Balamuthia mandrillaris, free-living ameba and opportunistic agent of encephalitis, is a potential host for Legionella pneumophila bacteria

Balamuthia mandrillaris is a free-living ameba and an opportunistic agent of granulomatous encephalitis in humans and other mammalian species. Other free-living amebas, such as Acanthamoeba and Hartmannella, can provide a niche for intracellular survival of bacteria, including the causative agent of Legionnaires' disease, Legionella pneumophila. Infection of amebas by L. pneumophila enhances the bacterial infectivity for mammalian cells and lung tissues. Likewise, the pathogenicity of amebas may be enhanced when they host bacteria. So far, the colonization of B. mandrillaris by bacteria has not been convincingly shown. In this study, we investigated whether this ameba could host L. pneumophila bacteria. Our experiments showed that L. pneumophila could initiate uptake by B. mandrillaris and could replicate within the ameba about 4 to 5 log cycles from 24 to 72 h after infection. On the other hand, a dotA mutant, known to be unable to propagate in Acanthamoeba castellanii, also did not replicate within B. mandrillaris. Approaching completion of the intracellular cycle, L. pneumophila wild-type bacteria were able to destroy their ameboid hosts. Observations by light microscopy paralleled our quantitative data and revealed the rounding, collapse, clumping, and complete destruction of the infected amebas. Electron microscopic studies unveiled the replication of the bacteria in a compartment surrounded by a structure resembling rough endoplasmic reticulum. The course of intracellular infection, the degree of bacterial multiplication, and the ultrastructural features of a L. pneumophila-infected B. mandrillaris ameba resembled those described for other amebas hosting Legionella bacteria. We hence speculate that B. mandrillaris might serve as a host for bacteria in its natural environment.     

Balamuthia mandrillaris, agent of amebic encephalitis: detection of serum antibodies and antigenic similarity of isolates by enzyme immunoassay

ABSTRACT. We report the development of an enzyme-linked immunosorbent assay (ELISA) for detecting antibodies to Balamuthia mandrillaris , a free-living ameba that is an etiologic agent of granulomatous amebic encephalitis (GAE). As part of the California Encephalitis Project (CEP), we have tested serum and cerebrospinal fluid (CSF) samples from a subgroup of 130 hospitalized encephalitis patients (out of ∼430 samples) over a 16-month period. Case criteria were based on clinical, laboratory, and occupational/recreational histories. All serum samples initially underwent screening by immunofluorescent antibody (IFA) staining with results ranging from no detectable ameba antibodies to titers of 1:256. In addition to the 130 samples tested prospectively, sera and/or CSF from 11 previously confirmed cases of balamuthiasis, six healthy individuals, and earlier CEP submissions with high IFA antibody titers were also tested retrospectively. Among the 130 samples, two cases of balamuthiasis were identified by ELISA and confirmed by the polymerase chain reaction (PCR). The availability of sera from human and animal cases and from varied geographic areas allowed comparisons of serologic similarities of the different Balamuthia strains and human sera. All sera, whether from human or other mammals, reacted with all strains of Balamuthia , as they did with Balamuthia amebae from different geographic areas. Enzyme-linked immunosorbent assay results were consistent with the IFA results. Differences between readings were likely due to cross-reactivity between Balamuthia antigens and unidentified antibodies in serum.     

Pathogenic and opportunistic free-living amoebae: Acanthamoeba spp., Balamuthia mandrillaris, Naegleria fowleri, and Sappinia diploidea

Among the many genera of free-living amoebae that exist in nature, members of only four genera have an association with human disease: Acanthamoeba spp., Balamuthia mandrillaris, Naegleria fowleri and Sappinia diploidea. Acanthamoeba spp. and B. mandrillaris are opportunistic pathogens causing infections of the central nervous system, lungs, sinuses and skin, mostly in immunocompromised humans. Balamuthia is also associated with disease in immunocompetent children, and Acanthamoeba spp. cause a sight-threatening infection, Acanthamoeba keratitis, mostly in contact-lens wearers. Of more than 30 species of Naegleria, only one species, N. fowleri, causes an acute and fulminating meningoencephalitis in immunocompetent children and young adults. In addition to human infections, Acanthamoeba, Balamuthia and Naegleria can cause central nervous system infections in animals. Because only one human case of encephalitis caused by Sappinia diploidea is known, generalizations about the organism as an agent of disease are premature. In this review we summarize what is known of these free-living amoebae, focusing on their biology, ecology, types of disease and diagnostic methods. We also discuss the clinical profiles, mechanisms of pathogenesis, pathophysiology, immunology, antimicrobial sensitivity and molecular characteristics of these amoebae.     

