Azole-Resilient Biofilms and Non-wild Type C. albicans Among Candida Species Isolated from Agricultural Soils Cultivated with Azole Fungicides: an Environmental Issue?

This study aimed to identify Candida spp. from agricultural soils cultivated with azole fungicides and investigate their susceptibility to clinical (fluconazole, itraconazole, voriconazole, and amphotericin B) and agricultural (tetraconazole and tebuconazole) antifungals in planktonic form. Additionally, Candida biofilm-forming ability and biofilm susceptibility to agricultural antifungals and voriconazole were analyzed. Species identification was performed by phenotypic and molecular assays. The susceptibility of planktonic cells was evaluated by the broth microdilution method. The biofilm metabolic activity was evaluated by the XTT reduction assay. The recovered Candida spp. were identified as C. parapsilosis sensu stricto (n = 14), C. albicans (n = 5), C. tropicalis (n = 2), C. fermentati (n = 1), and C. metapsilosis (n = 2). Minimum inhibitory concentration ranges for clinical and agricultural antifungals were ≤ 0.03-4 µg/mL and 1-128 µg/mL, respectively. Two and one C. albicans strains were considered non-wild type for voriconazole and fluconazole, respectively. All strains were biofilm producers. The minimum biofilm inhibitory concentration ranges for tetraconazole and tebuconazole were 128-> 1024 µg/mL, while for voriconazole was 512-> 1024 µg/mL. In summary, this study shows that non-wild type and azole-resilient biofilm-producing Candida species colonize agricultural soils cultivated with azole fungicides.

Ex vivo biofilm-forming ability of dermatophytes using dog and cat hair: an ethically viable approach for an infection model.

The aim of this study was to establish an ex vivo model for dermatophyte biofilm growth, using hair from dogs and cats. Strains of Microsporum canis, M. gypseum, Trichophyton mentagrophytes and T. tonsurans were assessed for in vitro and ex vivo biofilm production. All T. mentagrophytes and T. tonsurans isolates and 8/12 M. canis and 1/7 M. gypseum isolates formed biofilms in vitro, while all tested isolates presented biofilm growth on ex vivo models. T. mentagrophytes and M. canis formed more homogeneous and better-structured biofilms with greater biomass production on cat hair but T. tonsurans formed more biofilm on dog hair. Confocal and scanning electron microscopy demonstrated fungal hyphae colonizing and perforating the hair shaft, abundant fungal conidia, biofilm extracellular matrix and biofilm water channels. The present study demonstrated an ex vivo model for the performance of studies on biofilm formation by dermatophytes, using dog and cat hair.

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.

Farnesol inhibits planktonic cells and antifungal-tolerant biofilms of Trichosporon asahii and Trichosporon inkin.

Trichosporon species have been considered important agents of opportunistic systemic infections, mainly among immunocompromised patients. Infections by Trichosporon spp. are generally associated with biofilm formation in invasive medical devices. These communities are resistant to therapeutic antifungals, and therefore the search for anti-biofilm molecules is necessary. This study evaluated the inhibitory effect of farnesol against planktonic and sessile cells of clinical Trichosporon asahii (n = 3) andTrichosporon inkin (n = 7) strains. Biofilms were evaluated during adhesion, development stages and after maturation for metabolic activity, biomass and protease activity, as well as regarding morphology and ultrastructure by optical microscopy, confocal laser scanning microscopy, and scanning electron microscopy. Farnesol inhibited Trichosporon planktonic growth by 80% at concentrations ranging from 600 to 1200 µM for T. asahii and from 75 to 600 µM for T. inkin. Farnesol was able to reduce cell adhesion by 80% at 300 µM for T. asahii and T. inkin at 600 µM, while biofilm development of both species was inhibited by 80% at concentration of 150 µM, altering their structure. After biofilm maturation, farnesol decreased T. asahii biofilm formation by 50% at 600 µM concentration and T. inkin formation at 300 µM. Farnesol inhibited gradual filamentation in a concentration range between 600 and 1200 µM. Farnesol caused reduction of filament structures of Trichosporon spp. at every stage of biofilm development analyzed. These data show the potential of farnesol as an anti-biofilm molecule.

