Relationship between Legionella pneumophila and Acanthamoeba polyphaga: physiological status and susceptibility to chemical inactivation.

Survival studies were conducted on Legionella pneumophila cells that had been grown intracellularly in Acanthamoeba polyphaga and then exposed to polyhexamethylene biguanide (PHMB), benzisothiazolone (BIT), and 5-chloro-N-methylisothiazolone (CMIT). Susceptibilities were also determined for L. pneumophila grown under iron-sufficient and iron-depleted conditions. BIT was relatively ineffective against cells grown under iron depletion; in contrast, iron-depleted conditions increased the susceptibilities of cells to PHMB and CMIT. The activities of all three biocides were greatly reduced against L. pneumophila grown in amoebae. PHMB (1 x MIC) gave 99.99% reductions in viability for cultures grown in broth within 6 h and no detectable survivors at 24 h but only 90 and 99.9% killing at 6 h and 24 h, respectively, for cells grown in amoebae. The antimicrobial properties of the three biocides against A. polyphaga were also determined. The majority of amoebae recovered from BIT treatment, but few, if any, survived CMIT treatment or exposure to PHMB. This study not only shows the profound effect that intra-amoebal growth has on the physiological status and antimicrobial susceptibility of L. pneumophila but also reveals PHMB to be a potential biocide for effective water treatment. In this respect, PHMB has significant activity, below its recommended use concentrations, against both the host amoeba and L. pneumophila.     

Relationship between Legionella pneumophila and Acanthamoeba polyphaga: physiological status and susceptibility to chemical inactivation.

Survival studies were conducted on Legionella pneumophila cells that had been grown intracellularly in Acanthamoeba polyphaga and then exposed to polyhexamethylene biguanide (PHMB), benzisothiazolone (BIT), and 5-chloro-N-methylisothiazolone (CMIT). Susceptibilities were also determined for L. pneumophila grown under iron-sufficient and iron-depleted conditions. BIT was relatively ineffective against cells grown under iron depletion; in contrast, iron-depleted conditions increased the susceptibilities of cells to PHMB and CMIT. The activities of all three biocides were greatly reduced against L. pneumophila grown in amoebae. PHMB (1 x MIC) gave 99.99% reductions in viability for cultures grown in broth within 6 h and no detectable survivors at 24 h but only 90 and 99.9% killing at 6 h and 24 h, respectively, for cells grown in amoebae. The antimicrobial properties of the three biocides against A. polyphaga were also determined. The majority of amoebae recovered from BIT treatment, but few, if any, survived CMIT treatment or exposure to PHMB. This study not only shows the profound effect that intra-amoebal growth has on the physiological status and antimicrobial susceptibility of L. pneumophila but also reveals PHMB to be a potential biocide for effective water treatment. In this respect, PHMB has significant activity, below its recommended use concentrations, against both the host amoeba and L. pneumophila.     

Biguanide-induced changes in Acanthamoeba castellanii: an electron microscopic study

Trophozoites and cysts of Acanthamoeba castellanii were exposed to chlorhexidine diacetate (CHA) and polyhexamethylene biguanide (PHMB); changes in cell ultrastructure and surface structure were examined by both transmission and scanning electron microscopy. PHMB caused a greater degree of structural and membrane damage; the cytoplasmic contents were severely depleted and there were clusters of densely stained precipitates on the cell surface. Concentrations of CHA greater than 100 μg ml⁻¹ produced shrinkage from the cyst wall. At high concentrations, PHMB induced a slight withdrawal of the cytoplasm from the wall and, unlike CHA, induced swelling of the cysts. These findings do not define the mechanisms of action of CHA and PHMB, but provide evidence that a major target site for both agents is the plasma membrane. However, additional intracellular damage undoubtedly contributes to the lethal effects. The greater resistance of cysts may be associated with reduced biguanide uptake.     