Cryptococcosis as an opportunistic infection in immunodeficiency secondary to paracoccidioidomycosis

We describe the case reports of two patients with immunodeficiency secondary to paracoccidioidomycosis (PCM) and opportunistic Cryptococcus neoformans infections. Secondary immunodeficiency likely occurred as a consequence of the intestinal loss of proteins and lymphocytes associated with malabsorption syndrome due to obstructed lymphatic drainage. Both patients had had severe abdominal involvement during the acute PCM disease. Immunological evaluation showed cellular and humoral immunity impairment. Cryptococcosis manifested as relatively well circumscribed lesions: osteolytic lesions of the skull in one patient, and pulmonary nodules in the other. The latter was treated surgically and with amphotericin B, whereas the other was treated with the combination amphotericin-B and flucytosine. Both patients had a good response to treatment with complete regression of the lesions. They have now 2 and 4 years of follow-up with maintenance therapy and no indication of reactivation of the infection. PCM also did not reactivate. The clinical and immunological characteristics of these patients are discussed and compared to the opportunistic C. neoformans infections of AIDS and transplant patients.     

Isolation of opportunistic fungi from bronchoalveolar lavage of compromised hosts in Isfahan,Iran

In this study, bronchoalveolar lavage (BAL) specimens from 247 immunocompromised patients were investigated for the incidence of opportunistic fungi. In the direct examination and culture of the specimens, 5 (2.02%) of filamentous fungi and 55 (22.26%) yeasts were isolated and identified as follows: Aspergillus fumigatus (2), A. terreus (1), A. nidulans (1), Mucorsp. (1), Candida albicans (29), C. glabrata (3), C. Parapsilosis (1), Trichosporon beigelii (1), Candida sp. (13) and unknown yeasts (8). In addition, one strain of Nocardia asteroides and two strains of Streptomyces sp. were isolated.     

In-vitro activity of miltefosine and voriconazole on clinical isolates of free-living amebas: Balamuthia mandrillaris, Acanthamoeba spp., and Naegleria fowleri

The anticancer agent miltefosine and the antifungal drug voriconazole were tested in vitro against Balamuthia mandrillaris, Acanthamoeba spp., and Naegleria fowleri. All three amebas are etiologic agents of chronic (Balamuthia, Acanthamoeba) or fulminant (Naegleria) encephalitides in humans and animals and, in the case of Acanthamoeba, amebic keratitis. Balamuthia exposed to <40 microm concentrations of miltefosine survived, while concentrations of >or=40 microM were generally amebacidal, with variation in sensitivity between strains. At amebastatic drug concentrations, recovery from drug effects could take as long as 2 weeks. Acanthamoeba spp. recovered from exposure to 40 microM, but not 80 microM miltefosin. Attempts to define more narrowly the minimal inhibitory (MIC) and minimal amebacidal concentrations (MAC) for Balamuthia and Acanthamoeba were difficult due to persistence of non-proliferating trophic amebas in the medium. For N. fowleri, 40 and 55 microM were the MIC and MAC, respectively, with no trophic amebas seen at the MAC. Voriconazole had little or no inhibitory effect on Balamuthia at concentrations up to 40 microg/ml, but had a strong inhibitory effect upon Acanthamoeba spp. and N. fowleri at all drug concentrations through 40 microg/ml. Following transfer to drug-free medium, Acanthamoeba polyphaga recovered within a period of 2 weeks; N. fowleri amebas recovered from exposure to 1 microg/ml, but not from higher concentrations. All testing was done on trophic amebas; drug sensitivities of cysts were not examined. Miltefosine and voriconazole are potentially useful drugs for treatment of free-living amebic infections, though sensitivities differ between genera, species, and strains.     

Identification of 3-phenylaminoquinolinium and 3-phenylaminopyridinium salts as new agents against opportunistic fungal pathogens

Previous studies on the indoloquinoline alkaloid, cryptolepine (2), revealed that it has antii-nfective properties among other activities. Using Structure-activity relationship (SAR) techniques, several ring-opened analogs of cryptolepine (3-phenylaminopyridinium and 3-phenylaminoquinolinium derivatives) were designed to improve the potency and lower the cytotoxicity shown by several of the precursor agents. Results indicate that these ring-opened analogs constitute new anti-infective agents with over a 100-fold potency and several fold lower cytotoxicity than cryptolepine from which they are derived.     

