Viable But Non Culturable Legionella Bacteria

What are Viable But Non Culturable Legionella bacteria?

Viable But Non Culturable (VBNC) bacteria are those which are in alive but for some reason they cannot be cultured in a laboratory sample. It is generally claimed that Legionella laboratory cultures are the “gold standard” when it comes to monitoring for Legionella bacteria however a laboratory culture technique will not identify VBNC bacteria, as the name suggests.

It is therefor important to be aware of these bacteria because they are present in your water system, whether that be a cooling system, process water system or domestic hot and cold water systems.

This means that there could be more Legionella bacteria in your water system than you thought after having a laboratory culture sample taken.

An example of what can cause Legionella bacteria to be VBNC is starvation, for example if environmental conditions are such that the Legionella bacteria do not have sufficient food, another example is cold water temperatures below 20oC.

Legionella Life Cycle

Environmentally Legionella bacteria require to live in association with Amoeba as it is within these amoebae that Legionella can find nutrients, divide, and grow into larger numbers. Within a human host Legionella bacteria do not encounter amoebae but they do encounter “macrophages” which are part of the body’s defense mechanism with Legionella treating them in exactly the same way as they treat amoebae.

Does VBNC mean they are harmless?

The question then becomes if Legionella bacteria cannot be grown in a laboratory culture because they are VBNC are they harmless once inside the human host? Unfortunately, there are not enough scientific studies to effectively answer this however there are scientific papers that show VBNC Legionella bacteria can infect amoebae and macrophages.

One example of a scientific study is a 2018 paper “Starved viable but non-culturable (VBNC) legionella strains can infect and replicate in amoebae and human macrophages” by Dietersdorfer et al. (https://DOI.org/10.1016/j.watres.2018.01.058).

This paper showed that Legionella pneumophila sg1, sg6 and Legionella micdadei can all “directly infect different primary human macrophage cell types and amoeba.” It went on to say that they did so with reduced efficacy which is seen as lower percentages of infected cells. This work was carried out in a laboratory so it is unclear whether human macrophages would be infected in vivo. They also showed that VBNC Legionella bacteria could infect amoebae for example in a biofilm.

Amoebae in environmental waters

Amoebae are ubiquitous in man-made water systems such as a cooling system or domestic hot and cold water systems and studies have shown that they can be resistant to disinfection treatments. A scientific paper in 2004 “Amoebae in domestic water systems: resistance to disinfection treatments and implication in Legionella persistence” by Thomas et al. (doi:10.1111/j.1365-2672.2004.02391.x) showed that Amoebae resisted all the disinfection treatments applied, such as two common biocides chlorine and chlorine dioxide, and “probably acted as reservoirs for Legionella pneumophila, allowing quick re-colonisation of the system once the treatments were interrupted”.

A 2018 scientific paper “Detection of viable but non-culturable legionella in hospital water network following monochloramine disinfection” by Casini et al (https://doi.org/10.1016/j.jhin.2017.09.006) showed that Legionella bacteria can enter a VBNC state as a result of biocide addition.

The implications of this are clear, that Legionella bacteria could become VBNC due to the addition of a biocide to the system, the biocide may not be effective against amoebae which can then act as a defense mechanism for Legionella bacteria allowing them to grow and overcome their VBNC stage. This means that Legionella bacteria can then become a problem again.

So, dosing a biocide may mean Legionella bacteria are no longer measurable in a laboratory culture test because they are either VBNC or surviving inside amoebae.

Can VBNC Legionella be measured in some other way?

There are technologies that can be used to measure VBNC Legionella, for example qPCR. It has been shown that this technology can identify alive VBNC Legionella bacteria, for example in a 2019 paper “Validation and in-field testing of a new on-site qPCR system for quantification of Legionella  pneumophila according to ISO/TS 12869:2012 in HVAC cooling towers” by Ahmed et al. (https://iwaponline.com/jwh/article/17/2/237/66176/Validation-and-in-field-testing-of-a-new-on-site).

This therefore represents a means of identifying what was previously not possible in a laboratory culture technique.

Conclusion

It is important to understand what a sample result is telling you, whether that is a laboratory culture test or a rapid test analysis such as qPCR. It is also important to understand we do not test for all organisms that can contribute to an understanding of managing Legionella bacteria as we do not routinely test for amoebae in water systems. As a result we only ever have a partial picture of what is happening in man-made water systems and so should take a holistic approach to managing Legionella and err on the side of caution when interpreting laboratory results.

If you would like further information or help in interpreting Legionella sample results and what that means for your system then contact Collaton Consultancy via our email address general@collatonconsultancy.com or phone us on +44 (0)7958 124563.

Collaton Consultancy Limited

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