scholarly journals Increased Sensitivity of Amoeba-Grown Francisella Species to Disinfectants

2020 ◽  
Vol 8 (9) ◽  
pp. 1260 ◽  
Author(s):  
Maša Knežević ◽  
Valentina Marečić ◽  
Mateja Ožanič ◽  
Nikolina Špoljarić ◽  
Ina Kelava ◽  
...  
Keyword(s):  
Legionella Pneumophila ◽  
Benzalkonium Chloride ◽  
Isopropyl Alcohol ◽  
Acanthamoeba Castellanii ◽  
Bacterial Membrane ◽  
Bacterial Viability ◽  
Colony Forming Unit ◽  
Transmission Electron ◽  
Didecyldimethylammonium Chloride ◽  
Increased Sensitivity

Francisella tularensis is a highly infectious, intracellular bacterium and it is the causative agent of tularemia. The bacterium has been isolated from more than 250 species, including protozoa. Previous studies have shown that the growth of Legionella pneumophila within the amoeba results in a dramatic increase in the resistance to disinfectants. Since Francisella persists in the environment for years, this study investigates whether Acanthamoeba castellanii-grown F. novicida exhibits an alteration in the resistance to disinfectants. The disinfectants used are didecyldimethylammonium chloride (DDAC) combined with isopropyl alcohol (D1), benzalkonium chloride combined with DDAC and formic acid (D2), and polyhexamethylene biguanide (PHMB, D3). The effect of disinfectants on the bacterial viability is determined by a colony-forming unit (CFU), by transmission electron microscopy (TEM), by fluorescence microscopy, and the damage of the bacterial membrane. Our data has shown that only a one-log10 loss in bacterial viability is exhibited upon treatment of agar-grown Francisella, while in amoeba-grown Francisella there was a three-log10 difference with D3. The D1 disinfectant sterilized the bacteria within 10 s. The treatment of agar-grown F. novicida with D2 reduces bacterial viability by seven-log10 within 10 s and 15 min, respectively. Surprisingly, the treatment of amoeba-grown F. novicida with D2 results in a total loss of bacterial viability. In conclusion, A. castellanii-grown F. novicida is more susceptible to many disinfectants.

scholarly journals Development of a Fluorescent Tool for Studying Legionella bozemanae Intracellular Infection

2021 ◽  
Vol 9 (2) ◽  
pp. 379
Author(s):  
Breanne M. Head ◽  
Christopher I. Graham ◽  
Teassa MacMartin ◽  
Yoav Keynan ◽  
Ann Karen C. Brassinga
Keyword(s):  
Growth Kinetics ◽  
Legionella Pneumophila ◽  
Fluorescent Protein ◽  
Disease Incidence ◽  
Acanthamoeba Castellanii ◽  
Infection Model ◽  
Intracellular Infection ◽  
Green Fluorescent

Legionnaires’ disease incidence is on the rise, with the majority of cases attributed to the intracellular pathogen, Legionella pneumophila. Nominally a parasite of protozoa, L. pneumophila can also infect alveolar macrophages when bacteria-laden aerosols enter the lungs of immunocompromised individuals. L. pneumophila pathogenesis has been well characterized; however, little is known about the >25 different Legionella spp. that can cause disease in humans. Here, we report for the first time a study demonstrating the intracellular infection of an L. bozemanae clinical isolate using approaches previously established for L. pneumophila investigations. Specifically, we report on the modification and use of a green fluorescent protein (GFP)-expressing plasmid as a tool to monitor the L. bozemanae presence in the Acanthamoeba castellanii protozoan infection model. As comparative controls, L. pneumophila strains were also transformed with the GFP-expressing plasmid. In vitro and in vivo growth kinetics of the Legionella parental and GFP-expressing strains were conducted followed by confocal microscopy. Results suggest that the metabolic burden imposed by GFP expression did not impact cell viability, as growth kinetics were similar between the GFP-expressing Legionella spp. and their parental strains. This study demonstrates that the use of a GFP-expressing plasmid can serve as a viable approach for investigating Legionella non-pneumophila spp. in real time.

scholarly journals Subinhibitory Concentrations of Antimicrobial Agents Reduce the Uptake of Legionella pneumophila into Acanthamoeba castellanii and U937 Cells by Altering the Expression of Virulence-Associated Antigens

1998 ◽  
Vol 42 (11) ◽  
pp. 2870-2876 ◽  
Author(s):  
P. Christian Lück ◽  
Jürgen W. Schmitt ◽  
Arne Hengerer ◽  
Jürgen H. Helbig
Keyword(s):  
Monoclonal Antibodies ◽  
Membrane Protein ◽  
Outer Membrane ◽  
Outer Membrane Protein ◽  
Legionella Pneumophila ◽  
Antimicrobial Agents ◽  
Acanthamoeba Castellanii ◽  
Host Cells ◽  
Uncharacterized Protein ◽  
Subinhibitory Concentrations

ABSTRACT We determined the MICs of ampicillin, ciprofloxacin, erythromycin, imipenem, and rifampin for two clinical isolates of Legionella pneumophila serogroup 1 by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay and by quantitative culture. To test the influence of subinhibitory concentrations (sub-MICs) of antimicrobial agents on Legionella uptake into Acanthamoeba castellanii and U937 macrophage-like cells, both strains were pretreated with 0.25 MICs of the antibiotics for 24 h. In comparison to that for the untreated control, subinhibitory concentrations of antibiotics significantly reducedLegionella uptake into the host cells. Measurement of the binding of monoclonal antibodies against several Legionellaantigens by enzyme-linked immunoassays indicated that sub-MIC antibiotic treatment reduced the expression of the macrophage infectivity potentiator protein (Mip), the Hsp 60 protein, the outer membrane protein (OmpM), an as-yet-uncharacterized protein of 55 kDa, and a few lipopolysaccharide (LPS) epitopes. In contrast, the expression of some LPS epitopes recognized by monoclonal antibodies 8/5 and 30/4 as well as a 45-kDa protein, a 58-kDa protein, and the major outer membrane protein (OmpS) remained unaffected.

scholarly journals Peptidyl-Prolyl-cis/trans-Isomerases Mip and PpiB ofLegionella pneumophilaContribute to Surface Translocation, Growth at Suboptimal Temperature, and Infection

2018 ◽  
Vol 87 (1) ◽  
Author(s):  
J. Rasch ◽  
C. M. Ünal ◽  
A. Klages ◽  
Ü. Karsli ◽  
N. Heinsohn ◽  
...  
Keyword(s):  
Legionella Pneumophila ◽  
Acanthamoeba Castellanii ◽  
Temperature Tolerance ◽  
Lung Damage ◽  
Atypical Pneumonia ◽  
Content Type ◽  
Wide Range ◽  
Legionnaires Disease ◽  
Environmental Fitness ◽  
Sliding Motility

ABSTRACTThe gammaproteobacteriumLegionella pneumophilais the causative agent of Legionnaires’ disease, an atypical pneumonia that manifests itself with severe lung damage.L. pneumophila, a common inhabitant of freshwater environments, replicates in free-living amoebae and persists in biofilms in natural and man-made water systems. Its environmental versatility is reflected in its ability to survive and grow within a broad temperature range as well as its capability to colonize and infect a wide range of hosts, including protozoa and humans. Peptidyl-prolyl-cis/trans-isomerases (PPIases) are multifunctional proteins that are mainly involved in protein folding and secretion in bacteria. InL. pneumophilathe surface-associated PPIase Mip was shown to facilitate the establishment of the intracellular infection cycle in its early stages. The cytoplasmic PpiB was shown to promote cold tolerance. Here, we set out to analyze the interrelationship of these two relevant PPIases in the context of environmental fitness and infection. We demonstrate that the PPIases Mip and PpiB are important for surfactant-dependent sliding motility and adaptation to suboptimal temperatures, features that contribute to the environmental fitness ofL. pneumophila. Furthermore, they contribute to infection of the natural hostAcanthamoeba castellaniias well as human macrophages and human explanted lung tissue. These effects were additive in the case of sliding motility or synergistic in the case of temperature tolerance and infection, as assessed by the behavior of the double mutant. Accordingly, we propose that Mip and PpiB are virulence modulators ofL. pneumophilawith compensatory action and pleiotropic effects.

scholarly journals Effects of Fragrance Ingredients on the Uptake of Legionella pneumophila into Acanthamoeba castellanii

2018 ◽  
Vol 23 (4) ◽  
pp. 241-244
Author(s):  
HARUE NOMURA ◽  
SAKIKO TAKAHASHI ◽  
YUKI TOHARA ◽  
YASUNORI ISSHIKI ◽  
KEISUKE SAKUDA ◽  
...  
Keyword(s):  
Legionella Pneumophila ◽  
Acanthamoeba Castellanii

The amoebae plate test implicates a paralogue of lpxB in the interaction of Legionella pneumophila with Acanthamoeba castellanii

Microbiology ◽  
2005 ◽  
Vol 151 (1) ◽  
pp. 167-182 ◽  
Author(s):  
Urs Albers ◽  
Katrin Reus ◽  
Howard A. Shuman ◽  
Hubert Hilbi
Keyword(s):  
Legionella Pneumophila ◽  
Lipid A ◽  
Sequence Similarity ◽  
Acanthamoeba Castellanii ◽  
Growth Defect ◽  
Wild Type ◽  
Intracellular Growth ◽  
Plate Test ◽  
Raw264.7 Macrophages ◽  
Mutant Strains

Legionella pneumophila is a bacterial parasite of freshwater amoebae which also grows in alveolar macrophages and thus causes the potentially fatal pneumonia Legionnaires' disease. Intracellular growth within amoebae and macrophages is mechanistically similar and requires the Icm/Dot type IV secretion system. This paper reports the development of an assay, the amoebae plate test (APT), to analyse growth of L. pneumophila wild-type and icm/dot mutant strains spotted on agar plates in the presence of Acanthamoeba castellanii. In the APT, wild-type L. pneumophila formed robust colonies even at high dilutions, icmT, -R, -P or dotB mutants failed to grow, and icmS or -G mutants were partially growth defective. The icmS or icmG mutant strains were used to screen an L. pneumophila chromosomal library for genes that suppress the growth defect in the presence of the amoebae. An icmS suppressor plasmid was isolated that harboured the icmS and flanking icm genes, indicating that this plasmid complements the intracellular growth defect of the mutant. In contrast, different icmG suppressor plasmids rendered the icmG mutant more cytotoxic for A. castellanii without enhancing intracellular multiplication in amoebae or RAW264.7 macrophages. Deletion of individual genes in the suppressor plasmids inserts identified lcs (Legionella cytotoxic suppressor) -A, -B, -C and -D as being required for enhanced cytotoxicity of an icmG mutant strain. The corresponding proteins show sequence similarity to hydrolases, NlpD-related metalloproteases, lipid A disaccharide synthases and ABC transporters, respectively. Overexpression of LcsC, a putative paralogue of the lipid A disaccharide synthase LpxB, increased cytotoxicity of an icmG mutant but not that of other icm/dot or rpoS mutant strains against A. castellanii. Based on sequence comparison and chromosomal location, lcsB and lcsC probably encode enzymes involved in cell wall maintenance and peptidoglycan metabolism. The APT established here may prove useful to identify other bacterial factors relevant for interactions with amoeba hosts.

scholarly journals Biodiversity of Amoebae and Amoeba-Resisting Bacteria in a Hospital Water Network

2006 ◽  
Vol 72 (4) ◽  
pp. 2428-2438 ◽  
Author(s):  
Vincent Thomas ◽  
Katia Herrera-Rimann ◽  
Dominique S. Blanc ◽  
Gilbert Greub
Keyword(s):  
Legionella Pneumophila ◽  
Strong Association ◽  
Acanthamoeba Castellanii ◽  
Control Measures ◽  
Water Network ◽  
Water Control ◽  
Domestic Water ◽  
Mycobacterium Xenopi ◽  
Hartmannella Vermiformis ◽  
Human Infections

ABSTRACT Free-living amoebae (FLA) are ubiquitous organisms that have been isolated from various domestic water systems, such as cooling towers and hospital water networks. In addition to their own pathogenicity, FLA can also act as Trojan horses and be naturally infected with amoeba-resisting bacteria (ARB) that may be involved in human infections, such as pneumonia. We investigated the biodiversity of bacteria and their amoebal hosts in a hospital water network. Using amoebal enrichment on nonnutrient agar, we isolated 15 protist strains from 200 (7.5%) samples. One thermotolerant Hartmannella vermiformis isolate harbored both Legionella pneumophila and Bradyrhizobium japonicum. By using amoebal coculture with axenic Acanthamoeba castellanii as the cellular background, we recovered at least one ARB from 45.5% of the samples. Four new ARB isolates were recovered by culture, and one of these isolates was widely present in the water network. Alphaproteobacteria (such as Rhodoplanes, Methylobacterium, Bradyrhizobium, Afipia, and Bosea) were recovered from 30.5% of the samples, mycobacteria (Mycobacterium gordonae, Mycobacterium kansasii, and Mycobacterium xenopi) were recovered from 20.5% of the samples, and Gammaproteobacteria (Legionella) were recovered from 5.5% of the samples. No Chlamydia or Chlamydia-like organisms were recovered by amoebal coculture or detected by PCR. The observed strong association between the presence of amoebae and the presence of Legionella (P < 0.001) and mycobacteria (P = 0.009) further suggests that FLA are a reservoir for these ARB and underlines the importance of considering amoebae when water control measures are designed.

C60 fullerene tubes as removable templates

2006 ◽  
Vol 21 (2) ◽  
pp. 529-534 ◽  
Author(s):  
Jun-ichi Minato ◽  
Kun'ichi Miyazawa
Keyword(s):  
Methyl Alcohol ◽  
Isopropyl Alcohol ◽  
Optical Observations ◽  
C60 Fullerene ◽  
Outer Diameter ◽  
Solution Deposition ◽  
Transmission Electron ◽  
Inner Diameter ◽  
Ultrasonic Pulverization ◽  
Capillary Attraction

Crystalline microtubes (inner diameter 240–2100 nm) consisting of C60 fullerene molecules were prepared in the mixture of C60-saturated pyridine and isopropyl alcohol kept at 0 °C. Characterization by transmission electron microscopy showed a linear relationship between the outer diameter and the inner diameter for the C60 fullerene tubes. Optical observations suggested that the specimens already had tubular structure when they were grown in the solution. Deposition of guest crystals inside the C60 fullerene tubes was performed by the following procedure: (i) ultrasonic pulverization to obtain the C60 fullerene tubes with open ends, (ii) absorption of methyl alcohol solution of KBr into the tubes by the capillary attraction, and (iii) evaporation of methyl alcohol to precipitate KBr inside the tubes. Columnar KBr crystals were then obtained by dissolving the fullerene tube walls in toluene.

scholarly journals Mitigation of Expression of Virulence Genes in Legionella pneumophila Internalized in the Free-Living Amoeba Willaertia magna C2c Maky

Pathogens ◽  
2020 ◽  
Vol 9 (6) ◽  
pp. 447 ◽  
Author(s):  
Rayane Mouh Mameri ◽  
Jacques Bodennec ◽  
Laurent Bezin ◽  
Sandrine Demanèche
Keyword(s):  
Gene Expression ◽  
Legionella Pneumophila ◽  
Virulence Genes ◽  
Natural Habitat ◽  
Acanthamoeba Castellanii ◽  
Severe Form ◽  
Aquatic Environments ◽  
Free Living ◽  
Intracellular Parasites ◽  
Free Living Amoeba

Legionella pneumophila is a human pathogen responsible for a severe form of pneumonia named Legionnaire disease. Its natural habitat is aquatic environments, being in a free state or intracellular parasites of free-living amoebae, such as Acanthamoeba castellanii. This pathogen is able to replicate within some amoebae. Willaertia magna C2c Maky, a non-pathogenic amoeba, was previously demonstrated to resist to L. pneumophila and even to be able to eliminate the L. pneumophila strains Philadelphia, Lens, and Paris. Here, we studied the induction of seven virulence genes of three L. pneumophila strains (Paris, Philadelphia, and Lens) within W. magna C2c Maky in comparison within A. castellanii and with the gene expression level of L. pneumophila strains alone used as controls. We defined a gene expression-based virulence index to compare easily and without bias the transcript levels in different conditions and demonstrated that W. magna C2c Maky did not increase the virulence of L. pneumophila strains in contrast to A. castellanii. These results confirmed the non-permissiveness of W. magna C2c Maky toward L. pneumophila strains.

scholarly journals Behavior of Yersinia enterocolitica in the Presence of the Bacterivorous Acanthamoeba castellanii

2013 ◽  
Vol 79 (20) ◽  
pp. 6407-6413 ◽  
Author(s):  
E. Lambrecht ◽  
J. Baré ◽  
I. Van Damme ◽  
W. Bert ◽  
K. Sabbe ◽  
...  
Keyword(s):  
Yersinia Enterocolitica ◽  
Pathogenic Bacteria ◽  
Acanthamoeba Castellanii ◽  
Ecological Niches ◽  
Free Living ◽  
Content Type ◽  
Food Borne ◽  
Transmission Electron ◽  
Set Up ◽  
The Impact

ABSTRACTFree-living protozoa play an important role in the ecology and epidemiology of human-pathogenic bacteria. In the present study, the interaction betweenYersinia enterocolitica, an important food-borne pathogen, and the free-living amoebaAcanthamoeba castellaniiwas studied. Several cocultivation assays were set up to assess the resistance ofY. enterocoliticatoA. castellaniipredation and the impact of environmental factors and bacterial strain-specific characteristics. Results showed that allY. enterocoliticastrains persist in association withA. castellaniifor at least 14 days, and associations withA. castellaniienhanced survival ofYersiniaunder nutrient-rich conditions at 25°C and under nutrient-poor conditions at 37°C. Amoebae cultivated in the supernatant of oneYersiniastrain showed temperature- and time-dependent permeabilization. Intraprotozoan survival ofY. enterocoliticadepended on nutrient availability and temperature, with up to 2.8 log CFU/ml bacteria displaying intracellular survival at 7°C for at least 4 days in nutrient-rich medium. Transmission electron microscopy was performed to locate theYersiniacells inside the amoebae. AsYersiniaandAcanthamoebashare similar ecological niches, this interaction identifies a role of free-living protozoa in the ecology and epidemiology ofY. enterocolitica.

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