Simplified automated image analysis for detection and phenotyping of Mycobacterium tuberculosis on porous supports by monitoring growing microcolonies.

BACKGROUND: Even with the advent of nucleic acid (NA) amplification technologies the culture of mycobacteria for diagnostic and other applications remains of critical importance. Notably microscopic observed drug susceptibility testing (MODS), as opposed to traditional culture on solid media or automated liquid culture, has shown potential to both speed up and increase the provision of mycobacterial culture in high burden settings. METHODS: Here we explore the growth of Mycobacterial tuberculosis microcolonies, imaged by automated digital microscopy, cultured on a porous aluminium oxide (PAO) supports. Repeated imaging during colony growth greatly simplifies "computer vision" and presumptive identification of microcolonies was achieved here using existing publically available algorithms. Our system thus allows the growth of individual microcolonies to be monitored and critically, also to change the media during the growth phase without disrupting the microcolonies. Transfer of identified microcolonies onto selective media allowed us, within 1-2 bacterial generations, to rapidly detect the drug susceptibility of individual microcolonies, eliminating the need for time consuming subculturing or the inoculation of multiple parallel cultures. SIGNIFICANCE: Monitoring the phenotype of individual microcolonies as they grow has immense potential for research, screening, and ultimately M. tuberculosis diagnostic applications. The method described is particularly appealing with respect to speed and automation

Growth of strains H37Ra and HR1 in the presence of RIF.

<p>Microcolonies of RIF susceptible strain H37Ra and resistant strain HR1 were grown without RIF for 7 days, then moved to fresh agar with or without RIF from day 7 onwards. A, C: Averaged sizes of H37Ra (A) and HR1 (C) colonies incubated with (open symbols) and without RIF (closed symbols). Average colony size is plotted; bars indicate ±1 standard deviation of measured colony sizes. A statistically significant difference (indicated by an asterisk (*), p<0.001) between the groups at day 7 and 8 (unpaired T-test) was found between H37Ra with and without RIF at day 8. B: Growth rate of individual H37Ra (B) and HR1 (D) colonies incubated with (white bars) and without (black bars) RIF. Statistical analysis was performed using a paired T-test on colony growth between day 6 and day 7, and day 6 and day 8. Statistical significance is indicated by an asterisk (*; p<0.001). Although there is a statistically significant difference between the growth rate of HR1 at day 6 and 7 in the presence of RIF, the change in growth rate is so small compared to the sensitive strain that we consider this effect irrelevant.</p

Coefficient of variation in object size vs. average object size over 8 separate measurements of approx.

<p>500 objects.</p

Colony formation on PAO.

<p>A–D; H37Ra cultured for 1, 2, 3 and 7 days on PAO were fixed and stained with Nile Red and examined with a 40X objective using fluorescent epi-illumination. Representative microcolonies for each time point are shown.</p

Growth of microcolonies on PAO.

<p>A: Growth curves of H37Ra on PAO on agar (continuous black line), or directly on agar (dashed grey line). B: Growth curves of H37Ra (▪) and HR1 (•) on PAO on agar. Average colony size is plotted; bars indicate ±1 standard deviation of measured colony sizes. Statistically significant differences between (A) PAO and agar or (B) H37Ra and HR1 (tested at days 7–10) are indicated by an asterisk (*; p<0.001).C: Example images of a single microscope field (705 µm×705 µm) taken at inoculation (d0) and daily between 3 and 10 days after inoculation.</p

Growth of mixed strains H37Ra and HR1 in the presence of RIF.

<p>Microcolonies of mixed cultures of H37Ra and HR1 were grown without RIF for 7 days, then moved to fresh agar with or without RIF from day 7 onwards. A: Average colony sizes of total mixed population incubated with (▪) and without (□) RIF. Average colony size is plotted; bars indicate ±1 standard deviation of measured colony sizes. B, C: Distribution of growth rates (% increase/day) at day 7 (B) and 8 (C) of the total mixed population without RIF exposure. D, E: Distribution of growth rates (% increase/day) at day 7 (D) and 8 (E) of the total mixed population after RIF exposure. In E the vertical dashed line indicates the cut-off value (25%) for growth defined on basis of measured growth rate at day 8. F: Averaged colony sizes of the mixed population with RIF separated into the growing (RIF resistant, black line) and non-growing (RIF susceptible, grey line) populations on basis of the cut-off value shown in E. Average colony size is plotted; bars indicate ±1 standard deviation of measured colony sizes. For reference, data for the complete mixed population is indicated by the dashed line.</p

Nasal commensal Staphylococcus epidermidis counteracts influenza virus

Several microbes, including Staphylococcus epidermidis (S. epidermidis), a Gram-positive bacterium, live inside the human nasal cavity as commensals. The role of these nasal commensals in host innate immunity is largely unknown, although bacterial interference in the nasal microbiome may promote ecological competition between commensal bacteria and pathogenic species. We demonstrate here that S. epidermidis culture supernatants significantly suppressed the infectivity of various influenza viruses. Using high-performance liquid chromatography together with mass spectrometry, we identified a giant extracellular matrix-binding protein (Embp) as the major component involved in the anti-influenza effect of S. epidermidis. This anti-influenza activity was abrogated when Embp was mutated, confirming that Embp is essential for S. epidermidis activity against viral infection. We also showed that both S. epidermidis bacterial particles and Embp can directly bind to influenza virus. Furthermore, the injection of a recombinant Embp fragment containing a fibronectin-binding domain into embryonated eggs increased the survival rate of virus-infected chicken embryos. For an in vivo challenge study, prior Embp intranasal inoculation in chickens suppressed the viral titres and induced the expression of antiviral cytokines in the nasal tissues. These results suggest that S. epidermidis in the nasal cavity may serve as a defence mechanism against influenza virus infection