Chemical reactivity of some isothiazolone biocides

Chemical reactions between the isothiazolone biocides, N-methylisothiazol-3-one (MIT), benzisothiazol-3-one (BIT) and 5-chloro-N-methylisothiazol-3-one (CMIT) with cysteine have been investigated by u.v. and NMR spectroscopy. At physiological pH all three agents interacted oxidatively with thiols to form disulphides. Further interaction with thiols caused the release of cystine and formation of a reduced, ring-opened form of the biocide (mercaptoacrylamide). In an analogous fashion to the initial reaction the mercaptoacrylamide reacted with another molecule of biocide to give biocide dimers. NMR spectral studies indicated that for CMIT the mercaptoacrylamide form is capable of tautomerization to a highly reactive thio-acyl chloride. Formation of mercaptoacrylamide was in all cases highly pH-dependent. Alcohol dehydrogenase was insensitive to all three agents but was highly sensitive to CMIT when co-administered with dithiothreitol. Capacity to form a thioacyl chloride from the mercaptoacrylamide is suggested to account for much of this enhanced activity. Stopped-flow spectroscopic studies showed rates of reaction with glutathione (GSH) to directly parallel antimicrobial activity. Additionally, CMIT was able to react directly with both ionization states of GSH (pH 7-10) whilst BIT and MIT appeared only to interact when the glutamyl-nitrogen of GSH was charged (pH 8.5).     

Growth inhibitory and biocidal activity of some isothiazolone biocides

C ollier , P.J., R amsey , A.J., A ustin , P. & G ilbert , P. 1990. Growth inhibitory and biocidal activity of some isothiazolone biocides. Journal of Applied Bacteriology 69 , 569–577.
Similar patterns of growth inhibition were observed for the three biocides, benzisothiazol-3-one (BIT), 5-chloro-N-methylisothiazol-3-one (CMIT) and N-methylisothiazol-3-one (MIT) against Escherichia coli ATCC 8739 and Schizo-saccharomyces pombe NCYC 1354. After periods of induced stasis, proportional to biocide concentration, growth proceeded at an inhibited rate. Extrapolation of the static periods and inhibited growth rates against biocide concentration gave minimum growth inhibitory concentration estimates of 0.1–0.5 μg/ml for CMIT, 15–20 μg/ml for BIT and 40–250 μg/ml for MIT. Patterns of growth inhibition by CMIT and induced morphological changes in inhibited cultures suggested this com-pound to also inhibit initiation of DNA replication. Growth inhibitory activity was rapidly quenched by the addition of thiol-containing materials such as glutathione and cysteine. The activity of CMIT was additionally quenched by the presence of the non-thiol amino acids valine and/or histidine. These results suggest that the chlorinated isothiazolones can react with amines as well as with essential thiol groups.     

Growth inhibitory and biocidal activity of some isothiazolone biocides

Similar patterns of growth inhibition were observed for the three biocides, benzisothiazol-3-one (BIT), 5-chloro-N-methylisothiazol-3-one (CMIT) and N-methylisothiazol-3-one (MIT) against Escherichia coli ATCC 8739 and Schizosaccharomyces pombe NCYC 1354. After periods of induced stasis, proportional to biocide concentration, growth proceeded at an inhibited rate. Extrapolation of the static periods and inhibited growth rates against biocide concentration gave minimum growth inhibitory concentration estimates of 0.1-0.5 micrograms/ml for CMIT, 15-20 micrograms/ml for BIT and 40-250 micrograms/ml for MIT. Patterns of growth inhibition by CMIT and induced morphological changes in inhibited cultures suggested this compound to also inhibit initiation of DNA replication. Growth inhibitory activity was rapidly quenched by the addition of thiol-containing materials such as glutathione and cysteine. The activity of CMIT was additionally quenched by the presence of the non-thiol amino acids valine and/or histidine. These results suggest that the chlorinated isothiazolones can react with amines as well as with essential thiol groups.     

Relationship between Legionella pneumophila and Acanthamoeba polyphaga: physiological status and susceptibility to chemical inactivation.

