Sit bath systems: A new source of Legionella infection.

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

Cutaneous Legionella infections in allogeneic hematopoietic cell transplantation recipients.

To date, only twenty cases of cutaneous legionellosis have been reported. Cutaneous legionellosis has heterogeneous manifestations including abscesses, nodules, and cellulitis. The detection of most cutaneous Legionella species requires specific diagnostic cultures and assays. Herein, we report a case of cutaneous legionella in a hematopoietic cell transplantation recipient with culture-negative nodules unresponsive to empiric antibiotics. We also discuss the varied morphology of cutaneous legionellosis and important diagnostic considerations.

Emerging respiratory infections: The infectious disease pathology of SARS, MERS, pandemic influenza, and Legionella.

Lower respiratory infections remain one of the top global causes of death and the emergence of new diseases continues to be a concern. In the first two decades of the 21st century, we have born witness to the emergence of newly recognized coronaviruses that have rapidly spread around the globe, including severe acute respiratory syndrome virus (SARS) and Middle Eastern respiratory syndrome virus (MERS). We have also experienced the emergence of a novel H1N1 pandemic influenza strain in 2009 that caused substantial morbidity and mortality around the world and has transitioned into a seasonal strain. Although we perhaps most frequently think of viruses when discussing emerging respiratory infections, bacteria have not been left out of the mix, as we have witnessed an increase in the number of infections from Legionella spp. since the organisms' initial discovery in 1976. Here, we explore the basic epidemiology, clinical presentation, histopathology, and clinical laboratory diagnosis of these four pathogens and emphasize themes in humans' evolving relationship with our natural environment that have contributed to the infectious burden. Histology alone is rarely diagnostic for these infections, but has been crucial to bettering our understanding of these diseases. Together, we rely on the diagnostic acumen of pathologists to identify the clinicopathologic features that raise the suspicion of these diseases and lead to the early control of the spread in our populations.

Bacterial secretion system skews the fate of Legionella-containing vacuoles towards LC3-associated phagocytosis.

The evolutionarily conserved processes of endosome-lysosome maturation and macroautophagy are established mechanisms that limit survival of intracellular bacteria. Similarly, another emerging mechanism is LC3-associated phagocytosis (LAP). Here we report that an intracellular vacuolar pathogen, Legionella dumoffii, is specifically targeted by LAP over classical endocytic maturation and macroautophagy pathways. Upon infection, the majority of L. dumoffii resides in ER-like vacuoles and replicate within this niche, which involves inhibition of classical endosomal maturation. The establishment of the replicative niche requires the bacterial Dot/Icm type IV secretion system (T4SS). Intriguingly, the remaining subset of L. dumoffii transiently acquires LC3 to L. dumoffii-containing vacuoles in a Dot/Icm T4SS-dependent manner. The LC3-decorated vacuoles are bound by an apparently undamaged single membrane, and fail to associate with the molecules implicated in selective autophagy, such as ubiquitin or adaptors. The process requires toll-like receptor 2, Rubicon, diacylglycerol signaling and downstream NADPH oxidases, whereas ULK1 kinase is dispensable. Together, we have discovered an intracellular pathogen, the survival of which in infected cells is limited predominantly by LAP. The results suggest that L. dumoffii is a valuable model organism for examining the mechanistic details of LAP, particularly induced by bacterial infection.

Legionella longbeachae Is Immunologically Silent and Highly Virulent In Vivo.

BACKGROUND: Legionella longbeachae (Llo) and Legionella pneumophila (Lpn) are the most common pneumonia-causing agents of the genus. Although both species can be lethal to humans and are highly prevalent, little is known about the molecular pathogenesis of Llo infections. In murine models of infection, Lpn infection is self-limited, whereas Llo infection is lethal. METHODS: We used mouse macrophages, human macrophages, human epithelial cells, and mouse infections in vivo to evaluate multiple parameters of the infection. RESULTS: We determined that the Llo Dot/Icm secretion system is critical for virulence. Different than Lpn, Llo disseminates and the animals develop a severe pulmonary failure, as demonstrated by lung mechanics and blood oxygenation assays. As compared to Lpn, Llo is immunologically silent and fails to trigger the production of cytokines in human pulmonary epithelial cells and in mouse and human macrophages. Infections in Tnfr1-/-, Ifng-/-, and Il12p40-/- mice supported the participation of cytokines for the resistance phenotype. CONCLUSIONS: Both Lpn and Llo require the Dot/Icm system for pathogenesis, but the infection outcome is strikingly different. Llo is immunologically silent, highly virulent, and lethal. The differences reported herein may reflect unappreciated clinical differences in patients infected with Lpn or Llo.