Biguanide-induced changes in Acanthamoeba castellanii: an electron microscopic study

Trophozoites and cysts of Acanthamoeba castellanii were exposed to chlorhexidine diacetate (CHA) and polyhexamethylene biguanide (PHMB); changes in cell ultrastructure and surface structure were examined by both transmission and scanning electron microscopy. PHMB caused a greater degree of structural and membrane damage; the cytoplasmic contents were severely depleted and there were clusters of densely stained precipitates on the cell surface. Concentrations of CHA greater than 100 microg ml(-1) produced shrinkage from the cyst wall. At high concentrations, PHMB induced a slight withdrawal of the cytoplasm from the wall and, unlike CHA, induced swelling of the cysts. These findings do not define the mechanisms of action of CHA and PHMB, but provide evidence that a major target site for both agents is the plasma membrane. However, additional intracellular damage undoubtedly contributes to the lethal effects. The greater resistance of cysts may be associated with reduced biguanide uptake.     

The lethal effects of biguanides on cysts and trophozoites of Acanthamoeba castellanii

The effects of a range of biocides on trophozoite and encysted forms of Acanthamoeba castellanii were investigated. Viable acanthamoebae were enumerated by a plaque assay technique. The cyst form of Acanthamoeba castellanii was more resistant to all biocides tested than the trophozoite form. Of the biocides tested, chlorhexidine diacetate (CHA) and polyhexamethylene biguanide (PHMB) were the most effective. Their lethal effects were time- and concentration-dependent. CHA was very effective when formulated in 0.1% EDTA combined with Tris buffer pH 7.8 whereas PHMB activity was reduced by 0.1% EDTA. Three per cent dimethylsulphoxide (DMSO) enhanced the activity of CHA but not of PHMB.     

Resistance, biguanide sorption and biguanide-induced pentose leakage during encystment of Acanthamoeba castellanii

AIMS: This study investigates the effects of biguanides during encystment of Acanthamoeba castellanii. METHODS AND RESULTS: A non-nutrient encystment system was used to investigate the changes in the levels of sorption (uptake) of three non-cysticidal concentrations (10, 20 and 50 microg ml(-1)) of chlorhexidine diacetate (CHA) and polyhexamethylene biguanide (PHMB) as well as their effects on viability and leakage of pentose sugars during the first 36 h of encystment. Trophozoites treated with CHA or PHMB were more sensitive and generally sorbed more of each biocide than cysts. During encystment, the largest increases in resistance developed between 18 and 36 h for both biguanides with the resistance emerging to biguanide concentrations of 10 or 20 microg ml(-1) between 18 and 24 h. At 50 microg ml(-1) resistance emerged between 24 and 36 h. There was a general decrease in biocide sorption during encystment between 0-24 and 0-21 h for CHA and PHMB, respectively, at a concentration of 50 microg ml(-1). The greatest decline in biguanide-induced pentose leakage was between 0 and 12 h. CONCLUSIONS: The results suggest that during encystment some of the changes in the susceptibility to CHA or PHMB may be related to decreases in the levels of biocide sorption, which is limited by the developing cyst wall. SIGNIFICANCE AND IMPACT OF THE STUDY: During encystation, changes occur in biguanide sensitivity. The physical barrier of the cyst wall may be an important factor in limiting biocide sorption.     

Aspects of the mechanisms of action of biguanides on trophozoites and cysts of Acanthamoeba castellanii

A non-radioactive method was used to investigate the uptake by Acanthamoeba castellanii of chlorhexidine diacetate (CHA) and polyhexamethylene biguanide (PHMB). Based on the Giles et al. (1974) hypothesis, the uptake of CHA by trophozoites appeared to be of the L3 pattern whereas that of cysts was C2. Unlike CHA, trophozoites took up PHMB with an L2 pattern at low concentrations followed by a C-type pattern at higher concentrations, the uptake by cysts was found to be of the C2 pattern with a plateau effect at high concentrations. A diphasic leakage effect was found in trophozoites whereas a relatively low peak of maximal leakage occurred from cysts treated with high biocide concentrations. The amount of pentose release depended on the formulation ingredients. No correlation between pentose leakage and trophozoicidal or cysticidal activity was found.     

