Effect of Ethylenediaminetetraacetic Acid and Related Chelating Agents on Whole Cells of Gram-Negative Bacteria

Haque, Hasna Fahmima; Russell, A. D.

doi:10.1128/aac.5.5.447

Cited by 40 publications

(25 citation statements)

References 12 publications

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“…Since sodium tripolyphosphate is a chelating agent, it is plausible, as with other chelators such as EDTA, that this antibacterial activity occurs by disruption of the bacterial cell envelope through the sequestration of stabilizing divalent cations. Such cations normally link bacterial lipopolysaccharides to the outer membrane and interference with this process can destabilize the outer membrane in Gram-negative bacteria, impairing barrier function (50,51,52). Furthermore, strong chelating agents may inhibit bacterial growth by sequestering trace minerals required for bacterial metabolism (51,53).…”

Section: Discussionmentioning

confidence: 99%

Effects of Formulation on Microbicide Potency and Mitigation of the Development of Bacterial Insusceptibility

Cowley

Forbes

Amézquita

et al. 2015

Appl Environ Microbiol

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bRisk assessments of the potential for microbicides to select for reduced bacterial susceptibility have been based largely on data generated through the exposure of bacteria to microbicides in aqueous solution. Since microbicides are normally formulated with multiple excipients, we have investigated the effect of formulation on antimicrobial activity and the induction of bacterial insusceptibility. We tested 8 species of bacteria (7 genera) before and after repeated exposure (14 passages), using a previously validated gradient plating system, for their susceptibilities to the microbicides benzalkonium chloride, benzisothiozolinone, chlorhexidine, didecyldimethyl ammonium chloride, DMDM-hydantoin, polyhexamethylene biguanide, thymol, and triclosan in aqueous solution (nonformulated) and in formulation with excipients often deployed in consumer products. Susceptibilities were also assessed following an additional 14 passages without microbicide to determine the stability of any susceptibility changes. MICs and minimum bactericidal concentrations (MBC) were on average 11-fold lower for formulated microbicides than for nonformulated microbicides. After exposure to the antimicrobial compounds, of 72 combinations of microbicide and bacterium there were 19 >4-fold (mean, 8-fold) increases in MIC for nonformulated and 8 >4-fold (mean, 2-fold) increases in MIC for formulated microbicides. Furthermore, there were 20 >4-fold increases in MBC (mean, 8-fold) for nonformulated and 10 >4-fold (mean, 2-fold) increases in MBC for formulated microbicides. Susceptibility decreases fully or partially reverted back to preexposure values for 49% of MICs and 72% of MBCs after further passage. In summary, formulated microbicides exhibited greater antibacterial potency than unformulated actives and susceptibility decreases after repeated exposure were lower in frequency and extent. Microbicides are broad-spectrum chemical agents that inactivate microorganisms (1-3). They are widely deployed throughout health care (4-6), domestic (7,8), and industrial (9-11) environments, where their application includes antisepsis (12), hard surface disinfection (13), and pharmaceutical product preservation (14). They may also be incorporated into medical device coatings, for instance in sutures (15), wound dressings (16), and urinary catheters (17) to inhibit bacterial adhesion and subsequent biofilm formation.It has been hypothesized that the use of microbicides could select for bacterial adaptation, resulting in reduced efficacy of the primary agent, as well as potentially decreasing bacterial susceptibility to chemically unrelated agents such as other microbicides and antibiotics (18). While there have been reports documenting the laboratory selection of bacteria with decreased microbicide sensitivity following repeated exposure to microbicides in highly selective conditions, it remains unclear whether this commonly occurs in the environment (19-24).The majority of studies reporting reductions in microbicide susceptibility have used the active compo...

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Section: Discussionmentioning

confidence: 99%

Effects of Formulation on Microbicide Potency and Mitigation of the Development of Bacterial Insusceptibility

Cowley

Forbes

Amézquita

et al. 2015

Appl Environ Microbiol

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“…Agar plates containing concentrations of 0 to 2 ,ug of cetrimide, benzalkonium chloride, and chlorhexidine per ml were "spotted" with low numbers of viable cells of each organism, and the colonies were counted after incubation. Counts on drug-containing plates were not significantly different from those on control plates (no drug present), and it was concluded that the concentrations in agar of cetrimide, benzalkonium chloride, and chlorhexidine should not exceed 2 ,ug/ml. In the actual viable counting for determining survivor levels (below), levels of these compounds in agar were 1/106 of those used in Tables 2 to 5 and thus were negligible.…”

Section: Mics Of the Antibacterial Agents Thementioning

confidence: 99%

“…This slow initial rate of death could be related to the magnitude of the effect of the chelating agent. The two P. aeruginosa strains are the most susceptible to EDTA and related substances (2), and thus pretreated cells would be expected to be killed most rapidly by a sublethal drug concentration; strain Kl is the most resistant to these chelating agents (2), and thus pretreated cells would be expected to be killed least rapidly by such a drug concentration. These, in fact, occur (Tables 2-4), and it is thus tempting to speculate that cell wall cations, by virtue of their binding effect, are involved to considerable extent in cell resistance.…”

Section: Mics Of the Antibacterial Agents Thementioning

confidence: 99%

“…Haque and Russell (2) have recently shown that two strains of Pseudomonas aeruginosa were considerably more susceptible to ethylenediaminetetraacetic acid (EDTA) and related chelating agents than were other, nonPseudomonas strains of gram-negative bacteria. Of the chelating agents studied, nitriloacetic acid (NTA) and iminodiacetic acid (IDA) had little or no inhibitory activity even when used at concentrations 10 times higher than the other substances.…”

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confidence: 99%

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Effect of Chelating Agents on the Susceptibility of Some Strains of Gram-Negative Bacteria to Some Antibacterial Agents

Haque

Russell

1974

Antimicrob Agents Chemother

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The effects of ethylenediaminetetraacetic acid and related chelating agents on the susceptibility of Pseudomonas aeruginosa and other strains of gram-negative bacteria to some fi-lactam antibiotics and some non-antibiotic antibacterial agents have been studied by two methods: (i) the determination of minimal inhibitory concentrations in the presence and absence of a chelating agent; and (ii) pretreatment with a chelating agent, followed by exposure for up to 120 min at 37 C to a fl-lactam drug in a nutrient medium or to the other type of antibacterial agent in a non-nutrient environment. The pretreatment technique gave the more significant results, especially with chlorhexidine, cetrimide, and benzalkonium chloride. Chelating agents that had previously been found to give a low order of activity as measured by effects on bacterial viability nevertheless appeared to induce some changes in the surface layers of the treated organisms, rendering them susceptible to sub-inhibitory concentrations of non-antibiotic drugs.Haque and Russell (2) have recently shown that two strains of Pseudomonas aeruginosa were considerably more susceptible to ethylenediaminetetraacetic acid (EDTA) and related chelating agents than were other, nonPseudomonas strains of gram-negative bacteria. Of the chelating agents studied, nitriloacetic acid (NTA) and iminodiacetic acid (IDA) had little or no inhibitory activity even when used at concentrations 10 times higher than the other substances. It was also found (2) that the two strains of P. aeruginosa were the most resistant to nonchelating antibacterial agents such as cetrimide, chlorhexidine, and benzalkonium chloride.Leive (3-5) has shown that pretreatment of Escherichia coli with EDTA rendered the cells susceptible to actinomycin D; this method has since been adopted by other workers using E. coli or other strains and various antibacterial agents (11). Thus a study has been made of the effect of pretreatment, or simultaneous treatment, with EDTA or other chelating agents on the susceptibility of P. aeruginosa and some other gram-negative strains to some fi-lactam antibiotics (cephaloridine, ampicillin, carbenicillin) and to other antibacterial agents (benzalkonium chloride, cetrimide, chlorhexidine).MATERIALS AND METHODS Bacterial strains. These consisted of P. aeruginosa NCTC 6750 and NCTC 1999, E. coli R+TEM and R-TEM, Enterobacter cloacae P99 and P99M, and Klebsiella aerogenes KJ. Cultures were grown overnight at 37 C on 150 ml of nutrient agar (Oxoid, Ltd., London) in Roux flasks; the cells were collected, washed twice with tris(hydroxymethyl)aminomethane (Tris) buffer (10-2 M, pH 7.8), and resuspended to a density of about 10' viable cells/ml in the same buffer.Antibacterial agents. Carbenicillin sodium and ampicillin sodium were purchased from Beecham

