Comparison of bacterial diversity and distribution on the gills of Atlantic salmon (Salmo salar L.): an evaluation of sampling techniques.

AIMS: Assess bacterial diversity and richness in mucus samples from the gills of Atlantic salmon in comparison to preserved or fixed gill filament tissues. Ascertain whether bacterial diversity and richness are homogeneous upon different arches of the gill basket. METHODS AND RESULTS: Bacterial communities contained within gill mucus were profiled using 16S rRNA gene sequencing. No significant difference in taxa richness, alpha (P > 0·05) or beta diversity indices (P > 0·05) were found between the bacterial communities of RNAlater preserved gill tissues and swab-bound mucus. A trend of lower richness and diversity indices were observed in bacterial communities from posterior hemibranchs. CONCLUSIONS: Non-lethal swab sampling of gill mucus provides a robust representation of bacterial communities externally upon the gills. Bacterial communities from the fourth arch appeared to be the least representative overall. SIGNIFICANCE AND IMPACT OF THE STUDY: The external mucosal barriers of teleost fish (e.g. gill surface) play a vital role as a primary defence line against infection. While research effort on the role of microbial communities on health and immunity of aquaculture species continues, the collection and sampling processes to obtain these data require evaluation so methodologies are consistently applied across future studies that aim to evaluate the composition of branchial microbiomes.

Complete genome sequence and bioinformatics analyses of Bacillus thuringiensis strain BM-BT15426.

OBJECTIVES: This study aimed to investigate the genetic characteristics of Bacillus thuringiensis strain BM-BT15426. METHODS: B. thuringiensis strain was identified by sequencing the PCR product (amplifying 16S rRNA gene) using ABI Prism 377 DNA Sequencer. The genome was sequenced using PacBio RS II sequencers and assembled de novo using HGAP. Also, further genome annotation was performed. RESULTS: The genome of B. thuringiensis strain BM-BT15426 has a length of 5,246,329 bp and contains 5409 predicted genes with an average G + C content of 35.40%. Three genes were involved in the "Infectious diseases: Amoebiasis" pathway. A total of 21 virulence factors and 9 antibiotic resistant genes were identified. CONCLUSIONS: The major pathogenic factors of B. thuringiensis strain BM-BT15426 were identified through complete genome sequencing and bioinformatics analyses which contributes to further study on pathogenic mechanism and phenotype of B. thuringiensis.

Azole Antifungal Agents To Treat the Human Pathogens Acanthamoeba castellanii and Acanthamoeba polyphaga through Inhibition of Sterol 14α-Demethylase (CYP51).

In this study, we investigate the amebicidal activities of the pharmaceutical triazole CYP51 inhibitors fluconazole, itraconazole, and voriconazole against Acanthamoeba castellanii and Acanthamoeba polyphaga and assess their potential as therapeutic agents against Acanthamoeba infections in humans. Amebicidal activities of the triazoles were assessed by in vitro minimum inhibition concentration (MIC) determinations using trophozoites of A. castellanii and A. polyphaga. In addition, triazole effectiveness was assessed by ligand binding studies and inhibition of CYP51 activity of purified A. castellanii CYP51 (AcCYP51) that was heterologously expressed in Escherichia coli. Itraconazole and voriconazole bound tightly to AcCYP51 (dissociation constant [Kd] of 10 and 13 nM), whereas fluconazole bound weakly (Kd of 2,137 nM). Both itraconazole and voriconazole were confirmed to be strong inhibitors of AcCYP51 activity (50% inhibitory concentrations [IC50] of 0.23 and 0.39 µM), whereas inhibition by fluconazole was weak (IC50, 30 µM). However, itraconazole was 8- to 16-fold less effective (MIC, 16 mg/liter) at inhibiting A. polyphaga and A. castellanii cell proliferation than voriconazole (MIC, 1 to 2 mg/liter), while fluconazole did not inhibit Acanthamoeba cell division (MIC, >64 mg/liter) in vitro. Voriconazole was an effective inhibitor of trophozoite proliferation for A. castellanii and A. polyphaga; therefore, it should be evaluated in trials versus itraconazole for controlling Acanthamoeba infections.

