The inhibitory effect of copper, zinc, and manganese on Legionella longbeachae and other Legionella spp. in vitro.

Legionella longbeachae is an important cause of Legionnaires' disease in Australasia and is associated with exposure to potting soils. Our aim was to identify ways to reduce the load of L. longbeachae in potting soils. Inductively-coupled plasma optical emission spectrometry (ICP-OES) of an all-purpose potting mix showed copper (Cu) concentrations (mg/kg) range from 15.8 to 23.6. Zinc (Zn) and manganese (Mn) were significantly higher than Cu ranging from 88.6-106 to 171-203, respectively. Minimal inhibitory and bactericidal concentrations of 10 salts used in the horticultural industry were determined for Legionella species in buffered yeast extract (BYE) broth. For L. longbeachae (n = 9) the median (range) minimum inhibitory concentration (MIC) (mg/L) of copper sulfate was 31.25 (15.6-31.25), zinc sulfate 31.25 (7.81-31.25), and manganese sulfate 31.25 (7.81-62.5). The MIC and minimum bactericidal concentration (MBC) were within one dilution of each other. Susceptibility to Cu and Zn salts increased as the concentration of pyrophosphate iron in the media decreased. The MIC values for these three metals against Legionella pneumophila (n = 3) and Legionella micdadei (n = 4) were similar. Combinations of Cu, Zn, and Mn were additive. Legionella longbeachae has similar susceptibility to Cu and other metal ions in comparison to L. pneumophila.

Legionella longbeachae wound infection: case report and review of reported Legionella wound infections.

Extrapulmonary manifestations of infection with Legionella species, of which 24 may cause disease in humans, are very rare. Here, we describe a case of a 61-year-old woman with no history of immunosuppression presenting with pain and swelling of her index finger after a prick by rose thorns during gardening. Clinical examination showed fusiform swelling of the finger with mild redness, warmth, and fever. The blood sample revealed a normal white blood cell count and a slight increase in C-reactive protein. Intraoperative observation showed extensive infectious destruction of the tendon sheath, while the flexor tendons were spared. Conventional cultures were negative, while 16S rRNA PCR analysis identified Legionella longbeachae that also could be isolated on buffered charcoal yeast extract media. The patient was treated with oral levofloxacin for 13 days, and the infection healed quickly. The present case report, with a review of the literature, indicates that Legionella species wound infections may be underdiagnosed due to the requirement for specific media and diagnostic methods. It emphasizes the need for heightened awareness of these infections during history taking and clinical examination of patients presenting with cutaneous infections.

Legionella longbeachae effector protein RavZ inhibits autophagy and regulates phagosome ubiquitination during infection.

Legionella organisms are ubiquitous environmental bacteria that are responsible for human Legionnaires' disease, a fatal form of severe pneumonia. These bacteria replicate intracellularly in a wide spectrum of host cells within a distinct compartment termed the Legionella-containing vacuole (LCV). Effector proteins translocated by the Dot/Icm apparatus extensively modulate host cellular functions to aid in the biogenesis of the LCV and intracellular proliferation. RavZ is an L. pneumophila effector that functions as a cysteine protease to hydrolyze lipidated LC3, thereby compromising the host autophagic response to bacterial infection. In this study, we characterized the RavZ (RavZLP) ortholog in L. longbeachae (RavZLLO), the second leading cause of Legionella infections in the world. RavZLLO and RavZLP share approximately 60% sequence identity and a conserved His-Asp-Cys catalytic triad. RavZLLO is recognized by the Dot/Icm systems of both L. pneumophila and L. longbeachae. Upon translocation into the host, it suppresses autophagy signaling in cells challenged with both species, indicating the functional redundancy of RavZLLO and RavZLP. Additionally, ectopic expression of RavZLLO but not RavZLP in mammalian cells reduces the levels of cellular polyubiquitinated and polyneddylated proteins. Consistent with this process, RavZLLO regulates the accumulation of polyubiquitinated species on the LCV during L. longbeachae infection.

Extensive epigenetic modification with large-scale chromosomal and plasmid recombination characterise the Legionella longbeachae serogroup 1 genome.

