Legionella Serositis: A Rare Presentation.

BACKGROUND: Legionella has a higher prevalence in India than in the world. Legionaries' disease most commonly involves the lungs but because of increased awareness, extrapulmonary manifestations are also being diagnosed more frequently. CASE DESCRIPTION: We present a case of a young female with acute onset of fever and chest pain. On initial investigation, an electrocardiogram (ECG) reported widespread pulse rate (PR) depression suggestive of pericarditis which was confirmed by ECG. High-resolution computed tomography (HRCT) thorax suggested mild bilateral pleural effusion with normal lung parenchyma. elevated erythrocyte sedimentation rate (ESR), C-reactive protein (CRP) added to the diagnosis of serositis. Serological study for atypical organisms was remarkable for positive immunoglobulin M (IgM) for Legionella. She was treated with a high dose of steroids and azithromycin successfully. CONCLUSION: Isolated extrapulmonary presentation of legionaries disease is often overlooked and is common. So it should be always included in the diagnostic armamentarium as treatment is highly efficacious if started early.

Legionella anisa or Legionella bozemanii? Traditional and molecular techniques as support in the environmental surveillance of a hospital water network.

Understanding the actual distribution of different Legionella species in water networks would help prevent outbreaks. Culture investigations followed by serological agglutination tests, with poly/monovalent antisera, still represent the gold standard for isolation and identification of Legionella strains. However, also MALDI-TOF and mip-gene sequencing are currently used. This study was conducted to genetically correlate strains of Legionella non pneumophila (L-np) isolated during environmental surveillance comparing different molecular techniques. Overall, 346 water samples were collected from the water system of four pavilions located in a hospital of the Apulia Region of Italy. Strains isolated from the samples were then identified by serological tests, MALDI-TOF, and mip-gene sequencing. Overall, 24.9% of water samples were positive for Legionella, among which the majority were Legionella pneumophila (Lpn) 1 (52.3%), followed by Lpn2-15 (20.9%), L-np (17.4%), Lpn1 + Lpn2-15 (7.1%), and L-np + Lpn1 (2.3%). Initially, L-np strains were identified as L. bozemanii by monovalent antiserum, while MALDI-TOF and mip-gene sequencing assigned them to L. anisa. More cold water than hot water samples were contaminated by L. anisa (p < 0.001). PFGE, RAPD, Rep-PCR, and SAU-PCR were performed to correlate L. anisa strains. Eleven out of 14 strains identified in all four pavilions showed 100% of similarity upon PFGE analysis. RAPD, Rep-PCR, and SAU-PCR showed greater discriminative power than PFGE.

Diagnostic Study to Assess the Performance of a New Urinary Legionella Antigen Test-A National Study in Three Referral University Hospitals in Austria during 2014-2017.

BACKGROUND: We evaluated the performance of a rapid diagnostic antigen test (Coris) as an index test versus the urinary Antigen ELISA (Bartels) as the reference test. METHODS: Prospective diagnostic accuracy study (2014-2017) at three university hospitals in Austria. RESULTS: A total of 996 patients were included in the study. Legionellosis was diagnosed in 49/996 (4.9%) using the reference test. The sensitivity and specificity of the Coris test were 75.5% (95% CI 61.1-86.7%) and 100% (95%CI 99.6-100%), respectively. The PPV was 100% and when using the lower 95% CI limit of the estimate for sensitivity, the resulting PPV was 61.1%. The NPV was 98.7% and the accuracy was 98.8%. The index test showed a PPV > 97% during the period of summer and autumn (May through November) and ≥88% during winter (December through February). The NPV was >97% during all of the periods. The median of the monthly incidence in the general population was 0.1 per 100,000 (IQR 0.1; 0.3). CONCLUSION: The new rapid test gave a high level of diagnostic accuracy in a rapid fashion. The test can be applied at the bedside by non-laboratory staff.

A microfluidic biosensor for rapid simultaneous detection of waterborne pathogens.

