Detection of Legionella pneumophila by real-time PCR for the mip gene

A real-time PCR assay for the mip gene of Legionella pneumophila was tested with 27 isolates of L. pneumophila, 20 isolates of 14 other Legionella species, and 103 non-Legionella bacteria. Eight culture-positive and 40 culture-negative clinical specimens were tested. This assay was 100% sensitive and 100% specific for L. pneumophila.     

Nucleotide sequence analysis of the Legionella micdadei mip gene, encoding a 30-kilodalton analog of the Legionella pneumophila Mip protein.

After the demonstration of analogs of the Legionella pneumophila macrophage infectivity potentiator (Mip) protein in other Legionella species, the Legionella micdadei mip gene was cloned and expressed in Escherichia coli. DNA sequence analysis of the L. micdadei mip gene contained in the plasmid pBA6004 revealed a high degree of homology (71%) to the L. pneumophila mip gene, with the predicted secondary structures of the two Mip proteins following the same pattern. Southern hybridization experiments, with the plasmid pBA6004 as the probe, suggested that the mip gene of L. micdadei has extensive homology with the mip-like genes of several Legionella species. Furthermore, amino acid sequence comparisons revealed significant homology to two eukaryotic proteins with isomerase activity (FK506-binding proteins).     

Procalcitonin kinetics in Legionella pneumophila pneumonia

Little is known about procalcitonin (PCT) levels in patients with community-acquired pneumonia (CAP) caused by Legionella pneumophila . The aim of the present study was to investigate this infection marker in patients admitted with L. pneumophila pneumonia in relation to conventional inflammatory parameters, severity of pneumonia upon admission and clinical outcome. Eighteen patients admitted with CAP caused by L. pneumophila serogroup 1 were retrospectively examined. PCT measurements were carried out during the first week of admission in addition to measurements of C-reactive protein (CRP), white blood cell (WBC) count and registration of severity of pneumonia upon admission (CURB-65 score). The mean PCT level upon admission in patients with L. pneumophila pneumonia was 13.5 ng/mL (range 0.3–55.7 ng/mL). Mean CRP level was 397 mg/L (range 167–595 mg/L) and mean WBC count 11.7 × 10⁹/L (range 4.5–20.4 × 10⁹/L). Initial high PCT levels were indicative of more severe disease as reflected by prolonged intensive care unit (ICU) stay and/or in-hospital death. Patients admitted to the ICU showed significantly higher PCT levels compared with the remaining patients [26.7 ng/mL (range 4.6–55.7 ng/mL) vs. 6.9 ng/mL (range 0.3–29.3 ng/mL); p 0.019]. There was a significant correlation between Acute Physiology and Chronic Health Evaluation-II scores upon ICU admission and initial PCT levels upon hospital admission ( r  = 0.86; p 0.027). Persistently increased PCT levels during treatment were indicative of unfavourable clinical outcome. Conventional inflammatory parameters (CRP and WBC) and the CURB-65 score lacked this discriminatory capacity in our study population. PCT may therefore be a valuable tool in the initial clinical assessment and follow-up of patients with L. pneumophila pneumonia.     

The occurrence of Legionella species other than Legionella pneumophila in clinical and environmental samples in Denmark identified by mip gene sequencing and matrix-assisted laser desorption ionization time-of-flight mass spectrometry

In Denmark, several laboratories use PCR as a routine diagnostic method for Legionnaires' disease, and almost all PCR-positive samples are investigated by culture. From 1993 to 2010, isolates of Legionella species other than Legionella pneumophila were obtained from respiratory samples from 33 patients, and from 1997 to 2010, 42 isolates of Legionella non-pneumophila species were obtained and saved from water samples from 39 different sites in Denmark. Macrophage infectivity potentiator gene (mip) sequencing was used as a reference method to identify the Legionella non-pneumophila species. Only one of the 75 isolates did not meet the acceptance criterion of a similarity of ≥98% to sequences in the database. The species distribution between clinical and environmental isolates varied. For the former, four species were detected, with Legionella bozemanae and Legionella micdadei predominating (both 44%). For the latter, eight species were detected, with Legionella anisa predominating (52%). The distribution among the Danish clinical isolates was different from the general distribution both in Europe and outside Europe, where L. bozemanae and Legionella longbeachae are the most commonly found clinical Legionella non-pneumophila species. The 75 isolates were also investigated by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS): 64 were correctly identified, with a score of ≥2.0; eight had a score of <2.0, but only two of these were wrongly identified; and three gave no results with MALDI-TOF MS. Both mip sequencing and MALDI-TOF MS are robust methods for Legionella species identification.     

Genetic typing in a cluster of Legionella pneumophila infections.

