Hartmannella vermiformis Inhibition of Legionella pneumophila Cultivability

Hartmannella vermiformis and Acanthamoeba polyphaga are frequently isolated from drinking water and permissive to Legionella pneumophila parasitization. In this study, extracellular factor(s) produced by H. vermiformis and A. polyphaga were assessed for their effects on cultivability of L. pneumophila. Page’s amoeba saline (PAS) was used as an encystment medium for H. vermiformis and A. polyphaga monolayers, and the culture supernatants (HvS and ApS, respectively) were assessed against L. pneumophila growth. Compared to PAS and ApS, HvS significantly inhibited L. pneumophila strain Philadelphia-1 (Ph-1) cultivability by 3 log₁₀ colony forming unit (CFU) mL^?1 after 3 days of exposure compared to <0.5 log₁₀ CFU mL^?1 reduction of strain Lp02 (P?<?0.001). Flow cytometric analysis revealed changes in the percentage and cultivability of three bacterial subpopulations: intact/slightly damaged membrane (ISM), undefined membrane status (UD), and mixed type (MT). After 3 days of HvS exposure, the MT subpopulation decreased significantly (31.6 vs 67.2 %, respectively, P?<?0.001), while the ISM and UD subpopulations increased (+26.7 and +6.9 %, respectively) with the ISM subpopulation appearing as viable but nonculturable (VBNC) cells. HvS was separated into two fractions based on molecular weight, with more than 99 % of the L. pneumophila inhibition arising from the <5 kDa fraction (P?<?0.001). Liquid chromatography indicated the inhibitory molecule(s) are likely polar and elute from a Novapak C18 column between 6 and 15 min. These results demonstrate that H. vermiformis is capable of extracellular modulation of L. pneumophila cultivability and probably promote the VBNC state for this bacterium.     

Cytopathogenicity of Naegleria fowled for Cultured Rat Neuroblastoma Cells1

The cytopathogenicity of Naegleria fowleri strain LEE (ATCC-30894) for cultured rat neuroblastoma cells (B-103) has been investigated. Both live N. fowleri amoebae and Naegleria lysates added to ⁵¹Cr-labeled B-103 cells caused release of radiolabel, which was dependent upon the ratio of amoebae to target cells or to the lysate concentration. Lysates of N. fowleri strains LEE, NF-66, NF-69, and HB-4 were equally injurious to B-103 target cells whereas lysates of strains 6088 and KUL were less cytotoxic. Highly pathogenic mouse-passaged strain LEE were less cytotoxic than axenically grown amoebae. Maximum cytotoxicity was observed in lysates from amoebae in late exponential or early stationary phase of growth. Cytopathogenicity of lysates was reduced after heating at 44°C for 60 min or at 60°C for 30 min. Cytotoxicity was stable during storage at 4°C or at ?20°C for 26 h. Neither live amoebae nor lysates injured B-103 target cells at 4°C. Live amoebae and lysates injured B-103 by a time, temperature, and concentration dependent process.     

Hartmanella vermiformis can be permissive for Pseudomonas aeruginosa

Aims: The amoebae of the genus Hartmanella are frequently recovered from hospital water taps, whereas Pseudomonas aeruginosa is often implicated in nosocomial infections. Previous works suggested that free living amoebae can act as vehicles of bacterial transmission. The present work investigates the relationships between a strain of Hartmanella vermiformis and three strains of P. aeruginosa: a reference strain, a strain from a patient and an environmental strain. Methods and Results: In a saline medium, H. vermiformis is not able to favour for the development of P. aeruginosa. In a rich co‐cultivation medium, only the environmental strain has shown a growth. Conclusions: We showed that P. aeruginosa is not a good nutrient source for H. vermiformis. Significance and Impact of the Study: Nevertheless, in particular conditions and with particular strains, the presence of H. vermiformis could represent a possibility of growth for P. aeruginosa.     

