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Applied and Environmental Microbiology, March 2009, p. 1465-1469, Vol. 75, No. 5
0099-2240/09/$08.00+0     doi:10.1128/AEM.02447-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Physicochemical Basis for the Inhibitory Effects of Organic and Inorganic Salts on the Growth of Pectobacterium carotovorum subsp. carotovorum and Pectobacterium atrosepticum

Elian-Simplice Yaganza,¹ Russell J. Tweddell,^2* and Joseph Arul¹

Department of Food Science and Nutrition and Horticultural Research Centre, Université Laval, Québec, Québec, Canada G1V 0A6,¹ Department of Plant Science and Horticultural Research Centre, Université Laval, Québec, Québec, Canada G1V 0A6²

Received 24 October 2008/ Accepted 22 December 2008

Top ABSTRACT INTRODUCTION REFERENCES

 
Twenty-one salts were tested for their effects on the growth of Pectobacterium carotovorum subsp. carotovorum and Pectobacterium atrosepticum. In liquid medium, 11 salts (0.2 M) exhibited strong inhibition of bacterial growth. The inhibitory action of salts relates to the water-ionizing capacity and the lipophilicity of their constituent ions.

Top ABSTRACT INTRODUCTION REFERENCES

 
Different biochemical mechanisms have been put forth to explain the antimicrobial activity of organic and inorganic salts, including inhibition of several steps of the energy metabolism (benzoate, bicarbonate, propionate, sorbate, and sulfite salts) (2, 3, 11, 16, 17, 19, 25) and complexation to DNA and RNA (aluminum and sulfites) (12, 13, 15, 20, 27, 28). However, little is known about the physicochemical basis for the general antimicrobial action of salts. The objective of this work was to gain an understanding of the relationship between the inhibitory action of salts on bacterial growth and their physicochemical properties by using the bacteria Pectobacterium carotovorum subsp. carotovorum (formerly Erwinia carotovora subsp. carotovora) and Pectobacterium atrosepticum (formerly Erwinia carotovora subsp. atroseptica). These bacteria are responsible for soft rot, a disease of economic importance affecting numerous stored vegetable crops (14, 22).

Pectobacterium carotovorum subsp. carotovorum (strain Ecc 1367) and P. atrosepticum (strain Eca 709), provided by the Laboratoire de Diagnostic en Phytoprotection (MAPAQ, Québec, Canada), were grown in 250-ml flasks containing 50 ml of 20% tryptic soy broth (Difco Laboratories, Becton Dickinson, Sparks, MD) amended with salts (200 mM) or unamended (control), by incubation at 24°C with agitation (150 rpm; Lab-Line Instruments Inc., Melrose Park, IL) for 24 h. The pHs of the media were not adjusted but varied with the type of salts, unless stated otherwise. Flasks were inoculated with 100 µl of each bacterial suspension (1 x 10⁷ CFU/ml). Bacterial growth was determined by turbidimetry at 600 nm with a UV/visible spectrophotometer (Ultrospec 2000; Pharmacia Biotech Ltd, Cambridge, United Kingdom), using appropriate blanks. Results were expressed as the percentage of growth inhibition compared with the growth of the control. A completely randomized experimental design with three replicates was used, the experimental unit being a flask. Analysis of variance was carried out with the GLM (general linear model) procedure of SAS (SAS Institute, Cary, NC) software. When they were significant (P < 0.05), treatment means were compared using Fisher's protected least-significant-difference test.

Among the 21 salts tested, sodium carbonate, sodium metabisulfite, trisodium phosphate, aluminum lactate, aluminum chloride, sodium bicarbonate, sodium propionate, ammonium acetate, aluminum dihydroxy acetate, potassium sorbate, and sodium benzoate exhibited strong inhibition (97%) of the growth of both P. carotovorum subsp. carotovorum and P. atrosepticum (Table 1). Calcium chloride, sodium formate, sodium acetate, ammonium hydrogen phosphate, and sodium hydrogen phosphate exhibited a moderately inhibitory effect; sodium lactate and tartrate had no effect. On the other hand, ammonium chloride, potassium chloride, and sodium chloride stimulated the growth of P. atrosepticum.

