The assessment of the antibacterial and antifungal activities of aspirin, EDTA and aspirin–EDTA combination and their effectiveness as antibiofilm agents

Aims:  To evaluate the antimicrobial activities of aspirin, EDTA and an aspirin-EDTA (A-EDTA) combination against Pseudomonas aeruginosa , Escherichia coli and Candida albicans in planktonic and biofilm cultures.
Methods and Results:  Minimal inhibitory concentrations (MIC) and minimal biocidal concentrations (MBC) were determined using twofold broth microdilution and viable counting methods, respectively. Aspirin's recorded MIC values ranged from 1·2 to 2·7 mg ml⁻¹. Checkerboard assay demonstrated a synergism in antimicrobial activity upon combination. Aspirin's minimal biofilm eradication concentration values (MBEC) against the established biofilms ranged between 1·35 and 3·83 mg ml⁻¹. A complete eradication of bacterial biofilms was achieved after a 4-h treatment with the A-EDTA combination.
Conclusion:  Both aspirin and EDTA possess broad-spectrum antimicrobial activity for both planktonic and biofilm cultures. Aspirin used at the MBEC for 24 h was successful in eradicating P. aeruginosa , E. coli and C. albicans biofilms established on abiotic surfaces. Moreover, the exposure to the A-EDTA combination (4 h) effected complete bacterial biofilm eradication.
Significance and Impact of the Study:  There is a continuous need for the discovery of new antimicrobial agents. Aspirin and EDTA are 'nonantibiotic drugs', the combination of which can be used successfully to treat and eradicate biofilms established on abiotic surfaces.     

Effect of divalent cations on permeabilizer-induced lysozyme lysis of Pseudomonas aeruginosa

The presence of divalent (Mg²⁺) ions greatly reduced the lysis of Pseudomonas aeruginosa strain G48 in a system at pH 7·8 or 9·0 consisting of ethylenediamine tetraacetic acid (EDTA), lysozyme and tris. Similar reductions in lysis occurred when EDTA was replaced by nitrilotriacetic acid, sodium citrate or sodium polyphosphate. The effect depended on the cation concentration. Mg²⁺ may replace cations removed from the outer membrane, or may effectively remove the permeabilizer from the system. The results suggest that the permeabilizing activity associated with these agents against this organism has a common basis in affecting the outer membrane.     

The synergistic effect of EDTA/antimicrobial combinations on Pseudomonas aeruginosa

AIMS: To demonstrate that the nonlinear concentration-dependent inhibition of Pseudomonas aeruginosa to EDTA can be used to successfully model and predict the potentiation of antimicrobials by EDTA. METHODS AND RESULTS: A model used successfully to describe the concentration-dependent inhibition of bacterial growth caused by many antimicrobials was unable to describe the inhibition of P. aeruginosa by EDTA. Examination of the inhibition profiles for EDTA against P. aeruginosa revealed a biphasic inhibitory pattern suggesting different mechanisms of action at different concentrations. A modelled, two-stage inhibitory process was shown to fit the observations. This model was then used to examine the effect of combining EDTA with other antimicrobials. The apparent synergy of mixtures of EDTA with quaternary ammonium surfactants (QAC) and specific antibiotics was successfully modelled. Minimum inhibitory concentrations (MIC) of the QAC and that of oxacillin and cefamandole were reduced by a factor of 3-10, whereas ampicillin was reduced by a factor of 70 from an MIC of 1524 to 21 mg l(-1) in the presence of 500 mg l(-1) of EDTA. CONCLUSIONS: A nonlinear concentration-dependent inhibition of P. aeruginosa by EDTA gives rise to apparent observation of synergy with other antimicrobials. SIGNIFICANCE AND IMPACT OF THE STUDY: This is a further example where the current methodology for the examination of antimicrobial synergy (the summed fractional inhibitory concentrations) leads to false conclusions.     

