Detection of <i>Legionella</i> spp. in Natural and Man-made Water Systems Using Standard Guidelines

Abstract

Infections caused by Legionella spp. are considered at the present time, an emerging public health problem and are linked to high rates of mortality and morbidity, if not properly treated. In this study were analyzed 54 samples of water from 8 counties at Northern Portugal, with the aim of obtaining a collection of strains of the genus Legionella and to characterize them genetically and phenotypically. Another objective of this study was to evaluate the effectiveness of the technique of cultivation, a standard method according to International Organization for Standardization ISO 11731:1998, for detection and enumeration of species of Legionella. For laboratory processing, after the filtration of samples (1 L), the filtrate was resuspended in sterile distilled water (5 ml). Heat treatment for selective inhibition of non-Legionella bacteria was performed. Subsequently, 100 μl of the suspension was spread in GVPC selective agar medium, and incubated (7 to 10 days) at 37 ℃. Colonies that were morphologically characteristic of the genus were sub-cultured onto BCYE agar and blood agar for verification. According to the procedure recommended by the standard method, only the colonies which grew in BCYE agar and not on blood agar were considered as suspected Legionella strains. The identification of these initially selected colonies was performed by sequencing the 16S rRNA gene, which revealed that none of the isolates were identified as belonging to the genus Legionella. However, through the ISO 11731:1998 they were interpreted as positive, corresponding therefore to "false-positive" results. The methods used in this study allowed the isolation of a number of isolates (40), which form an independent group of all genus of the family Chitinophagaceae outlined so far, and that by their phylogenetic distance might be a genus not yet described and therefore a new species. The results obtained, highlighted the importance of using culture and genetic methods in parallel for the proper identification of microorganisms.

Legionella, water and biotechnology

“…Among the standardized methods for the detection of Legionella spp. in water samples are those reported by the International Organization for Standardization (ISO) 11731: 1998 (Detection and enumeration of Legionella), ISO 11731-2: 2004 (Detection and enumeration of Legionella, Direct membrane filtration method for waters with low bacterial counts), and the method of the Association Française de Normalisation (AFNOR) NF T 90-431/2003 (Pelaz, 2006;Borges et al, 2012;Rodriguez et al, 2015;Lösch & Merino, 2016;Cortés et al, 2018). The different methods of microbiological analysis of water share some similarities, being generally constituted in five phases: a.…”

“…The activities of a microbiology laboratory commonly apply various techniques including phenotypic, molecular and proteomic that allow the isolation, identification and/or detection of microorganisms associated with infectious processes detrimental to health (Bou et al, 2011). In the case of species of Legionella, different methods have been reported for the detection and identification in samples from aquatic systems, based on culture by conventional methodologies and phenotypic characteristics of these bacteria (morphology, development, biochemical and metabolic properties), which also allow further studies of antimicrobial sensitivity, typing and epidemiology (Ausina et al, 2005;Pelaz, 2006;Bou et al, 2011;Borges et al, 2012;Lösch & Merino, 2016;Cortés et al, 2018). L. pneumophila, the main causative agent of legionellosis, presents different phenotypic characteristics for its identification in culture, as it is a rod-shaped microorganism of 1.5-5.0 μm long by 0.5-0.7 μm wide, mobile, catalase positive, uses amino acids as the main source of energy, presents positive gelatin liquefaction, and requires iron salts and L-cysteine for growth.…”

“…Decontamination and reduction of associated microbiota in the concentrated sample, which is carried out by thermal and acid treatment; c. Inoculation in selective culture media; d. Incubation, and e. Counting, confirmation and typing of isolated colonies (Fig. 1) (Pelaz, 2006;Borges et al, 2012;Rodriguez et al, 2015;Lösch & Merino, 2016;Cortés et al, 2018). In addition to the conventional method of analysis, there are other alternatives, that can reduce the analysis time, obtain results and perform actions corresponding to control, prevention or treatment, such as the enzyme-linked immunoassay in water with analogous characteristics in the detection of cell wall lipopolysaccharide antigen of L. pneumophila serogroup 1 in urine, and the molecular assays like the Polymerase Chain Reaction (PCR), conventional or real time and both qualitative and quantitative, based on studies of 5S rRNA genes, 16S rRNA and 23S rRNA, mip (macrophage infectivity potentiator) that encodes the infectivity enhancer gene of Legionella in mac-rophages, or the dotA infectivity gene (defective organelle trafficking) among others (Ausina et al, 2005;Pelaz, 2006;Borges et al, 2012;Lu et al, 2016;Cortés et al, 2018).…”

“…1) (Pelaz, 2006;Borges et al, 2012;Rodriguez et al, 2015;Lösch & Merino, 2016;Cortés et al, 2018). In addition to the conventional method of analysis, there are other alternatives, that can reduce the analysis time, obtain results and perform actions corresponding to control, prevention or treatment, such as the enzyme-linked immunoassay in water with analogous characteristics in the detection of cell wall lipopolysaccharide antigen of L. pneumophila serogroup 1 in urine, and the molecular assays like the Polymerase Chain Reaction (PCR), conventional or real time and both qualitative and quantitative, based on studies of 5S rRNA genes, 16S rRNA and 23S rRNA, mip (macrophage infectivity potentiator) that encodes the infectivity enhancer gene of Legionella in mac-rophages, or the dotA infectivity gene (defective organelle trafficking) among others (Ausina et al, 2005;Pelaz, 2006;Borges et al, 2012;Lu et al, 2016;Cortés et al, 2018). In addition, molecular methods such as Whole Genome Sequencing (WGS), Amplified Fragment Length Polymorphism (AFLP), Pulsed-field Gel Electrophoresis (PFGE), and Multilocus Sequence Typing (MLST) are used in the typing and characterrization of strains associated with outbreaks (Ausina, 2005;Rojas & Figueras, 2006;Borges et al, 2016;Khodr et al, 2016;Raphael et al, 2016;Cortés et al, 2018;Herwaldt & Marra, 2018).…”

Uncertainties associated with assessing the public health risk from Legionella

“…Inaccuracies with culture enumeration may also occur if final confirmation of all Legionella isolates are not performed using an alternative method such as 16s RNA sequencing, polymerase chain reaction (PCR), latex agglutination test, or immunofluorescence antibody test. Borges et al (2012) used the standard Legionella culturing method and found that 40 isolates from natural and artificial water samples grew on GVPC selective Legionella agar, had the same morphological “ground glass” appearance of Legionella , and when restreaked onto blood agar isolates did not grow. However, 16s RNA sequencing confirmed that the isolates were not Legionella and in fact were from the Chitinophagaceae family.…”