A microfluidic based biosensor was investigated for rapid and simultaneous detection of Salmonella, Legionella, and Escherichia coli O157:H7 in tap water and wastewater. The biosensor consisted of two sets of focusing electrodes connected in parallel and three sets of interdigitated electrodes (IDE) arrays. The electrodes enabled the biosensor to concentrate and detect bacteria at both low and high concentrations. The focusing region was designed with vertical metal sidewall pairs and multiple tilted thin-film finger pairs to generate positive dielectrophoresis (p-DEP) to force the bacteria moving toward the microchannel centerline. As a result, the bacterial pathogens were highly concentrated when they reached the detection electrode arrays. The detection IDE arrays were coated with three different antibodies against the target bacterial pathogens and a cross-linker to enhance the binding of antibodies to the detection electrode. As the binding of bacterial pathogen to its specific antibodies took place, the impedance value changed. The results demonstrated that the biosensors were capable of detecting Salmonella, Legionella, and E. coli 0157:H7 simultaneously with a detection limit of 3 bacterial cells/ml in 30 - 40 min.

The use of BCYE medium for the detection of Legionella in environmental water samples: an appropriate update to ISO 11731:2017 standard?

We evaluated the diagnostic performances of 2 media (BCYE, MWY) on 951 Legionella-positive hospital water samples. MWY allowed detecting Legionella in 89.2% of samples, but in 10.8% (103/951) Legionella was found on BCYE plates only. In samples where Legionella was isolated with other microorganisms (663/951), MWY was essential to detect the majority of positive samples (349/663, 52.6%), as fewer plates resulted unreadable; however, in those containing Legionella only, a higher frequency of positive samples was recorded with BCYE (94.8%, 273/288) compared to MWY (85.1%, 245/288). Considering the 484 concordant positive samples, overall Legionella counts were significantly higher on BCYE (P = 0.0029), with 47% of samples showing higher counts on BCYE compared to MWY plates. Furthermore, discordant samples (positive on only one medium) showed different relative proportions between Legionella pneumophila and non-pneumophila, the latter being found more frequently on BCYE only (P = 0.0296).Our findings confirm the appropriateness of the ISO 11731:2017 update.

New Insight regarding Legionella Non-Pneumophila Species Identification: Comparison between the Traditional mip Gene Classification Scheme and a Newly Proposed Scheme Targeting the rpoB Gene.

The identification of Legionella non-pneumophila species (non-Lp) in clinical and environmental samples is based on the mip gene, although several studies suggest its limitations and the need to expand the classification scheme to include other genes. In this study, the development of a new classification scheme targeting the rpoB gene is proposed to obtain a more reliable identification of 135 Legionella environmental isolates. All isolates were sequenced for the mip and rpoB genes, and the results were compared to study the discriminatory power of the proposed rpoB scheme. Complete concordance between the mip and rpoB results based on genomic percent identity was found for 121/135 (89.6%) isolates; in contrast, discordance was found for 14/135 (10.4%) isolates. Additionally, due to the lack of reference values for the rpoB gene, inter- and intraspecies variation intervals were calculated based on a pairwise identity matrix that was built using the entire rpoB gene (∼4,107 bp) and a partial region (329 bp) to better evaluate the genomic identity obtained. The interspecies variation interval found here (4.9% to 26.7%) was then proposed as a useful sequence-based classification scheme for the identification of unknown non-Lp isolates. The results suggest that using both the mip and rpoB genes makes it possible to correctly discriminate between several species, allowing possible new species to be identified, as confirmed by preliminary whole-genome sequencing analyses performed on our isolates. Therefore, starting from a valid and reliable identification approach, the simultaneous use of mip and rpoB associated with other genes, as it occurs with the sequence-based typing (SBT) scheme developed for Legionella pneumophila, could support the development of multilocus sequence typing to improve the knowledge and discovery of Legionella species subtypes. IMPORTANCELegionella spp. are a widely spread bacteria that cause a fatal form of pneumonia. While traditional laboratory techniques have provided valuable systems for Legionella pneumophila identification, the amplification of the mip gene has been recognized as the only useful tool for Legionella non-pneumophila species identification both in clinical and environmental samples. Several studies focused on the mip gene classification scheme showed its limitations and the need to improve the classification scheme, including other genes. Our study provides significant advantages on Legionella identification, providing a reproducible new rpoB gene classification scheme that seems to be more accurate than mip gene sequencing, bringing out greater genetic variation on Legionella species. In addition, the combined use of both the mip and rpoB genes allowed us to identify presumed new Legionella species, improving epidemiological investigations and acquiring new understanding on Legionella fields.

