Virulence and in vitro antifungal susceptibility of Candida albicans and Candida catenulata from laying hens.

Funder: Università degli Studi di Bari Aldo MoroIn spite of evidence that domestic and wild birds may act as carriers of human pathogenic fungi, data on the role of laying hens as reservoirs of drug resistant and virulent yeasts is lacking. Here, we assess several virulence factors (phospholipase and haemolysin activity) and the antifungal susceptibility profiles of 84 Candida albicans and 17 Candida catenulata strains isolated from cloacae (group A), faeces (group B) and eggs (group C) of laying hens. Of these strains, 95% C. albicans and 23% C. catenulata strains displayed phospholipase and haemolytic activities. For C. albicans, the highest values of phospholipase (Pz = 0.62) and haemolytic activities (Hz = 0.49) were recorded among the strains from group C whilst for C. catenulata (Pz = 0.54; Hz = 0.49) among those from group A. High minimum inhibitory concentration (MIC) values for azoles and amphotericin B (AmB) were recorded irrespective of their sources in all C. albicans strains. A total of 22 C. albicans strains were multidrug resistant, displaying resistance to fluconazole, itraconazole (ITZ), voriconazole (VOR) and posaconazole (POS). All C. catenulata strains from group C were resistant to ITZ, POS, micafungin and anidulafungin and susceptible to AmB. In this study, C. albicans and C. catenulata isolated from the cloacae, faeces and eggs of laying hens produced phospholipase and haemolysin and might be multidrug resistant. In the environment (faeces) or in eggs, C. albicans and C. catenulata strains might acquire pathogenic virulence traits and/or show multidrug resistance profiles. Based on these results, breeding and handling of laying hens and/or eggs may have implications for human and animal health

Virulence and in vitro antifungal susceptibility of Candida albicans and Candida catenulata from laying hens

Funder: Università degli Studi di Bari Aldo MoroAbstract: In spite of evidence that domestic and wild birds may act as carriers of human pathogenic fungi, data on the role of laying hens as reservoirs of drug resistant and virulent yeasts is lacking. Here, we assess several virulence factors (phospholipase and haemolysin activity) and the antifungal susceptibility profiles of 84 Candida albicans and 17 Candida catenulata strains isolated from cloacae (group A), faeces (group B) and eggs (group C) of laying hens. Of these strains, 95% C. albicans and 23% C. catenulata strains displayed phospholipase and haemolytic activities. For C. albicans, the highest values of phospholipase (Pz = 0.62) and haemolytic activities (Hz = 0.49) were recorded among the strains from group C whilst for C. catenulata (Pz = 0.54; Hz = 0.49) among those from group A. High minimum inhibitory concentration (MIC) values for azoles and amphotericin B (AmB) were recorded irrespective of their sources in all C. albicans strains. A total of 22 C. albicans strains were multidrug resistant, displaying resistance to fluconazole, itraconazole (ITZ), voriconazole (VOR) and posaconazole (POS). All C. catenulata strains from group C were resistant to ITZ, POS, micafungin and anidulafungin and susceptible to AmB. In this study, C. albicans and C. catenulata isolated from the cloacae, faeces and eggs of laying hens produced phospholipase and haemolysin and might be multidrug resistant. In the environment (faeces) or in eggs, C. albicans and C. catenulata strains might acquire pathogenic virulence traits and/or show multidrug resistance profiles. Based on these results, breeding and handling of laying hens and/or eggs may have implications for human and animal health

Chemical Composition, Antibacterial and Antifungal Activities of Crude Dittrichia viscosa (L.) Greuter Leaf Extracts

The small amount of data regarding the antifungal activity of Dittrichia viscosa (L.) Greuter against dermatophytes, Malassezia spp. and Aspergillus spp., associated with the few comparative studies on the antimicrobial activity of methanolic, ethanolic, and butanolic extracts underpins the study herein presented. The total condensed tannin (TCT), phenol (TPC), flavonoid (TFC), and caffeoylquinic acid (CQC) content of methanol, butanol, and ethanol (80% and 100%) extracts of D. viscosa were assessed and their bactericidal and fungicidal activities were evaluated. The antibacterial, anti-Candida and anti-Malassezia activities were evaluated by using the disk diffusion method, whereas the anti-Microsporum canis and anti-Aspergillus fumigatus activities were assessed by studying the toxicity effect of the extracts on vegetative growth, sporulation and germination. The methanolic extract contained the highest TPC and CQC content. It contains several phytochemicals mainly caffeoylquinic acid derivatives as determined by liquid chromatography with photodiode array and electrospray ionisation mass spectrometric detection (LC/PDA/ESI-MS) analysis. All extracts showed an excellent inhibitory effect against bacteria and Candida spp., whereas methanolic extract exhibited the highest antifungal activities against Malassezia spp., M. canis and A. fumigatus strains. The results clearly showed that all extracts, in particular the methanolic extract, might be excellent antimicrobial drugs for treating infections that are life threatening (i.e., Malassezia) or infections that require mandatory treatments (i.e., M. canis or A. fumigatus)