农村生活污水处理厂中阿米巴原虫的定量检测

【目的】自由生活的棘阿米巴属(Acanthamoeba spp.)和哈曼属原虫(Hartmannella vermiformis)普遍存在于自然界的土壤和各种水体中,这两个属中的某些种类被认为对人和动物具有潜在的致病性,应用染料法实时荧光定量PCR技术建立特异性强、灵敏度高及重复性好的快速检测阿米巴虫的方法具有实际意义。【方法】采用非培养方法选择适合低拷贝基因检测的荧光染料BRYT Green? dye用于农村生活污水处理厂不同工艺阶段水样中Acanthamoeba spp.和H. Vermiformis 18S rRNA基因的检测和定量分析。【结果】在整个处理工艺流程中均检测到Acanthamoeba spp.和H. Vermiformis,并呈现出不同的变化趋势,进水中分别达到8.70×105拷贝/L和1.84×106拷贝/L。与进水相比,调节池、好氧池和膜池中阿米巴原虫的数量均降低了1?2个数量级,但是出水中Acanthamoeba spp. 则出现增加趋势。【结论】对阿米巴原虫可能造成的潜在健康危害应引起重视,并有必要作为污水处理达标的补充标准。     

Pathogen- and Host-Directed Antileishmanial Effects Mediated by Polyhexanide (PHMB)

Background

Cutaneous leishmaniasis (CL) is a neglected tropical disease caused by protozoan parasites of the genus Leishmania. CL causes enormous suffering in many countries worldwide. There is no licensed vaccine against CL, and the chemotherapy options show limited efficacy and high toxicity. Localization of the parasites inside host cells is a barrier to most standard chemo- and immune-based interventions. Hence, novel drugs, which are safe, effective and readily accessible to third-world countries and/or drug delivery technologies for effective CL treatments are desperately needed.

Methodology/Principal Findings

Here we evaluated the antileishmanial properties and delivery potential of polyhexamethylene biguanide (PHMB; polyhexanide), a widely used antimicrobial and wound antiseptic, in the Leishmania model. PHMB showed an inherent antileishmanial activity at submicromolar concentrations. Our data revealed that PHMB kills Leishmania major (L. major) via a dual mechanism involving disruption of membrane integrity and selective chromosome condensation and damage. PHMB’s DNA binding and host cell entry properties were further exploited to improve the delivery and immunomodulatory activities of unmethylated cytosine-phosphate-guanine oligodeoxynucleotides (CpG ODN). PHMB spontaneously bound CpG ODN, forming stable nanopolyplexes that enhanced uptake of CpG ODN, potentiated antimicrobial killing and reduced host cell toxicity of PHMB.

Conclusions

Given its low cost and long history of safe topical use, PHMB holds promise as a drug for CL therapy and delivery vehicle for nucleic acid immunomodulators.     

X-ray microanalysis of chlorine and phosphorus content in biguanide-treated Acanthamoeba castellanii

Energy dispersive analysis of X-rays (EDAX) was used to study the effects of chlorhexidine diacetate (CHA) and polyhexamethylene biguanide (PHMB) on Acanthamoeba castellanii. A high variation of elements occurred in untreated individual cells and only two elements, Cl (a biocide marker) and P, were investigated. X-ray dot mapping of untreated trophozoites and cysts revealed that Cl in cells was uniformly distributed throughout the cytoplasm, whereas P was less dense in the vacuoles. X-ray dots of Cl in biocide-treated trophozoites and cysts appeared denser and evenly distributed within the cells as the biguanide concentration increased. Quantitative analysis of either CHA or PHMB within the cells using Cl as an elemental marker was unsatisfactory because of the high Cl levels in untreated cells. The apparent increases of P in some experiments with treated cells might be associated with reduced permeability, protein coagulation or aggregation of phospholipids.     

Biodegradability of end-groups of the biocide polyhexamethylene biguanide (PHMB) assessed using model compounds

Polyhexamethylene biguanide (PHMB), a biocide used in a wide variety of disinfection and preservation applications, is a polydisperse mixture in which the end-groups may be any combination of amine, guanidine and cyanoguanidine. Using PHMB model compounds (1,6-diaminohexane; 1,6-diguanidinohexane; 1,6-di(cyanoguanidino)hexane; 4-guanidinobutyric acid), we have determined the biodegradation characteristics of each end-group in several strains of bacteria isolated for their ability to utilise PHMB as a sole source of nitrogen. Bacteria were screened for growth at the expense of each model compound (at non-inhibitory concentrations) as sole nitrogen source. None of the isolated bacteria was capable of utilising a cyanoguanidine end-group as growth substrate, whereas several bacteria were shown to utilise amine or guanidine end-groups. In particular, a strain of Pseudomonas putida was capable of extensive growth with 1,6-diguanidinohexane as a sole nitrogen source, with complete removal of guanidine groups from culture medium within 2 days, and with concomitant formation of unsubstituted urea, which in turn was also utilised by the organism. We conclude that whilst amine and guanidine end-groups in PHMB are likely to be susceptible to biodegradation, cyanoguanidine end-groups are likely to be recalcitrant.     

