Hi guys, I am working with the mtb-infected mouse model. and i am thinking about whether we can monitor the bacterial burden with mouse blood. the current way we do for the bacterial burden monitoring is to sac the mice and harvest the organ. It is expensive and time-costing.
I do not have a clear clue on which components I should examine in blood. CFU I can try but I doubt it will work. antibodies either. Maybe small RNA from MTB or specific IFN+ T cell population of the mice. the limitation here is that there will be only 200ul blood every time we can use for the monitoring.
Hi Wenxi,
What you would like to do would clearly be very valuable but nobody has yet come up with a workable method. I think the most advanced method is using a fluorescent substrate activated by the mycobacterial beta lactamase, and with that the limit of detection is several hundred CFU with a correlation coefficient of 0.87. Here's the paper:
https://www.ncbi.nlm.nih.gov/pubmed/27421748
An alternative approach from serum would be using LCMS to look for Mtb-specific small molecules in serum, as demonstrated in this paper:
https://www.ncbi.nlm.nih.gov/pubmed/26014799
I don't know how quantitative that would be but probably not very.
Both of these methods would require expensive instrumentation, the imager in the BSL3, the LCMS outside but still expensive.
Some investigators use qPCR but I am skeptical of the accuracy of such methods, no doubt they are also counting genomes of dead cells.
I think for the time being we will need to stick with the expensive, messy and time consuming work of plating for CFU.
Best regards,
Brian
Hi Wenxi,
What you would like to do would clearly be very valuable but nobody has yet come up with a workable method. I think the most advanced method is using a fluorescent substrate activated by the mycobacterial beta lactamase, and with that the limit of detection is several hundred CFU with a correlation coefficient of 0.87. Here's the paper:
https://www.ncbi.nlm.nih.gov/pubmed/27421748
An alternative approach from serum would be using LCMS to look for Mtb-specific small molecules in serum, as demonstrated in this paper:
https://www.ncbi.nlm.nih.gov/pubmed/26014799
I don't know how quantitative that would be but probably not very.
Both of these methods would require expensive instrumentation, the imager in the BSL3, the LCMS outside but still expensive.
Some investigators use qPCR but I am skeptical of the accuracy of such methods, no doubt they are also counting genomes of dead cells.
I think for the time being we will need to stick with the expensive, messy and time consuming work of plating for CFU.
Best regards,
Brian
Hi Brian,
It is so nice to get an answer from you! You know everything, I mean it.
I agree that the most canonical way is plating for CFU, which is a way derived from the clinical diagnosis. it may be the main reason why so many years we stick on it only, in terms of bacterial burden assay. Yet I think it also has us ignoring the non-culturable part of mtb such as persisters, which is really a pain for TB research.
I will read these papers and we can talk later.
thanks a lot!
Wen
Hi Wenxi,
Yet, no blood assay has been shown to correlate with lungs, spleen or liver CFUs. The mouse model will depends upon the mouse strain being used. I may suggest that will compare occurence of Blood CFU vz RT PCR during the following of the infection (either IV or aerosol infected!) and compare with organ CFU and survival.
Best regards
Phillipe H Lagrange.
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