Survival of protozoa in cooling tower biocides

Protozoa from cooling towers may serve as hosts for legionellae, but such protozoa have not been examined with respect to effects of cooling tower biocides. In this study, two ciliate species,Tetrahymena sp andColpoda sp, and two amoebae species,Vannella miroides andAcanthamoeba hatchetti, were isolated from a cooling tower and tested for survival in the presence of four cooling tower biocides. The protozoa were exposed for 24 h to a thiocarbamate compound, an isothiazolone compound, quaternary ammonium compounds (QAC), and tributyltin neodecanoate with quarternary ammonium compounds (TBT/QAC). After exposure, cells were examined for viability. The highest concentration of each biocide in which cells could survive was compared to the manufacturers' recommended maintenance dosage (MRMD) of the biocides.Tetrahymena andColpoda survived concentrations within the range of the MRMD of thiocarbamate and QAC.Vannella andAcanthamoeba survived concentrations within the MRMD of thiocarbamate, isothiazolone, and QAC.Colpoda cysts andAcanthamoebae cysts remained viable after exposure to concentrations much greater than the MRMD of thiocarbamate, isothiazolone, and QAC. None of the protozoa in any stage could survive the MRMD of TBT/QAC. These results show that protozoa indigenous to cooling towers may survive the recommended concentration of certain biocides, and this information may be important in devising procedures for eradicating hosts for legionellae.     

Stimulatory effect of cooling tower biocides on amoebae.

Two species of amoebae were isolated from the cooling tower of an air-conditioning system and examined for effects of exposure to four cooling tower biocides, a thiocarbamate compound, tributyltin neodecanoate mixed with quaternary ammonium compounds, another quaternary ammonium compound alone, and an isothiazolin derivative. The amoebae isolated were Acanthamoeba hatchetti and a Cochliopodium species. Two other amoeba cultures, an A. hatchetti culture and Cochliopodium bilimbosum, were obtained from the American Type Culture Collection (ATCC) and were also tested. The cooling tower isolates were more resistant to most of the biocides than the ATCC isolates were. The isothiazolin derivative was the least inhibitory to all four amoeba isolates, and tributyltin neodecanoate mixed with quaternary ammonium compounds was the most inhibitory to three of the four isolates. After exposure to lower concentrations of the biocides, including for one strain the manufacturer's recommended concentration of one biocide, the cooling tower amoeba populations increased significantly compared with unexposed controls, whereas the ATCC isolates were not stimulated at any of the concentrations tested. In some cases, concentrations which stimulated cooling tower amoebae inhibited the growth of the ATCC isolates. These results suggest that cooling tower amoebae may adapt to biocides, underscoring the need to use freshly isolated cooling tower organisms rather than organisms from culture collections for testing the efficacy of such biocides. The stimulatory effect of biocides on amoeba populations is an alarming observation, since these organisms may be reservoirs for legionellae. Biocides used to control microbial growth may actually enhance populations of host organisms for pathogenic bacteria.     

Exposure of Salmonella enterica serovar Typhimurium to high level biocide challenge can select multidrug resistant mutants in a single step

Background

Biocides are crucial to the prevention of infection by bacteria, particularly with the global emergence of multiply antibiotic resistant strains of many species. Concern has been raised regarding the potential for biocide exposure to select for antibiotic resistance due to common mechanisms of resistance, notably efflux.

Methodology/Principal Findings

Salmonella enterica serovar Typhimurium was challenged with 4 biocides of differing modes of action at both low and recommended-use concentration. Flow cytometry was used to investigate the physiological state of the cells after biocide challenge. After 5 hours exposure to biocide, live cells were sorted by FACS and recovered. Cells recovered after an exposure to low concentrations of biocide had antibiotic resistance profiles similar to wild-type cells. Live cells were recovered after exposure to two of the biocides at in-use concentration for 5 hours. These cells were multi-drug resistant and accumulation assays demonstrated an efflux phenotype of these mutants. Gene expression analysis showed that the AcrEF multidrug efflux pump was de-repressed in mutants isolated from high-levels of biocide.

Conclusions/Significance

These data show that a single exposure to the working concentration of certain biocides can select for mutant Salmonella with efflux mediated multidrug resistance and that flow cytometry is a sensitive tool for identifying biocide tolerant mutants. The propensity for biocides to select for MDR mutants varies and this should be a consideration when designing new biocidal formulations.     

