Factors involved in bactericidal activities of formaldehyde and formaldehyde and formaldehyde condensate/isothiazolone mixtures

Enhanced antimicrobial activities of formaldehyde (FA)_and 5-choloro-2-methyl-4-isothiazolin-3-one (IT) mixtures were determined in tryptic soy broth (TSB), minimal salt-based medium, saline, and soluble oil emulsion using a Pseudomonas aeruginosa strain as a test organism. The apparent higher activity of mixture was the result of simultaneous and combined action of the biocides. The involvement of sulfhydryl groups as targets in the mode of action of both biocides was investigated using S-nitroso thioglycollic acid as a covalent modifier of exterior sulfhydryl groups of the cells. The results suggest their involvement in the mode of action of IT. These studies also indicate a considerable independent action by both biocides when the mixture is used with absence of cross-resistance in resistant isolates and independent induction of resistance in Pseudomonas aeruginosa to these biocides. Replacement of FA in the mixture with 1, 3,5-tris (2-hydroxyethyl)-hexahydrotriazine at equimolar concentrations of FA in TSB showed essentially the same levels of increased activity with no apparent adverse effects on the antimicrobial activity of IT from the amine portion of the molecule. Furthermore, in-vitro studies also indicated direct protection of IT by FA and FA condensate biocides in the presence of nucleophiles.     

The effect of fifteen biocides on formaldehyde-resistant strains ofPseudomonas aeruginosa

Summary Evaluation of formaldehyde and fifteen biocides in formaldehyde sensitive (S) and resistant (R) strains ofPseudomonas aeruginosa revealed a pattern of response that allowed a comparison of the mode of action of these biocides. The response of these strains to the various biocides, as well as the induction of transient resistance or cross-resistance in the (S) strain, allowed a grouping of biocides based on this pattern of response. Group 1 biocides acted in a manner indistinguishable from formaldehyde for both the (S) and (R) strains. Group 2 biocides were not effective against either the (S) or (R) strains at concentrations calculated to release equimolar concentrations of formaldehyde. However, treatment of the (S) strain with formaldehyde or Group 2 biocides resulted in the development of cross-resistance. Group 3 biocides were equally effective against the (S) and (R) strain, but the (S) strain survivors of treatment with Group 3 biocides were resistant to formaldehyde. Group 4 biocides (controls) had no presumed connection to formaldehyde mode of action. These four groupings, based on pattern of response, also resulted in groupings of biocides based on chemical structure.     

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.     

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.     

Assessing the risk of resistance to cationic biocides incorporating realism-based and biophysical approaches

The control of microorganisms is a key objective in disease prevention and in medical, industrial, domestic, and food-production environments. Whilst the effectiveness of biocides in these contexts is well-evidenced, debate continues about the resistance risks associated with their use. This has driven an increased regulatory burden, which in turn could result in a reduction of both the deployment of current biocides and the development of new compounds and formulas. Efforts to balance risk and benefit are therefore of critical importance and should be underpinned by realistic methods and a multi-disciplinary approach, and through objective and critical analyses of the literature. The current literature on this topic can be difficult to navigate. Much of the evidence for potential issues of resistance generation by biocides is based on either correlation analysis of isolated bacteria, where reports of treatment failure are generally uncommon, or laboratory studies that do not necessarily represent real biocide applications. This is complicated by inconsistencies in the definition of the term resistance. Similar uncertainties also apply to cross-resistance between biocides and antibiotics. Risk assessment studies that can better inform practice are required. The resulting knowledge can be utilised by multiple stakeholders including those tasked with new product development, regulatory authorities, clinical practitioners, and the public. This review considers current evidence for resistance and cross-resistance and outlines efforts to increase realism in risk assessment. This is done in the background of the discussion of the mode of application of biocides and the demonstrable benefits as well as the potential risks.     

