Factors Associated with Legionella Detection in the Water Systems of National Lodging Organization Facilities with Water Management Programs in the United States.

A better understanding of risk factors and the predictive capability of water management program (WMP) data in detecting Legionella are needed to inform the efforts aimed at reducing Legionella growth and preventing outbreaks of Legionnaires' disease. Using WMPs and Legionella testing data from a national lodging organization in the United States, we aimed to (1) identify factors associated with Legionella detection and (2) assess the ability of WMP disinfectant and temperature metrics to predict Legionella detection. We conducted a logistic regression analysis to identify WMP metrics associated with Legionella serogroup 1 (SG1) detection. We also estimated the predictive values for each of the WMP metrics and SG1 detection. Of 5435 testing observations from 2018 to 2020, 411 (7.6%) had SG1 detection, and 1606 (29.5%) had either SG1 or non-SG1 detection. We found failures in commonly collected WMP metrics, particularly at the primary test point for total disinfectant levels in hot water, to be associated with SG1 detection. These findings highlight that establishing and regularly monitoring water quality parameters for WMPs may be important for preventing Legionella growth and subsequent disease. However, while unsuitable water quality parameter results are associated with Legionella detection, this study found that they had poor predictive value, due in part to the low prevalence of SG1 detection in this dataset. These findings suggest that Legionella testing provides critical information to validate if a WMP is working, which cannot be obtained through water quality parameter measurements alone.

Legionella pneumophila cell surface RtxA release by LapD/LapG and its role in virulence.

BACKGROUND: Legionella pneumophila is a Gram-negative intracellular bacillus and is the causative agent of a severe form of pneumonia called Legionnaires' disease which accounts for 2-9% of cases of community acquired pneumonia. It produces an extremely large protein belonging to the RTX (Repeats in ToXin) family, called RtxA, and we previously reported that RtxA is transported by a dedicated type 1 secretion system (T1SS) to the cell surface. RTX proteins have been shown to participate in the virulence or biofilm formation of various bacteria, the most studied models being the pore forming hemolysin A (HlyA) of Escherichia coli and the biofilm associated protein LapA of P. fluorescens. LapA localization depends on the enzymatic release by LapD/LapG complex activity. This study aimed to elucidate the dual localization (cell surface associated or released state) of L. pneumophila RTX protein (RtxA) and whether this released versus sequestered state of RtxA plays a role in L. pneumophila virulence. RESULTS: The hereby work reveals that, in vitro, LapG periplasmic protease cleaves RtxA N-terminus in the middle of a di-alanine motif (position 108-109). Consistently, a strain lacking LapG protease maintains RtxA on the cell surface, whereas a strain lacking the c-di-GMP receptor LapD does not exhibit cell surface RtxA because of its continuous cleavage and release, as in the LapA-D-G model of Pseudomonas fluorescens. Interestingly, our data point out a key role of RtxA in enhancing the infection process of amoeba cells, regardless of its location (embedded or released); therefore, this may be the result of a secondary role of this surface protein. CONCLUSIONS: This is the first experimental identification of the cleavage site within the RTX protein family. The primary role of RtxA in Legionella is still questionable as in many other bacterial species, hence it sounds reasonable to propose a major function in biofilm formation, promoting cell aggregation when RtxA is embedded in the outer membrane and facilitating biofilm dispersion in case of RtxA release. The role of RtxA in enhancing the infection process may be a result of its action on host cells (i.e., PDI interaction or pore-formation), and independently of its status (embedded or released).

Genotypic and phenotypic profiling of 127 Legionella pneumophila strains: Insights into regional spread.

