Journal of Applied Bacteriology 1991. 71, 191-201

ADONIS OO218&4791oO1116

A REVIEW

Mechanisms of bacterial resistance to non-antibiotics: food additives and food and pharmaceutical preservatives A.D. Russell Welsh School of Pharmacy, University of Wales College of Cardiff, Wales 3487/09/90: accepted 28 December 1990

Introduction, 191 Comparative responses of bacteria to non-antibiotics, 192 2.1 Preservatives, 192 2.2 Food additives, 193 Mechanisms of bacterial resistance, 195 3.1 Resistance of bacterial spores, 195

1. INTRODUCTION

Bacterial resistance and tolerance to antibiotics are well established and the mechanisms widely studied. In contrast, mechanisms of insusceptibility to non-antibiotic agents, such as preservatives and antiseptics, are less well understood (Russell & Gould 1988). The slow but steady progress, however, suggests (Day & Russell 1991) that bacterial resistance to non-antibiotics can be as sophisticated as the mechanisms of resistance expressed to antibiotics. It is the purpose of this paper to examine the mechanisms whereby bacteria resist the effects of non-antibiotic antibacterial agents. The types of organisms that will be considered are essentially bacterial spores and Grampositive and -negative non-spore-formers. Gilbert (1983) has listed organisms responsible for bacterial food poisoning, they include Clostridium spp. (Cl. botulinum, C l . perfringens), Bacillus cereus, Salmonella spp., Vibrio parahaemolyticus and Staphylococcus aureus. T o these may be added Yersinia enterocolitica (Aulisio et al. 1980; Brackett 1986), Listeria monocytogenes and Enterobacteriaceae such as Escherichia coli. Some of these organisms will be considered in this paper. Preservatives are an important means of limiting microbial growth in various types of pharmaceutical, cosmetic and food products, as well as in other specialized areas. Antimicrobial agents used as preservatives in sterile pharmaceutical products such as multiple-dose parenteral and Correspondence t o : Dr A.D. Russell, Welsh School of Pharmaqy, University oJ Wales College of Card@, PO Box I.?, Cardrff CFI .?XF, Wales.

3.2 Resistance of non-sporing bacteria, 196 3.2.1 Intrinsic resistance, 196 3.2.2 Acquired resistance, 197 4. Revival of injured microbes, 197 5. Possible ways of preventing or overcoming resistance, 198 6. References, 199

ophthalmic products include phenolics, organomercury compounds, a quaternary ammonium compound (benzalkonium chloride) and chlorhexidine (Table 1). For non-sterile pharmaceutical products, some of these may also be employed, together with others such as parabens (esters of para(4)-hydroxybenzoic acids) and organic acids (Table 2). Organic acids, as acidulants, and esters may also be employed as food preservatives, together with sulphites and carbon dioxide (Table 3; Gomez & Herrero 1983). Food additives, as discussed in Section 2, may also have a role to play in limiting or preventing microbial proliferation. Obviously, the number of chemical compounds permitted to be used as food and pharmaceutical preservatives is limited, due in no small part to the problem of actual or potential toxicity to users. It is clear that care is needed in choosing a preservative and in employing it to the best posTable 1 Antimicrobial agents commonly used as preservatives in sterile pharmaceutical products

Product classification

Example(s)

Preservatives

Parenteral

Multiple-dose injectables

Phenolics Phenylmercuric salts Thiomersal (immunological products)

Ophthalmic

Multiple-dose eye-drops

Phenylmercuric salts Benzalkonium chloride Chlorhexidine

192 A . D . RUSSELL

Table 2 Examples of preservatives used in non-sterile pharmaceutical products

Preservative

Concentration (%)

Use(s)

Phenolics Sorbic acid/potassium sorbate Benzoic acid/sodium benzoate Para bens

0.1-0.5

Phenylmercuric nitrate Alcohol Sulphur dioxide Chloroform* Formaldehyde-releasing agents

0.00 1 12-20 400 PPm 0.25 0.1-0.5

Creams Gums, mucilages and syrups Oral preparations Emulsions, creams and lotions; liquid oral preparations Creams Liquid oral preparations Liquid oral preparations Liquid oral preparations Creams

