Direct Quantitative Evaluation of the Effects of Biocides on Pseudomonas fluorescens in Various Nutrient Media

A method for quantitative evaluation of the effects of biocides is presented. The method was tested in experiments with Pseudomonas fluorescens grown on various agar nutrient media. The effective concentrations of biocides that decreased the maximum specific rate of the colony biomass growth ("_m) were called S (suppressing) concentrations, and concentrations that decreased the number of colony-forming units (CFU) were taken as L (sublethal) concentrations. The efficiency of the reported approach was demonstrated in experiments with three biocides tested in four nutrient media. It was found that the biocide sensitivity of Pseudomonas fluorescens varied by a factor of 30, depending on the amount and type of the nutrient substrate.     

Direct quantitative method for evaluating the effect of biocides on Pseudomonas fluorescens in different culture media

A method for quantitative evaluation of the effects of biocides is presented. The method was tested in experiments with Pseudomonas fluorescens grown on various agar nutrient media. The effective concentrations of biocides that decreased the maximum specific rate of the colony biomass growth (mu'm) were called S (suppressing) concentrations, and concentrations that decreased the number of colony-forming units (CFU) were taken as L (sublethal) concentrations. The efficiency of the reported approach was demonstrated in experiments with three biocides tested in four nutrient media. It was found that the biocide sensitivity of Pseudomonas fluorescens varied by a factor of 30, depending on the amount and the type of the nutrient substrate.     

Practical aspects of biofouling control in industrial water systems

Both water consumption and discharge in industrial water systems are currently minimised. The circulation of such water results in concentration of dissolved and suspended substances, promoting the growth of waterborne microbes, biofouling and subsequent macrofouling of the system and concomitant microbially induced corrosion.A number of reviews have been published on the mechanisms of microbially induced corrosion and the organisms involved. The subject of biofilm formation has also been well covered in the literature. A lack of information on the community structure and physiology is, however, apparent. Many advances have, nevertheless, been made in biofouling control. Probably the most important is the shift in emphasis from planktonic bacterial monitoring to sessile bacterial monitoring. This led to the introduction of a variety of different sessile monitoring techniques. Much experience has since been obtained on the use and limitations of these techniques and, to date, one of the main problem areas remaining is the monitoring of biofouling.Research has also indicated the problem of microbial resistance to nonoxidising biocides. This has suggested that some of these compounds may be mutagens. From an environmental point of view, it has become very important to verify this. This has also indicated the need to develop biocides which do not induce resistance in micro-organisms, and to investigate whether oxidising biocides are also capable of inducing resistance in micro-organisms. Recent studies have indicated that biofilm ecosystems respond to stress (i.e. biocides) in ways similar to macro-ecosystems. Generally, there is a decline in species diversity and a selection of more tolerant isolates.These developments have placed the spotlight on alternative technologies, like biodispersants, which have shown potential as biofouling control agents, and which should be investigated further. Physical control measures are currently still limited to pigging, although a number of other technologies show promise. Although fluid dynamics and their effect on biofouling control programmes have been well reported in the literature, it remains an aspect which is neglected by industry in terms of practical applications.     

Booster biocides and microfouling

Antifouling (AF) paints are used to prevent the attachment of living organisms to the submerged surfaces of ships, boats and aquatic structures, usually by the release of biocides. Apart from copper, organic booster biocides are the main active components in AF paints, but their use can have a negative impact on the marine environment. The direct effects of biocides on marine bacteria are poorly known. This work investigates the impact of two biocides, viz. diuron and tolylfluanid, on the growth and the viability of marine microorganisms and on their ability to form biofilms. The biocides in solution were found to inhibit growth of two strains of marine bacteria, viz. Pseudoalteromonas and Vibrio vulnificus, at a high concentration (1000 μg ml^?1), but only a small effect on viability was observed. Confocal laser scanning microscopy (CLSM) showed that the booster biocides decreased biofilm formation by both bacteria. At a concentration of 10 μg ml^?1, the biocides inhibited cell attachment and reduced biofilm thickness on glass surfaces. The percentage of live cells in the biofilms was also reduced. The effect of the biocides on two diatoms, Fragilaria pinnata and Cylindrotheca closterium, was also evaluated in terms of growth rate, biomass, chlorophyll a content and attachment to glass. The results demonstrate that diuron and tolylfluanid are more active against diatoms than bacteria.     

