Legionella bozemanii sp. nov. andLegionella dumoffii sp. nov.: Classification of two additional species ofLegionella associated with human pneumonia

Deoxyribonucleic acid (DNA) relatedness was used to distinguish strains ofLegionella-like organisms (LLO) fromLegionella pneumophila. Two of these LLO strains, WIGA and MI 15, showed sufficient DNA relatedness to one another to be classified in the same species. The nameLegionella bozemanii species nova is proposed for this new species. The type strain ofL. bozemanii is WIGA (=ATCC 33217) Two other LLO strains, NY 23 and Tex-KL, were shown to represent a new species. The nameLegionella dumoffii species nova is proposed for this species. The type strain ofL. dumoffii is NY 23 (=ATCC 33279). These two species joinL. pneumophila andL. micdadei in the genusLegionella.     

Signal Transduction in the Protozoan Host Hartmannella vermiformis upon Attachment and Invasion by Legionella micdadei

The intracellular pathogens Legionella micdadei and Legionella pneumophila are the two most common Legionella species that cause Legionnaires’ disease. Intracellular replication within pulmonary cells is the hallmark of Legionnaires’ disease. In the environment, legionellae are parasites of protozoans, and intracellular bacterial replication within protozoans plays a major role in the transmission of Legionnaires’ disease. In this study, we characterized the initial host signal transduction mechanisms involved during attachment to and invasion of the protozoan host Hartmannella vermiformis by L. micdadei. Bacterial attachment prior to invasion of H. vermiformis by L. micdadei is associated with tyrosine dephosphorylation of multiple host cell proteins, including a 170-kDa protein. We have previously shown that this 170-kDa protein is the galactose N-acetylgalactosamine (Gal/GalNAc)-inhibitable lectin receptor that mediates attachment to and invasion of H. vermiformis by L. pneumophila. Subsequent bacterial entry targets L. micdadei into a phagosome that is not surrounded by the rough endoplasmic reticulum (RER). In contrast, uptake of L. pneumophila mediated by attachment to the Gal/GalNAc lectin is followed by targeting of the bacterium into an RER-surrounded phagosome. These results indicate that despite similarities in the L. micdadei and L. pneumophila attachment-mediated signal transduction mechanisms in H. vermiformis, the two bacterial species are targeted into morphologically distinct phagosomes in their natural protozoan host.     

Screening-level assays for potentially human-infectious environmental <Emphasis Type="Italic">Legionella</Emphasis> spp.

In spite of the fact that various Legionella species are isolated from nonclinical water settings, there is no standard method to determine whether environmental legionellae may be infectious to humans. Here we provide a screening-level approach based on an in vivo murine (A/J mouse) model and three in vitro proliferation assays using Acanthamoeba polyphaga, and THP-1 human and J774 murine macrophage cell lines to identify potentially human-infectious legionellae. As an initial demonstration the infectivity potential of three clinical (Legionella pneumophila, L, longbeacheae, and L. micdadei) and three environmental (L. dumoffii, L. maceachernii, and L. sainthelensi) legionellae were evaluated. A/J mice were intranasally infected and by 6 h post infection (p.L), there were significant bacterial titers in the lungs. L. pneumophila, L. dumoffii, and L. micdadei densities were higher than L. longbeacheae, L. maceacherni, and L. sainthelensi at 24 h p.i. However, only L. pneumophila and L. micdadei persisted in the lungs after 48 h, indicating that the other isolates were rapidly cleared. Results from the in vitro assays showed that only L. pneumophila significantly multiplied within A. polyphaga, THP-1 and J774 cells after 72 h, but lysis of any of the in vitro hosts also flagged the strains for potential concern (e.g. L. dumoffii and L. micdadei). The results demonstrate the value of using multiple approaches to assess the potential level of pathogenicity of Legionella strains isolated from different environmental matrices.     