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TABLE 1. Effect of salts on the growth of P. atrosepticum and P. carotovorum subsp. carotovorum

Several factors in the salt solutions can contribute to bacterial growth inhibition. Elevated osmolarity due to salt addition may trigger the osmoregulatory process, causing an increased maintenance metabolism and leading to reduction in bacterial growth. Thus, we calculated the osmotic pressure () of salt solutions using van't Hoff's equation (26). As shown in Table 1, salts with comparable osmolarities displayed complete or no bacterial growth inhibition, indicating that osmotic stress or reduction in water activity alone may not have brought about the inhibition of the bacterial growth. Therefore, other factors may play a role.

The acidity or alkalinity of the medium resulting from the addition of some of the salts can have profoundly adverse effects on bacterial growth. Extreme pH conditions can lead to denaturation of proteins like enzymes present on the cell surface, depolarization of transport for essential ions and nutrients, modification of cytoplasmic pH, and DNA damage (12, 18). Table 1 shows that the addition of aluminum lactate, aluminum chloride, and sodium metabisulfite, whose pHs (pH = |7.5 [the optimal pH for growth] – the pH of the salt-amended medium|) are 3, strongly acidified the medium, whereas the addition of sodium carbonate and trisodium phosphate strongly increased the pH (pH 3.1). Except for ammonium acetate, sodium acetate, sodium bicarbonate, and the preservative salts (potassium sorbate, sodium benzoate, and sodium propionate), whose pHs are <1, all the other salts generally display inhibitory effects when pH values are 1 (Fig. 1). Based on this result, the effect of the highly acidic or alkaline salts (which strongly affected the pH of the medium) on the growth of P. atrosepticum was evaluated at pH 7.5. Sodium carbonate and sodium metabisulfite completely inhibited bacterial growth at pH 7.5, as they did at pHs 10.6 and 4.5, respectively; trisodium phosphate (pH 11.9) exhibited a slightly lower inhibitory effect (growth inhibition of 83.2%) at pH 7.5. These observations suggest that growth inhibition by sodium carbonate, sodium metabisulfite, and trisodium phosphate cannot be attributed solely to extreme pH and passive proton transfer (extreme pH) across the bacterial membrane. Since aluminum salts precipitate at pH 7.5 (due to formation of hydrated aluminum hydroxide), it was not possible to test their inhibitory effect at pH 7.5.

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FIG. 1. Relationship between pH (|7.5 [the optimal pH for growth] – the pH of the salt-amended medium|) and growth inhibition of Pectobacterium atrosepticum. 1, Sodium chloride; 2, potassium chloride; 3, ammonium chloride; 4, sodium tartrate; 5, sodium lactate; 6, sodium formate; 7, ammonium hydrogen phosphate; 8, sodium acetate; 9, sodium hydrogen phosphate; 10, calcium chloride; 11, ammonium acetate; 12, sodium benzoate; 13, sodium propionate; 14, potassium sorbate; 15, sodium bicarbonate; 16, aluminum dihydroxy acetate; 17, sodium metabisulfite; 18, sodium carbonate; 19, aluminum lactate; 20, aluminum chloride; 21, trisodium phosphate.

The dissociation of salts in aqueous medium generates ionic species which can participate in proton exchange reactions with water molecules. The capacity of an ion to dissociate water is an intrinsic characteristic, determined by its pK value (pK_a for acidic species or pK_b for basic ones) (4, 21, 24). For an ionic strength of >0.1 M, pK_a and pK_b values of the ions are more accurate when they are defined as apparent constants (pK'_a or pK'_b) in terms of the activities of hydronium and hydroxyl ions, ionic species concentrations and activity coefficients (6). Thus, for the acidic ions, we have the equation ), and for the basic anions, pK'_b = pK_b + log(_HB/B^–), where pK'_a and pK'_b are the apparent acidity constant and basicity constant, respectively; is the activity coefficient of the conjugate base (B^–); and _HB is that of the acidic (HB) species. The activity coefficient () of the species i can be expressed as a function of ionic strength (µ), using the Güntelberg approximation of the Debye-Hückel equation (21), as follows: –log _i=[(0.51Z_i² µ^1/2)/(1 + µ^1/2)], where Z_i is the charge on the species i, and µ is the ionic strength. Thus, log(/_HB) = [(0.51µ^1/2)/(1 + µ^1/2)] (), and log(_HB/) = –[(0.51µ^1/2)/(1 + µ^1/2)] ().