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“…Additionally, the formulation included chelating agents such as sodium tripolyphosphate, which may, as is the case with many chelators, have antimicrobial activity due to its ability to disrupt the bacterial cell envelope (39). This may occur through the sequestering of stabilizing divalent cations, causing the release of lipopolysaccharides from the cell envelope and consequently impairing barrier function (40,41). This provides a plausible explanation for the heightened antimicrobial activity observed with the BAC formulation compared to the simple aqueous BAC solution.…”

Section: Discussionmentioning

confidence: 99%

Formulation of Biocides Increases Antimicrobial Potency and Mitigates the Enrichment of Nonsusceptible Bacteria in Multispecies Biofilms

Forbes

Cowley

Humphreys

et al. 2017

Appl Environ Microbiol

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The current investigation aimed to generate data to inform the development of risk assessments of biocide usage. Stabilized domestic drain biofilm microcosms were exposed daily over 6 months to increasing concentrations (0.01% to 1%) of the biocide benzalkonium chloride (BAC) in a simple aqueous solution (BAC-s) or in a complex formulation (BAC-f) representative of a domestic cleaning agent. Biofilms were analyzed by culture, differentiating by bacterial functional group and by BAC or antibiotic susceptibility. Bacterial isolates were identified by 16S rRNA sequencing, and changes in biofilm composition were assessed by high-throughput sequencing. Exposure to BAC-f resulted in significantly larger reductions in levels of viable bacteria than exposure to BAC-s, while bacterial diversity greatly decreased during exposure to both BAC-s and BAC-f, as evidenced by sequencing and viable counts. Increases in the abundance of bacteria exhibiting reduced antibiotic or BAC susceptibility following exposure to BAC at 0.1% were significantly greater for BAC-s than BAC-f. Bacteria with reduced BAC and antibiotic susceptibility were generally suppressed by higher BAC concentrations, and formulation significantly enhanced this effect. Significant decreases in the antimicrobial susceptibility of bacteria isolated from the systems before and after longterm BAC exposure were not detected. In summary, dose-dependent suppression of bacterial viability by BAC was enhanced by formulation. Biocide exposure decreased bacterial diversity and transiently enriched populations of organisms with lower antimicrobial susceptibility, and the effects were subsequently suppressed by exposure to 1% BAC-f, the concentration most closely reflecting deployment in formulated products.IMPORTANCE Assessment of the risks of biocide use has been based mainly on the exposure of axenic cultures of bacteria to biocides in simple aqueous solutions. The current investigation aimed to assess the effects of formulation on the outcome of biocide exposure in multispecies biofilms. Formulation of the cationic biocide BAC significantly increased antimicrobial potency. Bacteria with lower antimicrobial susceptibility whose populations were enriched after low-level biocide exposure were more effectively suppressed by the biocide at in-use concentrations (1% [wt/vol]) in a formulation than in a simple aqueous solution. These observations underline the importance of simulating normal deployment conditions in considering the risks and benefits of biocide use.

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Effect of Ethylenediaminetetraacetic Acid and Related Chelating Agents on Whole Cells of Gram-Negative Bacteria

Cited by 40 publications

References 12 publications

Effects of Formulation on Microbicide Potency and Mitigation of the Development of Bacterial Insusceptibility

Effects of Formulation on Microbicide Potency and Mitigation of the Development of Bacterial Insusceptibility

Effect of Chelating Agents on the Susceptibility of Some Strains of Gram-Negative Bacteria to Some Antibacterial Agents

Formulation of Biocides Increases Antimicrobial Potency and Mitigates the Enrichment of Nonsusceptible Bacteria in Multispecies Biofilms

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