Antibiotic and biocide resistance in bacteria: introduction

Drug resistance in bacteria is increasing and the pace at which new antibiotics are being produced is slowing. It is now almost commonplace to hear about methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant enterococci (VRE), multi-drug resistance in Mycobacterium tuberculosis (MDRTB) strains and multi-drug-resistant (MDR) gram-negative bacteria. So-called new and emerging pathogens add to the gravity of the situation. Reduced susceptibility to biocides is also apparently increasing, but is more likely to be low level in nature and to concentrations well below those used in hospital, domestic an industrial practice. A particular problem, however, is found with bacteria and other micro-organisms present in biofilms, where a variety of factors can contribute to greater insusceptibility compared with cells in planktonic culture. Also of potential concern is the possibility that widespread usage of biocides is responsible for the selection and maintenance of antibiotic-resistant bacteria. The basic mechanisms of action of, and bacterial resistance to, antibiotics are generally well documented, although data continue to accumulate about the nature and importance of efflux systems. In contrast, the modes of action of most biocides are poorly understood and consequently, detailed evaluation of bacterial resistance mechanisms is often disappointing. During this Symposium, the mechanisms of bacterial resistance to antibiotics and biocides are discussed at length. It is hoped that this knowledge will be used to develop newer, more effective drugs and biocides that can be better and perhaps, on occasion, more logically used to combat the increasing problem of bacterial resistance.     

Biocide resistance mechanisms

Information about the mechanisms of action and resistance of biocides used as preservatives, sanitizers, and disinfectants continues to accumulate. While biocides are generally not as well-studied as antibiotics, it is becoming clear that bacteria employ the same major resistance strategies against both groups of antibacterial chemicals. The resistance strategies—inhibitor inactivation, target site alteration, and target site exclusion—are described in the context of the presumed mechanisms of action of the biocides using examples drawn from the literature.     

A novel approach to mode of action of cationic biocides morphological effect on antibacterial activity

A. KANAZAWA, T. IKEDA AND T. ENDO. 1995 A new concept for the mode of action of cationic biocides is proposed in which the antibacterial activity of cationic disinfectants is ascribed essentially to molecular organizations of cations within aggregates, i.e. the activity is determined by the size of aggregates and number of active molecules comprising the aggregate. On the basis of the new concept, the morphological effect of disinfectants in aqueous solution on the antibacterial activity is discussed for low molecular weight phosphonium salts with single and double long alkyl chains (carbon number 14). The proposed new concept can be applied to all phenomena reported previously in antibacterial activity of cationic biocides and this concept is very important from the viewpoint of molecular design of more active cationic biocides.     

Effects of biocides on gene expression in the sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough

Although sulfate-reducing bacteria (SRB), such as Desulfovibrio vulgaris Hildenborough (DvH) are often eradicated in oil and gas operations with biocides, such as glutaraldehyde (Glut), tetrakis (hydroxymethyl) phosphonium sulfate (THPS), and benzalkonium chloride (BAC), their response to these agents is not well known. Whole genome microarrays of D. vulgaris treated with biocides well below the minimum inhibitory concentration showed that 256, 96, and 198 genes were responsive to Glut, THPS, and BAC, respectively, and that these three commonly used biocides affect the physiology of the cell quite differently. Glut induces expression of genes required to degrade or refold proteins inactivated by either chemical modification or heat shock, whereas BAC appears to target ribosomal structure. THPS appears to primarily affect energy metabolism of SRB. Mutants constructed for genes strongly up-regulated by Glut, were killed by Glut to a similar degree as the wild type. Hence, it is difficult to achieve increased sensitivity to this biocide by single gene mutations, because Glut affects so many targets. Our results increase understanding of the biocide's mode of action, allowing a more intelligent combination of mechanistically different agents. This can reduce stress on budgets for chemicals and on the environment.     