Legionella longbeachae is an environmental bacterium that is the most clinically significant Legionella species in New Zealand (NZ), causing around two-thirds of all notified cases of Legionnaires' disease. Here we report the sequencing and analysis of the geo-temporal genetic diversity of 54 L. longbeachae serogroup 1 (sg1) clinical isolates, derived from cases from around NZ over a 22-year period, including one complete genome and its associated methylome. The 54 sg1 isolates belonged to two main clades that last shared a common ancestor between 95 BCE and 1694 CE. There was diversity at the genome-structural level, with large-scale arrangements occurring in some regions of the chromosome and evidence of extensive chromosomal and plasmid recombination. This includes the presence of plasmids derived from recombination and horizontal gene transfer between various Legionella species, indicating there has been both intra- and inter-species gene flow. However, because similar plasmids were found among isolates within each clade, plasmid recombination events may pre-empt the emergence of new L. longbeachae strains. Our complete NZ reference genome consisted of a 4.1 Mb chromosome and a 108 kb plasmid. The genome was highly methylated with two known epigenetic modifications, m4C and m6A, occurring in particular sequence motifs within the genome.

Diagnosis and treatment for a case of Legionella longbeachae severe pneumonia and literature review. / 1ä¾‹é•¿æ»©å†›å›¢èŒæ€§é‡ç—‡è‚ºç‚Žè¯Šæ²»åˆ†æžåŠæ–‡çŒ®å¤ä¹ .

As a type of Legionella bacteria, Legionella longbeachae bacteria can lead to very rare legionella disease case in China with clinical characteristics, such as no typical early clinical symptoms, strong toxicity, high mortality, and not easy to detect by conventional etiology. A case of severe pneumonia caused by Legionella longbeachae infection was confirmed by bronchoalveolar lavage fluid pathogen metagenomics, and the patient's condition was improved after targeted anti-infection treatment. At present, our understanding in Legionella longbeachae severe pneumonia is limited. The diagnosis and treatment process of the patient with severe pneumonia of Legionella longbeachaeis retrospectively analyzed and the relevant literature was reviewed to provide the experience for its future diagnosis and treatment.

How safe are gloves and masks used for protection against Legionella longbeachae infection when gardening?

Legionella longbeachae has been frequently identified in composted plant material and can cause Legionnaires' disease (LD). We wanted to determine how frequently L. longbeachae DNA was present on gardeners' gloves, and how long L. longbeachae could persist on inoculated gloves and masks. Volunteers completed a survey of gardening practices and their gardening gloves were tested for L. longbeachae DNA by qPCR. The persistence of viable L. longbeachae was assessed by timed subcultures after inoculation of gardening gloves and masks. Gloves but not masks were used regularly. L. longbeachae was detected on 11 (14%; 95% CI 8-24%) gloves. Viable organisms were recovered from 25-50% of inoculated cotton, leather and PU coated gloves but not rubber gloves after 8 h incubation. There was a difference in dose-response curve slopes by glove material (P = 0·001) and time to 50% sterility (P = 0·036). There were differences in persistence of L. longbeachae between mask types from analysis of the slopes and 50% sterility on the decay curves (P = 0·042, P < 0·001 respectively). Gardening gloves and masks may act as a vector for transmission of L. longbeachae during gardening. Washing gardening gloves and prompt disposal of masks could reduce risk of LD.

An outbreak investigation of Legionella non-pneumophila Legionnaires' disease in Sweden, April to August 2018: Gardening and use of commercial bagged soil associated with infections.

