Enzyme immunoassay for detection of antibodies against isolated flagella ofLegionella pneumophila

Flagella ofLegionella pneumophila serogroup 1, strain Philadelphia (ATCC 33152), were isolated and used as antigen to detect antibodies by enzyme immunoassay (EIA) in sera of patients with significant titers againstLegionella as determined by the indirect immunofluorescence assay (IFA). Healthy blood donors served as a control group. Whereas the sera of blood donors were negative in both assays, sera of patients showed two patterns of reactivity. Sera with elevated titers againstLegionella pneumophilasero-group 1 orLegionella gormanii in IFA usually demonstrated high titers in EIA. However, high IFA titers againstLegionella bozemanii were associated with low titers in EIA. The data show that flagella protein ofLegionella pneumophila serogroup 1 strain Philadelphia is of limited value for the detection of antibodies againstLegionella other thanLegionella pneumophila serogroup 1, although in vitro studies revealed genus-specific epitopes. This might be due to unpredictable variabilities in the expression of flagella in vivo.     

Occurrence and immunogenicity of proteinases fromLegionella species

Proteinases produced by variousLegionella species were studied by means of caseinate precipitation. Proteinase production was particularly high in strains ofLegionella pneumophila (serogroups 1–6), much lower in otherLegionella species, and absent inLegionella micdadei strain Tatlock. Immunoglobulins against proteinases ofLegionella pneumophila (serogroup 1, strain Philadelphia 1) inhibited the proteinase activity of all strains ofLegionella pneumophila, Legionella bozemanii, Legionella dumoffi andLegionella gormanii. There was no cross-reactivity between these antibodies and proteinases from bacteria belonging to other genera. Antibodies againstLegionella pneumophila proteinases were not found in human convalescent sera. The proteinases ofLegionella species could possibly be associated with pathogenicity factors.     

Entry and intracellular localization ofLegionella dumoffiiin Vero cells

Organisms of someLegionellaspecies are known to internalize and multiply within epithelial cell lines. During the study on interaction betweenLegionellaspp. and Vero cells, we found thatL. dumoffiiTex-KL (ATCC 33343) can enter into Vero cells approximately four to 20 times more often than five other strains of four species of legionella. The mode of entry betweenL. dumoffiiTex-KL andL. pneumophilaPhiladelphia-1 was compared and studied by treating Vero cells with reagents which inhibit phagocytosis and endocytosis. Monodansylcadaverine, cytochalasin D and nocodazol were used as inhibitors of receptor-mediated endocytosis, microfilament-dependent phagocytosis and polymerization of microtubules, respectively. The uptake ofL. dumoffiiTex-KL required receptor-mediated endocytosis by Vero cells, while the uptake ofL. pneumophilaPhiladelphia-1 used mainly microfilament-dependent phagocytosis. Polymerization of microtubules was necessary for Vero cells for the uptake of both strains of legionella. An electron microscopic examination revealed that some organisms of theL. dumoffiistrain Tex-KL escaped from endosomal vacuoles into cytoplasm in the early stage of infection, and proliferated in the cytoplasm. At that period, most of the bacteria were surrounded by rough endoplasmic reticula. In contrast,L. pneumophilaPhiladelphia-1 proliferated only within ribosome-lined endosome. We suggest thatL. dumoffiiTex-KL internalize and proliferate in Vero cells in a different way toL. pneumophilaPhiladelphia-1, and that there is a variety of the mode of interaction betweenLegionellaspp. and epithelial cells.     

Legionella pneumophila urinary antigen subtyping using monoclonal antibodies as a tool for epidemiological investigations

Legionnaires’ disease is diagnosed predominantly by urinary antigen detection, and patient isolates are rarely available. The lipopolysaccharide (LPS) epitope pattern of isolates detected by monoclonal antibodies is an accepted marker for the phenotyping of L. pneumophila serogroup 1 strains into monoclonal subgroups. L. pneumophila LPS is the dominant antigen in patients’ urinary specimens. By using commercially available microtiter wells coated with rabbit anti-Legionella serogroup 1 IgG as the catching antibody, LPS components in urine specimens were bound and detected separately by corresponding monoclonal antibodies of the Dresden Panel. The subtyping of LPS on urinary antigen molecules by using enzyme-linked immunosorbent assay (ELISA) allows deducing of first evidences for the identity/non-identity of environmental isolates and the legionellosis pathogen. Most importantly in our study, urinary antigen typing possesses high probability to distinguish (or does not distinguish) if the pathogen belongs to the MAb 3/1-negative L. pneumophila strains, which are widespread contaminants of water systems, but represent the minority of patient isolates.     

