Legionella Isolates Research Articles

Two Sporadic Cases of Legionellosis Associated with the Use of Domestic Ultrasonic Humidifiers

Two sporadic cases of legionellosis occurring in consecutive years were confirmed by positive antigenuria to Legionella pneumophila serogroup 1 in individuals with limited mobility who were confined to their homes. Both cases had a history of using ultrasonic humidifiers and of low exposure to other possible sources of infection. This study was conducted through an expanded epidemiological survey and home inspection. Samples were collected from domestic hot water and humidifiers. Environmental isolates were characterised by immunoagglutination and immunofluorescence. The Dresden panel is employed for the determination of groups and subgroups of serogroup 1. The amoebae were isolated by water filtration and subsequent cultivation of the filters. Identification of the isolates was conducted through the sequencing of PCR products. In both cases, epidemiological studies identified the ultrasonic humidifiers as the probable source of infection. The presence of Legionella pneumophila was confirmed in the sanitary water installation and in the humidifiers, where high values of Legionella pneumophila serogroup 1 counts were also obtained. The isolates were characterised as Olda Oxford and Olda OLDA. Furthermore, in addition to the Legionella isolates, the presence of Vermamoeba vermiformis was also identified in samples obtained from domestic hot water and humidifiers in one of the residential addresses under investigation. It has been demonstrated that humidifiers can act as an amplification mechanism for pathogenic microorganisms when they are not cleaned and maintained correctly. This can pose a health danger, especially to people with previous respiratory pathologies, the immunosuppressed, and the elderly. Therefore, it is of the utmost importance that professionals who recommend the therapeutic use of this equipment should issue warnings regarding the importance of its correct use, cleaning, and disinfection. Finally, humidifiers should be considered in epidemiological surveys and in the study of sporadic cases.

Virulence Genes and Biofilm Formation Among Legionella pneumophila Isolates Collected from Hospital Water Sources.

Legionella pneumophila can be transmitted to people, especially immunocompromised patients, via hospital water pipe systems and cause severe pneumonia. The aim of our study was to investigate the presence of major virulence factor genes, ability of biofilms formation, and correlation between presence of Legionella isolates and temperature, pH, and residual chlorine of water. Hundred water samples were collected from nine hospitals in Tehran, Iran. Temperature, pH, and residual chlorine were determined during sampling. Different virulence genes and the ability to form biofilms were subsequently analyzed among the L. pneumophila isolates. Results showed that 12 (12%) samples were positive in culture method and all of the isolates were positive as L. pneumophila species (mip). A correlation was found between Legionella culture positivity and temperature and pH of water, but there was no significant correlation between residual chlorine of water samples and the presence of Legionella. The isolation of Legionella rate in summer and spring was higher than winter and autumn. Twelve (100%) isolates were positive for mip genes, 9 (75%) for dot genes, 8 (66.66%) for hsp, 6 (50%) for lvh, and 4 (33.33%) for rtx. All of the isolates displayed strong ability for biofilm production every three days. Two of these isolates (16.6%) displayed weak ability to form biofilm on the first day of incubation. This study revealed that water sources in hospitals were colonized by virulent Legionella and should be continuously monitored to avoid elevated concentrations of Legionella with visible biofilm formation.

A novel hybridization capture method for direct whole genome sequencing of clinical specimens to inform Legionnaires' disease investigations.

