In a previous blog post, ‘Legionella sampling; does “Not detected” mean there are no Legionella present?’, I described how not finding Legionella bacteria in a culture sample could be explained by biofilms and limits of detection.
This post develops that subject and discusses why starving Legionella bacteria may be the reason.
Kirschner, ‘Determination of Viable Legionellae in Engineered Water Systems’. wrote:
In many cases culture-negative results are obtained despite the presence of viable legionellae, and clinical cases of legionellosis cannot be traced back to their respective contaminated water source. Among the various explanations for these discrepancies, the presence of viable but non-culturable (VBNC) Legionella cells.
This theme was further developed by Schrammel et al., in ‘Differential Development of Legionella Sub-Populations during Short- and Long-Term Starvation’.
Under conditions of stress, such as nutrient scarcity, high temperature orthe presence of oxidizing agents, legionellae are able to enter a reversible dormant state, which is described as a viable but non-culturable (VBNC) state (extensively reviewed by Kirschner, 2016). After alleviation of the stress, the cells may be resuscitated via passage through an amoeba host cell to become culturable again (e.g., Al-Bana et al., 2014; Epalle et al., 2014; Steinert et al.,1997). In the case of oxidative damage, it was shown that scavengers of reactive oxygen species enhance the resuscitation of injured cells (Ducret et al., 2014). Different stress conditions, such as starvation (Al-Bana et al., 2014;Steinert et al., 1997), treatment with reactive oxidizing chlorine species (Alleron et al., 2008; Wang et al.,2010), or heat treatment (Epalle et al., 2014), may result in differing stress responses and abilities to recover (reviewed in Kirschner,2016).
VBNC legionellae may regain their ability to infect human or human cell-lines after passage through amoebae. To date, direct infection of or resuscitation in humans or human cell lines has never been reported
However the need for amoeba is not always required as described by Dietersdorfer et al. in ‘Starved viable but non-culturable (VBNC) Legionella strains can infect and replicate in amoebae and human macrophages’
Several starved VBNC Legionella strains (four L. pneumophila serogroup 1 strains, a serogroup 6 strain and a L. micdadei strain) can directly infect different types of human macrophages and amoebae even after one year of starvation in ultrapure water. However, under these conditions, the strains caused infection with reduced efﬁcacy, as represented by the lower percentages of infected cells, prolonged time in co-culture and higher multiplicities of infection required. Interestingly, the VBNC cells remained mostly non-culturable even after multiplication within the host cells. Amoebal infection by starved VBNC Legionella, which likely occurs in oligotrophic bioﬁlms, would result in an increase in the bacterial concentration in drinking-water systems. If cells remain in the VBNC state, the real number of active legionellae will be underestimated by the use of culture-based standard techniques.
The problem of identifying these VBNC Legionella bacteria, whether they have been through amoeba or not remains, especially if there are only a few VBNC Legionella bacteria present.
If low numbers of culturable legionellae and a high number of VBNC cells are observed, further measures have to be considered. In outbreak scenarios, a ten-day analysis period is much too long and culture-independent methods have to be applied immediately. As above, rapid screening of all potential sources should be done with qPCR, FCM or SPC, obtaining results within one or two working days. All positive sources should be checked in parallel with standard cultivation in order to (hopefully) isolate the causative strain. The availability of a multiplex PCR targeting the most important sequence types may help to direct efforts of source identification.Kirschner, ‘Determination of Viable Legionellae in Engineered Water Systems’.
Consequently the problem remains, in the UK as in most of the world, determining Legionella bacteria using culture techniques remains the “gold standard”, and is the method supported by the HSE in HSG274, however this method will not find VBNC Legionella as VBNC refers to “viable but not culturable”.
We need, therefore, other tools to determine the presence of Legionella in water systems. These methods are increasingly available and commercially viable, all that is required is the confidence of industry to support and promote these tools so that we can find bacteria that a culture method cannot, and in a timescale that is more suitable for infection prevention and control.
A further requirement will be the support of the UK’s HSE, with, ideally, the inclusion of these tools in an update of HSG274.
If you want to discuss the application of the information above to your systems then contact Collaton Consultancy via: firstname.lastname@example.org or phone on +44 (0)7958 124563.
Collaton Consultancy Limited are expert Legionella consultants working for both water treatment companies and end users alike, Expert Witness services are also offered should a legal case arise. If you have any specific issues relating to the above you would like help with then contact Collaton Consultancy Limited