Survival studies were conducted on Legionella pneumophila cells that had been grown intracellularly in Acanthamoeba polyphaga and then exposed to polyhexamethylene biguanide (PHMB), benzisothiazolone (BIT), and 5-chloro-N-methylisothiazolone (CMIT). Susceptibilities were also determined for L. pneumophila grown under iron-sufficient and iron-depleted conditions. BIT was relatively ineffective against cells grown under iron depletion; in contrast, iron-depleted conditions increased the susceptibilities of cells to PHMB and CMIT. The activities of all three biocides were greatly reduced against L. pneumophila grown in amoebae. PHMB (1 x MIC) gave 99.99% reductions in viability for cultures grown in broth within 6 h and no detectable survivors at 24 h but only 90 and 99.9% killing at 6 h and 24 h, respectively, for cells grown in amoebae. The antimicrobial properties of the three biocides against A. polyphaga were also determined. The majority of amoebae recovered from BIT treatment, but few, if any, survived CMIT treatment or exposure to PHMB. This study not only shows the profound effect that intra-amoebal growth has on the physiological status and antimicrobial susceptibility of L. pneumophila but also reveals PHMB to be a potential biocide for effective water treatment. In this respect, PHMB has significant activity, below its recommended use concentrations, against both the host amoeba and L. pneumophila.     

Relationship between Legionella pneumophila and Acanthamoeba polyphaga: physiological status and susceptibility to chemical inactivation.

Survival studies were conducted on Legionella pneumophila cells that had been grown intracellularly in Acanthamoeba polyphaga and then exposed to polyhexamethylene biguanide (PHMB), benzisothiazolone (BIT), and 5-chloro-N-methylisothiazolone (CMIT). Susceptibilities were also determined for L. pneumophila grown under iron-sufficient and iron-depleted conditions. BIT was relatively ineffective against cells grown under iron depletion; in contrast, iron-depleted conditions increased the susceptibilities of cells to PHMB and CMIT. The activities of all three biocides were greatly reduced against L. pneumophila grown in amoebae. PHMB (1 x MIC) gave 99.99% reductions in viability for cultures grown in broth within 6 h and no detectable survivors at 24 h but only 90 and 99.9% killing at 6 h and 24 h, respectively, for cells grown in amoebae. The antimicrobial properties of the three biocides against A. polyphaga were also determined. The majority of amoebae recovered from BIT treatment, but few, if any, survived CMIT treatment or exposure to PHMB. This study not only shows the profound effect that intra-amoebal growth has on the physiological status and antimicrobial susceptibility of L. pneumophila but also reveals PHMB to be a potential biocide for effective water treatment. In this respect, PHMB has significant activity, below its recommended use concentrations, against both the host amoeba and L. pneumophila.     

Outer membrane protein shifts in biocide-resistant Pseudomonas aeruginosa PAO1

Benzisothiazolone (BIT), N-methylisothiazolone (MIT) and 5-chloro-N-methylisothiazolone (CMIT) are highly effective biocidal agents and are used as preservatives in a variety of cosmetic preparations. The isothiazolones have proven efficacy against many fungal and bacterial species including Pseudomonas aeruginosa. However, some species are beginning to exhibit resistance towards this group of compounds after extended exposure. This experiment induced resistance in cultures of Ps. aeruginosa exposed to incrementally increasing sub-minimum inhibitory concentrations (MICs) of the isothiazolones in their pure chemical forms. The induced resistance was observed as a gradual increase in MIC with each new passage. The MICs for all three test isothiazolones and a thiol-interactive control compound (thiomersal) increased by approximately twofold during the course of the experiment. The onset of resistance was also observed by reference to the altered presence of an outer membrane protein, designated the T-OMP, in SDS-PAGE preparations. T-OMP was observed to disappear from the biocide-exposed preparations and reappear when the resistance-induced cultures were passaged in the absence of biocide. This reappearance of T-OMP was not accompanied by a complete reversal of induced resistance, but by a small decrease in MIC. The induction of resistance towards one biocide resulted in the development of cross-resistance towards other members of the group and the control, thiomersal. It has been suggested that the disappearance of T-OMP from these preparations is associated with the onset of resistance to the isothiazolones in their Kathon form (CMIT and MIT).     