0.1

0.05-0.1 0.1-0.2

* Limitations as to use. Table 3 Antimicrobial agents commonly used as food preservatives

Class

Example(s)

Organic acids

Benzoic acid Sorbic acid Dehydroacetic acid Formic acid Propionic acid

Comment -l

Activity depends mainly on undissociated acid

Organic acid esters

Esters of Chydroxybenzoic acid (the parabens)

Solubility decreases but activity increases as homologous series ascended (from methyl to butyl)

Nitrites

Sodium nitrite

Toxicity a problem

Sulphites/sulphur dioxide

Sodium sulphite/metabisulphite

sible advantage (Branen 1983; Hugo & Russell 1991). A comprehensive list of antimicrobial preservatives has been published (Wallhausser 1984). 2. COMPARATIVE RESPONSES OF BACTERIA TO NON-ANTIBIOTICS 2.1 Preservatives

Bacteria show a wide response to preservatives and other typcs of biocides (Table 4). T h i s response is determined mainly by the nature of the antimicrobial agent and the type of organism. Other (extraneous) factors must also be considcrcd, the most important of which are the p H of the environment, the temperature at which contact is effected and whether organic (or other binding) matter is present (Russell 1990a, 1991). Environmental p H is often a particularly important parameter since it can modify the practical application of preservatives. T h e most resistant of all types of bacteria are bacterial spores (Russell 1990b; Russell & Chopra 1990). T h 'IS statement is an over-simplification, however. Although spores

are not usually killed by preservatives, their growth (and possibly toxin production) may be inhibited by virtue of preservative effects on germination or outgrowth at concentrations equivalent to those that prevent the growth of nonsporulating organisms. T h e most important food spoilage and food-poisoning spore-formers are B. cereus and Clostridium spp. Bacterial spores are not killed by any of the preservatives listed in Tables 1-3. Whilst at first sight this may prove to be an insurmountable problem, it must be pointed out that (1) many pharmaceutical products are sterile (Table 1) but may contain a preservative to prevent accidental contamination during use of multiple-dose containers and (2) in foods, one of the major requirements may be to inhibit spore germination and outgrowth (Section 3.1) and toxin production. T h e resistance of mycobacteria is considered as being intermediate between bacterial spores and non-sporing, non-acid-fast bacteria (Rubin 1983), although wide divergences in response may occur. Quaternary ammonium compounds, organomercurials and bisbiguanides have an inhibitory rather than a lethal action. Other non-sporulating Gram-positive bacteria such as

P R E S E R V A T I V E S A N D B A C T E R I A L R E S I S T A N C E 193

Table 4 Comparative responses of different bacteria to some preservatives and other non-antibiotic agents

MIC @g/ml) vs Antibacterial

agent

Staphylococcus aureus

Pseudomonas aeruginosa

Escherichia cola

Klebsiella pneumoniae

Bronopol Propionic acid Sorbic acid (PH 6) Benzoic acid (PH 6) Hexachlorophane Propamidine

