Differential growth of Legionella pneumophila strains within a range of amoebae at various temperatures associated with in-premise plumbing

Aims: The potential effect of in‐premise plumbing temperatures (24, 32, 37 and 41°C) on the growth of five different Legionella pneumophila strains within free‐living amoebae (Acanthamoeba polyphaga, Hartmannella vermiformis and Naegleria fowleri) was examined. Methods and Results: Compared with controls that actively fed on Escherichia coli prey, when Leg. pneumophila was used as prey, strains Lp02 and Bloomington‐2 increased in growth at 30, 32 and 37°C while strains Philadelphia‐1 and Chicago 2 did not grow at any temperature within A. polyphaga. Strains Lp02, Bloomington‐2 and Dallas 1E did not proliferate in the presence of H. vermiformis nor did strain Philadelphia‐1 in the presence of N. fowleri. Yet, strain Bloomington‐2 grew at all temperatures examined within N. fowleri, while strain Lp02 proliferated at all temperatures except 41°C. More intriguing, strain Chicago 2 only grew at 32°C within H. vermiformis and N. fowleri suggesting a limited temperature growth range for this strain. Conclusions: Identifying the presence of pathogenic legionellae may require the use of multiple host amoebae and incubation temperatures. Significance and Impact of the Study: Temperature conditions and species of amoeba host supported in drinking water appear to be important for the selection of human‐pathogenic legionellae and point to future research required to better understand Legionella ecology.     

The importance of morphological versus chemical defences for the bloom-forming cyanobacterium Microcystis against amoebae grazing

Amoebae grazing can be an important loss factor for blooms of the common cyanobacterium Microcystis. Some Microcystis strains seem to be protected against amoebae grazing, but it is unclear whether this is achieved by their colony morphology or biochemically. These factors were investigated in grazing experiments using two Microcystis-grazing amoebae (Korotnevella sp. and Vannella sp.) and two Microcystis strains with differing colony morphology (aeruginosa and viridis morphotype) and different sensitivity to amoebae grazing. Amoebae did not increase in density and failed to reduce the growth rate of cultures of the amoebae insensitive viridis strain, irrespective of whether the Microcystis strain was colonial or unicellular. This suggests that the extended mucilage matrix surrounding viridis colonies is not the main defence mechanism against amoebae grazing. At the same time, the growth rate of both unicellular and colonial cultures of the amoebae-sensitive aeruginosa strain was heavily reduced by the growing amoebae. The addition of filtered viridis-conditioned medium to aeruginosa cultures significantly decreased both amoebae growth and its effect on aeruginosa growth rates, which indicates that extracellular compounds constitutively produced by viridis are at least partially responsible for their insensitivity to amoebae grazing. These results demonstrate the potential importance of chemical interactions between lower trophic levels (protists) for Microcystis bloom dynamics.     

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.     

Taxonomic Criteria for Limax Amoebae,with Descriptions of 3 New Species of Hartmannella and 3 of Vahlkampfia*

SYNOPSIS. Seven species of limax amoebae were isolated into clonal, monoxenic cultures with Aerobacter aerogenes from material collected from freshwater habitats. Studies were made of their trophic structure, nuclear division, cyst structure, some aspects of cytochemistry, and other characteristics. Six new species are described: Vahlkampfia inornata, V. avara, V. jugosa, Hartmannella limacoides, H. vermiformis, and H. exundans. The well-known species Naegleria gruberi (Schardinger, 1899) is re-described on the basis of 8 strains; its flagellated phase was found to be biflagellate, with rare exceptions. A correlation exists between the manner of locomotion and the pattern of nuclear division in the limax amoebae in the family Vahlkampfiidae and those in the genus Hartmannella. Trophic amoebae of all species had a PAS-positive surface layer, altho results with H. vermiformis and H. exundans were less definite than with other species. All species except H. limacoides formed cysts in culture. The cyst walls of all cyst-forming species were strongly PAS-positive, but results of the zinc chloroiodide test for cellulose were negative with the method used. The genus Hartmannella Alexeieff, 1912, is re-defined to include those species which assume a simple, monopodial limax-like form during locomotion and have nuclear division similar to that of metazoan cells and to distinguish it from the genus Acanthamoeba Volkonsky, 1931.     

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.     