Impact of Chlorine and Heat on the Survival of Hartmannella vermiformis and Subsequent Growth of Legionella pneumophila

Hartmannella vermiformis, a common amoebal inhabitant of potable-water systems, supports intracellular multiplication of Legionella pneumophila and is probably important in the transportation and amplification of legionellae within these systems. To provide a practical guide for decontamination of potable-water systems, we assessed the chlorine and heat resistance of H. vermiformis. H. vermiformis cysts and trophozoites were treated independently with chlorine at concentrations of 2.0 to 10.0 ppm for 30 min and then cocultured with L. pneumophila. Both cysts and trophozoites were sensitive to concentrations between 2.0 and 4.0 ppm and above (trophozoites somewhat more so than cysts), and 10.0 ppm was lethal to both forms. Hartmannellae treated with chlorine up to a concentration of 4.0 ppm supported the growth of legionellae. To determine whether heat would be an effective addendum to chlorine treatment of amoebae, hartmannellae were subjected to temperatures of 55 and 60°C for 30 min and alternatively to 50°C followed by treatment with chlorine at a concentration of 2 ppm. Fewer than 0.05% of the amoebae survived treatment at 55°C, and there were no survivors at 60°C. Pretreatment at 50°C appeared to make hartmannella cysts more susceptible to chlorine but did not further reduce the concentration of trophozoites.     

Rapid quantification of viable legionellae in water and biofilm using ethidium monoazide coupled with real‐time quantitative PCR

Aims: To optimize ethidium monoazide (EMA) coupled with real‐time quantitative PCR (qPCR) and to evaluate its environmental applicability on quantifying viable legionellae in water and biofilm of cooling towers and hot water systems. Methods and Results: EMA (0·9–45·5 μg ml^?1) and propidium monoazide (PMA, 0·9 and 2·3 μg ml^?1) combined with qPCR (i.e. EMA‐qPCR and PMA‐qPCR, respectively) were applied to unheated and heated (70°C for 30 min) Legionella pneumophila to quantify viable cells, which was also simultaneously determined by BacLight Bacterial Viability kit with epifluorogenic microscopic enumeration (BacLight‐EM). The effects of nontarget microflora and sample matrix on the performance of EMA‐qPCR were also evaluated. In comparison with BacLight‐EM results, qPCR with EMA at 2·3 μg ml^?1 was determined as the optimal EMA‐qPCR assay, which performed equally well as PMA‐qPCR for unheated Leg. pneumophila but better than PMA‐qPCR for heated Leg. pneumophila (P < 0·05). Moreover, qPCR with EMA at 2·3 μg ml^?1 accurately quantified viable Leg. pneumophila, Legionella anisa and Legionella‐like amoebal pathogens 6 (LLAP 6) without interferences by heated legionellae, unheated nonlegionellae cells and cooling tower water matrix (P > 0·05). As for water and biofilm samples collected from cooling towers and hot water systems, the viable legionellae counts determined by EMA‐qPCR were mostly greater than the culturable counts by culture assay but consistently lower than the total cell counts quantified by qPCR. Conclusions: The qPCR with EMA at 2·3 μg ml^?1 may accurately quantify viable legionellae (including fastidious LLAP 6) and Leg. pneumophila pretreated with superheating and is applicable for water and biofilm samples obtained from cooling towers and hot water systems. Significance and Impact of the Study: The EMA‐qPCR assay may be useful in environmental surveillance for viable legionellae and in evaluation of superheating efficacy against legionellae.     

Influence of Acanthamoeba castellanii on Intracellular Growth of Different Legionella Species in Human Monocytes

Previous studies using a murine model of coinhalation of Legionella pneumophila and Hartmannella vermiformis have shown a significantly enhanced intrapulmonary growth of L. pneumophila in comparison to inhalation of legionellae alone (J. Brieland, M. McClain, L. Heath, C. Chrisp, G. Huffnagle, M. LeGendre, M. Hurley, J. Fantone, and C. Engleberg, Infect. Immun. 64:2449–2456, 1996). In this study, we introduce an in vitro coculture model of legionellae, Mono Mac 6 cells (MM6) and Acanthamoeba castellanii, using a cell culture chamber system which separates both cell types by a microporous polycarbonate membrane impervious to bacteria, amoebae, and human cells. Whereas L. pneumophila has shown a maximal 4-log-unit multiplication within MM6, which could not be further increased by coculture with Acanthamoeba castellanii, significantly enhanced replication of L. gormanii, L. micdadei, L. steigerwaltii, L. longbeachae, and L. dumoffii was seen after coculture with amoebae. This effect was seen only with uninfected amoebae, not with Legionella-infected amoebae. The supporting effect for intracellular multiplication in MM6 could be reproduced in part by addition of a cell-free coculture supernatant obtained from a coincubation experiment with uninfected A. castellanii and Legionella-infected MM6, suggesting that amoeba-derived effector molecules are involved in this phenomenon. This coculture model allows investigations of molecular and biochemical mechanisms which are responsible for the enhancement of intracellular multiplication of legionellae in monocytic cells after interaction with amoebae.     