Use of Biocides as Agents against Microorganism-Induced Corrosion Increases Ecological Safety

Five synergistic combinations of biocides have been identified, of which kathon plus copper sulfate is the most efficient against Serratia marcescens. Depending on the ratio of the biocides, the active concentrations needed to maintain the synergistic effect can be decreased 4- to 20-fold. Combinations of biocides with salts promoting steel passivation (such as carbonates and phosphates) have been developed, which are more potent in decreasing corrosion-induced losses of mild steel than biocides and salts taken in isolation. Biocides should be introduced into systems susceptible to biocorrosion at the start of their exploitation, otherwise higher concentrations or synergistic compositions have to be used.     

Damage to Pseudomonas aeruginosa PAO1 bacteriophage F116 DNA by biocides

The mechanism of action of biocides against viruses has not been widely studied, although two main targets are viral proteins (capsids, enzymes) and the viral genome. This study was undertaken in order to investigate the efficacy of several disinfectants against the nucleic acid of the Pseudomonas aeruginosa PAO bacteriophage F116. Of all the biocides tested, only peracetic acid affected significantly the phage genome. However, it is not clear whether the nucleic acid was damaged inside the phage capsid or when released into the surrounding medium.     

Increase in the ecological danger upon the use of biocides for fighting corrosion induced by microorganisms

Five synergistic combinations of biocides were found, among which the combination of kathon + copper sulfate was the most efficient against Serratia marcescens. Depending on the ratio of these biocides, the synergistic effect of this pair allowed 4-20-fold decreases in the effective concentrations. Combinations of biocides with salts (carbonates and phosphates) that facilitate passivation of steel were found, which considerably decreased the corrosion losses of mild steel in comparison to isolated treatment with biocides or salts. The data showed that biocides must be added to corrosion-prone systems simultaneously with the beginning of their exploitation. Otherwise, considerably excessive amounts of biocides or their synergistic compositions are needed.     

Decreased biocide susceptibility of adherent Legionella pneumophila.

In a study of the in vitro effectiveness of biocides against Legionella pneumophila, some aspects of the cooling tower environment were replicated in the laboratory, paying particular attention to water hardness and pH. Pieces of Douglas fir and polyvinyl chloride were colonized in a recirculating system and the comparative efficacy of two biocides (Bronopol and Kathon) against the sessile and planktonic populations was examined. While the biocides were relatively effective against the planktonic L. pneumophila population over a short period of time (minimum 9-12 h), substantially longer periods of time (maximum greater than 48 h) were required to reduce the number of cultivable bacteria to below detectable levels in the adherent population. The results indicate that failure to monitor the sessile population of L. pneumophila in laboratory studies of biocides may result in the use of incorrect dosages and/or contact times in field trials with apparently reduced in situ efficacy.     

Biocidal activity of formaldehyde and nonformaldehyde biocides toward Mycobacterium immunogenum and Pseudomonas fluorescens in pure and mixed suspensions in synthetic metalworking fluid and saline

The microbicidal activity of four different biocides was studied in synthetic metalworking fluid (MWF) against Mycobacterium immunogenum, a suspected causative agent for hypersensitivity pneumonitis, and Pseudomonas fluorescens, a representative for the predominant gram-negative bacterial contaminants of MWF. The results indicated that M. immunogenum is more resistant than P. fluorescens to the tested formaldehyde-releasing biocides (Grotan and Bioban), isothiazolone (Kathon), and phenolic biocide (Preventol). Kathon was effective against mycobacteria at lower concentrations than the other three test biocides in MWF. In general, there was a marked increase in biocidal resistance of both the test organisms when present in MWF matrix compared to saline. Increased resistance of the two test organisms to biocides was observed when they were in a mixed suspension (1:1 ratio). The results indicate the protective effect of the MWF matrix against the action of commonly used biocides on the MWF-colonizing microbial species of occupational health significance, including mycobacteria.     

The Use of Machine Learning Methodologies to Analyse Antibiotic and Biocide Susceptibility in Staphylococcus aureus

Background

The rise of antibiotic resistance in pathogenic bacteria is a significant problem for the treatment of infectious diseases. Resistance is usually selected by the antibiotic itself; however, biocides might also co-select for resistance to antibiotics. Although resistance to biocides is poorly defined, different in vitro studies have shown that mutants presenting low susceptibility to biocides also have reduced susceptibility to antibiotics. However, studies with natural bacterial isolates are more limited and there are no clear conclusions as to whether the use of biocides results in the development of multidrug resistant bacteria.

