chemokines in patients with tuberculosis and latent tuberculosis infection

S68 Our objective was to understand the role of PPK1 in bacterial survival under stress. Methods: To evaluate the role of PPK1 in mycobacterial survival, the gene encoding PPK1 was disrupted in M. smegmatis through homologous recombination. The effect of stress on the wild type and the mutant were then analysed. ppk1 was also silenced by antisensing in M. tuberculosis. Results: The PPK1-deleted strain of M. smegmatis exhibited significantly reduced intracellular survival in murine RAW 264.7 macrophages compared to the survival of its wild type counterpart. It exhibited an extended lag phase of growth when shifted to a low phosphate-containing medium, was unable to grow in nitrogen-depleted medium and impaired in its ability to survive in anaerobic conditions. It showed decreased transcription of the stringent response regulator relA. M. tuberculosis ppk1 could complement the mutant. Antisensing of the ppk1 gene in M. tuberculosis also showed reduced levels of relA transcription. The ppk1 mutant was defective in its ability to form biofilms. Conclusion: ppk1 likely plays a role in mycobacterial persistence and is a potential target for drug development. O326 Cholesterol oxidase as a virulence factor of Mycobacterium tuberculosis A. Brzostek, B. Dziadek, J. Pawelczyk, A. Rumijowska-Galewicz, J. Dziadek (Lodz, PL) Objectives: The objective of this study was to evaluate cholesterol oxidase (ChoD) as a putative virulence factor of Mycobacterium tuberculosis. Methods: A two-step recombination protocol was used to replace the cholesterol oxidase gene with a non-functional copy. The pathogenicity of resultant strains was analysed in vitro using peritoneal macrophages and in vivo by mice infection. Results: Cholesterol oxidase is known to be a key enzyme initiating cholesterol degradation processes in many soil bacteria, including fast growing mycobacteria. Pathogenic mycobacteria accumulate cholesterol without its utilisation and express cholesterol oxidase as an extracellular enzyme. A homologous recombination was used to construct an M. tuberculosis strain with an unmarked deletion within the choD gene. The wild type M. tuberculosis strain (Mt-wt), choD-mutant (mutchoD) and mutant complemented with choD gene controlled by a heat shock protein promoter (mut-choD-PhspchoD) were used to study the function of cholesterol oxidase in mycobacterial pathogenesis. Peritoneal macrophages (106 /well) were infected with Mt-wt, mut-choD and mutchoD-PhspchoD M. tuberculosis strains (105 /well) and incubated for four and six days. The numbers of Mt-wt and mut-choD-PhspchoD viable bacteria recovered from macrophages were at least an order of magnitude higher compared to mut-cho strain as revealed by cfu analysis. The same strains were used for infection of mice. Ten weeks post infection, lungs and spleens were isolated from euthanised mice. The numbers of viable bacteria in each organ were detected by cfu. Analysing at least 10 mice of each group, the strong attenuation of choD mutants was observed compared to wild type and complementation strains. Conclusion: Cholesterol oxidase is an important virulence factor during infection by M. tuberculosis. This work was supported with KBN grant: contract no. 3P05A14024 O327 Ex vivo profiling of cytokines/chemokines in patients with tuberculosis and latent tuberculosis infection T. Bodmer, D. Grandgirard, S. Bigler, C. Daubenberger, S. Leib (Berne, Basle, CH) Objectives: While in most cases infection with Mycobacterium tuberculosis (Mtb) is contained (latent Mtb infection; LTBI), it progresses to overt disease (TB) in a small proportion of those infected. We studied the cytokine/chemokine profiles associated with LTBI and TB in order to gain insight into ongoing immune activation events in Mtb-infection.