Given the recent global surge in Legionnaires' disease cases, the monitoring of Legionella pneumophila becomes increasingly crucial. Epidemiological cases often stem from local outbreaks rather than widespread dissemination, emphasizing the need to study the characteristics of this pathogen at a local level. This study focuses on isolates of L. pneumophila in the Italian region of Friuli Venezia Giulia to assess specific genotype and phenotype distribution over time and space. To this end, a total of 127 L. pneumophila strains isolated between 2005 and 2017 within national surveillance programs were analysed. Rep-PCR, RAPD, and Sau-PCR were used for genotypic characterization, while phenotypic characterization was conducted through fatty acids analysis. RAPD and Sau-PCR effectively assessed genetic characteristics, identifying different profiles for the isolates and excluding the presence of clones. Although Sau-PCR is rarely used to analyse this pathogen, it emerged as the most discriminatory technique. Phenotypically, hierarchical cluster analysis categorized strains into three groups based on varying membrane fatty acid percentages. However, both phenotypic and genotypic analyses revealed a ubiquitous profile distribution at a regional level. These results suggest an absence of correlations between strain profiles, geographical location, and isolation time, indicating instead high variability and strain dissemination within this region.

Legionella maintains host cell ubiquitin homeostasis by effectors with unique catalytic mechanisms.

The intracellular bacterial pathogen Legionella pneumophila modulates host cell functions by secreting multiple effectors with diverse biochemical activities. In particular, effectors of the SidE family interfere with host protein ubiquitination in a process that involves production of phosphoribosyl ubiquitin (PR-Ub). Here, we show that effector LnaB converts PR-Ub into ADP-ribosylated ubiquitin, which is further processed to ADP-ribose and functional ubiquitin by the (ADP-ribosyl)hydrolase MavL, thus maintaining ubiquitin homeostasis in infected cells. Upon being activated by actin, LnaB also undergoes self-AMPylation on tyrosine residues. The activity of LnaB requires a motif consisting of Ser, His and Glu (SHxxxE) present in a large family of toxins from diverse bacterial pathogens. Thus, our study sheds light on the mechanisms by which a pathogen maintains ubiquitin homeostasis and identifies a family of enzymes capable of protein AMPylation.

Can genomics and meteorology predict outbreaks of legionellosis in urban settings?

Legionella pneumophila is ubiquitous and sporadically infects humans causing Legionnaire's disease (LD). Globally, reported cases of LD have risen fourfold from 2000 to 2014. In 2016, Sydney, Australia was the epicenter of an outbreak caused by L. pneumophila serogroup 1 (Lpsg1). Whole-genome sequencing was instrumental in identifying the causal clone which was found in multiple locations across the city. This study examined the epidemiology of Lpsg1 in an urban environment, assessed typing schemes to classify resident clones, and investigated the association between local climate variables and LD outbreaks. Of 223 local Lpsg1 isolates, we identified dominant clones with one clone isolated from patients in high frequency during outbreak investigations. The core genome multi-locus sequence typing scheme was the most reliable in identifying this Lpsg1 clone. While an increase in humidity and rainfall was found to coincide with a rise in LD cases, the incidence of the major L. pneumophila outbreak clone did not link to weather phenomena. These findings demonstrated the role of high-resolution typing and weather context assessment in determining source attribution for LD outbreaks in urban settings, particularly when clinical isolates remain scarce.IMPORTANCEWe investigated the genomic and meteorological influences of infections caused by Legionella pneumophila in Sydney, Australia. Our study contributes to a knowledge gap of factors that drive outbreaks of legionellosis compared to sporadic infections in urban settings. In such cases, clinical isolates can be rare, and thus, other data are needed to inform decision-making around control measures. The study revealed that core genome multi-locus sequence typing is a reliable and adaptable technique when investigating Lpsg1 outbreaks. In Sydney, the genomic profile of Lpsg1 was dominated by a single clone, which was linked to numerous community cases over a period of 40 years. Interestingly, the peak in legionellosis cases during Autumn was not associated with this prevalent outbreak clone. Incorporating meteorological data with Lpsg1 genomics can support risk assessment strategies for legionellosis in urban environments, and this approach may be relevant for other densely populated regions globally.

The Legionella collagen-like protein employs a distinct binding mechanism for the recognition of host glycosaminoglycans.