Legionella pneumophila strains isolated from six patients, three air-conditioning- and cooling tower-derived strains, and three hot water supply-derived strains were analyzed by three genetic typing methods. The results of the whole-cell DNA restriction endonuclease analysis and the restriction patterns based on genes coding for rRNA correlated with each other and demonstrated that the patient isolates were indistinguishable from the air-conditioning- and cooling tower-derived isolates but differed markedly from the hot water supply-derived isolates. The patient and air-conditioning- and cooling tower-derived strains contained plasmids of the same molecular weight; the hot water supply-derived strains were plasmidless. These results indicated that the cooling tower or the air-conditioning system was the environmental source for the examined cluster of Legionnaires disease strains.     

Use of partial 16S rRNA gene sequencing for identification of Legionella pneumophila and non-pneumophila Legionella spp

We examined 49 Legionella species, 26 L. pneumophila and 23 non-pneumophila Legionella spp., using partial 16S rRNA gene sequencing. This approach accurately identified all the L. pneumophila isolates, characterized all non-pneumophila Legionella isolates as such within this genus, and classified most (20/23; 87%) of the non-pneumophila Legionella isolates to the species level.     

Characterization of a Legionella micdadei mip mutant.

The pathogenesis of Legionella micdadei is dependent upon its ability to infect alveolar phagocytes. To better understand the basis of intracellular infection by this organism, we examined the importance of its Mip surface protein. In Legionella pneumophila, Mip promotes infection of both human macrophages and freshwater protozoa. Southern hybridization and immunoblot analyses demonstrated that mip sequences were present and expressed within a panel of virulent L. micdadei strains. Using allelic exchange mutagenesis, we then constructed an L. micdadei strain that completely and specifically lacked Mip. Although unimpaired in its ability to grow in bacteriologic media, this Mip mutant was defective in its capacity to infect U937 cells, a human macrophage-like cell line. Most significantly, the Mip- organism displayed a 24-fold reduction in survivability immediately after its entry into the phagocyte. Similarly, the mutant was less able to parasitize Hartmannella amoebae. Taken together, these data argue that Mip specifically potentiates intracellular growth by L. micdadei.     

Characterization of the Legionella pneumophila gene ligA

Legionella pneumophila is a bacterial pathogen that resides and multiplies in macrophages as well as in its natural aquatic hosts, the protozoa. Different bacterial factors contribute to pathogenicity and accompanying eukaryotic intracellular events. Sequencing of mip flanking regions revealed a gene of 2610 bp, ligA, that has no significant similarity to any of the genes identified previously. Epidemiological studies indicate that this gene is present in Legionella pneumophila, the species most often associated with cases of the Legionnaires' disease, but not in Legionella species other than L. pneumophila. The isogenic ligA deletion mutant was resistant to NaCl, and showed decreased cytotoxicity to human monocytes and decreased hemolytic activity to red blood cells. However, the most prominent effect of the L. pneumophila ligA mutant strain LEPF1 was the nearly completely reduced replication within the natural host Acanthamoeba castellanii. Since this gene is L. pneumophila specific and regulates numerous bacterial properties we designated this gene ligA for Legionella pneumophila infectivity gene A.     

Microbiological investigations on a nosocomial case of Legionella pneumophila pneumonia associated with water birth and review of neonatal cases

A case of Legionella pneumophila 1 pneumonia, confirmed by positive serology and urinary antigen, occurred in a 7-day old neonate after water birth in hospital. As respiratory samples were not available for culture, further microbiological investigations were performed in neonate and environment, in order to recognize the source of infection. The hospital water supply was contaminated by L. pneumophila 1 strains (300-2000 cfu/L) of two monoclonal subtypes of Pontiac subgroup. L. spiritensis (10-225 cfu/L) was isolated from cold tap water of the patient's home. PCR from tap and humidifiers water of the patient's home was positive for Legionella spp, but not for L. pneumophila. Because L. pneumophila 1, responsible of child infection, was only isolated from the hospital pool water for waterbirthing, we conclude that the infant acquired the nosocomial legionellosis by prolonged delivery in contaminated water, perhaps by aspiration. Infection control measures for waterbirthing are highly recommended. A review of neonatal case of legionellosis is also presented. As this rare infection may have a high fatality rate if unrecognized, pediatricians should be aware of the possibility of the legionellosis in newborns.     

Roles of interleukin-17 in an experimental Legionella pneumophila pneumonia model

Interleukin-17 (IL-17) is a key factor in T helper type 17 (Th17) lineage host responses and plays critical roles in immunological control of a variety of infectious diseases. Although Legionella pneumophila, an intracellular bacterium found widely in the environment, often causes a serious and life-threatening pneumonia in humans, the contribution of IL-17 to immune function during Legionella pneumonia is unknown. In the present study, we used an experimental Legionella pneumonia infection to clarify the role of IL-17 in the resulting immune response. We observed robust production of pulmonary IL-17A and IL-17F (IL-17A/F), peaking on day 1 and declining thereafter. Upregulated production of tumor necrosis factor alpha (TNF-α), IL-6, and IL-1β, but not monocyte chemotactic protein 1 (MCP-1), was observed in Legionella-infected bone marrow-derived macrophages from BALB/c mice that had been stimulated with IL-17A or IL-17F. A significant decrease in the production of proinflammatory cytokines IL-6 and TNF-α was observed in IL-17A/F-deficient mice (BALB/c background) infected with L. pneumophila. Moreover, we found impaired neutrophil migration and lower numbers of chemokines (KC, LIX, and MIP-2) in IL-17A/F-deficient mice. IL-17A/F-deficient mice also eliminated L. pneumophila more slowly and were less likely to survive a lethal challenge. These results demonstrate that IL-17A/F plays a critical role in L. pneumophila pneumonia, probably through induction of proinflammatory cytokines and accumulation of neutrophils at the infection site.     

