Interaction of Mycobacterium avium with environmental amoebae enhances virulence.

Environmental mycobacteria are a common cause of human infections. Recently, contaminated domestic water supplies have been suggested as a potential environmental source of several mycobacterial diseases. Since many of these mycobacterial species replicate best intracellularly, environmental hosts have been sought. In the present study, we examined the interaction of Mycobacterium avium with a potential protozoan host, the water-borne amoeba Acanthamoeba castellanii. We found that M. avium enters and replicates in A. castellanii. In addition, similar to that shown for mycobacteria within macrophages, M. avium inhibits lysosomal fusion and replicates in vacuoles that are tightly juxtaposed to the bacterial surfaces within amoebae. In order to determine whether growth of M. avium in amoebae plays a role in human infections, we tested the effects of this growth condition on virulence. We found that growth of M. avium in amoebae enhances both entry and intracellular replication compared to growth of bacteria in broth. Furthermore, amoeba-grown M. avium was also more virulent in the beige mouse model of infection. These data suggest a role for protozoa present in water environments as hosts for pathogenic mycobacteria, particularly M. avium.     

Isolation of two strains of Acanthamoeba castellanii from human tissue and their pathogenicity and isoenzyme profiles

Two strains of amoebae, one (CDC:0180:1) from the lung tissue of a patient who died of granulomatous amoebic encephalitis and the other (CDC:0179:1) from the debrided tissue of a mandibular autograft, were isolated and identified as Acanthamoeba castellanii based on the morphological and immunofluorescent staining characteristics of the trophozoites and cysts. Both strains of amoebae caused cytopathic effects in mammalian cell cultures and destroyed the cell sheet. However, only the CDC:0180:1 strain, on intranasal instillation into mice, produced the disease manifested by ruffled fur and aimless wandering, followed by coma and death within 30 days. The CDC:0180:1 strain also differed consistently from CDC:0179:1 and another nonpathogenic A. castellanii strain (ATCC 30,011) in isoenzyme makeup, a dissimilarity which probably reflects its pathogenic potential.     

Clinical implications of Mycobacterium kansasii species heterogeneity: Swiss National Survey

Several subtypes of Mycobacterium kansasii have been described, but their respective pathogenic roles are not clear. This study investigated the distribution of subtypes and the pathogenicity of M. kansasii strains (n = 191) isolated in Switzerland between 1991 and 1997. Demographic, clinical, and microbiological information was recorded from clinical files. Patients were classified as having an infection according to the criteria of the American Thoracic Society. Subtypes were defined by PCR-restriction enzyme analysis of the hsp65 gene. Subtype 1 comprised 67% of the isolates (n = 128), while subtypes 2 and 3 comprised 21% (n = 40) and 8% (n = 15), respectively. Other subtypes (subtypes 4 and 6 and a new subtype, 7) were recovered from only 4% of patients (n = 8). M. kansasii subtype 1 was considered pathogenic in 81% of patients, while M. kansasii subtype 2 was considered pathogenic in 67% of patients and other subtypes were considered pathogenic in 6% of patients. The majority of patients with M. kansasii subtype 2 were immunocompromised due to the use of corticosteroids (21% of patients) or coinfection with HIV (62.5% of patients). Subtyping M. kansasii may improve clinical management by distinguishing pathogenic from nonpathogenic subtypes.     

Interaction of Blastomyces dermatitidis, Sporothrix schenckii, and Histoplasma capsulatum with Acanthamoeba castellanii

Several dimorphic fungi are important human pathogens, but the origin and maintenance of virulence in these organisms is enigmatic, since an interaction with a mammalian host is not a requisite for fungal survival. Recently, Cryptococcus neoformans was shown to interact with macrophages, slime molds, and amoebae in a similar manner, suggesting that fungal pathogenic strategies may arise from environmental interactions with phagocytic microorganisms. In this study, we examined the interactions of three dimorphic fungi with the soil amoeba Acanthameobae castellanii. Yeast forms of Blastomyces dermatitidis, Sporothrix schenckii, and Histoplasma capsulatum were each ingested by amoebae and macrophages, and phagocytosis of yeast cells resulted in amoeba death and fungal growth. H. capsulatum conidia were also cytotoxic to amoebae. For each fungal species, exposure of yeast cells to amoebae resulted in an increase in hyphal cells. Exposure of an avirulent laboratory strain of H. capsulatum to A. castellanii selected for, or induced, a phenotype of H. capsulatum that caused a persistent murine lung infection. These results are consistent with the view that soil amoebae may contribute to the selection and maintenance of certain traits in pathogenic dimorphic fungi that confer on these microbes the capacity for virulence in mammals.     