Bacterial adhesion is a fundamental process which enables colonisation of niche environments and is key for infection. However, in Legionella pneumophila, the causative agent of Legionnaires' disease, these processes are not well understood. The Legionella collagen-like protein (Lcl) is an extracellular peripheral membrane protein that recognises sulphated glycosaminoglycans on the surface of eukaryotic cells, but also stimulates bacterial aggregation in response to divalent cations. Here we report the crystal structure of the Lcl C-terminal domain (Lcl-CTD) and present a model for intact Lcl. Our data reveal that Lcl-CTD forms an unusual trimer arrangement with a positively charged external surface and negatively charged solvent exposed internal cavity. Through molecular dynamics simulations, we show how the glycosaminoglycan chondroitin-4-sulphate associates with the Lcl-CTD surface via distinct binding modes. Our findings show that Lcl homologs are present across both the Pseudomonadota and Fibrobacterota-Chlorobiota-Bacteroidota phyla and suggest that Lcl may represent a versatile carbohydrate-binding mechanism.

Automated cooling tower detection through deep learning for Legionnaires' disease outbreak investigations: a model development and validation study.

BACKGROUND: Cooling towers containing Legionella spp are a high-risk source of Legionnaires' disease outbreaks. Manually locating cooling towers from aerial imagery during outbreak investigations requires expertise, is labour intensive, and can be prone to errors. We aimed to train a deep learning computer vision model to automatically detect cooling towers that are aerially visible. METHODS: Between Jan 1 and 31, 2021, we extracted satellite view images of Philadelphia (PN, USA) and New York state (NY, USA) from Google Maps and annotated cooling towers to create training datasets. We augmented training data with synthetic data and model-assisted labelling of additional cities. Using 2051 images containing 7292 cooling towers, we trained a two-stage model using YOLOv5, a model that detects objects in images, and EfficientNet-b5, a model that classifies images. We assessed the primary outcomes of sensitivity and positive predictive value (PPV) of the model against manual labelling on test datasets of 548 images, including from two cities not seen in training (Boston [MA, USA] and Athens [GA, USA]). We compared the search speed of the model with that of manual searching by four epidemiologists. FINDINGS: The model identified visible cooling towers with 95·1% sensitivity (95% CI 94·0-96·1) and a PPV of 90·1% (95% CI 90·0-90·2) in New York City and Philadelphia. In Boston, sensitivity was 91·6% (89·2-93·7) and PPV was 80·8% (80·5-81·2). In Athens, sensitivity was 86·9% (75·8-94·2) and PPV was 85·5% (84·2-86·7). For an area of New York City encompassing 45 blocks (0·26 square miles), the model searched more than 600 times faster (7·6 s; 351 potential cooling towers identified) than did human investigators (mean 83·75 min [SD 29·5]; mean 310·8 cooling towers [42·2]). INTERPRETATION: The model could be used to accelerate investigation and source control during outbreaks of Legionnaires' disease through the identification of cooling towers from aerial imagery, potentially preventing additional disease spread. The model has already been used by public health teams for outbreak investigations and to initialise cooling tower registries, which are considered best practice for preventing and responding to outbreaks of Legionnaires' disease. FUNDING: None.

Outbreak of Legionnaires' disease in Rzeszów in 2023. / Ognisko choroby legionistów w Rzeszowie w 2023 roku.

