Davide Immediato

Azole susceptibility of Malassezia pachydermatis and Malassezia furfur and tentative epidemiological cut-off values.

This study aims to determine the minimal inhibitory concentration (MIC) distribution and the epidemiological cut-off values (ECVs) of Malassezia pachydermatis and Malassezia furfur isolates for fluconazole (FLZ), itraconazole (ITZ), posaconazole (POS), and voriconazole (VOR). A total of 62 M. pachydermatis strains from dogs with dermatitis and 78 M. furfur strains from humans with bloodstream infections (BSI) were tested by a modified broth microdilution Clinical and Laboratory Standards Institute (CLSI) method. ITZ and POS displayed lower MICs than VOR and FLZ, regardless of the Malassezia species. The MIC data for azoles of M. pachydermatis were four two-fold dilutions lower than those of M. furfur. Based on the ECVs, about 94% of Malassezia strains might be categorized within susceptible population for all azoles, except for FLZ, and azole cross-resistance was detected in association with FLZ in M. pachydermatis but not in M. furfur.The study proposes, for the first time, tentative azole ECVs for M. pachydermatis and M. furfur for monitoring the emergence of isolates with decreased susceptibilities and shows that the azole MIC distribution varied according to the Malassezia species tested, thus suggesting the usefulness of determining the susceptibility profile for effective treatment of each species.

Species Distribution and In Vitro Azole Susceptibility of Aspergillus Section Nigri Isolates from Clinical and Environmental Settings

ABSTRACT Aspergillus section Nigri includes species of interest for animal and human health, although studies on species distribution are limited to human cases. Data on the antifungal susceptibilities and the molecular mechanism of triazole resistance in strains belonging to this section are scant. Forty-two black Aspergillus strains from human patients (16 isolates), animals (14 isolates), and the environment (12 isolates) were molecularly characterized and their in vitro triazole susceptibilities investigated. Aspergillus tubingensis was isolated from humans, animals, and environmental settings, whereas Aspergillus awamori and Aspergillus niger were isolated exclusively from humans. Phylogenetic analyses of β-tubulin and calmodulin gene sequences were concordant in differentiating A. tubingensis from A. awamori and A. niger. Voriconazole and posaconazole (PSZ) were the most active triazoles. One A. tubingensis strain was resistant to itraconazole and PSZ and one A. niger strain to PSZ. Sequence analysis of the cyp51A gene revealed different sequence types within a species, and A. tubingensis strains were also phylogenetically distinct from A. awamori/A. niger strains according to the strain origin and susceptibility profile. Genetic analysis of the cyp51A sequences suggests that two nonsynonymous mutations resulting in amino acid substitutions in the CYP51A protein (changes of L to R at position 21 [L21R] and of Q to R at position 228 [Q228R]) might be involved in azole resistance. Though azole resistance in black Aspergillus isolates from animals and rural environments does not represent a threat to public health in Southern Italy, the use of triazoles in the clinical setting needs to better monitored. The cyp51A sequence is useful for the molecular identification of black Aspergillus, and point mutations in protein sequences could be responsible for azole resistance phenomena.

In vitro and in vivo activity of a killer peptide against Malassezia pachydermatis causing otitis in dogs

In order to overcome the limitations inherent in current pharmacological treatments for Malassezia pachydermatis, the cause of otitis externa in dogs, the efficacy of a killer decapeptide (KP) was evaluated in vitro and in vivo. Sixteen dogs with naturally occurring M. pachydermatis otitis externa were enrolled, and the in vitro fungicidal activity of KP was evaluated using yeasts recovered from these animals. The therapeutic activity was evaluated in four groups of four animals each. The dogs were topically treated with KP (150 μl, 2 mg/ml) three times per week (group A) or every day (group B), treated with a scramble peptide every day (group C), or left untreated (group D). Assessment of clinical signs (pruritus, erythema, and lichenification and/or hyperpigmentation), expressed as mean of the total clinical index score (mTCIS), the population size of M. pachydermatis at the cytological examination (mean number of yeast cells at 40× magnification [mYC]), and culture testing (mean number of log10 CFU/swab [mCFU]), were conducted daily from the first day of treatment (T0) until two consecutive negative cultures (mCFU ≤ 2). KP showed an in vitro fungicidal effect against M. pachydermatis isolates, with an MFC90 value of 1 μg/ml. The mTCIS, mYC and mCFU were negative only in animals in group B after T8. Daily administration of KP for 8 days was safe and effective in controlling both clinical signs and the population size of M. pachydermatis causing otitis externa, thus offering an alternative to the currently available therapeutic or prophylactic protocols for recurrent cases of Malassezia otitis in dogs.

The role of drug efflux pumps in Malassezia pachydermatis and Malassezia furfur defence against azoles

This study aims to evaluate the effect of efflux pump modulators (EPMs) on the minimal inhibitory concentration (MIC) of fluconazole (FLZ) and voriconazole (VOR) in Malassezia furfur and Malassezia pachydermatis. The in vitro efficacy of azoles, in combination with EPMs (ie haloperidol‐HAL, promethazine‐PTZ and cyclosporine A‐CYS), against 21 M. furfur from bloodstream infection patients and 14 M. pachydermatis from the skin of dogs with dermatitis, was assessed using a broth microdilution chequerboard analysis. Data were analysed using the model‐fractional inhibitory concentration index (FICI) method. The MIC of FLZ and VOR of Malassezia spp. decreased in the presence of sub‐inhibitory concentrations of HAL and/or PTZ. The synergic effect was observed only in strains with FLZ MIC≥128 μg/mL for M. furfur, FLZ MIC≥64 μg/mL for M. pachydermatis and VOR MIC≥4 μg/mL in both Malassezia spp. These results suggest that the drug efflux pumps are involved as defence mechanisms to azole drugs in Malassezia yeast. The synergism might be related to an increased expression of efflux pump genes, eventually resulting in azole resistance phenomena. Finally, the above FLZ and VOR MIC values might be considered the cut‐off to discriminate susceptible and resistant strains.

Native strains of Beauveria bassiana for the control of Rhipicephalus sanguineus sensu lato

BackgroundRhipicephalus sanguineus sensu lato ticks are widespread worldwide due to their adaptability to survive under different environmental conditions. They may act as vectors of a wide range of pathogens to humans and animals and their control is based on the use of chemical products on dogs and in the environment. Alternative control strategies, such as the use of entomopathogenic fungi as bio-control agents have also been investigated. The ability of native strains of Beauveria bassiana sensu lato in causing mortality in different tick species (e.g., Amblyomma cajennense and Rhipicephalus microplus) has been demonstrated. However, limited studies have assessed the use of B. bassiana for the control of R. sanguineus s.l. and none of them have employed native strains of this fungus. Here we investigated the pathogenicity of a native strain of B. bassiana (CD1123) against all developmental stages of R. sanguineus s.l..MethodsBatches of eggs, larvae, nymphs and adult ticks were immersed in a suspension of 107 conidia/ml of B. bassiana s.l., isolated from a R. sanguineus s.l. engorged female. All treatment and control groups were observed for 20 days, and the biological parameters (i.e., mortality, hatching, moulting percentage, pre-oviposition period, oviposition period and rate, eggs production efficiency, reproductive efficiency and fitness indexes) were assessed.ResultsThe effect of the B. bassiana strain tested herein on eggs, larvae, nymphs and adults showed a significantly higher mortality than those of the control groups (p < 0.05) at 5 days post-infection. No infected eggs hatched and no infected larvae moulted. