PMMB 2021, 4, 1; a0000193. doi: a0000193 http://journals.hh-publisher.com/index.php/pmmb Review Article Legionella pneumophila — The causative agent of Legionnaires’ disease Loh Teng Hern Tan1,2†, Wei Yu Tee2†, Tahir Mehmood Khan3,4, Long Chiau Ming5, Vengadesh Letchumanan2* Article History 1Clinical School Johor Bahru, Jeffrey Cheah School of Medicine and Health Sciences, Monash University Malaysia, Johor Bahru 80100, Malaysia; loh.teng.hern@monash.edu (LT-HT) 2Novel Bacteria and Drug Discovery (NBDD) Research Group, Microbiome and Bioresource Research Strength (MBRS), Jeffrey Cheah School of Medicine and Health Sciences, Monash University Malaysia, 47500, Selangor Darul Ehsan, Malaysia; weiyuwy1@gmail.com (W-YT) 3Institute of Pharmaceutical Sciences (IPS), University of Veterinary & Animal Sciences (UVAS), Pakistan; tahir.khan@uvas.edu.pk (TMK) 4School of Pharmacy, Monash University Malaysia, 47500 Bandar Sunway, Selangor Darul Ehsan, Malaysia 5PAP Rashidah Sa’adatul Bolkiah Institute of Health Sciences, Universiti Brunei Darussalam, Gadong, Brunei Darussalam; long.ming@ubd.edu.bn (LCM) *Corresponding author: Vengadesh Letchumanan, vengadesh.letchumanan1@monash.edu Received: 3rd March 2021; Received in Revised Form: 3rd April 2021; Accepted: 5th April 2021; Available Online: 8th April 2021 † These authors contributed equally to the writing. Abstract: Over the years, Legionella pneumophila has increasingly become a public health threat that causes sporadic and epidemic community-acquired and nosocomial-acquired pneumonia. Thus, this review aims to discuss the current knowledge of L. pneumophila, focusing on the global epidemiology, clinical features, diagnosis and treatment of Legionnaires’ disease (LD). Legionella bacteria are Gram-negative rod-shaped bacteria that are ubiquitous in aquatic environments. L. pneumophila was first discovered in 1976 and recognized as the causative agent of LD. L. pneumophila is a facultative intracellular pathogen that infects and replicates within eukaryotic host cells such as macrophages and protozoan. Diagnosis of LD remains a significant challenge as the clinical manifestation of LD is hardly distinguishable from pneumonia caused by other respiratory pathogens. Therefore, early testing and appropriate treatment are keys to alleviating the rising morbidity and mortality caused by LD. Keywords: Legionella; pneumonia; amoeba; Legionnaires’ disease; respiratory disease PMMB 2020, 4, 1; a0000193 2 of 12 1. Introduction Throughout history, human populations have encountered several serious epidemics caused by pathogens[1–3]. Pathogens are various organisms that cause severe illnesses to their hosts, including bacteria, viruses, fungi and protozoa[4–11]. Among the various illnesses, respiratory infections represent the largest category of human disease and the leading cause of mortality globally[12–16]. Apart from the pneumonic plague caused by Yersinia pestis as one of the most significant outbreaks in human history, Legionella is another pathogenic bacteria that were discovered from investigations of an epidemic of a mysterious pneumonia outbreak in Philadelphia in 1976[17]. At the time of writing, there are more than 60 species and serogroups under the genus Legionella, a great variety of them can inflict human disease or legionellosis. Legionellosis is defined as an infection caused by bacteria under the genus Legionella, of which there are two distinct clinical manifestations in humans — Pontiac fever (PF) and Legionnaires’ disease (LD)[18]. Legionella pneumophila serogroup 1 is the clinically most relevant species that cause LD in human, accounting for 90% of the total reported human infections. Meanwhile, L. bozemanii, L. longbeachae, L. dumoffii are the several species also known to cause LD[19]. Primarily, LD is caused by inhalation of aerosols or aspiration of water containing Legionella bacteria. Given the continued increasing incidence of LD globally, Legionella bacteria present a significant threat to human health. There was a 286% increase in legionellosis cases reported in the United States from the year 2000 to 2014[20]. Furthermore, a steady increase of LD cases was also observed in European countries, whereby more than 80% of the infections contributed by L. pneumophila serogroup 1[21]. Therefore, this review aims to update the current knowledge of Legionella bacteria prevalence, clinical manifestations, pathogenesis, detection and treatment strategies (Figure 1). 