Exposure of Candida parapsilosis complex to agricultural azoles: An overview of the role of environmental determinants for the development of resistance.

This work investigated the phenotypic behavior of Candida parapsilosis species complex in response to exposure to agricultural azoles and fluconazole. Three fluconazole-susceptible strains of C. parapsilosis sensu stricto, C. orthopsilosis and C. metapsilosis were used. Initial minimum inhibitory concentrations (iMICs) for agricultural and clinical azoles were determined by broth microdilution. Then, the strains were exposed to tebuconazole, tetraconazole and fluconazole for 15 days, at concentrations that were two-folded daily, starting at one-eighth the iMIC (iMIC/8) up to 64 times iMIC (64xiMIC). After 15-day-exposure, antifungal susceptibility, biofilm formation, CDR, MDR and ERG expression were evaluated. The three cryptic species developed tolerance to the antifungals they were exposed and presented reduction (P < 0.05) in fluconazole susceptibility. In addition, C. parapsilosis sensu stricto and C. metapsilosis also presented reduced susceptibility to voriconazole, after fluconazole exposure. Azole exposure decreased (P < 0.05) biofilm production by C. parapsilosis sensu stricto and C. orthopsilosis and increased (P < 0.05) the expression of ERG11 in all tested strains. The results show that exposure to agricultural azoles and fluconazole induces changes in the phenotypic behavior and gene expression by the three cryptic species of C. parapsilosis complex, highlighting the importance of environmental determinants for the development of antifungal resistance.

ß-lactam antibiotics & vancomycin increase the growth & virulence of Candida spp.

AIM: To investigate the direct effect of antibiotics on growth and virulence of the major Candida species associated with invasive infections. MATERIALS & METHODS: Cefepime, imipenem, meropenem, amoxicillin and vancomycin were tested at twofold the peak plasma concentration (2× PP) and the peak plasma concentration (PP). The effects of antibiotics on Candida albicans, Candida parapsilosis, Candida krusei and Candida tropicalis were investigated by colony counting, flow cytometry, proteolytic activity and virulence in Caenorhabditis elegans. RESULTS: Antibiotics increase growth and proteolytic activity of Candida spp; In addition, amoxicillin potentiates virulence of C. krusei and C. tropicalis against Caenorhabditis elegans. CONCLUSION: These results suggest that antimicrobial therapy may have a direct effect on the pathophysiology of invasive fungal infections in patients at risk.

Malassezia pachydermatis from animals: Planktonic and biofilm antifungal susceptibility and its virulence arsenal.

The yeast Malassezia pachydermatis is a component of the microbiota of dogs and cats, however it can cause otitis and seborrheic dermatitis in these animals. The objective of this study was to determine the antifungal susceptibility, and evaluate virulence and pathogenicity of 25 M. pachydermatis strains from animals. Susceptibility to ketoconazole, fluconazole, itraconazole, voriconazole, terbinafine, and amphotericin B was evaluated by broth microdilution assay. In addition, biofilm-forming ability, protease, phospholipase, hemolysin and melanin production and adhesion to epithelial cells by this yeast species were assessed. Finally, strain pathogenicity was investigated using the nematode Caenorhabditis elegans. Concerning the planktonic susceptibility, minimum inhibitory concentrations varied from <0.03 to>64 µg/mL for azole derivatives, 1 to >16 µg/mL for amphotericin B and 0.03 to 0.25 µg/mL for terbinafine. All strains were classified as strong biofilm producers, and ketoconazole, fluconazole and amphotericin B presented the best inhibitory effect against mature biofilms. All fungal isolates produced proteases, whereas 14/25 strains were positive for phospholipase production. Hemolytic activity was not observed and 18/25 strains showed dark pigmentation in the presence of L-DOPA. Regarding adhesion to epithelial cells, a low adhesion rate was observed in 10/12 evaluated strains. C. elegans mortality rate reached 95.9% after 96 h of exposure of the worms to M. pachydermatis. This yeast species produces important virulence factors and presents high pathogenicity, corroborating its clinical importance.