Bis-benzimidazole hits against Naegleria fowleri discovered with new high-throughput screens.

Naegleria fowleri is a pathogenic free-living amoeba (FLA) that causes an acute fatal disease known as primary amoebic meningoencephalitis (PAM). The major problem for infections with any pathogenic FLA is a lack of effective therapeutics, since PAM has a case mortality rate approaching 99%. Clearly, new drugs that are potent and have rapid onset of action are needed to enhance the treatment regimens for PAM. Diamidines have demonstrated potency against multiple pathogens, including FLA, and are known to cross the blood-brain barrier to cure other protozoan diseases of the central nervous system. Therefore, amidino derivatives serve as an important chemotype for discovery of new drugs. In this study, we validated two new in vitro assays suitable for medium- or high-throughput drug discovery and used these for N. fowleri. We next screened over 150 amidino derivatives of multiple structural classes and identified two hit series with nM potency that are suitable for further lead optimization as new drugs for this neglected disease. These include both mono- and diamidino derivatives, with the most potent compound (DB173) having a 50% inhibitory concentration (IC50) of 177 nM. Similarly, we identified 10 additional analogues with IC50s of <1 µM, with many of these having reasonable selectivity indices. The most potent hits were >500 times more potent than pentamidine. In summary, the mono- and diamidino derivatives offer potential for lead optimization to develop new drugs to treat central nervous system infections with N. fowleri.

Primary amebic meningoencephalitis: a case report and literature review.

Primary amebic meningoencephalitis (PAM) is a rare but nearly always fatal disease caused by infection with Naegleria fowleri, a thermophilic, free-living ameba found in freshwater environments. Cases of N. fowleri infection have been reported from many of the southern-tier states in the United States, with Florida and Texas disproportionately represented among them. Primary amebic meningoencephalitis presents clinically in a fashion that may be indistinguishable from bacterial and viral meningitis. Unfortunately, because the disease is so rare, PAM is often excluded from the differential diagnosis of children with meningitis resulting in delayed diagnostic and therapeutic efforts.Pediatric acute care practitioners in emergency departments, general pediatric wards, and critical care units, especially those practicing in the southern United States, should be familiar with the risk factors for acquisition of PAM, its clinical presentation, and the fact that common empiric treatment of bacterial meningitis will not treat N. fowleri. Herein, we present the case of an adolescent who died of PAM and review the (a) epidemiology, (b) pathophysiology, (c) available diagnostic modalities, (d) treatment options, and (e) outcomes of patients treated for N. fowleri infection of the central nervous system.

Epizootic fatal amebiasis in an outdoor group of Old World monkeys.

BACKGROUND: Entamoeba (E.) histolytica is an obligate parasite of humans and non-human primates. METHODS: This report describes the pathomorphological, immunohistological, and microbiological findings of fatal E. histolytica infection in two mantled guerezas (Colobus guereza) and one Hanuman langur (Semnopithecus entellus) from an epizootic outbreak of amebiasis in an open-range recreation park. RESULTS: Pathomorphological examination revealed multifocal necrotizing and granulomatous hepatitis with intralesional protozoan trophozoites in all three cases. In addition, necrotizing and ulcerative gastritis was detected in both mantled guerezas. Furthermore, oligofocal acute pulmonary embolization was detected in one of these cases. No extra-hepatic lesions were observed in the Hanuman langur. Immunohistological examination confirmed the etiologic diagnosis of E. histolytica-induced lesions. CONCLUSIONS: Although E. histolytica is a rarely diagnosed pathogen in Western European countries, veterinarians and animal keepers involved in handling and care taking of non-human primates should be aware of the potential threat caused by this zoonotic parasite.

Contrasting microbiota profiles observed in children carrying either Blastocystis spp. or the commensal amoebas Entamoeba coli or Endolimax nana.

