Discovering novel agents of pneumonia

45th ICAAC
A meeting of the American Society for Microbiology

December 16-19, 2005, Washington, DC
For more information on any presentation at the 45th ICAAC contact Jim Sliwa, ASM Office of Communications at
jsliwa@asmusa.org

EMBARGOED UNTIL: Friday, December 16, 12:15 p.m. EST
(Session 20, Paper 206)
Gilbert Greub
Univ. of Lausanne
Lausanne, Switzerland
Phone: 00 33 6 22 49 57 62
gilbert.greub@hospvd.ch

Pneumonia is a common disease associated with considerable morbidity and mortality. Identifying the causal agent of pneumonia is critical to  choose the optimal antibiotic treatment and to guide public health decisions. However, using conventional microbiological diagnostic approaches, an etiological agent of pneumonia is only identified in about 50% of cases.

Fastidious intracellular bacteria which grow poorly or not at all on media used routinely for the detection of human pathogens could be the agents of these pneumonia of unknown etiology. Thus, specific diagnostic approaches should be used to detect intracellular bacteria, such as Chlamydia, Mycoplasma and Legionella. Though Legionella is not transmitted from person to person, it may cause large outbreaks of pneumonia, being transmitted through aerosols or microaspiration of contaminated water. 

Eradication of Legionella from water network system and cooling towers represent thus a public health priority. However, free-living amoebae, which are widespread in water, represent a huge aquatic reservoir of Legionella and may, especially when encysted, protect the internalized bacteria from the action of biocides and other sanitation procedures. 

Free-living amoebae generally feed on bacteria. However, Legionella and other amoebae-resisting intracellular bacteria evolved to resist to the microbicidal effectors of free-living amoebae. In addition to the their role of reservoir and protective armour, free-living amoebae should also be considered as a potent evolutionary crib or as a training ground, that lead the bacteria to select virulence traits and adapt to survive to another professional phagocyte: the human alveolar macrophage. Thus, amoebae-resisting intracellular bacteria are good candidates as agent of pneumonia of unexplained etiology. 

Discovering new agents of pneumonia is the main objective of Gilbert Greub’s research group (i.e., the Laboratory for Research on Intracellular Bacteria of the Institute of Microbiology of the University of Lausanne, Lausanne, Switzerland). To reach this ambitious goal, his group is searching new bacterial species, that will then be further studied for their pathogenic potential (Figure). 

                            Figure: Strategy used by Greub’s group to discover new agents of pneumonia

To isolate new bacterial species, they employ an innovative cell culture system that uses free-living amoebae as cellular background. This amoebal co-culture technique selectively grows amoebae-resisting bacteria, i.e. potential pathogens to which humans are frequently exposed (since amoebae are widespread in water), that are highly resistant to water decontamination, and that resist destruction by human macrophages.

Then, to test the pathogenic potential of the newly discovered amoebae-resisting bacterial species, Gilbert Greub’s group use a three step strategy. First, the strains are tested in vitro for their pathogenic potential by looking at interactions with human macrophages. To be pathogenic to humans, the amoebae-resisting bacteria should be able to circumvent the microbicidal machinery of these cells, and might even replicate within their cells. This has been demonstrated by Greub et al. for Parachlamydia acanthamoebae, one emerging agent of pneumonia his research group is working on. Second, once a bacteria has been identified as a potential human pathogen,  various diagnostic tools are developed, such as new molecular tools (PCR). Using PCR, the presence of a given bacteria may be detected with high sensitivity, and its prevalence compared between patients with and without pneumonia. Similarly, specific serodiagnostic tools are developed and used to determine and compare the seroprevalence between patients with and without pneumonia. Novel culture approaches are also developed to increase the sensitivity of growing a given emerging pathogen from patients with pneumonia. Third, to study the bacterial pathogenesis, an animal model is developed. 

By applying amoebal co-culture to 200 tap water of a hospital water network, amoebae-resisting bacteria were isolated from as many as 87 sampling points (see also poster K-866). Among them, 4 new bacterial species that have been recovered by culture, are now studied for their role in pneumonia. Analysis also identified Legionella and Mycobacteria species, including the Legionella pneumophila bacteria, the agent of Legionnaire disease. There was an association between the presence of amoebae and the presence of Legionella and Mycobacteria in water, suggesting that amoebae present in the water networks may also play the role of reservoir for Mycobacteria. Using amoebal co-culture in other settings, Greub’s group successfully isolated several Chlamydia-like organisms that are also currently investigated for their role in pneumonia.

In conclusion, discovering novel agents of pneumonia is a major challenge. Free-living amoebae co-culture systems represents a new promising approach.