BACKGROUND: Legionnaires' disease is a type of severe pneumonia caused by Legionella bacteria. The case fatality rate in this disease is 5-10%. People with various comorbidities, smokers and the elderly are at greater risk of developing the disease. OBJECTIVE: The aim of the work is to present the results of an epidemiological investigation into the outbreak of Legionnaires' disease that occurred in the city of Rzeszów and the surrounding area in August and September 2023 and to present the threat related to the presence of Legionella bacteria in water supply installations and networks. MATERIAL AND METHODS: The material for this publication was data from an epidemiological investigation conducted in the outbreak of Legionnaires disease in Rzeszów in 2023. RESULTS: Epidemiological investigation revealed 165 cases of Legionnaires' disease in the outbreak, including 152 confirmed cases and 13 probable cases. The case fatality rate in a legionellosis outbreak was 15%. Environmental tests were carried out in residential and public buildings and industrial installations during the investigation. As part of environmental tests, 187 water samples were collected, including 87 warm water samples. CONCLUSIONS: The outbreak of Legionnaires' disease in the city of Rzeszów draws attention to the potential threat from the Legionella bacteria to the health and life of especially elderly people suffering from chronic diseases. The environmental tests carried out confirmed the highest number of Legionella bacteria at medium and high levels in water samples taken in the private apartments of sick people. Despite the lack of strict legal regulations clearly specifying the obligations regarding periodic disinfection of internal hot water supply installations, cooperation with their owners should be undertaken to enforce plans and actions in this area.

Efficacy of omadacycline in the treatment of Legionella pneumonia: a case report.

Legionella, one of the main pathogens that causes community-acquired pneumonia, can lead to Legionella pneumonia, a condition characterized predominantly by severe pneumonia. This disease, caused by the bacterium Legionella pneumophila, can quickly progress to critical pneumonia and is often associated with damage to multiple organs. As a result, it requires close attention in terms of clinical diagnosis and treatment. Omadacycline, a new type of tetracycline derivative belonging to the aminomethylcycline class of antibiotics, is a semi-synthetic compound derived from minocycline. Its key structural feature, the aminomethyl modification, allows omadacycline to overcome bacterial resistance and broadens its range of effectiveness against bacteria. Clinical studies have demonstrated that omadacycline is not metabolized in the body, and patients with hepatic and renal dysfunction do not need to adjust their dosage. This paper reports a case of successful treatment of Legionella pneumonia with omadacycline in a patient who initially did not respond to empirical treatment with moxifloxacin. The patient also experienced electrolyte disturbance, as well as dysfunction in the liver and kidneys, delirium, and other related psychiatric symptoms.

Innate immune responses and monocyte-derived phagocyte recruitment in protective immunity to pathogenic bacteria: insights from Legionella pneumophila.

Legionella species are Gram-negative intracellular bacteria that evolved in soil and freshwater environments, where they infect and replicate within various unicellular protozoa. The primary virulence factor of Legionella is the expression of a type IV secretion system (T4SS), which contributes to the translocation of effector proteins that subvert biological processes of the host cells. Because of its evolution in unicellular organisms, T4SS effector proteins are not adapted to subvert specific mammalian signaling pathways and immunity. Consequently, Legionella pneumophila has emerged as an interesting infection model for investigating immune responses against pathogenic bacteria in multicellular organisms. This review highlights recent advances in our understanding of mammalian innate immunity derived from studies involving L. pneumophila. This includes recent insights into inflammasome-mediated mechanisms restricting bacterial replication in macrophages, mechanisms inducing cell death in response to infection, induction of effector-triggered immunity, activation of specific pulmonary cell types in mammalian lungs, and the protective role of recruiting monocyte-derived cells to infected lungs.

Metagenomic next-generation sequencing reveals co-infection with Legionella pneumophila and Fusobacterium necrophorum in a patient with severe pneumonia: a case report.

BACKGROUND: Legionella pneumonia is one of the most severe types of atypical pneumonia, impairing multiple organ systems, posing a threat to life. Diagnosing Legionella pneumonia is challenging due to difficulties in culturing the bacteria and limitations in immunoassay sensitivity and specificity. CASE PRESENTATION: This paper reports a rare case of sepsis caused by combined infection with Legionella pneumophila and Fusobacterium necrophorum, leading to respiratory failure, acute kidney injury, acute liver injury, myocardial damage, and electrolyte disorders. In addition, we systematically reviewed literature on patients with combined Legionella infections, analyzing their clinical features, laboratory results and diagnosis. CONCLUSIONS: For pathogens that require prolonged incubation periods and are less sensitive to conventional culturing methods, metagenomic next-generation sequencing (mNGS) can be a powerful supplement to pathogen screening and plays a significant role in the auxiliary diagnosis of complex infectious diseases.