Recent studies have shown how intestinal parasites can modulate gut microbiota. This observation is not surprising since the human intestinal lumen, like any other niche, is a battlefield of microbial competition, and Eukaryotes can affect bacterial populations. Intestinal pathogenic protist has been associated with reshaping the microbial community structure; however, the interactions between the colonic bacterial communities and parasites like Blastocystis spp., Entamoeba coli, and Endolimax nana have been poorly studied. In this work, we studied the distal intestinal bacterial microbiota of 49 children attending 7 public daycare centers in Medellin, Colombia, and compared the bacterial microbiota structure in the presence or absence of the protists Blastocystis spp., E. coli, and E. nana. Parasite colonization was associated with an increase in bacterial richness. Moreover, Blastocystis spp. presented a positive relationship with Prevotella, since this bacterium was selectively enriched in children carrying it. Remarkably, the E. coli colonized children showed a microbial profile that was closer to uninfected controls, although some bacterial taxa displayed to be enriched. This is the case for Akkermansia, which showed to be favored in E. coli colonized individuals, while notably reduced in the Blastocystis spp. parasitized group.

Pathogenic amoebas from brackish and ocean sediments, with a description of Acanthamoeba hatchetti, n. sp.

Acanthamoeba culbertsoni was isolated from a sewage-spoil dump site near Ambrose Light, New York Bight. A second species, Acanthamoeba hatchetti, n. sp., was isolated from Brewerton Channel, Baltimore Harbor, Maryland. Both species killed laboratory mice after infection by the intranasal route.

The role of proteases in the differentiation of Acanthamoeba castellanii.

Proteases are significant determinants of protozoan pathogenicity and cytolysis of host cells. However, there is now growing evidence of their involvement in cellular differentiation. Acanthamoeba castellanii of the T4 genotype elaborates a number of proteases, which are inhibited by the serine protease inhibitor phenylmethylsulphonyl fluoride. Using this and other selective protease inhibitors, in tandem with siRNA primers, specific to the catalytic site of Acanthamoeba serine proteases, we demonstrate that serine protease activity is crucial for the differentiation of A. castellanii. Furthermore, both encystment and excystment of A. castellanii was found to be dependent on serine protease function.

Therapeutic effect of rokitamycin in vitro and on experimental meningoencephalitis due to Naegleria fowleri.

Inhalation of freshwater containing the free-living amoeba Naegleria fowleri leads to a potentially fatal infection known as primary amoebic meningoencephalitis (PAME). Amphotericin B is the only agent with clinical efficacy in the treatment of PAME in humans, however this drug is often associated with adverse effects on the kidney and other organs. In an attempt to select other useful therapeutic agents for treating PAME, the amoebicidal activities of antibacterial agents including clarithromycin, erythromycin, hygromycin B, neomycin, rokitamycin, roxithromycin and zeocin were examined. Results showed that the growth of amoeba was effectively inhibited by treatment with hygromycin B, rokitamycin and roxithromycin. Notably, when N. fowleri trophozoites were treated with rokitamycin, the minimal inhibitory concentration was 6.25 microg/mL on Day 2. In the treatment of experimental meningoencephalitis due to N. fowleri, survival rates of mice treated with roxithromycin and rokitamycin were 25% and 80%, respectively, over 1 month. The mean time to death for roxithromycin and rokitamycin treatment was 16.2 days and 16.8 days, respectively, compared with 11.2 days for control mice. Finally, rokitamycin showed both in vitro and in vivo therapeutic efficacy against N. fowleri and may be a candidate drug for the treatment of PAME.

Emerging diseases in tropical dermatology.

All dermatologists worldwide should have at least passing familiarity with various tropical maladies that generate cutaneous manifestions. In addition to the standard infectious ailments associated with tropical environs, the authors have described herein five "emerging" illnesses that are gaining increasing attention for their capacity to cause human disease in those immigrating from, or traveling to, the tropical and subtropical world.

'Candidatus Cochliophilus cryoturris' (Coxiellaceae), a symbiont of the testate amoeba Cochliopodium minus.