Effect of Bacteria on Survival and Growth of Acanthamoeba castellanii

The growth and survival of Acanthamoeba castellanii in the presence of Gram-negative bacteria such as Pseudomonas aeruginosa, Escherichia coli, Serratia marcescens, and Stenotrophomonas maltophilia varied with the densities and species of bacteria. All species of bacteria suspended in a buffered saline at densities of 10⁵ to 10⁶/ml supported the growth and survival of 10⁶/ml trophozoites of Acanthamoeba castellanii in a buffered saline solution. At densities of bacteria to amoebae of 100:1 or greater, growth and survival of A. castellanii were suppressed, particularly by P. aeruginosa. In an enrichment medium, the rapid growth of most co-inoculated bacteria inhibited the growth and survival of the amoeba. Received: 12 September 1996 / Accepted: 20 September 1996     

The Occurrence of Multidrug-Resistant Pseudomonas aeruginosa on Hydrocarbon-Contaminated Sites

The main aim of this paper was the comprehensive estimation of the occurrence rate and the antibiotic-resistance conditions of opportunistic pathogen Pseudomonas aeruginosa in hydrocarbon-contaminated environments. From 2002 to 2007, 26 hydrocarbon-contaminated sites of Hungary were screened for the detection of environmental isolates. Altogether, 156 samples were collected and examined for the determination of appearance, representative cell counts, and antibiotic-resistance features of P. aeruginosa. The detected levels of minimal inhibitory concentrations of ten different drugs against 36 environmental strains were compared to the results of a widely used reference strain ATCC 27853 and four other clinical isolates of P. aeruginosa. Based on our long-term experiment, it can be established that species P. aeruginosa was detectable in case of 61.5% of the investigated hydrocarbon-contaminated sites and 35.2% of the examined samples that shows its widespread occurrence in polluted soil–groundwater systems. In the course of the antibiotic-resistance assay, our results determined that 11 of the examined 36 environmental strains had multiple drug-resistance against several clinically effective antimicrobial classes: cephalosporins, wide spectrum penicillins, carbapenems, fluoroquinolones, and aminoglycosides. The fact that these multiresistant strains were isolated from 8 different hydrocarbon-contaminated sites, mainly from outskirts, confirms that multiple drug-resistance of P. aeruginosa is widespread not only in clinical, but also in natural surroundings as well.     

Co-Existence of Multidrug-Resistant and -Susceptible Strains of Pseudomonas aeruginosa from a Single Clinical Isolate

Pseudomonas aeruginosa is a leading cause of hospital-acquired infections and difficult to treat due to acquired-resistance to multiple antibiotics. A pair of strains, M38100A and M38100B, previously identified from a single clinical isolate of P. aeruginosa was investigated to understand phenotypic and genotypic characteristics. Results revealed that the pair of strains was very similar for serum susceptibility, growth rate in a complex medium (Luria–Bertani), RAPD-genotype profiles, status of genes encoding type III secretion toxins, and no extra-chromosomal DNA. However, antibiotic susceptibility of the strain M38100B showed resistant to all tested-antibiotics while the strain M38100A showed susceptible to the same tested-antibiotics as similar levels of P. aeruginosa PAO1. The strain M38100B exhibited no growth in a minimal medium as a sole carbon and nitrogen source of glutamate while the strain M38100A grew well in the same minimal medium. These results suggest that multidrug resistance of the strain M38100B may be caused by multiple mutations on its genomic DNA and a precursor stage for a homogeneous multidrug resistant population.     

Grazing on <Emphasis Type="Italic">Microcystis</Emphasis> (Cyanophyceae) by testate amoebae with special reference to cyanobacterial abundance and physiological state

We examined the growth of testate amoebae preying on Microcystis whose physiological states were different in laboratory experiments and a hypertrophic pond. We prepared three experimental systems using water samples dominated by Microcystis aeruginosa: light incubation (control), dark incubation (dark), and light incubation with addition of nitrogen and phosphorus (+NP). In all the systems, the colony density of M. aeruginosa decreased slightly during incubation. Physiological activity of phytoplankton as determined by chlorophyll fluorescence was high and almost constant in the control and +NP systems, whereas it decreased in the dark system. Cell densities of testate amoebae increased in the control and +NP systems, whereas in the dark system they remained low. Thus, growth of the amoebae was low in the systems where physiological activity of Microcystis was low. In a hypertrophic pond, cell density of testate amoebae increased and remained high when M. aeruginosa predominated. Cell density of testate amoebae increased remarkably, simultaneously with the increases in M. aeruginosa colony density and phytoplankton physiological activity. We also found a significant correlation between densities of M. aeruginosa colonies and testate amoebae. We suggested that the physiological activity of Microcystis is one important factor affecting the growth of testate amoebae grazing on Microcystis.     