Plastid origin of an extrachromosomal DNA molecule from Plasmodium, the causative agent of malaria.

Several species of Plasmodium have been shown to contain a circular extrachromosomal DNA molecule which is widely supposed to be mitochondrial DNA. However, it has recently been shown to have a number of features in common with chloroplast DNA. Here, a phylogenetic analysis of RNA polymerase coding sequences from the Plasmodium molecule has been carried out using distance matrix, maximum likelihood, parsimony and operator invariant methods. The analysis indicates that the molecule is in fact derived from an oxygenic photosynthetic organism and should be regarded as plastid DNA. This suggests that Plasmodium originated from a phototroph that has lost the capacity to photosynthesize.     

Ubiquinone systems of Coccidioides immitis, the causative agent of coccidioidomycosis

Abstract The ubiquinone (coenzyme Q) systems of eleven strains of Coccidioides immitis were determined by high performance liquid chromatography (HPLC). The ubiquinone profile of the fungi was shown to be homogeneous: in all of the strains, ubiquinone-10 (Q-10) was demonstrated to be the major component, with Q-9 as a minor component. The results imply that the ubiquinone system may serve as an additional phenotypic criterion for identifying the fungus.     

Indigenous microflora and opportunistic pathogens of the freshwater zebra mussel, Dreissena polymorpha

Freshwater fouling invertebrate zebra mussels (Dreissena polymorpha) harbor a diverse population of microorganisms in the Great Lakes of North America. Among the indigenous microorganisms, selective species are opportunistic pathogens to zebra mussels. Pathogenicity to zebra mussels by opportunistic bacteria isolated from the mussels was investigated in this study. Among the more than 30 bacteria isolated from temperature-stressed mussels, Aeromonas media, A. veronii, A. salmonicida subsp. salmonicida, and Shewanella putrefaciens are virulent pathogens to juvenile zebra mussels. Inoculation of a bacterial concentration of A. media, A. salmonicida subsp. salmonicida and S. putrefaciens at 10⁷ cells per zebra mussel resulted in 100% mortality within 5 days, and only 64.9% for A. veronii. In contrast, mortality was less than 12.3% following inoculation of a sterile phosphate buffer solution as a control. In addition, mortality was dependent on the size of the pathogen population used in inoculation and the incubation temperature, indicating the close relationship between the bacterial population and subsequent death. On the mussel tissue, a dense microbial population was evident from the moribund mussels viewed with Scanning Electron Microscope (SEM). Opportunistic bacteria invaded and destroyed the D. polymorpha tissue after 7 days of incubation when the bacterial inoculation was larger than 10⁵ per zebra mussel. Our results suggest that mussels are reservoirs of opportunistic pathogenic microorganisms to aquatic organisms and humans and a better understanding of the microbial ecology of the mussels will provide insights to the possible health hazards from these microorganisms.     

Bioactive molecules of Taenia solium metacestode,a causative agent of neurocysticercosis

Neurocysticercosis (NC), an infection of the CNS with Taenia solium metacestode, exemplifies formidable public health concerns associated with significant morbidity and mortality. The disease is a complex phenomenon involving molecular cell biological cross-talks between the parasite and human host. To effectively combat NC, specific diagnosis and proper management are prerequisites. Bioactive molecules implicated in host–parasite interactions and parasitic homeostasis should be elucidated. This article provides an overview of currently available serological biomarkers, especially those comprising low-molecular-weight proteins, and discusses available immunoproteomics for identification of such molecules. T. solium metacestode bioactive molecules, which might be critically implicated in the progression of NC disease, are summarized. Comprehensive understanding of the biochemical properties and biological functions of bioactive molecules may contribute to the development of novel intervention strategies against NC.     

Identification of the causative agent of cutaneous leishmaniasis in Chichaoua province, Morocco

Cutaneous leishmaniasis (CL) in Morocco is caused by three species, Leishmania major, L. tropica and L. infantum. CL has been known in Chichaoua province since 2000. Using DNA extracted from microscopic slides and parasite cultures, collected in the years 2006 and 2009, we identified for the first time L. tropica as the causative agent of CL in this region. Species identification was achieved by performing the ITS1-PCR-RFLP approach. By using this method it was possible to identify parasites in Giemsa stained slides containing less than five parasites per oil-immersion field even they were conserved for up to four months.     

