Ilkka M. Helander

Lactic Acid Permeabilizes Gram-Negative Bacteria by Disrupting the Outer Membrane

ABSTRACT The effect of lactic acid on the outer membrane permeability ofEscherichia 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 MgCl2. 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. aeruginosawas 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.

Berry Phenolics: Antimicrobial Properties and Mechanisms of Action Against Severe Human Pathogens

Abstract: Antimicrobial activity and mechanisms of phenolic extracts of 12 Nordic berries were studied against selected human pathogenic microbes. The most sensitive bacteria on berry phenolics were Helicobacter pylori and Bacillus cereus. Campylobacter jejuni and Candida albicans were inhibited only with phenolic extracts of cloudberry, raspberry, and strawberry, which all were rich in ellagitannins. Cloudberry extract gave strong microbicidic effects on the basis of plate count with all studied strains. However, fluorescence staining of liquid cultures of virulent Salmonella showed viable cells not detectable by plate count adhering to cloudberry extract, whereas Staphylococcus aureus cells adhered to berry extracts were dead on the basis of their fluorescence and plate count. Phenolic extracts of cloudberry and raspberry disintegrated the outer membrane of examined Salmonella strains as indicated by 1-N-phenylnaphthylamine (NPN) uptake increase and analysis of liberation of [14C]galactose- lipopolysaccharide. Gallic acid effectively permeabilized the tested Salmonella strains, and significant increase in the NPN uptake was recorded. The stability of berry phenolics and their antimicrobial activity in berries stored frozen for a year were examined using Escherichia coli and nonvirulent Salmonella enterica sv. Typhimurium. The amount of phenolic compounds decreased in all berries, but their antimicrobial activity was not influenced accordingly. Cloudberry, in particular, showed constantly strong antimicrobial activity during the storage.

Polyethyleneimine is an effective permeabilizer of Gram-negative bacteria

The effect of the polycation polyethyleneimine (PEI) on the permeability properties of the Gram-negative bacterial outer membrane was investigated using Escherichia coli, Pseudomonas aeruginosa and Salmonella typhimurium as target organisms. At concentrations of less than 20 micrograms ml-1, PEI increased the bacterial uptake of 1-N-phenylnaphthylamine, which is a hydrophobic probe whose quantum yield is greatly increased in a lipid environment, indicating increased hydrophobic permeation of the outer membrane by PEI. The effect of PEI was comparable to that brought about by the well-known permeabilizer EDTA. Permeabilization by PEI was retarded but not completely inhibited by millimolar concentrations of MgCl2. PEI also increased the susceptibility of the test species to the hydrophobic antibiotics clindamycin, erythromycin, fucidin, novobiocin and rifampicin, without being directly bactericidal. PEI sensitized the bacteria to the lytic action of the detergent SDS in assays where the bacteria were pretreated with PEI. In assays where PEI and SDS were simultaneously present, no sensitization was observed, indicating that PEI and SDS were inactivating each other. In addition, a sensitizing effect to the nonionic detergent Triton X-100 was observed for P. aeruginosa. In conclusion, PEI was shown to be a potent permeabilizer of the outer membrane of Gram-negative bacteria.

Characterization of the lipopolysaccharide from the polymyxin-resistant pmrA mutants of Salmonella typhimurium.

Lipopolysaccharide (LPS, endotoxin) is a major constituent of the outer membrane (OM) of Gramnegative bacteria and is essential for the proper assembly, organization, and function of this membrane [ 1,2]. The complex chemical structure of US involves the O-specific chain, the core oligosaccharide, and the lipid A component and has been largely elucidated in many Gram-negative bacteria, mainly enteric bacteria (reviewed in [3-S]). Lipopolysaccharides, e.g., those of Salmonella, exhibit structural heterogeneity of thus far unknown significance. One striking example is the wide heterogeneity in the length of the O-specific chain in smooth bacteria [6-91. Another example is the partial substitution of phosphate residues located in the core oligosaccharide and the lipid A component by residues such as phosphorylethanolamine (PEtn) and 4-amino4-deoxy-L-arabinose (II-ARAN) [ lo,1 11. We have isolated S. typhimurium mutants (pmrA mutants) which have outer membranes with increased resistance to polymyxin, a basic, amphipathic antibiotic as well as to other cationic agents and TrisEDTA ([ 12-141 and M. V., submitted). Here we show that these pmrA mutants represent a novel type of mutation affecting the lipid A structure: the esterlinked phosphate in their lipid A is almost completely substituted with 4-ARAN. Their LPS has also an increased content of ethanolamine.