62.5 1000 5CL-100

31.25 3000 1W300

31.25 2000 50-100

62.5 1250 50-100

50-100

200-500

100-200

100-200

0.5 2

250 256

12.5 64

12.5 256

0.5- 1 4 0.2

5560 64-128 8 1-5

1 16 4 0.5

5-10 16 4 0.5

isethionate

Chlorhexidine Cetrimide Thiomersal Phenylmercuric nitrate

0.1

staphylococci and streptococci are generally more sensitive to preservatives than are Gram-negative organisms (Russell & Gould 1988). A particular problem may exist, however, with methicillin-resistant Staph. aureus (MRSA) strains which may show resistance to several antibiotics as well as to cationic-type preservatives. MRSA strains are a potential hazard in the hospital environment, but have not been associated with foods or pharmaceutical products. Apart from MRSA strains, other staphylococci and streptococci are considered to be susceptible to the action of quaternary ammonium compounds, bisbiguanides, organomercurials and phenolics. Gram-negative bacteria are usually more resistant than Gram-positive cocci to many biocides. A particularly important organism in the medical context is Pseudomonas aeruginosa, but other Gram-negative organisms must also be considered (Table 4), including those implicated as food contaminants. Gram-negative bacteria are invariably less susceptible to quaternary ammonium compounds, bisbiguanides, organomercurials and some phenolics. Organic acids added as food acidulants are almost equally effective in inhibiting the growth of Gram-positive and Gram-negative bacteria (Section 2.2). They are most effective at low pH values and their activity depends mainly on the undissociated form although the anion is now believed to contribute to the overall effect (Eklund 1980, 1983, 1985a,b; Chipley 1983; Gould et al. 1983; Cherrington et al. 1990). When added to certain meat products, sodium metabisulphite or sulphite delays microbial spoilage. Although sulphur dioxide (SO,) was long considered to be solely responsible for the antimicrobial activity, the current view is that bisulphite (HSO,) and sulphite (SO:-) also con-

tribute (Hammond & Carr 1976; Banks et al. 1987). Activity is thus demonstrated only by the free (unbound) form. Gram-negative bacteria including Enterobacteriaceae are inhibited to the greatest extent in meat products, extending shelf-life and favouring eventual growth of less potentiallyhazardous Gram-positive bacteria. 2.2 Food additives

Many chemical agents are potentially inhibitors of microbial growth (Davidson et al. 1983), although only very few are permitted by regulatory authorities to be included in food and pharmaceuticals. Other compounds might also be antimicrobial, notably those occurring naturally in foods. These have been listed by Davidson et al. (1983) and include spices and their oils (Paster et al. 1990), onions, garlic and the lactoperoxidase system of milk (other constituents such as casein, lactoferrin and casein may be important). Most of these appear to be inhibitory rather than lethal, although lactoperoxidase (which requires hydrogen peroxide, as well as thiocyanate, for optimal activity and which is thus primarily active against H,O,-producing bacteria such as streptococci and lactobacilli) may be bactericidal (Davidson et al. 1983). Peroxide is generated via microbial metabolism. Other food additives include hydrogen peroxide (e.g. in the USA in raw milk to be used in making certain types of cheese, for fish marinades and to treat packages for aseptic packing of foods; Busta & Foegeding 1983), the disodium and disodium calcium salts of ethylenediamine tetraacetic acid (EDTA), sodium chloride, citric acid, ascorbic acid, fatty acids and antioxidants. Of these, H,O, is bactericidal, fungicidal and sporicidal, whereas the other chemicals are

194 A . D . R U S S E L L

not normally considered as antimicrobial compounds. EDTA, however, can potentiate the activity of food and pharmaceutical preservatives against Gram-negative bacteria (Russell 1971; Hart 1984). Sodium chloride at organoleptically acceptable levels inhibits the growth of many, but not all, types of bacteria, including B. cereus and CI. botulinum in certain foods, such as cured products (Troller 1983). Citric acid inhibits the growth of proteolytic strains of Cl. botulinum (Graham & Lund 1986), to some extent because of its chelating properties which decrease as pH falls and the inhibition can be overcome by saturation with cations such as Ca2+ ions. Citric acid appears to act also as a permeabilizing agent since it potentiates the effect of monolaurin against Gram-negative bacteria (Shibasaki & Kato 1978), and is obviously worthy of further study. Ascorbic acid (vitamin C) and isoascorbic acid are reducing agents that enhance the anticlostridial action of nitrite in canned meats but are not inhibitory alone. The antibacterial activity of fatty acids has been known for many years (reviewed by Kabara 1978, 1983, 1984a). These compounds can be considered as being aliphatic monocarboxylic acids, some of which, e.g. sorbic, propionic (propanoic) acids, are important preservatives (Table 5). Examples of saturated straight-chain monocarboxylic acids are formic (methanoic), acetic (ethanoic), propionic (propanoic), caproic (hexanoic) and undecylic (hendecanoic) acids. Unsaturated (mono-olefinic) straight-chain monocarboxylic acids include oleic acid (cis-A9-octadecenoic acid) whereas unsaturated (poly-olefinic) straight-chain monocarboxylic acids are exemplified by sorbic (A2,4hexadienoic), linoleic (cis-cis-A9, 12-octadecadienoic) and linolenic (A9,12,15-octadecatrienoic) acids. Escherichia coli and B. subtilis are equally inhibited by similar concentra-

