Fungal strains and the development of tolerance against natamycin.

Antimicrobial resistance is a relevant theme with respect to both antibacterial and antifungal compounds. In this study we address the possible development of tolerance against the antifungal food preservative natamycin. A selection of 20 fungal species, originating from a medical as well as a food product context, was subjected to increasing concentrations of natamycin for prolonged time, a procedure designated as "training". The range of Minimum Inhibitory Concentrations (M.I.C.) before (1.8-19.2µM) and after (1.8-19.8µM) training did not change significantly, but natamycin-exposure caused an increase of M.I.C. in 13 out of 20 tested strains. The average M.I.C. increased from 6.1 to 8.6µM and 4 strains showed a >2-fold increase of tolerance after training. One strain (of Aspergillus ochraceus) also showed increased tolerance to amphotericin B and nystatin. However, two Fusarium strains showed similar or even decreased tolerance for these other polyene antifungals. The work reported here shows that a continuous and prolonged increasing selection pressure induced natamycin tolerance in individual strains. This implies that such a selection pressure should be avoided in the technical application of natamycin to ensure its continued safe use as a food preservative.

The decarboxylation of the weak-acid preservative, sorbic acid, is encoded by linked genes in Aspergillus spp.

The ability to resist anti-microbial compounds is of key evolutionary benefit to microorganisms. Aspergillus niger has previously been shown to require the activity of a phenylacrylic acid decarboxylase (encoded by padA1) for the decarboxylation of the weak-acid preservative sorbic acid (2,4-hexadienoic acid) to 1,3-pentadiene. It is now shown that this decarboxylation process also requires the activity of a putative 4-hydroxybenzoic acid (3-octaprenyl-4-hydroxybenzoic acid) decarboxylase, encoded by a gene termed ohbA1, and a putative transcription factor, sorbic acid decarboxylase regulator, encoded by sdrA. The padA1,ohbA1 and sdrA genes are in close proximity to each other on chromosome 6 in the A. niger genome and further bioinformatic analysis revealed conserved synteny at this locus in several Aspergillus species and other ascomycete fungi indicating clustering of metabolic function. This cluster is absent from the genomes of A. fumigatus and A. clavatus and, as a consequence, neither species is capable of decarboxylating sorbic acid.

A rapid microbial inhibition-based screening strategy for fluoroquinolone and quinolone residues in foods of animal origin.

A rapid, high-throughput antimicrobial screening assay for the detection of fluoroquinolone and 4-quinolone residues in foods of animal origin has been developed in ampoule format. The assay employs a single Escherichia coli species sensitive to those Gram-negative inhibitiory antimicrobial compounds and is presented in a comparable format to the existing commercially available Premi Test and Delvotest ampoule-based microbial inhibition tests (DSM, Delft, The Netherlands). In the novel E. coli assay the microorganism, in vegetative state, is inoculated into a nutrient agar pellet containing a pH sensitive acid-base indicator dye. A simple extraction protocol that is selective for fluoroquinolone and quinolone compounds was developed to recover, cleanup and concentrate the target analyte(s) from a variety of tissue types and matrices prior to screening analysis. The method detected 16 target compounds at concentrations equal to or below the maximum residue limits (where applicable). The method has been validated using the prototype assay in accordance with the 2002/657/EC guidelines for the validation of qualitative screening assays. False positive and false negative responses rates for the procedure have been determined as less than 5%. The stability of a selection of representative target analytes has been demonstrated for a 20-week period under a variety of storage conditions both in tissue and in extract.