Epidemiological and Environmental Investigations of Legionella pneumophila Infection in Cattle and Case Report of Fatal Pneumonia in a Calf

A fatal pneumonia due to Legionella pneumophila was diagnosed in a young calf reared in a dairy herd located in northern Italy. Clinical symptoms consisted of watery diarrhea, hyperthermia, anorexia, and severe dyspnea. The pathological and histological findings were very similar to those observed in human legionellosis. Legionella pneumophila serogroup 1 (SG1) and SG10 were isolated from the calf’s lung, and L. pneumophila SG1 was isolated from the calf’s liver. L. pneumophila SG1 was also demonstrated in the lung tissue by immunofluorescence and immunohistochemical examinations. Nine of 10 L. pneumophila SG1 isolates belonged to the Olda subtype, and 1 belonged to the Camperdown subtype. A very low prevalence of antibodies to Legionella was detected in cows and calves reared in the same herd. Cultures of aqueous sediment of an old electric water heater which supplied hot water for the feeding of the calves yielded L. pneumophila SG1. Four of the colonies tested belonged to the Olda subtype. Ten clinical and four environmental isolates were examined for the presence of plasmids. Nine of them were also examined by pulsed-field gel electrophoresis assay, and the same patterns were found for L. pneumophila SG1 Olda strains isolated from the calf and from the electric heater. This is the first report of a documented case of a naturally occurring Legionella pneumonia in an animal. Cattle probably act as accidental hosts for legionellae, much the same as humans.     

Dual detection of Legionella pneumophila and Legionella species by real-time PCR targeting the 23S-5S rRNA gene spacer region

Although the majority of cases of Legionnaires’ disease (LD) are caused by Legionella pneumophila, an increasing number of other Legionella species have been reported to cause human disease. There are no clinical presentations unique to LD and hence accurate laboratory tests are required for early diagnosis. Therefore, we designed a real-time PCR assay that targets the 23S-5S rRNA intergenic spacer region (23S-5S PCR) and allows for detection of all Legionella species and discrimination of L. pneumophila from other Legionella species. In total, 271 isolates representing 50 Legionella species were tested and the assay was validated using 39 culture-positive and 110 culture-negative patient specimens collected between 1989 and 2006. PCR-positive results were obtained with all 39 culture-positive samples (100% sensitivity). Specimens that tested positive according to 23S-5S PCR, but were culture-negative, were further analysed by DNA sequencing of the amplicon or the macrophage infectivity potentiator (mip) gene. In addition to L. pneumophila, Legionella longbeachae, Legionella cincinnatiensis and Legionella micdadei were identified in the specimens. The assay showed a 7-log dynamic range displaying a sensitivity of 7.5 CFU/mL or three genome equivalents per reaction. Sixty-one specimens containing viruses or bacteria other than Legionellae were negative according to 23S-5S PCR, demonstrating its specificity. Use of this assay should contribute to the earlier detection of respiratory disease caused by Legionella species, as well as to increased rates of detection.     

Legionella spp. in UK composts—a potential public health issue?

Over the past 5 years, a number of cases of legionellosis in Scotland have been associated with compost use; however, studies investigating sources of infection other than water systems remain limited. This study delivers the first comprehensive survey of composts commonly available in the UK for the presence of Legionella species. Twenty-two store-bought composts, one green-waste compost and one home-made compost were tested for Legionella by culture methods on BCYE-α medium, and the findings were confirmed by macrophage infectivity potentiator (mip) speciation. Twenty-two of the samples were retested after an enrichment period of 8 weeks. In total, 15 of 24 composts tested positive for Legionella species, a higher level of contamination than previously seen in Europe. Two isolates of Legionella pneumophila were identified, and Legionella longbeachae serogroup 1 was found to be one of the most commonly isolated species. L. longbeachae infection would not be detected by routine Legionella urinary antigen assay, so such testing should not be used as the sole diagnostic technique in atypical pneumonia cases, particularly where there is an association with compost use. The occurrence of Legionella in over half of the samples tested indicates that compost could pose a public health risk. The addition of general hygiene warnings to compost packages may be beneficial in protecting public health.     