Comparative analysis of virulence traits between a Legionella feeleii strain implicated in Pontiac fever and a strain that caused Legionnaires' disease.

Legionella strains of the same species and serogroup are known to cause Legionnaires' disease (a potentially fatal atypical pneumonia) or Pontiac fever (a mild, flu-like disease), but the bacterial factors that define these dramatic differences in pathology have not been elucidated. To gain a better understanding of these factors, we compared the characteristics of Legionella feeleii strains that were isolated from either a sample of freshwater implicated in an outbreak of Pontiac fever (ATCC 35072, serogroup 1, LfPF), or a patient with Legionnaires' disease (ATCC 38549, serogroup 2, LfLD). Growth of LfPF and LfLD in BYE broth was slower than the positive control, Legionella pneumophila strain JR32. However, LfLD grew faster than LfPF at 42 °C. After in vitro infection to J774 murine or U937 human macrophage cell lines and A549 human lung epithelial cell line, LfLD showed a higher cell infection rate, stronger internalization by host cells, and greater cytotoxicity than that of LfPF. Large amounts of IL-6 and IL-8 were secreted by human host cells after infection with LfLD, but not with LfPF. LfLD possessed mono-polar flagellum while LfPF was unflagellated. When LfLD was cultured at 25, 30 and 37 °C, the bacteria had higher motility rate at lower temperatures. Based on our results, this is the first study that showed distinct characteristics between LfPF and LfLD, which may give important leads in elucidating differences in their virulence.

Microbiological and Clinical Studies of Legionellosis in 33 Patients with Cancer.

Legionella, a large group of environmental Gram-negative bacteria, represents an occasional cause of pneumonia. We analyzed the microbiological and clinical features of 33 consecutive cases of Legionella infections that occurred at the University of Texas MD Anderson Cancer Center, Houston, TX, from 2002 to 2014. The Legionella strains were isolated from bronchoscopy specimens (32 strains) and a blood culture (1 strain) and were identified by sequencing analysis of the full-length 16S rRNA gene. The 33 strains involved 12 Legionella species or subspecies: 15 strains of L. pneumophila subsp. pneumophila, 3 strains of L. pneumophila subsp. fraseri or L. pneumophila subsp. pascullei, 4 strains of "L. donaldsonii," 3 strains of L. micdadei, and one each of L. bozemanae, L. feeleii, L. gormanii, L. longbeachae, L. maceachernii, L. parisiensis, L. sainthelensi, and Legionella sp. strain D5382. All patients except one asymptomatic carrier showed pneumonia, including one with concurrent bacteremia. Nine patients died, with this infection being the immediate cause of death in six. Twenty-seven patients had underlying hematologic malignancies. Twenty-three patients were leukopenic. Six patients were recipients of allogeneic hematopoietic stem cell transplant, with their infections caused by five Legionella species. Together, these results suggest that diverse Legionella species infect patients with cancer in the Houston area and its vicinity. The five cases of pneumonia due to L. donaldsonii and Legionella sp. D5382 are likely the first reports of human infection with these organisms.

Different STAT Transcription Complexes Drive Early and Delayed Responses to Type I IFNs.

IFNs, which transduce pivotal signals through Stat1 and Stat2, effectively suppress the replication of Legionella pneumophila in primary murine macrophages. Although the ability of IFN-γ to impede L. pneumophila growth is fully dependent on Stat1, IFN-αß unexpectedly suppresses L. pneumophila growth in both Stat1- and Stat2-deficient macrophages. New studies demonstrating that the robust response to IFN-αß is lost in Stat1-Stat2 double-knockout macrophages suggest that Stat1 and Stat2 are functionally redundant in their ability to direct an innate response toward L. pneumophila. Because the ability of IFN-αß to signal through Stat1-dependent complexes (i.e., Stat1-Stat1 and Stat1-Stat2 dimers) has been well characterized, the current studies focus on how Stat2 is able to direct a potent response to IFN-αß in the absence of Stat1. These studies reveal that IFN-αß is able to drive the formation of a Stat2 and IFN regulatory factor 9 complex that drives the expression of a subset of IFN-stimulated genes, but with substantially delayed kinetics. These observations raise the possibility that this pathway evolved in response to microbes that have devised strategies to subvert Stat1-dependent responses.