Table 5 Antibacterial activity of lipophilic acids

MIC (mmol/l) us Lipophilic acid*

Gram-positive bacteria

Gram-negative bacteria

Acetic (methanoic) Propionic (propanoic) Caproic (hexanoic; 6 : 0) Caprylic (octanoic; 8 : 0) Capric (decanoic; 10 : 0) Lauric (12 : 0) Linolenic (18 : 2)

65 40 11 1.8 0.25 0.062.5 O.O&NI

58 42 8 >4 > 10 NI NI

* Figures in brackets refer to carbon chain length and number of unsaturated linkages. NI, No inhibition at maximum concentration tested.

tions of compounds containing up to six carbon atoms (Freese et al. 1973; Sheu & Freese 1973; Freese 1978; Freese & Levin 1978; Doores 1983; Kabara 1983, 1984a). Caprylic acid (octanoic acid), a C8 acid, requires higher concentrations to inhibit the Gram-negative organism (Doores 1983) and decanoic acid, a C10 acid, is considerably less effective against E . coli than it is against the Gram-positive organism (Sheu et al. 1975). Kabara et al. (1972) likewise found a greater effect of straight-chain fatty acids against Gram-positive bacteria than against enterobacteria (E. coli, Proteus spp., Serratia marcescens, Klebsiella, Salmonella typhimurium) and Ps. aeruginosa, but further demonstrated that a fatty acid derivative, laurylamine (dodecylamine) hydrochloride, was active against Gram-positive oganisms and enterobacteria. Other authors have likewise shown a greater effect of fatty acids against Gram-positive than Gram-negative organisms (Galbraith et al. 1971; Galbraith & Miller 1973a, b, c; Lueck 1980; Davidson 1983). Lauricidin (a preparation of monolaurin ; glycerol monolaurate) is a non-ionic emulsifier with antibacterial properties (Kabara 1984b). Low concentrations inhibited the growth of Gram-positive bacteria and of fungi and mycoplasma, whereas Gram-negative bacteria were insusceptible. Lauricidin has been approved in the USA for food use as an emulsifier and has been recommended for use in cosmetics. I t has been recommended as a ‘preservative potentiator’ in some foods and cosmetics (Kabara 1984b). Before 1975, butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) were used solely as antioxidants for the prevention of oxidation and rancidity in lipids and lipid-containing foods (Davidson 1983), but they do also exhibit antimicrobial activity. Butylated hydroxyanisole is less effective against E . coli and Salm. typhimurium than against Staph. aureus (Pierson et al. 1980; Davidson 1983), and B H T is considerably less active against Gram-negative organisms. Davidson (1983), for example, quotes M I C values for B H T of 150 ppm against Staph. aureus and > 10000 ppm against Salm. senftenberg. Vitamin A (retinol) shows antibacterial activity; it lyses protoplasts of B. megaterium K M but not those of the mould Neurospora crassa, i.e. it acts on cells containing little or no membrane cholesterol (Dingle & Lucy 1965). Carbon dioxide is an unusual type of additive. As pointed out by Law & Mabbitt (1983) it is unthinkable to add a preservative to milk unless it can be shown to be effective and can be removed after its job is completed. CO, gas (active as HCO;) is inhibitory to many foodspoilage organisms including psychrotrophs and has been used for preserving fruit, vegetables and meats. The gas can easily be removed. CO,-enriched atmospheres in the form of gas-flush packs have been used in the meat industry.

