Legionella has emerged as major pathogen and may consequence to death. Knowledge of its affliction in India is limited to a few clinical studies^14-17 and fewer environmental studies^15,18. There is a need to prioritize studies on this unexplored pathogen as the rates of ventilator associated pneumonia is fairly larger in India^19,20. Though conventional culture technique is considered the gold standard for detection, use of molecular techniques like PCR have proven to increase the sensitivity for clinical diagnosis of Legionella^21-24 . Respiratory equipment like nebulizers and humidifiers have been implicated as sources of Legionella resulting in hospital-acquired Legionellosis. Surveillance of hospital water has been a primary prevention method of hospital-acquired legionellosis. L. pneumophila cell populations can potentially survive as free organisms for long periods by maintaining metabolic activity but temporarily losing cultivability²⁵. Considering the fact that, L. pneumophila and other respiratory pathogens are able to enter into the viable but non culturable (VBNC) state easily, conventional culture techniques may no longer be the ideal method for detection. A lot of speculation over false positives being reported due to PCR have limited the applications of this technique.

Swabs from surfaces have yielded better recovery of Legionella than water samples¹⁴. A comparative account for the detection of Legionella by conventional and direct PCR method is our study depicted in table 1. Higher sensitivity for detection was observed in the direct PCR detection of genes from samples compared to culture technique which is in concordance to many previous reports²⁶.

Table (1):
Isolation of Legionella by conventional method and detection by molecular methods.

The JFP-JRP primer pair has been repeatedly reported to be specific for the genus Legionella and the same is proven based on sequence alignment (Supplementary Figure S1). Many studies have reported the concomitant occurrence of both JFP-JRP region and dotA in clinical samples²⁷ . In our study, we observed many samples that were positive for dotA but not JFP-JRP region, hence the eubacterial 16S rDNA was amplified and attempted for sequencing. Mixed reads were obtained which indicated that these surfaces contain a mixture of bacteria. The non-specific growth observed in 23 of 35 samples on GVPC reiterates that these surfaces harbor many viable bacteria.  The detection of dotA gene alone and absence of L. pneumophila 16S rRNA in some samples brings to light the need to elucidate the importance of this virulence gene in other potentially infectious organisms. The phylogenetic tree analysis of the dotA sequences revealed that this gene was not clonal and have been derived from many different strains. There was no geographical clonality among these sequences since though 4 of 11 (MT953188 to MT953191) clustered together they were not 100% identical. The other sequences were similar to dotA sequences reported from many other geographical locations of the world including Europe and America. The higher rate of detection of dotA compared to JFP-JRP region can be attributed to the fact that dotA detection was by a semi-nested PCR method.

Horizontal gene transfer has come to be of prime importance in the rise of new age pathogens. The Dot/Icm is the major virulence mechanism present in the pathogens like, Helicobacter pylori, Bordetella pertussis, Brucella sp, L. pneumophila and Coxiella burnetii utilize type IV secretion system for pathogenesis^28. Of these, L. pneumophila and Coxiella burnetii are evolutionarily closely related organisms and both are human intracellular pathogens transmitted to humans through aerosols. Both organisms reside in alveolar macrophages inside an intact phagosome during infection which is mediated by the Dot/Icm.

Though in this study Legionella species have been detected only in two samples by the conventional culture technique, it is necessary to highlight that the presence of virulence genes like dotA on the therapy equipment surfaces may indicate the presence of VBNC forms of the pathogen L.pneumophila. Additionally, the persistence of the virulence gene on these surfaces can result in the mobilization of these genes to other species and hence give rise to virulent forms. The dotA regions from the different samples were not identical indicating that these genes were from a variety of strains of L. pneumophila that are present on the various RTE within a healthcare setup.

RTEs are often used in individuals with compromised immunity and hence better cold sterilization techniques and routine monitoring using culture-independent techniques have to be made mandatory in hospitals.

The present study reveals the occurrence of Legionella pneumophila and associated species in respiratory equipment. To the best of our knowledge, this is the first report on the occurrence Legionella spp. in respiratory therapy equipment in tertiary care hospitals from this geographical location. This evidences that these RTE may serve as potential reservoirs of Legionella species and the usage of such respiratory equipment could transmit pathogenic Legionella spp in to the respiratory tract of patients.

Our observations support the need for concerted efforts to improve the proper maintenance of respiratory equipment as well as awareness among hospital personnel on the potential for aerosolizing pulmonary pathogens. Apart from routine monitoring of water systems for Legionella, we recommend monitoring swabs of respiratory therapy instruments for Legionella and other respiratory pathogens by culture-independent methods as an additional preventive measure against Legionellosis.