The unprecedented precision and resolution of whole genome sequencing (WGS) can provide definitive identification of infectious agents for epidemiological outbreak tracking. WGS approaches, however, are frequently impeded by low pathogen DNA recovery from available primary specimens or unculturable samples. A cost-effective hybrid capture assay for Legionella pneumophila WGS analysis directly on primary specimens was developed. DNA from a diverse range of sputum and autopsy specimens PCR-positive for L. pneumophila serogroup 1 (LPSG1) was enriched with this method, and WGS was performed. All tested specimens were determined to be enriched for Legionella reads (up to 209,000-fold), significantly improving the discriminatory power to compare relatedness when no clinical isolate was available. We found the WGS data from some enriched specimens to differ by less than five single-nucleotide polymorphisms (SNPs) when compared to the WGS data of a matched culture isolate. This testing and analysis retrospectively provided previously unconfirmed links to environmental sources for clinical specimens of sputum and autopsy lung tissue. The latter provided the additional information needed to identify the source of these culture-negative cases associated with the South Bronx 2015 Legionnaires' disease (LD) investigation in New York City. This new method provides a proof of concept for future direct clinical specimen hybrid capture enrichment combined with WGS and bioinformatic analysis during outbreak investigations.IMPORTANCELegionnaires' disease (LD) is a severe and potentially fatal type of pneumonia primarily caused by inhalation of Legionella-contaminated aerosols from man-made water or cooling systems. LD remains extremely underdiagnosed as it is an uncommon form of pneumonia and relies on clinicians including it in the differential and requesting specialized testing. Additionally, it is challenging to obtain clinical lower respiratory specimens from cases with LD, and when available, culture requires specialized media and growth conditions, which are not available in all microbiology laboratories. In the current study, a method for Legionella pneumophila using hybrid capture by RNA baiting was developed, which allowed us to generate sufficient genome resolution from L. pneumophila serogroup 1 PCR-positive clinical specimens. This new approach offers an additional tool for surveillance of future LD outbreaks where isolation of Legionella is not possible and may help solve previously unanswered questions from past LD investigations.

Evaluating Fourier-transform infrared spectroscopy with IR Biotyper as a faster and simpler method for investigating the sources of an outbreak of legionellosis.

Fourier-transform infrared (FTIR) spectroscopy using the IR Biotyper and core genome singlenucleotide polymorphism (cgSNP) analysis were performed on 12 Legionella isolates associated with an outbreak at a spa house in Kanagawa Prefecture, Japan, and 3 non-outbreak isolates. The discriminative power of FTIR spectroscopy for 48-h incubation conditions of L. pneumophila in this outbreak was lower than cgSNP-based typing but higher than serogroup typing. FTIR spectroscopy could screen outbreak isolates from a group of genetically related isolates and may be useful as an initial typing method in Legionella outbreak investigations.

Legionella pneumophila Presence in Dental Unit Waterlines: A Cultural and Molecular Investigation in the West Bank, Palestine.

A Legionella pneumophila bacterium is ubiquitous in water distribution systems, including dental unit waterlines (DUWLs). Legionellosis is atypical pneumonia, including Legionnaires' disease (LD) and the less acute form of Pontiac fever. Legionellosis occurs as a result of inhalation/aspiration of aerosolized Legionella-contaminated water by susceptible patients, health workers, and dentists. In this study, we undertook to determine the prevalence of Legionella in water and biofilm samples from Tap and DUWLs collected from five sites of dental clinics and faculties across the West Bank. Water samples were tested for physical and chemical parameters. The study samples included 185 samples, 89 (48%) water samples, and 96 (52%) biofilm swabs, which were analyzed by cultivation-dependent analysis (CDA) and by the cultivation-independent technique (CIA). Also, partial sequencing of the 16S rRNA gene for fifteen L. pneumophila isolates was performed for quality assurance and identification. L. pneumophila was isolated from 28 (15%) of 185 samples using CDA and was detected in 142 (77%) of 185 samples using CIA. The abundance of culturable L. pneumophila was low in DUWL of the sampling sites (range: 27-115 CFU/Liter). PCR was 5× more sensitive than the culture technique. L. pneumophila Sg 1 was detected in (75%) of the isolates, while (25%) isolates were L. pneumophila Sg 2-14. All fifteen sequenced Legionella isolates were identified as L. pneumophila ≥ 94.5%. The analysis of phylogenetic tree showed that L. pneumophila branch clearly identified and distinguished from other branches. These results show that DUWLs of the examined dental clinics and faculties are contaminated with L. pneumophila. This finding reveals a serious potential health risk for infection of immunocompromised patients and dentists' post-exposure.

