Legionella Pneumophila Serogroup Research Articles

Cell-SELEX for aptamer discovery and its utilization in constructing electrochemical biosensor for rapid and highly sensitive detection of Legionella pneumophila serogroup 1

This study introduces an innovative electrochemical aptasensor designed for the highly sensitive and rapid detection of Legionella pneumophila serogroup 1 (L. pneumophila SG1), a particularly virulent strain associated with Legionellosis. Employing a rigorous selection process utilizing cell-based systematic evolution of ligands by exponential enrichment (cell-SELEX), we identified new high-affinity aptamers specifically tailored for L. pneumophila SG1. The selection process encompassed ten rounds of cell-SELEX cycles with live L. pneumophila, including multiple counter-selection steps against the closely related Legionella sub-species. The dissociation constant (Kd) of the highest affinity sequence to L. pneumophila SG1 was measured at 14.2 nM, representing a ten-fold increase in affinity in comparison with the previously reported aptamers. For the development of electrochemical aptasensor, a gold electrode was modified with the selected aptamer through the formation of self-assembled monolayers (SAMs). The newly developed aptasensor exhibited exceptional sensitivity, and specificity in detecting and differentiating various Legionella sp., with a detection limit of 5 colony forming units (CFU)/mL and an insignificant/negligible cross-reactivity with closely related sub-species. Furthermore, the aptasensor effectively detected L. pneumophila SG1 in spiked water samples, demonstrating an appreciable recovery percentage. This study shows the potential of our aptamer-based electrochemical biosensor as a promising approach for detecting L. pneumophila SG1 in diverse environments.

Expect the unexpected: endocarditis caused by Legionella feeleii.

We report a fatal case of Legionella feeleii endocarditis in a post-lung transplant patient. The diagnosis was delayed, as routine microbiological testing of nonrespiratory specimens does not account for extrapulmonary Legionella, and urine antigen testing only reliably detects Legionella pneumophila serogroup 1. This case also illustrates the utility of molecular sequencing for blood culture-negative endocarditis.

Legionnaires’ disease Surveillance in Italy

In the report presented, data on legionellosis diagnosed in the year 2003 in Italy and notified to the National Surveillance System are analysed. Overall, 617 cases were notified, of which 517 were confirmed and 46 were presumptive. The characteristics of the patients are very similar to those reported in the previous years in terms of male/female ratio, age–specific distribution, occupation, etc. Legionella pneumophila serogroup 1 was responsible for approximately 90% of the cases.

Legionnaires’ disease and occupational risk: a case report

We report results of environmental microbiological sampling in an office building whose occupants referred different symptoms ascribable to the air conditioning system. Legionella pneumophila serogroup 1 was detected in water samples drawn from the cooling tower with counts that ranged from 1.4 x105 to 1.5x107 CFU/L. This biological agent is classified in the second risk group according to Italian law (D.Lgs. 626/94), consequently proper control measures in order to re-establish acceptable hygienic environment conditions and to assure healthy workplace are needed.

Legionella pneumophila serogroup 5 infection in the presence of multiple environmental contamination. The importance of a bacteriological diagnosis

Legionella pneumophila is a pathogen that causes severe pneumonia in humans; L. pneumophila serogroup 1 accounts for at least 90% of infections. This is not linked to an environmental predominance of Legionella pneumophila 1, but may be due to a greater virulence of the strain. L. pneumophila sg 5 has also been reported, albeit less frequently, to be a cause of the disease. We report a case of L. pneumophila sg 5 occurring in a large hospital in southern Italy (Apulia region), where both L. pneumophila sg 1 and sg 5 were detected in the water supply; the nosocomial origin was demonstrated by molecular subtyping (PFGE). An environmental investigation, performed immediately after diagnosis of the case of legionellosis, identified a ow L. pneumophila sg 5 contamination level. Our experience highlights that in hospital, risk assessment, in rder to institute control measures for Legionella, should be carried out not only in response to a case of the disease and/or in risk wards only, as described in the Italian Guidelines, but periodically in every ward. The present study confirms that, although in the community L. pneumophila sg 1 is the most frequent strain isolated in both outbreaks and isolated cases, in hospital other serogroups and species may often cause infection because of the high susceptibility of the hosts.

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

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. 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. 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. 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.

Recurrence, Microevolution, and Spatiotemporal Dynamics of Legionella pneumophila Sequence Type 1905, Portugal, 2014-2022.

We investigated molecular evolution and spatiotemporal dynamics of atypical Legionella pneumophila serogroup 1 sequence type 1905 and determined its long-term persistence and linkage to human disease in dispersed locations, far beyond the large 2014 outbreak epicenter in Portugal. Our finding highlights the need for public health interventions to prevent further disease spread.

