Legionella-like Amoebal Pathogens Research Articles

Diverse Legionella-Like Bacteria Associated with Testate Amoebae of the Genus Arcella (Arcellinida: Amoebozoa).

Diverse species of Legionella and Legionella-like amoebal pathogens (LLAPs) have been identified as intracellular bacteria in many amoeboid protists. There are, however, other amoeboid groups such as testate amoeba for which we know little about their potential to host such bacteria. In this study, we assessed the occurrence and diversity of Legionella spp. in cultures and environmental isolates of freshwater arcellinid testate amoebae species, Arcella hemispherica, Arcella intermedia, and Arcella vulgaris, via 16S rRNA gene sequence analyses and fluorescent insitu hybridization (FISH). Analysis of the 16S rRNA gene sequences indicated that A.hemispherica, A.intermedia, and A.vulgaris host Legionella-like bacteria with 94-98% identity to other Legionella spp. based on NCBI BLAST search. Phylogenetic analysis placed Legionella-like Arcella-associated bacteria (LLAB) in three different clusters within a tree containing all other members of Legionella and LLAPs. The intracellular localization of the Legionella within Arcella hosts was confirmed using FISH with a Legionella-specific probe. This study demonstrates that the host range of Legionella and Legionella-like bacteria in the Amoebozoa extends beyond members of "naked" amoebae species, with members of the testate amoebae potentially serving an ecological role in the dispersal, protection, and replication of Legionella spp. in natural environments.

Surveillance of parasitic Legionella in surface waters by using immunomagnetic separation and amoebae enrichment

Free-living amoebae (FLA) are potential reservoirs of Legionella in aquatic environments. However, the parasitic relationship between various Legionella and amoebae remains unclear. In this study, surface water samples were gathered from two rivers for evaluating parasitic Legionella. Warmer water temperature is critical to the existence of Legionella. This result suggests that amoebae may be helpful in maintaining Legionella in natural environments because warmer temperatures could enhance parasitisation of Legionella in amoebae. We next used immunomagnetic separation (IMS) to identify extracellular Legionella and remove most free Legionella before detecting the parasitic ones in selectively enriched amoebae. Legionella pneumophila was detected in all the approaches, confirming that the pathogen is a facultative amoebae parasite. By contrast, two obligate amoebae parasites, Legionella-like amoebal pathogens (LLAPs) 8 and 9, were detected only in enriched amoebae. However, several uncultured Legionella were detected only in the extracellular samples. Because the presence of potential hosts, namely Vermamoeba vermiformis, Acanthamoeba spp. and Naegleria gruberi, was confirmed in the samples that contained intracellular Legionella, uncultured Legionella may survive independently of amoebae. Immunomagnetic separation and amoebae enrichment may have referential value for detecting parasitic Legionella in surface waters.

Amoeba-Resistant Bacteria: Their Role in Human Infections

Free-living amoebae in water supply systems harbor bacterial endosymbionts. Legionella pneumophila, the agent of Legionnaires' disease, was one of the first agents that was demonstrated to survive and multiply within amoebae. Similar bacteria, termed Legionella-like amoebal pathogens (LLAP's), were subsequently described and implicated in human respiratory tract infections. Amoebal co-cultivation/enrichment techniques and molecular methods (e.g., PCR) have elucidated a variety of alphaproteobacteria (e.g., Afipia, Bosea, Mesorhizobium, and Rasbobacterium species) and Chlamydia-like bacteria (e.g., Parachlamydia, Simkania, Protochlamydia, and Waddlia species) within free-living amoebae in hospital water systems. These agents are also able to survive and multiply within macrophages, pneumocytes, and lung fibroblasts. Sero-epidemiological studies and application of molecular technology to clinical specimens have revealed these agents as rare causes of community- and health care-associated pneumonia, particularly among immunocompromised patients. Additional studies have implicated these agents in other infectious processes. This article reviews these newly described microorganisms and the evidence for their roles in human infections and disease.

Amoebal pathogens as emerging causal agents of pneumonia

Despite using modern microbiological diagnostic approaches, the aetiological agents of pneumonia remain unidentified in about 50% of cases. Some bacteria that grow poorly or not at all in axenic media used in routine clinical bacteriology laboratory but which can develop inside amoebae may be the agents of these lower respiratory tract infections (RTIs) of unexplained aetiology. Such amoebae-resisting bacteria, which coevolved with amoebae to resist their microbicidal machinery, may have developed virulence traits that help them survive within human macrophages, i.e. the first line of innate immune defence in the lung. We review here the current evidence for the emerging pathogenic role of various amoebae-resisting microorganisms as agents of RTIs in humans. Specifically, we discuss the emerging pathogenic roles of Legionella-like amoebal pathogens, novel Chlamydiae (Parachlamydia acanthamoebae, Simkania negevensis), waterborne mycobacteria and Bradyrhizobiaceae (Bosea and Afipia spp.).

Molecular evidence for the ubiquitous presence of Legionella species in Dutch tap water installations

Our aim was to investigate the occurrence and identity of Legionella spp. in Dutch tap water installations using culture, real-time PCR and sequence analysis. The PCR assays used were a 16S rRNA gene based PCR with both a Legionella species specific probe and a L. pneumophila specific probe and a L. pneumophila-specific PCR based on the sequence of the mip gene. A total of 357 water samples from 250 locations in The Netherlands was investigated. The detection rates of Legionella spp. were 2,2% (8 of 357) by culture, and 87,1% (311 of 357) by PCR. The majority of samples was found to contain Legionella species other than L. pneumophila. These comprised of Legionella Like Amoebal Pathogens (LLAPs), L. busanensis, L. worsliensis and others. Fourteen (3,9%) samples were positive for L. pneumophila by either culture, 16S rRNA based PCR and/or mip based PCR. It is apparent from this study that Legionella spp. DNA is ubiquitous in Dutch potable water samples. Our findings further suggest that LLAPs and viable but nonculturable (VBNC) Legionella represent a large proportion of the population in man-made environments.

Phylogenetic characterization ofLegionella‐like endosymbiotic X‐bacteria inAmoeba proteus: a proposal for ‘CandidatusLegionella jeonii’ sp. nov

The X-bacteria which initiated organismic association with the D strain of Amoeba proteus in 1966 as parasites have changed to obligate endosymbionts on which the host depends for survival. Owing to the difficulty in cultivating the bacteria in vitro, the identity of X-bacteria has not been determined. As the life cycle of X-bacteria is similar to that of Legionella spp. in soil amoebae, we applied the polymerase chain reaction method with specific primers aimed at Legionella spp. for the detection and cloning of 16S rRNA gene. The identity and intracellular localization of the endosymbiont were confirmed by the application of a specific fluorescently labelled 16S rRNA-targeted probe. In addition we cloned RNA polymerase beta-subunit gene (rpoB) of X-bacteria by genomic library tagging. A phylogenetic analysis of the 16S rRNA gene placed the bacterium within a unique monophyletic group containing all other members of the genus Legionella. Phylogeny from rpoB and mip genes further confirmed the taxonomic context of X-bacteria to be a Legionella sp. In all three phylogenic analyses, X-bacterium was placed apart from Legionella-like amoebal pathogens present in soil amoebae. Thus, we propose the name 'Candidatus Legionella jeonii' sp. nov. for the endosymbiotic X-bacteria in Amoeba proteus.

MP066 Characterization of potential virulence genes in the environmental chlamydia isolate UWE25

Free-living amoebae in water supply systems harbor bacterial endosymbionts. Legionella pneumophila, the agent of Legionnaires' disease, was one of the first agents that was demonstrated to survive and multiply within amoebae. Similar bacteria, termed Legionella-like amoebal pathogens (LLAP's), were subsequently described and implicated in human respiratory tract infections. Amoebal co-cultivation/enrichment techniques and molecular methods (e.g., PCR) have elucidated a variety of alphaproteobacteria (e.g., Afipia, Bosea, Mesorhizobium, and Rasbobacterium species) and Chlamydia-like bacteria (e.g., Parachlamydia, Simkania, Protochlamydia, and Waddlia species) within free-living amoebae in hospital water systems. These agents are also able to survive and multiply within macrophages, pneumocytes, and lung fibroblasts. Sero-epidemiological studies and application of molecular technology to clinical specimens have revealed these agents as rare causes of community- and health care-associated pneumonia, particularly among immunocompromised patients. Additional studies have implicated these agents in other infectious processes. This article reviews these newly described microorganisms and the evidence for their roles in human infections and disease.

Various bacterial pathogens and symbionts infect the amoeba Dictyostelium discoideum

The haploid soil amoeba Dictyostelium discoideum is a suitable model organism to study host-pathogen interactions with Legionella pneumophila. In this study we show that D. discoideum AX2 is also susceptible to infection with other important human pathogens and obligate intracellular symbionts. Infection assays demonstrated that Legionella-like amoebal pathogens (LLAP K62), Mycobacterium avium and the obligate intracellular endosymbionts of Acanthamoeba sp. strains TUME1, UWE25 and UWC6 were able to multiply within Dictyostelium. Salmonella typhimurium and Pseudomonas aeruginosa also invaded Dictyostelium, however were degraded shortly after uptake. Comitin-minus host cells were more permissive to infections with L. pneumophila and LLAP K62. Furthermore, this mutation significantly delayed the degradation of S. typhimurium. Accompanying electron and fluorescence microscopy of infected AX2 cells revealed that L. pneumophila and M. avium replicate within vacuoles, while LLAP K62, TUME1 and UWE25 were tightly enclosed by membranous structures within the cytoplasm. The beta-proteobacterium UWC6 was found to persist in the cytoplasm. The observed subcellular locations which correspond to the locations within the respective natural hosts suggest that D. discoideum is a representative model system for these pathogens and symbionts.