Prevalence and Characterization of Legionella pneumophila and Related Species from Water-Based Recreational Sites

Abstract Background and Objectives Legionella is a genus of gram-negative bacteria common in soil and aquatic systems and is associated with protists. They have emerged as a pathogenic group due to the increased use and poor maintenance of artificial water environments. This study aims at phenotypic and molecular identification of Legionella in water and swab samples collected from water-based recreational sites. The biofilm-forming ability of Legionella on exposure to various temperatures and iron concentrations was also studied. Methods A total of 60 samples including 30 swab samples and 30 water samples (decorative fountain ponds, swimming pools, garden sprinklers, drip irrigation system) were collected from in and around Mangalore, Karnataka, India. From each source, swab and water samples were collected as per the Indian standard IS: 1622. The collected samples were processed within 4 hours of collection. The samples were subjected to microbiological and chemical estimation followed by filtration through a 0.2 µm membrane filter. Isolation of Legionella from collected samples was performed as per US Centers for Disease Control and Prevention guidelines 2005. The positive isolates were then checked for biofilm-forming ability at various temperatures (25°C, 35°C, and 45°C) and iron concentration (3 mg/L, 30 mg/L, and 300 mg/L) using crystal violet assay. Results Out of 30, water and swab samples tested, one water sample from a garden sprinkler, swimming pool, and one from both water and swab samples from a decorative fountain showed the presence of Legionella. A biofilm study of Legionella at various temperatures and iron concentrations categorized the bacteria as a moderate biofilm former. Conclusion This study revealed that most of the water and swab samples were found to be negative for Legionella that is quite encouraging and the contamination of water systems in recreational facilities can be reduced by decontamination techniques and proper hygienic practices.

Antibiotic susceptibility pattern of Portuguese environmental Legionella isolates.

Legionnaires' Disease is a pneumonia caused by Legionella spp., currently treated empirically with fluoroquinolones and macrolides. In this study, we aim to describe the antibiotic susceptibility pattern of environmental Legionella recovered in the south of Portugal. Minimal inhibitory concentration (MIC) determination of 57 Legionella isolates (10 Lp sg 1, 32, Lp sg 2-14 15 L. spp) was achieved by broth microdilution, as described by EUCAST, for azithromycin, clarithromycin, ciprofloxacin, levofloxacin, and doxycycline. Fluoroquinolones were the most active antibiotic, displaying the lowest MIC values in contrast to doxycycline which had the highest. MIC90 and epidemiological cut-off (ECOFF) values were, respectively, 0.5/1 mg/L for azithromycin, 0.125/0.25 mg/L for clarithromycin, 0.064/0.125 mg/L for ciprofloxacin, 0.125/0.125 mg/L for levofloxacin and 16/32 mg/L for doxycycline. MIC distributions were higher than reported by EUCAST for all antibiotics. Interestingly, two phenotypically resistant isolates with high-level quinolone resistance were identified. This is the first time that MIC distributions, lpeAB and tet56 genes have been investigated in Portuguese environmental isolates of Legionella.

Tap water as the source of a Legionnaires' disease outbreak spread to several residential buildings and one hospital, Finland, 2020 to 2021.

In Finland, all microbiology laboratories notify Legionella findings and physicians notify Legionnaires' disease (LD) cases to the National Infectious Disease Register. All cases are interviewed, and water samples obtained from potential places of exposure. Legionella isolates from humans and water are compared by whole genome sequencing (WGS). In March 2021, Legionella pneumophila serogroup 1 (Lp 1) pneumonia cases increased in one Finnish city (120,000 inhabitants) where single LD cases are detected annually. We identified 12 LD cases, nine living in different residential buildings and three nosocomial, linked by identical human and/or water isolates. Three of these cases were from January 2020, October 2020 and February 2021 and identified retrospectively. Eleven were diagnosed by urinary antigen test, 10 by PCR and five by culture; age ranged between 52 and 85 years, and 10 had underlying diseases. Nine of 12 homes of LD cases and 15 of 26 water samples from the hospital were positive for Lp 1, with concentrations up to 640,000 cfu/L. Water samples from regional storage tanks were negative. Positivity in homes and the hospital suggested inadequate maintenance measures. Enhanced surveillance combined with WGS was crucial in detecting this unusual LD outbreak related to domestic and hospital water systems.