Free-living amoebae are well known for their role in controlling microbial community composition through grazing, but some groups, namely Acanthamoeba species, also frequently serve as hosts for bacterial symbionts. Here we report the first identification of a bacterial symbiont in the testate amoeba Cochliopodium. The amoeba was isolated from a cooling tower water sample and identified as C. minus. Fluorescence in situ hybridization and transmission electron microscopy revealed intracellular symbionts located in vacuoles. 16S rRNA-based phylogenetic analysis identified the endosymbiont as member of a monophyletic group within the family Coxiellaceae (Gammaprotebacteria; Legionellales), only moderately related to known amoeba symbionts. We propose to tentatively classify these bacteria as 'Candidatus Cochliophilus cryoturris'. Our findings add both, a novel group of amoeba and a novel group of symbionts, to the growing list of bacteria-amoeba relationships.

Experimental demonstration of the possible role of Acanthamoeba polyphaga in the infection and disease progression in Buruli Ulcer (BU) using ICR mice.

The transmission of Buruli ulcer (BU), caused by Mycobacterium ulcerans (MU), remains puzzling although a number of hypothesis including through bites of infected aquatic insects have been proposed. We report the results of experiments using ICR mice that give credence to our hypothesis that Acanthamoeba species may play a role in BU transmission. We cocultured MU N2 and MU 1615 which expresses red fluorescent protein (RFP) and Acanthamoeba polyphaga (AP), and confirmed infected AP by Ziehl-Neelsen (ZN) staining. We tested for viability of MU inside AP and observed strong RFP signals inside both trophozoites and cysts after 3 and 42 days of coculturing respectively. ICR mice were topically treated, either on shaved intact or shaved pinpricked rumps, with one of the following; MU N2 only (2.25 x 106 colony forming units [CFU] / ml), MU N2:AP coculture (2.96 x 104 CFU: 1.6 x 106 cells/ml), AP only (1.6 x 106 cells/ml), PYG medium and sterile distilled water. Both MU N2 only and MU N2:AP elicited reddening on day (D) 31; edema on D 45 and D 44 respectively, and ulcers on D 49 at pinpricked sites only. To ascertain infectivity and pathogenicity of MU N2 only and MU N2:AP, and compare their virulence, the standard mouse footpad inoculation method was used. MU N2:AP elicited reddening in footpads by D 3 compared to D 14 with MU N2 only of the same dose of MU N2 (2.96 x 104 CFU). ZN-stained MU were observed in both thin sectioned and homogenized lesions, and aspirates from infected sites. Viable MU N2 were recovered from cultures of the homogenates and aspirates. This study demonstrates in ICR mice MU transmission via passive infection, and shows that punctures in the skin are prerequisite for infection, and that coculturing of MU with AP enhances pathogenesis.

September 2004: a 6-year-old girl with headache and stiff neck.

Free-living amebas in the genera Naegleria, Acanthamoeba and Balamuthia are known to cause CNS infections. Here we report a case of fatal granulomatous amebic meningoencephalitis (GAE) caused by Balamuthia mandrillaris in a 6-year-old previously healthy girl who presented with headache and stiff neck. She was treated medically for brain abscess after a CT scan identified a ring-enhancing lesion in the right temporo-parietal area. A brain biopsy showed necrosis and granulomatous inflammation. Subsequently, multiple new lesions appeared in the brain bilaterally. A second brain biopsy revealed viable amebic trophozoites that were most abundant in perivascular spaces, accompanied by neutrophils, macrophages and eosinophils. Immunofluorescence study confirmed the amoeba as Balamuthia mandrillaris. This case demonstrates that making diagnosis of GAE pre-mortem requires a high index of suspicion. Amebic infection should be included in the differential diagnosis of any granulomatous lesion in CNS; and careful search for amebic parasites should be carried out especially when necrosis predominates in the pathological material.

Influence of salmonid gill bacteria on development and severity of amoebic gill disease.