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.

Fulminant septic shock due to community-acquired pneumonia caused by Legionella pneumophila SG1 Olda OLDA ST1. Case report

Legionellers’ desease accounts for 1–8 % of cases of severe community-acquired pneumonia (CAP). Legionella spp. Is the causative organism that can result in respiratory failure, multi-organ dysfunction, sepsis, and death. Therefore, rapid diagnosis and efficient treatment are crucial. We report the clinical and microbiology study of a patient with community-acquired pneumonia caused by Legionella pneumophila, with fatal outcome. After death, the strain causing the infection was identified as Legionella pneumophila serogroup 1, Olda OLDA phenotype and sequence-type 1. This is the first reported case of septic shock and death associated with an isolate of these characteristics.

CRP under 130 mg/L rules out the diagnosis of Legionella pneumophila serogroup 1 (URINELLA Study)

IntroductionIn case of pneumonia, some biological findings are suggestive for Legionnaire’s disease (LD) including C-reactive protein (CRP). A low level of CRP is predictive for negative Legionella Urinary-Antigen-Test (L-UAT).MethodObservational retrospective study in Nord-Franche‐Comté Hospital with external validation in Besançon University Hospital, France which included all adults with L-UAT performed during January 2018 to December 2022. The objective was to determine CRP optimal threshold to predict a L-UAT negative result.ResultsURINELLA included 5051 patients (83 with positive L-UAT). CRP optimal threshold was 131.9 mg/L, with a negative predictive value (NPV) at 100%, sensitivity at 100% and specificity at 58.0%. The AUC of the ROC-Curve was at 88.7% (95% CI, 86.3–91.1). External validation in Besançon Hospital patients showed an AUC at 89.8% (95% CI, 85.5–94.1) and NPV, sensitivity and specificity was respectively 99.9%, 97.6% and 59.1% for a CRP threshold at 131.9 mg/L; after exclusion of immunosuppressed patients, index sensitivity and NPV reached also 100%.ConclusionIn case of pneumonia suspicion with a CRP level under 130 mg/L (independently of the severity) L-UAT is useless in immunocompetent patients with a NPV at 100%. We must remain cautious in patients with symptoms onset less than 48 h before CRP dosage.

Identification rate of Legionella species in non-purulent sputum culture is comparable to that in purulent sputum culture in Legionella pneumonia.

Many Legionella pneumonia patients do not produce sputum, and it is unknown whether purulent sputum is required for the identification of Legionella species. This study aimed to evaluate the identification rate of Legionella species based on sputum quality and the factors predictive of Legionella infection. This study included Legionella pneumonia patients at Kurashiki Central Hospital from November 2000 to December 2022. Sputum quality, based on gram staining, was classified as the following: Geckler 1/2, 3/6 and 4/5. Geckler 4/5 was defined as purulent sputum. The sputa of 104 of 124 Legionella pneumonia patients were cultured. Fifty-four patients (51.9%) were identified with Legionella species, most of which were Legionella pneumophila serogroup 1 (81.5%). The identification rates of Legionella species according to sputum quality were 57.1% (16/28) in Geckler 1/2 sputum, 50.0% (34/68) in Geckler 3/6 sputum, and 50.0% (4/8) in Geckler 4/5 sputum, which were not significantly different (P = 0.86). On multivariate analysis, pre-culture treatment with anti-Legionella antimicrobials (odds ratio [OR] 0.26, 95% confidence interval [CI] 0.06-0.91), Pneumonia Severity Index class ≥IV (OR 2.57 [95% CI 1.02-6.71]), and intensive care unit admission (OR 3.08, 95% CI 1.06-10.09) correlated with the ability to identify Legionella species, but sputum quality did not (OR 0.88, 95% CI 0.17-4.41). The identification rate of Legionella species in non-purulent sputum was similar to that in purulent sputum. For the diagnosis of Legionella pneumonia, sputum should be collected before administering anti-Legionella antibiotics and cultured regardless of sputum quality.

Inactivation efficacy and mechanisms of wavelength-specific UV sources for various strains of Legionella pneumophila serogroup 1

Infections of Legionnaires' disease in the United States caused by Legionella have increased ninefold between the years 2000–2018. Legionella harbored in biofilms or inside amoeba within premise plumbing can be more resistant to disinfectants, thus causing treatment challenges. Ultraviolet-light emitting diodes (UV-LEDs) are an emerging water disinfection technology with several advantages over conventional UV lamps. In this study, we evaluated the effects of UV-LEDs (255, 265, and 285 nm), a low-pressure (LP) mercury UV lamp (254 nm), and a bandpass filtered medium-pressure (MP) mercury UV lamp (220 nm) on properties and inactivation of three strains of L. pneumophila serogroup 1. The UV-LEDs emitting at 255 and 265 nm showed greater inactivation performance against all the strains compared to the UV-LED at 285 nm and the LP UV lamp at 254 nm. Our results showed that strains of the same serogroup exhibited different UV sensitivities. Analyses of DNA and protein damage revealed that UV exposure using 254, 255, and 265 nm predominantly causes DNA damage, while protein damage is predominant at 220 nm. Both DNA and protein damage were observed at 285 nm, but the extent of DNA damage was relatively less significant compared to the other wavelengths. Electric energy consumption analysis showed that water treatment using UV-LEDs is currently unsatisfactory compared to conventional LP UV lamps due to the mediocre wall plug efficiency (WPE) of UV-LEDs. However, recent studies indicate that the WPE of UV-LEDs is continuously improving. Overall, our study highlights that UV-LEDs are a promising technology for inactivating waterborne pathogens and have the potential to replace existing UV mercury lamps for water disinfection applications.

Cell-based SELEX aptamer selection for electrochemical detection of Fluoribacter bozemanae bacteria

Fluoriobacter bozemanae (F. bozemanae) can cause Legionnaires' disease. The conventional methods for the detection of F. bozemanae are laborious, costly and has to be performed in centralized labs by skilled personnel. Therefore, there is an urgent need for rapid detection methods for F. bozemanae. The present work reports the first electrochemical aptasensor for the rapid and specific detection of the most virulent type of Legionellosis. Firstly, high-affinity aptamer was selected against the F. bozemanae using cell-based systematic evolution of ligands by exponential enrichment (Cell SELEX). The aptamers selections were based on 10 rounds of cell-SELEX with live F. bozemanae cells, and the counter-selection was performed against five other closely related Legionella sub-species. The selected oligonucleotide sequences were characterized using the dissociation constant (Kd). Apt-FB6 sequence gave a Kd value as low as 9.516 nM. This selected aptamer was integrated with electrochemical impedance spectroscopy (EIS). The impedimetric aptasensor for F. bozemanae exhibited a detection limit down to 10 CFU/mL. Finally, the aptasensor was tested against Legionella anisa, Legionella micdadei, Fluoriobacter dumoffii, and Legionella pneumophila serogroup 1 and 3. The biosensor didn't show significant cross-reactivity with the closely-related sub-species of Legionella. The selected aptasensor can be further exploited for further biosensing applications for clinical, environmental, and diagnostic purposes.

Lipopolysaccharide of Legionella pneumophila Serogroup 1 Facilitates Interaction with Host Cells.

Legionella pneumophila is the primary causative agent of Legionnaires' disease. The mutant-type strain interrupted in the ORF7 gene region responsible for the lipopolysaccharide biosynthesis of the L. pneumophila strain Heysham-1, lacking the O-acetyl groups attached to the rhamnose of the core part, showed a higher surface polarity compared with the wild-type strain. The measurement of excitation energy transfer between fluorophores located on the surface of bacteria and eukaryotic cells showed that, at an early stage of interaction with host cells, the mutant exhibited weaker interactions with Acanthamoeba castellanii cells and THP-1-derived macrophages. The mutant displayed reduced adherence to macrophages but enhanced adherence to A. castellanii, suggesting that the O-acetyl group of the LPS core region plays a crucial role in facilitating interaction with macrophages. The lack of core rhamnose O-acetyl groups made it easier for the bacteria to multiply in amoebae and macrophages. The mutant induced TNF-α production more strongly compared with the wild-type strain. The mutant synthesized twice as many ceramides Cer(t34:0) and Cer(t38:0) than the wild-type strain. The study showed that the internal sugars of the LPS core region of L. pneumophila sg 1 can interact with eukaryotic cell surface receptors and mediate in contacting and attaching bacteria to host cells as well as modulating the immune response to infection.

A multi-center validation study on the discrimination of Legionella pneumophila sg.1, Legionella pneumophila sg. 2-15 and Legionella non-pneumophila isolates from water by FT-IR spectroscopy.

This study developed and validated a method, based on the coupling of Fourier-transform infrared spectroscopy (FT-IR) and machine learning, for the automated serotyping of Legionella pneumophila serogroup 1, Legionella pneumophila serogroups 2-15 as well as their successful discrimination from Legionella non-pneumophila. As Legionella presents significant intra- and inter-species heterogeneities, careful data validation strategies were applied to minimize late-stage performance variations of the method across a large microbial population. A total of 244 isolates were analyzed. In details, the method was validated with a multi-centric approach with isolates from Italian thermal and drinking water (n = 82) as well as with samples from German, Italian, French, and British collections (n = 162). Specifically, robustness of the method was verified over the time-span of 1 year with multiple operators and two different FT-IR instruments located in Italy and Germany. Moreover, different production procedures for the solid culture medium (in-house or commercial) and different culture conditions (with and without 2.5% CO2) were tested. The method achieved an overall accuracy of 100, 98.5, and 93.9% on the Italian test set of Legionella, an independent batch of Legionella from multiple European culture collections, and an extra set of rare Legionella non-pneumophila, respectively.

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.

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.

Sensitive immunoassay of Legionella using multivalent conjugates of engineered VHHs.

VHH antibodies or nanobodies, which are antigen-binding domains of heavy chain antibodies from camelid species, have several advantageous characteristics, including compact molecular size, high productibility in bacteria and easy engineering for functional improvement. Focusing on these advantages of VHHs, we attempted to establish an immunoassay system for detection of Legionella, the causative pathogen of Legionnaires' disease. A VHH phage display library was constructed using cDNA from B cells of alpacas immunized with Legionella pneumophila serogroup1 (LpSG1). Through biopanning, two specific VHH clones were isolated and used to construct a Legionella detection system based on the latex agglutination assay. After engineering the VHHs and improving the assay system, the sensitive detection system was successfully established for the LpSG1 antigen. The immunoassay developed in this study should be useful in easy and sensitive detection of Legionella, the causative agent of Legionnaires' disease, which is a potentially fatal pneumonia.

Two Cases of Legionnaires’ disease following exposure to a hot tub at a private residence – Ontario, 2021

On September 10, 2021, the Haliburton, Kawartha, Pine Ridge District Health Unit in Ontario, Canada, received a case report of Legionella pneumophila serogroup 1 in a patient (case 1) hospitalized for pneumonia. A second case (case 2) within the same household was also identified. Case 1 was laboratory diagnosed by urine antigen testing, whereas case 2 was diagnosed by PCR and culture from a respiratory specimen collected from case 2, both at Public Health Ontario Laboratory, Ontario, Canada. Cases were linked by household and both cases were exposed via a newly purchased but previously used hot tub. Samples from the hot tub were culture positive when tested at PHO’s Laboratory. Given a respiratory culture isolate was available, together with an environmental isolate from the hot tub, sequence based typing was undertaken. Having both a clinical and an environmental culture isolate is essential for molecular investigations. The sequence type for the clinical isolate was the same as the environmental isolate, providing additional evidence that the hot tub was the source of infection for both cases.

Validation of MICA Legionella for Enumeration of Legionella pneumophila in Sanitary Waters and Cooling Tower Waters: AOAC Performance Tested MethodSM 032201.

Frequent testing for Legionella concentration in water is required by most health risk monitoring organizations worldwide. Domestic hot water and cooling tower water networks must be regularly controlled to prevent Legionnaires' disease, a potentially deadly lung infection. MICA Legionella is the fastest culture-based detection method for all serogroups of Legionella pneumophila, with automatic enumeration in 48 h and no need for confirmation. This study compares the performance and robustness of MICA Legionella with the reference method ISO 11731:2017 for the enumeration of culturable L. pneumophila. MICA Legionella and ISO 11731:2017 results were compared for domestic hot water and cooling tower water. Inclusivity and exclusivity were tested on reference and environmental strains. Ruggedness, lot-to-lot consistency, and stability of the reagents kit were also studied. Enumeration of L. pneumophila by MICA Legionella was statistically equivalent to ISO 11731:2017 in both matrixes. In cooling tower waters, MICA Legionella showed better sensitivity than ISO 11731:2017. It presented a 94% sensitivity and a 97% specificity. MICA Legionella is a highly sensitive and specific method for culturable L. pneumophila enumeration. It presents, in 48 hours, equivalent or better results than ISO 11731:2017. Its protocol is robust to variations. Its reagents kit is stable for up to 18 months. MICA Legionella is a robust and reliable method for the enumeration of culturable L. pneumophila in domestic and cooling tower water. It reduces significantly the number of sample pretreatments required in ISO 11731:2017. Automatic identification and enumeration of L. pneumophila microcolonies eliminates the requirement to have skilled analysts and limits the results variability. It also greatly reduces the time to results to 48 h instead of 7-10 days with ISO 11731:2017 while providing statistically equivalent results.