Legionella-Like and Other Amoebal Pathogens as Agents of Community-Acquired Pneumonia

We tested serum specimens from three groups of patients with pneumonia by indirect immunofluorescence against Legionella-like amoebal pathogens (LLAPs) 1-7, 9, 10, 12, 13; Parachlamydia acanthamoeba strains BN 9 and Hall's coccus; and Afipia felis. We found that LLAPs play a role (albeit an infrequent one) in community-acquired pneumonia, usually as a co-pathogen but sometimes as the sole identified pathogen.

Legionella drozanskii sp. nov., Legionella rowbothamii sp. nov. and Legionella fallonii sp. nov.: three unusual new Legionella species.

Seven strains of Legionella-like amoebal pathogens (LLAPs) were characterized on the basis of their cultural and staining characteristics, biochemical reactions, serology, cellular fatty acids (CFAs), isoprenoid quinone composition, total DNA relatedness, analysis of 16S rRNA and macrophage infectivity potentiator (mip) gene sequence analyses. All seven strains exhibited limited growth on buffered charcoal yeast extract alpha (BCYE) agar, required cysteine for growth and contained branched-chain CFAs and quinones typical of Legionella species. The bacilli were Gram-negative and catalase-positive. There were varying degrees of serological cross-reactions between these LLAP strains and other previously described Legionella species. Results from the various tests revealed that four LLAP strains represent three unusual new species of Legionella: Legionella drozanskii sp. nov., type strain LLAP-1T; Legionella rowbothamii sp. nov., type strain LLAP-6T; and Legionella fallonii sp. nov., type strain LLAP-10T. Three other LLAP strains, designated LLAP-7FL, LLAP-7NF and LLAP-9, were shown to be members of the species Legionella lytica. The deductions made from the phenetic characteristics of these bacteria were consistent with the phylogenetic relationships inferred from 16S rRNA and mip gene sequence analyses. This study is the first to speciate LLAP strains on the basis of data including quantitative DNA hybridization.

Potential importance of Legionella Species as etiologies in community acquired pneumonia (CAP)

Large percentages of patients with community acquired pneumonia (CAP) do not have a defined etiology. Between 1992–1993, 99 acute and convalescent sera were collected from patients with CAP of unknown etiology. The sera were tested using an indirect immunofluorescence antibody assay (IFA) against the following antigens: Legionella pneumophila, serogroups 3,5,6 and 7 and L. longbeachae, L. anisa, L. bozemanii and Legionella-Like Amoebal Pathogens (LLAP). A four-fold rise in titer to at least one of the antigens tested, was seen in 14% of patients; 8% to L. bozemanii, 4% to L. anisa, 2% to S. lyticum, 2% to LLAP 10 and 1% each to LLAP 1, 6 and 9. Two patients reacted to several antigens. These results indicate that other species of legionella may be important in the etiology of CAP. L. bozemanii was the organism identified in the majority of these infections. Better diagnostic studies i.e. cultures, serologies and urinary antigen testing, which recognize legionella isolates other than L. pneumophila serogroup 1 need to be developed.

Intracellular Bacteria of Acanthamoebae Resembling Legionefla spp. Turned Out to be Cytophaga sp.

Acanthamoeba sp. isolated from the drinking water system of a hospital harboured gram-negative bacteria multiplying inside phagosomes and within the cytoplasm of their host cells. According to their morphology demonstrated by electron microscopy they resembled Llaps (Legionella-like amoebal pathogens) but turned out to be Cytophaga sp. as shown by a typical profile of cellular fatty acids obtained by means of gas-liquid chromatography.

Use of heteroduplex analysis to classify legionellae on the basis of 5S rRNA gene sequences.

Seventeen different species of Legionella, 12 serogroups of Legionella pneumophila, and 2 Legionella-like amoebal pathogens (LLAP1 and Sarcobium lyticum) were examined by heteroduplex analysis of PCR products of the 5S rRNA gene. Eight different banding patterns were identified, indicating that heteroduplex analysis of this gene can be used to classify these bacteria according to base substitutions between species. This classification may have future applications in clinical and epidemiological studies.

Incidence, etiologic pathogens, and diagnostic testing of community-acquired pneumonia.

Determination of the etiologic pathogens of community-acquired pneumonia has been problematic because of the lack of reliable rapid laboratory diagnostic tools as well as the controversy concerning diagnostic criteria. In the studies reviewed here, a specific pathogen was identified in 39% to 88% of patients. Streptococcus pneumoniae remains the most common cause of community-acquired pneumonia. Depending on the demographics of the study, between 2% to 43% of cases have been attributed to legionella or Chlamydia pneumoniae. More recently, other pathogens have emerged, including respiratory syncytial virus in adults, hantavirus, and possibly legionella-like amoebal pathogens and Streptococcus milleri group. Treatment guidelines published by various societies of experts have been helpful, but they cannot replace the need for better and rapid diagnostic techniques.