Detection of Mycobacteria in Water

Background:

Although usually harmless, environmental mycobacteria are a concern in clinical settings because they can cause severe disease in immunocompromised patients. The bacteria can colonise hospital pipework and medical equipment that contains reservoirs of still water, from which it can be expelled into the patient environment. Healthcare regulations mandate that hospitals must be able to detect mycobacteria at concentrations as low as 1cfu per 100ml of water. However, mycobacteria are difficult to detect using traditional culture-based approaches because they grow very slowly and become crowded out and concealed by other, quicker growing microbes. This makes the standard method of testing water for mycobacteria very unreliable and can mean that medical equipment is quarantined for 6-8 weeks while awaiting results. Molecular-based alternatives are more sensitive, but analysers can typically only process very small amounts of liquid (just a few millilitres), and regulations require that the entire 100ml water sample must be screened.

Process:

James optimised the mycobacteria testing capabilities of a national contract testing laboratory by firstly developing a novel, anti-fungal agar supplement for the selective culture of mycobacteria species, followed by the development and validation of a molecular method of detection capable of screening 100ml quantities of water.

Mycobacteria are grown on agar that is supplemented with antibiotics that will kill other bacterial species, meaning that only mycobacterial species should be visible. However, mycobacteria are extremely slow-growing microorganisms requiring a minimum of 28 days incubation, during which time the agar can become overgrown with mould colonies arising from fungal spores that were present in the original sample and unaffected by the antibiotics in the agar. Fungal growth can become so dense that the mycobacteria can no longer be seen and quantified, and so the test is invalid. Standard process steps, such as acid treatment of the capture membrane through which the water sample is filtered, are not wholly effective. To address this issue, James developed a novel anti-fungal supplement that can be added to the standard M7H10/M7H11 mycobacterial culture media, validated its effectiveness, and gained UKAS accreditation for its usage. The supplement proved 100% effective in suppressing mould growth whilst not affecting or delaying the development of mycobacterial colonies.

James then began development of molecular testing approaches that could equate to the sensitivity of traditional culture-based techniques. The primary challenge was the recovery of very low numbers of microorganisms from large volumes of liquid. Unlike culture methods that can test litres of water using membrane filtration, molecular methods such as quantitative polymerase chain reaction (qPCR) or loop-mediated isothermal amplification (LAMP) assays require very small volumes of sample that are prepared in such a way that a large amount of genomic material can become lost before the analysis begins, leading to inaccurate results that do not represent the true condition of the sample. Over the course of a few years, James developed and refined a highly efficient and lossless workflow that could capture low numbers of waterborne bacteria, transfer them through a nucleic acid extraction process and retain the resultant genomic material while condensing it into the very small volumes of liquid required by nucleic acid amplification tests. This method was then made commercially available as an alternative option to the standard culture test.

Outcomes:

Mycobacteria culture tests demonstrated improved accuracy and reliability resulting in fewer invalid test results, fewer delays due to repeat testing, and leading to a reduction in the length of time that medical devices were quarantined whilst waiting for test results.
Delivery of an additional commercial offering in the form of a rapid, qPCR test for the detection of mycobacteria in water samples.