Chitosan�CEDTA New Combination is a Promising Candidate for Treatment of Bacterial and Fungal Infections

Chitosan is an attractive preparation widely used as a pharmaceutical excipient. This study aimed to evaluate the antimicrobial activities of chitosan derivatives, EDTA, and the newly developed chitosan-EDTA combination against Gram-negative and Gram-positive bacteria as well as Candida albicans. Antimicrobial activity was studied. Both minimal Inhibitory Concentrations (MIC) and minimal biocidal concentrations (MBC) were determined. Chitosan acetic acid recorded lower MIC values against Enterococcus faecalis, Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Candida albicans than those exhibited by EDTA. EDTA failed to have inhibitory activity against Enterococcus faecalis as well as MBC against any of the studied microorganisms. Chitosan acetic acid's MBC were recorded to all examined species. Checkerboard assay results indicated a synergistic antimicrobial activity of the new combination against Staphylococcus aureus and an additive effect against other microorganisms. Moreover, a short microbial exposure to chitosan-EDTA (20-30 min) caused complete eradication. Due to the continuous emergence of resistant strains, there is an urgent need to discover new antimicrobial agents. Our findings suggest the use of chitosan as an enhancing agent with antibacterial and antifungal properties in combination with EDTA in pharmaceutical preparations.     

Lactic acid permeabilizes gram-negative bacteria by disrupting the outer membrane

The effect of lactic acid on the outer membrane permeability of Escherichia coli O157:H7, Pseudomonas aeruginosa, and Salmonella enterica serovar Typhimurium was studied utilizing a fluorescent-probe uptake assay and sensitization to bacteriolysis. For control purposes, similar assays were performed with EDTA (a permeabilizer acting by chelation) and with hydrochloric acid, the latter at pH values corresponding to those yielded by lactic acid, and also in the presence of KCN. Already 5 mM (pH 4.0) lactic acid caused prominent permeabilization in each species, the effect in the fluorescence assay being stronger than that of EDTA or HCl. Similar results were obtained in the presence of KCN, except for P. aeruginosa, for which an increase in the effect of HCl was observed in the presence of KCN. The permeabilization by lactic and hydrochloric acid was partly abolished by MgCl(2). Lactic acid sensitized E. coli and serovar Typhimurium to the lytic action of sodium dodecyl sulfate (SDS) more efficiently than did HCl, whereas both acids sensitized P. aeruginosa to SDS and to Triton X-100. P. aeruginosa was effectively sensitized to lysozyme by lactic acid and by HCl. Considerable proportions of lipopolysaccharide were liberated from serovar Typhimurium by these acids; analysis of liberated material by electrophoresis and by fatty acid analysis showed that lactic acid was more active than EDTA or HCl in liberating lipopolysaccharide from the outer membrane. Thus, lactic acid, in addition to its antimicrobial property due to the lowering of the pH, also functions as a permeabilizer of the gram-negative bacterial outer membrane and may act as a potentiator of the effects of other antimicrobial substances.     

Effects of disodium salt of ethylenediaminetetraacetic acid (Na2EDTA) and tetracyclines on drug resistant bacteria. Studies in vitro

An effect of Na2EDTA and tetracycline (oxytetracycline and doxycycline) resistant strains of Staphylococcus aureus and Pseudomonas aeruginosa was tested. The strains were isolated from clinical specimens. The tests were performed in vitro by serial dilutions of the drugs in liquid medium. MIC for Na2EDTA, tetracyclines and a combination of Na2EDTA and tetracyclines was determined. It was shown that the combination of oxytetracycline or doxycycline with Na2EDTA caused changes in sensitivity of Staphylococcus aureus and Pseudomonas aeruginosa resistant to these antibiotics. After an application of the mixture of various concentrations of tetracycline and Na2EDTA it was observed that, with the reduction of the effective Na2EDTA dose by about half, the lowest concentrations of tetracyclines inhibiting the growth of resistant bacteria were 2-64 times lower than MIC values of antibiotics without Na2EDTA.     

Offline Controlling of Pseudomonas aeruginosa Resistant to protein Inhibitor Antibiotics Using Combination of EDTA and Na- citrate or Disinfectant (s)

P·aeruginosahave a number of virulence factors like extracellular toxins[1], protea-ses[2,3], haemolysins[4,5], and exopolysaccharide[6,7], which adapt the infection of specifichost tissues[8], causing severe problems·P·aeruginosacan survive in a numbe…     

Ginseng aqueous extract attenuates the production of virulence factors, stimulates twitching and adhesion, and eradicates biofilms of Pseudomonas aeruginosa

This study was carried out to examine the antimicrobial activity of the aqueous extract of Panax quinquefolius from North American ginseng (NAGE) root against Pseudomonas aeruginosa . The minimum inhibitory concentrations of reference and clinical isolates of Pseudomonas aeruginosa were measured by a standard agar-dilution method. At subinhibitory NAGE concentrations, the secretion of virulence factors, motility on agar, and adhesion to 96-well microplates were studied on the nonmucoid Pseudomonas aeruginosa O1 strain. At suprainhibitory concentrations, the activity of NAGE against mature biofilm complexes formed in the Calgary Biofilm Device and the Stovall flow cell were assessed. NAGE possessed an antibacterial activity against all the Pseudomonas aeruginosa strains at 1.25%-2.5% w/v. NAGE also significantly attenuated pyocyanin, pyoverdine, and lipase concentrations, stimulated twitching, and attenuated swarming and swimming motility. At 1.25% w/v, NAGE augmented adhesion, and at 5% w/v detached 1-day-old biofilms in microplates. The extract also eradicated 6-day-old mature biofilms (5% w/v), and fluorescence microscopy displayed a reduction of live cells and biofilm complexes compared with nontreated biofilms. These data suggest that the aqueous extract from North American ginseng possesses antimicrobial activities in vitro.     

Antibacterial activity in vivo and in vitro in the hemolymph of Galleria mellonella infected with Pseudomonas aeruginosa

The antibacterial activity of hemolymph from Galleria mellonella infected with entomopathogenic strain of Pseudomonas aeruginosa and non-pathogenic bacterium Escherichia coli was studied. In vivo, the antimicrobial activity appeared shortly after P. aeruginosa infection, reached the maximum level 18 h postinjection, while 30 h later only trace activity was noted. The activity induced by E. coli sustained on the high level until 48 h after infection. We also noted that the antimicrobial activity level induced by the non-pathogenic bacterium was higher in comparison to that measured in insects infected with the pathogenic strain of P. aeruginosa. The results of our in vitro studies indicated that inducible antimicrobial peptides of G. mellonella larvae were digested by P. aeruginosa elastase B. After 1 h incubation of cell-free hemolymph of immune-challenged larvae with elastase B, no antibacterial activity was observed. It was also shown that elastase B degraded synthetic cecropin B while in the presence of 6 mM EDTA antibacterial activity of cell-free hemolymph as well as cecropin B, was not changed which confirmed that the activity was abolished by the metalloprotease.     

Bacterial killing is enhanced by expression of lysozyme in the lungs of transgenic mice

To assess the role of lysozyme in pulmonary host defense in vivo, transgenic mice expressing rat lysozyme cDNA in distal respiratory epithelial cells were generated. Two transgenic mouse lines were established in which the level of lysozyme protein in bronchoalveolar (BAL) lavage fluid was increased 2- or 4-fold relative to that in WT mice. Lung structure and cellular composition of BAL were not altered by the expression of lysozyme. Lysozyme activity in BAL was significantly increased (6.6- and 17-fold) in 5-wk-old animals from each transgenic line. To determine whether killing of bacteria was enhanced by expression of rat lysozyme, 5-wk-old transgenic mice and WT littermates were infected with 10(6) CFU of group B streptococci or 10(7) CFU of a mucoid strain of Pseudomonas aeruginosa by intratracheal injection. Killing of group B streptococci was significantly enhanced (2- and 3-fold) in the mouse transgenic lines at 6 h postinfection and was accompanied by a decrease in systemic dissemination of pathogen. Killing of Pseudomonas aeruginosa was also enhanced in the transgenic lines (5- and 30-fold). Twenty-four hours after administration of Pseudomonas aeruginosa, all transgenic mice survived, whereas 20% of the WT mice died. Increased production of lysozyme in respiratory epithelial cells of transgenic mice enhanced bacterial killing in the lung in vivo, and was associated with decreased systemic dissemination of pathogen and increased survival following infection.     

Effect of ovotransferrin, protamine sulfate and EDTA combination on biofilm formation by catheter-associated bacteria

AIMS: To determine the effect of a composition comprising ovotransferrin (OT), protamine sulfate (PS) and ethylenediaminetetraacetic acid (EDTA) on biofilm formation by catheter-associated bacteria. METHODS AND RESULTS: The in vitro activity of OT, PS and EDTA alone and in combinations against biofilm formation by Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Proteus mirabilis, Enterococcus faecalis and Staphylococcus epidermidis was investigated. All the three compounds either alone or in combinations failed to inhibit the growth completely at the concentrations tested. However, the subinhibitory concentrations of three compounds in a composition showed synergistic inhibitory effect on biofilm formation by K. pneumoniae, Ps. aeruginosa and S. epidermidis. Furthermore, 79-95% reduction in Ps. aeruginosa and S. epidermidis biofilm formation was observed in a clear vinyl urinary catheter treated with the composition. CONCLUSION: The subinhibitory concentrations of OT, PS and EDTA in a composition were effective in reducing biofilm formation by catheter-associated bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows that a synergistic composition-comprising non-antibiotic generally regarded as safe (GRAS) compounds such as OT, PS and EDTA may be used in the prevention of catheter-related infections.     

Antimicrobial properties of highly fluorinated tris(pyrazolyl)borates

Highly fluorinated tris(pyrazolyl)borates were tested for their antimicrobial activity against various bacterial species. Both the silver(I) tris(pyrazolyl)borate [HB(3,5-(CF(3))(2)Pz)(3)]Ag(THF) (THF=tetrahydrofuran) and the sodium analog [HB(3,5-(CF(3))(2)Pz)(3)]Na(THF) appeared highly effective at inhibiting the growth of two different species of Gram-positive bacteria (i.e. being 12 and 21 fold more effective, respectively, (on a molar basis, based on the minimum inhibitory concentrations) against Staphylococcus aureus than silver sulfadiazine, a currently used silver antimicrobial). This suggests that the ligand portion of these molecules is responsible for the observed high effectiveness against the Gram-positive species. Furthermore, it appeared that the fluorinated substituents on the tris(pyrazolyl)borate were important for this high level of growth inhibition. Against two species of Gram-negative bacteria, including Pseudomonas aeruginosa, the fluorinated silver(I) tris(pyrazolyl)borate exhibited a moderate level of growth inhibition (similar to that of silver sulfadiazine), while the sodium analog showed very little ability to inhibit growth, indicating that for the Gram-negative species, the apparent responsible antimicrobial portion is the silver ion.     

Resistance of Pseudomonas to Quaternary Ammonium Compounds. I. Growth in Benzalkonium Chloride Solution

Resistant cells of Pseudomonas aeruginosa and a waterborne Pseudomonas sp. (strain Z-R) were able to multiply in nitrogen-free minimal salts solution containing various concentrations of commercially prepared, ammonium acetate-buffered benzalkonium chloride (CBC), a potent antimicrobial agent. As the CBC concentration increased, growth increased until a point was reached at which the extent of growth leveled off or was completely depressed. Minimal salts solutions of pure benzalkonium chloride (PBC) containing no ammonium acetate did not support bacterial growth. When ammonium acetate was added to PBC solutions in the same concentrations found in CBC solutions, growth patterns developed that were comparable to those found with CBC. Likewise, (NH(4))(2)SO(4) added to PBC solutions supported growth of both organisms. P. aeruginosa was initially resistant to CBC levels of 0.02% and it was adapted to tolerate levels as high as 0.36%. Strain Z-R was naturally resistant to 0.4% CBC. Since ammonium acetate, carried over by the CBC used in drug formulations and disinfectant solutions, has the potential to support the growth of resistant bacteria and thus make possible the risk of serious infection, it is suggested that regulations allowing the presence of ammonium acetate in CBC solution be reconsidered.     

Antimicrobial activity and effectiveness of a combination of sodium hypochlorite and hydrogen peroxide in killing and removing Pseudomonas aeruginosa biofilms from surfaces

AIMS: To evaluate both the antimicrobial activity and the effectiveness of a combination of sodium hypochlorite and hydrogen peroxide (Ox-B) for killing Pseudomonas aeruginosa ATCC 19142 cells and removing P. aeruginosa biofilms on aluminum or stainless steel surfaces. METHODS AND RESULTS: Pseudomonas aeruginosa biofilms were developed in tryptic soy broth containing vertically suspended aluminium or stainless steel plates. Biofilms were exposed to a mixed sodium hypochlorite and hydrogen peroxide solution as a sanitizer for 1, 5 and 20 min. The sanitizer was then neutralized, the cells dislodged from the test surfaces, and viable cells enumerated. Cell morphologies were determined using scanning (SEM) and transmission electron microscopy (TEM). Cell viability was determined by confocal scanning laser microscopy (CSLM). Biofilm removal was monitored by Fourier transform infrared (FTIR) spectrophotometry. Cell numbers were reduced by 5-log to 6-log after 1 min exposure and by 7-log after 5 min exposure to Ox-B. No viable cells were detected after a 20 min exposure. Treatment with equivalent concentrations of sodium hypochlorite reduced viable numbers by 3-log to 4-log after 1 min exposure and by 4-log to 6-log after 5 min, respectively. A 20 min exposure achieved a 7-log reduction. Hydrogen peroxide at test concentration treatments showed no effect. FTIR analysis of treated pseudomonad biofilms on aluminium or stainless steel plates showed either a significant reduction or complete removal of biofilm material after a 5 min exposure to the mixed sodium hypochlorite and hydrogen peroxide solution. SEM and TEM images revealed damage to cell wall and cell membranes. CONCLUSIONS: A combination of sodium hypochlorite and hydrogen peroxide effectively killed P. aeruginosa cells and removed biofilms from both stainless steel and aluminium surfaces. SIGNIFICANCE AND IMPACT OF THE STUDY: The combination of sodium hypochlorite and hydrogen peroxide can be used as an alternative disinfectant and/or biofilm remover of contaminated food processing equipment.     

Examination of bacterial resistance to exogenous nitric oxide

While much research has been directed to harnessing the antimicrobial properties of exogenous NO, the possibility of bacteria developing resistance to such therapy has not been thoroughly studied. Herein, we evaluate potential NO resistance using spontaneous and serial passage mutagenesis assays. Specifically, Staphylococcus aureus, Methicillin-resistant S. aureus (MRSA), Staphylococcus epidermidis, Escherichia coli, and Pseudomonas aeruginosa were systematically exposed to NO-releasing 75mol% MPTMS-TEOS nitrosothiol particles at or below minimum inhibitory concentration (MIC) levels. In the spontaneous mutagenesis assay, bacteria that survived exposure to lethal concentrations of NO showed no increase in MIC. Similarly, no increase in MIC was observed in the serial passage mutagenesis assay after exposure of these species to sub-inhibitory concentrations of NO through 20 d.     

The metabolically active subpopulation in Pseudomonas aeruginosa biofilms survives exposure to membrane-targeting antimicrobials via distinct molecular mechanisms

Biofilms are reported to be inherently refractory toward antimicrobial attack and, therefore, cause problems in industrial and medical settings. Pseudomonas aeruginosa biofilms contain subpopulations that exhibit high metabolic activity and subpopulations that exhibit low metabolic activity. We have found that membrane-targeting antimicrobials such as colistin, EDTA, SDS, and chlorhexidine specifically kill the inactive subpopulation in P.?aeruginosa biofilms, whereas the active subpopulation survives exposure to these compounds. Because treatment of P.?aeruginosa biofilms with the membrane-targeting compounds colistin, EDTA, SDS, and chlorhexidine resulted in the same spatial distribution of live and dead bacteria, we investigated whether tolerance to these compounds originated from the same molecular mechanisms. Development of colistin-tolerant subpopulations was found to depend on the pmr genes encoding lipopolysaccharide modification enzymes, as well as on the mexAB-oprM, mexCD-oprJ, and muxABC-opmB genes encoding antimicrobial efflux pumps, but does not depend on the mexPQ-opmE efflux pump genes. Development of chlorhexidine-tolerant subpopulations was found to depend on the mexCD-oprJ genes, but does not depend on the pmr, mexAB-oprM, mexPQ-opmE, or muxABC-opmB genes. Tolerance to SDS and EDTA in P. aeruginosa biofilms is linked to metabolically active cells, but does not depend on the pmr, mexAB, mexCD, mexPQ, or muxABC genes. Our data suggest that the active subpopulation in P.?aeruginosa biofilms is able to adapt to exposure to membrane-targeting agents through the use of different genetic determinants, dependent on the specific membrane-targeting compound.     

Ethylenediaminetetraacetate-extractable protein-lipopolysaccharide complex of Pseudomonas aeruginosa: characterization of protein components

Five major outer membrane proteins (D1, D2, E, G, and H1) of Pseudomonas aeruginosa, but not proteins F (porin), I (lipoprotein), and H2, were detected in high-molecular-weight protein-lipopolysaccharide complex(es) solubilized from sucrose-stabilized cells on exposure to ethylenediaminetetraacetate and tris(hydroxymethyl)aminomethane.     

Changes in <Emphasis Type="Italic">Galleria mellonella</Emphasis> lysozyme level and activity during <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> infection

The level of lysozyme in fat body, hemocytes and cell-free hemolymph from Galleria mellonella larvae infected with Pseudomonas aeruginosa was determined and evaluated. In the samples of fat body and hemocytes, an increase in lysozyme content was detected 1 d after infection and then a significant decrease was observed after a prolonged infection time. In the case of cell-free hemolymph, an increase in the lysozyme level was noticeable during the first 30 h post injection and stayed at a similar level for 42 h. The smaller decrease of the lysozyme level after 42 h might be associated with the development of bacteremia of P. aeruginosa in insects. In addition, the gradual increase in the content of lysozyme correlated with the increase of its activity in the hemolymph of the infected larvae as a response to injection with P. aeruginosa. The G. mellonella lysozyme appeared to be insensitive to extracellular proteinases produced in vivo by P. aeruginosa.     

Interactions between Pseudomonas aeruginosa and iodophor germicides in milking parlor udder wash water systems

In a field study of 29 dairy farms, Pseudomonas aeruginosa was isolated more frequently (P = 0.05) from milking parlor udder wash water systems containing iodophor germicides than from those with no germicide. Most available iodine (AI2) concentrations were below the recommended level of 25 ppm (25 microgram/ml). Rubber and polyvinyl chloride hoses caused rapid decreases in the AI2 concentrations of 25 ppm iodophor solutions. AI2 dropped from 25 ppm to 6 ppm or less in 240 min for solutions contained in either polyvinyl chloride or rubber, compared with solutions in glass, which were unchanged in 240 min. Addition of inactivated iodophor solution to aqueous cultures resulted in significantly higher (P less than 0.05) numbers of P. aeruginosa at 10 and 24 h postinoculation. P. aeruginosa was grown in polyvinyl chloride tubing and exposed twice daily to 0, 10, or 25 ppm of AI2. None of the exposure concentrations eliminated the bacteria from the hoses, and bacterial numbers were not significantly different in hoses exposed to 0 and 10 ppm by the eighth treatment day. Bacteria taken from the water in these hoses were exposed to different concentrations of iodophor solution. Iodophor concentrations which will kill 50% of P. aeruginosa cultures previously exposed to 0, 10, and 25 ppm of AI2 were predicted to be 3.0, 11.8, and 20.8 ppm, respectively.