.0 0.0 0.0 5000 0.0 0.0 0.0 0.0 0.0 2000 0.0 0.0 0.0 0.0 0.0 1000 0.0 0.0 0.0 0.0 0.0 100 0.0 0.0 0.0 0.0 0.0 10 0.0 0.0 0.0 0.0 0.0 1 3.0 0.0 0.0 0.0 0.0 S.E.: Standard Error. 124 A. Kimiran-Erdem et al. -2 TM TABLE 2 - Survival of strains of Legionella pneumophila (log CFU cm ) exposed to Gemacide PN-50 at different contact times and concentrations Sessile strains Gemacide (mg/L) Survivors (Log of no. ± S.E.) 0 h 3 h 6 h 24 h 168 h C1 Control 5.7 ± 0.03 5.0 ± 0.02 5.4 ± 0.03 5.0 ± 0.05 5.5 ± 0.03 10000 4.5 ± 0.01 4.1 ± 0.01 3.6 ± 0.01 2.2 ± 0.02 0.0 5000 5.3 ± 0.04 4.8 ± 0.01 4.3 ± 0.02 3.3 ± 0.02 1.9 ± 0.01 2000 5.2 ± 0.01 4.9 ± 0.01 4.7 ± 0.01 3.6 ± 0.02 2.3 ± 0.02 1000 5.2 ± 0.01 5.0 ± 0.01 5.3 ± 0.01 4.7 ± 0.01 3.9 ± 0.01 C2 Control 4.7 ± 0.03 4.5 ± 0.05 4.9 ± 0.02 4.7 ± 0.01 4.6 ± 0.02 10000 3.5 ± 0.01 3.2 ± 0.04 3.0 ± 0.2 1.6 ± 0.02 0.0 5000 4.4 ± 0.01 3.8 ± 0.04 3.3 ± 0.07 2.5 ± 0.03 1.2 ± 0.01 2000 4.5 ± 0.01 4.0 ± 0.02 3.8 ± 0.02 2.8 ± 0.02 1.5 ± 0.01 1000 4.5 ± 0.01 4.3 ± 0.01 4.2 ± 0.01 3.8 ± 0.02 2.8 ± 0.02 Standard strain Control 4.9 ± 0.02 4.4 ± 0.01 4.7 ± 0.02 5.2 ± 0.02 5.0 ± 0.03 10000 3.5 ± 0.01 3.0 ± 0.02 2.4 ± 0.01 1.1 ± 0.01 0.0 5000 3.5 ± 0.03 3.2 ± 0.02 2.9 ± 0.03 2.4 ± 0.01 0.9 ± 0.01 2000 4.4 ± 0.01 3.9 ± 0.01 3.8 ± 0.01 2.3 ± 0.01 1.2 ± 0.02 1000 4.6 ± 0.01 4.3 ± 0.01 4.1 ± 0.01 2.6 ± 0.01 1.9 ± 0.02 S.E.: Standard Error. mental conditions. In microorganisms, development of of 24-hour contact time with non-oxidizing agents. Our result is coherent with Elsmore’s criteria and shows that reduced susceptibility to antimicrobial agents increases the tested biocide at 100 mg/L can be used for Legionella probability of further disinfection failures, leading severe decontamination. On the other hand, this dosage was problems in various industrial areas, food preparation, and unable to kill all biofilm bacteria and higher dosages (above human health areas (Chapman, 2003). Novel biocides were the recommended amount) of biocide were prepared to kill quested due to the emerging resistance of microorganisms sessile populations of Legionella strains (Table 2). With against antibacterial compounds. biofilms, after 168-hours contact time, 5000 mg/L achieved In respect to contact time and log reduction, Gemacide a > 3 log for all strains, while 10000 mg/L achieved ≥ 5 log TM PN-50 was found to be effective below the recommended for strain C1 and standard strain, >4 log for strain C2. Our dosages against planktonic legionellae according to results are in harmony with other investigator’s results in relation to biofilm resistance (Green and Pirrie, 1993; Elsmore’s criteria. The biocide (10000 mg/L) also achieved Surman et al., 1993; Murga et al., 2001; Donlan et al., significant log decreases for biofilm bacteria (> 4 log) for all 2002; Kool, 2002). strains in 168-hour contact time. Due to the assessment of The sensitivity of the strains against QAC was in the TM the stability of Gemacide PN-50 in long contact time (168- order of: standard strain > C1 > C2, both in suspension and hour) and the fact that it does not allow microorganismal biofilm states. This result is also consistent with those of Fux resistance it could be recommended as an alternative bio- et al. (2005), who reported that in the course of sequential cide for decontamination of legionellae from various man- in vitro passage, laboratory reference strains might differ- made water systems. entiate significantly from non-passaged samples. Although Prior to their commercial application in decontamination we did not passage the strains more than 3 times, we cer- of water systems, different biocides need to be identified tainly did not know the