Presence of qacEΔ1 gene and susceptibility to a hospital biocide in clinical isolates of Pseudomonas aeruginosa resistant to antibiotics

Biocides play an important role in healthcare-associated infection control by either minimizing or preventing microorganism dissemination. This study evaluated the susceptibility of Pseudomonas aeruginosa clinical isolates to a quaternary ammonium (QAC) disinfectant and antibiotics, and verified the presence of qacEΔ1, a determinant of resistance to QAC. The disinfectant test was the Association of Official Analytical Chemists Use-Dilution Test, and polymerase chain reaction was used to examine for qacEΔ1. The qacEΔ1 gene was detected in 48% of the isolates. Eighty-eight percent of the multiresistant isolates carried qacEΔ1 gene, while 35% of the non-multiresistant isolates was positive to this gene, and multiresistance well correlated with its presence. Among isolates tested for the disinfectant, 46% showed a reduced susceptibility to the disinfectant. qacEΔ1 gene was present in 70% of the susceptible isolates to the biocide, whereas 90% of the less susceptible strains harbored this gene. Reduced susceptibility to the disinfectant was independent of presence of qacEΔ1 suggesting that it does not play an important role in biocide resistance in P. aeruginosa. As far as we know, it is the first report confirming this fact and testing with disinfectant at its in-use concentration. The evidence of less susceptible strains than the reference bacterium used in disinfectant testing, and the high percentage of qacEΔ1 gene detected are of special concern and suggests continued investigation in laboratory and in situ, not only in healthcare settings, but also in all areas of biocide usage, including different micro-organisms and biocides.     

The efficacy of biocides and other chemical additives in cooling water systems in the control of amoebae

Aims:  In vitro experiments were undertaken to evaluate biocide formulations commonly used in cooling water systems against protozoa previously isolated from cooling towers. The investigations evaluated the efficacy of these formulations against amoebic cysts and trophozoites.
Methods and Results:  Laboratory challenges against protozoa isolated from cooling towers using chlorine, bromine and isothiazolinone biocides showed that all were effective after 4 h. The presence of molybdate and organic phosphates resulted in longer kill times for bromine and isothiazolinones. All treatments resulted in no detectable viable protozoa after 4 h of exposure.
Conclusions:  The chemical disinfection of planktonic protozoa in cooling water systems is strongly influenced by the residence time of the formulation and less so by its active constituent. Bromine and isothiazolinone formulations may require higher dosage of concentrations than currently practiced if used in conjunction with molybdate- and phosphate-based scale/corrosion inhibitors.
Significance and Impact of the Study:  Cooling water systems are complex microbial ecosystems in which predator–prey relationships play a key role in the dissemination of Legionella . This study demonstrated that at recommended dosing concentrations, biocides had species-specific effects on environmental isolates of amoebae that may act as reservoirs for Legionella multiplication in cooling water systems.     

<Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> PAO1 adapted to 2-phenoxyethanol shows cross-resistance to dissimilar biocides and increased susceptibility to antibiotics

The growth adaptability to increasing concentration of the biocide 2-phenoxyethanol (PE) was determined in Pseudomonas aeruginosa PAO1 (P.a.) as part of efforts to understand and control the biocide tolerance and its effect on cross-resistance to other biocides and resistance to antibiotics. After repeated subculturing in media containing increasing sub-minimum-inhibitory PE concentration, P.a. exhibited an adaptive resistance indicated by two-fold increase in MIC at the 10th passage. The resistance was stable and remained after passaging the strain in further 7 successive passages in PE-free growth media. The strain showed cross-resistance towards dissimilar biocides and displayed increased susceptibility to antibiotics, which was not influenced by the presence of the efflux inhibitor ‘carbonyl cyanide m-chlorophenyl hydrazone’. Outer membranes of adapted strain showed altered protein profile when examined by SDS-PAGE.     

Field trial of biocides for control of Legionella in cooling towers

Legionella and amebae populations in 16 cooling towers were challenged with three commercially available biocide formulations. The active agents were: a chlorinated phenolic thioether (CPTE), bromo-nitro-propane-diol (BNPD), and bromo-chloro-dimethylhydantoin (BCD, in briquette form). The towers were dosed with these biocides for approximately 4 weeks. BCD was effective against Legionella in each of nine challenge experiments, and CPTE in eight of nine challenges. BNPD was effective in only five of 11 challenges. None of the biocides had any significant effect in reducing planktonic amebae concentrations during the challenges.     