Antifungal susceptibility and virulence of Candida parapsilosis species complex: an overview of their pathogenic potential.

PURPOSE: Antifungal resistance and several putative virulence factors have been associated with the pathogenicity of the Candida parapsilosis species complex. The objective of this study was to evaluate the antifungal susceptibility, the production of virulence factors and the pathogenicity of the C. parapsilosis complex. METHODOLOGY: Overall, 49 isolates of C. parapsilosis sensu stricto, 19 C. orthopsilosis and nine C. metapsilosis were used. The planktonic and biofilm susceptibility to fluconazole, itraconazole, voriconazole, amphotericin B and caspofungin was assessed using a broth microdilution assay. Finally, the production of biofilm and hydrolytic enzymes and the fungal pathogenicity against Caenorhabditis elegans were investigated.Results/Key findings. Overall, one C. orthopsilosis was resistant to caspofungin and susceptible-dose-dependent to itraconazole, the other two C. orthopsilosis were susceptible-dose-dependent to fluconazole and itraconazole, and one C. metapsilosis was susceptible-dose-dependent to azoles. A total of 67.5 % of the isolates were biofilm producers. Amphotericin B and caspofungin caused the greatest reduction in the metabolic activity and biomass of mature biofilms. Phospholipase and protease production was observed in 55.1 % of C. parapsilosis sensu stricto, 42.1 % of C. orthopsilosis and 33.3 % of C. metapsilosis isolates. Moreover, 57.9 % of C. orthopsilosis and 20.4 % of C. parapsilosis sensu stricto isolates were ß-haemolytic, and all C. metapsilosis were α-haemolytic. Finally, the C. parapsilosis complex caused high mortality of C. elegans after 96 h of exposure. CONCLUSION: These results reinforce the heterogeneity of these cryptic species for their antifungal susceptibility, virulence and pathogenic potential, emphasizing the relevance of monitoring these emerging pathogens.

Candida parapsilosis complex in veterinary practice: A historical overview, biology, virulence attributes and antifungal susceptibility traits.

The Candida genus is composed by yeast that commensally live as part of human and animal microbiota. In the last years, C. parapsilosis complex, composed by the cryptic species C. parapsilosis sensu stricto, C. orthopsilosis and C. metapsilosis, has been frequently implicated in human nosocomial infections in Europe and Latin America. In veterinary medicine, C. parapsilosis sensu lato infections have been reported in different animal species. Several putative virulence factors have been associated with the pathogenicity of this species complex, including biofilm formation and the production of proteases, phospholipases, lipases and other hydrolytic enzymes. Additionally, these species have developed antifungal resistance, especially to azole derivatives and echinocandins. Thus, considering the pathogenic potential of the C. parapsilosis species complex, along with the emergence of antifungal resistant strains, this review was designed to approach historical and biological aspects, microbiological features, virulence factors and antifungal susceptibility traits of C. parapsilosis complex from animals.

Research advances on the multiple uses of Moringa oleifera: A sustainable alternative for socially neglected population.

Moringa oleifera Lam (Moringaceae) is a plant with high nutritional and medicinal value. Native to India, it is now widely distributed throughout tropical and subtropical regions of the world. Its different parts are sources of proteins, vitamins and minerals and present different pharmacological and biotechnological potential. Moreover, M. oleifera seeds are widely used in water and effluent treatment, for their coagulation, flocculation and sedimentation properties, their ability of improving water quality, by reducing organic matter and microbial load, with special applicability in intensive animal production systems, such as aquaculture. In addition, due to its high nutritional value and several medicinal properties, this tree may act as a nutritional and medical alternative for socially neglected populations. In this context, this review gathers information on M. oleifera, emphasizing its chemical constituents, nutritional, pharmacological and antimicrobial properties, applications in the treatment of water effluents, and ecological and social aspects.