Occurrence of the Legionella species in the respiratory samples of patients with pneumonia symptoms from Ahvaz, Iran; first detection of Legionella cherrii.

BACKGROUND: This study aimed to investigate the occurrence of Legionella species in the respiratory samples of patients with pneumonia symptoms from Ahvaz, Iran by culture and the real-time PCR of 23S-5S rRNA gene spacer region. METHODS AND RESULTS: A total of 123 clinical respiratory samples including 63 pleural aspirates, 57 bronchoalveolar lavage (BAL), and 3 sputum were collected from 65 males and 58 females with pneumonia symptoms. All samples were cultured on the Modified Wadowsky-Yee (MWY) agar. The Legionella species was identified by routine bacteriological tests. The presence of the 16S-23S rRNA spacer region gene was investigated by real-time PCR. The Legionella species were differentiated by sequencing of 16S-23S rRNA gene. A total of 2 (1.6%) BAL specimens were positive for Legionella species by culture method. No Legionella spp. were identified in pleural aspirates and sputum samples by the culture method. Using real-time PCR, 9 (7.3%) samples including 6 BAL, 1 sputum, and 2 pleural aspirates were positive for legionella species. These species were detected in 3 (5.2%) females and 6 males (9.2%). The results of sequencing showed that eight species were L. pneumophila while one was L. cherrii. Also, the 2 isolates that were identified by culture method, were confirmed as L. pneumophila by sequencing. CONCLUSIONS: The results showed that using the real-time PCR has a more efficacy for detecting of Legionella species in respiratory samples. Also, L. pneumophila was the most prevalent species circulating in the southwest region of Iran. So, periodic monitoring programs is recommended to prevent epidemics due to this bacterium.

Characterization of the First Cultured Psychrotolerant Representative of Legionella from Antarctica Reveals Its Unique Genome Structure.

Culture-independent analysis shows that Legionella spp. inhabit a wide range of low-temperature environments, but to date, no psychrotolerant or psychrophilic strains have been reported. Here, we characterized the first cultivated psychrotolerant representative, designated strain TUM19329T, isolated from an Antarctic lake using a polyphasic approach and comparative genomic analysis. A genome-wide phylogenetic tree indicated that this strain was phylogenetically separate at the species level. Strain TUM19329T shared common physiological traits (e.g., Gram-negative, limited growth on buffered charcoal-yeast extract α-ketoglutarate [BCYEα] agar with l-cysteine requirements) with its relatives, but it also showed psychrotolerant growth properties (e.g., growth at 4°C to 25°C). Moreover, this strain altered its own cellular fatty acid composition to accumulate unsaturated fatty acid at a lower temperature, which may help maintain the cell membrane fluidity. Through comparative genomic analysis, we found that this strain possessed massive mobile genetic elements compared with other species, amounting to up to 17% of the total genes. The majority of the elements were the result of the spread of only a few insertion sequences (ISs), which were spread throughout the genome by a "copy-and-paste" mechanism. Furthermore, we found metabolic genes, such as fatty acid synthesis-related genes, acquired by horizontal gene transfer (HGT). The expansion of ISs and HGT events may play a major role in shaping the phenotype and physiology of this strain. On the basis of the features presented here, we propose a new species-Legionella antarctica sp. nov.-represented by strain TUM19329T (= GTC 22699T = NCTC 14581T). IMPORTANCE This study characterized a unique cultivated representative of the genus Legionella isolated from an Antarctic lake. This psychrotolerant strain had some common properties of known Legionella species but also displayed other characteristics, such as plasticity in fatty acid composition and an enrichment of mobile genes in the genome. These remarkable properties, as well as other factors, may contribute to cold hardiness, and this first cultivated cold-tolerant strain of the genus Legionella may serve as a model bacterium for further studies. It is worth noting that environmentally derived 16S rRNA gene phylotypes closely related to the strain characterized here have been detected from diverse environments outside Antarctica, suggesting a wide distribution of psychrotolerant Legionella bacteria. Our culture- and genome-based findings may accelerate the ongoing studies of the behavior and pathogenicity of Legionella spp., which have been monitored for many years in the context of public health.