Mycobacterium abscessus Phospholipase C Expression Is Induced during Coculture within Amoebae and Enhances M. abscessus Virulence in Mice

Mycobacterium abscessus is a pathogenic, rapidly growing mycobacterium involved in pulmonary and cutaneo-mucous infections worldwide, to which cystic fibrosis patients are exquisitely susceptible. The analysis of the genome sequence of M. abscessus showed that this bacterium is endowed with the metabolic pathways typically found in environmental microorganisms that come into contact with soil, plants, and aquatic environments, where free-living amoebae are frequently present. M. abscessus also contains several genes that are characteristically found only in pathogenic bacteria. One of them is MAB_0555, encoding a putative phospholipase C (PLC) that is absent from most other rapidly growing mycobacteria, including Mycobacterium chelonae and Mycobacterium smegmatis. Here, we report that purified recombinant M. abscessus PLC is highly cytotoxic to mouse macrophages, presumably due to hydrolysis of membrane phospholipids. We further showed by constructing and using an M. abscessus PLC knockout mutant that loss of PLC activity is deleterious to M. abscessus intracellular survival in amoebae. The importance of PLC is further supported by the fact that M. abscessus PLC was found to be expressed only in amoebae. Aerosol challenge of mice with M. abscessus strains that were precultured in amoebae enhanced M. abscessus lung infectivity relative to M. abscessus grown in broth culture. Our study underlines the importance of PLC for the virulence of M. abscessus. Despite the difficulties of isolating M. abscessus from environmental sources, our findings suggest that M. abscessus has evolved in close contact with environmental protozoa, which supports the argument that amoebae may contribute to the virulence of opportunistic mycobacteria.     

High-catalase strains of Mycobacterium kansasii isolated from water in Texas.

Isolation techniques with membrane-filtered potable water samples resulted in the isolation of potentially pathogenic high-catalase strains of Mycobacterium kansasii from 8 of 19 representative outlets in a small central Texas town. Mycobacterium gordonae was isolated from all samples, and Mycobacterium fortuitum was isolated from two samples. Data on chlorine levels are presented along with a possible explanation for the unusually high numbers of mycobacteria in these potable water samples. Findings suggest that water is a source of M. kansasii and may be an important link in the epidemiological picture of the disease.     

Interaction of lectin pathway of complement-activating pattern recognition molecules with Mycobacteria

We have demonstrated that mannose-binding lectin (MBL) recognizes various slow-growing, pathogenic mycobacteria [Mycobacterium tuberculosis (MTB), M. bovis, M. kansasii, M. gordonae] as well as non-pathogenic M. smegmatis. Recognition resulted in activation of the lectin pathway (LP) of complement and an enhancement of phagocytosis (shown for M. tuberculosis). Although MBL may be considered the main factor activating the LP upon recognition of mycobacteria, involvement of ficolins has also to be considered. Interaction of ficolin-3 with M. tuberculosis, M. bovis and M. kansasii, and ficolin-1 with M. tuberculosis and M. bovis was shown for the first time. Binding of recombinant MBL or ficolin-3 to MTB H₃₇Rv led to the agglutination of bacteria and promoted their phagocytosis, but little effect was apparent with ficolin-1 or ficolin-2. Data from Western blots suggest mannosylated lipoarabinomannan (ManLAM) to be one of the main cell components of slow-growing mycobacteria, involved in LP activation. However, the LP was also activated by other cell fractions. Results presented here supplement considerably the data concerning the ability of complement-activating lectins to interact with mycobacteria. Ficolins (especially ficolin-3) might influence host response to infection and thus have clinical significance, at least as disease modifiers.     

Identification of mycobacteria in solid-culture media by matrix-assisted laser desorption ionization-time of flight mass spectrometry

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has recently been introduced into the clinical microbiology laboratory as a rapid and accurate method to identify bacteria and yeasts. In this paper we describe our work on the use of MALDI-TOF MS for the identification of mycobacterial isolates. We developed a protocol for protein extraction from mycobacteria and utilized it to construct a database containing 42 clinically relevant type and reference strains of mycobacteria. The database was used to identify 104 clinical isolates of mycobacteria. All members of the Mycobacterium tuberculosis complex were identified accurately at the complex level but could not be separated at the species level. All other organisms were identified at the species level, with the exception of one strain of M. kansasii (accurately identified but with a low spectral score) and three pairs of closely related strains: M. abscessus and M. massiliense, M. mucogenicum and M. phocaicum, and M. chimaera and M. intracellulare. These pairs of organisms can currently be identified only by multilocus gene sequence analysis. We conclude that MALDI-TOF MS analysis can be incorporated into the work flow of the microbiology laboratory for rapid and accurate identification of most strains of mycobacteria isolated from solid growth media.     

Environmental mimics and the Lvh type IVA secretion system contribute to virulence-related phenotypes of Legionella pneumophila

Legionella pneumophila, the causative organism of Legionnaires' disease, is a fresh-water bacterium and intracellular parasite of amoebae. This study examined the effects of incubation in water and amoeba encystment on L. pneumophila strain JR32 and null mutants in dot/icm genes encoding a type IVB secretion system required for entry, delayed acidification of L. pneumophila-containing phagosomes, and intracellular multiplication when stationary-phase bacteria infect amoebae and macrophages. Following incubation of stationary-phase cultures in water, mutants in dotA and dotB, essential for function of the type IVB secretion system, exhibited entry and delay of phagosome acidification comparable to that of strain JR32. Following encystment in Acanthamoeba castellanii and reversion of cysts to amoeba trophozoites, dotA and dotB mutants exhibited intracellular multiplication in amoebae. The L. pneumophila Lvh locus, encoding a type IVA secretion system homologous to that in Agrobacterium tumefaciens, was required for restoration of entry and intracellular multiplication in dot/icm mutants following incubation in water and amoeba encystment and was required for delay of phagosome acidification in strain JR32. These data support a model in which the Dot/Icm type IVB secretion system is conditionally rather than absolutely required for L. pneumophila virulence-related phenotypes. The data suggest that the Lvh type IVA secretion system, previously thought to be dispensable, is involved in virulence-related phenotypes under conditions mimicking the spread of Legionnaires' disease from environmental niches. Since environmental amoebae are implicated as reservoirs for an increasing number of environmental pathogens and for drug-resistant bacteria, the environmental mimics developed here may be useful in virulence studies of other pathogens.     

Intracellular growth of Legionella pneumophila within Acanthamoeba castellanii Neff

Acanthamoeba castellanii Neff supports the intracellular growth of Legionella pneumophila. When acanthamoebae were exposed to L. pneumophila for 1 h and then washed free of unassociated bacteria and placed in liquid culture, levels of viable amoeba-associated legionellae and legionellae free in the culture medium increased by three to four orders of magnitude in 48 to 72 h. However, most of the legionellae remained amoeba-associated and could be cultured only after disruption of the amoebae. Furthermore, legionella viability declined rapidly in amoeba culture medium alone or when bacteria and amoebae were separated by a microporous membrane. Therefore, direct amoeba-legionella contact is required for this growth. Infected acanthamoebae treated with cold acetone to permeabilize them to fluorescent-labeled anti-L. pneumophila antibody appeared to contain far more legionellae than amoebae fixed with glutaraldehyde so as to prevent antibody penetration. Electron micrographs of infected A. castellanii showed numerous bacteria, including some dividing forms, within vacuoles in the cytoplasm. These results together show that A. castellanii is able to provide an intracellular niche for the growth of L. pneumophila.     

Cryptococcus neoformans var. gattii can exploit Acanthamoeba castellanii for growth.

It has recently been proposed that the origin and maintenance of virulence in certain environmental fungi is influenced by their interactions with non-vertebrate hosts such as amoebae and nematodes. In prior studies we have shown that the interactions of the soil amoebae Acanthamoeba castellanii with Cryptococcus neoformans varieties neoformans and grubii resemble those with macrophages. Here we extend those studies to C. neoformans variety gattii and describe quantitative differences in the type and outcome of the interactions observed relative to the other varieties. C. neoformans var. gattii proliferated in the presence of A. castellanii but the interaction was primarily extracellular with a paucity of phagocytic events. Experiments with acapsular cells coated with polysaccharide suggest that differences in the capsule structure may be responsible for the different interactions between cells of varieties neoformans, grubii, and gattii with amoebae. The ability of C. neoformans var. gattii to exploit amoebae indicates that despite major biological differences between C. neoformans varieties, all retain the ability to be pathogenic for A. castellanii.     