Gluten,a lectin with oligomannosyl specificity and the causative agent of gluten-sensitive enteropathy

The pathogenesis of gluten-sensitive enteropathy or coeliac disease is as yet unknown. We can demonstrate by laser nephelometric measurements that gluten has lectin-like properties. Gluten binds ‘high-mannose type’ glycoproteins and the complex formation is inhibitable by mannan. As known for other lectins the reaction is absolutely Ca-dependent. Glycoproteins from the immature crypt cells from the small intestine are highly more reactive than glycoproteins from the mature villous zone. The possibility of a genetically determined deficiency of the growth-dependent N-acetyl-glucosaminyltransferase-1 as the pathogenic factor of the gluten-sensitive enteropathy is discussed.     

The organization of the muscle system of the causative agent of dicrocoeliosis,Dicrocoelium dendriticum

The musculature of parasitic flatworms plays a central role in locomotory movement, attachment to the host, and in the function of the digestive, reproductive, and excretory systems. We examine for the first time the muscle system of the flatworm Dicrocoelium dendriticum, a causative agent of the parasitic disease dicrocoeliosis, by use of fluorescently labeled phalloidin and confocal laser scanning microscopy. Somatic musculature of D. dendriticum consists of the circular, longitudinal, and diagonal muscles. The distribution of the muscle fibers in the body wall differed among the anterior, middle, and posterior body regions of the worm. The musculature of the attachment organs, the oral and ventral suckers, includes several types of muscles: the external equatorial and meridional muscles, internal circular and semicircular muscles, and radial muscles. Inside of the ventral sucker the diagonally located muscles were revealed and the supplementary u-shaped muscles were found adjoined to the base of the sucker from outside. The musculature of the internal organs composed of the excretory, reproductive, and digestive systems were characterized. Our results increase our knowledge of the morphology of trematodes and the arrangement of their muscle system.     

The core structure of the lipopolysaccharide from the causative agent of plague, Yersinia pestis

The rough-type lipopolysaccharide (LPS) of the plague pathogen, Yersinia pestis, was studied after mild-acid and strong-alkaline degradations by chemical analyses, NMR spectroscopy and electrospray-ionization mass spectrometry, and the following structure of the core region was determined:where L-alpha-D-Hep stands for L-glycero-alpha-D-manno-heptose, Sug1 for either 3-deoxy-alpha-D-manno-oct-2-ulosonic acid (alpha-Kdo) or D-glycero-alpha-D-talo-oct-2-ulosonic acid (alpha-Ko), and Sug2 for either beta-D-galactose or D-glycero-alpha-D-manno-heptose. A minority of the LPS molecules lacks GlcNAc.     

Genes for the type IV secretion system in an intracellular symbiont, Wolbachia, a causative agent of various sexual alterations in arthropods

Wolbachia species are intracellular bacteria known to cause reproductive abnormalities in their hosts. In this study, we identified Wolbachia genes encoding homologs to the type IV secretion system by which many pathogenic bacteria secrete macromolecules. The genes identified encoded most of the essential components of the secretion system and were cotranscribed as an operon.     

Anti-saprolegnia potency of some plant extracts against Saprolegnia diclina,the causative agent of saprolengiasis

Saprolegnosis of fresh water fishes caused by Saprolegnia diclina often results in serious economic losses to fish hatcheries. Despite the proven efficiency of malachite green as a potential fungicide in prevention and control of fish saprolegnosis, there is a strong debate about its safety aspects in use since it was documented to be responsible for many carcinogenic and teratogenic attributes. Bioactivity of four ethanolic plant extracts were assessed to attain a natural alternative to the traditional fungicide currently used in saprolegnosis control. Ethanolic extracts of Punica granatum and Thymus vulgaris exhibited a potential efficacy in suppressing mycelial growth of S. diclina at concentration of 0.5 mg/ml while extracts of Nigella sativa and Zingiber officinales were not effective respectively. The extract of pomegranate showed the highest antifungal potency with minimum inhibitory concentration (MIC) of 200 ppm while thyme extract was less effective and recorded MIC of 400 ppm against S. diclina. The acute fish toxicity of the plant extracts indicated the low toxicity of P. granatum and T. vulgaris extracts as no fish mortalities were detected at aquaria containing 200, 400 and 800 ppm of plant extracts respectively. Considering the low toxicity of these plant extracts, it may be concluded that 200 and 400 ppm of pomegranate and thyme extracts which suppressed the mycelial growth of the S. diclina could be safely used for saprolegniasis control.Both of pomegranate and thyme extracts which proved to possess a potential antifungal activity can be considered as a natural alternative fungicides to control saprolegniasis avoiding carcinogenic malachite green application.