Subtyping Listeria monocytogenes through the combined analyses of genotype and expression of the hlyA virulence determinant.

AIMS: A major challenge for Listeria monocytogenes diagnostics is that this bacterium is ubiquitous in the environment, and that only a small fraction of the lineages are potential human pathogens. The aim of this work was to obtain a better subtyping of L. monocytogenes through utilization of combined analyses of genotype and the expression of the virulence determinant hlyA. METHODS AND RESULTS: We investigated the effect of growth temperature and medium on the hlyA expression. The gene expression levels were determined by real-time quantitative reverse transcription PCR. The expression pattern of hlyA was highly diverse among the different strains tested. The expression ranged from repression to a 1000-fold induction for growth at 42 degrees C, as compared with 0 degrees C. The expression patterns were compared with the corresponding genotypes. There were surprisingly low correlations between the expression patterns and the genotype clusterings. This is exemplified for the virulent type strain NTNC 7973 and non-virulent type strain DSMZ 20600. These strains are genetically nearly identical, while the hlyA gene expression patterns are very different. CONCLUSIONS: The hlyA gene expression was highly diverse even within genetically clustered subgroups of L. monocytogenes. Consequently, the gene expression patterns can be used to further differentiate the strains within these genetic subgroups. SIGNIFICANCE AND IMPACT OF THE STUDY: A major limitation in the control of L. monocytogenes is that the current tools for subtyping are not accurate enough in determining the potential virulent strains. The impact of this study is that we have developed a subtyping approach that actually targets a virulence property.

Industrial application of an antilisterial strain of Lactobacillus sakei as a protective culture and its effect on the sensory acceptability of cooked, sliced, vacuum-packaged meats.

The application of a protective lactic acid bacterium (LAB) during the commercial production of cooked meat products is described. The LAB, a strain of Lactobacillus sakei, was previously isolated from cooked ham and inhibited growth of Listeria monocytogenes and Escherichia coli O157:H7 in this product. L. sakei was applied to the cooked products at a concentration of 10(5)-10(6) cfu/g immediately before slicing and vacuum-packaging using a hand-operated spraying bottle. The LAB strain inhibited growth of 10(3) cfu/g of a cocktail of three rifampicin resistant mutant L. monocytogenes strains both at 8 degrees C and 4 degrees C. Consumer acceptance tests of cooked ham and of servelat sausage, a Norwegian non-fermented cooked meat sausage, showed that control and inoculated products were equally acceptable. The products were still acceptable after storage for 28 days at 4 degrees C and, after opening the packages, for a further 5 days at 4 degrees C. The findings presented here confirm that the L. sakei strain is suitable for use as a protective culture and may technically easily be implemented in the commercial production of cooked meat products.

Comparison between the growth of Yersinia enterocolitica, listeria monocytogenes, escherichia coli O157:H7 and salmonella spp. in ground beef packed by three commercially used packaging techniques.

Growth of the pathogens Yersinia enterocolitica, Listeria monocytogenes, Escherichia coli O157:H7 and strains of Salmonella were compared in ground beef packed in modified atmospheres of 60% CO2/40% N2/0.4% CO (high CO2/low CO mixture), 70% O2/30% CO2 (high O2 mixture) and in chub packs (stuffed in plastic casings). The ground beef was inoculated with rifampicin-resistant or nalidixic acid/streptomycin-resistant strains of the pathogens (final concentration 10(2) - 10(3) bacteria/g) and stored at 4 and 10 degrees C for up to 14 days. At 4 degrees C the shelf life, based on colour stability and background flora development, was prolonged for the high CO2/low CO mixture compared to the two other packaging methods, but at 10 degrees C the shelf life was < 8 days for all the packaging methods. Growth of Y. enterocolitica was nearly totally inhibited both at 4 and 10 degrees C in the high CO2/low CO mixture, while the bacterial numbers in the samples packed in the high O2 mixture increased from about 5 x 10(2) bacteria/g at day 0 to about 10(4) at day 5 at 4 degrees C and to 10(5) at 10 degrees C. Growth in the chub packs was even higher. L. monocytogenes showed very little growth at 4 degrees C in all treatments. At 10 degrees C there was slow growth from about 5 x 10(3) bacteria/g to about 10(4) at day 5 in the high CO2/low CO mixture, while the numbers in the high O2 mixture and the chub packs were about 10 times higher. Growth of E. coli O157:H7 at 10 degrees C in the ground beef was nearly totally inhibited in both the high CO2/low CO mixture and the high O2 mixture. Growth in the chub packs was higher, as the number of bacteria increased 3 log in 5 days. The Salmonella strains (S. typhimurium, S. dublin, S. enteritidis and S. enterica 61:k:1,5,(7)) in the ground beef stored at 10 degrees C for 5 and 7 days grew to a higher number in the high CO2/low CO mixture than in the high O2 mixture. This study shows that the growth of Y. enterocolitica and L. mononcytogenes in ground beef stored in the high CO2 /low CO mixture was not increased as a result of prolonging the shelf life. However, the observed growth of strains of Salmonella at 10 degrees C in this mixture and in chub packs does emphasise the importance of temperature control during storage.