Antimicrobial susceptibility changes and T-OMP shifts in pyrithione-passaged planktonic cultures of Pseudomonas aeruginosa PAO1

AIMS: The aim of this study was to determine whether passaging Pseudomonas aeruginosa PAO1 with sub-MICs of the pyrithione biocides results in both the induction of decreased susceptibility towards these antimicrobials and associated outer membrane profile changes. METHODS AND RESULTS: Previous work by this group has shown that it is possible to induce susceptibility changes towards the isothiazolone biocides in Ps. aeruginosa PAO1 by successive passages in the presence of increasing sub-MICs of biocide. This procedure was accompanied by the loss of a 35 kDa outer membrane protein, T-OMP. In this experiment, this process was repeated with the biocides sodium pyrithione (NaPT), zinc pyrithione (ZnPT) and cetrimide. The pattern of susceptibility was similar to that observed with the isothiazolone biocides. Upon removal of biocide, the observed MIC did not return to the original pre-exposure value. The onset and development of resistance was accompanied by the loss of T-OMP from outer membrane profiles, which suggests that this is a non-specific membrane channel whose production within the cell is sensitive to biocide presence. The T-OMP reappeared when the cells were passaged in the absence of pyrithione. Cross-resistance studies indicated that induced resistance to one biocide yields partial resistance towards other members of the group and the positive control. CONCLUSIONS: These results indicate that the pyrithione biocides have similar susceptibility profiles in Ps. aeruginosa to those exhibited by the isothiazolones, but that the acquired changes in susceptibility to the pyrithiones is largely irreversible. SIGNIFICANCE AND IMPACT OF THE STUDY: This study indicates that acquired susceptibility changes towards sub-MICs of selected biocides are multifactorial in nature.     

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.     

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.     

Differential biocide susceptibility of the multiple genotypes of <Emphasis Type="Italic">Mycobacterium immunogenum</Emphasis>

The non-tuberculous mycobacterium Mycobacterium immunogenum colonizes industrial metalworking fluids (MWFs) presumably due to its relative resistance to the currently practiced biocides and has been implicated in occupational respiratory hazards, particularly hypersensitivity pneumonitis. With an aim to understand its inherent biocide susceptibility profile and survival potential in MWF, five different genotypes of this organism, including a reference genotype (700506) and four novel test genotypes (MJY-3, MJY-4, MJY-10 and MJY-12) isolated in our recent study from diverse MWF operations were evaluated. For this, two commercial biocide formulations, Grotan (Hexahydro-1,3,5-tris(2-hydroxyethyl)-s-triazine) and Kathon (5-chloro-2-methyl-4-isothiazolin-3-one) currently practiced for the control of microorganisms, including mycobacteria, in MWF operations were tested. Effect of the fluid matrix on the biocide susceptibility was investigated for the synthetic (S) and semi-synthetic (SS) MWF matrices. In general, the minimum inhibitory concentration values were higher for the HCHO-releasing biocide Grotan than the isothiazolone biocide Kathon. All genotypes (except the reference genotype) showed lower susceptibility in SS as compared to S fluid matrix for Grotan. However, in case of Kathon, a greater susceptibility was observed in SS fluid for majority of the test genotypes (MJY-3, 4 and 10). The test genotypes were more resistant than the reference genotype to either biocide in both fluid types. Furthermore, the individual genotypes showed differential biocidal susceptibility, with MJY-10 being the most resistant. These observations emphasize the importance of using the resistant genotypes of M. immunogenum as the test strains for formulation or development and evaluation of existing and novel biocides, for industrial applications.     

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.     

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.     

The function of gamma-glutamylcysteine and bis-gamma-glutamylcystine reductase in Halobacterium halobium

gamma-Glutamylcysteine and bis-gamma-glutamylcystine reductase appear to function in the halobacteria in a fashion analogous to GSH and glutathione reductase in other cells. Bis-gamma-glutamylcystine reductase (GCR), a NADPH-dependent dimer of Mr 122,000 recently purified to homogeneity from Halobacterium halobium (Sundquist, A.R., and Fahey, R.C. (1988) J. Bacteriol., 170, 3459-3467), was found to be highly specific for bis-gamma-glutamylcystine and to be present in cell extract at a level sufficient to maintain gamma-glutamylcysteine predominantly in its thiol form [( thiol]/[disulfide] approximately 50). Bis-gamma-glutamylcystine reductase is similar to glutathione reductase in many respects; GCR demonstrated a FAD:subunit stoichiometry of 1, inhibition by heavy metal ions, and a pH optimum near neutrality. However, GCR exhibited no activity with GSSG and was most active at salt levels exceeding 2 M. A turnover number of 1,700 mumol min-1 mumol-1 FAD and apparent Km values of 0.8 mM for bis-gamma-glutamylcystine and 0.29 mM for NADPH were determined for GCR. The effect of salt on the autoxidation rates of gamma-glutamylcysteine, GSH, and Cys was also studied. In the absence of added salt, Cys oxidized more rapidly than gamma-glutamylcysteine, which in turn oxidized more rapidly than GSH. The presence of 4.3 M chloride (K+ and Na+) significantly slowed the autoxidation of all three thiols. The rate of autoxidation of gamma-glutamylcysteine in 4.3 M chloride proved slower than that of GSH in the absence of added chloride. Thus, gamma-glutamylcysteine is at least as stable under halophilic conditions as GSH is under nonhalophilic conditions, explaining why halobacteria utilize gamma-glutamylcysteine rather than GSH.     

Analysis of biocide transport limitation in an artificial biofilm system

An alginate gel bead artificial biofilm system was used to assay biofilm susceptibility to four biocides and to analyse the extent to which each agent penetrated the biofilm. Chlorine, glutaraldehyde, an isothiazolone, and a quaternary ammonium compound were tested on alginate-entrapped Enterobacter aerogenes in gel beads ranging from 1·8 to 6 mm in diameter. Gel-entrapped bacteria were less susceptible to all four antimicrobial agents than were planktonic micro-organisms. The degree of kill measured in artificial biofilm gel beads depended on the size of the gel bead and the cell density at which it was loaded. Disinfection efficacy decreased as gel bead radius or cell density increased. The manifest dependence of biofilm disinfection efficacy on the physical properties of the artificial biofilm (radius and cell density) suggests the impingement of transport limitation of biocide transport into the biofilm. A previously developed theory of biocide reaction and diffusion in biofilm was tested by calculating an appropriate Thiele modulus. In accordance with the theory, the efficacy of all four biocides decreased, albeit noisily, as the Thiele modulus exceeded 1. This result demonstrates that transport limitation can impact antimicrobial performance against biofilms not only of oxidizing biocides but also of non-oxidizing agents.     

Resistance of Pseudomonas aeruginosa to isothiazolone

This investigation was to determine whether Pseudomonas aeruginosa could acquire resistance to the bactericide isothiazolone, and what the nature of such a resistance mechanism would be. The Pseudomonas was cultured in nutrient-limited broth in the presence of sub-inhibitory concentrations of isothiazolone (a mixture of 1.15% 5-chloro- N -methylisothiazolone (CMIT) and 0.35% N -methylisothiazolone (MIT)). Three cultures tested in parallel adapted gradually during exposure for 15 d from an initial minimum inhibitory concentration (MIC) of 300 μl 1^-1 to 607 μl ^-1. The three parallel cultures adapted at similar rates, so the adaptation was not ascribed to mutation but to a specific mechanism. Resistant cells did not produce any extracellular isothiazolone-quenching compounds nor undergo detectable alterations in their lipopolysaccharide layer. In wild cells, a 35 kDa outer membrane protein (protein T) was detectable, whereas resistant cells lacked this protein. Production of protein T was suppressed within 24 h of exposure to isothiazolone. It was still suppressed after 72 h of growth in isothiazolone-free medium. It is proposed that Ps. aeruginosa acquires resistance to isothiazolone by a process of adaptation where the outer membrane protein T is suppressed.     

ppGpp accumulation in Pseudomonas aeruginosa and Pseudomonas fluorescens subjected to nutrient limitation and biocide exposure

The effect of a commonly used biocide, 1,2-benzisothiazolin-3-one (BIT) on ppGpp accumulation in the pathogen, Pseudomonas aeruginosa PAO1, and an environmental isolate, Ps. fluorescens, was examined. It is concluded that BIT is able to induce a stringent response in Ps. aeruginosa and Ps. fluorescens, determined by the rapid accumulation of ppGpp following addition of BIT to exponentially-growing cells. Western blot analysis of whole-cell extracts with anti-RelA antibody demonstrated that both species contain a RelA homologue. This is the first report of a RelA-like protein in pseudomonads.     