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.     

Adaptation of amoebae to cooling tower biocides

Adaptation of amoebae to four cooling tower Biocides, which included a thiocarbamate compound, tributyltin neodecanoate mixed with quaternary ammonium compounds (TBT/QAC), another QAC alone, and an isothiazolin derivative, was studied. Previously we found that amoebae isolated from waters of cooling towers were more resistant to cooling tower biocides than amoebae from other habitats. Acanthamoeba hatchetti and Cochliopodium bilimbosum, obtained from American Type Culture Collection and used in the previous studies, were tested to determine whether they could adapt to cooling tower Biocides. A. hatchetti was preexposed to subinhibitory concentrations of the four Biocides for 72h, after which they were tested for their resistance to the same and other biocides. C. bilimbosum was exposed to only two biocides, as exposure to the other two was lethal after 72 h. Preexposure to the subinhibitory concentrations of the Biocides increased the resistance of the amoebae, as indicated by a significant increase in the minimum inhibitory concentration (up to 30-fold). In addition, cross-resistance was also observed, i.e., exposure to one biocide caused resistance to other biocides. These results show that amoebae can adapt to biocides in a short time. The phenomenon of cross-resistance indicates that regularly alternating biocides, as is done to control microbial growth in cooling towers, may not be effective in keeping amoeba populations in check. On the contrary, exposure to one biocide may boost the amoebae's resistance to a second biocide before the second biocide is used in the cooling tower. Since amoebae may harbor Legionella, or alone cause human diseases, these results may be important in designing effective strategies for controlling pathogens in cooling towers. Correspondence to: S.G. Berk.     

Evaluation of strategies to prevent algal fouling on white architectural and cellular concrete

In this study, different strategies have been examined for the prevention of algal fouling on two types of concrete with different bioreceptivity, i.e. white architectural and autoclaved aerated concrete (AAC). These strategies are aimed towards the decrease of the bioreceptivity and comprised the application of water repellents and/or biocides. Both traditional (stearates, silanes, silane siloxane mixtures and a pyridine biocide) and innovative compounds (quaternary ammonium silane-based biocides, silver copper zeolites and silver nanoparticles) have been assessed for their performance by means of an accelerated water run-off test. A modular setup was designed which allowed the simultaneous evaluation of 12 different product formulations. Algal fouling was evaluated by means of colorimetric and image analysis, for which two new evaluation criteria have been proposed.For white concrete, contrary to untreated specimens which had 40% of the surface covered with algae, no fouling was observed for surface treated specimens after 12 weeks of exposure to algae under the test conditions.For AAC, the different strategies examined were unable to completely prevent the algal fouling. The use of water repellents resulted in green algal streaks along the surface. Biocide treated specimens showed a delay of onset of fouling of two to four weeks under the test conditions. The best performance was obtained with a combination of a silane-based water repellent and a chlorinated pyridine-based biocide, for which only limited fouling was observed after eight weeks of intensive fouling exposure.     