Protective cultures inhibit growth of Listeria monocytogenes and Escherichia coli O157:H7 in cooked, sliced, vacuum- and gas-packaged meat.

Contamination of cooked meat products with Listeria monocytogenes poses a constant threat to the meat industry. The aim of this study was therefore to investigate the use of indigenous lactic acid bacteria (LAB) as protective cultures in cooked meat products. Cooked, sliced, vacuum- or gas-packaged ham and servelat sausage from nine meat factories in Norway were inoculated with 10(3) cfu/g of a mixture of three rifampicin resistant (rif-mutant) strains of L. monocytogenes and stored at 8 degrees C for four weeks. Growth of L. monocytogenes and indigenous lactic acid flora was followed throughout the storage period. LAB were isolated from samples where L. monocytogenes failed to grow. Five different strains growing well at 3 degrees C. pH 6.2, with 3% NaCl, and producing moderate amounts of acid were selected for challenge experiments with the rif-resistant strains of L. monocytogenes. a nalidixic acid/streptomycin sulphate-resistant strain of Escherichia coli O157:H7 and a mixture of three rif-resistant strains of Yersinia enterocolitica O:3. All five LAB strains inhibited growth of both L. monocytogenes and E. coli O157:H7. No inhibition of Y. enterocolitica O:3 was observed. A professional taste panel evaluated cooked, sliced, vacuum-packaged ham inoculated with each of the five test strains after storage for 21 days at 8 degrees C. All samples had acceptable sensory properties. The five LAB strains hybridised to a 23S rRNA oligonucleotide probe specific for Lactobacillus sakei. These indigenous LAB may be used as protective cultures to inhibit growth of L. monocytogenes and E. coli O157:H7 in cooked meat products.

Caldicellulosiruptor kristjanssonii sp. nov., a cellulolytic, extremely thermophilic, anaerobic bacterium.

A cellulolytic anaerobic bacterium, strain I77R1BT, was isolated from a biomat sample of an Icelandic, slightly alkaline, hot spring (78 degrees C). Strain I77R1BT was rod-shaped, non-spore-forming, non-motile and stained Gram-negative at all stages of growth. It grew at 45-82 degrees C, with an optimum growth temperature around 78 degrees C. At 70 degrees C, growth occurred at pH 5.8-8.0, with an optimum near pH 7.0. At the optimum temperature and pH, with 2 g cellobiose l-1 as substrate, strain I77R1BT had a generation time of 2 h. During growth on Avicel, strain I77R1BT produced acetate, hydrogen and carbon dioxide as major fermentation products together with small amounts of lactic acid and ethanol. The strain fermented many substrates, including cellulose, xylan, starch and pectin, but did not grow with casein peptone, pyruvate, D-ribose or yeast extract and did not reduce thiosulfate to H2S. The G+C ratio of the cellular DNA was 35 mol%. Comparative 16S rDNA analysis placed strain I77R1BT among species of Caldicellulosiruptor. The closest relative was Caldicellulosiruptor lactoaceticus. Hybridization of total DNA showed 42% hybridization to C. lactoaceticus and 22% hybridization to Caldicellulosiruptor saccharolyticus. A new species, Caldicellulosiruptor kristjanssonii sp. nov. (I77R1BT) is proposed.

Extremely thermophilic cellulolytic anaerobes from Icelandic hot springs.

Anaerobic enrichment cultures with Avicel as substrate and inoculated with biomat samples from Icelandic hot springs were cultured at 70 degrees C or 78 degrees C and examined for the presence of microorganisms that produce extracellular cellulolytic and xylanolytic enzymes. From four enrichments grown at 78 degrees C eighteen strains were isolated. Five of the strains were screened for their substrate utilization, and on the basis of differences in morphology and substrates used, the two most unique strains were selected for further characterization. All cellulolytic cultures were rod-shaped and non-sporeforming. Motility was not observed. Cells stained gram-negative at various stages of the growth phase. During growth on Avicel, most cultures produced acetate as the major fermentation product, with smaller amounts of lactic acid and ethanol. Carbon dioxide and hydrogen were also produced. The phenotypic characteristics of the enrichment cultures and of isolates are described and assessed in relation to temperature and pH in the hot spring environment. A comparison is made between Icelandic strains isolated in our laboratory and strains isolated from hot springs from other parts of the world. The biotechnological potential of this group of bacteria is briefly discussed.