An update on iron acquisition by Legionella pneumophila: new pathways for siderophore uptake and ferric iron reduction.

Iron acquisition is critical for the growth and pathogenesis of Legionella pneumophila, the causative agent of Legionnaires' disease. L. pneumophila utilizes two main modes of iron assimilation, namely ferrous iron uptake via the FeoB system and ferric iron acquisition through the action of the siderophore legiobactin. This review highlights recent studies concerning the mechanism of legiobactin assimilation, the impact of c-type cytochromes on siderophore production, the importance of legiobactin in lung infection and a newfound role for a bacterial pyomelanin in iron acquisition. These data demonstrate that key aspects of L. pneumophila iron acquisition are significantly distinct from those of long-studied, 'model' organisms. Indeed, L. pneumophila may represent a new paradigm for a variety of other intracellular parasites, pathogens and under-studied bacteria.

Metabolism of the vacuolar pathogen Legionella and implications for virulence.

Legionella pneumophila is a ubiquitous environmental bacterium that thrives in fresh water habitats, either as planktonic form or as part of biofilms. The bacteria also grow intracellularly in free-living protozoa as well as in mammalian alveolar macrophages, thus triggering a potentially fatal pneumonia called "Legionnaires' disease." To establish its intracellular niche termed the "Legionella-containing vacuole" (LCV), L. pneumophila employs a type IV secretion system and translocates ~300 different "effector" proteins into host cells. The pathogen switches between two distinct forms to grow in its extra- or intracellular niches: transmissive bacteria are virulent for phagocytes, and replicative bacteria multiply within their hosts. The switch between these forms is regulated by different metabolic cues that signal conditions favorable for replication or transmission, respectively, causing a tight link between metabolism and virulence of the bacteria. Amino acids represent the prime carbon and energy source of extra- or intracellularly growing L. pneumophila. Yet, the genome sequences of several Legionella spp. as well as transcriptome and proteome data and metabolism studies indicate that the bacteria possess broad catabolic capacities and also utilize carbohydrates such as glucose. Accordingly, L. pneumophila mutant strains lacking catabolic genes show intracellular growth defects, and thus, intracellular metabolism and virulence of the pathogen are intimately connected. In this review we will summarize recent findings on the extra- and intracellular metabolism of L. pneumophila using genetic, biochemical and cellular microbial approaches. Recent progress in this field sheds light on the complex interplay between metabolism, differentiation and virulence of the pathogen.

Live cell imaging of phosphoinositide dynamics during Legionella infection.

The "accidental" pathogen Legionella pneumophila replicates intracellularly in a distinct compartment, the Legionella-containing vacuole (LCV). To form this specific pathogen vacuole, the bacteria translocate via the Icm/Dot type IV secretion system approximately 300 different effector proteins into the host cell. Several of these secreted effectors anchor to the cytoplasmic face of the LCV membrane by binding to phosphoinositide (PI) lipids. L. pneumophila thus largely controls the localization of secreted bacterial effectors and the recruitment of host factors to the LCV through the modulation of the vacuole membrane PI pattern. The LCV PI pattern and its dynamics can be studied in real-time using fluorescently labeled protein probes stably produced by the soil amoeba Dictyostelium discoideum. In this chapter, we describe a protocol to (1) construct and handle amoeba model systems as a tool for observing PIs in live cell imaging, (2) capture rapid changes in membrane PI patterning during uptake events, and (3) observe the dynamics of LCV PIs over the course of a Legionella infection.

Biofilms: the stronghold of Legionella pneumophila.

Legionellosis is mostly caused by Legionella pneumophila and is defined as a severe respiratory illness with a case fatality rate ranging from 5% to 80%. L. pneumophila is ubiquitous in natural and anthropogenic water systems. L. pneumophila is transmitted by inhalation of contaminated aerosols produced by a variety of devices. While L. pneumophila replicates within environmental protozoa, colonization and persistence in its natural environment are also mediated by biofilm formation and colonization within multispecies microbial communities. There is now evidence that some legionellosis outbreaks are correlated with the presence of biofilms. Thus, preventing biofilm formation appears as one of the strategies to reduce water system contamination. However, we lack information about the chemical and biophysical conditions, as well as the molecular mechanisms that allow the production of biofilms by L. pneumophila. Here, we discuss the molecular basis of biofilm formation by L. pneumophila and the roles of other microbial species in L. pneumophila biofilm colonization. In addition, we discuss the protective roles of biofilms against current L. pneumophila sanitation strategies along with the initial data available on the regulation of L. pneumophila biofilm formation.

Intra-amoeba multiplication induces chemotaxis and biofilm colonization and formation for Legionella.

Legionella pneumophila, a facultative intracellular bacterium, is the causative agent of legionellosis. In the environment this pathogenic bacterium colonizes the biofilms as well as amoebae, which provide a rich environment for the replication of Legionella. When seeded on pre-formed biofilms, L. pneumophila was able to establish and survive and was only found at the surface of the biofilms. Different phenotypes were observed when the L. pneumophila, used to implement pre-formed biofilms or to form mono-species biofilms, were cultivated in a laboratory culture broth or had grown intracellulary within the amoeba. Indeed, the bacteria, which developed within the amoeba, formed clusters when deposited on a solid surface. Moreover, our results demonstrate that multiplication inside the amoeba increased the capacity of L. pneumophila to produce polysaccharides and therefore enhanced its capacity to establish biofilms. Finally, it was shown that the clusters formed by L. pneumophila were probably related to the secretion of a chemotaxis molecular agent.

Osteomyelitis of the patella caused by Legionella anisa.

A 51-year-old man with a history of stage IV angioimmunoblastic T-cell lymphoma was diagnosed with osteomyelitis of the patella. Legionella anisa was identified by 16S rRNA gene sequencing and culture. The patient had pneumonia 2 months prior to this osteomyelitis episode. L. anisa was retrospectively detected in his lung tissue by 16S rRNA gene sequencing and was considered the source of the L. anisa that caused his patella osteomyelitis.

VipD of Legionella pneumophila targets activated Rab5 and Rab22 to interfere with endosomal trafficking in macrophages.

Upon phagocytosis, Legionella pneumophila translocates numerous effector proteins into host cells to perturb cellular metabolism and immunity, ultimately establishing intracellular survival and growth. VipD of L. pneumophila belongs to a family of bacterial effectors that contain the N-terminal lipase domain and the C-terminal domain with an unknown function. We report the crystal structure of VipD and show that its C-terminal domain robustly interferes with endosomal trafficking through tight and selective interactions with Rab5 and Rab22. This domain, which is not significantly similar to any known protein structure, potently interacts with the GTP-bound active form of the two Rabs by recognizing a hydrophobic triad conserved in Rabs. These interactions prevent Rab5 and Rab22 from binding to downstream effectors Rabaptin-5, Rabenosyn-5 and EEA1, consequently blocking endosomal trafficking and subsequent lysosomal degradation of endocytic materials in macrophage cells. Together, this work reveals endosomal trafficking as a target of L. pneumophila and delineates the underlying molecular mechanism.

Legionella micdadei prosthetic valve endocarditis complicated by brain abscess: case report and review of the literature.

Legionella endocarditis is extremely uncommon, and embolic phenomena have never been reported. We report the first case of Legionella micdadei prosthetic valve endocarditis complicated by brain abscess. A 57-y-old immunocompromised woman with a history of mitral valve replacement developed confusion and left-sided weakness. Brain magnetic resonance imaging showed a 3-cm peripheral-enhancing mass. Transoesophageal echocardiography suggested a perivalvular abscess. Blood cultures and valve cultures were negative. She was diagnosed with 16S rRNA polymerase chain reaction and silver stain, and was discharged with levofloxacin after a redo mitral valve replacement. Twelve cases of Legionella endocarditis were reviewed. Only one case had a native valve, and her endocarditis occurred after pneumonia. All cases were cured. The duration of antibiotic therapy was variable. Legionella species should be considered in the differential diagnosis of culture-negative endocarditis in both immunocompetent and immunocompromised patients. Molecular techniques and silver impregnation stains are useful, especially when cultures using buffered charcoal-yeast extract agar are negative.

Legionella infections and travel associated legionellosis.

Travel associated Legionnaires' disease represents a significant cause of travel associated respiratory tract infections and impacts disproportionately on otherwise healthy individuals as a consequence of their travel abroad or within their own country. Because of the propensity of these bacteria to colonize man-made water systems, legionellosis are frequently reported in travelers who stayed in accommodations sites such as hotels or cruise ships. Since the discovery of this new pathogen and the creation of surveillance networks, the number of reported travel associated legionellosis cases have increased regularly. Education of physicians about the association of Legionnaires' disease with travel and the use of appropriate diagnostic tests and therapy can result in a reduction in morbidity and mortality due to this important cause of community-acquired pneumonia.