Bacterial target sites for biocide action

Although biocides have been used for a century, the number of products containing biocides has recently increased dramatically with public awareness of hygiene issues. The antimicrobial efficacy of biocides is now well documented; however, there is still a lack of understanding of their antimicrobial mechanisms of action. There is a wide range of biocides showing different levels of antimicrobial activity. It is generally accepted that, in contrast to chemotherapeutic agents, biocides have multiple target sites within the microbial cell and the overall damage to these target sites results in the bactericidal effect. Information about the antimicrobial efficacy of a biocide (i.e. the eta-value) might give some useful indications about the overall mode of action of a biocide. Bacteriostatic effects, usually achieved by a lower concentration of a biocide, might correspond to a reversible activity on the cytoplasmic membrane and/or the impairment of enzymatic activity. The bacteriostatic mechanism(s) of action of a biocide is less documented and a primary (unique?) target site within the cell might be involved. Understanding the mechanism(s) of action of a biocide has become an important issue with the emergence of bacterial resistance to biocides and the suggestion that biocide and antibiotic resistance in bacteria might be linked. There is still a lack of understanding of the mode of action of biocides, especially when used at low concentrations (i.e. minimal inhibitory concentration (MIC) or sublethal). Although this information might not be required for highly reactive biocides (e.g. alkylating and oxidizing agents) and biocides used at high concentrations, the use of biocides as preservatives or in products at sublethal concentrations, in which a bacteriostatic rather than a bactericidal activity is achieved, is driving the need to better understand microbial target sites. Understanding the mechanisms of action of biocides serves several purposes: (i) it will help to design antimicrobial formulations with an improved antimicrobial efficacy and (ii) it will ensure the prevention of the emergence of microbial resistance.     

Antagonistic activity of Staphylococcus siderophores and chemical biocides against Bacillus subtilis in a paper-machine environment

The antagonistic potential of nonpathogenic Staphylococcus strains against Bacillus subtilis wild and type strains were studied under conditions simulating a paper- and board-machine environment. The antimicrobial activity was measured by growth inhibition in an automated turbidimeter. The antagonistic potential was compared with that of generally used chemical biocides in a paper- and board-machine environment. The siderophore-containing extracts of Staphylococcus strains significantly inhibited vegetative growth of B. subtilis and delayed the germination of spores both in synthetic and in white-water media. The mill strains were more resistant than type strain against Staphylococcus siderophores and against chemical biocides. The Staphylococcus siderophore-containing extracts did not interfere with the bacteriostatic effect of chemical biocides, but no synergy was detected. The results indicate the potential for application of Staphylococcus siderophore-containing extracts as biocontrol agents in paper- and boardmachine environment. Received 2 January 1998/ Accepted in revised form 4 December 1998     

The use of biocides to control sulphate‐reducing bacteria in biofilms on mild steel surfaces

Three different types of biocides, viz. formaldehyde (FM), glutaraldehyde (GA) and isothiozolone (ITZ) were used to control planktonic and sessile populations of two marine isolates of sulphate‐reducing bacteria (SRB). The influence of these biocides on the initial attachment of cells to mild steel surfaces, on subsequent biofilm formation and on the activity of hydrogenase enzymes within developed biofilms was evaluated. In the presence of biocides the rate and degree of colonization of mild steel by SRB depended on incubation time, bacterial isolate and the type of biocide used. Although SRB differed in their susceptibility to biocides, for all isolates the biofilm population was more resistant to the treatment than the planktonic population. GA showed highest efficiency in controlling planktonic and sessile SRB compared with the other two biocides. The activity of the enzyme hydrogenase measured in SRB biofilms varied between isolates and with the biocide treatment. No correlation was found between the number of sessile cells and hydrogenase activity.     

Adaptation of Campylobacter jejuni to biocides used in the food industry affects biofilm structure,adhesion strength,and cross-resistance to clinical antimicrobial compounds

The emergence of biocide-adapted Campylobacter jejuni strains that developed into biofilms and their potential to develop clinical resistance to antimicrobial compounds was studied. C. jejuni was grown in sub-lethal concentrations of five biocides used in the food industry. C. jejuni exhibited adaptation to these biocides with increased minimum inhibitory concentrations. The 3-D structures of the biofilms produced by the biocide-adapted cells were investigated by atomic force microscopy (AFM). The results revealed marked variability in biofilm architecture, including ice-crystal-like structures. Adaptation to the biocides enhanced biofilm formation, with significant increases in biovolume, surface coverage, roughness, and the surface adhesion force of the biofilms. Adaptation to commercial biocides induced resistance to kanamycin and streptomycin. This study suggests that the inappropriate use of biocides may lead to cells being exposed to them at sub-lethal concentrations, which can result in adaptation of the pathogens to the biocides and a subsequent risk to public health.     

Pyridine nucleotide synthesis in 3T3 cells.

