Selection of Test Strains for Sanitizer Efficacy and Challenge Studies

The strain or strains of a particular microorganism selected for studies designed to determine the efficacy of a decontamination treatment or survival and growth in challenge studies are extremely important. The use of well-characterized reference strains enhances the comparative assessment of a given method among laboratories. Five or more strains, preferably recently isolated from produce or other plant materials, and from patients suffering from illness associated with consumption of a raw fruit or vegetable, are preferred. Approximately equal populations of each strain in a mixed inoculum should be used. If there are differences in the ability of one or more of these strains to survive or grow on produce subjected to various environmental conditions during storage, or if there are differences in susceptibility to decontamination treatments, the most robust strain(s) will prevail. If only one strain is used in the inoculum, it should be first evaluated against several other strains for its ability to survive or grow under the proposed test conditions. The use of a single strain that may be less tolerant to test conditions could result in an inaccurate assessment of the behavior of the test microorganism. Strains used to prepare mixed-strain suspensions should be examined for potential reactions against each other that may be caused by bactoriocins, killer proteins, and other inhibitors they may produce.

Test microorganisms should be cultured in a standard broth or on a defined agar medium at a specific temperature for a specific time. The temperature at which microorganisms are grown for preparing inocula should be representative of the temperature at which they had grown before contaminating produce or the temperature at which inoculated produce will be stored after inoculation, in the case of a challenge study. Several transfers of cultures should be made preceding the day of inoculum preparation. The time elapsed between the last transfer and collecting cells to prepare the inoculum will depend on the test microorganism. Although this practice may result in strains with reduced environmental stress tolerance as a result of adaptation to a nutrient-rich medium, it is desirable to prepare inoculum of uniform cell type. The type of study being conducted should be considered in terms of potential impact of genetic selection of test strains on the predictive value of the results. This is particularly important for pathogenic bacteria that may have originated from diverse sources such as clinical specimens, foods, or the environment. Stationary phase bacterial and yeast cells are generally more tolerant than are logarithmic growth phase cells to environmental stresses [43]. For this reason, cells in stationary growth phase should be used in studies to develop optimum procedures to assess their behavior on or in inoculated produce.

The use of markers such as antibiotic resistance may be desirable to facilitate the recovery of cells in enrichment broth or counting colonies on selective or nonselective direct plating media. Otherwise, these media may support the growth of large numbers of background microflora which interfere with growth of the test microorganism. Adaptation of Gram-negative pathogens to nalidixic acid (50 ^/ml) has been used to achieve this objective. In a study to determine survival of five strains of nalidixic acid-resistant and refampicin-resistant Salmonella Poona on cantaloupes it was observed that average reductions in the number of control and antibiotic-resistant cells were not significantly different (P > 0.05) [44]. Resistance of test cells to rifampicin (80 ^/ml) can also be successfully used as a marker, particularly for isolating pathogens from inoculated fruits and vegetables that have significant adhering soil. Plasmid-borne or chromosomally stabilized markers such as fluorescent proteins with various chromophoric properties have also been used. It is important to assess the impact of markers on the growth rate, stress tolerance, and recovery efficiency of cells on enumeration media before subjecting them to sanitizer efficacy or challenge studies. Characterization of the stability of the marker over at least ten generations, without selection, is needed for challenge studies in which growth may occur and for recovery methods that include preenrichment or enrichment procedures.

Different strains of the same bacterial species may release byproducts that inhibit or kill other strains. Colicins produced by Escherichia coli and killer toxins produced by some species of yeasts are examples. When an inoculum containing several strains of the same microorganism is used, each strain should be tested for its potential to inhibit all other strains in the inoculum. This can be done by cross streaking cultures of individual strains on an appropriate agar medium and examining incubated plates for inhibition of growth at intersections of the streaked cultures.

Determination of the survival characteristics of viruses and parasites on produce poses unique problems. Survival of enteric viruses has been studied but obstacles still remain in standardizing methodology to determine the efficacy of sanitizers in killing or removing viruses that may occasionally contaminate produce. Freshly isolated viruses such as hepatitis A and noro-viruses are not culturable, and thus cannot be propagated in sufficient quantities to prepare inocula, nor can they be quantitated by plaque assay. A few viruses, e.g., poliovirus, have been adapted to grow in tissue culture in the laboratory and can be quantitated by plaque assays. Although some of these viruses belong to the same family, they can vary greatly in their level of resistance to chemical and physical stresses. Adapted strains may be representative of their parental wild type but not other members in the same family. Propagation of foodborne viruses has been limited to only a few adapted strains. The behavior of these strains may or may not be similar in behavior to other isolates of the same virus. Rapid molecular methods to detect viruses in foods are sensitive but cannot be used to quantitate viruses or to differentiate between infectious and noninfectious strains [45]. These attributes pose unique challenges in developing and standardizing methodology for detecting and quantitating viruses on produce.

Survival of parasites on raw produce as affected by treatment with sani-tizers or exposure to various environmental stress factors during storage has not been well defined. A major constraint to investigating survival characteristics is the limited supply of oocysts [46]. Infected humans are the only source of Cyclospora cayetanensis oocysts in quantities needed in studies to determine susceptibility to stress or lethal conditions that may be imposed by decontamination treatment or storage conditions. The lack of sensitive laboratory methods for quantitating and assessing the viability of oocysts hampers progress in developing methods to determine the efficacy of saniti-zation treatments and the influence of processing, packaging, and storage conditions on their survival.

Vehicles of pathogens and spoilage microorganisms for contaminating fruits and vegetables include dust, rain water, irrigation water, sewage, soil, feces, decayed plant material, contact surfaces, workers at any point from harvesting through preparation in foodservice, and home settings [47] Vegetative cells, spores, cysts, and other propagules of microorganisms are likely to be entrapped in organic material. To simulate practical conditions of surface contamination of produce, the carrier for the inoculum should contain organic material. Horse serum (5%) and aqueous peptone solution (0.1%) have been used as carriers with fairly defined composition in studies to determine the efficacy of sanitizers. Buffer solutions and other carriers containing salts or other chemicals that could be detrimental to cells after the inoculum has dried on the surface of test produce are not recommended for use as carriers. Cells in broth cultures of bacteria or yeasts should be washed in peptone water and resuspended in the organic carrier shortly before using as an inoculum. For challenge studies designed to determine the survival or growth of pathogenic and spoilage microorganisms on or in produce, the carrier may provide a source of nutrients, thus complicating interpretation of results. The use of two carriers, one with and one without organic material (deionized or distilled water) for test cells may be useful in generating information to enable the effects of carrier nutrients on survival and growth of test microorganisms to be discerned.

The desired population of test cells in the inoculum depends on the objective of the study. Two or three levels of inocula, ranging from 100 to 107CFU/g or CFU/cm2, may be applied to facilitate the determination of efficiency of retrieval, efficacy of sanitizers, or survival and growth during subsequent storage. High numbers of cells in the inoculum are needed in decontamination studies to enable measurement of several log10 reductions in population. Challenge studies require inocula containing low numbers of cells to enable measurement of growth during storage under conditions simulating practices to which produce is subjected in commercial distribution, retail, foodservice, and home settings.

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