Microbial degradation of the biocide polyhexamethylene biguanide: isolation and characterization of enrichment consortia and determination of degradation by measurement of stable isotope incorporation into DNA

AIMS: To isolate micro-organisms capable of utilizing polyhexamethylene biguanide (PHMB) as a sole source of nitrogen, and to demonstrate biodegradation of the biocide. METHODS AND RESULTS: Two consortia of bacteria were successfully enriched at the expense of PHMB, using sand from PHMB-treated swimming pools as inoculum. Both consortia were shown to contain bacteria belonging to the genera Sphingomonas, Azospirillum and Mesorhizobium. It was shown that the presence of both Sphingomonas and Azospirillum spp. was required for extensive growth of the consortia. In addition, the Sphingomonads were the only isolates capable of growth in axenic cultures dosed with PHMB. Using a stable isotope (15N)-labelled PHMB, metabolism of the biocide by both consortia was demonstrated. By comparing the level of 15N atom incorporation into bacterial DNA after growth on either 15N-PHMB or 15N-labelled NH4Cl, it was possible to estimate the percentage of PHMB biodegradation. CONCLUSIONS: The microbial metabolism of nitrogen from the biguanide moiety of PHMB has been demonstrated. It was revealed that Sphingomonas and Azospirillum spp. are the principal organisms responsible for growth at the expense of PHMB. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study to demonstrate the microbial metabolism of PHMB.     

Weak cytotoxic activity of miltefosine against clinical isolates of Acanthamoeba spp

Hexadecylphosphocholine (miltefosine) is an anticancer drug active in vitro against various protozoan parasites, and recently used for the treatment of disseminated Acanthamoeba infection. In the present study, we present results of weak cytotoxic activity of this potential amoebicidal agent for 2 of 3 clinical isolates of Acanthamoeba spp. Although the inhibition effect for all tested concentrations was apparent, and showed 100% eradication of trophozoites of Acanthamoeba castellanii strain at a concentration of 62.5 μM after 24 hr, the strains Acanthamoeba sp. and Acanthamoeba lugdunensis exhibited low sensitivity to hexadecylphosphocholine, even in high concentrations. The determined minimal trophocidal concentrations were 250 μM for Acanthamoeba sp. and 500 μM for A. lugdunensis after 24 hr of exposure. Although hexadecylphosphocholine is a potential agent for treatment of Acanthamoeba keratitis and systemic infections, in clinical practice the possible insusceptibility of the amoebic strain should be considered for optimizing therapy.     

Area 51: How do Acanthamoeba invade the central nervous system?

Acanthamoeba granulomatous encephalitis generally develops as a result of haematogenous spread, but it is unclear how circulating amoebae enter the central nervous system (CNS) and cause inflammation. At present, the mechanisms which Acanthamoeba use to invade this incredibly well-protected area of the CNS and produce infection are not well understood. In this paper, we propose two key virulence factors: mannose-binding protein and extracellular serine proteases as key players in Acanthamoeba traversal of the blood-brain barrier leading to neuronal injury. Both molecules should provide excellent opportunities as potential targets in the rational development of therapeutic interventions against Acanthamoeba encephalitis.     