The biosynthesis of NAD has been examined in 3T3 cells. The net synthesis of pyridine nucleotides does not occur when cells are cultured in the absence of performed pyridine ring compounds; however, growth continues normally for up to four cell doublings resulting in cells with a total pyridine nucleotide content that is reduced by as much as 12-fold. The mechanism that adjust the relative amounts of NADP and NAD are also altered such that the amount of NADP relative to NAD increases 5-fold. Both nicotinate and nicotinamide can be used as a precursor for NAD biosynthesis, however nicotinate is utilized less efficiently than nicotinamide. The presence of functional pathways for the biosynthesis of NAD from nicotinate via nicotinate mononucleotide and nicotinate adenine dinucleotide and from nicotinamide via nicotinamide mononucleotide has been demonstrated by identification of biosynthetic intermediates following short term exposure of cells to radiolabelled precursors. When cells are grown in Dulbecco's modified Eagle's medium which contains 33 μM nicotinamide the biosynthesis of NAD proceeds by a single pathway with nicotinamide mononucleotide as the only intermediate. Nicotinamide ribonucleoside which previously has been postulated to be an intermediate in the conversion of nicotinamide to NAD is not an intermediate in NAD biosynthesis.     

The role of melanin in Aspergillus tolerance to biocides and photosensitizers

Cationic biocides are widely utilized for surface disinfection. Photosensitizers such as toluidine blue O (TBO) produce reactive oxygen species following light excitation and are being investigated as novel biocides for similar applications. Aspergillus brasiliensis conidia contain melanin which protects against environmental stressors. The negative charge and antioxidant properties of melanin may confer resistance to photosensitizers and other biocides. In this study, the yeasticidal and fungicidal activity benzalkonium chloride (BZC), sodium dichloroisocyanurate (NaDCC) and TBO with red light were examined using quantitative suspension tests. All three biocides were highly effective against Candida albicans and > 5·0 log₁₀ reductions in viability were attainable within 5 minutes. Wild-type A. brasiliensis conidia were highly tolerant to treatment and 0·4 log₁₀ reductions in viability were observed within the same time frame when treated with TBO or BZC. NaDCC was markedly more effective. Inhibition of melanin biosynthesis by culturing with 100 μg ml⁻¹ kojic acid resulted in a hypopigmented phenotype with significantly increased sensitivity to all three biocides. These observations indicate that melanin is a significant contributor towards A. brasiliensis tolerance of biocides and photosensitizers and demonstrate that cationic biocides are poorly suited to applications where the control of A. brasiliensis is required.     

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.     

Conditioned medium enhances the fusion capability of rat bone marrow mesenchymal stem cells and cardiomyocytes

Mesenchymal stem cells (MSCs) show accelerated regeneration potential when these cells experience hypoxic stress. This “preconditioning” has shown promising results with respect to cardio-protection as it stimulates endogenous mechanisms resulting in multiple cellular responses. The current study was carried out to analyze the effect of hypoxia on the expression of certain growth factors in rat MSCs and cardiomyocytes (CMs). Both cell types were cultured and assessed separately for their responsiveness to hypoxia by an optimized dose of 2,4,-dinitrophenol (DNP). These cells were allowed to propagate under normal condition for either 2 or 24 h and then analyzed for the expression of growth factors by RT-PCR. Variable patterns of expression were observed which indicate that their expression depends on the time of re-oxygenation and extent of hypoxia. To see whether the growth factors released during hypoxia affect the fusion of MSCs with CMs, we performed co-culture studies in normal and conditioned medium. The conditioned medium is defined as the medium in which CMs were grown for re-oxygenation till the specified time period of either 2 or 24 h after hypoxia induction. The results showed that the fusion efficiency of cells was increased when the conditioned medium was used as compared to that in the normal medium. This may be due to the presence of certain growth factors released by the cells under hypoxic condition that promote cell survival and enhance their fusion or regenerating ability. This study would serve as another attempt in designing a therapeutic strategy in which conditioned MSCs can be used for ischemic diseases and provide more specific therapy for cardiac regeneration.     

<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.     