Genomic characterization and assessment of pathogenic potential of Legionella spp. isolates from environmental monitoring.

Several species in the genus Legionella are known to cause an acute pneumonia when the aerosols containing the bacteria from man-made water systems are inhaled. The disease is usually caused by Legionella pneumophila, but other species have been implicated in the infection. The disease is frequently manifested as an outbreak, which means several people are affected when exposed to the common source of Legionella contamination. Therefor environmental surveillance which includes isolation and identification of Legionella is performed routinely. However, usually no molecular or genome-based methods are employed in further characterization of the isolates during routine environmental monitoring. During several years of such monitoring, isolates from different geographical locations were collected and 39 of them were sequenced by hybrid de novo approach utilizing short and long sequencing reads. In addition, the isolates were typed by standard culture and MALDI-TOF method. The sequencing reads were assembled and annotated to produce high-quality genomes. By employing discriminatory genome typing, four potential new species in the Legionella genus were identified, which are yet to be biochemically and morphologically characterized. Moreover, functional annotations concerning virulence and antimicrobial resistance were performed on the sequenced genomes. The study contributes to the knowledge on little-known non-pneumophila species present in man-made water systems and establishes support for future genetic relatedness studies as well as understanding of their pathogenic potential.

Enfermedad del legionario en Argentina: evolución de la estrategia diagnóstica en el laboratorio

Legionnaires’ disease (LD) is severe acute pneumonia that occurs in sporadic or epidemic form, and generally requires hospitalization. The objective of this work was to describe the experience in the LD laboratory diagnostic approach in Argentina during the period 2016–2021. The laboratory analyzed 168 clinical specimens from 93 cases of suspected LD pneumonia. Laboratory tests included the detection of the soluble antigen of Legionella pneumophila serogroup 1 in urine sample, detection of DNA of Legionella spp. in lower respiratory secretions by conventional and commercial molecular methods and isolation in selective medium. LD was confirmed in 12 patients. The urinary antigen allowed the diagnosis for 8 patients. L. pneumophila was isolated from the respiratory material of 6 patients suffering from health care-associated pneumonia, who had been previously diagnosed using the commercial molecular method. Fifty percent of these cases did not show detectable urinary antigen. A single patient did not shows neither detectable antigenuria nor isolation of Legionella from the respiratory sample and was diagnosed as a confirmed case of LD by the detection of DNA of Legionella spp. by PCR directly from the respiratory secretion and the epidemiological link with another case of confirmed LD by culture. Urinary antigen detection is the first-line diagnostic test. However, the incorporation of complementary molecular methods has proved to avoid false negatives and contributed to a better understanding of the true incidence of the disease.

Strain-Level Characterization of Legionella Environmental Isolates via MALDI-TOF-MS.

As a waterborne pathogen of increasing concern, techniques for cost-effective and rapid characterization of Legionella are vital. This study examines the development of a Matrix Assisted Laser Desorption/Ionization-Time of Flight Mass Spectrometry (MALDI-TOF-MS) analysis methodology for this microbe. First, optimal sample preparation methods for the analysis of environmental Legionella isolates via MALDI-TOF-MS were determined. These methods were then implemented to perform strain-level characterization of environmental Legionella isolates from central Arizona. Results demonstrate that a MALDI-TOF-MS method involving BCYE agar-based culturing and protein extraction-based sample preparation yield high-quality mass spectra. Twenty-eight environmental Legionella isolates originating from two separate drinking water distribution systems were analyzed. Multiple species were detected, and strain-level characterization was achieved, with 12 unique strains distinguished. In addition, isolates of L. pneumophila, the most common species observed in the study, were correctly assigned to specific sampling sites. These results demonstrate the potential for this technique to be applied for sub-species characterization of Legionella with significant benefits over established methodologies.