Pathological evidence of rhabdomyolysis-induced acute tubulointerstitial nephritis accompanying Legionella pneumophila pneumonia

A case of Legionella pneumophila pneumonia with rhabdomyolysis-induced acute tubulointerstitial nephritis (ATIN) and prolonged renal dysfunction is presented. The patient was a 54-year-old man, admitted with high-grade fever, ataxia and muscle dysfunction; chest roentgenogram showed multilobular infiltrations. L pneumophila was detected in his sputum and urine, by PCR and by culture, and L pneumophila pneumonia was diagnosed. Despite antimicrobial treatment, he developed renal failure and rhabdomyolysis. Renal biopsy showed the presence of myoglobin casts that occluded the distal tubuli and tubulointerstitial nephritis, leading to the diagnosis of rhabdomyolysis-induced ATIN. Renal function subsequently normalised, and he was discharged. This is believed to be the first pathological evidence of involvement of rhabdomyolysis in legionellosis-associated ATIN.     

Thirteenth serogroup of Legionella pneumophila isolated from patients with pneumonia.

A Legionella-like organism (strain 82A3105; ATCC 43736) was isolated from a lung aspirate taken from a patient with pneumonia. Results of physiologic, gas-liquid chromatographic, genetic, and serologic tests showed that strain 82A3105 and four additional clinical isolates belong to a new Legionella pneumophila serogroup, serogroup 13.     

11th serogroup of Legionella pneumophila isolated from a patient with fatal pneumonia.

A Legionella-like organism (strain 797-PA-H; ATCC 43130) was isolated from a specimen taken from an endotracheal tube of a patient 4 days before death and from the left lung at autopsy. Growth characteristics were consistent with those for Legionella species. Strain 797-PA-H gave negative test results with available direct immunofluorescence assay conjugates and with slide agglutination test antisera prepared against the 22 Legionella species and 35 serogroups now recognized. Minimal reactivity (1 to 2 +) was observed with both tests by using reagents prepared against the Legionella-like organism Lansing 3. Reciprocal absorption studies, however, showed that the cross-reactive antibodies could be removed easily. Physiologic, gas-liquid chromatographic, and DNA hybridization tests revealed that the strain belonged to the species Legionella pneumophila. Therefore, strain 797-PA-H was designated as the type strain of a new L. pneumophila serogroup, serogroup 11.     

Legionella pneumophila serogroup 14 isolated from patients with fatal pneumonia.

Two Legionella-like organisms, one isolated from postmortem lung tissue and the other from a bronchial aspirate, were shown by growth, physiologic, and genetic characteristics to belong to the species Legionella pneumophila. Subsequent serologic testing indicated that both strains belonged to a new serogroup, serogroup 14.     

A Legionella pneumophila gene that promotes hemin binding.

The ability to bind and utilize hemin is a trait common to many human pathogens. Nevertheless, the relationship between Legionella pneumophila, the agent of Legionnaires' disease, and hemin has received little attention. Thus, we explored the capacity of a virulent, serogroup 1 strain of L. pneumophila to bind hemin and use it as an iron source. Hemin, but not protoporphyrin IX, restored bacterial growth in iron-limiting media, indicating that it can serve as an iron source for L. pneumophila. In support of this idea, we observed that wildtype legionellae were able to bind 50 to 60% of added hemin, a binding capacity that was comparable to those of other pathogens. To begin to identify proteins involved in hemin acquisition, we identified a Legionella locus that conferred hemin binding upon Escherichia coli. Subcloning and nucleotide sequence analysis determined that a single open reading frame, which was designated hbp for hemin-binding promotion, was responsible for this binding activity. The hbp gene was predicted to encode a secreted, 15.5-kDa protein. To ascertain the importance of this gene in L. pneumophila biology, we used allelic exchange to construct an hbp mutant. Importantly, the mutant displayed a 42% reduction in hemin binding, confirming that hbp potentiates hemin acquisition by L. pneumophila. However, the strain was unaltered in its ability to grow within macrophage-like cells and freshwater amoebae, indicating that hbp is not required for intracellular infection. Despite this, Southern hybridization analysis and database searches demonstrated that hbp is nearly exclusive to the L. pneumophila species.