number of times the standard strain was passaged. These differences might be due to the fol- and investigated for their efficacy in various water systems. TM lowing: i) C1 and C2 environmental strains might be The current study proved that Gemacide PN-50 is effec- exposed antibacterial compounds and thus, may exhibit tive against both suspensions and biofilms of legionellae. improved resistance; ii) biocidal activity varies significantly The results indicated that studying the biocides against only among different types of microorganisms, moreover, it the planktonic populations of L. pneumophila might not be might also differ among different strains of the same species sufficient to provide the optimum dosage and contact time (Maillard, 2002), and since the L. pneumophila serogroup 1 information for field trials. strains used in our study were isolated from different sources, they might belong to different subtypes of L. pneu- Acknowledgements. This work was supported by the mophila serogroup 1. 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Efficacy of a quaternary ammonium compound against planktonic and sessile populations of differentLegionella pneumophila strains

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Springer Journals
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Copyright © 2007 by University of Milan and Springer
Subject
Life Sciences; Microbiology; Microbial Genetics and Genomics; Microbial Ecology; Fungus Genetics; Medical Microbiology; Applied Microbiology
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1590-4261
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1869-2044
DOI
10.1007/BF03175060
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Abstract

Annals of Microbiology, 57 (1) 121-125 (2007) Efficacy of a quaternary ammonium compound against planktonic and sessile populations of different Legionella pneumophila strains Ayten KIMIRAN-ERDEM*, Nazmiye Ozlem SANLI-YURUDU, Aysin COTUK Istanbul University, Faculty of Science, Department of Biology, 34418, Istanbul, Turkey Received 1 November 2006 / Accepted 21 December 2006 TM Abstract - Efficacy of Gemacide PN-50 (a quaternary ammonium compound) as a commercial formulation recommended for disin- fecting heat exchangers was determined for both planktonic and sessile populations of various Legionella pneumophila strains. The qua- ternary ammonium compound (QAC) was preferred as an alternative due to the emerging resistance of potentially pathogenic bacteria against different biocides. Planktonic L. pneumophila strains were suspended in tap water while sessile ones were grown on stainless steel that is used in construction of the cooling towers, then both group of strains were exposed to the biocide. The sensitivity of both planktonic and sessile populations of L. pneumophila strains to the biocide was different. The biocide was found effective below recom- mended dosages (1000-2000 mg/L) against planktonic populations of L. pneumophila, whereas it was determined that higher than the recommended dosages were required for sessile populations. The environmental isolates were more resistant to the biocide than the ATCC isolate was. The results indicated that studying only the planktonic populations of L. pneumophila for biocide tests might not be sufficient to provide the optimum dosage and contact time information for field trials. Therefore, biocidal activity of a water treatment chemical must be evaluated in terms of dosage and contact times on both planktonic and sessile bacteria. TM Key words: Legionella pneumophila, biocide, quaternary ammonium compounds (QACs), Gemacide PN-50 , biocidal activity, biofilm. INTRODUCTION