The relationship between salmonid gill bacteria and Neoparamoeba sp., the aetiological agent of amoebic gill disease (AGD) was determined in vivo. Fish were divided into 4 groups and were subjected to following experimental infections: Group 1, amoebae only; Group 2, Staphylococcus sp. and amoebae; Group 3, Winogradskyella sp. and amoebae; Group 4, no treatment (control). Fish (Groups 1, 2 and 3) were exposed to potassium permanganate to remove the natural gill microflora prior to either bacterial or amoebae exposure. AGD severity was quantified by histological analysis of gill sections to determine the percentage of lesioned filaments and the number of affected lamellae within each lesion. All amoebae infected groups developed AGD, with fish in Group 3 showing significantly more filaments with lesions than other groups. Typically lesion size averaged between 2 to 4 interlamellar units in all AGD infected groups. The results suggest that the ability of Neoparamoeba sp. to infect filaments and cause lesions might be enhanced in the presence of Winogradskyella sp. The possibility is proposed that the prevalence of more severe AGD is due to the occurrence of Winogradskyella sp. at high concentrations on the gills.

Acanthamoebiasis and immunosuppression. Case report.

Immunosuppression and debilitating illnesses are occasionally associated with multifocal brain lesions of Acanthamoebiasis, an encephalitis distinct from the acute, water-sport related meningoencephalitis caused by Naegleria fowler. A 38-year-old man with a renal transplant two and one-half years before his final illness developed pneumonia due to Legionella micdadei. Candida albicans was isolated from sputum and cytomegalovirus was found in lung and liver biopsies. He had continuous corticosteroids, broad spectrum antibiotics, and immunosuppressive therapies. Coma developed and computerized tomography of the head revealed multifocal areas of decreased density, interpreted as cerebral abscesses. Brain biopsy demonstrated encephalitis and amoebae (Acanthamoeba castellanii). Four days after brain biopsy, he died and multifocal hemorrhagic lesions were found in the cerebral hemispheres, brainstem, and cerebellum. This case supports the hypothesis that Acanthamoebiasis is an opportunistic infection.

Fulminant amebic meningoencephalitis due to Acanthamoeba.

A 57-year-old chronically immunosuppressed woman with systemic lupus erythematosus developed fulminant meningoencephalitis due to Acanthamoeba castellanii. Amebic trophozoites were also found in the lungs, suggesting a primary pulmonary focus of infection. This case illustrates that Acanthamoeba can cause a fulminant, rapidly fatal meningoencephalitis, as well as the previously reported chronic granulomatous meningoencephalitis. X

Susceptibility of Acanthamoeba to cryotherapeutic method.

Acanthamoeba keratitis is becoming an increasingly well-known clinical entity. The Acanthamoeba species in their encysted state are resistant to antimicrobial agents, resulting in corneal infections that are refractory to medical therapy. Corneal cryotherapy has been used to treat Acanthamoeba keratitis with varying and equivocal success. We subjected trophozoites and cysts of two species of Acanthamoeba recovered from corneal infections to trials of in vitro freeze-thaw-refreeze cryotherapeutic methods. The trophozoites of both species were killed in all trials. However, the cysts of both species survived all trials. Cryotherapy may not be an effective means to eliminate Acanthamoeba cysts from the cornea.

Endogenous amoebic endophthalmitis.

A 42-year-old man experienced abrupt reduction of vision in the left eye and had vitreous opacity and hypopyon on initial examination. He underwent an emergency vitrectomy and phacoemulsification with posterior capsulectomy, followed by a 2-week course of intravenous fluconazole and antibiotics. The final visual acuity was 20/20 OS. Amoebas, together with polymorphonuclear cells, were observed on examination of specimens of the aqueous humor and vitreous fluid obtained during the surgery. Staphylococcus epidermidis was also cultured from the vitreous fluid. The amoebas had a crescent-shaped periphery of the cytoplasm and ambiguous nuclei. Ultrastructurally, the cytoplasm was surrounded by a thick electron-dense coating. The early vitrectomy and combined use of antibiotics and antifungals might have led to the good visual outcome in this patient.