Resistance and Adaptation to Preservatives and Sanitizers

A recent Institute of Food Technologists' (IFT) Scientific Status Summary (Food Technology 56 2002 11) discusses in some detail the potential for microorganisms to become resistant to antimicrobials (preservatives) and sanitizers.

The authors list three reasons for concern in this area:

1. the increasing incidence of microorganisms exhibiting resistance to antibiotics.
2. the increasing reliance on preservatives and sanitizers as primary tools for controlling the outgrowth of pathogens in foods.
3. evidence that tolerance to preservatives and sanitizers may be generated within microorganisms exposed to certain stresses.

The second reason given is more pertinent to the US scene than it is to Australia. Preservatives used in foods in Australia must be sanctioned in the Food Standards Code and the foods permitted to contain a preservative are also subject to the same legislation. In general, in Australian law, foods which can support the growth of pathogens do not rely on a chemical preservative system for their safety. The exceptions to this are the cured meats which are permitted to contain nitrite or nitrate. No pathogenic spore-forming bacteria have been found to be resistant to the inhibitory properties of nitrite when heated.

However, it is true that sanitizers, defined as processing aids in the Food Standards Code, are used in some industries (e.g. fresh cuts and sprouted seeds) as primary mechanisms for controlling the initial load of microorganisms including potential pathogens on the product. The sanitizers mainly used for this purpose are chlorine and chlorine derivatives, peracetic acid, hydrogen peroxide and some quarternary ammonium compounds.

The authors of the summary note that while a great deal is known about the mechanisms of action of and resistance to antibiotics used therapeutically, the mechanisms and targets of most preservatives and sanitizers are not clearly understood. We are therefore less able to predict potential resistance to these groups of compounds. The following outlines some from the main points in the summary.

Resistance to preservatives
Any resistance of microorganisms to preservatives (or sanitizers) may be innate, apparent or acquired.

Innate resistance is a genetically controlled property that is naturally always associated with a particular microorganism. Differences in resistance demonstrated by different types of microorganism under identical conditions are most likely controlled innately say the authors. Apparent resistance is related to the application conditions. Food pH is the most important factor that influences the effectiveness of the common food preservatives which are themselves weak acids, e.g. sorbic acid and benzoic acid. They are most effective in their undissociated form. The lower the pH, the greater the proportion of the acid in the undissociated form and the greater the antimicrobial activity. Acquired resistance results from genetic changes in the microbial cell through mutation or acquisition from other cells.

Because antibiotics generally have specific target sites in microbial cells, they have greater potential to be rendered ineffective by mutations and the development of acquired resistance. Preservatives and sanitizers are non-specific and resistance to these compounds is caused primarily by innate factors.

Benzoic acid and its salts and sorbic acid and its salts have been approved food preservatives for many decades. The primary use of these compounds is to inhibit yeasts and moulds in acidic foods. Differences in microbial resistance to benzoates and sorbates occurs as a result of differences in innate tolerance. Therefore, the innate resistance of yeasts and moulds to benzoates and sorbates is of greater concern to the food industry than that of bacteria. A number of yeasts including Schizosaccharomyces pombe and Zygosaccharomyces bailii have been observed to grow in 500 mg/L of benzoic acid - significantly above the usual inhibitory concentration.

There is also strong evidence, cited in the summary, for acquired resistance to benzoate and sorbate by some yeasts. Work in this laboratory in the 1970s and 80s illustrated that Z. bailii and other yeasts grown in the presence of sub-inhibitory concentrations of benzoic or sorbic acid were able to tolerate concentrations of preservatives that were up to two times greater than concentrations used to inhibit unadapted cells.

Resistance of yeasts to these weak acid preservatives probably involves more than one metabolic system (International Journal of Food Microbiology 50 1999 1).

However, considering the length of time that sorbic and benzoic acids have been used in food products without the development of acquired resistance (i.e. in organisms where it was not previously present) the development of such resistance is very rare or non-existent the summary authors conclude.

Resistance to sanitizers
The summary also concludes that there is no evidence that proper use of sanitizers in food manufacturing will lead to development of resistant microorganisms. However with increasing use of sanitizers on food handling equipment and on raw products, the potential for emergence of such resistant organisms must continue to be evaluated.

It is recommended good hygiene practice to alternate from time to time the chemical sanitizers used in a food processing environment. This, however, is because certain microorganisms have innate resistance to some sanitizers including chlorine, the most commonly used sanitizer.

What this means is that persistent use of one sanitizer in a factory situation may lead to the selection and persistence of innately resistant microorganisms.

Bacterial spores have relatively high innate resistance to chlorine although this resistance varies considerably from strain to strain. This has caused problems in can cooling environments where sub-lethal concentrations of chlorine can select a resistant population which can build to high numbers in microenvironments. The antimicrobial activity of chlorine compounds is significantly reduced by organic matter and high pH and these conditions must be avoided to ensure the chlorine sanitizer is being delivered at the intended concentration. The summary cites an example of varying Salmonella resistance to chlorine in a poultry processing environment where chlorine remains the sanitizer of choice.

It is also noted that it is now common practice to use sanitizers, again primarily chlorine compounds, in the wash water for fresh and minimally processed fruits and vegetables. One of the pathogens associated with these products in foodborne illness incidents has been Listeria monocytogenes. The summary cites a report (Journal of Food Protection 59 1996 374) which describes the exposure of L. monocytogenes to sub-lethal concentrations of a range of commonly used sanitizers. Except for one (an acid ionic compound), there was no measurable difference in susceptibility of cells when subsequently challenged with commercial use concentrations of the sanitizers.


Resistance to naturally occurring preservatives
The summary states that most of the attention on acquired resistance has been focussed on microbiologically derived preservatives. There is greater similarity between these compounds and their mode of action with the medically important antibiotics. In contrast to antibiotics, however, these preservatives have a much narrower spectrum of activity.

The two important preservatives in this category which have been studied in relation to acquired resistance are natamycin and nisin.

Natamycin, usually referred to a pimaricin in this country, is effective against yeasts and moulds but has little or no effect on bacteria and is used primarily as an antifungal agent on cheese. Studies to investigate the potential for development of resistance to natamycin amongst fungi have concluded that such development is both rare and of a minor nature only.
This is attributed to the lethal, as opposed to static, activity of the compound together with its instability over time. The situation with nisin is rather different. Nisin has a narrow spectrum of activity against vegetative cells and spores of Gram- positive bacteria.

Target microorganisms exhibiting resistance to nisin may inactivate the peptide by enzyme activity or they may alter their membrane to block entry of the molecule. Microorganisms showing innate resistance to nisin have been found in a number of genera including Clostridium and Bacillus species and L. monocytogenes. The obvious implication of the emergence of pathogenic microorganisms resistant to this and similar compounds is the hazard in foods preserved by a single compound of this type and is a sound reason for avoiding such a situation.

The authors of the summary note that the most important question concerning the potential for target microorganisms to acquire resistance to this type of compound is whether such resistance conveys an advantage over non-resistant strains in food systems. They conclude there is no evidence to support this but more research is definitely warranted. Their overall conclusion is that, with the data presently available to us, resistance of microorganisms to preservatives and sanitizing systems does not appear to have 'a major negative impact on health'. However they believe it is critical to acquire more data that is relevant to real food processing situations.