P R E S E R V A T I V E S A N D B A C T E R I A L R E S I S T A N C E 195

3. MECHANISMS OF BACTERIAL RESISTANCE

Having considered the relative sensitivity of various types of bacteria to food and pharmaceutical preservatives and food additives, it is now pertinent to discuss the mechanisms involved in their response to these chemical agents. This aspect is not only of academic interest but also of potential practical significance. Bacterial resistance to preservatives is essentially of two types : (1) intrinsic resistance, a natural (innate) chromosomally-controlled property of an organism ; (2) acquired, resulting from genetic changes in a bacterial cell and arising either by mutation or by the acquisition of genetic material from another cell. 3.1 Resistance of bacterial spores

Spores are more resistant to preservatives than are nonsporulating bacteria (Gould 1985). Conversely, during spore germination and/or outgrowth, spores lose their resistance to both chemical and physical agents and show the same degree of susceptibility as vegetative cells. Intrinsic resistance of spores to preservatives is linked to changes in cell structure. Mechanisms of intrinsic resistance can be studied by using (1) a ‘step-down’ procedure in which vegetative cell cultures are transferred from a rich culture medium to a nutritionally poor medium to produce a 90% synchronously sporulating suspension ; (2) Spo- mutants that sporulate only as far as a genetically determined point in the sporulation process and (3) agents for removing the spore coat(s) (Russell 1990b). On the basis of these experiments, Knott et al. (1990) have summarized the development of resistance during sporulation to preservatives and other types of biocides (Table 6). In this, toluene, moist heat and lysozyme are Table 6 Development of resistance to Bacillus subtilis 168 during sporulation

Development of resistance Early

Intermediate

Late

Phenylmercuric

Lysozyme Glutaralehyde

~~

Toluene

Formaldehyde Anionic surfactant

Phenolics

nitrate

Chlorhexidine diacetate Cetylpyridinium chloride Moist heat Chlorine-releasing agents

taken as markers of early, intermediate and late events, respectively. T h e results demonstrate that intrinsic resistance of bacterial spores to chemical agents is a property essentially of the spore coats, although the cortex may be a contributory factor. In addition to knowing how resistance of spores is expressed towards preservatives and other types of biocides, it is also necessary to comment upon the changes that occur during germination and outgrowth that render spores more sensitive to such inhibitors. Apart from their medical significance of being aetiological agents of some infections, bacterial spores are also of importance as food-poisoning sources, e.g. Cl. botulinum, Cl. perfringens and B. cereus. Spore-forming agents are of particular concern in food products when they are capable of surviving food-processing treatments, of causing food spoilage and of being foodborne pathogens. Due to changes, e.g. palatability, nutritional, in a food it is not often possible to destroy all spores that may be present (Lueck 1980; Genigeorgis 1981; Cook & Pierson 1983; Foegeding & Fulp 1988). Consequently, specific preservatives are often included to prevent growth from spores. Sodium nitrite delays, but does not alone necessarily prevent, botulinal outgrowth (Ingram & Roberts 1971; Cook & Pierson 1983) but its potential toxicity is well known. Methyl and propyl parabens are commonly used as preservatives (Anon. 1984) with the propyl ester more effective in inhibiting Cl. botulinum growth and toxin production. Sorbic acid is a weak lipophilic acid widely used as a food preservative (Lueck 1980; Ronning & Frank 1987). It inhibits botulinal spore germination, a property that is pH-dependent and appears at pH values below 6.0 (Robach & Pierson 1978; Sofos et al. 1979; Sofos & Busta 1981). Potassium sorbate delays growth of Cl. botulinum and toxin production in cured meats (Foegeding & Busta 1981) has been suggested as a potential replacement for nitrite in such products and when added to acidic foods is hydrolysed to sorbic acid. In addition to its effect on germination, sorbate delays or prevents the outgrowth of Cl. botulinum spores (Blocher & Busta 1983, 1985). Antibacterial agents are also used as preservatives in pharmaceutical products but sporeforming organisms are not usually the major organisms of concern (Russell 1991). It is clear from the above that lack of toxicity and prevention of spore germination and outgrowth are two essential properties for any anti-spore preservative. Ideally, a sporicidal action is necessary but sporicides, e.g. glutaraldehyde and hypochlorites, are obviously too toxic for human consumption. Generally, during germination and/or outgrowth, sensitivity to preservatives increases, and the sites of action of some preservatives and other biocides are depicted in Fig. 1, together with the morphological and biochemical changes occurring in the cells. Of particular interest in this context is sorbic acid, which is believed to

196 A . D . RUSSELL

Loss of heat resistance Phase darkening Stainability t Dry weight J DPA release OD 1

Allosteric Bacterial spore

Trigger reaction

t I I I I I

Glutaraldehyde” Alcohols? Sorbate? Chlorocresol ?