Enhanced bactericidal activity of platelet β-lysin forE. coli in the presence of membrane perturbation

Inhibition by sodium chloride of the growth of 19 strains ofLegionella pneumophila and of 10 strains of otherLegionella spp. was studied. Results from growth in buffered -ketoglutarate cysteine yeast extract (BAYE) broth containing 0 to 2.0% sodium chloride indicated that 15/19 laboratory strains ofL. pneumophila were capable of growing in 1.0% to 1.5% sodium chloride, whereas 4 strains ofL. pneumophila and 10 strains of 6 other species were not.L. micdadei andL. longebeachae were the most inhibited in BAYE broth, growing only in concentrations of 0.5% sodium chloride. These in vitro studies indicate thatL. micdadei andL. longbeachae might be differentiated from other species by their low tolerance to salt in BAYE broth, and thatL. pneumophila may be more tolerant to salt concentrations found in brackish water environments.     

PCR methods for the rapid detection and identification of four pathogenic Legionella spp. and two Legionella pneumophila subspecies based on the gene amplification of gyrB

A total of 25 gyrB gene sequences from 20 Legionella pneumophila subsp. pneumophila strains and five L. pneumophila subsp. fraseri strains were obtained and analyzed, and a multiplex PCR for the simultaneous detection of Legionella bozemanae, Legionella longbeachae, Legionella micdadei and Legioenella pneumophila, and two single PCRs for the differentiation of L. pneumophila subsp. pneumophila and L. pneumophila subsp. fraseri were established. The multiplex PCR method was shown to be highly specific and reproducible when tested against 41 target strains and 17 strains of other bacteria species. The sensitivity of the multiplex PCR was also analyzed and was shown to detect levels as low as 1 ng of genomic DNA or 10 colony-forming units (CFUs) per milliliter in mock water samples. Sixty-three air conditioner condensed water samples from Shanghai City were examined, and the result was validated using 16S rRNA sequencing. The data reported here demonstrate that the multiplex PCR method described is efficient and convenient for the detection of Legionella species in water samples. Twenty L. pneumophila subsp. pneumophila strains and five L. pneumophila subsp. fraseri strains were used for the validation of the two L. pneumophila subspecies-specific PCR methods, and the results indicated that the two PCR methods were both highly specific and convenient for the identification of L. pneumophila at the subspecies level.     

Specific Detection of Legionella pneumophila: Construction of a New 16S rRNA-Targeted Oligonucleotide Probe

Based on comparative sequence analysis, we have designed an oligonucleotide probe complementary to a region of 16S rRNA of Legionella pneumophila which allows the differentiation of L. pneumophila from other Legionella species without cultivation. The specificity of the new probe, LEGPNE1, was tested by in situ hybridization to a total of four serogroups of six strains of L. pneumophila, five different Legionella spp. and three nonlegionella species as reference strains. Furthermore, L. pneumophila cells could be easily distinguished from Legionella micdadei and Pseudomonas aeruginosa cells by using in situ hybridization with probes LEGPNE1, LEG705, and EUB338 after infection of the protozoan Acanthamoeba castellanii.     

The human LL-37 peptide exerts antimicrobial activity against Legionella micdadei interacting with membrane phospholipids

Legionella micdadei is responsible for community- or nosocomial-acquired pneumonia as well as the influenza-like illness Pontiac fever. The aim of this study was to investigate the ability of L. micdadei to utilize extracellular choline for phosphatidylcholine (PC) synthesis and its consequences for the phospholipid composition of its membrane system and the interaction with the human LL-37 peptide. Comparative analysis of the PC content using isotopic labeling revealed that in presence of exogenous choline 98% of the total PC was synthesized via the Pcs pathway while the remaining 2% were generated via the PE-methylation (PmtA) pathway. PC species were to a greater extent defined by the Pcs pathway in the outer membrane than in the inner membrane. While no major changes in the bacterial lipid content were observed using ³¹P NMR, indication for utilization of longer acyl chains and slight increase of PG in response to choline addition was observed by a top-down lipidomics screen. The LL-37 peptide inhibited L. micdadei growth in a dose-dependent manner. Bacteria cultured with exogenous choline were more sensitive to the LL-37 peptide when compared to the standard culture condition. Our biophysical investigations show that the peptide perturbs bacterial-derived phospholipid monolayers and this interaction is dependent on the molar portion of PC. This interaction is responsible for the observed changes in the anti-L. micdadei activity of the LL-37 peptide.     

Biocide activity against Legionella and Pseudomonas

Four biocides were tested for their abilities to kill Legionella micdadei and Pseudomona fluorescens in a buffered saline solution. The most effective against both organisms, at concentrations of 50 ppm or above, was a phosphonium compound, but this biocide failed to produce a 99·9% kill of P. fluorescens within 24 h at concentrations below 50 ppm. All chemicals apart from an oxidisingbiocide were more effective against L. micdadei than P. fluorescens. Old cells of Legionella, but not of Pseudomonas, exhibited increased resistance against BNPD. This could be associated with an altered outer envelope, demonstrated by SDS-PAGE analysis.     

Detection ofLegionella antibodies by enzyme-linked immunosorbent assay (ELISA) using whole cell and carbohydrate antigens

The systematic study ofLegionella as a human pathogen and a bacterium widely disseminated in the environment requires simplification of present methodology. We describe a highly sensitive enzyme-linked immunosorbent assay (ELISA) for the detection of serum antibodies that can also be used for the detection of antigen.Legionella pneumophila serogroups 1 and 3 (Philadelphia 2 and Bloomington 2),L. bozemanii (WIGA), andL. micdadei (TATLOCK) were grown in diphasic medium consisting of charcoal yeast extract agar (CYE) overlayed with yeast extract medium (YEM) for the production of whole cell antigen and CYE for the extraction of carbohydrate antigen. The whole cells were inactivated with 0.5% formalin. The carbohydrate was obtained from the supernatant of cells resuspended twice in phosphate buffered saline (PBS). The antigen was sterilized and concentrated by filtration and purified by chromatography through a Sepharose 4B column. The highest molecular weight fractions were used for chemical characterization, which confirmed the carbohydrate nature of the antigen, and for micro-ELISA. Titers ranging from 5×10³ to 3×10⁵ (inverse of serum dilutions) were obtained from rabbit sera collected after 1, 2, or 3 injections of whole cells. The titers were somewhat higher and more consistent with the higher of 2 antigen concentrations used (5 or 15g/ml protein or dry weight), and with the carbohydrate rather than the whole cell antigen. The reactions were serogroup and species specific and only low titers were obtained with some of the heterologous antigens. The sensitivity and specificity of the reactions were not diminished when as many as 4 antigens were mixed in the same well. Thus, the micro-ELISA can be used as a test of highly specific antigens as well as a screening test with mixtures of antigens. A preliminary test withLegionella containing water specimen concentrates and high-titer rabbit sera indicated that the micro-ELISA can also be used for the detection of antigen. This investigation appears to have paved the way for the simplification of the serological methodology for the study ofLegionella. On temporary leave from: Department of Microbiology, University of Maryland School of Medicine, 660 West Redwood Street, Baltimore, MD 21201.     

Escherichia coli and other species of the enterobacteriaceae encode a protein similar to the family of Mip-like FK506-binding proteins

A newly identified gene in Escherichia coli, fkpA, encodes a protein with extensive similarity to the macrophage infectivity potentiator (Mip) proteins of Legionella pneumophila and Chlamydia trachomatis. The FkpA protein may be a new member of the family of FK506-binding proteins (FKBPs) because its carboxyl domain includes a sequence that matches the consensus FK506-binding motif in 40 of 48 positions. including those amino acids at the active site that form hydrogen bonds with the drug FK506. The amino acid sequence of the 29kDa FkpA protein is 30–35% identical to the Mip proteins of L. pneumophila, L. micdadei, and C. trachomatis. Of the 270 amino acids of FkpA, 113 (42%) are identical to the sequence of one or another of these Mip proteins. Overexpression of FkpA or deletion of fkpA from the E. coli chromosome had no detrimental effect on bacterial growth, indicating that fkpA is not an essential gene. Hybridization of fkpA-specific DNA probes to genomic blots révealed that similar genes exist in several representatives of the Enterobacteriaceae. Thus, mip-like genes are not found exelusively in bacteria having a predominately intracellular life style, but instead appear to be a new FKBP subfamily that is a common constituent of many bacteria.     

Altered rate of synthesis of specific peptides in the legionellae in response to growth temperature

The effect of a sharp temperature rise (30° to 42°C) on the rate of peptide synthesis inLegionella pneumophila (three strains),Tatlockia micdadei (Tatlock), andFluoribacter bozemanae (WIGA) was examined. Most striking was an increase in the rate of synthesis of 4–5 peptides of approximate molecular weight 85,000 daltons (85k), 78k, 70k, and either 60k (only in theL. pneumophila strains and WIGA) or 62.5k, and 40k (only in Tatlock). The response required new mRNA synthesis. In addition, the heat shock response of these organisms continued as long as the high temperature condition persisted (at least 24 h) and for at least 1 h after temperature downshift (to 30°C).     

Plasmid profiles ofLegionella species

Forty-six strains ofLegionella species were assayed for plasmid DNA content using routine laboratory procedures. Large-molecular-weight cryptic plasmids were detected inLegionella pneumophila serogroups 2, 3, and 4,L. bozemanii, L. dumoffii, L. micdadei, L. gormanii, L. longbeachae, and as yet unclassifiedLegionella-like organisms. No plasmids were found in strains ofL. pneumophila serogroups 1, 5, and 6. No correlations could be made between the possession of a specific plasmid profile, or lack of one, and any phenotypic markers such as virulence or antibiotic resistance. Several parameters were identified in this study as critical to the isolation of plasmid DNA fromLegionella: (i) DNA preparations obtained from frozen egg or animal materials had a higher incidence of detectable plasmid DNA than subcultures on bacteriologic media. (ii) A newly formulated broth supported exponential growth in all of the 46 strains; one strain required the addition of CO₂. (iii) Considerable heterogeneity was seen in cell susceptibility to various detergents. Since no single lytic agent was suitable for all strains, both ionic and noninic lysis methods were used with each strain. Within the limitations of both crude lysate preparations and the agarose gel electrophoresis method, this study identified a large 60–80 megadalton plasmid species in over 50% of the plasmid-containing strains.     

Genomic heterogenicity amongst phenotypically similar Legionella micdadei strains

Abstract Nine unrelated Legionella micdadei strains isolated from clinical and environmental samples have been characterized biochemically, serologically using polyclonal and monoclonal antibodies and by macrorestriction analyses using pulsed-field gel electrophoresis. All strains were positive in the Bromocresol purple spot test and grew as blue colonies on dye-containing media. They were positive for catalase, weakly positive for oxidase, and negative for sodium-hippurate hydrolysis, β-lactamase and gelatinase. None of the strains showed autofluorescence under long-wave ultraviolet light. A panel of six monoclonal antibodies raised against the ATCC strain TATLOCK revealed no significant differences in the surface antigen composition of the L. micdadei strains. None of these monoclonal antibodies reacted with L. maceachernii and L. longbeachae serogroup 2, the only species that cross-react with polyclonal antisera. Each of the nine L. micdadei strains showed individual restriction patterns of the genomic DNA when using both Sfi I and Not I restriction enzymes in the pulsed-field gel electrophoresis. Macrorestriction analysis is a valuable tool for studies on the molecular epidemiology of L. micdadei .     

Detection of culturable and nonculturable Legionella species from hot water systems of public buildings in Japan

Aims: To investigate the prevalence of culturable and nonculturable Legionella species in hot water systems of public buildings in Japan and assess the risk factors associated with Legionella contamination in hot water systems. Methods and Results: Legionella species were detected by conventional culture and molecular methods in 130 water samples collected from 40 buildings. A total of 26 (20·0%) water samples from 17 (42·5%) buildings were positive by culture, qualitative PCR or both methods: Legionella pneumophila and Leg. anisa were detected in four samples by a culture method, whereas 23 samples were positive by qualitative PCR, with the presence of various Legionella species confirmed by sequencing. Of these 23 samples, bacterial counts were quantifiable in 21 by real‐time PCR (from 1·7 × 10⁵ to 2·6 × 10¹¹ cells per litre). Phylogenetic analysis of amplified partial 16S rRNA gene showed close relations to various species of Legionella, including Leg. anisa and Leg. micdadei, all of which have been associated with respiratory diseases or increased antibody titres in human sera. Assessment of risk factors showed that turbidity, free chlorine concentration, iron concentration and heterotrophic plate count (HPC) were significantly associated with Legionella contamination (P < 0·05). Conclusions: Contamination of hot water systems of public buildings with culturable and nonculturable Legionella species may be a potential risk factor for Legionella infection in Japan. Adequate levels of chlorine, low levels of iron and HPC are important maintenance measures in the reduction of Legionella contamination in hot water systems. Significance and Impact of the Study: More than 40% of hot water systems in the Japanese public buildings examined were contaminated by not only culturable Leg. pneumophila and Leg. anisa but also by nonculturable pathogenic species. To our knowledge, this is the first report of both culturable and nonculturable Legionella contamination in hot water systems of public buildings in Japan.     

A New Oligonucleotide Microarray for Detection of Pathogenic and Non-Pathogenic Legionella spp.

Legionella pneumophila has been recognized as the major cause of legionellosis since the discovery of the deadly disease. Legionella spp. other than L. pneumophila were later found to be responsible to many non-pneumophila infections. The non-L. pneumophila infections are likely under-detected because of a lack of effective diagnosis. In this report, we have sequenced the 16S-23S rRNA gene internal transcribed spacer (ITS) of 10 Legionella species and subspecies, including L. anisa, L. bozemanii, L. dumoffii, L. fairfieldensis, L. gormanii, L. jordanis, L. maceachernii, L. micdadei, L. pneumophila subspp. fraseri and L. pneumophila subspp. pasculleii, and developed a rapid oligonucleotide microarray detection technique accordingly to identify 12 most common Legionella spp., which consist of 11 pathogenic species of L. anisa, L. bozemanii, L. dumoffii, L. gormanii, L. jordanis, L. longbeachae, L. maceachernii, L. micdadei, and L. pneumophila (including subspp. pneumophila, subspp. fraseri, and subspp. pasculleii) and one non-pathogenic species, L. fairfieldensis. Twenty-nine probes that reproducibly detected multiple Legionella species with high specificity were included in the array. A total of 52 strains, including 30 target pathogens and 22 non-target bacteria, were used to verify the oligonucleotide microarray assay. The sensitivity of the detection was at 1.0 ng with genomic DNA or 13 CFU/100 mL with Legionella cultures. The microarray detected seven samples of air conditioner-condensed water with 100% accuracy, validating the technique as a promising method for applications in basic microbiology, clinical diagnosis, food safety, and epidemiological surveillance. The phylogenetic study based on the ITS has also revealed that the non-pathogenic L. fairfieldensis is the closest to L. pneumophila than the nine other pathogenic Legionella spp.     

Legionella oakridgensis: unusual new species isolated from cooling tower water.

We describe a new species of Legionella represented by 10 strains isolated from industrial cooling towers. Legionella oakridgensis differed genetically from the other seven species of Legionella in DNA hybridization studies and differed serologically in direct fluorescent-antibody tests. The new species, unlike all other species except L. jordanis, did not require added L-cysteine for growth in serial transfer on charcoal-yeast extract agar. L. oakridgensis, as well as three other species tested, required L-cysteine for primary isolation from animal tissues. L. oakridgensis was the only species of Legionella that failed to produce alkaline phosphatase at pH 8.5. In all other respects, it resembled other species of Legionella, including having a high content of branched-chain cellular fatty acids and being pathogenic for guinea pigs. These bacteria have not yet been associated with human disease, but they are potential causes of legionellosis.     

Problems associated with identification of Legionella species from the environment and isolation of six possible new species

Following investigation of an outbreak of legionellosis in South Australia, numerous Legionella-like organisms were isolated from water samples. Because of the limited number of commercially available direct fluorescent-antibody reagents and the cross-reactions found with some reagents, non-pneumophila legionellae proved to be difficult to identify and these isolates were stored at -70 degrees C for later study. Latex agglutination reagents for Legionella pneumophila and Legionella anisa developed by the Institute of Medical and Veterinary Science, Adelaide, Australia, were found to be useful as rapid screening aids. Autofluorescence was useful for placing isolates into broad groups. Cellular fatty acid analysis, ubiquinone analysis, and DNA hybridization techniques were necessary to provide definitive identification. The species which were isolated most frequently were L. pneumophila, followed by L. anisa, Legionella jamestowniensis, Legionella quinlivanii, Legionella rubrilucens, Legionella spiritensis, and a single isolate each of Legionella erythra, Legionella jordanis, Legionella birminghamensis, and Legionella cincinnatiensis. In addition, 10 isolates were found by DNA hybridization studies to be unrelated to any of the 26 currently known species, representing what we believe to be 6 possible new species.     

Evaluation of the Duopath Legionella lateral flow assay for identification of Legionella pneumophila and Legionella species culture isolates

Duopath Legionella (Merck KGaA, Darmstadt, Germany) is a new immunochromatographic assay for the simultaneous identification of cultured L. pneumophila and Legionella species other than L. pneumophila. In tests of 89 L. pneumophila strains and 87 Legionella strains other than L. pneumophila representing 41 different species, Duopath and a widely used latex agglutination assay detected L. pneumophila with 100% and 98% accuracy, respectively, whereas the percentages differed significantly for other Legionella spp. (93% versus 37% [P < 0.001]). Since many countries' regulations require the identification of Legionella spp. in water and environmental samples, the use of Duopath Legionella to comply with those regulations could contribute to significantly fewer false-negative results.     

Problems associated with identification of Legionella species from the environment and isolation of six possible new species.

Following investigation of an outbreak of legionellosis in South Australia, numerous Legionella-like organisms were isolated from water samples. Because of the limited number of commercially available direct fluorescent-antibody reagents and the cross-reactions found with some reagents, non-pneumophila legionellae proved to be difficult to identify and these isolates were stored at -70 degrees C for later study. Latex agglutination reagents for Legionella pneumophila and Legionella anisa developed by the Institute of Medical and Veterinary Science, Adelaide, Australia, were found to be useful as rapid screening aids. Autofluorescence was useful for placing isolates into broad groups. Cellular fatty acid analysis, ubiquinone analysis, and DNA hybridization techniques were necessary to provide definitive identification. The species which were isolated most frequently were L. pneumophila, followed by L. anisa, Legionella jamestowniensis, Legionella quinlivanii, Legionella rubrilucens, Legionella spiritensis, and a single isolate each of Legionella erythra, Legionella jordanis, Legionella birminghamensis, and Legionella cincinnatiensis. In addition, 10 isolates were found by DNA hybridization studies to be unrelated to any of the 26 currently known species, representing what we believe to be 6 possible new species.     

High-performance liquid chromatographic analysis of ubiquinones from new Legionella species

Abstract The ubiquinone composition of Legionella feeleii, L. oakridgensis, L. rubrilucens, L. sainthelensi, L. wadsworthii and some environmental legionella isolates was investigated by high performance liquid chromatography. The results of the present study indicate ubiquinones are valuable criteria for the rapid identification of Legionella species and the recognition of new taxa.