Virulence factors of Gram-negative bacteria from free-ranging Amazon river dolphins (Inia geoffrensis).

Freshwater cetaceans play a significant role as sentinel animals, providing important data on animal species and aquatic ecosystem health. They also may serve as potential reservoirs of emerging pathogens and host virulence genes in their microbiota. In this study, we evaluated virulence factors produced by Gram-negative bacteria recovered from individuals belonging to two populations of free-ranging Amazon river dolphins (Inia geoffrensis). A total of 132 isolates recovered from the oral cavity, blowhole, genital opening and rectum of 21 river dolphins, 13 from Negro River and 8 from Tapajós River, Brazil, were evaluated for the production of virulence factors, such as biofilms and exoproducts (proteases, hemolysins and siderophores), in planktonic and biofilm forms. In planktonic form, 81.1% (107/132) of the tested bacteria of free-ranging Amazon river dolphins were able to produce virulence factors, with 44/132 (33.4%), 65/132 (49,2%) and 54/132 (40,9%) positive for protease, hemolysin and siderophore production, respectively. Overall, 57/132 (43.2%) of the isolates produced biofilms and, under this form of growth, 66/132 (50%), 88/132 (66.7%) and 80/132 (60.6%) of the isolates were positive for protease, hemolysin and siderophore production. In general, the isolates showed a higher release of exoproducts in biofilm than in planktonic form (P < 0.001). The present findings show that Amazon river dolphins harbor potentially pathogenic bacteria in their microbiota, highlighting the importance of monitoring the micro-organisms from wild animals, as they may emerge as pathogens for humans and other animals.

Effect of promethazine on biofilms of gram-positive cocci associated with infectious endocarditis.

This study evaluated the antimicrobial activity of promethazine against Staphylococcus aureus, Staphylococcus epidermidis and Streptococcus mutans and its effect on the antimicrobial susceptibility of biofilms grown in vitro and ex vivo on porcine heart valves. Promethazine was evaluated alone and in combination with vancomycin and oxacillin against Staphylococcus spp. and vancomycin and ceftriaxone against S. mutans in planktonic form and biofilms grown in vitro and ex vivo. Promethazine minimum inhibitory concentration range was 24.4-95.31 µg/mL and minimum biofilm eradication concentration range was 781.25-3.125 µg/mL. Promethazine interacted synergistically with vancomycin, oxacillin and ceftriaxone against biofilms in vitro. Promethazine alone reduced (p < 0.05) the CFU-counts of biofilms grown on heart valves for Staphylococcus spp., but not for S. mutans, and increased (p < 0.05) the activity of vancomycin, oxacillin and ceftriaxone against biofilms of Gram-positive cocci grown ex vivo. These findings bring perspectives for repurposing promethazine as adjuvant in the treatment of infective endocarditis.

Vancomycin enhances growth and virulence of Trichosporon spp. planktonic cells and biofilms.

Invasive fungal infections (IFIs) are important worldwide health problem, affecting the growing population of immunocompromised patients. Although the majority of IFIs are caused by Candida spp., other fungal species have been increasingly recognized as relevant opportunistic pathogens. Trichosporon spp. are members of skin and gut human microbiota. Since 1980's, invasive trichosporonosis has been considered a significant cause of fungemia in patients with hematological malignancies. As prolonged antibiotic therapy is an important risk factor for IFIs, the present study investigated if vancomycin enhances growth and virulence of Trichosporon. Vancomycin was tested against T. inkin (n = 6) and T. asahii (n = 6) clinical strains. Planktonic cells were evaluated for their metabolic activity and virulence against Caenorhabditis elegans. Biofilms were evaluated for metabolic activity, biomass production, amphotericin B tolerance, induction of persister cells, and ultrastructure. Vancomycin stimulated planktonic growth of Trichosporon spp., increased tolerance to AMB, and potentiates virulence against C. elegans. Vancomycin stimulated growth (metabolic activity and biomass) of Trichosporon spp. biofilms during all stages of development. The antibiotic increased the number of persister cells inside Trichosporon biofilms. These cells showed higher tolerance to AMB than persister cells from VAN-free biofilms. Microscopic analysis showed that VAN increased production of extracellular matrix and cells in T. inkin and T. asahii biofilms. These results suggest that antibiotic exposure may have a direct impact on the pathophysiology of opportunistic trichosporonosis in patients at risk. LAY ABSTRACT: This study showed that the vancomycin stimulated Trichosporon growth, induced morphological and physiological changes on their biofilms, and also enhanced their in vivo virulence. Although speculative, the stimulatory effect of vancomycin on fungal cells should be considered in a clinical scenario.

Anthraquinones from Aloe spp. inhibit Cryptococcus neoformans sensu stricto: effects against growing and mature biofilms.

This study aimed to evaluate the in vitro effect of aloe emodin, barbaloin and chrysophanol on growing and mature biofilms of Cryptococcus neoformans sensu stricto. The compounds were added at the moment of inducing biofilm growth or after growth for 72 h to evaluate their effects on growing and mature biofilms, respectively. Then, biofilm biomass was evaluated by crystal violet staining and metabolic activity by the XTT reduction assay. Morphological alterations were also evaluated by laser scanning confocal microscopy. Aloe emodin and barbaloin affected growing biofilms and disrupted mature biofilms, reducing metabolic activity by > 60% and biomass by > 70%. Chrysophanol only inhibited mature biofilms, but to a lesser extent. In conclusion, anthraquinones, especially aloe emodin and barbaloin, show a relevant effect against growing and mature biofilms of C. neoformans sensu stricto.