2. Sources of Legionella bacteria Legionella is a Gram-negative, facultative aerobic and intracellular waterborne bacterium present in natural aquatic environments, such as lakes, rivers and groundwater, but at low concentrations[22]. The widespread distribution of Legionella bacteria is attributed to their ability to proliferate within diverse types of niches, including live in planktonic form, infect and replicate within protozoan hosts as well as coexist within multi-organismal biofilms developed on the surfaces of water systems[23,24]. Given the ubiquitous nature of Legionella bacteria in aquatic habitats, Legionella bacteria can easily enter and proliferate in man-manufactured water systems, including cooling towers and water distribution system of large buildings[25]. A relatively high prevalence of Legionella bacteria has also been found in public facilities such as hospitals, showers, hotels and hot water systems[26–29]. Several factors that enhance the colonization of Legionella bacteria in the water systems include lukewarm water temperature, obstruction and stagnation of water flow, plumbing materials, biofilm formation and presence of amoeba, which support the growth of Legionella sp. These risk factors are commonly present in the water distribution system of antiquated buildings, PMMB 2020, 4, 1; a0000193 3 of 12 especially in old hospitals[30]. In fact, half of the sporadic cases of hospital-acquired pneumonia are attributed to Legionella bacteria, where patients are infected via inhalation of Legionella sp. contaminated aerosols from medical devices like respiratory equipment, misting devices, cooling towers and hot tubs[31]. Figure 1. The transmission sources, life cycle within water systems and human macrophage, the diagnostic tools and antibiotic treatment for Legionella pneumonia. Besides living in the planktonic phase, L. pneumophila utilizes amoebae as a host for survival and replication. Currently, there have been more than 40 species of Legionella sp. identified to possess the ability to parasitize amoebae, specifically Acanthamoeba and Naegleria[32]. Thus, these protozoans act as a vector of various pathogenic microorganisms[33], including Legionella, and facilitates pathogen dissemination. Being an intracellular pathogen, Legionella sp. also can infect and thrive within human macrophage [34]. A recent study demonstrated that the infection processes and the intracellular life cycle of Legionella within amoeba are remarkably similar to that occurred within human macrophages[35], although both are evolutionary distant hosts. Researchers believe that the L. pneumophila is primed for subsequent infection in the protist host and becoming more virulent and infectious after escaping from its environmental host, resulting in a more robust disease in humans[35,36]. In fact, a previous study indicated that intracellularly pre-grown L. pneumophila acquired higher resistance to disinfectants and antimicrobials, suggesting to confer increased infectivity to mammalian cells[37]. 3. Epidemiology of Legionnaires’ disease Legionella pneumophila is a significant bacterial pathogen that causes severe sporadic and epidemic community-acquired and nosocomial acquired pneumonia[38]. An PMMB 2020, 4, 1; a0000193 4 of 12 estimate of 12 to 15% of overall mortality rate among hospitalized patients[39], the number may go up further to 27% for those who failed to receive adequate antibiotic treatment. Furthermore, LD is also commonly associated with poorer outcomes with the increasing length of hospitalization[40]. Dated back to July 1976 in Philadelphia, the first-ever reported LD outbreak due to L. pneumophila resulted in 29 deaths out of the total 180 persons attending the 56th annual American Legion Convention[41]. Over the years, the burden of LD is increasing each year in the United States and European countries. In the United States, there was an incidence of 1.89 cases of LD per 100,000 populations in 2015. Similarly, 1.4 cases of LD per 100,000 populations were reported by the European Centre for Disease Prevention and Control in 2016. Recently, a study in Italy reported an incidence rate of 48.9 cases per 100,000 populations for legionellosis in 2018, of which 3.4% was of nosocomially acquired cases[42]. In Hong Kong, the incidence of LD was increasing from 0.16 cases/100,000 population in 2005 to 0.91 cases/100,000 population in 2015[43]. Among all the Legionella sp. identified to date, L. pneumophila has responsible for 80–90% of the LD cases reported in the United States and Europe. Within the similar Legionella species, L. pneumophila serogroup 1 accounts for approximately 90% of cases. Generally, the sources of outbreaks for nosocomial acquired LD are attributed to the contaminated water in the hospital plumbing system and the exposure to contaminated cooling towers. From 2006 to 2017, cooling waters, air conditions and evaporative condensers were reported to have contributed a major portion (60%) of outbreak-associated deaths due to LD or PF. Moreover, building water systems contributed to approximately 13% and 17% of the outbreak-related cases and deaths, respectively[44]. Besides that, The European Working Group for Legionella Infections and the United States Centers for Disease Control and Prevention (CDC) have identified numerous cases of travel-associated LD where the most common source is the contaminated water in hotels. Travel-associated LD has also been linked to cruise ships[45]. The mode of transmission of LD to human is mainly by the inhalation of contaminated aerosols droplets where the source can disseminate water droplets such as the cooling towers. Due to the small sizes of water droplets containing the Legionella sp., which can be deeply inhaled into the respiratory airways, it has increased the ease of transmission of LD. Besides that, there is limited evidence to indicate the person-to-person transmission of LD. Not until recently, the first strong evidence of person-to-person transmission of LD was reported in Vila Franca de Xira, Portugal, in 2014[46]. The host-related risk factors for LD infection may be associated with factors such as old age, cigarette smoking, organ transplantation, the use of immunosuppressive medications, obstructive pulmonary disease as well as patients with acquired immune deficiency syndrome. Moreover, the elevated risk of nosocomial infection may attribute to general anaesthesia and endotracheal intubation. Besides that, there are reports of infection in children and premature neonates[45]. In addition, those workers who maintain the water PMMB 2020, 4, 1; a0000193 5 of 12 cooling towers and air-conditioning system are the most at-risk group to be infected with LD where the circulated air that contaminated with water droplets containing Legionella sp. can be inhaled easily into the respiratory system[45,47]. 4. Pathogenesis of Legionella pneumophila Predominantly, human exposure occurs when L. pneumophila are inhaled into the lungs through inhalation of contaminated aerosols which results in pneumonic respiratory disease. A combination of bacterial virulence factors and host immunity determines the outcome of Legionella infection, whether leading to a self-limiting and mild respiratory disease (PF) or severe pneumonia (LD)[48]. Once L. pneumophila gets into the lung, the bacteria are phagocytosed by the alveolar macrophages, where they can multiply intracellularly. This is due to the ineffective killing of L. pneumophila, which has been demonstrated through several in vitro models of infection by specific antibody and neutrophils[49]. Meanwhile, the pathogenesis of PF may involve a host response to endotoxin from the lipopolysaccharide of Legionella without replication of the bacteria in the host[50]. The infection process of L. pneumophila on protozoa and mammalian phagocytic cells are shown to be similar where Legionella uses common gene and gene products to infect mammalian and protozoan cells[35]. The uptake of L. pneumophila by monocytes and macrophages is by the conventional and coiling phagocytosis which is an important feature in the pathogenesis of the bacteria[51]. After internalization of the pathogen by phagocytosis, L. pneumophila evades endocytic degradation, controls the innate immune response, and triggers the Legionella-containing vacuole (LCV) biogenesis. The LCV biogenesis involves the recruitment of the rough endoplasmic reticulum and mitochondria result in formation of a specialized vacuole for the intracellular replication of L. pneumophila. This mechanism is directed by the type IV secretion system encoded by the Dot/Icm genes. The Dot/Icm type IV secretion system is the main virulence system of L. pneumophila which consists of bacterial proteins that promote the secretion of bacterial virulence factors suggested to be responsible for inhibition of phagosome-lyzosome fusion[52]. Hence, L. pneumophila can multiply intracellularly in human macrophages by avoiding phagosome-lyzosome fusion[53]. As Legionella becomes vigorously motile in the massive intracellular vesicle, it causes the host cell to become overwhelmed by the infection resulting in lysis of the host cell. This has led to the death of the host cell macrophage and the release of the progeny of the pathogen from the cell to the environment where new host cells are found. 5. Detection and diagnosis of Legionella pneumonia Today, various diagnostic techniques are available for Legionella pneumonia, including urinary antigen tests, respiratory specimen cultures, molecular detection tests, and serum antibody tests[54]. Despite being the diagnostic gold standard, culture of Legionella bacteria isolated from the clinical respiratory sample is limited by the requirement of a specific culture medium[55] and long laboratory turnaround time, which requires at least 3 to 5 days to identify culture positivity[56]. PMMB 2020, 4, 1; a0000193 6 of 12 Serological test for antibodies against Legionella bacteria was the principal diagnostic tool for LD in the early 1980s. However, serological test has been replaced by more rapid and definitive analyses in the modern laboratory, such as the urinary antigen test (UAT) and molecular methods. The use of serology was reported to have declined from 61% to 6% from 1995 to 2010[57], due to the widespread adoption of the less technically demanding UAT. The UAT has been widely utilized globally due to its straight-forward procedure and rapid turnaround time. In Europe and the United States, UAT represents more than 80% to 90% of the diagnostic tools used for LD confirmation. The specific urinary antigens of Legionella can be detected in most of the L. pneumophila infections in the first few days of clinical symptom onset and up to several days to more than ten months. Typically, the specific urinary Legionella antigen is no longer detected in most cases after 1 to 2 months of antibiotic therapy. However, the current commercially available UAT for non-L. pneumophila serogroup 1 exhibit lower sensitivity and highly variable than those for L. pneumophila associated disease when tested with urine from confirmed LD patients[58,59]. Remarkably, a recent study demonstrated a novel urinary antigen test kit (LAC-116) that can detect non-L. pneumophila serogroup 1 pneumonia on top of showing comparable accuracy with the other existing kits for Legionella pneumonia[54]. With the advances of molecular biology and the development of new technologies, the field of diagnostic and detection of pathogens has been improved tremendously, with many new molecular methods being developed to generate rapid, sensitive and accurate results[60]. Currently, real-time PCR is regarded as the molecular method of choice for the detection of Legionella bacteria. Targeting the macrophage inhibitor protein mip gene, highly conserved in all L. pneumophila isolates, has been widely employed by numerous published studies, showing a 15% increased yield in detecting any L. pneumophila serogroup than that of culture method[61]. Some studies performed PCR targeting 16S rDNA gene of Legionella sp. which was suggested to offer greater sensitivity. However, the CAPNETZ study demonstrated a low detection rate of legionellosis (10%) with Legionella sp. PCR targeting 16S rDNA and only 60% of these samples were confirmed by DNA sequencing. Although Legionella nucleic acid-based detection methods offer higher sensitivity and rapid turnaround time, there are several notable disadvantages and limitations. For instance, non- lower respiratory tract samples, such as urine and serum, are not suitable for the PCR test, and the inherent limitation of PCR which cannot assess bacterial viability. Furthermore, nucleic acid amplification technologies also require highly trained personnel to handle sophisticated equipment to perform the diagnostic test[62] but are increasingly accessible to many laboratories on a moderate budget[61]. Nevertheless, nucleic acid-based methods are valuable additions to LD diagnostic and detection methods and complement well with the use of other testing modalities to enhance the rate of successful detection[61]. In the United States, a clinical case of Legionella pneumonia is considered when laboratory results show positive UAT, bacterial culture and serology test for L. pneumophila serogroup 1. Meanwhile, a suspected or probable case is considered when Legionella PMMB 2020, 4, 1; a0000193 7 of 12 antigens in respiratory specimens are detected by serology test, detection of seroconversion to non-serogroup 1 or non-L. pneumophila, and/or detection of Legionella nucleic acid[63]. 6. Clinical manifestation and management of Legionella pneumonia Legionellosis presents two distinct clinical manifestations in humans: milder respiratory disease PF and severe pneumonia LD[64]. PF manifests as a milder form of LD that involves respiratory illness without pneumonia, which resembles the self-limiting flu- like illness with symptoms lasting for approximately a week. Meanwhile, LD is a more severe form of pneumonia that could also affect other organs, including the liver and kidneys[65,66]. Typically, LD is a severe pneumonia that often occurs in susceptible persons, including old ages, smokers and those with comorbid conditions or immunosuppression[67]. LD does not present any specific or defining clinical features but shows a range of clinical manifestations and symptoms. LD basically begins with a headache, muscle pain and general feeling of sickness. Then, these typical symptoms are followed by severe high fever, dry cough, shaking chills, nausea, vomiting and diarrhoea. The infected person also might experience chest pain and difficulty in breathing. In some rare incidences, a persistent or relapsing LD may develop into slow or non- resolving pneumonia which is characterized by the persistence of pulmonary infiltrates for more than 30 days since the disease onset[68]. Majority of the previously reported cases occurred in immunocompromised patients as a result of intracellular survival of Legionella pathogens due to ineffective antibiotic treatment[69,70] rather than de novo reinfection via exposure to contaminated sources. These slowly or non-resolving LD patients experience severe pulmonary complications, including pleural effusion, abscess formation and empyema, over the course of the infection[71]. Considering the high mortality and morbidity associated with untreated LD, early diagnosis and prompt treatment with effective antibiotics are extremely crucial, together with appropriate clinical management of complications and underlying comorbidities should be prioritized for LD patients[72]. Given that LD does not present any defining clinical features, empiric antibiotic therapy is well justifiable for the initial management of all moderate-to- severe community- and nosocomial-acquired pneumonia prior to LD confirmation made from microbiological diagnosis. Primarily, therapy for LD is the antibiotic treatment of the infection and management of complications. In general, all Legionella sp. tested are found to be susceptible to commonly prescribed macrolides (erythromycin, azithromycin) and fluoroquinolones (levofloxacin)[73]. Antibiotics, such as macrolides and fluoroquinolones, are also known to achieve therapeutic intracellular concentration within tissue and alveolar macrophages where L. pneumophila resides. Since 1976, erythromycin had been the treatment of choice for L. pneumonia but only until 1990s, because of its side effects when given intravenously. To date, azithromycin and levofloxacin have become the mainstay LD treatment in both healthy and immunocompromised patients[18]. A recent meta-analysis revealed that levofloxacin PMMB 2020, 4, 1; a0000193 8 of 12 resulted in a significant reduction in the length of hospitalization, lowered mortality, shorter time to apyrexia, and reduced risks of adverse effects compared to macrolides[74]. World Health Organization (WHO) has developed a framework that can be used to assess and manage the risks posed by Legionella[31]. The framework includes health-based targets, water safety plans and surveillance. In addition to the framework proposed by WHO, many approaches of disinfection have been tried over the past 13 years with variable success. Superheating of water up to 70°C and 80°C, hyperchlorination[75] and copper-silver oxidation method were also implemented to halt the LD outbreak. 7. Conclusion Legionella pathogen is one of the top causative agents of severe respiratory disease, which requires swift treatment and rapid diagnosis to achieve successful disease resolution with appropriate antibiotic therapy. Legionella pathogen replicates intracellularly in the host cells, especially amoeba and macrophages. The process of invasion, development and replication of Legionella sp. in the environment has been well studied. LD occurs as outbreaks or as sporadic cases in the community and hospital. The transmission of this disease is through inhalation of contaminated aerosols or aspiration of contaminated water. Furthermore, the diagnosis and treatment of LD have changed over the years, where the urinary antigen detection test and molecular methods are commonly used nowadays. Early testing and appropriate treatment are keys to mortality and morbidity caused by LD. Ultimately, greater effort is required from all facets of Legionella research to facilitate better disease surveillance, improve disease diagnosis and detection, and improve clinical outcomes for LD patients. Author Contributions: LT-HT and W-YT performed literature search and manuscript writing. TMK, LCM and VL provided critical review and proofreading of this manuscript. LT-HT and VL conceptualize this review writing project. Funding: The SEED Funding funded this work from Microbiome and Bioresource Research Strength (MBRS), Jeffrey Cheah School of Medicine and Health Sciences, (Vote Number: MBRS/JCSMHS/02/2020) awarded to VL. 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