Characterization of renatured profilin purified by urea elution from poly-L-proline agarose columns

We present evidence that native profilin can be purified from cellular extracts of Acanthamoeba, Dictyostelium, and human platelets by affinity chromatography on poly-L-proline agarose. After applying cell extracts and washing the column with 3 M urea, homogeneous profilin is eluted by increasing the urea concentration to 6-8 M. Acanthamoeba profilin-I and profilin-II can subsequently be separated by cation exchange chromatography. The yield of Acanthamoeba profilin is twice that obtained by conventional methods. Several lines of evidence show that the profilins fully renature after removal of the urea by dialysis: 1) dialyzed Acanthamoeba and human profilins rebind quantitatively to poly-L-proline and bind to actin in the same way as native, conventionally purified profilin without urea treatment; 2) dialyzed profilins form 3-D crystals under the same conditions as native profilins; 3) dialyzed Acanthamoeba profilin-I has an NMR spectrum identical with that of native profilin-I; and 4) dialyzed human and Acanthamoeba profilins inhibit actin polymerization. We report the discovery of profilin in Dictyostelium cell extracts using the same method. Based on these observations we conclude that urea elution from poly-L-proline agarose followed by renaturation will be generally useful for preparing profilins from a wide variety of cells. Perhaps also of general use is the finding that either myosin-II or alpha-actinin in crude cell extracts can be bound selectively to the poly-L-proline agarose column depending on the ionic conditions used to equilibrate the column. We have purified myosin-II from both Acanthamoeba and Dictyostelium cell extracts and alpha-actinin from Acanthamoeba cell extracts in the appropriate buffers. These proteins are retained as complexes with actin by the agarose and not by a specific interaction with poly-L-proline. They can be eluted by dissociating the complexes with ATP and separated from actin by gel filtration if necessary.     

Acanthamoeba: keratopathogenicity of isolates from domestic tap water in Korea

In a previous study, we reported on the contamination rate of free living amoeba, including Acanthamoeba, isolated from contact lens storage cases (CLSC) and domestic tap water in Korea. In an effort to evaluate the potential kerato-pathogenicity of 5 isolates from CLSC and 17 isolates from domestic tap water, we have conducted an investigation into the morphological features, mitochondrial DNA (mtDNA) restriction fragment length polymorphism (RFLP) phenotypes, 18S rDNA sequences, and drug sensitivities of these isolates, and have compared the results with those of 20 amoebic keratitis (AK) isolates from Korea, as well as 14 reference strains. Cysts from 22 isolates obtained from CLSC and domestic tap water showed typical characteristics of morphological group 2. A total of three and five mtDNA RFLP patterns generated by EcoRI were found in 5 of the isolates from CLSC and 17 of the isolates from domestic tap water, respectively. The mtDNA RFLP patterns of four of the five isolates from the CLSC were found to be identical to those of the isolates from domestic tap water of students who had contaminated CLSC. The majority had mtDNA RFLP patterns identical to those of AK isolates in Korea. The results of 18S rDNA sequencing analysis were also shown to coincide with the results of mtDNA RFLP analysis. KA/WP12 was determined to be profoundly sensitive to chlorhexidine (MCC; 6.25microg/ml), and KAWP2 was the most sensitive strain to polyhexamethylene biguanide (PHMB) (MCC; 4.69microg/ml). Some difference in the cytopathic effects of isolates against human corneal epithelial cells was observed according to their mtDNA genotypes. In conclusion, domestic tap water may constitute a source of Acanthamoeba contamination of CLSC, and most isolates from CLSC and domestic tap water appear to be potentially keratopathogenic.     

三种消毒剂对大黄鱼致病性弧菌的体外杀灭作用研究

为了解不同消毒剂对大黄鱼中致病性弧菌的抑菌效果。采用纸片扩散法比较了聚维酮碘(PVP-I)、聚六亚甲基双胍(PHMB)和过氧化氢(H2O2)对溶藻弧菌和副溶血性弧菌的抑菌效果,再以肉汤稀释法测定了三种消毒剂的最低抑菌浓度(MIC)、最低杀菌浓度(MBC)及MBC/MIC比值,在此基础上,获得了抑菌效果相对较优的H2O2、PHMB对溶藻弧菌、副溶血性弧菌及其混菌的杀菌动力学曲线。结果表明,H2O2(3%)对两种弧菌具有最大的抑菌圈直径(19~21mm),PHMB(30mg/mL)次之(约为15mm),PVP-I(10%)的抑菌效果最弱(小于10mm)。3种消毒剂的MIC值由小到大依次为PHMB相似文献   