Increased substitution of phosphate groups in lipopolysaccharides and lipid A of the polymyxin‐resistant pmrA mutants of Salmonella typhimurium: a 31P‐NMR study

De‐O‐acylated lipopolysaccharides (LPS) of three polymyxin‐resistant Salmonella typhimurium pmrA mutants and their parent strains were analysed by 31P‐NMR (nuclear magnetic resonance) in order to assess, in relation to polymyxin resistance, the types and degree of substitution of phosphates of the LPS and lipid A. in the pmrA mutant LPS phosphate diesters predominated over phosphate monoesters, whereas the latter were more abundant in the parent wild‐type LPS. The increase in the proportion of phosphate diesters was traced to both the core oligosaccharide and the lipld A part. In the latter, the ester‐linked phosphate at position 4’was to a large extent (79–88%) substituted with 4‐amino‐4‐deoxy‐l‐arabinose, whereas in the wild‐type LPS the 4′‐phosphate was mainly present as monoester. In each LPS, regardless of the pmrA mutation, the glycosidically linked phosphate of lipid A was largely unsubstituted.

Lack of O‐antigen is essential for plasminogen activation by Yersinia pestis and Salmonella enterica

The O‐antigen of lipopolysaccharide (LPS) is a virulence factor in enterobacterial infections, and the advantage of its genetic loss in the lethal pathogen Yersinia pestis has remained unresolved. Y. pestis and Salmonella enterica express β‐barrel surface proteases of the omptin family that activate human plasminogen. Plasminogen activation is central in pathogenesis of plague but has not, however, been found to be important in diarrhoeal disease. We observed that the presence of O‐antigen repeats on wild‐type or recombinant S. enterica, Yersinia pseudotuberculosis or Escherichia coli prevents plasminogen activation by PgtE of S. enterica and Pla of Y. pestis; the O‐antigen did not affect incorporation of the omptins into the bacterial outer membrane. Purified His6‐Pla was successfully reconstituted with rough LPS but remained inactive after reconstitution with smooth LPS. Expression of smooth LPS prevented Pla‐mediated adhesion of recombinant E. coli to basement membrane as well as invasion into human endothelial cells. Similarly, the presence of an O‐antigen prevented PgtE‐mediated bacterial adhesion to basement membrane. Substitution of Arg‐138 and Arg‐171 of the motif for protein binding to lipid A 4′‐phosphate abolished proteolytic activity but not membrane translocation of PgtE, indicating dependence of omptin activity on a specific interaction with lipid A. The results suggest that Pla and PgtE require LPS for activity and that the O‐antigen sterically prevents recognition of large‐molecular‐weight substrates. Loss of O‐antigen facilitates Pla functions and invasiveness of Y. pestis; on the other hand, smooth LPS renders plasminogen activator cryptic in S. enterica.

Permeability barrier of the gram-negative bacterial outer membrane with special reference to nisin.

The effect of nisin pretreatment on organic acid-induced permeability increase in strains of Escherichia coli, Pseudomonas aeruginosa, P. marginalis, and Salmonella enterica sv. Typhimurium was investigated, using assays based on the uptake of a fluorescent dye 1-N-phenylnaphthylamine (NPN) and on the bacterial susceptibility to detergent-induced bacteriolysis. The outer membrane of bacteria which had been pretreated with nisin was shown to be less stable against 1 mM EDTA, as indicated by their significantly higher NPN uptake levels as compared to untreated bacteria. Upon challenge with a tenfold lower concentration of EDTA (0.1 mM) some nisin-treated strains (Typhimurium, P. marginalis) exhibited, however, NPN uptake levels which were lower than those seen in control bacteria, suggesting that nisin had stabilized their outer membrane. Nisin pretreatment also decreased the NPN uptake induced by citric or lactic acid or both in E. coli, P. marginalis, and Typhimurium, whereas in P. aeruginosa the pretreatment resulted in increased NPN uptake in response to citric and lactic acid. These results suggest that, with the exception of P. aeruginosa, nisin could protect bacteria from the outer membrane-disrupting effect caused by the acids. P. aeruginosa was, however, shown to be protected against bacteriolysis induced by the detergents sodium dodecylsulfate and Triton X-100. With a pair of isogenic mutants of Typhimurium differing in their cell surface charge it was shown that the NPN uptake response to I mM EDTA of the abnormally cationic strain was not significantly affected by nisin, whereas in the normal anionic strain nisin strongly strengthened the uptake. Our hypothesis based on these findings is that the normally anionic cell surface of Gram-negative bacteria has a tendency to bind the cationic nisin. The binding of nisin to the surface does not proceed to the cytoplasmic membrane, but in the outer membrane the bound nisin actually stabilizes its structure through electrostatic interactions. With the exception of EDTA, the organic acids at pH 4 did not cause leakage of cell contents from Typhimurium, indicating that these acids do not permeabilize the outer membrane to an extent required for cytoplasmic pore formation by nisin.

Permeabilizing action of polyethyleneimine on Salmonella typhimurium involves disruption of the outer membrane and interactions with lipopolysaccharide

Polyethyleneimine (PEI), a polycationic polymer substance used in various bioprocesses as a flocculating agent and to immobilize enzymes, was recently shown to make Gram-negative bacteria permeable to hydrophobic antibiotics and to detergents. Because this suggests impairment of the protective function of the outer membrane (OM), the effect of PEI on the ultrastructure of Salmonella typhimurium was investigated. Massive alterations in the OM of PEI-treated and thin-sectioned bacteria were observed by electron microscopy. Vesicular structures were seen on the surface of the OM, but no liberation of the membrane or its fragments was evident. Since a potential mechanism for the action of PEI could be its binding to anionic LPSs on the OM surface, the interaction of PEI with isolated LPSs was assayed in vitro. The solubility of smooth-type LPSs of Salmonella, regardless of the sugar composition of their O-specific chains, was not affected by PEI, nor was that of Ra-LPS (lacking O-specific chains but having a complete core oligosaccharide). PEI strongly decreased the solubility of rough-type LPSs of the chemotypes Rb2 and Re, whereas it had only a weak effect on the abnormally cationic Rb2-type pmrA mutant LPS, suggesting that the negative charge to mass ratio of LPS plays a critical role in the interaction.

Weakening Effect of Cell Permeabilizers on Gram-Negative Bacteria Causing Biodeterioration

ABSTRACT Gram-negative bacteria play an important role in the formation and stabilization of biofilm structures on stone surfaces. Therefore, the control of growth of gram-negative bacteria offers a way to diminish biodeterioration of stone materials. The effect of potential permeabilizers on the outer membrane (OM) properties of gram-negative bacteria was investigated and further characterized. In addition, efficacy of the agents in enhancing the activity of a biocide (benzalkonium chloride) was assessed. EDTA, polyethylenimine (PEI), and succimer (meso-2,3-dimercaptosuccinic) were shown to be efficient permeabilizers of the members of Pseudomonas and Stenotrophomonas genera, as indicated by an increase in the uptake of a hydrophobic probe (1-N-phenylnaphthylamine) and sensitization to hydrophobic antibiotics. Visualization of Pseudomonas cells treated with EDTA or PEI by atomic force microscopy revealed damage in the outer membrane structure. PEI especially increased the surface area and bulges of the cells. Topographic images of EDTA-treated cells were compatible with events assigned for the effect of EDTA on outer membranes, i.e., release of lipopolysaccharide and disintegration of OM structure. In addition, the effect of EDTA treatment was visualized in phase-contrast images as large areas with varying hydrophilicity on cell surfaces. In liquid culture tests, EDTA and PEI supplementation enhanced the activity of benzalkonium chloride toward the target strains. Use of permeabilizers in biocide formulations would enable the use of decreased concentrations of the active biocide ingredient, thereby providing environmentally friendlier products.

Chemical structure of the lipopolysaccharide of Haemophilus influenzae strain I-69 Rd−/b+

The chemical structure of the lipopolysaccharide of a deep-rough mutant (strain I-69 Rd−/b+) of Haemophilus influenzae was investigated. The hydrophilic backbone of lipid A was shown to consist of a β-(1′,6)-linked D-glucosamine disaccharide with phosphate groups at C-1 of the reducing D-glucosamine and at C-4′ of the nonreducing one. Four molecules of (R)-3-hydroxytetradecanoic acid were found directly linked to the lipid A backbone, two by amide and two by ester linkage (positions 2, 2′ and 3, 3′, respectively). Laser-desorption mass spectrometry showed that both 3-hydroxytetradecanoic acids linked to the non-reducing glucosamine carry tetradecanoic acid at their 3-hydroxyl group, so that altogether six molecules of fatty acid are present in lipid A. The lipopolysaccharide was the first described to contain only one sugar unit linked to lipid A. This, sugar in accordance with a previous report [Zamze et al. (1987) Biochem. J. 245, 583–587], was shown to be a dOclA phosphate. The phosphate group was found at position 4, but the analytical procedures employed (permethylation and methanolysis followed by gas-liquid chromatography/mass spectrometry) also revealed dOclA 5-phosphate. Since a cyclic 4,5-phosphate could be ruled out by 31P-NMR, we conclude that, in this lipopolysaccharide, a mixture of dOclA 4- and 5-phosphate is present. By methylation analysis of the dephosphorylated, deacylated and reduced lipopolysaccharide the attachment site of the dOclA was assigned to position C-6′ of the nonreducing glucosamine of lipid A. The anomeric linkages present in the lipopolysaccharide were assessed by 1H-NMR and 13C-NMR of deacylated lipopolysaccharide. The saccharide backbone of this Haemophilus influenzae lipopolysaccharide possesses the following structure: