Differential expression of lipoprotein genes in Mycoplasma pneumoniae after contact with human lung epithelial cells, and under oxidative and acidic stress.

BACKGROUND: Mycoplasma pneumoniae is a human pathogen that is a common cause of community-acquired pneumonia. It harbours a large number of lipoprotein genes, most of which are of unknown function. Because of their location on the cell surface, these proteins are likely to be involved in the bacterial response to environmental changes, or in the initial stages of infection. The aim of this study was to determine if genes encoding surface lipoproteins are differentially expressed after contact with a human cell line, or after exposure to oxidative or acidic stress. RESULTS: Using qRT-PCR assays, we observed that the expression of a number of lipoprotein genes was up-regulated when M. pneumoniae was placed in contact with human cells. In contrast, lipoprotein expression was generally down-regulated or unchanged when exposed to either hydrogen peroxide or low pH (5.5). When exposed to low pH, the mRNA levels of four polycistronically transcribed genes in Lipoprotein Multigene Family 6 formed a gradient of decreasing quantity with increasing distance from a predicted promoter. CONCLUSION: The demonstrated transcriptional changes provide evidence for the functionality of these mostly unassigned genes and indicate that they are regulated in response to changes in environmental conditions. In addition we have shown that the members of Lipoprotein Gene Family 6 may be expressed polycistronically.

Detection of Bacillus cereus group bacteria from cardboard and paper with real-time PCR.

The aim of this study was to develop a PCR-based rapid method to detect Bacillus cereus group cells from paper and cardboard. Primers targeting the 16S rDNA and real-time PCR with SYBR green I detection were used in order to be able to also quantify the target. Both autoclaved cardboard samples spiked with B. cereus vegetative cells or spores and naturally contaminated paper and cardboard samples were studied. Results were compared with culturing verified by commercial (API) tests. Several different methods were tested for DNA isolation from the paper and cardboard samples. Two commercial kits intended for soils, the UltraClean soil DNA kit and the FastDNA spin kit for soil, gave the most reproducible results. In spiked samples, the average yield was 50% of added vegetative cells, but spore yield was only about 10%. PCR results from adding vegetative cells correlated with added colony-forming unit (cfu) values ( r=0.93, P <0.001) in the range 100-10,000 cfu g(-1). Three out of nine studied paper and cardboard samples contained B. cereus group bacteria, based both on culturing and real-time PCR. The numbers were 10(2)-10(3) bacteria g(-1); and PCR gave somewhat higher results than culturing. Thus, real-time PCR can be used as a rapid semi-quantitative method to screen paper and cardboard samples for contamination with B. cereus group bacteria.