Antifungal effects of the flavonoids kaempferol and quercetin: a possible alternative for the control of fungal biofilms.

This study aimed to determine the minimum inhibitory concentration (MIC) of kaempferol and quercetin against planktonic and biofilm forms of the Candida parapsilosis complex. Initially, nine C. parapsilosis sensu stricto, nine C. orthopsilosis and nine C. metapsilosis strains were used. Planktonic susceptibility to kaempferol and quercetin was assessed. Growing and mature biofilms were then exposed to the flavonoids at MIC or 10xMIC, respectively, and theywere also analyzed by confocal laser scanning microscopy. The MIC ranges were 32-128 µg ml-1 for kaempferol and 0.5-16 µg ml-1 for quercetin. Kaempferol and quercetin decreased (P < 0.05) the metabolic activity and biomass of growing biofilms of the C. parapsilosis complex. As for mature biofilms, the metabolic effects of the flavonoids varied, according to the cryptic species, but kaempferol caused an overall reduction in biofilm biomass. Microscopic analyses showed restructuring of biofilms after flavonoid exposure. These results highlight the potential use of these compounds as sustainable resources for the control of fungal biofilms.

Biofilms of Candida spp. from the ocular conjunctiva of horses with reduced azole susceptibility: a complicating factor for the treatment of keratomycosis?

OBJECTIVES: This study aimed to assess the biofilm-forming ability of Candida spp. from the ocular conjunctiva of horses and to investigate the antifungal susceptibility of these biofilms. PROCEDURES: Initially, the biofilm-forming ability of 15 strains was assessed by crystal violet staining, which reveals the fungal biomass adhered to the polystyrene plates, and scanning electron microscopy. Then, the minimum inhibitory concentrations (MICs) of amphotericin B, fluconazole, itraconazole, and caspofungin were initially determined against strains in planktonic form. Afterward, antifungal susceptibility of mature biofilms was evaluated by exposing them to 10 × MIC and 50 × MIC of the tested drugs, followed by the assessment of their metabolic activity, using the oxidoreduction indicator XTT. Results were analyzed through ANOVA and Tukey's post-test, and P-values below 5% led to significant conclusions. RESULTS: Eight strains produced biofilms and were classified as strong (1/15), moderate (3/15) and weak (4/15) producers, according to the amount of crystal violet retained by the adhered fungal biomass. Biofilm metabolic activity of one C. tropicalis did not decrease after exposure to the tested antifungals, while biofilm metabolic activity of five strains was reduced by amphotericin B, but not the other drugs. One C. parapsilosis sensu stricto and one C. glabrata showed significant reduction in biofilm metabolic activity after exposure to fluconazole, itraconazole, and caspofungin, but not amphotericin B. CONCLUSIONS: The results demonstrate that Candida from the ocular conjunctiva of horses can pose as a risk to animal health as they are capable of forming biofilms, which are commonly involved in fungal keratitis.

Bipolaris hawaiiensis as an emerging cause of cutaneous phaeohyphomycosis in an Antillean manatee Trichechus manatus manatus.

Phaeohyphomycoses are emerging and opportunistic diseases caused by dematiaceous fungi that infect many animal species. This paper describes a case of cutaneous phaeohyphomycosis in an Antillean manatee Trichechus manatus manatus caused by Bipolaris hawaiiensis. Blackish skin lesions were observed in an Antillean manatee calf held captive in Brazil. Direct examination of skin scraping from the affected areas revealed the presence of dematious hyphae. Culture of skin fragments led to the isolation and subsequent identification of B. hawaiiensis as the etiologic agent. Treatment with itraconazole for 14 d was effective. Infections by Bipolaris spp. are rare in animals, and this is the first report of B. hawaiiensis in veterinary medicine.

Synthesis and antifungal activity in vitro of isoniazid derivatives against histoplasma capsulatum var. capsulatum.

Histoplasmosis is a severe infection that affects millions of patients worldwide and is endemic in the Americas. Amphotericin B (AMB) and itraconazole are highly effective for the treatment of severe and milder forms of the disease, but AMB is toxic, and the bioavailability of itraconazole is erratic. Therefore, it is important to investigate new classes of drugs for histoplasmosis treatment. In this study, a series of nine isoniazid hydrazone derivatives were synthesized and evaluated for their antifungal activities in vitro against the dimorphic fungus Histoplasma capsulatum var. capsulatum. The drugs were tested by microdilution in accordance with CLSI guidelines. The compound N'-(1-phenylethylidene)isonicotinohydrazide had the lowest MIC range of all the compounds for the yeast and filamentous forms of H. capsulatum. The in vitro synergy of this compound with AMB against the planktonic and biofilm forms of H. capsulatum cells was assessed by the checkerboard method. The effects of this hydrazone on cellular ergosterol content and membrane integrity were also investigated. The study showed that the compound alone is able to reduce the ergosterol content of planktonic cells and can alter the membrane permeability of the fungus. Furthermore, the compound alone or in combination with AMB showed inhibitory effects against mature biofilms of H. capsulatum. N'-(1-Phenylethylidene)isonicotinohydrazide alone or combined with AMB might be of interest in the management of histoplasmosis.