Effect of dissolved oxygen regime on growth dynamics of Pseudomonas spp during benzene degradation

We investigated the effect of different oxygen regimes on growth patterns of Pseudomonas spp. during benzene degradation in microcosm batch studies. Benzene degradation was induced by limiting oxygen available for microbial activity, which consists of three initial-dissolved oxygen (DO) levels of oxic, hypoxic, and anoxic conditions. Batch experiments were performed for cell growth and benzene degradation by inoculating three strains of Pseudomonas spp. (Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonas putida) in mineral salt medium containing aqueous benzene. Results showed that all strains were capable to grow and degrade benzene under all oxygen regimes but in a different manner. The highest cell growth of P. aeruginosa and P. fluorescens was achieved under oxic and anoxic condition, respectively, but there was no substantial difference on benzene degradation between the oxygen treatments with about 25% reduction for both strains. P. putida showed a facultative process for both cell growth and benzene degradation. This reveals that care should be taken in selection of microorganisms with regard to environmental studies since they exhibit different responses for given environmental conditions such as DO levels.     

Copper Uptake by <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> Isolated from Infected Burn Patients

Pseudomonas aeruginosa was isolated from infected burn patients and characterized by standard biochemical tests. The in vitro copper uptake was compared between this isolated pathogenic strain and two non-pathogenic control strains of Gram positive bacteria Bacillus thuringiensis strain Israelis as well as Gram negative bacteria Enterobacter aerogenes. Maximum copper uptake of 470 ppm/g biomass was obtained by P. aeruginosa strain, while the control strains B. thuringiensis and Enterobacter aerogenes had copper uptake of 350 and 383 ppm/g biomass, respectively. However, the lowest copper uptake (60 ppm/g biomass) was observed with another control the saprophytic strain Pseudomonas (Shewanella) putrefaciens. A further investigation regarding the effect of copper toxicity on bacterial growth, gave an MIC score of 600 ppm for P. aeruginosa strain compared to 460 and 300 ppm for the two Gram positive and Gram negative control strains, respectively. In tandem with these in vitro findings, blood analysis on burn patients infected with P. aeruginosa has indicated a selective decrease of copper (hypocupremia) and ceruloplasmin plasma levels. The iron metabolism was also affected by this copper deprivation leading to a similar decrease in plasma levels of PCV, iron, total iron binding capacity, and transferrin. All these hematological changes were significantly different (P < 0.05) from the matched group of non-infected burn patients. The observed hypocupremia in infected burn patients was attributed to demanding scavenger ability by P. aeruginosa strain for the copper of plasma.     

Evaluation of the ability of C. albicans to form biofilm in the presence of phage-resistant phenotypes of P. aeruginosa

Pseudomonas aeruginosa and Candida albicans are disparate microbial species, but both are known to be opportunistic pathogens frequently associated with nosocomial infections. The aim of this study was to provide a better understanding of the interactions between these microorganisms in dual-species biofilms. Several bacteriophage-resistant P. aeruginosa phenotypes have been isolated and were used in dual-species mixed-biofilm studies. Twenty-four and 48 h mixed-biofilms were formed using the isolated phenotypes of phage-resistant P. aeruginosa and these were compared with similar experiments using other P. aeruginosa strains with a defined lipopolysaccharide (LPS) deficiency based on chromosomal knockout of specific LPS biosynthetic genes. Overall, the results showed that the variants of phage-resistant P. aeruginosa and LPS mutants were both less effective in inhibiting the growth of C. albicans in mixed-biofilms compared to the wild-type strains of P. aeruginosa. Conversely, the proliferation of P. aeruginosa was not influenced by the presence of C. albicans. In conclusion, the ability of strains of P. aeruginosa to inhibit the formation of a biofilm of C. albicans appears to be correlated with the LPS chain lengths of phenotypes of P. aeruginosa, suggesting that LPS has a suppressive effect on the growth of C. albicans.     