-

Germination

I I I I I I

Phenols Cresols Parabens Alcohols HgZ+ Glutaraldehyde”

Biosynthesis (mRNA Protein DNA Cell wall)

Outgrowth

I I

I I

I I

Nisin QACs Biguanides Ethylene oxide” Glutaraldehyde” Organomercurials

Fig. 1 Changes occurring during germination and outgrowth of bacterial spores and effects of preservatives and other biocides. t denotes increase, 1decrease. OD, Optical density; DPA, dipicolinic acid; QACs, quaternary ammonium compounds. “Sporicidalagents also. (Based on Russell & Chopra 1990.)

inhibit the trigger mechanism in germination (Sofos & Busta 1981, 1982; Blocher & Busta 1985; Sofos et al. 1986). Other preservatives that inhibit germination are alcohols, phenols and cresols and parabens and this inhibition occurs a t concentrations that are closely related to those that inhibit the growth of vegetative bacteria. In many instances, however, the effects of preservatives may be reversible, sugesting a fairly loose binding to a site(s) on the spore surface (Russell 1990a, b). Some preservatives allow germination to proceed, but inhibit outgrowth. These include chlorhexidine, quaternary ammonium compounds and organomercurials. Sorbic acid inhibits outgrowth as well as germination.

3.2 Resistance of non-sporlng bacterla

3.2.1 Intrinsic resistance

In Gram-positive cocci there are no specific receptor molecules or permeases to assist preservation penetration. The exclusion limit of the cell wall of vegetative cells of B. megaterium has been calculated as being at least 30 000 (Lambert 1983). Consequently, preservatives enter these cells readily and intrinsic resistance is therefore low. In contrast, the outer membrane of Gram-negative bacteria plays an important role in limiting the entry of preservatives into the cell (Nikaido & Vaara 1985). T h e cell surface of smooth, wild-type Gram-negative bacteria is hydrophilic in nature; deep rough (heptoseless) mutants are much more hydrophobic because of the appearance of phospholipid (PL) patches on the cell surface. In wild-type bacteria, low

molecular weight ( < ca 600-650) hydrophilic molecules cross the outer membrane via the aqueous porins, but lipopolysaccharide (LPS) molecules prevent ready access of hydrophobic preservatives to the PL. I n deep rough strains, lacking the 0-specific side-chain and most of the core polysaccharide and in EDTA-treated cells, the PL patches at the cell surface have their head groups orientated towards the exterior (Fig. 2). Lipopolysaccharides act as a barrier to the entry of longchain fatty acids into Gram-negative cells (Sheu & Freese 1973; Freese & Levin 1978; Kabara 1983, 1984a). Studies with specific mutants of E. coli and Salm. typhimurium have been of use in evaluating the role of the outer membrane as a barrier to the entry of a homologous series of esters (the parabens) of para(4)-hydroxybenzoic acid (Russell et al. 1985, 1987; Russell & Furr 1986). These have increasing hydrophobicities (methyl to butyl) and activity increases against smooth strains and especially against deep rough strains as the homologous series is ascended (Table 7). Some preservatives such as chlorhexidine and quaternary ammonium compounds are believed to damage the outer membrane of Gram-negative bacteria thereby promoting their own uptake (Hancock 1984). However, the quaternaries are considerably less active against wild-type strains than they are against deep rough ones, whereas chlorhexidine has the same order of activity against both. This therefore suggests that LPS acts as an important barrier against entry of the former but not of the latter. Another type of intrinsic resistance is the possession of constitutive enzymes that enable bacteria to degrade preservatives. Although this mechanism is rare at in-use preservative concentrations, degradation of methyl para-

P R E S E R V A T I V E S A N D B A C T E R I A L R E S I S T A N C E 197

(a)

( b ) HYDROPHILIC

HYDROPHOBIC

H YDROPH I LIC

,I *

,

I

I

HYDROPHOBIC

I

Flg. 2 Comparison of the penetration of hydrophilic and hydrophobic preservatives and biocides into Gram-negative bacteria. (a) Smooth cells; (b) deep rough. P, porin; LPS, lipopolysaccharide; PL, phospholipid ; PS, periplasmic space; PTG, peptidoglycan;IM, inner membrane. a, Inner membrane protein; barrier. (Reprinted from Russell & Chopra 1990. Published by permission of Ellis Horwood, a Division of Simon & Schuster International Group).

