Detection of Balamuthia mandrillaris DNA by real-time PCR targeting the RNase P gene

<p>Abstract</p> <p>Background</p> <p>The free-living amoeba <it>Balamuthia mandrillaris </it>may cause fatal encephalitis both in immunocompromised and in – apparently – immunocompetent humans and other mammalian species. Rapid, specific, sensitive, and reliable detection requiring little pathogen-specific expertise is an absolute prerequisite for a successful therapy and a welcome tool for both experimental and epidemiological research.</p> <p>Results</p> <p>A real-time polymerase chain reaction assay using TaqMan^®probes (real-time PCR) was established specifically targeting the RNase P gene of <it>B. mandrillaris </it>amoebae. The assay detected at least 2 (down to 0.5) genomes of <it>B. mandrillaris </it>grown in axenic culture. It did not react with DNA from closely related <it>Acanthamoeba </it>(3 species), nor with DNA from <it>Toxoplasma gondii</it>, <it>Leishmania major</it>, <it>Pneumocystis murina</it>, <it>Mycobacterium bovis </it>(BCG), human brain, various mouse organs, or from human and murine cell lines. The assay efficiently detected <it>B. mandrillaris </it>DNA in spiked cell cultures, spiked murine organ homogenates, <it>B. mandrillaris</it>-infected mice, and CNS tissue-DNA preparations from 2 patients with proven cerebral balamuthiasis. This novel primer set was successfully combined with a published set that targets the <it>B. mandrillaris </it>18S rRNA gene in a duplex real-time PCR assay to ensure maximum specificity and as a precaution against false negative results.</p> <p>Conclusion</p> <p>A real-time PCR assay for <it>B. mandrillaris </it>amoebae is presented, that is highly specific, sensitive, and reliable and thus suited both for diagnosis and for research.</p

Characterisation of porin genes from Mycobacterium fortuitum and their impact on growth

<p>Abstract</p> <p>Background</p> <p>Highly pathogenic mycobacteria like <it>Mycobacterium tuberculosis </it>are characterised by their slow growth and their ability to reside and multiply in the very hostile phagosomal environment and a correlation between the growth rate of mycobacteria and their pathogenicity has been hypothesised. Here, porin genes from <it>M. fortuitum </it>were cloned and characterised to address their impact on the growth rate of fast-growing and pathogenic mycobacteria.</p> <p>Results</p> <p>Two genes encoding porins orthologous to MspA from <it>M. smegmatis, porM1 </it>and <it>porM2</it>, were cloned from <it>M. fortuitum </it>strains, which were originally isolated from human patients. Both porin genes were at least partially able to complement the mutations of a <it>M. smegmatis </it>mutant strain lacking the genes <it>mspA </it>and <it>mspC </it>with respect to the growth rate. <it>PorM1 </it>and <it>porM2 </it>were present in different strains of <it>M. fortuitum </it>including the type strain. Comparative expression analysis of <it>porM </it>genes revealed divergent porin expression among analysed <it>M. fortuitum </it>strains. Repression of the expression of porins by antisense technique decreased the growth rates of different <it>M. fortuitum</it>. The effects of over-expression of <it>porM1 </it>as well as <it>porM2 </it>varied depending on the strain and the concentration of antibiotic added to the medium and indicated that PorM1 and PorM2 enhance the growth of <it>M. fortuitum </it>strains, but also the diffusion of the antibiotic kanamycin into the cells.</p> <p>Conclusion</p> <p>This study demonstrates the important role of porin expression in growth as well as antibiotic susceptibility of the opportunistic bacterium <it>M. fortuitum</it>.</p

Viral promoters can initiate expression of toxin genes introduced into Escherichia coli