In early June 2018, an increase in non-travel-related cases of Legionella non-pneumophila Legionnaires' disease (LD) was observed in Sweden and a national outbreak investigation was started. Outbreak cases were defined as notified confirmed or probable cases of L. non-pneumophila LD, with symptom onset after 1 April 2018. From April to August 2018, 41 cases were reported, 30 of whom were identified as L. longbeachae. We conducted a case-control study with 27 cases and 182 matched controls. Results from the case-control study indicated that gardening and handling commercial bagged soil, especially dusty dry soil, were associated with disease. L. longbeachae was isolated in soils from cases' homes or gardens, but joint analysis of soil and human specimens did not identify any genetic clonality. Substantial polyclonality was noted between and within soil samples, which made finding a genetic match between soil and human specimens unlikely. Therefore, whole genome sequencing may be of limited use to confirm a specific soil as a vehicle of transmission for L. longbeachae. Handling soil for residential gardening was associated with disease and the isolation of L. longbeachae in different soils provided further evidence for Legionella non-pneumophila infection from soil.

Legionella longbeachae pneumonia: A case report and literature review in Japan.

Herein, we report the case of a 74-year-old man diagnosed with Legionella pneumonia detected by Loop-Mediated Isothermal Amplification (LAMP) method, which was suspected to have been transmitted from the potting soil. Legionella longbeachae was identified in the sputum culture. The patient was intubated and maintained on mechanical ventilation. Antimicrobial therapy with azithromycin was also administered. His symptoms were resolved and he was discharged after 26 days of hospitalization. Legionella longbeachae pneumonia rarely occurs in Japan, and published literature of Legionella longbeachae pneumonia cases in Japan was reviewed. Patients with severe pneumonia exposed to potting soils, but with negative urinary antigen test results, should be examined by LAMP method.

Epileptic Seizure after Use of Moxifloxacin in Man with Legionella longbeachae Pneumonia.

Legionellosis caused by Legionella longbeachae is diagnosed mainly by PCR. We report a case of L. longbeachae infection in mainland China, which was diagnosed by metagenomic next-generation sequencing, in a man who developed an epileptic seizure after using moxifloxacin. Metagenomic next-generation sequencing may be a useful tool to detect Legionella spp.

Enhancement of Culture of Legionella longbeachae from Respiratory Samples by Use of Immunomagnetic Separation and Antimicrobial Decontamination.

Legionella longbeachae is the commonest Legionella species identified in patients with community-acquired pneumonia in New Zealand. Isolation of the organism on culture is the gold standard for the diagnosis of Legionnaires disease, but it has poor sensitivity (40%) compared with quantitative PCR (qPCR). We have developed a selective decontamination process using glycine, vancomycin, polymyxin, and cycloheximide (GVPC) with immunomagnetic separation (IMS) for culturing L. longbeachae A polyclonal antibody specific for L. longbeachae was produced from New Zealand White rabbits and coupled to tosyl-activated magnetic beads. Stored L. longbeachae qPCR-positive respiratory samples were retrieved from -80°C storage for testing. One portion of test samples was mixed with GVPC and the antibody bead complex, separated, washed, and cultured on modified Wadowsky and Yee agar (MWY) agar. Another portion was exposed to HCl-KCl acidic buffer (pH 2.2) before incubation on MWY agar. qPCR used probes specific for the ITS (internal transcribed spacer) region of the L. longbeachae genome. Cultures were positive in 10/53 (19%) samples after acid wash and 26/53 (49%) after GVPC-IMS (P = 0.001). Growth of contaminants was rare. The mean qPCR threshold cycle values were lower in culture-positive samples after acid wash than in the culture-negative samples (mean, 29.9 versus 34.8; difference, 4.9; 95% confidence interval [CI], ±2.9; P = 0.001) but not after GVPC-IMS (mean, 33.0 versus 34.7; difference, 1.7; 95% CI, ±2.48; P = 0.16). The sensitivity of culture for L. longbeachae in respiratory specimens may be improved by using GVPC-IMS rather than acid wash for decontamination, but this should be confirmed in a prospective study of fresh specimens.

Genetic Manipulation of Non-pneumophila Legionella: Protocols Developed for Legionella longbeachae.