nate legionellae. Therefore, in order to prevent legionellosis outbreaks, contaminated water systems must be disinfected Legionella pneumophila, the causative agent of and the most effective and practical biocide need to be eval- Legionnaires’ disease and Pontiac fever, can be isolated uated (Elsmore, 1986). Various biocides have been exam- from a variety of potential human contact areas, such as ined for their efficacies in both laboratory simulations and heat rejection systems (cooling tower systems, evaporative field treatment of Legionella contaminations on cooling tow- condensers) and potable water sources (Wright et al., 1991; ers and in potable water systems (Skaliy et al., 1980; Grace Yamamoto et al., 1991; Fields et al., 2002). It has been et al., 1981; Elsmore, 1986; Yamamoto et al., 1991; suggested that the primary mode of dispersion of the etio- Elsmore, 1993; Green and Pirrie, 1993; Surman et al., logical agent is an aerosol; inhalation of contaminated 1993; Lin et al., 1998; Viera et al., 1999; Walker et al., aerosols may result in outbreaks (Wright et al., 1991; 1999; Gao et al., 2000; Donlan et al., 2002; Fields et al., Mcnally et al., 2000). In addition to aerosol forms, in man- 2002; Kim et al., 2002; Kool, 2002). made water systems, legionellae, like most other aquatic In this study, we preferred a quaternary ammonium TM bacteria often exist as attached complex consortia (Green compound (Gemacide PN-50 ), which is a commercial for- and Pirrie, 1993; Murga et al., 2001; Petti et al., 2004). mulation, recommended by manufacturer for decontamina- Biofilms and sediments might create significant problems tion of heat exchangers. QACs have been actively deployed such as fouling (Johansen et al., 1997), heat transfer resist- since the 1930s with no apparent reduction in their effec- ance (Wright et al., 1991), filter plugging (Laopaiboon et al., tiveness (McBain, 2004). These are a widely used group of 2002) and metal corrosion (Momba and Binda, 2002), as non-oxidizing biocides for control of microbial growth in a well as increased resistance to antimicrobial compounds variety of applications, such as cooling tower systems, dis- (Green and Pirrie, 1993; Surman et al., 1993; Viera et al., infection and sterilisation, corrosion inhibition, wood preser- 1999; Kool, 2002) in water systems. vation, dispersion agents and in food production (Lutey, Control measures need to be considered for all water 1995; Laopaiboon et al., 2002; Majtàn and Majtànovà, TM systems, because they can harbour, amplify and dissemi- 2003; Korukoglu et al., 2006). Gemacide PN-50 is an alternative compound to oxidizing agents, safe for humans, stable in solution even at elevated temperatures and a wide TM range of pH. Gemacide PN-50 has no corrosive effect on common industrial materials such as stainless steel or alu- * Corresponding author. Phone: +90 212 455 57 00; TM minium (Gemsan, Undated). Gemacide PN-50 does not Fax: +90 212 528 05 27; E-mail: aytenkimiran@yahoo.com, kimiran@istanbul.edu.tr need to get inside the microbial cells like many other antimi- 122 A. Kimiran-Erdem et al. crobial agents do. It is semi-soluble in membranes and it Determination of biocidal activity with qualitative test exhibits its effect by disrupting the membrane integrity. method. TM Although Gemacide PN-50 biocide is already recommend- Planktonic bacteria. In the current study, the qualitative ed for disinfecting heat exchangers, there is a lack of pub- suspension test method by Skaliy et al. (1980) was modi- lished data about the efficacy of this compound against both fied based on ASTM (1991) E 645–91 standard test method. planktonic and