Efficacy of biocides used in the modern food industry to control salmonella enterica, and links between biocide tolerance and resistance to clinically relevant antimicrobial compounds

Biocides play an essential role in limiting the spread of infectious disease. The food industry is dependent on these agents, and their increasing use is a matter for concern. Specifically, the emergence of bacteria demonstrating increased tolerance to biocides, coupled with the potential for the development of a phenotype of cross-resistance to clinically important antimicrobial compounds, needs to be assessed. In this study, we investigated the tolerance of a collection of susceptible and multidrug-resistant (MDR) Salmonella enterica strains to a panel of seven commercially available food-grade biocide formulations. We explored their abilities to adapt to these formulations and their active biocidal agents, i.e., triclosan, chlorhexidine, hydrogen peroxide, and benzalkonium chloride, after sequential rounds of in vitro selection. Finally, cross-tolerance of different categories of biocidal formulations, their active agents, and the potential for coselection of resistance to clinically important antibiotics were investigated. Six of seven food-grade biocide formulations were bactericidal at their recommended working concentrations. All showed a reduced activity against both surface-dried and biofilm cultures. A stable phenotype of tolerance to biocide formulations could not be selected. Upon exposure of Salmonella strains to an active biocidal compound, a high-level of tolerance was selected for a number of Salmonella serotypes. No cross-tolerance to the different biocidal agents or food-grade biocide formulations was observed. Most tolerant isolates displayed changes in their patterns of susceptibility to antimicrobial compounds. Food industry biocides are effective against planktonic Salmonella. When exposed to sublethal concentrations of individual active biocidal agents, tolerant isolates may emerge. This emergence was associated with changes in antimicrobial susceptibilities.     

Efficacy of Colloidal Silver-Hydrogen Peroxide and 2-Bromo-2-nitroporopane-1,3-diol Compounds Against Different Serogroups of <Emphasis Type="Italic">Legionella pneumophila</Emphasis> Strains

Cooling towers are considered as amplifier and disseminator sources for Legionella spp. despite preventive treatments. Information which was obtained from biocidal tests could improve the effectiveness of treatments. Therefore, the choice of appropriate biocides and the applying of biocides in correct dosages and contact times are important. Various oxidizing and non-oxidizing biocides have been investigated in vitro for their effectiveness against legionellae. Colloidal silver-hydrogen peroxide (CSHP) and 2-bromo-2-nitroporopane-1,3-diol (BNPD) biocides were selected as an example for oxidizing and non-oxidizing agents, respectively, in view of bactericidal action against different serogroups of L. pneumophila strains [serogroup 1 (S1) and serogroup 2–14 (S2)] which are isolated different cooling towers in the vicinity of Istanbul, Turkey and reference strain. In the current study, oxidizing biocide was found more effective than non-oxidizing biocide in terms of contact times, log reductions and recommended dosages. At the recommended concentrations for cooling towers (100 ppm), while CSHP compound killed all strains in 3 h contact time, BNPD compound killed S2 and reference strain in the same contact time, S1 strain after 6 h contact time. The results of the present study showed that effective biocide applications can be achieved by pre-determining minimum inhibitory concentration (MIC) and minimum contact time of different biocides to kill target bacteria.     

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

Efficacy of Gemacide PN-50^TM (a quaternary ammonium compound) as a commercial formulation recommended for disinfecting heat exchangers was determined for both planktonic and sessile populations of variousLegionella pneumophila strains. The quaternary ammonium compound (QAC) was preferred as an alternative due to the emerging resistance of potentially pathogenic bacteria against different biocides. PlanktonicL. 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 ofL. pneumophila strains to the biocide was different. The biocide was found effective below recommended dosages (1000–2000 mg/L) against planktonic populations ofL. 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 ofL. 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.     

