VBNC Legionella - now you see me! now you don't!

The famous 'poem' from boxing great Muhammad Ali ends 'the hands can't hit, what the eyes don't see!'. This is a lead in to explaining VBNC Legionella - that is Viable But Not Culturable bacteria. Sounds like a bit of a leap from boxing to bacteria? The common thread is missing your target!

Bacteria like all living things have survival responses and react to changes in their environment. For most bacteria this reaction is a change of physiological state. Environmental changes may stimulate bacteria to become dormant (some form spores) or to become motile (grow tails and swim away) or produce slime to coat their surface. Other changes are more sinister - produce toxins, switch on resistance genes, or a combination of everything I just listed! These are all evasion / survival strategies to make sure they remain a moving target. Many of them are stress responses.

VBNC is an environmental response that is still a bit of a mystery. It is a stress response. Once triggered, cells enter a semi-dormant stage and a whole heap of metabolic and physiological changes take place. Changes in the environment can then cause the cells to revert to business as usual. So why do we care what state bacteria are in?

About VBNC

The name Viable But Non Culturable explains what the problem is for us. Microbiology was built on, and still relies heavily on being able to culture bacteria. Individual cells are placed on a nutrient medium where they divide and multiply into colonies we can see. We count the colonies and get a handle on the number of bacteria in the original sample.

This all sounds good but there are a few assumptions in the mix. Firstly, we assume all the bacteria land in individual spots way from each other. So long as the sample is reasonably dilute this is a reasonable assumption. Secondly, we assume the bacteria will eat the nutrients we have provided. This can be tricky as some bacteria can be fussy eaters and may not grow (known as fastidious, and Legionella is one of those). Thirdly, bacteria may be in the VBNC state, in which case they are alive but won't grow on our nutrient medium i.e. they're viable but not culturable.

Sadly, there is little doubt that Legionella don't particularly like the agar medium they are grown on. Reports have estimated that culture results detect anywhere between 10 and 70% of the actual cells present. Inter-laboratory validation programs have shown that Legionella may not even be detected in spiked samples by accredited laboratories. Is this shaking your confidence? read on...

Legionella and VBNC

Recently some published research has delved into the VBNC Legionella story. The research uses a combination of two technologies to identify and count Legionella in the VBNC state. The method uses viability based flow cytometry cell sorting followed by qPCR. What dos that mean?

Both 'spiked' water samples and samples from anonymous hospital water systems were used. Cell cultures were stained with dyes that attach to different parts of a bacteria cell. These included parts of the cell only present in viable (living) cells eg membranes. So cells could be stained as 'total' 'dead' 'healthy' or 'VBNC' Flow cytometry 'optically' sorts the cells based on their dye characteristics. The collected cells are then counted using quantitative PCR (moecular methods). The same cell cultures were also inoculated onto Legionella agar (BCYE) and counted by the usual colony count method (cfu/mL).

VBNC cells were confirmed by testing for ATP, a short lived high energy molecule present in all living cells. They were also tested for their ability to infect and predate free living amoebae - proving they were still infectious. If you want to read in more detail there's a link to the paper at the bottom of this post.

The results have been published in international journals and point to a few key facts that we're going to have to live with.

We are missing the target

(Photo courtesy Jonny Sweeney)

Legionella culture testing is hit and miss

Results have shown a few things worth noting:

VBNC Legionella may still be infectious

Legionella are induced into the VBNC state by high temperatures (50 Celsius)
VBNC state is induced by some chemical treatments
ISO and AS/NZS Legionella culture methods induce the VBNC state
VBNC Legionella can be present when culture results are not detectable.

Also worth noting:

Legionella are significantly reduced in numbers by higher temperatures
Legionella are significantly reduced in numbers by chemical treatments

Those statements take a bit of absorbing. Do they suggest that our treatments don't work and neither does our means of monitoring them? Well, not really - the study showed that things like temperature control and disinfection (operational controls) do have a significant effect. Nevertheless, they don't remove the problem but they do reduce it's magnitude. The most unreliable step in the whole process is the culture results; and they will always underestimate the problem.

Don't Panic!

The results really only confirm what many of us already knew - We can't rely on culture results to manage or control Legionella. Water Safety Risk Management Plans rely on a well implemented hierarchy of control and evidence based interventions. Getting the barriers in place, developing your swiss cheese, or adjusting your bow tie are still the most important steps in planning.

Legionella testing as a verification step is still worth the effort. From the number of positive results we see from health and aged care there are still plenty of fallen barriers, burrowed cheeses and wonky bow ties out there! Getting a positive result should stimulate an immediate reaction. Getting a negative result is satisfying ONLY if all your operational controls are where they should be.

Make sure the verification cart is sat behind the operational horse!