BACKGROUND: The expression of recombinant proteins in eukaryotic cells requires the fusion of the coding region to a promoter functional in the eukaryotic cell line. Viral promoters are very often used for this purpose. The preceding cloning procedures are usually performed in Escherichia coli and it is therefore of interest if the foreign promoter results in an expression of the gene in bacteria. In the case molecules toxic for humans are to be expressed, this knowledge is indispensable for the specification of safety measures. RESULTS: We selected five frequently used viral promoters and quantified their activity in E. coli with a reporter system. Only the promoter from the thymidine kinase gene from HSV1 showed no activity, while the polyhedrin promoter from baculovirus, the early immediate CMV promoter, the early SV40 promoter and the 5' LTR promoter from HIV-1 directed gene expression in E. coli. The determination of transcription start sites in the immediate early CMV promoter and the polyhedrin promoter confirmed the existence of bacterial -10 and -35 consensus sequences. The importance of this heterologous gene expression for safety considerations was further supported by analysing fusions between the aforementioned promoters and a promoter-less cytotoxin gene. CONCLUSION: According to our results a high percentage of viral promoters have the ability of initiating gene expression in E. coli. The degree of such heterologous gene expression can be sufficient for the expression of toxin genes and must therefore be considered when defining safety measures for the handling of corresponding genetically modified organisms

Mutation on lysX from Mycobacterium avium hominissuis impacts the host–pathogen interaction and virulence phenotype

The lysX gene from Mycobacterium avium hominissuis (MAH) is not only involved in cationic antimicrobial resistance but also regulates metabolic activity. An MAH lysX deficient mutant was shown to exhibit a metabolic shift at the extracellular state preadapting the bacteria to the conditions inside host-cells. It further showed stronger growth in human monocytes. In the present study, the LysX activity on host–pathogen interactions were analyzed. The lysX mutant from MAH proved to be more sensitive toward host-mediated stresses such as reactive oxygen species. Further, the lysX mutant exhibited increased inflammatory response in PBMC and multinucleated giant cell (MGC) formation in human macrophages during infection studies. Coincidentally, the lysX mutant strain revealed to be more reproductive in the Galleria mellonella infection model. Together, these data demonstrate that LysX plays a role in regulating the bacillary load in host organisms and the lack of lysX gene facilitates MAH adaptation to intracellular host-habitat, thereby suggesting an essential role of LysX in the modulation of host–pathogen interaction.Peer Reviewe

Translating research into policy and practice in developing countries: a case study of magnesium sulphate for pre-eclampsia.

BACKGROUND: The evidence base for improving reproductive health continues to grow. However, concerns remain that the translation of this evidence into appropriate policies is partial and slow. Little is known about the factors affecting the use of evidence by policy makers and clinicians, particularly in developing countries. The objective of this study was to examine the factors that might affect the translation of randomised controlled trial (RCT) findings into policies and practice in developing countries. METHODS: The recent publication of an important RCT on the use of magnesium sulphate to treat pre-eclampsia provided an opportunity to explore how research findings might be translated into policy. A range of research methods, including a survey, group interview and observations with RCT collaborators and a survey of WHO drug information officers, regulatory officials and obstetricians in 12 countries, were undertaken to identify barriers and facilitators to knowledge translation. RESULTS: It proved difficult to obtain reliable data regarding the availability and use of commonly used drugs in many countries. The perceived barriers to implementing RCT findings regarding the use of magnesium sulphate for pre-eclampsia include drug licensing and availability; inadequate and poorly implemented clinical guidelines; and lack of political support for policy change. However, there were significant regional and national differences in the importance of specific barriers. CONCLUSION: The policy changes needed to ensure widespread availability and use of magnesium sulphate are variable and complex. Difficulties in obtaining information on availability and use are combined with the wide range of barriers across settings, including a lack of support from policy makers. This makes it difficult to envisage any single intervention strategy that might be used to promote the uptake of research findings on magnesium sulphate into policy across the study settings. The publication of important trials may therefore not have the impacts on health care that researchers hope for

Illegitimate recombination: An efficient method for random mutagenesis in Mycobacterium avium subsp. hominissuis

Background: The genus Mycobacterium (M.) comprises highly pathogenic bacteria such as M. tuberculosis as well as environmental opportunistic bacteria called non-tuberculous mycobacteria (NTM). While the incidence of tuberculosis is declining in the developed world, infection rates by NTM are increasing. NTM are ubiquitous and have been isolated from soil, natural water sources, tap water, biofilms, aerosols, dust and sawdust. Lung infections as well as lymphadenitis are most often caused by M. avium subsp. hominissuis (MAH), which is considered to be among the clinically most important NTM. Only few virulence genes from M. avium have been defined among other things due to difficulties in generating M. avium mutants. More efforts in developing new methods for mutagenesis of M. avium and identification of virulence-associated genes are therefore needed. Results: We developed a random mutagenesis method based on illegitimate recombination and integration of a Hygromycin-resistance marker. Screening for mutations possibly affecting virulence was performed by monitoring of pH resistance, colony morphology, cytokine induction in infected macrophages and intracellular persistence. Out of 50 randomly chosen Hygromycin-resistant colonies, four revealed to be affected in virulence-related traits. The mutated genes were MAV_4334 (nitroreductase family protein), MAV_5106 (phosphoenolpyruvate carboxykinase), MAV_1778 (GTP-binding protein LepA) and MAV_3128 (lysyl-tRNA synthetase LysS). Conclusions: We established a random mutagenesis method for MAH that can be easily carried out and combined it with a set of phenotypic screening methods for the identification of virulence-associated mutants. By this method, four new MAH genes were identified that may be involved in virulence