Improved technique for isolation of Mycobacterium kansasii from water.

A new concentration procedure, together with a new isolation medium, resulted in a 10- to 100-fold increase in the possibility of identifying Mycobacterium kansasii from water samples in comparison to a previously used procedure. In a survey which included both potable and natural water samples from many sites within the state of Texas, nine isolations of high-catalase strains of M. kansasii were obtained from 232 water samples tested. Acid-fast smear results were compared with mycobacterial isolations. An isolate from a river in central Texas is the first high-catalase strain of M. kansasii encountered in a natural water supply. Surveys of water samples from two Texas towns indicate that chlorine levels may influence the number of recoverable mycobacteria in water supplies.     

Enzyme immunoassay for identification of heat-killed mycobacteria belonging to the Mycobacterium tuberculosis and Mycobacterium avium complexes and derived from early cultures.

A simple enzyme-linked immunosorbent assay was developed for the identification of cultured mycobacteria belonging to the Mycobacterium tuberculosis complex, the Mycobacterium avium complex, and Mycobacterium kansasii. Six monoclonal antibodies were used: two (F23-49 and F24-2) were specific for the M. tuberculosis complex, two (F85-2 and F85-10) were specific for the identification of the M. avium complex, one (F126-22) was specific for M. kansasii, and one (F141-3) was broadly reactive and distinguished mycobacteria from other bacteria. In the enzyme-linked immunosorbent assay only 10(6) to 10(7) bacteria derived from early cultures (2 to 3 weeks) were needed for each monoclonal antibody. For the M. tuberculosis complex the sensitivity and specificity were both 100%. For the M. avium complex the specificity was 100% and the sensitivity was 70%. The three M. kansasii strains tested could all be identified as M. kansasii.     

Interaction of Legionella pneumophila with Acanthamoeba castellanii: uptake by coiling phagocytosis and inhibition of phagosome-lysosome fusion.

Legionella pneumophila is a facultative intracellular parasite able to survive within both human monocytes and amoebae. We have demonstrated that processing of L. pneumophila by the free-living amoeba Acanthamoeba castellanii shows many similarities to the processing of L. pneumophila by monocytes. These similarities include uptake of L. pneumophila by coiling phagocytosis and the subsequent confinement of L. pneumophila in a ribosome-studded phagosome. In addition, as in monocytes, inhibition of lysosomal fusion with phagosomes containing L. pneumophila was detected in amoebae. With all clinical isolates, inhibition of phagosomes-lysosome fusion correlated with virulence. However, with one of the environmental isolates tested, no significant difference in phagosome-lysosome fusion was observed between the virulent and avirulent forms. These results indicate that the avirulent form of this isolate differed from the virulent form in some other respect critical to intracellular survival. Therefore, intracellular multiplication of L. pneumophila within A. castellanii may not be solely dependent upon the inhibition of lysosomal fusion.     

Positive correlation between in vivo and in vitro assays for the evaluation of Pseudomonas virulence

Some bacterial phenotypes measured in vitro can be used to access bacterial virulence, on the premise that they are positively correlated with data from in vivo experiments. We show here that in vitro assessment of bacterial phenotypes, such as adherence and cytotoxicity, are positively correlated with data from in vivo experiments in Drosophila and can be used to assess bacterial virulence in vivo. Manipulation of environmental parameters, such as iron availability, induced changes in the phenotypes measured in vitro that correlated with changes in vivo virulence of all strains tested. Applying these assays, we demonstrate the pathogenic potential of a Pseudomonas fluorescens strain, initially isolated as a non-pathogenic milk contaminant. This strain displayed adherence and cytotoxicity comparable to those of the Pseudomonas aeruginosa pathogenic strain PAK, and colonized the infected flies as rapidly as the PAK strain. These results indicate that this "a priori" non-pathogenic bacterium is capable of escaping the host immune response, supporting the use of in vitro tests for screening of potential pathogens.     

Presence of CTX gene cluster in environmental non-O1/O139 Vibrio cholerae and its potential clinical significance

Purpose: The aim of this study was to understand the epidemiological linkage of clinical and environmental isolates of Vibrio cholerae and to determine their genotypes and virulence genes content. Materials and Methods: A total of 60 V. cholerae strains obtained from clinical specimens (n = 40) and surface waters (n = 20) were subjected to genotyping using PFGE and determination of their virulence-associated gene clusters. Result: PCR analysis showed the presence of chromosomally located hly and RTX genetic elements in 100% and 90% of the environmental isolates, respectively. The phage-mediated genetic elements such as CTX, TLC and VPI were detected in 5% of the environmental isolates suggesting that the environmental isolates cannot acquire certain mobile gene clusters. A total of 4 and 18 pulsotypes were obtained among the clinical and environmental V. cholerae isolates, respectively. Non-pathogenic environmentally isolated V. cholerae constituted a distinct cluster with one single non-O1, non-O139 strain (EP6) carrying the virulence genes similar to the epidemic strains. This may suggest the possible potential of conversion of non-pathogenic to a pathogenic environmental strain. Conclusions: The emergence of a single environmental isolate in our study containing the pathogenicity genes amongst the diverse non-pathogenic environmental isolates needs to be further studied in the context of V. cholerae pathogenicity sero-coversion.     

Free-living amoebae, a training field for macrophage resistance of mycobacteria

Mycobacterium species evolved from an environmental recent common ancestor by reductive evolution and lateral gene transfer. Strategies selected through evolution and developed by mycobacteria resulted in resistance to predation by environmental unicellular protists, including free-living amoebae. Indeed, mycobacteria are isolated from the same soil and water environments as are amoebae, and experimental models using Acanthamoeba spp. and Dictyostelium discoideum were exploited to analyse the mechanisms for intracellular survival. Most of these mechanisms have been further reproduced in macrophages for mycobacteria regarded as opportunistic and obligate pathogens. Amoebal cysts may protect intracellular mycobacteria against adverse conditions and may act as a vector for mycobacteria. The latter hypothesis warrants further environmental and clinical studies to better assess the role of free-living amoebae in the epidemiology of infections caused by mycobacteria.     

Neutrophil-macrophage cooperation in the host defence against mycobacterial infections

CD-1 mice inoculated intraperitoneally with Mycobacterium avium, M. bovis, M. microti or M. kansasii showed a persistent peritoneal granulocytosis (above 10(6) cells, i.e. more than 15% of total cells) throughout the 3 month period of infection studied. By contrast, in mice inoculated with the non-pathogenic M. aurum or with heat-killed M. avium the number of granulocytes decreased progressively after the first 15 days. No mycobacteria were found in granulocytes except in the first 2 days of infection. The mycobacteria-induced chronic granulocytosis was accompanied by phagocytosis of granulocytes by macrophages. Throughout the 3 months of infection, macrophages were found to contain intracellular lactoferrin. Macrophages with lactoferrin were also found in subcutaneous infection caused by M. marinum and in systemic infection caused by M. avium or M. kansasii. The in vitro activity of mouse peritoneal macrophages against M. avium and M. microti was increased after ingestion of granulocyte material by macrophages. These results lead us to propose that granulocytes participate in the host response to mycobacterial infections, not as phagocytes but rather through an indirect mechanism, as a source for the macrophages of molecules involved in antimicrobial mechanisms (e.g., lactoferrin and myeloperoxidase) lacking in the mature macrophage.     

IgA protease production as a characteristic distinguishing pathogenic from harmless neisseriaceae.

IgA proteases are extracellular enzymes of bacteria that have human immunoglobulin A of the IgA1 subclass as their only known substrate. The identification of this enzyme in neisseria prompted us to determine whether IgA protease production correlates with pathogenicity within this genus. Multiple clinical isolates of Neisseria gonorrhoeae, N. meningitidis and eight species of non-pathogenic neisseria that commonly colonize the normal human nasopharynx were examined for IgA protease activity. All N. gonorrhoeae and N. meningitidis strains were enzyme positive; all non-pathogenic strains were negative. Among meningococci, the enzyme occurred in strains carried harmlessly in the nasopharynx as well as those isolated from systemic infections. Because mucosal immune defense is largely mediated by antibodies of the IgA isotype, the finding that IgA protease activity is linked specifically to the pathogenic neisseria suggests that the enzyme may be involved in the pathogenesis of neisserial infection.