Assessment of hygienic conditions of recreational facility restrooms: an integrated approach.

INTRODUCTION: Microbiological quality of recreational environments included restrooms, is generally assessed by water and surface monitoring. In this study, an environmental monitoring, conducted in spring, of swimming pool restrooms of a recreation center located in the Marche region has been carried out. Seven water samples and seven surface swabs were collected. Moreover, six air samples have been included. The aim of this study was to evaluate if air microbiological monitoring, along with molecular detection in real-time PCR, could give additional useful information about the hygienic conditions of the facility. METHODS: Heterotrophic Plate Count (HPC) both at 22°C (psychrophilic) and 37°C (mesophilic) was determined by separate cultures in all samples. The presence of Legionella pneumophila and Pseudomonas aeruginosa was evaluated by both culture and real-time PCR. RESULTS: The analysis of shower water recorded a HPC load of mesophilic bacteria (37°C) more than 10-fold higher in men restroom, respect to women's one (> 100 vs < 10 CFU/ml), while in air samples was between < 100 and > 500. Concerning pathogen presence, both species Legionella pneumophila and Pseudomonas aeruginosa were detected only in men restroom, but in different sample types by using different methods (culture and real-time PCR). CONCLUSIONS: Air sampling may offer the advantage of giving more representative data about microbial presence in restrooms, including bacterial species transmitted through aerosol, like Legionella. Moreover, the concurrent use of molecular and microbiological detection in an integrated approach could offer the advantage of greater sensitivity.

Improvements to the Success of Outbreak Investigations of Legionnaires' Disease: 40 Years of Testing and Investigation in New York State.

Since 1978, the New York State Department of Health's public health laboratory, Wadsworth Center (WC), in collaboration with epidemiology and environmental partners, has been committed to providing comprehensive public health testing for Legionella in New York. Statewide, clinical case counts have been increasing over time, with the highest numbers identified in 2017 and 2018 (1,022 and 1,426, respectively). Over the course of more than 40 years, the WC Legionella testing program has continuously implemented improved testing methods. The methods utilized have transitioned from solely culture-based methods for organism recovery to development of a suite of reference testing services, including identification and characterization by PCR and pulsed-field gel electrophoresis (PFGE). In the last decade, whole-genome sequencing (WGS) has further refined the ability to link outbreak strains between clinical specimens and environmental samples. Here, we review Legionnaires' disease outbreak investigations during this time period, including comprehensive testing of both clinical and environmental samples. Between 1978 and 2017, 60 outbreaks involving clinical and environmental isolates with matching PFGE patterns were detected in 49 facilities from the 157 investigations at 146 facilities. However, 97 investigations were not solved due to the lack of clinical or environmental isolates or PFGE matches. We found 69% of patient specimens from New York State (NYS) were outbreak associated, a much higher rate than observed in other published reports. The consistent application of new cutting-edge technologies and environmental regulations has resulted in successful investigations resulting in remediation efforts. IMPORTANCE Legionella, the causative agent of Legionnaires' disease (LD), can cause severe respiratory illness. In 2018, there were nearly 10,000 cases of LD reported in the United States (https://www.cdc.gov/legionella/fastfacts.html; https://wonder.cdc.gov/nndss/static/2018/annual/2018-table2h.html), with actual incidence believed to be much higher. About 10% of patients with LD will die, and as high as 90% of patients diagnosed will be hospitalized. As Legionella is spread predominantly through engineered building water systems, identifying sources of outbreaks by assessing environmental sources is key to preventing further cases LD.