Legionella in industrial cooling towers: monitoring and control strategies

Aims: Legionella contamination of industrial cooling towers has been identified as the cause of sporadic cases and outbreaks of legionellosis among people living nearby. To evaluate and control Legionella contamination in industrial cooling tower water, microbiological monitoring was carried out to determine the effectiveness of the following different disinfection treatments: (i) continuous chlorine concentration of 0·01 ppm and monthly chlorine shock dosing (5 ppm) on a single cooling tower; (ii) continuous chlorine concentration of 0·4 ppm and monthly shock of biocide P3 FERROCID 8580 (BKG Water Solution) on seven towers. Methods and Results: Legionella spp. and total bacterial count (TBC) were determined 3 days before and after each shock dose. Both strategies demonstrated that when chlorine was maintained at low levels, the Legionella count grew to levels above 10⁴ CFU l^?1 while TBC still remained above 10⁸ CFU l^?1. Chlorine shock dosing was able to eliminate bacterial contamination, but only for 10–15 days. Biocide shock dosing was also insufficient to control the problem when the disinfectant concentration was administered at only one point in the plant and at the concentration of 30 ppm. On the other hand, when at a biocide concentration of 30 or 50 ppm was distributed throughout a number of points, depending on the plant hydrodynamics, Legionella counts decreased significantly and often remained below the warning limit. Moreover, the contamination of water entering the plant and the presence of sediment were also important factors for Legionella growth. Conclusions: For effective decontamination of outdoor industrial cooling towers, disinfectants should be distributed in a targeted way, taking into account the possible sources of contamination. Significance and Impact of the Study: The data of the research permitted to modify the procedure of disinfection for better reduce the water and aerosol contamination and consequently the exposure risk.     

Microbial community adaptation to quaternary ammonium biocides as revealed by metagenomics

Quaternary ammonium compounds (QACs) represent widely used cationic biocides that persist in natural environments. Although microbial degradation, sensitivity and resistance to QACs have been extensively documented, a quantitative understanding of how whole communities adapt to QAC exposure remain elusive. To gain insights into these issues, we exposed a microbial community from a contaminated river sediment to varied levels of benzalkonium chlorides (BACs, a family of QACs) for 3 years. Comparative metagenomic analysis showed that the BAC‐fed communities were dramatically decreased in phylogenetic diversity compared with the control (no BAC exposure), resulting presumably from BAC toxicity, and dominated by Pseudomonas species (> 50% of the total). Time‐course metagenomics revealed that community adaptation occurred primarily via selective enrichment of BAC‐degrading Pseudomonas populations, particularly P. nitroreducens, and secondarily via amino acid substitutions and horizontal transfer of a few selected genes in the Pseudomonas populations, including a gene encoding a PAS/PAC sensor protein and ring‐hydroxylating dioxygenase genes. P. nitroreducens isolates were reproducibly recoverable from communities after prolonged periods of no‐BAC exposure, suggesting that they are robust BAC‐degraders. Our study provides new insights into the mechanisms and tempo of microbial community adaptation to QAC exposure and has implications for treating QACs in biological engineered systems.     

Legionella spp. in Puerto Rico cooling towers

Water samples from air conditioning cooling towers receiving different treatment protocols on five large municipal buildings in San Juan, P.R., were assayed for various Legionella spp. and serogroups by using direct immunofluorescence. Several water quality parameters were also measured for each sample. Guinea pigs were inoculated with water samples to confirm pathogenicity and recover viable organisms. Legionella pneumophila serogroups 1 to 6, L. bozemanii, L. micdadei, L. dumoffii, and L. gormanii were observed in at least one of the cooling towers. L. pneumophila was the most abundant species; its density reached 10(5) cells per ml, which is within the range that is considered potentially pathogenic to humans. A significantly higher density of L. pneumophila was observed in the cooling tower water that was not being treated with biocides. Percent respiration (INT) and total cell activity (acridine orange direct count) were inversely correlated with bacterial density. This study demonstrates that Legionella spp. are present in tropical air-conditioning cooling systems and that, without continuous biocide treatment, they may reach densities that present a health risk.     

Legionella spp. in Puerto Rico cooling towers.

Water samples from air conditioning cooling towers receiving different treatment protocols on five large municipal buildings in San Juan, P.R., were assayed for various Legionella spp. and serogroups by using direct immunofluorescence. Several water quality parameters were also measured for each sample. Guinea pigs were inoculated with water samples to confirm pathogenicity and recover viable organisms. Legionella pneumophila serogroups 1 to 6, L. bozemanii, L. micdadei, L. dumoffii, and L. gormanii were observed in at least one of the cooling towers. L. pneumophila was the most abundant species; its density reached 10(5) cells per ml, which is within the range that is considered potentially pathogenic to humans. A significantly higher density of L. pneumophila was observed in the cooling tower water that was not being treated with biocides. Percent respiration (INT) and total cell activity (acridine orange direct count) were inversely correlated with bacterial density. This study demonstrates that Legionella spp. are present in tropical air-conditioning cooling systems and that, without continuous biocide treatment, they may reach densities that present a health risk.     