Novel Chlamydiales strains isolated from a water treatment plant

Chlamydiae are obligate intracellular bacteria infecting free-living amoebae, vertebrates and some invertebrates. Novel members are regularly discovered, and there is accumulating evidence supporting a very important diversity of chlamydiae in the environment. In this study, we investigated the presence of chlamydiae in a drinking water treatment plant. Samples were used to inoculate Acanthamoeba monolayers ( Acanthamoeba co-culture), and to recover autochthonous amoebae onto non-nutritive agar. Chlamydiae were searched for by a pan-chlamydia 16S rRNA gene PCR from both Acanthamoeba co-cultures and autochthonous amoebae, and phylotypes determined by 16S rRNA gene sequencing. Autochthonous amoebae also were identified by 18S rRNA gene amplification and sequencing. From a total of 79 samples, we recovered eight chlamydial strains by Acanthamoeba co-culture, but only one of 28 amoebae harboured a chlamydia. Sequencing results and phylogenetic analysis showed our strains belonging to four distinct chlamydial lineages. Four strains, including the strain recovered within its natural host, belonged to the Parachlamydiaceae ; two closely related strains belonged to the Criblamydiaceae ; two distinct strains clustered with Rhabdochlamydia spp.; one strain clustered only with uncultured environmental clones. Our results confirmed the usefulness of amoeba co-culture to recover novel chlamydial strains from complex samples and demonstrated the huge diversity of chlamydiae in the environment, by identifying several new species including one representing the first strain of a new family.     

The roles of thiols in the bacterial organomercurial lyase (MerB)

The bacterial plasmid-encoded organomercurial lyase, MerB (EC 4.99.1.2), catalyzes the protonolysis of organomercury compounds yielding Hg(II) and the corresponding protonated hydrocarbon. A small, soluble protein with no known homologues, MerB is widely distributed among eubacteria in three phylogenetically distinct subfamilies whose most prominent motif includes three conserved cysteine residues. We found that the 212-residue MerB encoded by plasmid R831b is a cytosolic enzyme, consistent with its high thiol requirement in vitro. MerB is inhibited by the nonphysiological dithiol DTT but uses the physiological thiols, glutathione and cysteine, equally well. Highly conserved Cys96 and Cys159 are essential for activity, whereas weakly conserved Cys160 is not. Proteins mutant in highly conserved Cys117 are insoluble. All MerB cysteines are DTNB-reactive in native and denatured states except Cys117, which fails to react with DTNB in the native form, suggesting it is buried. Mass spectrometric analysis of trypsin fragments of reduced proteins treated with N-ethylmaleimide or iodoacetamide revealed that all cysteines form covalent adducts and remain covalently modifiable even when exposed to 1:1 PHMB prior to treatment with NEM or IAM. Stable PHMB adducts were also observed on all cysteines in mutant proteins, suggesting rapid exchange of PHMB among the remaining protein thiols. However, PHMB exposure of reduced wild-type MerB yielded only Hg adducts on the Cys159/Cys160 peptide, suggesting a trapped reaction intermediate. Using HPLC to follow release of benzoic acid from PHMB, we confirmed that fully reduced wild-type MerB and mutant C160S can carry out a single protonolysis without exogenous thiols. On the basis of the foregoing we refine the previously proposed S(E)2 mechanism for protonolysis by MerB.     

Actin and myosin function in acanthamoeba

We have studied the functions of contractile proteins in Acanthamoeba by a combination of structural, biochemical and physiological approaches. We used electron microscopy and image processing to determine the three-dimensional structure of actin and the orientation of the molecule in the actin filament. We measured the rate constants for actin filament elongation and re-evaluated the effect of MgCl2 on the filament nucleation process. In Acanthamoeba actin polymerization is regulated, at least in part, by profilin, which binds to actin monomers, and by capping protein, which both nucleates polymerization and blocks monomer addition at the 'barbed' end of the filament. To test for physiological functions of myosin-II, we produced a monoclonal antibody that inhibits the actin-activated ATPase. When microinjected into living cells, this active-site-specific antibody inhibits amoeboid locomotion. We expect that similar experiments can be used to test for the physiological functions of the other components of the Acanthamoeba contractile system.