Kinetic evaluation of claimed synergistic paraben combinations using a factorial design.

The antimicrobial effects of methyl and propyl parabens are investigated, with Escherichia coli as test organism, with a view to determining whether the parabens act synergistically. At appropriate concentrations, the parabens killed E. coli cells according to first order kinetics and the bactericidal effects were quantified by the first order kill rate constants. Combinations of methyl or propyl parabens, at concentrations which slow down or inhibit bacterial growth when used singly, produced definite kill. In this sense, the parabens are therefore synergistic since in combination they produce an effect which is not observed when they are used singly. This effect is not true synergism as shown by the results of our experiments with a factorial design. Analysis of variance indicated no significant interaction between the two parabens.     

The bactericidal activity of a methyl and propyl parabens combination: isothermal and non-isothermal studies

D. GILLILAND, A. LI WAN PO AND E. SCOTT. 1992. The effect of temperature on the kill rate of Escherichia coli by methyl and propyl parabens was studied. The kill kinetics was first order. It was shown that the Arrhenius equation provided a good model for describing the relationship between the first order rate constant and the temperature. The activation energy was found to be 274 kJ/mol for exponential phase cells and 168 kJ/mol for stationary phase cells. Exponential phase cells were much more susceptible to the lethal effects of the parabens than were the stationary phase cells. For example, at 34°C stationary phase cells, in chemically defined media, had a kill rate constant of 0.072/h while the corresponding value for exponential phase cells was 0.238/h. In water the rate of kill for exponential phase cells was even faster giving a rate constant of 5.25/h at 34°C. Non-isothermal kinetic testing was not found to be useful for modelling bacterial kill kinetics because we could not achieve the precision required in bacterial enumeration.     

The bactericidal activity of a methyl and propyl parabens combination: isothermal and non-isothermal studies.

The effect of temperature on the kill rate of Escherichia coli by methyl and propyl parabens was studied. The kill kinetics was first order. It was shown that the Arrhenius equation provided a good model for describing the relationship between the first order rate constant and the temperature. The activation energy was found to be 274 kJ/mol for exponential phase cells and 168 kJ/mol for stationary phase cells. Exponential phase cells were much more susceptible to the lethal effects of the parabens than were the stationary phase cells. For example, at 34 degrees C stationary phase cells, in chemically defined media, had a kill rate constant of 0.072/h while the corresponding value for exponential phase cells was 0.238/h. In water the rate of kill for exponential phase cells was even faster giving a rate constant of 5.25/h at 34 degrees C. Non-isothermal kinetic testing was not found to be useful for modelling bacterial kill kinetics because we could not achieve the precision required in bacterial enumeration.     

Quantification of the urinary concentrations of parabens in humans by on-line solid phase extraction-high performance liquid chromatography-isotope dilution tandem mass spectrometry

Parabens (alkyl esters of p-hydroxybenzoic acid) are widely used as antimicrobial preservatives in cosmetic products, pharmaceuticals, and food processing. However, weak estrogenicity of some parabens has been revealed from several studies. Human exposure to parabens may be assessed by measuring the conjugated or free species of these compounds or their metabolites in urine. We have developed a method using on-line solid phase extraction-high performance liquid chromatography-isotope dilution tandem mass spectrometry with peak focusing to measure the urinary concentrations of methyl, ethyl, propyl, n- and iso- butyl, and benzyl parabens. This method has good reproducibility and accuracy with detection limits for all analytes below 0.2ng/mL in 100microL of urine, and permits quick and accurate analysis of a large number of samples in epidemiologic studies for assessing the prevalence of human exposure to parabens. Using this method, we detected methyl, ethyl, and propyl parabens, mostly as conjugated species, in 22 urine samples collected from anonymous adults.     

Antimicrobial efficacy of potassium salts of four parabens

Summary The antimicrobial activity of the soluble potassium salts of methyl, ethyl, propyl, and butyl parabens were evaluated to determine whether they would be more effective than their respective parabens (esters ofp-hydroxybenzoic acids). The potassium salts of the methyl and ethyl parabens as well as methyl and ethyl parabens were microbiocidal against the fungusAspergillus niger and five bacteria, whereas the potassium salts of propyl and butyl parabens and their respective parabens were not microbiocidal against all the test organisms. In the presence of several ingredients frequently used in pharmaceutical and cosmetic formulations, ethylenediaminetetraacetate (EDTA) and magnesium hydroxide did not interfere with the antimicrobial activity of the potassium salts of parabens and appeared to be microbiocidal against three of four test organisms. Simethicone and Tween 80 interfered with the antimicrobial activity of the preservatives. At pH 4–6, the potassium salt of butyl paraben, the only preservative tested, was active against more organisms than at pH 7–8. Overall, the highly soluble potassium salts of parabens showed microbiocidal activity against more of the test organisms than the less soluble parabens.     

The effect of parabens on DNA, RNA and protein synthesis in Escherichia coli and Bacillus subtilis

The effect of methyl, propyl and butyl esters of p -hydroxybenzoic acid on DNA and RNA synthesis has been tested in toluenized cells of Escherichia coli and Bacillus subtilis. Both RNA and DNA synthesis of these bacteria were inhibited. The inhibitory concentrations were higher than those previously reported for growth inhibition. Protein synthesis in cell-free extracts (S-30 fraction) of B. subtilis was even more sensitive to parabens than DNA and RNA synthesis, while protein synthesis in Esch. coli was largely unaffected.     

The effect of parabens on DNA, RNA and protein synthesis in Escherichia coli and Bacillus subtilis

The effect of methyl, propyl and butyl esters of p-hydroxybenzoic acid on DNA and RNA synthesis has been tested in toluenized cells of Escherichia coli and Bacillus subtilis. Both RNA and DNA synthesis of these bacteria were inhibited. The inhibitory concentrations were higher than those previously reported for growth inhibition. Protein synthesis in cell-free extracts (S-30 fraction) of B. subtilis was even more sensitive to parabens than DNA and RNA synthesis, while protein synthesis in Esch. coli was largely unaffected.     

prbA,a gene coding for an esterase hydrolyzing parabens in enterobacter cloacae and Enterobacter gergoviae strains

The new gene prbA encodes an esterase responsible for the hydrolysis of the ester bond of parabens in Enterobacter cloacae strain EM. This gene is located on the chromosome of strain EM and was cloned by several PCR approaches. The prbA gene codes for an immature protein of 533 amino acids, the first 31 of which represent a proposed signal peptide yielding a mature protein of a putative molecular mass of 54.6 kDa. This enzyme presents analogies with other type B carboxylesterases, mainly of eukaryotic origin. The cloning and expression of the prbA gene in a strain of Escherichia coli previously unable to hydrolyze parabens resulted in the acquisition of a hydrolytic capacity comparable to the original activity of strain EM, along with an increased resistance of the transformed strain to methyl paraben. The presence of homologues of prbA was tested in additional ubiquitous bacteria, which may be causative factors in opportunistic infections, including Enterobacter gergoviae, Enterobacter aerogenes, Pseudomonas agglomerans, E. coli, Pseudomonas aeruginosa, and Burkholderia cepacia. Among the 41 total strains tested, 2 strains of E. gergoviae and 1 strain of Burkholderia cepacia were able to degrade almost completely 800 mg of methyl paraben liter(-1). Two strains of E. gergoviae, named G1 and G12, contained a gene that showed high homology to the prbA gene of E. cloacae and demonstrated comparable paraben esterase activities. The significant geographical distance between the locations of the isolated E. cloacae and E. gergoviae strains suggests the possibility of an efficient transfer mechanism of the prbA gene, conferring additional resistance to parabens in ubiquitous bacteria that represent a common source of opportunistic infections.     

Novel natural parabens produced by a Microbulbifer bacterium in its calcareous sponge host Leuconia nivea

A broad variety of natural parabens, including four novel structures and known ethyl and butyl parabens, were obtained from culture of a Microbulbifer sp. bacterial strain isolated from the temperate calcareous marine sponge Leuconia nivea (Grant 1826). Their structures were elucidated from spectral analysis, including mass spectrometry and 1D and 2D nuclear magnetic resonance. Their antimicrobial activity evaluated against Staphylococcus aureus was characterized by much higher in vitro activity of these natural paraben compounds 3–9 than commercial synthetic methyl and propyl parabens, usually used as antimicrobial preservatives. Compounds 4 and 9 revealed a bacteriostatic effect and compounds 6 and 7 appeared as bactericidal compounds. Major paraben compound 6 was also active against Gram positive Bacillus sp. and Planococcus sp. sponge isolates and was detected in whole sponge extracts during all seasons, showing its persistent in situ production within the sponge. Moreover, Microbulbifer sp. bacteria were visualized in the sponge body wall using fluorescence in situ hybridization with a probe specific to L4-n2 phylotypes. Co-detection in the sponge host of both paraben metabolites and Microbulbifer sp. L4-n2 indicates, for the first time, production of natural parabens in a sponge host, which may have an ecological role as chemical mediators.     

Purification and characterization of PrbA,a new esterase from Enterobacter cloacae hydrolyzing the esters of 4-hydroxybenzoic acid (parabens)

The esterase PrbA from Enterobacter cloacae strain EM has previously been shown to confer additional resistance to the esters of 4-hydroxybenzoic acid (parabens) to two species of Enterobacter. The PrbA protein has been purified from E. cloacae strain EM using a three-step protocol resulting in a 60-fold increase in specific activity. The molecular mass of the mature enzyme was determined to be 54,619 +/- 1 Da by mass spectrometry. It is highly active against a series of parabens with alkyl groups ranging from methyl to butyl, with K(m) and V(max) values ranging from 0.45 to 0.88 mM and 0.031 to 0.15 mM/min, respectively. The K(m) and V(max) values for p-nitrophenyl acetate were 3.7 mM and 0.051 mM/min. PrbA hydrolyzed a variety of structurally analogous compounds, with activities larger than 20% relative to propyl paraben for methyl 3-hydroxybenzoate, methyl 4-aminobenzoate, or methyl vanillate. The enzyme showed optimum activity at 31 degrees C and at pH 7.0. PrbA was able to transesterify parabens with alcohols of increasing chain length from methanol to n-butanol, achieving 64% transesterification of 0.5 mm propyl paraben with 5% methanol within 2 h. PrbA was inhibited by 1-chloro-3-tosylamido-4-phenyl-2-butanone and 1-chloro-3-tosylamido-7-amino-2-heptanone (TLCK), with K(i) values of 0.29 and 0.20 mM, respectively, and was irreversibly inhibited by Diisopropyl fluorophosphate (DFP) or diethyl pyrocarbonate. The stoichiometry of addition of DFP to the enzyme was 1:1 and only 1 TLCK molecule was found in TLCK-modified enzyme, as measured by mass spectrometry. Analysis of the tryptic digest of the DFP-modified PrbA demonstrated that the addition of a DFP molecule occurred at Ser-189, indicating the location of the active serine.     

The membrane lateral pressure-perturbing capacity of parabens and their effects on the mechanosensitive channel directly correlate with hydrophobicity

Lipid bilayers provide a natural anisotropic environment for membrane proteins and can serve as apolar reservoirs for lipid-derived second messengers or lipophilic drugs. Partitioning of lipophilic agents changes the lateral pressure distribution in the bilayer, affecting integral proteins. p-Hydroxybenzoic acid esters (parabens) are amphipathic compounds widely used as food and cosmetics preservatives, but the mechanisms of their broad antibacterial action are unknown. Here we describe effects of ethyl, propyl, and butyl parabens on the gating of the bacterial mechanosensitive channel of small conductance (MscS) and compare them with the surface activity and lateral pressure changes measured in lipid monolayers in the presence of these substances. Near the bilayer-monolayer equivalence pressure of 35 mN/m, ethyl, propyl, or butyl paraben present in the subphase at 1 mM increased the surface pressure of the monolayer by 5, 12.5, or 20%, respectively. No spontaneous activation of MscS channels was observed in patch-clamp experiments with parabens added from either the cytoplasmic or periplasmic side. Increasing concentrations of parabens on the cytoplasmic side of excised patches shifted activation curves of MscS toward higher tensions. A good correlation between the pressure increases in monolayers and shifts in activation midpoints in patch-clamp experiments suggested that the more hydrophobic parabens partition more strongly into the lipid and exert larger effects on channel gating through changes in lateral pressure. We show that cytoplasmically presented ethyl or butyl parabens both hasten the process of desensitization of MscS and influence inactivation differently. The higher rate of desensitization is likely due to increased lateral pressure in the cytoplasmic leaflet surrounding the gate. Neither of the parabens strongly affects the rate of recovery and does not seem to penetrate the TM2-TM3 interhelical clefts in MscS. We conclude that the bacterial mechanosensitive channel MscS provides a sensitive readout of lateral membrane pressure exerted by amphipathic molecules but may not be the primary target for the parabens in their antimicrobial activity.     

Lack of effect of butylparaben and methylparaben on the reproductive system in male rats

BACKGROUND: Parabens are widely used preservatives in cosmetics and pharmaceutical products, and approved as food additives. Parabens have been considered safe for these uses for many years. Recently, adverse effects on male reproductive parameters in rats have been reported when parabens were given orally for 8 weeks starting at three weeks of age. Our studies used two representative parabens, methyl‐ and butylparaben, to try to replicate these studies and thereby evaluate potential reproductive effects in male Wistar rats. METHODS: Diets containing 0, 100, 1000 or 10,000 ppm of either butyl‐ or methylparaben were fed to male rats for eight weeks. Rats were 22 days of age at the start of exposure. Parameters evaluated included organ weights, histopathology of reproductive tissues, sperm production, motility, morphology and reproductive hormone levels (butylparaben only). RESULTS: None of the parameters evaluated for either paraben showed compound‐ or dosage‐dependent adverse effects. Metabolism experiments of butylparaben indicate that it is rapidly metabolized by non‐specific esterases to p‐hydroxybenzoic acid and butanol, neither of which is estrogenic. CONCLUSIONS: Exposure to methyl‐ or butylparaben in the diet for eight weeks did not affect any male reproductive organs or parameters at exposures as high as 10,000 ppm, corresponding to a mean daily dose of 1,141.1±58.9 or 1,087.6±67.8 mg/kg/day for methyl‐ and butylparaben, respectively. The rapid metabolism of parabens by esterases probably explains why these weakly estrogenic substances elicit no in vivo effects when administered by relevant exposure routes (i.e., topical and oral). Birth Defects Research (Part B) 2008. 2008 Wiley‐Liss, Inc.     

Antibacterial properties of eosinophil major basic protein and eosinophil cationic protein

We examined the bactericidal activity of two proteins that are abundant in the cytoplasmic granules of human eosinophils, major basic protein (MBP) and eosinophil cationic protein (ECP). Unlike the human neutrophil's peptide defensins, both MBP and ECP killed stationary phase Staphylococcus aureus 502A in a simple nutrient-free buffer solution. Although MBP also killed Escherichia coli ML-35 with considerable efficacy under these experimental conditions, the in vitro activity of ECP against E. coli was considerably enhanced if mid-logarithmic phase bacteria replaced stationary phase organisms or if the assay medium was enriched with trypticase soy broth. The antibacterial activity of both eosinophil proteins was modulated by incubation time, protein concentration, temperature and pH. A pBR322-transformed derivative of E. coli ML-35 was used to examine the effects of ECP and MBP on integrity of the bacterial inner membrane (IM) and outer membrane. Although both MBP and ECP caused outer and inner membrane permeabilization when nutrients were present, only MBP was effective under nutrient-free conditions. Two proton ionophores (DNP and carbonyl cyanide m-chlorophenyl hydrazone) protected E. coli from the bactericidal effects of ECP but not from MBP. These findings establish that MBP and ECP have bactericidal properties and suggest that these proteins kill E. coli by similar but nonidentical mechanisms marked by an attack on the target cell's membranes. In view of evidence that high concentrations of ECP and MBP exist in cytoplasmic granules whose contents are translocated to phagocytic vacuoles, we suggest that MBP and ECP contribute to the eosinophil's ability to kill ingested bacteria.     

Lactoferrin protects neonatal rats from gut-related systemic infection

Lactoferrin is a milk protein that reportedly protects infants from gut-related, systemic infection. Proof for this concept is limited and was addressed during in vivo and in vitro studies. Neonatal rats pretreated orally with recombinant human lactoferrin (rh-LF) had less bacteremia and lower disease severity scores (P < 0.001) after intestinal infection with Escherichia coli. Control animals had 1,000-fold more colony-forming units of E. coli per milliliter of blood than treated animals (P < 0.001). Liver cultures from control animals had a twofold increase in bacterial counts compared with cultures from rh-LF-treated pups (P < 0.02). Oral therapy with rh-LF + FeSO(4) did not alter the protective effect. In vitro studies confirmed that rh-LF interacted with the infecting bacterium and rat macrophages. An in vitro assay showed that rh-LF did not kill E. coli, but a combination of rh-LF + lysozyme was microbicidal. In vitro studies showed that rat macrophages released escalating amounts of nitric oxide and tumor necrosis factor-alpha when stimulated with increasing concentrations of rh-LF. The in vitro studies suggest that rh-LF may act with other "natural peptide antibiotics" or may prime macrophages to kill E. coli in vivo.     

Paralysis and killing of Caenorhabditis elegans by enteropathogenic Escherichia coli requires the bacterial tryptophanase gene

Pathogenic Escherichia coli, including enteropathogenic E. coli (EPEC), enterohaemorrhagic E. coli (EHEC), enteroinvasive E. coli (EIEC) and enterotoxigenic E. coli (ETEC) are major causes of food and water-borne disease. We have developed a genetically tractable model of pathogenic E. coli virulence based on our observation that these bacteria paralyse and kill the nematode Caenorhabditis elegans. Paralysis and killing of C. elegans by EPEC did not require direct contact, suggesting that a secreted toxin mediates the effect. Virulence against C. elegans required tryptophan and bacterial tryptophanase, the enzyme catalysing the production of indole and other molecules from tryptophan. Thus, lack of tryptophan in growth media or deletion of tryptophanase gene failed to paralyse or kill C. elegans. While known tryptophan metabolites failed to complement an EPEC tryptophanase mutant when presented extracellularly, complementation was achieved with the enzyme itself expressed either within the pathogen or within a cocultured K12 strains. Thus, an unknown metabolite of tryptophanase, derived from EPEC or from commensal non-pathogenic strains, appears to directly or indirectly regulate toxin production within EPEC. EPEC strains containing mutations in the locus of enterocyte effacement (LEE), a pathogenicity island required for virulence in humans, also displayed attenuated capacity to paralyse and kill nematodes. Furthermore, tryptophanase activity was required for full activation of the LEE1 promoter, and for efficient formation of actin-filled membranous protrusions (attaching and effacing lesions) that form on the surface of mammalian epithelial cells following attachment and which depends on LEE genes. Finally, several C. elegans genes, including hif-1 and egl-9, rendered C. elegans less susceptible to EPEC when mutated, suggesting their involvement in mediating toxin effects. Other genes including sek-1, mek-1, mev-1, pgp-1,3 and vhl-1, rendered C. elegans more susceptible to EPEC effects when mutated, suggesting their involvement in protecting the worms. Moreover we have found that C. elegans genes controlling lifespan (daf-2, age-1 and daf-16), also mediate susceptibility to EPEC. Together, these data suggest that this C. elegans/EPEC system will be valuable in elucidating novel factors relevant to human disease that regulate virulence in the pathogen or susceptibility to infection in the host.     

High-level expression of fully active yeast flavocytochrome b2 in Escherichia coli.

Wild-type flavocytochrome b2 (L-lactate dehydrogenase) from the yeast Saccharomyces cerevisiae and three singly substituted mutant forms (F254, R349 and K376) have been expressed in the bacterium Escherichia coli. The enzyme expressed in E. coli contains the protohaem IX and flavin mononucleotide (FMN) prosthetic groups found in the enzyme isolated from yeast, has an electronic absorption spectrum identical with that of the yeast protein and an identical Mr value of 57,500 estimated by SDS/polyacrylamide-gel electrophoresis. N-Terminal amino-acid-sequence data indicate that the flavocytochrome b2 isolated from E. coli begins at position 6 (methionine) when compared with mature flavocytochrome b2 from yeast. The absence of the first five amino acid residues appears to have no effect on the enzyme-catalysed oxidation of L-lactate, since Km values for the yeast- and E. coli-expressed wild-type enzymes were identical within experimental error. The F254 mutant enzyme expressed in E. coli also showed kinetic parameters essentially the same as those found for the enzyme from yeast. The R349 and K376 mutant enzymes had no activity when expressed in either yeast or E. coli. The yield of flavocytochrome b2 from E. coli is estimated to be between 500- and 1000-fold more than from a similar wet weight of yeast (this high level of expression results in E. coli cells which are pink in colour). The increased yield has allowed us to verify the presence of FMN in the R349 mutant enzyme. The advantages of E. coli as an expression system for flavocytochrome b2 are discussed.     

Interactions between cationic liposomes and bacteria: the physical-chemistry of the bactericidal action.

The bactericidal effect of dioctadecyldimethylammonium bromide (DODAB), a liposome forming synthetic amphiphile, is further evaluated for Escherichia coli, Salmonella typhimurium, Pseudomonas aeruginosa, and Staphylococcus aureus in order to establish susceptibilities of different bacteria species towards DODAB at a fixed viable bacteria concentration (2.5 x 10(7) viable bacteria/mL). For the four species, susceptibility towards DODAB increases from E. coli to S. aureus in the order above. Typically, cell viability decreases to 5% over 1 h of interaction time at DODAB concentrations equal to 50 and 5 microm for E. coli and S. aureus, respectively. At charge neutralization of the bacterial cell, bacteria flocculation by DODAB vesicles is shown to be a diffusion-controlled process. Bacteria flocculation does not yield underestimated counts of colony forming units possibly because dilution procedures done before plating cause deflocculation. The effect of vesicle size on cell viability demonstrates that large vesicles, due to their higher affinity constant for the bacteria (45.20 m(-)) relative to the small vesicles (0.14 m(-)), kill E. coli at smaller DODAB concentrations. For E. coli and S. aureus, simultaneous determination of cell viability and electrophoretic mobility as a function of DODAB concentration yields a very good correlation between cell surface charge and cell viability. Negatively charged cells are 100% viable whereas positively charged cells do not survive. The results show a clear correlation between simple adsorption of entire vesicles generating a positive charge on the cell surfaces and cell death.     

Nuclease colicins and their immunity proteins

It is more than 80 years since Gratia first described 'a remarkable antagonism between two strains of Escherichia coli'. Shown subsequently to be due to the action of proteins (or peptides) produced by one bacterium to kill closely related species with which it might be cohabiting, such bacteriocins have since been shown to be commonplace in the internecine warfare between bacteria. Bacteriocins have been studied primarily from the twin perspectives of how they shape microbial communities and how they penetrate bacteria to kill them. Here, we review the modes of action of a family of bacteriocins that cleave nucleic acid substrates in E. coli, known collectively as nuclease colicins, and the specific immunity (inhibitor) proteins that colicin-producing organisms make in order to avoid committing suicide. In a process akin to targeting in mitochondria, nuclease colicins engage in a variety of cellular associations in order to translocate their cytotoxic domains through the cell envelope to the cytoplasm. As well as informing on the process itself, the study of nuclease colicin import has also illuminated functional aspects of the host proteins they parasitize. We also review recent studies where nuclease colicins and their immunity proteins have been used as model systems for addressing fundamental problems in protein folding and protein-protein interactions, areas of biophysics that are intimately linked to the role of colicins in bacterial competition and to the import process itself.     

Evaluation of in vitro antibacterial activity of some disinfectants on Escherichia coli serotypes

Three disinfectants commonly used in poultry farms (formalin, TH4+, and Virkon-S) were chosen for the present study. The effect of disinfectant concentration and the duration of exposure to these disinfectants on the survival of Escherichia coli serotypes (O114:K-, O86, O55:K39, and O86:K60) were investigated. Formalin (0.6%), TH4+ (0.06%), and Virkon (0.5%) all killed the four serotypes within 5 min of exposure. As the disinfectant concentration decreases, the length of exposure time to kill serotype increases. At 0.03%, 0.007%, and 0.03% of formalin, TH4+ and Virkon-S concentrations failed to kill the four E. coli serotypes within 360 min, respectively. An improvement of the inhibitory effect of these disinfectants occurred when added together with the inoculum instead of an established population. The influence of formalin, TH4+, and Virkon-S on the cell morphology of E. coli O55:K39 was investigated by using transmission electron microscopy. Formalin-treated cells exhibited normal cell morphology, with the exception that the treated cell was less fimbriated, and more destruction of pili increased when formalin concentrations were doubled. Cells treated with TH4+ (0.03%) showed destruction of the cell wall and cell surface membrane after 5 min. Cell filamentation occurred at 0.015% and increased with the increase of exposure time to this drug. Spheroplasts were observed only when cells were treated with 0.125% Virkon-S for 60 min, and cell lysis started to occur when 0.25% Virkon-S was applied for 15 min. Scanning electron microscope study revealed that Virkon-S at 0.03% and TH4+ at 0.007% completely prevented the adherence of E. coli O55:K39 serotype to chicken tracheal organ, whereas formalin (0.03%) disinfection minimized the adherence of E. coli cells to tracheal explants after 360 min of incubation.