Production of Respirable Vesicles Containing Live Legionella pneumophila Cells by Two Acanthamoeba spp.

Two Acanthamoeba species, fed at three temperatures, expelled vesicles containing living Legionella pneumophila cells. Vesicles ranged from 2.1 to 6.4 μm in diameter and theoretically could contain several hundred bacteria. Viable L. pneumophila cells were observed within vesicles which had been exposed to two cooling tower biocides for 24 h. Clusters of bacteria in vesicles were not dispersed by freeze-thawing and sonication. Such vesicles may be agents for the transmission of legionellosis associated with cooling towers, and the risk may be underestimated by plate count methods.     

Chelators enhanced biocide inhibition of planktonic sulfate-reducing bacterial growth

Biocides are currently the primary mitigation method to control sulfate-reducing bacteria (SRB) in biofouling, reservoir souring and microbiologically influenced corrosion. Increasingly restrictive environmental regulations and safety concerns on biocide uses demand more efficient dosing of biocides. Chelators have been known to enhance antibiotics because of their properties such as increasing the permeability of the outer cell membrane of Gram-negative bacteria. Two readily biodegradable chelators, ethylenediaminedisuccinate (EDDS) and N-(2-hydroxyethyl)iminodiacetic acid (HEIDA) disodium salts that are touted as potential replacements of ethylenediaminetetraacetic acid (EDTA), were evaluated as potential biocide enhancers for glutaraldehyde and tetrakis hydroxymethyl phosphonium sulfate (THPS) in their inhibition of planktonic SRB growth. Desulfovibrio vulgaris ATCC 7757 and Desulfovibrio desulfuricans ATCC 14563 were grown in modified ATCC 1249 medium and in enriched artificial seawater, respectively. Laboratory tests in 100 ml anaerobic vials showed that EDDS or HEIDA alone did not inhibit SRB growth. However, when EDDS or HEIDA was combined with glutaraldehyde or THPS, each of them enhanced the biocide inhibition of planktonic SRB growth.     

Development of a new CARD-FISH protocol for quantification of Legionella pneumophila and its application in two hospital cooling towers

Aims: Open cooling towers are frequent sources of infections with Legionella pneumophila. The gold standard for the detection of Leg. pneumophila is based on cultivation lasting up to 10 days and detecting only culturable cells. Alternative fluorescence in situ hybridization (FISH) protocols have been proposed, but they result in faint fluorescence signals and lack specificity because of cross‐hybridization with other Legionella species. Our aim was thus to develop a new FISH protocol for rapid and specific detection of Leg. pneumophila in water samples. Methods and Results: A novel catalysed reporter deposition FISH (CARD‐FISH) protocol for the detection of Leg. pneumophila was developed, which significantly enhanced signal intensity as well as specificity of the probe through the use of a novel competitor probe. The developed protocol was compared with the culture method for monitoring the seasonal development of culturable and nonculturable Leg. pneumophila in two hospital cooling tower systems. Seasonal fluctuations of Leg. pneumophila concentrations detected via CARD‐FISH were related to the development of the total bacterial community in both cooling towers, with temperature and biocide as the main factors controlling this development. Conclusions: Our results clearly showed that the majority of the Leg. pneumophila cells were in a nonculturable state. Thus, detection of Leg. pneumophila with culture methods may underestimate the total numbers of Leg. pneumophila present. Significance and Impact of the Study: Rapid, sensitive and specific detection and quantification of Leg. pneumophila in water systems is prerequisite for reliable risk estimation. The new protocol significantly improves current methodology and can be used to monitor and screen for Leg. pneumophila concentrations in cooling towers or other water systems.     

Efficacy of biocides on laboratory-generated Legionella biofilms

Results of biocide efficacy testing on laboratory-generated microbial biofilms containing Legionella bozemanii is presented. These show that chlorine is effective at recommended concentrations in cooling towers; at least 48 h contact between free chlorine and the biofilm is necessary. Of five commercial biocides tested, those containing isothiazolinones and dibromonitrilo-proprionamide were most effective against sessile L. bozemanii.