Combining Traditional and Molecular Techniques Supports the Discovery of a Novel Legionella Species During Environmental Surveillance in a Healthcare Facility.

Legionella surveillance plays a significant role not only to prevent the risk of infection but also to study the ecology of isolates, their characteristics, and how their prevalence changes in the environment. The difficulty in Legionella isolation, identification, and typing results in a low notification rate; therefore, human infection is still underestimated. In addition, during Legionella surveillance, the special attention given to Legionella pneumophila leads to an underestimation of the prevalence and risk of infection for other species. This study describes the workflow performed during environmental Legionella surveillance that resulted in the isolation of two strains, named 8cVS16 and 9fVS26, associated with the genus Legionella. Traditional and novel approaches such as standard culture technique, MALDI-TOF MS, gene sequencing, and whole-genome sequencing (WGS) analysis were combined to demonstrate that isolates belong to a novel species. The strain characteristics, the differences between macrophage infectivity potential (mip), RNA polymerase β subunit (rpoB), and reference gene sequences, the average nucleotide identity (ANI) of 90.4%, and the DNA–DNA digital hybridization (dDDH) analysis of 43% demonstrate that these isolates belong to a new Legionella species. The finding suggests that, during the culture technique, special attention should be paid to the characteristics of the isolates that are less associated with the Legionella genus in order to investigate the differences found using more sensitive methods. The characterization of the two newly discovered isolates based on morphological, biochemical, and microscopic characteristics is currently underway and will be described in another future study.

Serotyping of Legionella Bacteria Isolated from Various Water Systems in the Central Anatolia Region

Objective: Legionella bacteria are waterborne environmental pathogens that are considered a public health problem because they cause Legionnaires' disease, which is a nationally notifiable disease. Materials and Methods: Legionella analysis was performed in a total of 651 water samples collected during the years 2015 (450) – 2016 (201). Water samples were collected from hospitals (64.66%), hotels (15.05%), the automotive industry (14.43%) and from the buildings (5.83%) in the Central Anatolia Region. After the isolation of Legionella by the filtration and culturing method, serogroup and subtypes were determined via latex agglutination tests and the direct fluorescent antibody method. Results: In 2015, the Legionella positivity rate was 8.6%, where 28.2% of detections were from L. pneumophila serogroup-1. Six isolates were found to be Philadelphia, four were Olda, and one was Bellingham subgroup. Overall, 64.1% were L. pneumophila serogroup 2-14. Moreover, 14 isolates were SG-5, 10 were SG-6, and one was SG-10. 7.7% were unidentified Legionella species. The Legionella species identified were L. micdadei and L. longbeachae. In 2016, the Legionella positivity rate was 10.4%, with 28.6% of them being from L. pneumophila serogroup-1. Four were found to be Olda and two were Philadelphia subgroups. Overall, 66.6% of them were L. pneumophila serogroup 2-14. Moreover, six of them were SG-5, four were SG-6, and four were SG-2. 4.7% were unidentified Legionella species. There was only one species detected as L. micdadei. Conclusions: It has been observed that the distribution of Legionella has exhibited diversity in different water systems throughout the Central Anatolia Region.

Presence of Viable, Clinically Relevant Legionella Bacteria in Environmental Water and Soil Sources of China.

