Molecular approaches and Diagnostic Strategies for Isolation and Identification of Legionella Pneumophila Serogroups 1 and 2-15 and associated Bacteria Isolated from Cancer Patients and Hospital Environment

Abstract

 Legionella pneumophila commonly leads to respiratory infections that get acquired in the community. The pathogenesis of legionnaire’s disease involves invasion of alveolar macrophages and cell mediated immunity is the primary means of immune control. Legionella pneumophila serotype1 is the most commonly reported cause of human Legionella infections. Sensitivity of urinary antigen testing is lower in immunocompromised patients because of higher likelihood of infections caused non-L. pneumophila species.

A total of 360 samples (Clinical 271 and 89 environmental samples) were collected from the Oncology Center at Al-Sadr hospital in Basrah city southern of Iraq, during January- March, 2020. (The clinical specimens included blood, urine and sputum, were taken from patients attending and /or admitting to the center. Meanwhile, the environmental samples were collected from air conditioners, hospital toilets and water). According to the morphpological features of L. pneumophila on Buffered charcoal yeast extract (BCYE) agar, and using 16S rDNA gene sequence revealed that forty seven species have been obtained in addition to95 legionella spp out of 47 species, 36 species belong to Bacillus cereus and B. anthracis, one genus of Alcaligenes aquatilis, Enterococcus sp., Klebsiella oxytoca, Escherichia sp. and Serratia sp.,in addition to4 genus of Pseudomonas sp. and 2 genus of Staphylococcus haemolyticus. All these isolates were isolated from samples of blood, urine, sputum, water and air. The idenfied Legionella spp. were more characterized using six specific primer. Using JFP and JRP primers, When the second specific primer L5SL9, L5SR93 were applied, the result showed that all isolates were positive. However, applying the third specific primer dnaJW19F, dnaJW19R for differentiate between all L. pneumophila serogroups and the strains that belong to the various species of Legionella.lt should be mentioned that the Legionella bacteria physiological tests were carried out in a separate study simultaneously with this one, pending its publication. The following tests are among them: using biochemical tests and some physiological experiment, which include: catalase, oxidase, Dnase, gelain liquefaction, hippurate hydrolysis, urease, biofilm forming (tube and Congo red methods), tissue culture plate method, starch hydrolysis, citrate utilization, himmagglutination activity, protease production, effect of pH, temperature and salinity and lecithinase and lipase production accomplished by serotyping the serogroup of Legionella pneumophila was performed using Hi Legionella Latex Test Kit, the identified legionella spp. subjected to nine types of antibiotics to determine the susceptibility which included; azithromycin 15 |ig, cefotaxime 30pg, ciprofloxacin 5pg, doxycycline 30 |ig, erythromycin 15 |ig,   levofloxacin 5pg, ofloxacin 10 |xg, norfloxacin 10 pg and Rifampicin 5 pg. antibiotics were subjected to detect minimum inhibitory concentrations using MIC strips which including azithromycin 0.016-256 mcgmL-1, cefotaxime 0.002-32 mcgmL-1, ciprofloxacin 0.016-256 mcgmL-1 and levofloxacin 0.002-32 mcgmL-1.