Lay Summary

DNA Thermo-Protection Facilitates Whole-Genome Sequencing of Mycobacteria Direct from Clinical Samples

Mycobacterim tuberculosis, the bug that causes tuberculosis or ‘TB’, is one of the leading causes of death due to infection. The World Health Organization estimates that 10 million new infections with TB bacteria, and 1.2 million deaths occurred worldwide in 2018.

Luckily, TB can be cured with antibiotics. Getting a quick diagnosis, so that patients receive the right antibiotics as soon as possible is really important, both for the patient and to stop them spreading the infection to other people. To treat people we need to find out the right antibiotics to give them. Choosing the right antibiotics has become trickier in recent years, because many TB bacteria have become resistant. This means that antibiotics which used to work, have become less effective treatments or stopped working.

Fortunately, thanks to recent advances in DNA sequencing technology, we can quickly sequence the whole genome of the TB bacteria infecting an individual patient. The aim of our study was to develop a simple, rapid, and cheap method of preparing TB bug DNA, directly from patient samples (sputum), for sequencing. This would avoid the slow process of growing TB bugs which takes weeks.

In our study, we invented a special solution, which is added to the patients’ sputum sample before the sample is heated for half an hour to kill the TB bugs. During heating, the solution protects the TB DNA from falling apart. Broken DNA is no good for sequencing! An essential ingredient in the solution is a high concentration of the salt potassium chloride.

This breakthrough allowed us to sequence complete TB bacteria genomes from 15/20 sputum samples tested and to find out the right antibiotics for the patients. The method is currently being tested in labs based in India and Madagascar, two locations with a high number of people suffering from TB infections.

High precision Neisseria gonorrhoeae variant and antimicrobial resistance calling from metagenomic Nanopore sequencing

Gonorrhoea is the second most common sexually transmitted infection. We have developed a new approach, using an exciting new technology, which promises to detect infections and provide the best treatment faster.

Gonorrhoea is caused by the bacteria called Neisseria gonorrhoeae. It can be treated with antibiotics, but the bacteria have developed resistance to many antibiotics, so that now only one antibiotic works reliably.

Gonorrhoea is usually detected by collecting a urine sample but can also be grown in a lab from swabs taken in a sexual health clinic. If bacteria do grow, further tests can be done to see which antibiotics will kill the bacteria. Scientists also do what are called ‘molecular’ tests, which involve trying to detect DNA from the gonorrhoea bacteria. It can take a while to get the results back from all of these tests – often bacteria take a few days to grow. A single test that could be done much faster, and which would both find out whether gonorrhoea bacteria are there and which antibiotics would kill them best, would let patients start the right treatment quicker. In turn, this would stop gonorrhoea being passed on.

A new molecular method, called Metagenomic Sequencing, identifies DNA from bacteria directly from patient samples, and is showing potential as a new diagnostic test. The bacterial DNA can be compared to a database of many known bacteria, to work out the cause of an infection – like a ‘paternity test’ for bugs. If we can get enough bacterial DNA from a sample, not only can we work out which type of bacteria is causing the infection but we can also find specific parts of its DNA that we know lead to antibiotic resistance. Metagenomic sequencing can also be faster than current tests, often finding out the cause of an infection and which treatments will work within a few hours. 

In our recent study we have been testing whether metagenomic sequencing can accurately find gonorrhoea infections in urine samples. We were trying to choose the best method for getting bacterial genetic material out of these urine samples, and we did many experiments in the laboratory to work this out. At first, we used artificial infections – taking uninfected urine and putting in known amounts of Neisseria gonorrhoeae bacteria – so we could measure how successful our experiments were at getting the bacterial genetic material (DNA) back. Once we had chosen the best method, we tested it on 10 urine samples collected from men with suspected gonorrhoea infections. We found that it was possible to detect almost all the complete genetic sequence (>90%) of gonorrhoea in all 10 of our samples. We were also able to find the parts of the genetic sequence that we know are involved in antibiotic resistance.

We have shown that with our optimised laboratory method we can detect gonorrhoea DNA directly in urine from men with suspected infections. These results provide a solid foundation on which to build, and we plan to test our method on a larger number of urine samples. We also plan to develop computer analysis methods that will allow us to take the metagenomic sequencing results and detect more genetic regions involved in antibiotic resistance.