Polytropic acid-potentiating ions (bicarbonate, carbonate, monohydrogen phosphate, phosphate, sulfite, and tartrate) in an aqueous solution can exist as (n + 1) possible species for which the parent acid is H_nA. These species may coexist in equilibrium under certain pH conditions. For these ions, pK'_a or pK'_b were expressed as the means of the coexisting species at a specified pH. Calculated values for pK'_a of acidic anions and cations and calculated values for pK'_b of basic anions are presented in Table 2. Figure 2A shows a sigmoidal relationship between the inhibitory effect of salts on bacterial growth and the pK'_b value of the basic ions (with a common cation, sodium or potassium, in the salt) and the pK'_a value of the acidic ions (with a common anion, chloride, in the salt). The plot exhibits a sharp linear relationship in the pK' range of 8.0 to 12.0. Below the pK' value of 8.0, inhibition is maximal, whereas above the pK' value of 11.0, ions appear to stimulate growth (growth was maximal above the pK' value of 12). This result demonstrates that the capacity of the constitutive ions of the salts to either donate or subtract protons to water molecules, either in the growth environment (as reflected in the modification of the medium pH) or in the developing cells, generally plays a role in their inhibitory action. The consequent transmembrane pH gradient generated leads to a passive H⁺ transport across the microbial membrane and to acidification (in the case of ions with low pK'_a) or alkalinization (in the case of ions with low pK'_b) of the cytoplasm, once the capacity for proton-coupled active transport is outstripped. In both cases, proton exchange with outer membrane proteins will destabilize these proteins, their interaction with membrane lipids, and ultimately, their function in solute transport, leading to growth inhibition. The modification of cytoplasmic pH can also alter nucleic acid structures and functions and contribute to growth inhibition (18).

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TABLE 2. Calculated apparent values for acidity, pK'a, and basicity, pK'ba

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FIG. 2. (A) Relationship between the growth inhibition of Pectobacterium atrosepticum and the apparent basicity constant (pK'b,•) of basic anions with common Na+ (or K+) cations in the salt, the apparent acidity constant (pK'a,) of acidic bisulfite anion (HSO3–), and the cations with common Cl– ions in the salt. (B) Relationship between the growth inhibition of Pectobacterium atrosepticum and the addition parameter (pK' + pPo/w) combining the partition coefficient (Po/w) and pK'b (•) of basic anions (common cation, Na+ or K+, in the salt) or pK'a () of cations (common anion, Cl–, in the salt) and the acidic bisulfite anion (HSO3–).

However, the water-ionizing capacity of the constituent ions of the salts and the consequent modification of the pH of the medium are not the sole factors accounting for growth inhibition, as suggested by the exceptional inhibitory actions of benzoate, propionate, and sorbate (Fig. 1 and 2A). These ions provide a higher inhibition than is expected from their pK' values (pK'_b values of 10.0, 9.3, and 9.4, respectively), while the pH of their solution is optimal for bacterial growth (pHs of 7.4, 7.4, and 7.7, respectively). This suggests that they possess additional characteristics mediating their action, in addition to their water-ionization property. In fact, these preservative agents have been shown to be active either as undissociated acids (like other weak acids) or as anions (7, 8), due to their possibly hydrophobic nature which would allow them to interact with lipid constituents of the cell envelope of gram-negative bacteria such as Pectobacterium spp., and to modify their functionality (5), resulting in growth inhibition. They can also cross the cell envelope due to their lipophilicity, and their acidification inside the cell can cause additional adverse effects.