sessile populations of legionellae. In various This method was used for determination of the efficacy of countries, alternative biocides have been searched due to microbicides used in cooling systems. the emerging resistance of the microorganisms against anti- The bactericidal activity of different biocide concentra- bacterial compounds. Therefore, in the current study, tions was assessed by adding 5 mL of bacterial suspension inhibitory characteristics of this biocide were investigated into 500 mL water containing different dosages of biocide, against both suspended and sessile bacteria. in a 1-litre glass Erlenmeyer flask. After shaking this mix- th rd th th ture gently, 1 mL aliquots were drawn at 0 , 3 , 6 , 24 th and 168 hours of incubation, and transferred into 9 mL of MATERIALS AND METHODS appropriate neutralizer (0.4 % sodium dodecyl sulphate in Buffered Yeast Extract Broth (BYEB). A 10-mL aliquot of the Test organisms. All tests were performed with three dif- neutralized sample was serially diluted (1:10) with sterile ferent L. pneumophila serogroup 1 strains confirmed by tap water. From each of three suitable dilutions, 0.1 mL was monoclonal antibody tests. C1 and C2 strains were isolated drawn and inoculated on BCYEA. After inoculation, plates from a cooling tower in the vicinity of Istanbul and potable were sealed in polyethylene bags to prevent dehydration water, respectively. A standard strain (SG 1 ATCC 33152) (Surman et al., 1993). After a 7-day incubation at 37 ºC, was obtained from Hertfordshire University Biodeterioration the number of survivors was determined by counting the Center. Since repeated subculturing on artificial media may colonies (CFU/mL) using a Colony Counter Device (åCOLyte affect the resistance of organisms to biocidal effect and may Super Colony Counter, Synbiosis). Plates on which colonies cause mutations, strains were maintained in phosphate did not develop were incubated for an additional 14 days buffer and glycerin suspension and stored at –70 ºC in cry- before discarding. At the time points given above, biocide- otubes. Freeze-dried cultures were not passaged more than bacteria suspensions were incubated at 25 ºC. At each time 3 times, as suggested (Skaliy et al. 1980; Elsmore, 1993). interval, 1 mL aliquots were processed as described above. 5 6 For the experiments, freeze-dried cultures of strains were The controls consisted of 10 -10 cells per mL in 500 mL grown on Buffered Charcoal Yeast Extract Agar (BCYEA, Cl -free sterile tap water containing no biocide. The rest of Oxoid) at 37 ºC in an atmosphere containing 2.5% CO . the experimental conditions were the same as above. Cultures were grown to late log phase, about 5-7 days of incubation, then cells were harvested and a suspension was Sessile bacteria. Bacterial suspensions (1 mL) were added prepared in Cl -free sterile tap water to a concentration of into 9 mL of BYEB in screw-capped bottles and then incu- Mc Farland 1 standard. Sterile tap water was preferred for bated at 37 ºC for 48 h in an atmosphere containing 2.5% dilutions because of the toxicity of sodium chloride against CO . Legionella inocula were transferred to beakers con- legionellae (Weiss and Westfall, 1984). The suspension was taining sterile stainless steel coupons to generate biofilm diluted in 1/10 ratio and used in the experiments. bacteria and the biofilm was allowed to develop on both sides of the coupons for 7 days at 37 ºC with slow stirring TM Biocide. Different dosages of Gemacide PN–50 , were (200 rpm, F 1.9 cm). After the incubation period, at 0, 3, 6 prepared in sterile demineralised water. Concentrations of and 24 h contact times, 6 coupons were aseptically 10000, 5000, 2000, 1000, 100, 10, 1 mg/L were used for removed, 3 of them were tested for biocidal activity while suspended bacteria and of 10000, 5000, 2000 and 1000 the other 3 were used as controls to determine the number mg/L were used for attached bacteria. Dosages for sus- of bacteria. After the incubation with biocide, test coupons pended bacteria were determined by MIC (minimum were transferred aseptically to 10 mL sterile neutralizer and inhibitory concentration) tests. were vortexed to detach bacteria. To determine the number of cells in biofilms, the content of tubes was diluted in 1/100 Toxicity test for neutralizer. Recommended neutralizer ratio and 0.1 mL of this dilution was cultivated in triplicate TM for Gemacide PN-50 by the manufacturer is a combination on BCYEA. The Petri plates were incubated for 7 days at 37 of Tween 80 (3%) and lecithin (0.3%). Instead of this com- ºC and following the incubation, colonies were counted. bination, sodium dodecyl sulphate (0.4%), which was Plates on which colonies did not develop were incubated for shown to have no toxicity effect on the organisms, was used an additional 14 days before discarding (Skaliy et al., 1980). as the quenching agent (Skaliy et al., 1980; Elsmore, 1993; Control coupons were transferred aseptically to tubes CEN, 1997; European Pharmacopeia, 2001; Johnston et al., containing 10 mL Cl -free sterile tap water and the tubes 2002). were vortexed to detach attached bacteria. The content of the tubes were diluted in 1/100 ratio and 0.1 mL of this Preparation of coupons. Stainless steel coupons (1 cm , solution was cultivated in BCYEA. 0.8 mm in thickness) were cleaned using a neutral deter- At the end of the qualitative suspension and biofilm gent (Triton) and then rinsed with water before autoclaving tests, the effect of biocide against C1 and C2 strains as well (120 °C, 20 min). After sterilisation and testing for the pres- as the standard strain were evaluated in respect to time and ence of any residual detergent, the coupons were placed biocide concentration. onto bacterial lawns on BCYEA plates using sterile forceps Statistical Analysis. Results were analysed statistically by and the plates were incubated at 37 ºC for 7 days. Plates were then examined for the presence of growth inhibition student’s t-test. Differences were considered significant when P <0.05. All the experiments described in this study zones around the coupons (Johansen et al., 1997). Ann. Microbiol., 57 (1), 121-125 (2007) 123 were done in triplicates. meric quaternary ammonium was found to be ineffective by Thomas et al. (1999) against Legionella bacteria. McCall et RESULTS AND DISCUSSION al. (1999) reported that poly[oxyethylene (dimethyliminio) ethylene-(dimethyliminio) ethylene dichloride] was not In the current study, inhibitory characteristics of Gemacide effective against Legionella bacteria (both planktonic and TM PN-50 , a QAC, were investigated against both suspended biofilm) when a single dosage of ~10 mg/L (maximum and biofilm bacteria. dosage recommended by a manufacturer) was applied in a It was found that the biocide of 10000, 5000, 2000, model plumbing system. 