Azole resistance in Candida albicans from animals: Highlights on efflux pump activity and gene overexpression.

This study investigated potential mechanisms of azole resistance among Candida albicans from animals, including efflux pump activity, ergosterol content and gene expression. For this purpose, 30 azole-resistant C. albicans strains from animals were tested for their antifungal susceptibility, according to document M27-A3, efflux pump activity by rhodamine 6G test, ergosterol content and expression of the genes CDR1, CDR2, MDR1, ERG11 by RT-qPCR. These strains were resistant to at least one azole derivative. Resistance to fluconazole and itraconazole was detected in 23 and 26 strains respectively. Rhodamine 6G tests showed increased activity of efflux pumps in the resistant strains, showing a possible resistance mechanism. There was no difference in ergosterol content between resistant and susceptible strains, even after fluconazole exposure. From 30 strains, 22 (73.3%) resistant animal strains overexpressed one or more genes. From this group, 40.9% (9/22) overexpressed CDR1, 18.2% (4/22) overexpressed CDR2, 59.1% (13/22) overexpressed MDR1 and 54.5% (12/22) overexpressed ERG11. Concerning gene expression, a positive correlation was observed only between CDR1 and CDR2. Thus, azole resistance in C. albicans strains from animals is a multifactorial process that involves increased efflux pump activity and the overexpression of different genes.

Yeasts from Scarlet ibises (Eudocimus ruber): A focus on monitoring the antifungal susceptibility of Candida famata and closely related species.

This study aimed to identify yeasts from the gastrointestinal tract of scarlet ibises (Eudocimus ruber) and from plant material collected from the environment where they live. Then, the isolates phenotypically identified as Candida famata were submitted to molecular identification of their closely related species and evaluated for their antifungal susceptibility and possible resistance mechanisms to antifungal drugs. Cloacal swabs from 20 scarlet ibises kept in captivity at Mangal das Garças Park (Brazil), pooled stool samples (n = 20) and samples of trunks and hollow of trees (n = 20) obtained from their enclosures were collected. The samples were seeded on Sabouraud agar supplemented with chloramphenicol. The 48 recovered isolates were phenotypically identified as 15 Candida famata, 13 Candida catenulata, 2 Candida intermedia, 1 Candida lusitaniae, 2 Candida guilliermondii, 1 Candida kefyr, 1 Candida amapae, 1 Candida krusei, 8 Trichosporon spp., and 4 Rhodotorula spp. The C. famata isolates were further identified as 3 C. famata, 8 Debaryomyces nepalensis, and 4 C. palmioleophila. All C. famata and C. palmioleophila were susceptible to caspofungin and itraconazole, while one D. nepalensis was resistant to fluconazole and voriconazole. This same isolate and another D. nepalensis had lower amphotericin B susceptibility. The azole resistant strain had an increased efflux of rhodamine 6G and an alteration in the membrane sterol content, demonstrating multifactorial resistance mechanism. Finally, this research shows that scarlet ibises and their environment harbor C. famata and closely related species, including antifungal resistant isolates, emphasizing the need of monitoring the antifungal susceptibility of these yeast species.

Aeromonas and Plesiomonas species from scarlet ibis (Eudocimus ruber) and their environment: monitoring antimicrobial susceptibility and virulence.