Outbreaks Associated with Treated Recreational Water - United States, 2015-2019.

Outbreaks associated with treated recreational water can be caused by pathogens or chemicals in aquatic venues such as pools, hot tubs, water playgrounds, or other artificially constructed structures that are intended for recreational or therapeutic purposes. For the pseriod 2015-2019, public health officials from 36 states and the District of Columbia (DC) voluntarily reported 208 outbreaks associated with treated recreational water. Almost all (199; 96%) of the outbreaks were associated with public (nonbackyard) pools, hot tubs, or water playgrounds. These outbreaks resulted in at least 3,646 cases of illness, 286 hospitalizations, and 13 deaths. Among the 155 (75%) outbreaks with a confirmed infectious etiology, 76 (49%) were caused by Cryptosporidium (which causes cryptosporidiosis, a gastrointestinal illness) and 65 (42%) by Legionella (which causes Legionnaires' disease, a severe pneumonia, and Pontiac fever, a milder illness with flu-like symptoms). Cryptosporidium accounted for 2,492 (84%) of 2,953 cases resulting from the 155 outbreaks with a confirmed etiology. All 13 deaths occurred in persons affected by a Legionnaires' disease outbreak. Among the 208 outbreaks, 71 (34%) were associated with a hotel (i.e., hotel, motel, lodge, or inn) or a resort, and 107 (51%) started during June-August. Implementing recommendations in CDC's Model Aquatic Health Code (MAHC) (1) can help prevent outbreaks associated with treated recreational water in public aquatic venues.

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

BACKGROUND: This 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. RESULTS: Legionella 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%. CONCLUSIONS: According 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.

LegionellaDB - A Database on Legionella Outbreaks.

LegionellaDB is the first database on Legionella outbreaks; it is based on a metadata analysis of peer-reviewed manuscripts from PubMed and SCOPUS. LegionellaDB is dynamic and extensible, allowing users to search for specific outbreaks, suggest additional information to be included after curation, visualize statistical representations on specific outbreaks, and download selected data. The database is maintained online.

Isolation and identification of Legionella spp. from hot spring water in Algeria by culture and molecular methods.

AIMS: Due to infectious risk associated with the presence of Legionella in warm water, we determined the prevalence of living Legionella spp. in hot spring water in Algeria. METHODS AND RESULTS: Detection of Legionella by culture was done by using two methods, direct culture on agar plates and co-culture with amoeba. Fifty samples were taken from different hot springs in northern Algeria, including swimming pools, showers and thermal sources. Legionella pneumophila serotypes were predominant, accounting for 60% of positive samples. Direct method allowed the isolation of 13 L. pneumophila only of 50 samples (26%), whereas co-culture using a panel of three free living amoeba allowed the isolation of 119 Legionella species from the same samples (80%) CONCLUSIONS: Amoeba co-culture allowed the isolation of several Legionella sp., while direct culture allowed the isolation of L. pneumophila only. Remarkably, Legionella longbeachae, usually isolated from soil and compost, was isolated for the first time in thermal water in three samples using Vermamoeba vermiformis co-culture. SIGNIFICANCE AND IMPACT OF THE STUDY: The presence of Legionella in the water of hot springs in Algeria, which are mainly frequented by individuals at risk of Legionellosis, requires urgent control measures.

Chasing Waterborne Pathogens in Antarctic Human-Made and Natural Environments, with Special Reference to Legionella spp.