Iron dependent degradation of an isothiazolone biocide (5-chloro-2-methyl-4-isothiazolin-3-one)

An isothiazolone biocide, 5-chloro-2-methyl-4-isothiazolin-3-one (CMI), was degraded in the presence of iron. According to the Fe-dependent degradation of CMI, stoichiometric production of chloride was observed. Copper and stainless steel did not enhance the physico-chemical degradation of CMI, whilst phosphate inhibited the Fe-dependent degradation. Neither aerobic nor anaerobic conditions influenced the Fe-dependent CMI degradation. Furthermore, FeO(OH)-powder and Fe(3)O(4)-powder did not lead to the physico-chemical degradation of CMI. Rapid disappearance of CMI was observed in an operating cooling water plant. CMI added to the cooling tower declined from 1.4 mg l(-1) to < 0.1 mg l(-1) in 2 d. This finding is important in optimising the use of CMI and combating resistance if encountered.     

Influence of biocide treatment regimen on resistance development to methylchloro-/methylisothiazolone in Pseudomonas aeruginosa

Resistance development among microbial populations exposed to industrial biocides intending to control microbial levels has received increasing attention in the last few years. Usually studies dealing with resistance development are done after the process has taken place. Characterization of resistant organisms by isolation of dominant populations reveals some details, but the steps leading to resistance usually remain unclear. In this study, development of resistance to methylchloro-/methylisothiazolone (IT) biocide under laboratory conditions is described. Results with experimental setups relevant to field dosing conditions demonstrated how the pattern of biocide treatment influenced the degree of resistance development. The induction of higher resistance in the laboratory populations exposed to different dosing patterns varied. Sequential treatment of culture with a constant increase in concentrations of isothiazolone (5–10 μl l⁻¹ of commercial product) resulted in increasing resistance, exceeding ten-fold. However, additional increases of 25–50 μl l⁻¹ in each step were lethal, suggesting threshold levels of resistance in populations tested. Extrapolation of laboratory data to field conditions appeared more relevant after Pseudomonas species, highly resistant to IT, were isolated from metalworking fluids. In these fluids the biocide treatment regimen in the field was similar to the one used in the laboratory. An understanding of the factors contributing to resistance development and selection in the field is emphasized.     

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.     

Analysis of Cell Wall Constituents of Biocide-Resistant Isolates from Dental-Unit Water Line Biofilms

In past years, the significance of microbial resistance to biocides has increased. Twenty biocide-resistant bacterial strains were isolated from dental-unit water line biofilm. All strains resisted high biocide concentrations (up to 100 mug ml(-)1): sodium dodecyl sulphate, hydrogen peroxide, sodium hypochlorite, phenol, Tween 20, ethylenediaminetetraacetic acid, chlorohexidine gluconate, and povidine iodine. Among bacteria, biocide sensitivity is based on permeability of biocides through the cell wall. Gram-positive bacteria are more permeable and susceptible to biocides, whereas Gram-negative bacteria have a more complex cell wall and are the least sensitive bacteria. The present study was designed to study the effect of biocides on the cell wall of biocide-resistant bacteria. Peptidoglycan (PG), diaminopimelic acid (DAP), and teichoic acid contents of the cell wall were determined in L-broth and L-broth supplemented with biocides at different temperatures (37 degrees C and 45 degrees C) and pH levels (7 and 9). In general and Gram staining-specific comparison, a significant increase (p < 0.05) in the DAP content of biocide-resistant bacteria was observed at pH 7 and at both temperatures. In tubing-specific comparison, a significant increase in the amount of teichoic acid in air water tubing (37 degrees C at pH 9) and DAP in patient tubing (pH 7 at both temperatures) was observed. In main water pipe, a significant decrease (p > 0.05) in PG content was noticed at 45 degrees C and pH 9. Overall, a significant increase in DAP content may be an important constituent in the manifestation of isolate resistance against various biocides.