ABSTRACTThe distribution of pathogenic Legionella in the environmental soil and water of China has not been documented yet. In this study, Legionella was detected in 129 of 575 water (22.43%) and 41 of 442 soil samples (9.28%) by culture. Twelve Legionella species were identified, of which 11 were disease-associated. Of the Legionella-positive samples, 109 of 129 (84.50%) water and 29 of 41 (70.73%) soil were positive for L. pneumophila, which accounted for about 75% of Legionella isolates in both water and soil, suggesting L. pneumophila was the most frequent species. Soil showed a higher diversity of Legionella spp. as compared with water (0.6279 versus 0.4493). In contrast, serogroup (sg) 1 was more prevalent among L. pneumophila isolates from water than from soil (26.66% versus 12.21%). Moreover, many disease-associated sequence types (STs) of L. pneumophila were found in China. Intragenic recombination was acting on L. pneumophila from both water and soil. Phylogeny, population structure, and molecular evolution analyses revealed a probable existence of L. pneumophila isolates with a special genetic background that is more adaptable to soil or water sources and a small proportion of genetic difference between water and soil isolates. The detection of viable, clinically relevant Legionella demonstrates soil as another source for harboring and dissemination of pathogenic Legionella bacteria in China. Future research should assess the implication in public health with the presence of Legionella in the soil and illustrate the genetic and pathogenicity difference of Legionella between water and soil, particularly the most prevalent L. pneumophila.IMPORTANCE Pathogenic Legionella spp. is the causative agent of Legionnaires’ disease (LD), and L. pneumophila is the most common one. Most studies have focused on L. pneumophila from water and clinical samples. However, the soil is another important reservoir for this bacterium, and the distribution of Legionella spp. in water and soil sources has not been compared and documented in China yet. Discovering the distribution of Legionella spp. and L. pneumophila in the two environments may help a deep understanding of the pathogenesis and molecular evolution of the bacterium. Our research systematically uncovered the distributions of Legionella spp. in different regions and sources (e.g., water and soil) of China. Moreover, phylogeny, population structure, and molecular evolution study revealed the possible existence of L. pneumophila with a special genetic background that is more adaptable to soil or water sources, and genetic difference may exist.

Isolation and identification of Legionella spp. from non-hospital facilities: a preliminary one-year surveillance study in the urban area of Pesaro-Urbino (Central Italy).

Legionella is considered one of the most important causes of potentially preventable morbid-ity and mortality. These microorganisms are ubiquitous, but incomplete information is available on the geographic distribution of Legionella species in our region. For the mentioned reasons, in this work the distribution of Legionella spp. in non-hospital facilities of the urban area of Pesaro-Urbino (Central Italy), including public fountains, residential build-ings, public and private offices and retirement homes, was investigated. A total of 298 water samples were collected from the different facilities and subjected to standard Legionella isolation and identification protocols. As reported, 17.8% of the collected water samples resulted positive for Legionella spp. (28.6% from retirement homes, 21.3% from residential buildings, 15.3% from private and public offices). The highest per-centage of positive samples (14.4%) was found in hot water from retirements homes (58.8%) and residential buildings (31.8%); the most frequent isolated serogroups were L. pneumophila 2-14 (71.7%). This work is the first describing the distribution of Legionella spp. in non-hospital facilities in the province of Pesaro-Urbino, and highlights a condition of potential risk for susceptible categories. From our data, we can point that a regular and constant control to prevent microbiological risk from legionellosis, particularly in facilities housing the elderly, is recommended.

Legionella Species Isolated in the Water Systems of Ships and Their Molecular Characterization