The increasing importance of Vermamoeba vermiformis

Vermamoeba vermiformis are one of the most prevalent free-living amoebae. These amoebae are ubiquitous and also thermotolerant. Of concern, V. vermiformis have been found in hospital water networks. Furthermore, associations between V. vermiformis and pathogenic bacteria have been reported, such as Legionella pneumophila. Moreover, V. vermiformis are well known to host viruses, bacteria, and other microorganisms and cases of keratitis due to V. vermiformis in conjunction with other amoebae have been reported. Despite the preceding, the medical importance of V. vermiformis is still an ongoing discussion and its genome has been only recently sequenced. Herein, we present a review of the current understanding of the biology and pathogenesis pertaining to V. vermiformis, as well as its’ role as an etiological agent and trojan horse. An approach known as theranostics which combines both diagnosis and therapy could be utilized to eradicate and diagnose keratitis cases caused by such amoebae. Given the rise in global warming, it is imperative to investigate these rarely studied amoebae and to understand their importance in human health.     

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.     

Mycobacterium avium Bacilli Grow Saprozoically in Coculture with Acanthamoeba polyphaga and Survive within Cyst Walls

Protozoans are gaining recognition as environmental hosts for a variety of waterborne pathogens. We compared the growth of Mycobacterium avium, a human pathogen associated with domestic water supplies, in coculture with the free-living amoeba Acanthamoeba polyphaga with the growth of M. avium when it was separated from amoebae by a 0.1-μm-pore-size polycarbonate membrane (in a parachamber). Although viable mycobacteria were observed within amoebal vacuoles, there was no significant difference between bacterial growth in coculture and bacterial growth in the parachamber. This suggests that M. avium is able to grow saprozoically on products secreted by the amoebae. In contrast, Legionella pneumophila, a well-studied intracellular parasite of amoebae, multiplied only in coculture. A comparison of amoebae infected with L. pneumophila and amoebae infected with M. avium by electron microscopy demonstrated that there were striking differences in the locations of the bacteria within amoebal cysts. While L. pneumophila resided within the cysts, M. avium was found within the outer walls of the double-walled cysts of A. polyphaga. These locations may provide a reservoir for the bacteria when environmental conditions become unfavorable.     

Mediators of Lipid A Modification, RNA Degradation, and Central Intermediary Metabolism Facilitate the Growth of Legionella pneumophila at Low Temperatures

Legionella pneumophila is an aquatic bacterium that is also the agent of Legionnaires’ disease pneumonia. Since L. pneumophila is transmitted directly from the environment to the lung, it is important to understand how legionellae survive at low temperatures. To identify genes that are needed for L. pneumophila growth at low temperature, we screened a population of mutagenized legionellae for strains that are specifically impaired for growth at 17°C. From the 7,400 mutants tested, 11 displayed defects ranging from ca. 10-fold to a complete inability to grow at the low temperature. PCR and sequence analysis were then utilized to identify the genes whose loss had compromised growth. The proteins thereby implicated in low-temperature growth included components of the type II secretion system (LspE, LspG, LspH), a lipid A biosynthetic enzyme (LpxP), a ribonuclease (RNAse R), an RNA helicase (CsdA/DeaD), TCA cycle enzymes (citrate synthase), enzymes linked to fatty acid (FadB) or amino acid (aspartate aminotransferase) catabolism, and two putative membrane proteins that were, based upon their sequences, unlike previously characterized proteins. Given the magnitude of their mutant’s defect, the aspartate aminotransferase, RNA helicase, and one of the putative membrane proteins were the factors most critical for L. pneumophila low-temperature growth. Thus, L. pneumophila not only employs some of the same processes and factors as other bacteria do in order to survive at low temperatures (e.g., LpxP, CsdA), but it also appears to possess novel modes of cold adaptation.     