Possible implications of biocide accumulation in the environment on the prevalence of bacterial antibiotic resistance

The lethality of biocides depends upon their interaction with a number of distinct biochemical targets. This often reflects reactive chemistry for any given agent, such as thiol oxidation. Susceptibility may vary markedly between different target organisms, and changes within the more sensitive targets can alter the inhibitory effect. The multiplicity of potential targets, however, usually dictates against the development of overt resistance to concentrations used for hygienic applications. Similarly, although changes in cellular permeability toward such agents, mediated either by envelope modification or the induction of efflux-pumps may reduce susceptibility, they rarely influence the outcome of treatments at use-concentration. It has recently been proposed that chronic exposure of the environment to biocides used in a variety of commercial products might expose some microbial communities to subeffective concentrations causing emergence of resistant clones. Such resistance might relate to mutational changes in the most susceptible target or to regulatory mutants that cause the constitutive expression of certain efflux pumps. Although selection of organisms with such modifications is unlikely to influence the effectiveness of the biocides, changes in their susceptibility to third-party antibiotics can be postulated. This is particularly the case where a cellular target is shared between a biocide and an antibiotic, or where induction of efflux is sufficient to confer antibiotic resistance in the clinic. Although such linkage has been demonstrated in the laboratory in pure culture, it has not been documented in environments commonly exposed to biocides. In nature, the effects of chronic stressing with biocides are complicated by competition between microbial community members that may result in clonal expansion of naturally insusceptible clones. Received 11 March 2002/ Accepted in revised form 16 August 2002     

A suggested standardised method for testing photocatalytic inactivation of aeroterrestrial algal growth on TiO2-coated glass

Aeroterrestrial green algae form conspicuous biofilms on man-made surfaces. The self-cleaning properties of photocatalytic coatings prevent their growth and can probably replace biocides. The aim of this study was to develop a laboratory method to investigate the efficiency of photocatalytic materials against algal growth. Two algal isolates (“Chlorella” luteoviridis, SAG 2196, and Coccomyxa sp., SAG 2040) functioned well as model organisms because they grew on almost all test specimens at 100% humidity and low UVA radiation. With these species, we examined algal growth prevention using photocatalytic glass. No effects on algal growth were detected, although the coated surfaces were photocatalytically active and degraded methylene blue. Because their cells are protected well against photocatalytically generated hydroxyl radicals, aeroterrestrial algae survive various stress factors. Nevertheless, the newly developed experimental design may be useful for assessing the biological function of other photocatalytic materials or stress factors.     

Biocide susceptibility and intracellular glutathione inEscherichia coli

Summary Inhibition of growth and speed of kill by biocides with different mechanisms of action was examined with respect to intracellular glutathione levels. strain deficient in intracellular glutathione was hypersusceptible to electrophilic biocides, with the exception of an isothiazolone biocide. Growth inhibition by quaternary ammonium compounds and radical-generating biocides was unaffected by intracellular glutathione levels. Speed of kill experiments demonstrated a faster rate of killing by formaldehyde in both log and stationary phase cultures of the glutathione-deficient strain as compared to its wild-type parent. Glutathione levels had no effect on the speed of kill by hydrogen peroxide in log phase cultures, but resulted in an increased rate of killing in stationary phase cultures. Stationary phase cultures of the glutathione-deficient strain were killed by a quaternary ammonium biocide at a slower than the glutathione-replete strain. These studies provide information about both the mechanism of action of biocides as well as the role of glutathione in determining microbicide susceptibility.     

The effect of proteins released by cultured fibroblasts on density-dependent agglutination

An important criterion for the designation of non-transformed cell lines is density-dependent regulation of growth. The mechanism for control of such growth is believed to be lodged in the cell membrane. To elucidate this mechanism, the density dependence of surface-related processes has been examined. This report on ConA-mediated agglutination extends our earlier findings on another surface phenomenon, cell to substratum adhesion. Both these processes are density dependent and both are stimulated by cell proteins released into serum-free media by cultured human fibroblasts. It is suggested that these released proteins are transferable between cells and that they play a critical role in the mediation of density dependent surface phenomenon and in the density dependent regulation of cell growth.