The present study aimed at evaluating the role of captive scarlet ibises (Eudocimus ruber) and their environment as reservoirs of Aeromonas spp. and Plesiomonas spp., and analyzing the in vitro antimicrobial susceptibility and virulence of the recovered bacterial isolates. Thus, non-lactose and weak-lactose fermenting, oxidase positive Gram-negative bacilli were recovered from cloacal samples (n = 30) of scarlet ibises kept in a conservational facility and from water samples (n = 30) from their environment. Then, the antimicrobial susceptibility, hemolytic activity and biofilm production of the recovered Aeromonas spp. and Plesiomonas shigelloides strains were assessed. In addition, the virulence-associated genes of Aeromonas spp. were detected. Ten Aeromonas veronii bv. sobria, 2 Aeromonas hydrophila complex and 10 P. shigelloides were recovered. Intermediate susceptibility to piperacillin-tazobactam and cefepime was observed in 2 Aeromonas spp. and 1 P. shigelloides, respectively, and resistance to gentamicin was observed in 4 P. shigelloides. The automated susceptibility analysis revealed resistance to piperacillin-tazobactam and meropenem among Aeromonas spp. and intermediate susceptibility to gentamicin among P. shigelloides. All Aeromonas isolates presented hemolytic activity, while 3 P. shigelloides were non-hemolytic. All Aeromonas spp. and 3/10 P. shigelloides were biofilm-producers, at 28 °C, while 10 Aeromonas spp. and 6/10 P. shigelloides produced biofilms, at 37 °C. The most prevalent virulence genes of Aeromonas spp. were asa1 and ascV. Scarlet ibises and their environment harbour potentially pathogenic bacteria, thus requiring monitoring and measures to prevent contamination of humans and other animals.

Cross-resistance to fluconazole induced by exposure to the agricultural azole tetraconazole: an environmental resistance school?

This study aimed to investigate the influence of tetraconazole and malathion, both used in agricultural activities, on resistance to fluconazole, itraconazole and voriconazole in Candida parapsilosis ATCC 22019. The susceptibility to tetraconazole, malathion, fluconazole, itraconazole and voriconazole, through broth microdilution. Then, 12 independent replicates, were separated and exposed to four treatment groups, each one containing three replicates: G1: tetraconazole; G2: malathion; G3: fluconazole (positive control); G4: negative control. Replicates from G1, G2 and G3, were exposed to weekly increasing concentrations of tetraconazole, malathion and fluconazole, respectively, ranging from MIC/2 to 32 × MIC, throughout 7 weeks. The exposure to tetraconazole, but not malathion, decreased susceptibility to clinical azoles, especially fluconazole. The tetraconazole-induced fluconazole resistance is partially mediated by the increased activity of ATP-dependent efflux pumps, considering the increase in antifungal susceptibility after the addition of the efflux pump inhibitor, promethazine, and the increase in rhodamine 6G efflux and CDR gene expression in the G1 replicates. Moreover, MDR expression was only detected in G1 and G3 replicates, suggesting that MDR pumps are also involved in tetraconazole-induced fluconazole resistance. It is noteworthy that tetraconazole and fluconazole-treated replicates behaved similarly, therefore, resistance to azoles of clinical use may be a consequence of using azoles in farming activities.

Enterobacteria and Vibrio from Macrobrachium amazonicum prawn farming in Fortaleza, Ceará, Brazil.

OBJECTIVE: To investigate the isolation of enterobacteria associated with Macrobrachium amazonicum (M. amazonicum) farming and evaluate the in vitro antimicrobial susceptibility of Vibrio strains. METHODS: Strains were isolated from female M. amazonicum prawns and environmental and hatchery water. Biochemical assays were used to identify bacterial genera and those belonging to the genus Vibrio were submitted to further analyses for species identification, through Vitek 2 automated system and serotyping. Susceptibility test was performed according to Clinical Laboratory Standards Institute. RESULTS: The following genera of enterobacteria were recovered: Enterobacter (n = 11), Citrobacter (n = 10), Proteus (n = 2), Serratia (n = 2), Kluyvera (n = 2), Providencia (n = 2), Cedecea (n = 1), Escherichia (n = 1), Edwardsiella (n = 1) and Buttiauxella (n = 1). As for Vibrio, three species were identified: Vibrio cholerae non-O1/non-O139 (n = 4), Vibrio vulnificus (V. vulnificus) (n = 1) and Vibrio mimicus (n = 1). Vibrio spp. showed minimum inhibitory concentrations values within the susceptibility range established by Clinical Laboratory Standards Institute for almost all antibiotics, except for V. vulnificus, which presented intermediate profile to ampicillin. CONCLUSIONS: Enterobacteria do not seem to be the most important pathogens associated with M. amazonicum farming, whereas the recovery of Vibrio spp. from larviculture, with emphasis on Vibrio cholerae and V. vulnificus, deserves special attention due to their role as potentially zoonotic aquaculture-associated pathogens. Furthermore, the intermediate susceptibility of V. vulnificus to ampicillin reflects the importance of monitoring drug use in prawn farming.