Chlamydial endocytobionts of free-living amoebae differentially affect the growth rate of their hosts

Bacteria closely related to chlamydiae live and multiply as endocytobionts within free-living amoebae, making these amoebae potential vehicles of new emerging bacterial pathogens of humans. Hartmannella vermiformis containing endobiotic Neochlamydia hartmannellae grew more rapidly than those without endobionts, whilst Acanthamoeba sp. harbouring the Parachlamydia-related endocytobiont UWE25 multiplied more slowly than those without endobionts. The cause for the opposite effect of chlamydial endocytobionts on the growth of their host cells remains unknown.     

Strong effects of amoebae grazing on the biomass and genetic structure of a Microcystis bloom (Cyanobacteria)

Despite its importance for bloom toxicity, the factors determining the population structure of cyanobacterial blooms are poorly understood. Here, we report the results of a two‐year field survey of the population dynamics of Microcystis blooms in a small hypertrophic urban pond. Microscopic enumeration of Microcystis and its predators and parasites was combined with pigment and microcystin analysis and denaturing gradient gel electrophoresis of the ITS rDNA region to assess population dynamics and structure. Two main Microcystis morpho‐ and ITS types were revealed, corresponding to M. aeruginosa and M. viridis. In both years, high population densities of naked amoebae grazing on Microcystis coincided with rapid decreases in Microcystis biomass. In one year, there was a shift from heavily infested M. aeruginosa to the less‐infested M. viridis, allowing the bloom to rapidly recover. The preference of amoebae for M. aeruginosa was confirmed by grazing experiments, in which several amoeba strains were capable of grazing down a strain of M. aeruginosa, but not of M. viridis. Zooplankton and chytrid parasites appeared to be of minor importance for these strong and fast reductions in Microcystis biomass. These findings demonstrate a strong impact of small protozoan grazers on the biomass and genetic structure of Microcystis blooms.     

Conservation of xcp genes, involved in the two-step protein secretion process, in different Pseudomonas species and other gram-negative bacteria

Summary The two-step protein secretion pathway in Pseudomonas aeruginosa is dependent on the xcp genes. We investigated whether a similar secretion mechanism is present in non-pathogenic Pseudomonas spp. and in other gram-negative bacteria. The plant growth stimulating Pseudomonas strains P. putida WCS358, P. fuorescens WCS374 and Pseudomonas 1310 appeared to secrete proteins into the extracellular medium. Southern hybridization experiments showed the presence of xcp genes in these strains and also in other gram-negative bacteria, including Xanthomonas campestris. Complementation experiments showed that the xcp gene cluster of P. aeruginosa restored protein secretion in an X. campestris secretion mutant. The secretion gene cluster of X. campestris however, restored secretion capacity in P. aeruginosa mutants only to a low degree. Two heterologous proteins were not secreted by P. fuorescens and P. aeruginosa. The results suggest the presence of a similar two-step protein secretion mechanism in different gram-negative bacteria, which however, is not always functional for heterologous proteins.     

Degradation of Benzene,Toluene and Xylene by Pseudomonas aeruginosa Engineered with the Vitreoscilla Hemoglobin Gene

This study concerns the potential use of Pseudomonas aeruginosa expressing the Vitreoscilla hemoglobin gene for the degradation of important harmful aromatic compounds such as benzene, toluene, and xylene (BTX). The use of these compounds by both strains was determined as the production of cell mass (viable cell number) in a minimal medium containing any one of the BTX compounds as the sole carbon and energy source. Furthermore, the BTX degradation capability of both strains was monitored by measuring the production of 3‐methylcatechol, a common intermediate. For the cells of the logarithmic phase, which were grown at high aeration/high agitation or low aeration/low agitation, the engineered strain showed a better growth rate than the host strain. With the benzene in the medium, the recombinant strain exhibited a higher (up to 4‐fold) cell density than the parental wild‐type strain at this phase. In contrast, regarding the cells of the late stationary phase under high aeration/high agitation conditions, the host strain had generally higher viable cell numbers than the recombinant strain. At this phase this difference was, however, less significant under the conditions of low aeration/low agitation. Similarly, in toluene containing medium (at high aeration/high agitation) the recombinant strain showed a higher cell density which was from a 15‐fold to almost one order of magnitude greater than its parental strain during the logarithmic phase where the cell density of P. aeruginosa remained nearly constant. Contrary to the results with benzene and toluene, both strains exhibited similar growth characteristics when they were grown in the presence of xylene. The positive effect of the oxygen uptake by the recombinant system on the BTX metabolizing activity was also apparent in a high accumulation of 3‐methylcatechol in the cultures of the recombinant strain. At certain points of incubation, the hemoglobin expressing strain showed a significantly (p < 0.05) higher 3‐methylcatechol accumulation than the host strain. These results demonstrated the possible potential of the Vitreoscilla hemoglobin as an efficient oxygen uptake system for the bioremediation of some compounds of environmental concern.     

Evaluation of rhamnolipid production capacity of <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> PAO1 in comparison to the rhamnolipid over-producer strains DSM 7108 and DSM 2874

A lack of understanding of the quantitative rhamnolipid production regulation in bioreactor cultivations of Pseudomonas aeruginosa and the absence of respective comparative studies are important reasons for achieving insufficient productivities for an economic production of these biosurfactants. The Pseudomonas strains DSM 7108 and DSM 2874 are described to be good rhamnolipid over-producers. The strain PAO1 on the other hand is the best analyzed type strain for genetic regulation mechanisms in the species P. aeruginosa. These three strains were cultivated in a 30-L bioreactor with a medium containing nitrate and sunflower oil as sole C-source at 30 and 37 °C. The achieved maximum rhamnolipid concentrations varied from 7 to 38 g/L, the volumetric productivities from 0.16 to 0.43 g/(L·h), and the cellular yield from 0.67 to 3.15 g/g, with PAO1 showing the highest results for all of these variables. The molar di- to mono-rhamnolipid ratio changed during the cultivations; it was strain dependent but not significantly influenced by the temperature. This study explicitly shows that the specific rhamnolipid synthesis rate per cell follows secondary metabolite-like courses coinciding with the transition to the stationary phase of typical logistic growth behavior. However, the rhamnolipid synthesis was already induced before N-limitation occurred.     

Heterologous expression of human paraoxonases in <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> inhibits biofilm formation and decreases antibiotic resistance

Quorum sensing (QS) regulates virulence and biofilm formation in Pseudomonas aeruginosa and other medically relevant bacteria. Human paraoxonases (hPONs) are a family of closely related enzymes with multiple functions, including inactivation of the QS signal molecule in P. aeruginosa. However, there is no direct evidence to show the functions of hPONs on biofilm formation and antibiotic resistance in P. aeruginosa. In the present study, hPONs (hPON1, hPON2, and hPON3) genes were respectively cloned into the pMEKm12 shuttle vector and transformed into P. aeruginosa strain PAO1. Expression of the three recombinant proteins was confirmed by Western blotting, and growth of the recombinant strains was not affected by the hPONs gene expression. Biofilm formation and antibiotics resistance of the hPONs recombinant strains were analyzed. Our results showed that biofilm formation was significantly inhibited in all of the three hPONs recombinant strains. Interestingly, this inhibition can be reverted by addition of the corresponding hPONs polyclonal antibodies in the culture media, further indicating that the inhibition of biofilm formation was due to hPONs protein expression. In addition, we also demonstrated that hPONs expression decreased resistance of P. aeruginosa to gentamicin and ceftazidima, two antibiotics clinically used for the treatment of P. aeruginosa infection.     

Protochlamydia phocaeensis sp. nov., a new Chlamydiales species with host dependent replication cycle

Chlamydiae are pathogenic and symbiotic bacteria, which form an important part of amoeba-associated microorganisms. In this paper, we report the isolation, developmental cycle and genome analysis of Protochlamydia phocaeensis sp. nov., an obligate intracellular parasite with a large host spectrum, able to infect Acanthamoeba castellanii, Acanthamoeba polyphaga, and Vermamoeba vermiformis. The genome size is 3,424,182 bp with a GC content of 42%. This bacterium displayed a particular developmental cycle depending on the infected host. The P. phocaeensis showed typical inclusion vacuoles in A. castellanii, while these were absent in V. vermiformis. Since “Chlamydiae–amoebae” interactions are supposed to depend on the chlamydial species, our findings speculate that variations in the developmental cycle of certain Chlamydiae are also host dependent.