Influence of Legionella pneumophila and other water bacteria on the survival and growth of Acanthamoeba polyphaga

We investigated in solid medium, in water microcosm co-cultures and by light and transmission electron microscopy the influence of Legionella pneumophila Lp-1, Pseudomonas aeruginosa ATCC 27853, Burkholderia cepacia ATCC 25416 and Pseudomonas fluorescens SSD35 on the growth and survival of Acanthamoeba polyphaga. The infection with L. pneumophila was microscopically characterized by the presence of few bacteria inside protozoa at 4th h, and by the beginning of disruptive effects in late phase of trial. In water microcosm studies, performed at different temperature, the more significant interactions were observed at 30°C. In these conditions, L. pneumophila caused a marked reduction in trophozoite and cyst counts from the 4th day until the end of incubation (11 days). B. cepacia showed, by microscopic observation, few and generally single rods within protozoan phagosomes and caused a light reduction of trophozoite viability and cyst formation in co-cultures. A more invasive type of endocytosis, characterized by an early invasion with the presence of a high bacteria number inside amoebae, was observed for Pseudomonas strains. P. fluorescens produced a violent lysis of the host, whereas P. aeruginosa did not cause lysis or suffering. These results underline that water bacteria other than legionella are capable of intracellular survival in Acanthamoeba, influencing the protozoa viable cycle.     

The Type II Secretion System of Legionella pneumophila Elaborates Two Aminopeptidases, as Well as a Metalloprotease That Contributes to Differential Infection among Protozoan Hosts

Legionella pneumophila, the agent of Legionnaires' disease, is an intracellular parasite of aquatic amoebae and human macrophages. A key factor for L. pneumophila in intracellular infection is its type II protein secretion system (Lsp). In order to more completely define Lsp output, we recently performed a proteomic analysis of culture supernatants. Based upon the predictions of that analysis, we found that L. pneumophila secretes two distinct aminopeptidase activities encoded by the genes lapA and lapB. Whereas lapA conferred activity against leucine, phenylalanine, and tyrosine aminopeptides, lapB was linked to the cleavage of lysine- and arginine-containing substrates. To assess the role of secreted aminopeptidases in intracellular infection, we examined the relative abilities of lapA and lapB mutants to infect human U937 cell macrophages as well as Hartmannella vermiformis and Acanthamoeba castellanii amoebae. Although these experiments identified a dispensable role for LapA and LapB, they uncovered a previously unrecognized role for the type II-dependent ProA (MspA) metalloprotease. Whereas proA mutants were not defective for macrophage or A. castellanii infection, they (but not their complemented derivatives) were impaired for growth upon coculture with H. vermiformis. Thus, ProA represents the first type II effector implicated in an intracellular infection event. Furthermore, proA represents an L. pneumophila gene that shows differential importance among protozoan infection models, suggesting that the legionellae might have evolved some of its factors to especially target certain of their protozoan hosts.     

Free-living amoebae (FLA) co-occurring with legionellae in industrial waters

Legionella pneumophila is known as the causative agent of Legionnaires’ disease and free-living amoebae (FLA) can serve as vehicles for legionellae. The aim of this study was to screen industrial waters for the occurrence of FLA and their co-occurrence with legionellae. A total of 201 water samples, including 129 cooling waters and 72 process waters, and 30 cooling lubricants were included in the study. Treated waters were screened periodically, pre and post treatment. Altogether, 72.6% of the water samples were positive for FLA, acanthamoebae being most prevalent (in 23.9% of the samples) followed by Vermamoeba vermiformis (19.4%). Only one cooling lubricant was positive (Acanthamoeba genotype T4). Legionella spp. were detected in 34.8% of the water samples and in 15% in high concentrations (>1000 CFU/100 ml). Altogether, 81.4% of the Legionella-positive samples were positive for FLA by standard methods. By applying a highly sensitive nested PCR to a representative set of random samples it was revealed that Legionella spp. always co-occurred with Acanthamoeba spp. Although the addition of disinfectants did influence amoebal density and diversity, treated waters showed no difference concerning FLA in the interphases of disinfection. It appears that FLA can re-colonize treated waters within a short period of time.     

Quantitative Detection and Differentiation of Free-Living Amoeba Species Using SYBR Green–Based Real-Time PCR Melting Curve Analysis

Real-time polymerase chain reaction melting curve analysis (MCA) allows differentiation of several free-living amoebae species. Distinctive characteristics were found for Naegleria fowleri, N. lovaniensis, N. australiensis, N. gruberi, Hartmanella vermiformis, and Willaertia magna. Species specificity of the amplicons was confirmed using agarose gel electrophoresis and sequence-based approaches. Amplification efficiency ranged from 91% to 98%, indicating the quantitative potential of the assay. This MCA approach can be used for quantitative detection of free-living amoebae after cultivation but also as a culture-independent detection method.     

The Biochemical Identification of Vahlkampfiid Amoebae1

ABSTRACT. Isoenyme electrophoresis of three different enzymes was used to compare 16 strains of vahlkampfiid amoebae and a strain identified as a slime mold. The strain designated as an Echinostelium sp. was found to be an isolate of Naegleria fowleri on the basis of zymogram type and other characters, confirming Cursons & Brown's similar conclusion drawn in 1975. The N. fowleri strains examined expressed the typical zymograms of the species. The N. gruberi strains in this study presented two distinctive groups of patterns that were different from the two previously reported types for N. gruberi. Each of the remaining species studied formed single distinctive groups by which they could be identified.     

Legionella clemsonensis sp. nov.: a green fluorescing Legionella strain from a patient with pneumonia

A novel Legionella species was identified based on sequencing, cellular fatty acid analysis, biochemical reactions, and biofilm characterization. Strain D5610 was originally isolated from the bronchial wash of a patient in Ohio, USA. The bacteria were gram‐negative, rod‐shaped, and exhibited green fluorescence under long wave UV light. Phylogenetic analysis and fatty acid composition revealed a distinct separation within the genus. The strain grows between 26–45°C and forms biofilms equivalent to L. pneumophila Philadelphia 1. These characteristics suggest that this isolate is a novel Legionella species, for which the name Legionella clemsonensis sp nov. is proposed.     

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.     

Differences in the respiratory burst of human polymorphonuclear leukocytes induced by virulent and avirulent Legionella pneumophila Serogroup 1

Two strains of Legionella pneumophila of different virulence were examined for their influence on the metabolic oxidative activity of human polymorphonuclear leukocytes. The leukocytes exhibited decreased rates of oxygen consumption and diminished chemiluminescence activity following phagocytosis of a virulent strain of L. pneumophila serogroup 1. In contrast, phagocytosis of its multipassaged derivative rendered avirulent, was accompanied by increased rates of both oxygen consumption and chemiluminescence activity. Although no differences were observed in oxygen uptake induced by the virulent legionellae compared to leukocytes at rest, statistically significant differences were observed in the chemiluminescence responses. These observations were not unexpected, since the luminol-enhanced chemiluminescence assay, is more sensitive than the oxygen uptake assay. In spite of decreased metabolic activity of PMN in the presence of virulent legionellae, electron microscope studies showed higher numbers of intracellular L. pneumophila than the avirulent subtype. Thus, virulent and avirulent L. pneumophila can be differentiated on the basis of oxygen consumption and chemiluminescence assays.     

Effects of Grazing by the Free-Living Soil Amoebae Acanthamoeba castellanii, Acanthamoeba polyphaga, and Hartmannella vermiformis on Various Bacteria

Cultures of 10 different bacteria were used to serve as food sources for axenically grown Acanthamoeba castellanii, Acanthamoeba polyphaga, and Hartmannella vermiformis. The nonpigmented enterobacteriaceae Escherichia coli K-12 and Klebsiella aerogenes appeared to be excellent feed to all three amoebae. Hardly any growth or ammonium production was observed in tests with Chromatium vinosum and Serratia marcescens, which share the presence of pigmented compounds. Distinct differences in net ammonium production were detected and were correlated to the amoebal growth yield. In general, growth of amoebae and ammonium production increased in the order A. polyphaga, A. castellanii, and H. vermiformis.