Current biomedical research into Legionnaires' disease is dominated by studies of Legionella pneumophila, largely because this pathogen is responsible for approximately 90% of clinical disease worldwide. However, in certain geographical regions, infections with non-pneumophila species are responsible for a significant proportion of diagnosed Legionnaires' disease. Understanding the pathogenesis of these non-pneumophila species of Legionella is an important step toward clinical intervention. The capacity to genetically manipulate these pathogens is essential in order to understand the genetic factors that contribute to infection and the environmental life cycle of these bacteria. The capacity to delete, mutate, and relocate genetic regions of interest allows molecular research into gene function and importance. In this chapter, methods are outlined to introduce plasmids into Legionella by electroporation. This technique is particularly useful as it is often the essential preliminary step to experiments that observe the behavior of the bacterium under altered conditions, for example, the transformation of bacteria with reporter plasmids to monitor Dot/Icm effector translocation. Electroporation is a well-established method for transformation of competent bacteria, and here specific protocols are provided, suiting a range of materials and conditions that have been successfully applied to L. longbeachae and L. dumoffii. Additionally, a homologous recombination approach to delete genetic regions of interest in L. longbeachae is outlined. The application of these techniques allows for identification of the genetic determinants of non-pneumophila Legionella virulence and for important comparative studies with other Legionella species.

MAIT cells protect against pulmonary Legionella longbeachae infection.

Mucosal associated invariant T (MAIT) cells recognise conserved microbial metabolites from riboflavin synthesis. Striking evolutionary conservation and pulmonary abundance implicate them in antibacterial host defence, yet their functions in protection against clinically important pathogens are unknown. Here we show that mouse Legionella longbeachae infection induces MR1-dependent MAIT cell activation and rapid pulmonary accumulation of MAIT cells associated with immune protection detectable in immunocompetent host animals. MAIT cell protection is more evident in mice lacking CD4+ cells, and adoptive transfer of MAIT cells rescues immunodeficient Rag2-/-γC-/- mice from lethal Legionella infection. Protection is dependent on MR1, IFN-γ and GM-CSF, but not IL-17A, TNF or perforin, and enhanced protection is detected earlier after infection of mice antigen-primed to boost MAIT cell numbers before infection. Our findings define a function for MAIT cells in protection against a major human pathogen and indicate a potential role for vaccination to enhance MAIT cell immunity.

Legionella longbeachae; don't miss it!

We here report on two immunocompetent patients admitted to our hospital within 3 weeks' time, both suffering from pneumonia caused by Legionella longbeachae (L. longbeachae). The pathogen was identified in broncho-alveolar lavage (BAL) liquid by Polymerase Chain Reaction (PCR), whereas sputum cultures remained negative. This organism is worldwide still relatively unknown and consequently underdiagnosed. However, with an increasing number of confirmed infections in Europe and more specifically in the Netherlands, early awareness and diagnostic measurements are indicated. As routine laboratory techniques like the urine antigen test do not detect L. longbeachae, we advocate early use of specific tests for non-pneumophila Legionella species such as PCR. Furthermore, we advocate the start of empirical antibiotic therapy (i.e. ciprofloxacin) and continuation in suspected cases.

In vitro activity of various antibiotics against clinical strains of Legionella species isolated in Japan.

The activities of various antibiotics against 58 clinical isolates of Legionella species were evaluated using two methods, extracellular activity (minimum inhibitory concentration [MIC]) and intracellular activity. Susceptibility testing was performed using BSYEα agar. The minimum extracellular concentration inhibiting intracellular multiplication (MIEC) was determined using a human monocyte-derived cell line, THP-1. The most potent drugs in terms of MICs against clinical isolates were levofloxacin, garenoxacin, and rifampicin with MIC90 values of 0.015 µg/ml. The activities of ciprofloxacin, pazufloxacin, moxifloxacin, clarithromycin, and azithromycin were slightly higher than those of levofloxacin, garenoxacin, and rifampicin with an MIC90 of 0.03-0.06 µg/ml. Minocycline showed the highest activity, with an MIC90 of 1 µg/ml. No resistance against the antibiotics tested was detected. No difference was detected in the MIC distributions of the antibiotics tested between L. pneumophila serogroup 1 and L. pneumophila non-serogroup 1. The MIECs of ciprofloxacin, pazufloxacin, levofloxacin, moxifloxacin, garenoxacin, clarithromycin, and azithromycin were almost the same as their MICs, with MIEC90 values of 0.015-0.06 µg/ml, although the MIEC of minocycline was relatively lower and that of rifampicin was higher than their respective MICs. No difference was detected in the MIEC distributions of the antibiotics tested between L. pneumophila serogroup 1 and L. pneumophila non-serogroup 1. The ratios of MIEC:MIC for rifampicin (8) and pazufloxacin (2) were higher than those for levofloxacin (1), ciprofloxacin (1), moxifloxacin (1), garenoxacin (1), clarithromycin (1), and azithromycin (1). Our study showed that quinolones and macrolides had potent antimicrobial activity against both extracellular and intracellular Legionella species. The present data suggested the possible efficacy of these drugs in treatment of Legionella infections.

Combining Environmental Investigation and a Dual-Analytical Strategy to Isolate the Legionella longbeachae Strain Linked to Two Occupational Cases of Legionellosis.

Legionella has a global distribution, mainly in aquatic and man-made environments. Under the right conditions, this bacterium is a notorious human pathogen responsible for severe pulmonary illnesses. Legionellosis outbreaks are reported around the world, and exposure to water droplet aerosols containing Legionella pneumophila is usually the mechanism of its transmission. Even if L. pneumophila causes most outbreaks, Legionella longbeachae also accounts for some cases. Unlike most other Legionella strains, L. longbeachae is typically found in soil. Given the wide diversity and high concentration of microorganisms found in soil, isolating L. longbeachae by culture can be challenging. Because the chances of successfully isolating the strain are low, it is often not even attempted. This study reports the strategies used to successfully isolate L. longbeachae strain that was responsible of the two occupational legionellosis in Quebec. Fifteen random samples were collected from the soil of the metal recycling plant where the diagnosed workers were employed, covering 1.5% of the accessible surface of the plant. All samples were analyzed with both the quantitative polymerase chain reaction (qPCR) and culture methods. Four qPCR detection systems targeting Legionella spp, L. pneumophila, L. pneumophila serogroup 1, and L. longbeachae were used. Acid, heat, and acid/heat treatments were used for the culture method. For the qPCR method, all samples were positives for Legionella spp but only four were positives for L. longbeachae. For the culture method, only one isolate could be confirmed to be L. longbeachae. However, that strain proves to be the same one that caused the occupational legionellosis. Detecting the presence of L. longbeachae using the qPCR method made it possible to target the right samples to enable the cultivable strain of L. longbeachae to be isolated from the soil of the metal recycling plant. The complementarity of the two methods was established. This paper demonstrated the advantages of selecting the proper sampling and analytical strategies to achieve the isolation of the strain responsible for the infections. It also highlights for the first time in Quebec the potential occupational risks associated with L. longbeachae from soil and should motivate questioning soil exposures when all sources of water contamination have been eliminated from the causal analysis of legionellosis.

[A Case of Severe Legionella longbeachae Pneumonia and Usefulness of LAMP Assay].

Urinary antigen test is frequently used as a routine laboratory test for early diagnosis of Legionella infection, which is especially suitable for ordinary Legionella pneumophila serogroup 1, but not for other types of Legionella. We report a case of severe pneumonia caused by Legionella longbeachae, where a method of loop-mediated isothermal amplification (LAMP) assay contributed an important role for the early detection. This case involved an 83-year-old man who developed fever, dyspnea, and productive cough. Since the medication of prescribed ceftriaxone had not been effective, he visited the emergency room of our hospital, where an X-ray revealed a severe pneumonia harboring a consolidation with air bronchogram in his right lower lung. His sputum and urine were subjected to the routine bacterial culture or the urinary antigen test for Legionella, which initially brought negative results. However, a positive result of LAMP assay enabled early diagnosis of Legionella pneumonia. Later, the bacterial cultures of sputum made some progress and 16S rRNA sequencing provided a proof of L. longbeachae. This LAMP assay may bring a benefit for the patients with Legionella pneumonia by enabling early detection of not only specific L. pneumophila serogroup 1, but also of the other Legionella species.