1000 or 100 mg/L concentrations killed all strains of bacte- It was observed in previous reports that QACs were also rial suspensions immediately (Table 1). At 10 mg/L concen- used in combination with different compounds such as iso- tration of biocide achieved > 5 log reduction for standard propanol (Skaliy et al., 1980) or organo-tin compounds strain and strain C1 in 168-hours contact time. Gemacide (Grace et al., 1981). Skaliy et al. (1980) showed that the TM PN-50 at 1 mg/L had no lethal effect on C1 and C2 strains mixture of didecyl dimethyl ammonium chloride and iso- in 168-hours contact time, while it killed the standard strain propanol at 72 mg/l and 144 mg/l respectively, achieved > in 3-hours contact time. The differences between the 5 log decrease after 3-hour contact time. Yet, it is not clear TM Gemacide PN-50 treated (10 mg/L, 168 h) and untreated whether the ammonium or the tin or isopropanol compound samples were statistically significant (P < 0.05) for C1 is the primary active ingredient. In the current study, it was strain. found that when the QAC was used alone, the effect Manufacturer’s recommended dosages for Gemacide obtained was not significantly different then that reported TM PN-50 was 0.1% (1000 mg/L) or 0.2% (2000 mg/L), by Skaliy et al. (1980). however we found that 100 mg/L dosage for all strains and Elsmore (1993) has stated that there is no standard 10 mg/L for standard strain achieved > 5 log reduction at method for the evaluation of biocides against legionellae, zero time. There are a few studies about the individual but some criteria must be noted; the contact time of the usage of QACs (Elsmore, 1986; McCall et al., 1999; Thomas biocide, the amount of bacteria in the initial inocula, and the et al., 1999). Elsmore (1986) determined that 800 mg/L validity and expression of the results. Elsmore (1993) has cationic poliquaternary ammonium compound was the min- also stated that a desirable water treatment biocide must imum inhibiting concentration for L. pneumophila. A poly- provide a 4-log reduction in bacteria, following a minimum -1 TM TABLE 1 - Survival of strains of Legionella pneumophila (log CFU mL ) exposed to Gemacide PN-50 at different contact times and concentrations Planktonic strains Gemacide Survivors (Log of no. ± S.E.) (mg/L) 0 h 3 h 6 h 24 h 168 h C1 Control 5.6 ± 0.03 5.5 ± 0.04 5.5 ± 0.02 5.7 ± 0.03 5.5 ± 0.01 10000 0.0 0.0 0.0 0.0 0.0 5000 0.0 0.0 0.0 0.0 0.0 2000 0.0 0.0 0.0 0.0 0.0 1000 0.0 0.0 0.0 0.0 0.0 100 0.0 0.0 0.0 0.0 0.0 10 3.7 ± 0.01 2.5 ± 0.03 2.5 ± 0.06 1 0.0 1 5.2 ± 0.03 4.7 ± 0.05 5.0 ± 0.04 4.8 ± 0.04 4.2 ± 0.02 C2 Control 5.8 ± 0.01 5.9 ± 0.03 5.8 5.8 ± 0.01 5.8 ± 0.01 10000 0.0 0.0 0.0 0.0 0.0 5000 0.0 0.0 0.0 0.0 0.0 2000 0.0 0.0 0.0 0.0 0.0 1000 0.0 0.0 0.0 0.0 0.0 100 0.0 0.0 0.0 0.0 0.0 10 5.6 ± 0.09 6.4 ± 0.01 5.0 ± 0.02 5.9 ± 0.02 5.9 ± 0.02 1 6.0 ± 0.03 6.4 ± 0.01 5.9 ± 0.04 5.5 ± 0.02 5.9 ± 0.04 Standard strain Control 5.7 ± 0.05 5.7 ± 0.01 5.7 ± 0.02 5.7 ± 0.03 5.7 ± 0.01 10000 0.0 0.0 0.0 0.0 0.0 5000 0.0 0.0 0.0 0.0 0.0 2000 0.0 0.0 0.0 0.0 0.0 1000 0.0 0.0 0.0 0.0 0.0 100 0.0 0.0 0.0 0.0 0.0 10 0.0 0.0 0.0 0.0 0.0 1 3.0 0.0 0.0 0.0 0.0 S.E.: Standard Error. 124 A. Kimiran-Erdem et al. -2 TM TABLE 2 - Survival of strains of Legionella pneumophila (log CFU cm ) exposed to Gemacide PN-50 at different contact times and concentrations Sessile strains Gemacide (mg/L) Survivors (Log of no. ± S.E.) 0 h 3 h 6 h 24 h 168 h C1 Control 5.7 ± 0.03 5.0 ± 0.02 5.4 ± 0.03 5.0 ± 0.05 5.5 ± 0.03 10000 4.5 ± 0.01 4.1 ± 0.01 3.6 ± 0.01 2.2 ± 0.02 0.0 5000 5.3 ± 0.04 4.8 ± 0.01 4.3 ± 0.02 3.3 ± 0.02 1.9 ± 0.01 2000 5.2 ± 0.01 4.9 ± 0.01 4.7 ± 0.01 3.6 ± 0.02 2.3 ± 0.02 1000 5.2 ± 0.01 5.0 ± 0.01 5.3 ± 0.01 4.7 ± 0.01 3.9 ± 0.01 C2 Control 4.7 ± 0.03 4.5 ± 0.05 4.9 ± 0.02 4.7 ± 0.01 4.6 ± 0.02 10000 3.5 ± 0.01 3.2 ± 0.04 3.0 ± 0.2 1.6 ± 0.02 0.0 5000 4.4 ± 0.01 3.8 ± 0.04 3.3 ± 0.07 2.5 ± 0.03 1.2 ± 0.01 2000 4.5 ± 0.01 4.0 ± 0.02 3.8 ± 0.02 2.8 ± 0.02 1.5 ± 0.01 1000 4.5 ± 0.01 4.3 ± 0.01 4.2 ± 0.01 3.8 ± 0.02 2.8 ± 0.02 Standard strain Control 4.9 ± 0.02 4.4 ± 0.01 4.7 ± 0.02 5.2 ± 0.02 5.0 ± 0.03 10000 3.5 ± 0.01 3.0 ± 0.02 