Vibrio spp. from Macrobrachium amazonicum prawn farming are inhibited by Moringa oleifera extracts.

OBJECTIVE: To investigate the in vitro antimicrobial potential of extracts of stem, leaves, flowers, pods and seeds of Moringa oleifera (M. oleifera) against Vibrio spp. from hatchery water and the prawn Macrobrachium amazonicum. METHODS: The ethanol extracts of stem, leaves, pods and seeds and chloroform extract of flowers of M. oleifera were tested against Vibrio cholerae (V. cholerae) serogroups non-O1/non-O139 (n = 4), Vibrio vulnificus (n = 1) and Vibrio mimicus (n = 1). Escherichia coli (E. coli) (ATCC(®) 25922) was used as quality control. Vibrio species were obtained from Macrobrachium amazonicum prawns and from hatchery water from prawn farming. The Minimum Inhibitory Concentration (MIC) was determined by broth microdilution method. RESULTS: The best result was obtained with the ethanol extract of pods, which inhibited three strains of the V. cholerae, Vibrio vulnificus, Vibrio mimicus and E. coli (MIC range 0.312-5.000 mg/mL). The chloroform extract of flowers was effective against all V. cholerae strains and E. coli (MIC range 0.625-1.250 mg/mL). However, the ethanol extracts of stem and seeds showed low effectiveness in inhibiting the bacterial growth. CONCLUSIONS: The extracts of pods, flowers and leaves of M. oleifera have potential for the control of Vibrio spp. Further studies are necessary to isolate the bioactive compounds responsible for this antimicrobial activity.

Virulence and antimicrobial susceptibility of clinical and environmental strains of Aeromonas spp. from northeastern Brazil.

The aims of the present study were to isolate and identify clinical and environmental strains of Aeromonas spp. by means of biochemical tests and the automated method VITEK 2 and to investigate the presence of the virulence genes cytotoxic enterotoxin (act), hemolysin (asa-1), and type III secretion system (ascV), and also the in vitro antimicrobial susceptibility of the strains. From the clinical isolates, 19 Aeromonas hydrophila, 3 Aeromonas veronii bv. sobria, and 1 Aeromonas caviae were identified, while from the environmental strains, 11 A. hydrophila, 22 A. veronii bv. sobria, 1 A. veronii bv. veronii, and 1 A. caviae were recovered. The gene act was detected in 69.5% of clinical isolates, asa-1 in 8.6%, and ascV in 34.7%. In the environmental strains, the detection rates were 51.4%, 45.7%, and 54.2% for the genes act, asa-1, and ascV, respectively. Resistance to amoxicillin-clavulanate and piperacillin-tazobactam was observed in 15 and 3 clinical strains, respectively, and resistance to ceftazidime, meropenem, imipenem, ciprofloxacin, and trimethoprim-sulfamethoxazole was observed in 1 strain for each drug. Resistance to amoxicillin-clavulanate and piperacillin-tazobactam was detected in 17 and 1 environmental strain, respectively. Higher resistance percentages were observed in clinical strains, but environmental strains also showed this phenomenon and presented a higher detection rate of virulence genes. Thus, it is important to monitor the antimicrobial susceptibility and pathogenic potential of the environmental isolates.