Malassezia spp. Yeasts of Emerging Concern in Fungemia

Malassezia spp. are lipid-dependent yeasts, inhabiting the skin and mucosa of humans and animals. They are involved in a variety of skin disorders in humans and animals and may cause bloodstream infections in severely immunocompromised patients. Despite a tremendous increase in scientific knowledge of these yeasts during the last two decades, the epidemiology of Malassezia spp. related to fungemia remains largely underestimated most likely due to the difficulty in the isolation of these yeasts species due to their lipid-dependence. This review summarizes and discusses the most recent literature on Malassezia spp. infection and fungemia, its occurrence, pathogenicity mechanisms, diagnostic methods,in vitro susceptibility testing and therapeutic approache

In Vitro Azole and Amphotericin B Susceptibilities of Malassezia furfur from Bloodstream Infections Using E-Test and CLSI Broth Microdilution Methods

The number of reports of Malassezia furfur bloodstream infections is constantly increasing and there is a need for more simple antifungal susceptibility methods for their management. In this study, a total of 39 M. furfur isolates collected from hospitalized patients with fungemia were screened for antifungal susceptibility to azole and amphotericin B (AmB) using Clinical and Laboratory Standards Institute broth microdilution (CLSI BMD) and E-test in Sabouraud dextrose agar + 1% Tween80 (SDAt) and mDixon agar (DIX). Essential agreement (EA) and discrepancies between the two methods were evaluated after 48 h and 72 h reading times. Itraconazole (ITZ) and posaconazole (POS) displayed the lowest MIC values whereas fluconazole (FLZ) and AmB the highest, regardless of the methods and the reading time. The EA between BMD was >95% for FLZ and voriconazole (VOR) regardless of the media in the E-tests and reading time. The EA between BMD with E-test for AmB was >97% only when E-test in SDAt was used. The EA between BMD and E-test for ITZ and POS varied according to the media in E-test procedures and the reading time and was higher than 66.6% (POS) or 72% (ITZ) only when SABt was used. Substantial discrepancies for ITZ and POS were >5.1% regardless of the media and the reading time. This study suggests that the E-test in SABt represents an alternative method to CLSI BMD to evaluate the susceptibility of M. furfur to FLZ, VOR and AmB and not for ITZ and POS

Chemical Composition, Antibacterial and Antifungal Activities of Crude Dittrichia viscosa (L.) Greuter Leaf Extracts

The small amount of data regarding the antifungal activity of Dittrichia viscosa (L.) Greuter against dermatophytes, Malassezia spp. and Aspergillus spp., associated with the few comparative studies on the antimicrobial activity of methanolic, ethanolic, and butanolic extracts underpins the study herein presented. The total condensed tannin (TCT), phenol (TPC), flavonoid (TFC), and caffeoylquinic acid (CQC) content of methanol, butanol, and ethanol (80% and 100%) extracts of D. viscosa were assessed and their bactericidal and fungicidal activities were evaluated. The antibacterial, anti-Candida and anti-Malassezia activities were evaluated by using the disk diffusion method, whereas the anti-Microsporum canis and anti-Aspergillus fumigatus activities were assessed by studying the toxicity effect of the extracts on vegetative growth, sporulation and germination. The methanolic extract contained the highest TPC and CQC content. It contains several phytochemicals mainly caffeoylquinic acid derivatives as determined by liquid chromatography with photodiode array and electrospray ionisation mass spectrometric detection (LC/PDA/ESI-MS) analysis. All extracts showed an excellent inhibitory effect against bacteria and Candida spp., whereas methanolic extract exhibited the highest antifungal activities against Malassezia spp., M. canis and A. fumigatus strains. The results clearly showed that all extracts, in particular the methanolic extract, might be excellent antimicrobial drugs for treating infections that are life threatening (i.e., Malassezia) or infections that require mandatory treatments (i.e., M. canis or A. fumigatus)

Subtyping Options for Microsporum canis Using Microsatellites and MLST: A Case Study from Southern Italy

Microsporum canis is considered one of the most common zoophilic dermatophyte species causing infections in animals and humans worldwide. However, molecular epidemiological studies on this dermatophyte are still rare. In this study, we aimed to analyse the population structure and relationships between M. canis strains (n = 66) collected in southern Italy and those isolated from symptomatic and asymptomatic animals (cats, dogs and rabbits) and humans. For subtyping purposes, using multilocus sequence typing (MLST) and multilocus microsatellite typing (MLMT), we first used a limited set of strains to screen for variability. No intraspecies variability was detected in six out of the eight reference genes tested and only the ITS and IGS regions showed two and three sequence genotypes, respectively, resulting in five MLST genotypes. All of eight genes were, however, useful for discrimination among M. canis, M. audouinii and M. ferrugineum. In total, eighteen microsatellite genotypes (A–R) were recognized using MLMT based on six loci, allowing a subdivision of strains into two clusters based on the Bayesian iterative algorithm. Six MLMT genotypes were from multiple host species, while 12 genotypes were found only in one host. There were no statistically significant differences between clusters in terms of host spectrum and the presence or absence of lesions. Our results confirmed that the MLST approach is not useful for detailed subtyping and examining the population structure of M. canis, while microsatellite analysis is a powerful tool for conducting surveillance studies and gaining insight into the epidemiology of infections due to this pathogen