Legionella oakridgensis ATCC 33761 genome sequence and phenotypic characterization reveals its replication capacity in amoebae

Legionella oakridgensis is able to cause Legionnaires’ disease, but is less virulent compared to L. pneumophila strains and very rarely associated with human disease. L. oakridgensis is the only species of the family legionellae which is able to grow on media without additional cysteine. In contrast to earlier publications, we found that L. oakridgensis is able to multiply in amoebae. We sequenced the genome of L. oakridgensis type strain OR-10 (ATCC 33761). The genome is smaller than the other yet sequenced Legionella genomes and has a higher G + C-content of 40.9%. L. oakridgensis lacks a flagellum and it also lacks all genes of the flagellar regulon except of the alternative sigma-28 factor FliA and the anti-sigma-28 factor FlgM. Genes encoding structural components of type I, type II, type IV Lvh and type IV Dot/Icm, Sec- and Tat-secretion systems could be identified. Only a limited set of Dot/Icm effector proteins have been recognized within the genome sequence of L. oakridgensis. Like in L. pneumophila strains, various proteins with eukaryotic motifs and eukaryote-like proteins were detected. We could demonstrate that the Dot/Icm system is essential for intracellular replication of L. oakridgensis. Furthermore, we identified new putative virulence factors of Legionella.     

Whole-Genome Mapping as a Novel High-Resolution Typing Tool for Legionella pneumophila

Legionella is the causative agent for Legionnaires'' disease (LD) and is responsible for several large outbreaks in the world. More than 90% of LD cases are caused by Legionella pneumophila, and studies on the origin and transmission routes of this pathogen rely on adequate molecular characterization of isolates. Current typing of L. pneumophila mainly depends on sequence-based typing (SBT). However, studies have shown that in some outbreak situations, SBT does not have sufficient discriminatory power to distinguish between related and nonrelated L. pneumophila isolates. In this study, we used a novel high-resolution typing technique, called whole-genome mapping (WGM), to differentiate between epidemiologically related and nonrelated L. pneumophila isolates. Assessment of the method by various validation experiments showed highly reproducible results, and WGM was able to confirm two well-documented Dutch L. pneumophila outbreaks. Comparison of whole-genome maps of the two outbreaks together with WGMs of epidemiologically nonrelated L. pneumophila isolates showed major differences between the maps, and WGM yielded a higher discriminatory power than SBT. In conclusion, WGM can be a valuable alternative to perform outbreak investigations of L. pneumophila in real time since the turnaround time from culture to comparison of the L. pneumophila maps is less than 24 h.     

Repression of Flagella Is a Common Trait in Field Isolates of Salmonella enterica Serovar Dublin and Is Associated with Invasive Human Infections

The nontyphoidal Salmonella enterica serovar Dublin is adapted to cattle but infrequently infects humans, very often resulting in invasive infections with high levels of morbidity and mortality. A Salmonella-induced intestinal acute inflammatory response is postulated as a mechanism to prevent bacterial dissemination to systemic sites. In S. enterica serovar Typhimurium, flagella contribute to this response by providing motility and FliC-mediated activation of pattern recognition receptors. In this study, we found 4 Salmonella enterica isolates, with the antigenic formula 9,12:−:−, that, based on fliC sequence and multilocus sequence type (MLST) analyses, are aflagellate S. Dublin isolates. Interestingly, all were obtained from human bloodstream infections. Thus, we investigated the potential role of flagella in the unusual invasiveness exhibited by S. Dublin in humans by analyzing flagellation and proinflammatory properties of a collection of 10 S. Dublin human clinical isolates. We found that 4 of 7 blood isolates were aflagellate due to significantly reduced levels of fliC expression, whereas all 3 isolates from other sources were flagellated. Lack of flagella correlated with a reduced ability of triggering interleukin-8 (IL-8) and CCL20 chemokine expression in human intestinal Caco-2 cells and with reduced early inflammation in the ceca of streptomycin-pretreated C57/BL6 mice. These results indicate that flagella contribute to the host intestinal inflammatory response to Salmonella serovar Dublin and suggest that their absence may contribute to its systemic dissemination through dampening of the gut immune response. Analysis of FliC production in a collection of cattle isolates indicated that the aflagellate phenotype is widely distributed in field isolates of S. Dublin.     