2.4 ± 0.01 1.1 ± 0.01 0.0 5000 3.5 ± 0.03 3.2 ± 0.02 2.9 ± 0.03 2.4 ± 0.01 0.9 ± 0.01 2000 4.4 ± 0.01 3.9 ± 0.01 3.8 ± 0.01 2.3 ± 0.01 1.2 ± 0.02 1000 4.6 ± 0.01 4.3 ± 0.01 4.1 ± 0.01 2.6 ± 0.01 1.9 ± 0.02 S.E.: Standard Error. mental conditions. In microorganisms, development of of 24-hour contact time with non-oxidizing agents. Our result is coherent with Elsmore’s criteria and shows that reduced susceptibility to antimicrobial agents increases the tested biocide at 100 mg/L can be used for Legionella probability of further disinfection failures, leading severe decontamination. On the other hand, this dosage was problems in various industrial areas, food preparation, and unable to kill all biofilm bacteria and higher dosages (above human health areas (Chapman, 2003). Novel biocides were the recommended amount) of biocide were prepared to kill quested due to the emerging resistance of microorganisms sessile populations of Legionella strains (Table 2). With against antibacterial compounds. biofilms, after 168-hours contact time, 5000 mg/L achieved In respect to contact time and log reduction, Gemacide a > 3 log for all strains, while 10000 mg/L achieved ≥ 5 log TM PN-50 was found to be effective below the recommended for strain C1 and standard strain, >4 log for strain C2. Our dosages against planktonic legionellae according to results are in harmony with other investigator’s results in relation to biofilm resistance (Green and Pirrie, 1993; Elsmore’s criteria. The biocide (10000 mg/L) also achieved Surman et al., 1993; Murga et al., 2001; Donlan et al., significant log decreases for biofilm bacteria (> 4 log) for all 2002; Kool, 2002). strains in 168-hour contact time. Due to the assessment of The sensitivity of the strains against QAC was in the TM the stability of Gemacide PN-50 in long contact time (168- order of: standard strain > C1 > C2, both in suspension and hour) and the fact that it does not allow microorganismal biofilm states. This result is also consistent with those of Fux resistance it could be recommended as an alternative bio- et al. (2005), who reported that in the course of sequential cide for decontamination of legionellae from various man- in vitro passage, laboratory reference strains might differ- made water systems. entiate significantly from non-passaged samples. Although Prior to their commercial application in decontamination we did not passage the strains more than 3 times, we cer- of water systems, different biocides need to be identified tainly did not know the number of times the standard strain was passaged. These differences might be due to the fol- and investigated for their efficacy in various water systems. TM lowing: i) C1 and C2 environmental strains might be The current study proved that Gemacide PN-50 is effec- exposed antibacterial compounds and thus, may exhibit tive against both suspensions and biofilms of legionellae. improved resistance; ii) biocidal activity varies significantly The results indicated that studying the biocides against only among different types of microorganisms, moreover, it the planktonic populations of L. pneumophila might not be might also differ among different strains of the same species sufficient to provide the optimum dosage and contact time (Maillard, 2002), and since the L. pneumophila serogroup 1 information for field trials. strains used in our study were isolated from different sources, they might belong to different subtypes of L. pneu- Acknowledgements. This work was supported by the mophila serogroup 1. 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Published: Nov 20, 2009

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