.,

-

I

PS+ PTG

hydroxybenzoate (Hugo & Foster 1964) and formaldehyde (Heinzel 1988) by Ps.aeruginosa has been demonstrated. 3.2.2 Acquired resistance

Acquired resistance to a preservative or other biocide results either by mutation or by the acquisition of genetic material (usually via a plasmid). General mechanisms have been considered by Heinzel(l988) and Russell (1990a). Plasmid-mediated resistance has been found to inorganic metal salts and organomercurials (Russell 1985; Russell et al. 1986; Silver & Misra 1988). In MRSA strains plasmidencoded resistance to quaternary ammonium compounds, acridines, ethidium bromide and diamidines has been

described (reviewed by Day 8i Russell 1991), and this has been transferred into E. COIL These findings are of possible clinical significance, although it must be noted that (1) only low-level resistance to quaternaries is expressed ; (2) the other compounds described, apart from the organomercurials, are not used as preservatives or other type of biocides and (3) there is as yet no suggestion that plasmidmediated resistance to preservatives poses a problem with food or pharmaceutical products. Resistance to preservatives determined by chromosomal gene mutation has been little studied in comparison to antibiotic resistance. It has long been known, however, that bacterial cells can adapt to grow in the presence of high

Table 7 Response of wild-type and lipopolysaccharide (LPS)-defective/porindeficient strains of Escherichiu coli to parabens*

Ratio of MICs (Me : other esters for each strain) E . coli strain

Protein deficiency

LPS deficiency

Me : Et

Me : Pr

Me : Bu

PC0479 D2 1 D2lf2 PC2040 CE1054 CE1055 CE 1056 CE1057 CE1058 CE1059 CEl122 CE1131

-

-

-

Rough Deep rough Heptoseless

1.46 1.75 2.62 2.2 1.74 2.19 1.74 2.18 1.74 2.19 1.64 1.74

2.5 3.15 7.1 4.73 3.16 4.73 2.71 5.92 2.7 1 9.46 2.84 2.7 1

3.83 4.08 19.17 10.19 6.38 16.97 5.1 15.97 5.1 25.53 3.82 5.1

-

OmpF OmpA OmpA OmpC OmpC OmpA OmpA OmpF OmpA

OmpF OmpF

-

Heptoseless -

OmpF OmpC OmpF OmpC OmpF

* Data of Russell el al. (1985). Ratios have been determined on a molar basis.

Heptoseless -

Heptoseless -

198 A . D . R U S S E L L

concentrations of a preservative. Increased resistance to chlorhexidine and to quaternary ammonium compounds has been described but is often unstable, because transfer of resistant cells to a preservative-free medium, i.e. removal of the selection pressure, usually leads to back-mutations and re-expression of a sensitive phenotype (Russell & Chopra 1990). Gould et al. (1983) described the adaptation of bacteria suddenly transferred to systems of (1) low water activity (a,) and (2) acidification, i.e. lower pH value. In both systems, the most important principle is homeostasis, the organisms reacting to maintain internal water contents or pH values constant. The main difference is that in the former, low a , , system tolerance is expressed via a long adaptation period whereas vegetative cells suddenly shifted to a lower pH value change growth rate. In this situation, there is an increased influx of protons into the cell interior and these must be extruded if internal acidification is to be prevented. This extrusion is energy-demanding. Phenotypically-acquired resistance to lipophilic acid preservatives such as benzoic acid and sorbic acid is well documented in yeasts (Warth 1977, 1978); this results from the enhanced ability of adapted cells to catalyse energydependent extrusion of the acids. No such mechanism has been claimed with bacteria. However, the levels of acquired resistance of bacteria to most organic acids rarely exceeds two- to threefold (Lueck 1980). Escherichia coli resistant to acridine antiseptics are as permeable as sensitive cells to these agents but can expel bound dye (Nakamura 1966; Kusher & Khan 1968). MRSA strains are also believed to extrude cationic preservatives and antiseptics (Russell & Chopra 1990) but these strains are unlikely to be of any significance in food and pharmaceutical preservation.