Effect of chlorogenic and gallic acids combined with azoles on antifungal susceptibility and virulence of multidrug-resistant Candida spp. and Malassezia furfur isolates

Chlorogenic acid (CHA) and gallic acid (GA) are safe natural phenolic compounds that are used as enhancers of some drugs in influencing antioxidant, anticancer, and antibacterial activities. Among fungi, Candida spp. and Malassezia spp. are characterized by an increasing prevalence of multidrug resistance phenomena and by a high morbidity and mortality of their infections. No data are available about the efficacy of CHA and GA combined with azoles on the antifungal susceptibility and on the virulence of both fungi. Therefore, their antifungal and antivirulence effects have been tested in combination with fluconazole (FLZ) or ketoconazole (KTZ) on 23 Candida spp. and 8 M. furfur isolates. Broth microdilution chequerboard, time-kill studies, and extracellular enzymes (phospholipase and hemolytic) activities were evaluated, displaying a synergistic antifungal action between CHA or GA and FLZ or KTZ on C. albicans, C. bovina, and C. parapsilosis, and antagonistic antifungal effects on M. furfur and Pichia kudriavzevii (Candida krusei) isolates. The time-kill studies confirmed the chequerboard findings, showing fungicidal inhibitory effect only when the GA was combined with azoles on Candida strains. However, the combination of phenolics with azoles had no effect on the virulence of the tested isolates. Our study indicates that the combination between natural products and conventional drugs could be an efficient strategy for combating azole resistance and for controlling fungistatic effects of azole drugs

Dittrichia viscosa L. Ethanolic Extract Based Ointment with Antiradical, Antioxidant, and Healing Wound Activities

Dittrichia viscosa which belongs to the Asteraceae family is frequently used to treat hematomas and skin disorders in Mediterranean herbal medicine. This study aims to validate its antioxidant effects and its potential on healing wounds. The ethanolic extract of D. viscosa leaves was formulated as 2.5% and 5% (w/w) in ointment bases on the beeswax and sesame oil. During this study, the ethanolic D. viscosa extract, ointments containing 2.5% and 5% of D. viscosa extract, and the vehiculum were assessed for their total phenol content (TPC), caffeoylquinic acid content (CQC), and antioxidant activities using complementary methods (TAC, the DPPH, ABTS, FRAP, and the BCB). The effects on wound healing of obtained ointments were evaluated by excision of the wound in a mice model for 12 days. Subsequently, the excised wound areas were measured at the 3rd, 9th, and 12th days. The skin tissues were isolated for histological studies. The ointments containing D. viscosa extract (2.5%, 5%) possessed a considerable TPC, CQC, radical scavenging potential, and antioxidant activities compared to the vehiculum. Treated animals with ointments containing D. viscosa extract at 2.5% and 5% showed almost and totally healed wounds compared to the vehiculum and control groups, evidenced by good skin regeneration and reepithelialization. The present work showed the role of D. viscosa antioxidants exerted by its polyphenolic compounds, in particular, caffeoylquinic acids, in enhancing wound healing

The best type of inoculum for testing the antifungal drug susceptibility of Microsporum canis: in vivo and in vitro results

Background Data correlating in vitro drug susceptibility of Microsporum canis with clinical outcomes of its infections are lacking as well as the most suitable inoculum and incubation time in broth microdilution assays. Objectives and Methods M. canis strains were collected from animal hosts that tested positive (Group I; n=13) and negative (Group II; n=14) to this pathogen following itraconazole (ITC) therapy. In vitro ITC susceptibility was assessed according to the Clinical Laboratory Standards Institute (CLSI M38-A2) methodology using conidia, hypha-conidia and arthroconidia at 3 and 7 days of incubation in order to assess the most suitable inoculum and incubation time. Successively, ketoconazole (KTC), voriconazole (VRC) terbinafine (TRB), posaconazole (PSZ), fluconazole (FLC) and griseofulvin (GRI) susceptibilities were assessed using the chosen inoculum. Results The MIC values of ITC after three day-incubation were equal than those recorded after seven day-incubation. ITC MICs were ≤1μg/ml for strains from Group II and >1μg/ml for those of Group II only when conidia were used. All strains showed high susceptibility to VRC, POS, TEB and low susceptibility to ITC, KTC, GRI and FLC regardless of the source and incubation time. Conclusions and clinical importance Results suggest that correlation between the in vitro results and clinical outcome was observed only by incubating conidia for 3 days at 30±2oC.These conditions might be most suitable to assess in vitro susceptibility of M. canis and assist in determining the occurrence of drug resistance and cross -resistance phenomena