Thus, we determined the octanol/water partition coefficient (P_o/w), an indicator of the lipophilic character of a compound, for the effective salts with common sodium (or potassium) or chloride ions. The P_o/w coefficients of the salts were determined in duplicate by using the general solvent-solvent separation procedure (9). Equal volumes (50 ml) of 1-octanol (Sigma Chemical Co., St. Louis, MO) and bidistilled water were poured into a separating flask and thoroughly shaken for 5 min. Four grams of each salt was then added, and the flask content was thoroughly mixed three times for 5 min each time, with a rest period of 5 min after each agitation. After complete separation (20 to 24 h at room temperature), the two phases were recovered separately in different flasks, and the concentration of the accompanying ion of the salt was measured in each phase by atomic absorption (model 3300 unit; Perkin-Elmer, Ueberlinger, Germany). The P_o/w coefficient was calculated as the ratio of the concentration of ion in 1-octanol to the concentration of ion in the aqueous phase. Sodium benzoate was found to be the most lipophilic (P_o/w = 1.41 x 10^–2), followed by potassium sorbate (P_o/w = 7.6 x 10^–3) and sodium metabisulfite (P_o/w = 2.0 x 10^–4). Most other salts, sodium chloride (reference salt), sodium bicarbonate and carbonate, sodium propionate, sodium acetate, calcium chloride, and aluminum chloride mainly remained in the aqueous phase (P_o/w = 2.0 x 10^–5 to 5.0 x 10^–5). This lipophilic characteristic of benzoate and sorbate ions would result from a reduced charge density in their molecules (due to the conjugated double bonds in their molecules). An addition parameter, pK' + pP_o/w, which combines the two properties of salts ions, i.e., the water-ionizing capacity (pK') and the lipophilicity (pP_o/w = –log P_o/w), appears to provide a more general basis for the inhibitory effect of salts (Fig. 2B). This suggests that while the dissociation constant of ions plays a major role in growth inhibition, as seen in Fig. 2A, the lipophilic character of the preservative-salt ions confers to them an added ability to penetrate the cell envelope and to inhibit bacterial growth (5, 10). The exclusion of ammonium (lower inhibition than expected from its pK'_a value) and calcium (higher inhibition than expected from its pK'_a value) ions from the sigmoidal pattern portrayed in Fig. 2B would have resulted from their interactions with water and other molecules (NH₄⁺) (1) or from cell membrane destabilization (Ca²⁺) (23).

In conclusion, the study has shown that several salts (0.2 M concentration), including aluminum dihydroxy acetate, aluminum chloride, aluminum lactate, ammonium acetate, potassium sorbate, sodium benzoate, sodium metabisulfite, sodium bicarbonate, sodium carbonate, sodium propionate, and trisodium phosphate, strongly inhibited the growth of P. carotovorum subsp. carotovorum and P. atrosepticum. In addition, the study has established for the first time a basic sigmoidal relationship between the antimicrobial activity of the salts and the physicochemical characteristics of their constituent ions, namely their water-ionizing capacity and their lipophilicity. The constituent ions of the highly inhibiting salts generally displayed a high capacity to ionize water molecules (low pK'_a or pK'_b values) (Al³⁺, CO₃^2–, PO₄^3–, HCO₃^–, and HSO₃^–) or a high lipophilicity (benzoate^– and sorbate^–), and these two parameters in combination with known biochemical activities of salts ions would affect bacterial growth.

 
This study was supported by Conseil des Recherches en Pêche et en Agroalimentaire du Québec (CORPAQ), Cultures H. Dolbec Inc., and Propur Inc.

We thank K. Belkacemi for assistance with data analysis.

 
* Corresponding author. Mailing address: Horticultural Research Centre, Pavillon de l'Envirotron, Université Laval, Québec, QC, Canada G1V 0A6. Phone: (418) 656-2131, ext. 4553. Fax: (418) 656-7871. E-mail: russell.tweddell{at}fsaa.ulaval.ca

Published ahead of print on 29 December 2008.

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Applied and Environmental Microbiology, March 2009, p. 1465-1469, Vol. 75, No. 5
0099-2240/09/$08.00+0     doi:10.1128/AEM.02447-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Yaganza, E.-S. Articles by Arul, J. Search for Related Content PubMed PubMed Citation Articles by Yaganza, E.-S. Articles by Arul, J. Agricola Articles by Yaganza, E.-S. Articles by Arul, J.