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.

Antimicrobial susceptibility testing reveals reduced susceptibility to azithromycin and other antibiotics in Legionella pneumophila serogroup 1 isolates from Portugal.

BACKGROUD: Although not fully investigated, studies show that Legionella pneumophila can develop antibiotic resistance. As there is limited data available for Portugal, we determined the antibiotic susceptibility profile of Portuguese L. pneumophila serogroup 1 (LpnSg1) isolates against antibiotics used in the clinical practice in Portugal. METHODS: Minimum inhibitory concentrations (MICs) were determined for LpnSg1 clinical (n = 100) and related environmental (n = 7) isolates, collected between 2006-2022 in the context of the National Legionnaire´s Disease Surveillance Programme, against azithromycin, clarithromycin, erythromycin, levofloxacin, ciprofloxacin, moxifloxacin, rifampicin, doxycycline, tigecycline, and amoxicillin/clavulanic acid, using three different assays. Isolates were also PCR-screened for the presence of the lpeAB gene. RESULTS: Twelve isolates had azithromycin MICs above the EUCAST tentative highest WT MIC, 9 of which were lpeAB negative; for erythromycin and clarithromycin, all isolates tested within the susceptible range. The number of isolates with MICs above the tentative highest WT MIC for the remaining antibiotics was: ciprofloxacin: 7; levofloxacin: 17; moxifloxacin: 8; rifampicin: 11; doxycycline: 82; tigecycline: 4. EUCAST breakpoints are not available for amoxicillin/clavulanic acid. We estimated the ECOFFs and one isolate had a MIC eightfold higher than the E-test ECOFF. Additionally, a clinical isolate generated three colonies growing on the E-test inhibition zone that resulted in MICs fourfold higher than for the parental isolate. CONCLUSIONS: We report, for the first time, elevated MICs against first-line and other antibiotics (including azithromycin, fluoroquinolones and amoxicillin/clavulanic acid commonly used to treat pneumonia patients in Portugal) in Portuguese L. pneumophila strains. Results point towards decreased susceptibility in circulating strains, justifying further investigation.

Legionella pneumophila exploits the endo-lysosomal network for phagosome biogenesis by co-opting SUMOylated Rab7.

Legionella pneumophila strains harboring wild-type rpsL such as Lp02rpsLWT cannot replicate in mouse bone marrow-derived macrophages (BMDMs) due to induction of extensive lysosome damage and apoptosis. The bacterial factor directly responsible for inducing such cell death and the host factor involved in initiating the signaling cascade that leads to lysosome damage remain unknown. Similarly, host factors that may alleviate cell death induced by these bacterial strains have not yet been investigated. Using a genome-wide CRISPR/Cas9 screening, we identified Hmg20a and Nol9 as host factors important for restricting strain Lp02rpsLWT in BMDMs. Depletion of Hmg20a protects macrophages from infection-induced lysosomal damage and apoptosis, allowing productive bacterial replication. The restriction imposed by Hmg20a was mediated by repressing the expression of several endo-lysosomal proteins, including the small GTPase Rab7. We found that SUMOylated Rab7 is recruited to the bacterial phagosome via SulF, a Dot/Icm effector that harbors a SUMO-interacting motif (SIM). Moreover, overexpression of Rab7 rescues intracellular growth of strain Lp02rpsLWT in BMDMs. Our results establish that L. pneumophila exploits the lysosomal network for the biogenesis of its phagosome in BMDMs.

High-glucose conditions attenuate the response of macrophages to Legionella pneumophila infection by inhibiting NOD1 and MAPK signaling.

BACKGROUND: Patients with diabetes are particularly susceptible to Legionella pneumophila (LP) infection, but the exact pathogenesis of LP infection in diabetic patients is still not fully understood. Herein, we investigated the effect of diabetes on immune function during LP infection in vitro and in vivo. METHODS: The time course of LP infection in macrophages under normal and high-glucose (HG) conditions was examined in vitro. Western blot was used to determine nucleotide-binding oligomerization domain 1 (NOD1), kinase 1/2 (ERK1/2), mitogen-activated protein kinase p38 (MAPK p38), and c-Jun N-terminal kinases (JNK). Enzyme-linked immunosorbent assay (ELISA) was used to assess the secretion of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6). Cell Counting Kit-8 (CCK8) assay assessed U937 cell viability after treating cells with different concentrations of high sugar medium and ML130 (NOD1 inhibitor). For the in vivo study, normal and streptozocin-induced diabetic guinea pigs were infected with LP for 6, 24, and 72 h, after which NOD1, MAPK-related signals, TNF-α, and IL-6 expression in lung tissues were assessed using immunohistochemistry, western blot, and RT-PCR. RESULTS: HG attenuated the upregulation of NOD1 expression and reduced TNF-α and IL-6 secretion caused by LP compared with LP-infected cells exposed to normal glucose levels (all p < 0.05). In diabetic guinea pigs, HG inhibited the upregulation of NOD1 expression in lung tissues and the activation of p38, ERK1/2, and cJNK caused by LP infection compared to control pigs (all p < 0.05). CONCLUSION: HG attenuates the response of macrophages to LP infection by inhibiting NOD1 upregulation and the activation of MAPK signaling.

Bacterial ubiquitin ligases hijack the host deubiquitinase OTUB1 to inhibit MTORC1 signaling and promote autophagy.

Many bacterial pathogens have evolved effective strategies to interfere with the ubiquitination network to evade clearance by the innate immune system. Here, we report that OTUB1, one of the most abundant deubiquitinases (DUBs) in mammalian cells, is subjected to both canonical and noncanonical ubiquitination during Legionella pneumophila infection. The effectors SidC and SdcA catalyze OTUB1 ubiquitination at multiple lysine residues, resulting in its association with a Legionella-containing vacuole. Lysine ubiquitination by SidC and SdcA promotes interactions between OTUB1 and DEPTOR, an inhibitor of the MTORC1 pathway, thus suppressing MTORC1 signaling. The inhibition of MTORC1 leads to suppression of host protein synthesis and promotion of host macroautophagy/autophagy during L. pneumophila infection. In addition, members of the SidE family effectors (SidEs) induce phosphoribosyl (PR)-linked ubiquitination of OTUB1 at Ser16 and Ser18 and block its DUB activity. The levels of the lysine and serine ubiquitination of OTUB1 are further regulated by effectors that function to antagonize the activities of SidC, SdcA and SidEs, including Lem27, DupA, DupB, SidJ and SdjA. Our study reveals an effectors-mediated complicated mechanism in regulating the activity of a host DUB.Abbreviations: BafA1: bafilomycin A1; BMDMs: bone marrow-derived macrophages; DUB: deubiquitinase; Dot/Icm: defective for organelle trafficking/intracellular multiplication; DEPTOR: DEP domain containing MTOR interacting protein; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; L. pneumophila: Legionella pneumophila; LCV: Legionella-containing vacuole; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MOI: multiplicity of infection; MTORC1: mechanistic target of rapamycin kinase complex 1; OTUB1: OTU deubiquitinase, ubiquitin aldehyde binding 1; PR-Ub: phosphoribosyl (PR)-linked ubiquitin; PTM: posttranslational modification; SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SidEs: SidE family effectors; Ub: ubiquitin.

An outbreak of Legionnaires' disease linked to a municipal and industrial wastewater treatment plant, The Netherlands, September-October 2022.

Wastewater treatment plants (WWTPs) are increasingly identified as Legionnaires' disease (LD) sources. An outbreak investigation was initiated following five LD cases reported in September 2022 in Houten, the Netherlands. Case identification was based on the European LD case definition, with symptom onset from 1 September 2022, residence in or within 5 km of Houten, or visit to Houten within the incubation period, without other likely sources. We sampled potential sources and genotyped environmental and clinical isolates. We identified 15 LD cases with onset between 13 September and 23 October 2022. A spatial source identification and wind direction model suggested an industrial (iWWTP) and a municipal WWTP (mWWTP) as potential sources, with the first discharging water into the latter. Both tested positive for Legionella pneumophila serogroups 1 and 6 with multiple sequence types (ST). We detected L. pneumophila sg1 ST42 in the mWWTP, matching with one of three available clinical isolates. Following control measures at the WWTPs, no further cases were observed. This outbreak underlines that municipal and industrial WWTPs can play an important role in community LD cases and outbreaks, especially those with favourable conditions for Legionella growth and dissemination, or even non-favourable conditions for growth but with the influx of contaminated water.

The increasing health burden of Legionella Pneumophila in NSW.

BACKGROUND: Legionella pneumophila can cause severe respiratory disease and is notifiable in NSW. An analysis of notifications linked to hospitalisation and death data over the period 2010-2022 was conducted to determine the burden of disease and any association with the introduction of NSW regulatory changes in 2018. METHODS: Cases were retrospectively identified from the Notifiable Conditions Records for Epidemiology and Surveillance (NCRES). Data on related morbidity and mortality were obtained from linked data within the NSW Communicable Disease Register (CDR). The impact of the regulatory change was evaluated by analysing monthly count data using an interrupted time series analysis. RESULTS: A total of 928 cases were notified with 84% admitted to hospital. Annual adjusted notification and admission rates increased over the period from 4.40 to 7.92 cases and 3.72 to 7.20 admissions, per 1,000,000 population, respectively. The mean length of hospital stay (LOS) was 14 days with a median of 8 days (range 1-262 days). Time series analysis identified an underlying increasing time trend in cases notified per month with an IRR of 1.069 (95% ci 0.751-1.523) post 2018 regulatory implementation. CONCLUSION: L. pneumophila is posing an increasing burden of disease with an underlying upward trend in notification incidence despite the introduction of regulatory changes in 2018. IMPLICATION FOR PUBLIC HEALTH PRACTICE: This study demonstrates how linking notification, hospitalisation and death data can measure the health burden of a notifiable condition. Furthermore, time-series analysis using these data is able to identify underlying temporal trends and evaluate policy changes.

Persistence of viable but nonculturable Legionella pneumophila state in hospital water systems: A hidden enemy?

There is little evidence of the long-term consequences of maintaining sanitary hot water at high temperatures on the persistence of Legionella in the plumbing system. The aims of this study were to describe the persistence and genotypic variability of L. pneumophila in a hospital building with two entirely independent hot water distribution systems, and to estimate the thermotolerance of the genotypic variants by studying the quantity of VBNC L. pneumophila. Eighty isolates from 55 water samples obtained between the years 2012-2017 were analyzed. All isolates correspond to L. pneumophila serogroup 6. The isolates were discriminated in four restriction patterns by pulsed-field gel electrophoresis. In one installation, pattern A + Aa predominated, accounting for 75.8 % of samples, while the other installation exhibited pattern B as the most frequent (81.8 % of samples; p < 0.001). The mean temperature of the isolates was: 52.6 °C (pattern A + Aa) and 55.0 °C (pattern B), being significantly different. Nine strains were selected as representative among patterns to study their thermotolerance by flow-cytometry after 24 h of thermic treatment. VBNC bacteria were detected in all samples. After thermic treatment at 50 °C, 52.0 % of bacteria had an intact membrane, and after 55 °C this percentage decreased to 23.1 %. Each pattern exhibited varying levels of thermotolerance. These findings indicate that the same hospital building can be colonized with different predominant types of Legionella if it has independent hot water installations. Maintaining a minimum temperature of 50 °C at distal points of the system would allow the survival of replicative L. pneumophila. However, the presence of Legionella in hospital water networks is underestimated if culture is considered as the standard method for Legionella detection, because VBNC do not grow on culture plates. This phenomenon can carry implications for the Legionella risk management plans in hospitals that adjust their control measures based on the microbiological surveillance of water.