17th ECCMID / 25th ICC, Oral presentations Methods: Blood was obtained from patients with culture-confirmed TB (n = 4), with recently acquired LTBI (n = 3), and from healthcare workers without Mtb exposure (n = 6). Interferon-gamma (IFN-g) that was released from sensitised lymphocytes upon ex vivo stimulation with Mtb-specific antigens was determined using the QuantiFERON-TB® Gold In Tube assay as recommended by the manufacturer (Cellestis Ltd., Carnegie, Australia). Microsphere-based multiplex assays (Lincoplex® , Linco Research Inc., St Charles, MA, USA) were used to assess the plasma concentrations of IFN-g, TNF-a, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p40, IL-12p70 IL-13, IL-15, Fraktalkine, GM-CSF, MCP-1, and IP-10 before (NIL) and after (TbAg) ex vivo stimulation. Values were determined using Bio-Plex Manager (Bio-Rad Laboratories, Hercules, CA, USA). The limit of detection was 3.2 pg/mL. Statistical analysis was performed using Prism 4.1 (GraphPad Software Inc., San Diego, CA, USA). The Kruskal-Wallis test was used for multiple group comparison, and the Mann-Whitney test for group to group comparison. Results: IFN-g, IP-10, IL-2, GM-CSF, IL-13 concentrations were significantly higher in patients with TB and LTBI as compared to controls (Table). DTb Ag − NIL, median (range), pg/mL]

IFN-g IP-10 IL-2 GM-CSF IL-13

ANOVA Mann−Whitney

Control

TB

LTBI

Kruskal- Control Wallis vs TB

Control vs LTBI

−0.2[−3.0 − 2.0] 212.0 [126.0 − 1409] 0.47 [−4.6 − 59.9] −2.3 [−12.9 − 11.0] −2.8 [−34.6 − 14.2]

175.3 [131.0 − 238.6] 18,188 [3,806 − 46,279] 630.5 [404.5 − 5,871] 27.6 [2.9 − 481.2] 61.6 [0.2 − 144.2]

115.9 [2.2 − 185.4] 12,996 [1,809 − 24,112] 413.5 [280.1 − 887.5] 20.8 [16.6 − 59.1] 57.6 [2.9 − 59.5]

P < 0.02 P < 0.02 P < 0.02 P < 0.02 P < 0.04

P < 0.03 P < 0.03 P < 0.03 P < 0.03 P < 0.05

P < 0.03 P < 0.01 P < 0.01 P < 0.02 P < 0.04

Conclusions: Mean plasma concentrations of IP-10 were 50 to 90-fold higher than those of IFN-g, both in patients with active TB and LTBI. Assessment of IP-10 might substantially increase the sensitivity of IFN-g release assays (IGRA) for detecting Mtb infection. Ex vivo profiling of cytokines/chemokines using IGRA in combination with flow-cytometer multiplex assays identified additional candidate parameters to confirm Mtb infection and will improve our understanding of TB pathogenesis.

Clostridium difficile O328 Community rather than hospital factors promote increase of Clostridium difficile associated diseases in Northern Bavaria S. Borgmann, A. Kick, T. Jakobiak, B. Schulte, M. Hamann, R. Busch, C. von Eichel-Streiber, O. Hasselmayer (Weiden, T¨ubingen, Erlangen, Mainz, DE) Objectives: Recently, the number of C. difficile associated diseases (CDAD) has increased. Prevalence data of CDAD particularly resulted from analyses performed at university hospitals, etc. In our lab we perform microbiological analyses for more than 3500 facilities comprising more than 40 hospitals in Northern Bavaria allowing examination of C. difficile prevalence in the local region. Methods: Stool samples were analysed by ELISA detecting toxins A and B of C. difficile (Cdt). The total number of ELISA performed in 2004, 2005, and 2006 and the number of positive results was determined by hybase analysis. All C. difficile positive patients identified in 2005 were documented. Analysis of toxin genes IStron types integrated into the genome (C. difficile integration profiles) was performed on C. difficile cultured from stool samples of 17 patients treated at 2 hospitals. Results: The number of Cdt positive stools samples increased from 372 in 2004 to 515 (2005) and 642 (Jan–Sept 2006). From October 2005 to January 2006 there was a marked increase from 31 to 103 Cdt positive stool samples per month. High numbers were also observed in the subsequent period until June 2006 (median 84/month). In July this number decreased to 39. When this analysis was performed on Cdt positive findings of patients from 4 harvested hospitals (I, II, III, IV) courses of each facility was very similar to that of the total collective suggesting that increase of CDAD in particular depended on community factors and secondarily on hygiene of certain hospitals. Typing analysis