Legionella species are generally found in nature and in water resources, and they are gram negative bacilli that can cause pneumonia by being transmitted from water systems to humans via aerosol or aspiration. Legionnaires' disease caused by this agent continues to be a public health problem in cruise ships. In this study, it was aimed to determine the prevalence of the colonization of Legionella species by culture method and to determine the molecular characterization of the isolated Legionella in water samples taken from the water systems of the ships docking in Mersin International Port. A total of 158 cold water samples were taken from 18 ferry and/or cargo ships docking in Mersin International Port between December 2014 and June 2015. Fifty-four of the samples were obtained from tanks, 68 from taps and 36 from shower heads. All samples were centrifuged and inoculated from the pellet onto "Buffered Coal Yeast Extract" (BCYE) (Oxoid, CM0655, UK) agar medium supplemented with iron pyrophosphate, L-cysteine and α-ketoglutarate (Oxoid, SR0110, UK). The culture plates were incubated for 10-15 days in microaerophilic environment in a desiccator at 37°C. The suspicious colonies grown in cultures were serogrouped by latex agglutination test (Oxoid, DR0800M, UK) and fluorescent antibody method (m-Tech Monoclonal Technologies, Inc., USA). For the molecular analysis of Legionella species grown in culture, DNA isolation was made from Legionella colonies and then polymerase chain reaction amplification was performed using specific primer sequences targeting the rpoB gene region of the Legionella genome. Direct DNA sequencing of rpoB gene products was performed in the "ABI PRISM 3130XL Genetic Analyzer" (Applied Biosystems, USA). The DNA sequences were typed by BLAST analysis and the determined types, and NCBI (National Center for Biotechnology Information) reference Legionella sequences were phylogenetically compared with the Neighbor-Joining comparison method by using the Mega 7 program. Legionella spp. was isolated in 18 (11.4%) of 158 samples. Of these, four (7.4%, 4/54) were detected from the tank, 11 (16.2%, 11/68) from the tap and three (8.33%, 3/36) from the shower head. After the latex agglutination test performed from the growing bacterial colonies, five (27.8%) were serogrouped as Legionella spp., four (22.2%) as Legionella pneumophila sg 5, two (11.1%, each) as L.pneumophila sg 1,L.pneumophila sg 8 and Legionella bozemanii and one (5.6%) as L.pneumophila sg 3. Two (11.1%) of the isolates grown in culture could not be serogrouped. Molecular characterization of 12 Legionella isolates could be performed. One of them was serologically serogrouped as L.bozemanii, and it was found to be 99% similar to Legionella rubrilucens when compared with NCBI Legionella sequence data in the BLAST program. One isolate that could not be differentiated by serogrouping was identified as Legionella erytra in the BLAST program after DNA sequence analysis. The remaining 10 isolates (55.6%, n= 18) were confirmed as L.pneumophilia after the comparison with reference NCBI sequences. In this study, it was determined that 11.4% of the water samples collected from the water systems of the ships docking in Mersin International Port were contaminated with Legionella species. The detected Legionella species have an important potential source of infection for the captain, ship workers and passengers travelling on the ships. In this respect, this study reveals the necessity of establishing studies to improve the risk management of Legionella in the water systems of ships.

A Legionnaires' Disease Cluster in a Private Building in Italy.

Legionnaires’ disease (LD) is a severe pneumonia caused by bacteria belonging to the genus Legionella. This is a major public health concern and infections are steadily increasing worldwide. Several sources of infection have been identified, but they have not always been linked to human isolates by molecular match. The well-known Legionella contamination of private homes has rarely been associated with the acquisition of the disease, although some patients never left their homes during the incubation period. This study demonstrated by genomic matching between clinical and environmental Legionella isolates that the source of an LD cluster was a private building. Monoclonal antibodies and sequence-based typing were used to type the isolates, and the results clearly demonstrated the molecular relationship between the strains highlighting the risk of contracting LD at home. To contain this risk, the new European directive on the quality of water intended for human consumption has introduced for the first time Legionella as a microbiological parameter to be investigated in domestic water systems. This should lead to a greater attention to prevention and control measures for domestic Legionella contamination and, consequently, to a possible reduction in community acquired LD cases.

Are pathogenic Legionella non-pneumophila a common bacteria in Water Distribution Networks?