Methods

The main goal is to perform an unbiased blind-based evaluation of the relationship between antibiotic and biocide reduced susceptibility in natural isolates of Staphylococcus aureus. One of the largest data sets ever studied comprising 1632 human clinical isolates of S. aureus originated worldwide was analysed. The phenotypic characterization of 13 antibiotics and 4 biocides was performed for all the strains. Complex links between reduced susceptibility to biocides and antibiotics are difficult to elucidate using the standard statistical approaches in phenotypic data. Therefore, machine learning techniques were applied to explore the data.

Results

In this pioneer study, we demonstrated that reduced susceptibility to two common biocides, chlorhexidine and benzalkonium chloride, which belong to different structural families, is associated to multidrug resistance. We have consistently found that a minimum inhibitory concentration greater than 2 mg/L for both biocides is related to antibiotic non-susceptibility in S. aureus.

Conclusions

Two important results emerged from our work, one methodological and one other with relevance in the field of antibiotic resistance. We could not conclude on whether the use of antibiotics selects for biocide resistance or vice versa. However, the observation of association between multiple resistance and two biocides commonly used may be of concern for the treatment of infectious diseases in the future.     

Decreased biocide susceptibility of adherent Legionella pneumophila

J.B. WRIGHT. I. RUSESKA AND J.W. COSTERTON. 1991. In a study of the in vitro effectiveness of biocides against Legionella pneumophila , some aspects of the cooling tower environment were replicated in the laboratory, paying particular attention to water hardness and pH. Pieces of Douglas fir and polyvinyl chloride were colonized in a recirculating system and the comparative efficacy of two biocides (Bronopol and Kathon) against the sessile and planktonic populations was examined. While the biocides were relatively effective against the planktonic L. pneumophila population over a short period of time (minimum 9-12 h), substantially longer periods of time (maximum > 48 h) were required to reducc the number of cultivable bacteria to below detectable levels in the adherent population. The results indicate that failure to monitor the sessile population of L. pneumophila in laboratory studies of biocides may result in the use of incorrect dosages and/or contact times in field trials with apparently reduced in situ efficacy.     

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     

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.     

Effect of some biocides on non-tuberculous mycobacteria

The incidence of disease and nosocomial infections produced by non tuberculosis mycobacteria (NTM) has increased in immunocompetent patients, but also and more frequently, in immunosuppressed patients. Several studies have disclosed that mycobacteria are more resistant to biocides than non-sporulating bacteria; in addition, some species are particularly resistant. The biocide action of sodium hypochloride and glutaraldehyde on Mycobacterium aviumintracellulare, Mycobacterium gordonae, Mycobacterium fortuitum and Mycobacterium chelonae was studied, using a modified Kelsey Maurer test. For the different species, both the minimal inhibitory concentration (MIC) and the minimal action time were determined. Effectiveness of sodium hypochloride and glutaraldehyde against the different mycobacterial species varied. The same was true for different isolates of the same species. Sodium hypochloride effective MIC and exposure time (killing of 99.99% of all NTM) were 0.2% and 5 minutes, respectively. In order to achieve 100% killing, 0.5% MIC and 15 minute exposure were needed. In the case of glutaraldehyde, 99.99% of the bacteria were killed with 1% MIC and a 15 minute exposure. An effectiveness of 100% was achieved with a 2% MIC of glutaraldehyde and a 15 minute exposure. Sodium hypochloride and glutaraldehyde are effective biocides for mycobacteria. The first biocide is cheap and effective at low concentrations, but its corrosive and oxidant nature makes it impossible for use in hospitals or with laboratory equipment. Glutaraldehyde (neither corrosive nor oxidant) is a safe alternative for disinfection of this type of equipment. However, it is important to bear in mind that these pathogens may develop resistance to biocides.     

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.