The mycobacterial DNA-binding protein 1 (MDP1) from Mycobacterium bovis BCG influences various growth characteristics

<p>Abstract</p> <p>Background</p> <p>Pathogenic mycobacteria such as <it>M. tuberculosis</it>, <it>M. bovis </it>or <it>M. leprae </it>are characterised by their extremely slow growth rate which plays an important role in mycobacterial virulence and eradication of the bacteria. Various limiting factors influence the generation time of mycobacteria, and the mycobacterial DNA-binding protein 1 (MDP1) has also been implicated in growth regulation. Our strategy to investigate the role of MDP1 in mycobacterial growth consisted in the generation and characterisation of a <it>M. bovis </it>BCG derivative expressing a MDP1-antisense gene.</p> <p>Results</p> <p>The expression rate of the MDP1 protein in the recombinant <it>M. bovis </it>BCG containing the MDP1-antisense plasmid was reduced by about 50% compared to the reference strain <it>M. bovis </it>BCG containing the empty vector. In comparison to this reference strain, the recombinant <it>M. bovis </it>BCG grew faster in broth culture and reached higher cell masses in stationary phase. Likewise its intracellular growth in mouse and human macrophages was ameliorated. Bacterial clumping in broth culture was reduced by the antisense plasmid. The antisense plasmid increased the susceptibility of the bacteria towards Ampicillin. 2-D protein gels of bacteria maintained under oxygen-poor conditions demonstrated a reduction in the number and the intensity of many protein spots in the antisense strain compared to the reference strain.</p> <p>Conclusion</p> <p>The MDP1 protein has a major impact on various growth characteristics of <it>M. bovis </it>BCG. It plays an important role in virulence-related traits such as aggregate formation and intracellular multiplication. Its impact on the protein expression in a low-oxygen atmosphere indicates a role in the adaptation to the hypoxic conditions present in the granuloma.</p

Genetic diversification of persistent Mycobacterium abscessus within cystic fibrosis patients

Mycobacterium (M.) abscessus infections in Cystic Fibrosis (CF) patients cause a deterioration of lung function. Treatment of these multidrug-resistant pathogens is associated with severe side-effects, while frequently unsuccessful. Insight on M. abscessus genomic evolvement during chronic lung infection would be beneficial for improving treatment strategies. A longitudinal study enrolling 42 CF patients was performed at a CF center in Berlin, Germany, to elaborate phylogeny and genomic diversification of in-patient M. abscessus. Eleven of the 42 CF patients were infected with M. abscessus. Five of these 11 patients were infected with global human-transmissible M. abscessus cluster strains. Phylogenetic analysis of 88 genomes from isolates of the 11 patients excluded occurrence of M. abscessus transmission among members of the study group. Genome sequencing and variant analysis of 30 isolates from 11 serial respiratory samples collected over 4.5 years from a chronically infected patient demonstrated accumulation of gene mutations. In total, 53 genes exhibiting non-synonymous variations were identified. Enrichment analysis emphasized genes involved in synthesis of glycopeptidolipids, genes from the embABC (arabinosyltransferase) operon, betA (glucose-methanol-choline oxidoreductase) and choD (cholesterol oxidase). Genetic diversity evolved in a variety of virulence- and resistance-associated genes. The strategy of M. abscessus populations in chronic lung infection is not clonal expansion of dominant variants, but to sustain simultaneously a wide range of genetic variants facilitating adaptation of the population to changing living conditions in the lung. Genomic diversification during chronic infection requires increased attention when new control strategies against M. abscessus infections are explored.Peer Reviewe