Relevance of Viable-But-Non-Culturable Legionellae

Natural freshwater environments are the major reservoirs of legionellae, but since their discovery in 1976, they have been isolated mainly from man-made aquatic environments. Anthropogenic water systems with elevated temperatures are the only source of legionellae associated with human disease, so far. Among these, showers, cooling towers, hot and cold water distribution systems or air conditioning systems, are most frequently and densely colonised by legionellae. The numbers of infections caused by legionellae in developed countries have been increasing over the past two decades. In Europe, 53,494 reported cases of Legionnaires Disease were recorded during the period from 1993-2008. It is assumed that the central survival strategy of Legionellae in the environment is the intracellular multiplication in environmental protozoa, particularly amoebae, but legionellae, specifically Legionella pneumophila, can also infect the human lung and replicate within alveolar macrophages. Legionellae are usually detected and quantified via standardised culture technique. However, it is common knowledge that legionellae, can switch into a viable-but-non-culturable (VBNC) state upon exposure to harsh environmental triggers. From our own data on cooling towers and a number of other reports it can be concluded that VBNC legionellae are abundant in various kinds of water systems but it is not clear whether they are of importance for human health. That VBNC cells can retain virulence and infectivity has been shown for a few pathogens, so far. It was demonstrated that VBNC legionellae can be resuscitated to the culturable state via transmission into amoebae and are supposed to regain their full infectivity and virulence potential after resuscitation. However, these few investigations have been performed only with a very limited number of Legionella strains, only a single method for proving viability and limited tests for infectivity/virulence. Moreover, it has not yet been investigated, whether VBNC legionellae are directly able to infect human host cells or cause illness. Thus a comprehensive picture on the potential threat emanating from VBNC legionellae cannot be drawn. Our central hypothesis is that the abundance (and maybe also diversity) of medically relevant legionellae in diverse anthropogenic water systems is being severely underestimated, mainly due the non-culturability of a considerable percentage of the Legionella populations. Our final aim is to isolate VBNC legionellae from environmental water systems (cooling towers, water distribution systems) and investigate their infectious potential. This shall provide the basis for inclusion of VBNC legionellae in future screening programs. To achieve these aims comprehensive preparation is necessary by building up a set of well-established model systems. These shall include: (i) a standardized system to push legionellae into the VBNC state and to test viability of legionellae (ii) A standardized system to isolate/separate legionellae from environmental samples via immunomagnetic bead sorting; (iii) a standardized human macrophage assay for infectivity and virulence testing. (iv) two host amoeba systems that can be used as "training grounds" for the legionellae and (v) an animal model for proof of principle. If our central hypothesis can be proven in the frame of our project, this will have highly significant impact on strategies for prevention of the disease and management of water systems as well as for the choice of the detection methods of legionellae in routine laboratories.

Legionellae belong to the most important waterborne pathogens in industrialized countries. They are transmitted via small water droplets from contaminated engineered water systems and cause a severe lung infection called Legionnaires disease. Surveillance of legionellae in engineered water systems is usually performed with microbiological cultivation methods, however in recent years it was found that a high number of viable but non-culturable (VBNC) legionellae may exist in many engineered water systems. In this project we wanted to assess the health relevance of these viable but non-culturable legionellae. Our most significant finding was that a variety of VBNC Legionella strains are able to directly infect and multiply in different human macrophage cell types. Up to now it was thought that prior resuscitation from the VBNC state via amoebae is a necessary precondition. Within this context we could also show that many virulence-related structures of the outer membranes of the bacteria are present at high levels even after several months in the VBNC state. Thus, VBNC cells as such (without resuscitation) may be of relevance for human health. Based on our results we can also deliver a model for the infection process: Upon the impact of the stressors triggering the transition to the VBNC state, different sub-populations of VBNC cells develop with the majority of cells showing only weak signs of viability. Nevertheless a small sub-population of highly active VBNC cells remains. It is highly likely that it is this sub-population that is able to cause infection of human macrophages. However, compared to culturable cells, these VBNC cells need much longer time and a higher multiplicity of infection to cause an infection. Moreover, the percentage of infected cells is considerably lower. The output of the project has been raising the awareness of the VBNC state of Legionella in particular (and of bacteria in general) within the scientific community but also in the practical field (physicians, hospital personnel, cooling tower operator, water engineers, water hygienists). Due to the raised awareness of the VBNC problem, the project will contribute to more effective disease prevention. This will be achieved on the one hand by a better monitoring of potentially contaminated water systems and on the other hand by a more effective treatment of Legionnaires disease patients. We recommend that VBNC Legionella cells should be included in future monitoring and surveillance activities of engineered water systems and in the case of recurrent or long-time legionellosis cases without a positive culture test, culture independent detection of Legionella in clinical samples (e.g. broncho-alveolar lavage) should be applied at least in suspicious cases.