4. REVIVAL OF INJURED M I C R O B E S

Although revival of injured microbes is not strictly a mechanism of resistance, failure to recognize that seemingly dead bactria in a food or pharmaceutical product can be revived could result in serious consequences. It is not unknown for bacterial spores or non-sporing bacteria to be able to repair sublethal injury after exposure to chemical and/or physical agents (Gilbert 1984; Gould 1984). For this reason, a few brief comments on this topic will be included. In any situation in which bacteria are exposed to an inhibitory chemical preservative (or possibly additive), or to a physical agent, it is possible that the bacterial population will then consist of uninjured (unaffected) cells at one extreme and irreversibly inactivated (dead) cells at the other. In between, various degrees of injury may occur, ranging from slightly damaged to severely damaged (Mossel & Van Netten 1984). Injured cells are particularly sensitive

to selective media, but this in itself can be used as a means of measuring their repair from injury. Revival of preservative-treated spores and vegetative cells has not, however, been as widely studied as that of heat-treated spores, and this is an area which has been rather neglected (Gilbert 1984). 5. POSSIBLE WAYS OF P R E V E N T I N G OR O V E R C O M I N G RESISTANCE

In considering ways of overcoming resistance or of preventing it from arising, due importance must be attached to the choice of a suitable preservative. Most Gram-positive nonsporing bacteria are susceptible to commonly-used preservatives in foods and pharmaceutical products and there is no evidence to suggest that MRSA strains are a problem in either type. When contamination with Gram-negative bacteria is likely to be a problem, preservatives that are hydrophilic, of low molecular weight and not degraded by microbial enzymes are required. Alternatively, Freese & Levin (1978) recommended the use of higher molecular weight compounds with higher partition coefficients, effective at much lower concentrations; on the basis of information presented in this paper, butyl para-hydroxybenzoate would fit into their category. With spore-formers, most preservatives are unlikely to be sporicidal, and effective inhibitors of germination and of toxin production are needed (Smoot & Pierson 1982). Another effective means of control, and therefore of preventing or overcoming resistance, is to use preservative systems that rely on the combined effects of several factors. For example, combinations of preservatives (Denyer et al. 1985, 1986; Lenhmann 1988) might increase the spectrum of activity, decrease resistance and reduce toxic effects. Other combined preservative systems must not be ignored, either ; these include suboptimal pH, temperature, osmotic pressure, a , and modified atmosphere packaging as well as the chemical preservative itself. For example, a combination of BHA and propyl paraben is synergistic against Staph. aureus, although much less so against Salm. typhimurium (Pierson et al. 1980). There are sound reasons for developing such combinations, because many of them act by placing additional energy demands on bacteria and other micro-organisms through interference with energyrequiring homeostatic mechanisms. Such mechanisms regulate cell water content when the environmental osmolarity is changed, regulate cytoplasmic pH and proton gradient across the cell membrane when the environmental pH is shifted, etc. (Gould et al. 1983; Russell & Gould 1988). It is noteworthy that many additives have a role to play in controlling bacterial growth and thereby rendering a preservative more effective. It is recommended that additional studies be made along these lines.

PRESERVATIVES AND BACTERIAL RESISTANCE

Table 8 Mechanisms of bacterial

resistance to preservatives*

199

Bacteria

Intrinsic resistance

Acquired resistance

Spores Staphylococci

Spore coats, mainly

-

-

Plasmids and MRSA strains;

Outer membrane Possible degradation of preservative

usually emux mechanism Mutation (training) : usually unstable resistance Plasmids : organomercurials, possibly formaldehyde

Gram-negative organisms

* -.

Not found to date.

A summary of mechanisms of resistance to preservatives is provided in Table 8.

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