Microenzyme-linked immunosorbent assay for detection of immunoglobulin G and immunoglobulin M antibodies to Legionella pneumophila

The microenzyme-linked immunosorbent assay (ELISA) for the detection of immunoglobulin M and G (IgM, IgG) antibodies to Legionella pneumophila serogroup 1 antigens was evaluated. IgM antibodies were measured by both double-sandwich and single-sandwich techniques. These assays were compared with the previously standardized indirect immunofluorescence test in four groups of subjects: (i) pneumonia patients with culture-proven Legionnaires disease with serogroup 1 isolates, (ii) pneumonia patients with serogroup 1 organisms detected by direct immunofluorescence testing of respiratory secretions but without culture confirmation, (iii) pneumonia patients with negative culture and direct immunofluorescence tests, and (iv) healthy hospital employees. In addition, the sensitivity and specificity of the IgG ELISA were evaluated with larger groups of controls and Legionnaires disease patients. The ELISA was more sensitive than the indirect immunofluorescence test. However, it detected antibody rises in pneumonia patients without culture or direct immunofluorescence evidence of L. pneumophila serogroup 1 infection, thereby suggesting that the specificity of the ELISA was slightly lower than that of the indirect immunofluorescence test. The double-sandwich ELISA was a sensitive method for detecting IgM antibodies and, as previously reported, appeared to be free from interference by rheumatoid factor. IgM anti-Legionella antibodies detected by the ELISA appeared earlier and were less persistent than IgG antibodies. In addition, the IgM ELISA was useful in detecting antibodies in necropsy serum samples obtained from patients dying acutely of Legionnaires disease. The data presented show that the ELISA is a reliable method for the detection of specific anti-Legionella antibodies.     

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.     

The origins of eukaryotic-like proteins in Legionella pneumophila

Legionella pneumophila, the causative agent of Legionnaires’ disease, is known to be an intracellular pathogen of multiple species of protozoa and is assumed to have co-evolved with these organisms for millions of years. Genome sequencing of L. pneumophila strains has revealed an abundance of eukaryotic-like proteins (ELPs). Here, we study the evolution of these ELPs, in order to investigate their origin. Thirty-four new ELPs were identified, based on a higher similarity to eukaryotic proteins than to bacterial ones. Phylogenetic analyses demonstrated that both lateral gene transfer from eukaryotic hosts and bacterial genes that became eukaryotic-like by gradual adaptation to the intracellular milieu or gene fragment acquisition, contributed to the existing repertoire of ELPs, which comprise over 3% of the putative proteome of L. pneumophila strains. A PCR survey of 72 L. pneumophila strains showed that most ELPs were conserved in nearly all of these strains, indicating that they are likely to play important roles in this species. Genes of different evolutionary origin have distinct patterns of selection, as reflected by their ratio of a synonymous vs. synonymous mutations. One ELP is common to several strains of Legionella, but outside this genus has homologs only in Acanthamoeba polyphaga mimivirus, indicating that gene exchange involving eukaryotic viruses and intracellular bacterial pathogens may also contribute to the evolution of virulence in either or both of these groups of organisms. Information on selection patterns and eukaryotic-like status was combined as a novel approach to predict type IV secretion system effectors of Legionella, which represent promising targets for future study.     

Clinical Application of a Multiplex Real-Time PCR Assay for Simultaneous Detection of Legionella Species,Legionella pneumophila,and Legionella pneumophila Serogroup 1

We developed a single-tube multiplex real-time PCR assay capable of simultaneously detecting and discriminating Legionella spp., Legionella pneumophila, and Legionella pneumophila serogroup 1 in primary specimens. Evaluation of 21 clinical specimens and 115 clinical isolates demonstrated this assay to be a rapid, high-throughput diagnostic test with 100% specificity that may aid during legionellosis outbreaks and epidemiologic investigations.     

Comparative Analysis of Infrequent-Restriction-Site PCR and Pulsed-Field Gel Electrophoresis for Epidemiological Typing of Legionella pneumophila Serogroup 1 Strains

Two methods were compared for the analysis of 48 unrelated and epidemiologically related Legionella pneumophila serogroup 1 isolates. These are the infrequent-restriction-site PCR (IRS-PCR) assay with adapters designed for XbaI and PstI restriction sites and the pulsed-field gel electrophoresis (PFGE) analysis determined after DNA restriction with SfiI. Both methods demonstrated a high level of discrimination with a similar capacity for differentiating 23 of the 24 unrelated isolates. PFGE analysis and IRS-PCR assay were both able to identify epidemiologically related isolates of L. pneumophila from three outbreaks. Hence, IRS-PCR assay appears to be a reproducible (intergel reproducibility, 100%) and discriminative (discriminatory index, ≥0.996) tool for typing of Legionella. Compared to PFGE, however, IRS-PCR presented an advantage through ease of performance and with attributes of rapidity and sensitivity of target DNA.     

Direct Sequencing of Legionella pneumophila from Respiratory Samples for Sequence-Based Typing Analysis

We have developed a procedure to test the efficiency and reliability of sequencing of Legionella pneumophila genes directly from respiratory samples and have compared the results with those derived from cultured isolates. We tried to obtain the nucleotide sequences of six protein-coding loci included in the sequence-based typing scheme for Legionella pneumophila and three intergenic regions from 132 samples corresponding to 106 patients positive for urine antigen. A seminested PCR approach was used to amplify and sequence these nine loci directly from respiratory samples. Nucleotide sequences were directly obtained for 23 Legionella isolates and also for 66 respiratory secretions from a total of 69 patients. The efficiency of sequencing from respiratory secretions was higher than that of sequencing after the isolation of the Legionella isolates. Moreover, the perfect match between the sequences obtained by both approaches when respiratory samples and cultured isolates from the same patient were available corroborates the suitability of the direct sequencing approach for the identification of Legionella species and molecular epidemiology studies with Legionella species.Legionella pneumophila is the etiological agent of Legionnaires'' disease (LD) and Pontiac fever and is also one of the causes of community-acquired pneumonia (CAP) (1, 12, 18) and severe pneumonia (22). Therefore, the rapid and sensitive diagnosis of infection with Legionella species is essential not only for treatment but also for the implementation of prevention procedures. The standard adopted by the European Working Group for Legionella Infections (EWGLI) for the typing and characterization of isolates related to outbreaks and sporadic cases of LD is based on the allelic profile obtained from the nucleotide sequences of seven protein-coding loci (7, 17). For clinical samples, the profiles are usually derived from sputum cultures. However, the efficiency of isolation of Legionella species from respiratory samples rarely reaches more than 20%, even when highly experienced laboratory personnel perform the isolation procedure. Molecular methods for the diagnosis of LD have focused on the extraction and specific PCR amplification of Legionella DNA from respiratory samples (10, 16). However, epidemiological investigations seldom depend only on the confirmation of infection by Legionella species, and more detailed characterization is usually necessary (11, 13). Due to the difficulties in culturing Legionella from sputum samples from patients with LD, it is desirable to test the suitability of obtaining nucleotide sequences directly from respiratory secretions from patients for whom a Legionella isolate is not available. Hence, our goal has been to obtain Legionella sequence information directly from sputum samples, thus avoiding dependence on the isolation of Legionella from cultures, and to compare the accuracy of multilocus sequence typing directly from lower respiratory secretions (bronchoalveolar fluid aspirated secretions [BAS] and sputum) to the accuracy of typing with cultured isolates.A large outbreak of Legionella affecting more than 200 people occurred in 1999 and 2000 in Alcoi, Comunidad Valenciana (CV), Spain (6). Public health authorities conducted an extensive epidemiological investigation, and strict regulations on the cleansing and maintenance of aerosol-producing devices were implemented. However, from 2001 to 2005 other minor outbreaks were detected in that area. The rate of legionellosis in the Alcoi area has been higher than that in the rest of Spain since 1999, with the only exception being 2001. The reasons for this high rate in this specific locality, despite public health authorities'' efforts to control and prevent further infections with Legionella species, remain unknown.     

First isolation of Legionella species,including L. pneumophila serogroup 1, in Greek potting soils: possible importance for public health

A total of 21 Legionella isolates were recovered from six out of 22 samples of potting soil from the Athens area, Greece. Legionella pneumophila (serogroups 1 and 2–15) and species and serotypes included in the group of L. longbeachae serogroups 1 and 2, L. bozemanii serogroups 1 and 2, L. dumoffii, L. gormanii, L. jordanis, L. micdadei and L. anisa were isolated on BCYEα agar containing cysteine, GVPC and natamycin and on BCYEα agar containing cysteine, Wadowsky Yee supplement and natamycin. The bacterial load was 4000–120 000 CFU/g of potting soil. The isolation of L. pneumophila serogroup 1 from Greek potting soils is reported here for the first time.     