The present study analyzes at the national level, the presence of circulating Legionella in the artificial aquatic systems of different facilities of all of them state-owned centers throughout Spain for 12 months. 1754 water samples from various state-owned centers were collected from January to December 2014. Samples were collected from the cooling towers and evaporative condensers (CTC), and water distribution networks such as domestic hot water (DHW), cold water for human consumption (CW), sprinkler irrigation systems (SIS), fire sprinkler systems (FSS), and water from decorative fountains (DF). All these facilities are considered, according to current regulations, as potential amplifying systems for bacteria and possible sources of infection by the generation of droplets and aerosols. The isolation and counting of Legionella in water samples was carried out using microbiological culture following the international normative UNE-EN-ISO 11,731:2007 (ISO 11,731:1998) and UNE-EN ISO 8199:2008 (ISO 8199:2005).The quantification of Legionella colonization, the annual distribution, and the geographical distribution of the Legionella isolates recovered in the water were analyzed. Besides, molecular techniques were used for the characterization of the Legionella non-pneumophila isolates. Legionella was recovered from 15.79% of the analyzed water samples. High colonization was more frequently detected in water samples from CTC, DHW, CW, and DF. Regarding the geographic distribution, positive samples of Legionella were obtained in 14 of the 18 Spanish locations analyzed. Legionella non-pneumophila was the most prevalent and was isolated from water samples from 13 different geographical locations (72%). Legionella anisa and Legionella jordanis were the most frequently non-pneumophila species isolated. Legionella donaldsonii was isolated for the first time in the water distribution networks in Spain. Legionella pneumophila sg 2–14 was detected in 13 locations and Legionella pneumophila sg 1 in 11 locations. Therefore, our study concludes that the presence of Legionella pneumophila and Legionella non-pneumophila species in these systems can be a potential threat to public health and should be examined thoroughly with complementary techniques, such as molecular techniques as a screen for routine diagnosis.

Comparison of BCYE\u03b1+AB agar and MWY agar for detection and enumeration of Legionella spp. in hospital water samples

BackgroundThis study illustrates for the first time the performance (sensitivity and selectivity) of the selective medium BCYEα +AB suggested by the new edition of ISO 11731 for legionella isolation and enumeration. We compared the efficacy of the selective BCYEα +AB medium with that of the highly selective MWY medium.ResultsLegionella spp. was detected in 48.2 and 47.1% of the samples by BCYEα +AB and MWY agar, respectively. For optimal detection of Legionella spp., most protocols recommend using selective media to reduce the number of non-Legionella bacteria. Agreement between the two media was 86.7%.ConclusionsAccording to the results, both media have a very similar performance and they both have advantages and disadvantages over each other. In AB medium there is the risk of being less selective so more interfering microbiota may grow but in MWY medium there is the risk of being too selective. The low selectivity of the AB medium could be resolved if other treatments are applied after filtration, e.g. acid and/or heat treatment, but it must be taken into account that these treatments still reduce the number of viable Legionella. In conclusion, we recommend using MWY as a selective medium for the detection of Legionella spp. as it is easier discern suspected colonies and facilitate the final Legionella spp. count.

Iron Content as an Indicator for Legionella Species in Artificial Water Systems

Background: Legionella have emerged as a pathogenic group due to increased use and poor maintenanceof artificial water environments. World Health Organization considers Legionella associated diseases tobe underestimated, especially in developing countries due to difficulty in detection. The aim of this studywas isolation and culture of Legionella spp. from artificial water systems and to test the importance of ironconcentration which can be developed as a reliable chemical marker.Method: Thirty samples each were collected from drinking water coolers, cooling towers and shower headsfitted in different houses of Mangaluru city. Isolation and identification of the bacteria were carried out as perthe standard protocols followed by determination of iron content prescribed by IS 3025 (Part 53).Conclusion: Our study shows that concentrations of iron, is an important factor that increases the likelihoodof Legionella spp. Statistically, a concentration of 300 mg Fe/L shows a positive correlation with Legionellapresence. Therefore, monitoring suspect sites for iron concentration and chelation of available iron, canbe developed as respective assays for identifying Legionella spp. Furthermore, we show that PCR baseddetection of Legionella is a more robust method than the classical 3-plate method, especially for virulentstrains.