Modulation of Transcriptional and Inflammatory Responses in Murine Macrophages by the Mycobacterium tuberculosis Mammalian Cell Entry (Mce) 1 Complex

The outcome of many infections depends on the initial interactions between agent and host. Aiming at elucidating the effect of the M. tuberculosis Mce1 protein complex on host transcriptional and immunological responses to infection with M. tuberculosis, RNA from murine macrophages at 15, 30, 60 min, 4 and 10 hrs post-infection with M. tuberculosis H37Rv or Δ-mce1 H37Rv was analyzed by whole-genome microarrays and RT-QPCR. Immunological responses were measured using a 23-plex cytokine assay. Compared to uninfected controls, 524 versus 64 genes were up-regulated by 15 min post H37Rv- and Δ-mce1 H37Rv-infection, respectively. By 15 min post-H37Rv infection, a decline of 17 cytokines combined with up-regulation of Ccl24 (26.5-fold), Clec4a2 (23.2-fold) and Pparγ (10.5-fold) indicated an anti-inflammatory response initiated by IL-13. Down-regulation of Il13ra1 combined with up-regulation of Il12b (30.2-fold), suggested switch to a pro-inflammatory response by 4 hrs post H37Rv-infection. Whereas no significant change in cytokine concentration or transcription was observed during the first hour post Δ-mce1 H37Rv-infection, a significant decline of IL-1b, IL-9, IL-13, Eotaxin and GM-CSF combined with increased transcription of Il12b (25.1-fold) and Inb1 (17.9-fold) by 4 hrs, indicated a pro-inflammatory response. The balance between pro-and anti-inflammatory responses during the early stages of infection may have significant bearing on outcome

Pyrazinamide Resistance among South African Multidrug-Resistant Mycobacterium tuberculosis Isolates▿

Pyrazinamide is important in tuberculosis treatment, as it is bactericidal to semidormant mycobacteria not killed by other antituberculosis drugs. Pyrazinamide is also one of the cornerstone drugs retained in the treatment of multidrug-resistant tuberculosis (MDR-TB). However, due to technical difficulties, routine drug susceptibility testing of Mycobacterium tuberculosis for pyrazinamide is, in many laboratories, not performed. The objective of our study was to generate information on pyrazinamide susceptibility among South African MDR and susceptible M. tuberculosis isolates from pulmonary tuberculosis patients. Seventy-one MDR and 59 fully susceptible M. tuberculosis isolates collected during the national surveillance study (2001 to 2002, by the Medical Research Council, South Africa) were examined for pyrazinamide susceptibility by the radiometric Bactec 460 TB system, pyrazinamidase activity (by Wayne's assay), and sequencing of the pncA gene. The frequency of pyrazinamide resistance (by the Bactec system) among the MDR M. tuberculosis isolates was 37 of 71 (52.1%) and 6 of 59 (10.2%) among fully sensitive isolates. A total of 25 unique mutations in the pncA gene were detected. The majority of these were point mutations that resulted in amino acid substitutions. Twenty-eight isolates had identical mutations in the pncA gene, but could be differentiated from each other by a combination of the spoligotype patterns and 12 mycobacterial interspersed repetitive-unit loci. A high proportion of South African MDR M. tuberculosis isolates were resistant to pyrazinamide, suggesting an evaluation of its role in patients treated previously for tuberculosis as well as its role in the treatment of MDR-TB

A rapid, 2-well, multiplex real-time polymerase chain reaction assay for the detection of SCCmec types I to V in methicillin-resistant Staphylococcus aureus

For us to assess the spread of methicillin-resistant Staphylococcus aureus (MRSA), typing of the staphylococcal cassette chromosome mec (SCCmec) is a valuable addition to existing typing methods, such as multilocus sequence typing (MLST). Traditional SCCmec typing assays, that is, that of Oliveira et al. and Ito et al., are polymerase chain reaction (PCR) based, requiring electrophoresis. We introduce a rapid, 2-well, multiplex real-time PCR assay that can be used directly on bacterial suspensions and is able to characterize SCCmec type I to V based on the detection of the ccr genes and the mec complex. The assay was evaluated on 212 clinical MRSA isolates from various countries, associated with MLST clonal complexes (CC) 1, 5, 8, 22, 30, and 45, as well as pig-associated CC398. When comparing the real-time PCR assay with traditional methods, the correct SCCmec element was identified in 209 (99%) of the 212 MRSA isolates. The new assay enables high-throughput analyses for SCCmec on large strain collections

Primers used for the PCR verification of deleted and extra regions as observed by CGH analysis.

*<p>:<a href="http://xrl.us/6cg3" target="_blank">http://xrl.us/6cg3</a></p><p>The annealing temperature for each primer set and the expected PCR product size based on the published sequences of <i>M. tuberculosis</i> H37Rv, <i>M. bovis</i> AF22197 or <i>M. tuberculosis</i> CDC1551 are also provided.</p

Gene graph of the <i>M.tuberculosis</i> H37Rv <i>1754c-1765c, Mb1785-1787</i>, <i>MT1808</i> and <i>MT1812-1813</i> regions.

<p>The graph is based on region z-scores, with each line representing one isolate analyzed by CGH. Region z-scores of more than ±4.42 (absolute value) occurring in one or more samples, corresponding to a standard normal P-value of 0.00001 (1e-5) indicate deletions (above the 0-line) or extra regions (below 0-line). Red lines represent isolates of the Beijing lineage, green lines; ST42 lineage and blue lines; non-Beijing isolates other than ST42.</p

Genetic diversity among 22 <i>Mycobacterium tuberculosis</i> isolates tested by CGH.

<p>The dendogram was produced by weighted pair group method (WPGMA) clustering using a Pearson correlation based distance measure defined on regional z-score values. The red color indicates deletions in the clinical isolates compared to the reference strain <i>M. tuberculosis</i> H37Rv, whereas the green color shows extra regions in the clinical isolates not present in the reference strain <i>M. tuberculosis</i> H37Rv.</p

Regions chosen for PCR verification based on the CGH array analysis.

<p>The dendogram based on CGH array analysis of 22 <i>M. tuberculosis</i> isolates from Myanmar (11 Beijing, 4 ST42, 2 ST89 and 5 previously unreported [UR] shared-types [ST]) was produced by weighted pair group method (WPGMA) clustering using a Pearson correlation based distance measure defined on regional z-score values, corresponding to P-values between 1e-3 and 1e-16. The green color indicates extra regions that are present in the clinical isolates, but not in the reference strain <i>M. tuberculosis</i> H37Rv, whereas the red color depicts deletions in the clinical isolates compared to the reference strain <i>M. tuberculosis</i> H37Rv.</p

PCR results from the 22 regions chosen for verification in 60 clinical <i>M. tuberculosis</i> isolates.

<p>The dendogram based on PCR results from 16 deletions and 6 extra regions chosen for verification on 60 clinical <i>M. tuberculosis</i> isolates (28 Beijing, 13 ST42 and 19 other non-Beijing) was produced by unweighted pair group method (UPGMA) clustering using a Pearson correlation based distance measure.</p