Detection of viable and dead Listeria monocytogenes on gouda-like cheeses by real-time PCR.

AIMS: Surface contamination by Listeria monocytogenes of gouda-like cheeses during processing represents a potential public health problem. The aim of this work was to develop novel real-time PCR diagnostics to detect the presence of viable, dead or viable but not culturable (VBNC) cells on gouda-like cheeses. METHODS AND RESULTS: We used ethidium monoazide bromide (EMA)-PCR for direct quantification of viable and dead cells, while semiquantitative detection of culturable cells below the PCR detection limit (c. 100 CFU g(-1)) was obtained by combining growth and real-time PCR. We were able to quantify the fraction of >0.5% viable cells in a background of dead cells by EMA-PCR, given that the viable cell concentration was above the PCR detection limit. The combined growth and real-time PCR complemented the EMA-PCR, and enabled semiquantitative detection of low levels of culturable cells (10 and 100 CFU g(-1)). SIGNIFICANCE AND IMPACT OF THE STUDY: The significance of this work is that we have developed a novel concept for detection of viable and potentially infectious L. monocytogenes.

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.

Pulmonary reaction associated with transfusion of plasma containing anti-5b.

A patient developed an acute respiratory distress syndrome with lung infiltrates during a transfusion of plasma. The transfused plasma was shown to contain a potent leucocyte agglutinating antibody of IgG type with anti-5b specificity, reactive with the patient's cells. A review of the literature on similar transfusion-related syndromes showed an association with more of less well-defined white cell antibodies, mainly in the donors' plasma. The majority of donors were multiparous women. The anti-5b in our donor was demonstrated only by granulocyte agglutination technique, as previously described antibodies in the five system. A long incubation procedure (20-25 h) seemed to be optimal for granulocyte agglutination.