In vitro activity,postantibiotic effect and human monocyte activity of grepafloxacin against Legionella species

ObjectiveTo determine the in vitro antimicrobial activity, postantibiotic effect (PAE) and human monocyte activity of grepafloxacin compared with sparfloxacin, ciprofloxacin, clarithromycin, erythromycin and rifampicin against 181 strains of Legionella pneumophila, nine strains of L. micdadei, 10 strains of L. dumoffii, seven strains of L. longbeachae and seven other Legionella strains.MethodsMICs were determined by standard agar dilution using buffered yeast extract (BYE) agar. PAE and human monocyte activity were determined by standard culture techniques.ResultsGrepafloxacin, sparfloxacin and rifampicin were the most active agents against L. pneumophila (MIC₉₀ ≥0.016 mg/L for most strains tested). Grepafloxacin was more active than erythromycin against L. dumoffii and L. longbeachae and more active than both erythromycin and clarithromycin against L. micdadei and isolates of other Legionella spp. The PAE of grepafloxacin against erythromycin-susceptible L. pneumophila (2.62 h) was higher than that of sparfloxacin (0.88 h), erythromycin (0.93 hours) and clarithromycin (0.72 h). Against erythromycin-resistant L. pneumophila, the PAE of grepafloxacin (4.18 h) was higher than those of all the other antibiotics tested. Grepafloxacin, sparfloxacin, ciprofloxacin and clarithromycin inhibited the growth of all L. pneumophila strains and other erythromycin-resistant Legionella spp. in human monocytes. However, only grepafloxacin and ciprofloxacin prevented regrowth or killed L. pneumophila after removal of extracellular antibiotic.ConclusionsGrepafloxacin showed effective antibacterial activity against the Legionella spp. tested, and has a PAE and activity within human monocytes that suggest it may be useful in the treatment of lower respiratory tract infections caused by Legionella spp.     

Molecular Pathogenesis of Infections Caused by Legionella pneumophila

Summary: The genus Legionella contains more than 50 species, of which at least 24 have been associated with human infection. The best-characterized member of the genus, Legionella pneumophila, is the major causative agent of Legionnaires'' disease, a severe form of acute pneumonia. L. pneumophila is an intracellular pathogen, and as part of its pathogenesis, the bacteria avoid phagolysosome fusion and replicate within alveolar macrophages and epithelial cells in a vacuole that exhibits many characteristics of the endoplasmic reticulum (ER). The formation of the unusual L. pneumophila vacuole is a feature of its interaction with the host, yet the mechanisms by which the bacteria avoid classical endosome fusion and recruit markers of the ER are incompletely understood. Here we review the factors that contribute to the ability of L. pneumophila to infect and replicate in human cells and amoebae with an emphasis on proteins that are secreted by the bacteria into the Legionella vacuole and/or the host cell. Many of these factors undermine eukaryotic trafficking and signaling pathways by acting as functional and, in some cases, structural mimics of eukaryotic proteins. We discuss the consequences of this mimicry for the biology of the infected cell and also for immune responses to L. pneumophila infection.     

Enhanced isolation of Legionella species from composted material

Legionella pneumophila and Legionella species were isolated from composted material when freshly prepared buffered charcoal yeast extract (BCYE) was supplemented with glycine (1.5 g/L), polymyxin B sulfate (40 000 IU/L), vancomycin hydrochloride (0.5 mg/L) and cycloheximide (40 mg/L) (GVPC medium) and Modified Wadowsky–Yee (MWY) (Oxoid, Cambridge, UK) plates were used for cultivation, but not with commercially sourced pre-poured GVPC and MWY plates (Oxoid). Legionella cincinnatiensis and pathogenic L. pneumophila serogroup (Sg) 1 Benidorm and France/Allentown were identified, as well as a non-typeable (NT) strain of L. pneumophila. As most laboratories no longer produce their own media, this may contribute to the lack of positive cultures from composted material. The antigenicity of the NT strain is discussed.