Preservative tolerance and resistance

Preservative tolerance is defined as a situation in which a formerly effective preservative system no longer controls microbial growth. Tolerance can have several potential causes: destabilization of the preservative, establishment of biofilms in the manufacturing system, and the development of resistance. As part of an effort to understand and control tolerance, a collection of preservative-resistant bacteria was established. Pseudomonas and related genera constitute 92% of the isolates in the collection. The collection includes bacteria isolated from products preserved with dimethoxy dimethyl hydantoin (DMDMH), dibromodicyanobutane (DBCB), glutaraldehyde (GLUT), benzisothiazolone (BIT), and methylchloroisothiazolone/methylisothiazolone (MCI/MI). Several isolates were also obtained from products preserved with combinations of DMDMH/iodopropargyl butylcarbamate (IPBC) and DMDMH/IBPC/MCI/MI. Bacteria were deemed resistant if they were isolated from a matrix in which the preservative was stable, and their minimal inhibitory concentrations (MIC) were significantly elevated as compared to a group of conspecific strains from the American Type Culture Collection. Isolates resistant to DMDMH, DBCB, or GLUT had normal outer membrane protein (OMP) profiles, and were cross-resistant to formaldehyde (FA) and several other FA-releasing preservatives, a pattern typical of strains producing elevated levels of FA dehydrogenase. The BIT-resistant isolates were cross-resistant to a number of structurally dissimilar preservatives but retained susceptibility to MCI. MCI-resistant isolates had altered OMP profiles, displayed reduced uptake of MCI, and were cross-resistant to some other preservatives. Restoration of sensitivity to MCI by treatment with EDTA confirmed the role of the outer membrane in MCI resistance.