Waterborne pathogenic diseases are public health issues, especially for people staying in remote environments, such as Antarctica. After repeated detection of Legionella by PCR from the shower room of Syowa Station, the Japanese Antarctic research station, we wanted to understand the occurrence of waterborne pathogens, especially Legionella, in the station and their potential sources. In this study, we analyzed water and biofilm samples collected from the water facilities of Syowa Station, as well as water samples from surrounding glacier lakes, by 16S rRNA gene-based amplicon sequencing. For Legionella spp., we further attempted to obtain a detailed community structure by using genus-specific primers. The results showed that potentially pathogenic genera were mostly localized in the station, while Legionella spp., Pseudomonas spp., and Mycobacterium spp. were also widely distributed in lakes. Genus-specific analysis of Legionella spp. within the lake environments confirmed the presence of diverse Legionella amplicon sequence variants (ASVs) that were distinctly different from the Legionella ASVs detected in the station. The majority of the Legionella ASVs inhabiting Antarctic lake habitats were phylogenetically distinct from known Legionella species, whereas the ASVs detected in the human-made station tended to contain ASVs highly similar to well-described mesophilic species with human pathogenicity. These data suggest that unexpected Legionella diversity exists in remote Antarctic cold environments and that environmental differences (e.g., temperature) in and around the station affect the community structure.IMPORTANCE We comprehensively examined the localization of potential waterborne pathogens in the Antarctic human-made and natural aquatic environment with special focus on Legionella spp. Some potential pathogenic genera were detected with low relative abundance in the natural environment, but most detections of these genera occurred in the station. Through detailed community analysis of Legionella spp., we revealed that a variety of Legionella spp. was widely distributed in the Antarctic environment and that they were phylogenetically distinct from the described species. This fact indicates that there are still diverse unknown Legionella spp. in Antarctica, and this genus encompasses a greater variety of species in low-temperature environments than is currently known. In contrast, amplicon sequence variants closely related to known Legionella spp. with reported pathogenicity were almost solely localized in the station, suggesting that human-made environments alter the Legionella community.

Legionella septentrionalis sp. nov., isolated from aquatic environments in the northern PR China.

Four strains (km711T, km714, km542 and km524), representing a novel Legionella species, were isolated from aquatic environments in northern PR China. Cells were Gram-stain-negative, rod-shaped, microaerobic, motile and growth depended on l-cysteine. They grew at 25‒42 °C (optimum, 35‒37 °C) and could tolerate up to 1.5 % (w/v) NaCl (optimum, 0.5 %). The major fatty acids (>5 %) of the type strain km711T were C17 : 0 anteiso, C15 : 0 anteiso, iso-C16 : 0 and C16 : 1 ω7c and/or iso-C15 : 0 2OH. The pairwise comparison values were <96.1 % for 16S rRNA gene sequences, 23.3‒28.7 % interspecies variation for mip gene sequences, <93.6 % average nucleotide identity and <72.8 % average amino acid identity between these four strains and related type strains within the genus Legionella. The phylogenetic tree based on the four concatenated genes (16S rRNA, mip, rpoB and rnpB) and protein-concatamer tree based on concatenation of 21 protein markers both revealed that these four strains formed a separate phylogenetic branch cluster within the genus Legionella. The results of phenotypic and genotypic features suggest that these four strains represent a novel species of the genus Legionella, for which the name Legionella septentrionalis sp. nov. is proposed (type strain km711T=KCTC 15655T=NBRC 113219T).

How Molecular Typing Can Support Legionella Environmental Surveillance in Hot Water Distribution Systems: A Hospital Experience.

In this study, we aimed to associate the molecular typing of Legionella isolates with a culture technique during routine Legionella hospital environmental surveillance in hot water distribution systems (HWDSs) to develop a risk map able to be used to prevent nosocomial infections and formulate appropriate preventive measures. Hot water samples were cultured according to ISO 11731:2017. The isolates were serotyped using an agglutination test and genotyped by sequence-based typing (SBT) for Legionella pneumophila or macrophage infectivity potentiator (mip) gene sequencing for non-pneumophila Legionella species. The isolates' relationship was phylogenetically analyzed. The Legionella distribution and level of contamination were studied in relation to temperature and disinfectant residues. The culture technique detected 62.21% of Legionella positive samples, characterized by L. pneumophila serogroup 1, Legionella non-pneumophila, or both simultaneously. The SBT assigned two sequence types (STs): ST1, the most prevalent in Italy, and ST104, which had never been isolated before. The mip gene sequencing detected L. anisa and L. rubrilucens. The phylogenetic analysis showed distinct clusters for each species. The distribution of Legionella isolates showed significant differences between buildings, with a negative correlation between the measured level of contamination, disinfectant, and temperature. The Legionella molecular approach introduced in HWDSs environmental surveillance permits (i) a risk map to be outlined that can help formulate appropriate disinfection strategies and (ii) rapid epidemiological investigations to quickly identify the source of Legionella infections.

Sit bath systems: A new source of Legionella infection.

Sit Bath Systems (SBSs) are the most common hygiene method for patients who are not self-sufficient. Therefore, the water quality of SBSs in the nosocomial environment plays a fundamental role in controlling infections for both patients and health-care workers. A long-term study on Legionella and Pseudomonas aeruginosa (P. aeruginosa) contamination was performed in SBSs (n = 20) of six Health Care Facilities (HCFs). A total of 254 water samples were analyzed following ISO procedures. The samples were positive for P. aeruginosa (46.85%) and Legionella (53.54%), respectively, both over the directive limits. Legionella isolates were identified as: Legionella pneumophila (L. pneumophila) serogroups 1, 3, and 6 and Legionella non-pneumophila species (L. anisa, L. londiniensis, L. rubrilucens, and L. nagelii). Moreover, the contamination found was studied with respect to median temperature measured (42 °C), from which two groups (A and B) could be distinguished. P. aeruginosa was found in both groups (100% of SBSs), while a higher percentage of Legionella positive samples was found in group A (75% of SBSs), compared to group B (50% of SBSs), showing how Legionella control could be carried out by using temperatures above 42 °C. An analysis of SBS water pipelines, maintenance, and disinfection treatments indicates SBSs as a new source of infection risk for both patients and health-care workers.

Rapid detection of Legionella spp in water samples by ScanVIT method: comparison of acid vs heat treatment.

BACKGROUND: A rapid detection of Legionella bacteria in water samples is crucial to minimize the risk of acquiring infections, especially in health care facilities. Different detection methods and different decontamination procedures have been reported to affect the recovery of Legionella spp. Our goal was to test the recovery of Legionella pneumophila and Legionella non-pneumophila species using a kit based on non-specific and species-specific probes to treat water samples after two different decontamination procedures. METHODS: The study was conducted with samples collected in the teaching hospital "Le Scotte" of Siena (Italy). Waters samples were analyzed by: i) ScanVIT method after treatment with acids; ii) ScanVIT method after heating; and iii) cultural standard method after heating. The results of the decontamination procedures and the detection methods were evaluated by comparing the number of Legionella-positive and -negative samples, and the recovery rates (CFU l-1) obtained by ScanVIT and the standard method. RESULTS: We find that ScanVIT method is highly sensitive with both decontamination treatments, yielding a higher recovery of L. pneumophila compared to the standard method. Conversely, ScanVIT associated with the acid-treatment yielded the highest recovery of L. non-pneumophila. CONCLUSIONS: The acid-treatment combined to the ScanVIT method increases the recovery of L. non-pneumophila in water samples compared to both ScanVIT associated with heat-treatment and standard culture method. Thus, this method may represent the best choice to detect L. non-pneumophila in water samples and reduce the risk of infection due to underestimation of Legionella loads.