Jarmila Vytřasová

Isolation ofArcobacter butzleri andA. cryaerophilus in samples of meats and from meat-processing plants by a culture technique and detection by PCR

A pilot survey of sources of contamination with arcobacters (representing a potential risk for humans) was done in a wide range of samples involved various kinds of meat (beef, pork, meat products, chilled chickens,etc.) from a retail level and domestic farming. Sanitary practices in slaughterhouses and production lines were checked in two different plants (a beef and pork production and a chicken processing plant). The method is based on a selective enrichment to isolate suspect strains, in combination with a PCR technique specific for arcobacters. The choice of a suitable enrichment broth and a plating agar was made with the use of pure bacterial strains and by means of real meat samples seeded withArcobacter butzleri. The PCR technique was optimized to allow differentiation of a 1223 bp product, typical of the genusArcobacter, and a product of 686 bp, specific forA. butzleri a total number of 198 samples were tested, of that 33 (17 %) were found to be positive for the genusArcobacter but only 22 (11 %) forA. butzleri.

The effect of acetic acid, citric acid, and trisodium citrate in combination with different levels of water activity on the growth ofArcobacter butzleri in culture

The influence of weak organic acids and trisodium citrate in combination with a high or a reduced water activity (aw) was investigated when a population ofArcobacter butzleri was exposed to a low concentration of acetic or citric acid, and trisodium citrate combined with high (0.993) and reduced (0.977)aw in culture broth at 30 °C. Regardless of water activity, acetic and citric acid (>0.2 %) inhibited the growth ofA. butzleri with no viable cells detected after 4–5 h of incubation. Enhanced survival was found at reducedaw with addition of acetic acid. In contrast, after exposure to citric acid in combination with reducedaw inactivation was more rapid than that after being exposed to high water activity. Incorporation of trisodium citrate in combination with reducedaw (0.977) would probably not confer any extra protection. Concentrations of organic acid widely used in meat decontamination processing represent feasible tools for reducingA. butzleri contamination and hence the risk ofArcobacter infection.

Identification of Arcobacter Species using Phospholipid and Total Fatty Acid Profiles

High-performance liquid chromatography (HPLC) and gas chromatography-mass spectrometry (GC-MS) were used to analyze the phospholipids and fatty acids of fourArcobacter species (becoming routinely isolated from a wide variety of food sources, especially of animal origin) to provide information for the identification within these species. Phospholipid differences were observed in the HPLC profiles. GC-MS analysis provided a complete fatty acid composition for each arcobacter that after pattern recognition analysis allows taxonomic classification of each species.

Effect of Selected Microorganisms on Fusarium Toxins Production

The aim of this study was to examine the effect of selected microorganisms on mycotoxins production by molds of the genus Fusarium, namely HT-2 and T-2 toxins. Appropriate nutritive media were inoculated with test microorganisms (Rhodotorula spp., Leuconostoc spp., Pantoea agglomerans), subsequently inoculated with Fusarium molds, then incubated under various conditions. Content of Fusarium mycotoxins in individual samples was determined using HPLC/MS/MS. Separation of mycotoxins was performed on a C18 stationary phase column using gradient elution. Total analysis time was less than 20 minutes. In examining the effect of accompanying microflora on the production of HT-2 and T-2 toxins, a decrease in production of both mycotoxins was observed under various experimental conditions. Greatest inhibitory effect was observed in the presence of Pantoea agglomerans CCM 298 bacteria. It was found that the amount of HT-2 and T-2 toxins produced by the examined mold strains also depends on cultivation conditions and the nutritive medium used.

Influence of chosen microbes and some chemical substances on the production of aflatoxins.

Aflatoxins are produced as secondary metabolites by A. flavus, A. parasiticus, A. nomius and A. tamarii . The aflatoxin biosynthetic pathway involves several enzymatic steps and genes ( apa-2 , ver- 1) that appear to be regulated by the aflR gene in these fungi. The aim of this work was the detection of aflatoxins by the HPLC method and the ascertainment of factors influencing their production. A. parasiticus CCM F-108, A. parasiticus CCF 141, A. parasiticus CCF 3137 and two isolates A. flavus were used. These toxigenic isolates were recovered from spice (strain 1) and wraps (strain 2). The gene for the production of aflatoxin B1 for each species of fungi was detected using an optimized PCR method. Rhodotorula spp. * , Lactococcus lactis subsp. lactis CCM 1881, Flavobacterium spp. and fungal strain Pythium oligandrum * were tested for inhibition of aflatoxins production and fungal growth. Having used the HPLC detection, various preservatives (propionic acid, citric acid, potassium sorbate) were tested from the viewpoint of their influence on the growth of aflatoxigenic fungi followed by the production of aflatoxins. The growth of A. flavus and A. parasiticus and aflatoxin production in Potato Dextrose Agar supplemented with propionic acid (1000-2000-3000 mg/kg), citric acid (2000-3000-4000 mg/kg) and potassium sorbate (500-800-1000 mg/kg) was tested by Agar Dilution Method. After 72 h of incubation was evaluated growth of fungi, all samples were frozen for later extraction and aflatoxins quantification by HPLC. Effect of peptone and sucrose additions were studied in yeast extract (2%) supplemented with peptone (5-10-15%) or sucrose (15%). Growth inhibition of Aspergillus by Pythium oligandrum was tested on wood surface. As shown, the highest inhibition effect on the aflatoxins production was obtained when propionic acid was applied in concentrations since 1000 mg/kg. A total inhibition of the fungi growth and aflatoxins production was observed in all samples containing peptone in the concentration range tested. Significant limitation of the growth and production of aflatoxins was also observed in the presence of other microorganisms such like Pythium oligandrum and Rhodotorula spp.

Electroimmunoassay for detection of bacterial cells

An amperometric method for Salmonella detection is presented as an example of electroimmunoassay of bacterial cells. The method is based on the reaction of salmonella with an enzyme-linked specific antibody (alkaline phosphatase), forming salmonella-antibody-alkaline phosphatase (SAAP) conjugates. After their hydrolysis, the products formed (phenols) are detected amperometrically using a carbon paste electrode; current signals were monitored at 0.65 V vs. Ag/AgCl/sat. KCl. A medium of pH = 10.00 was found as the best. Amount of phenol generated by SAAP was proportional to the number of Salmonella Enteritidis in a sample.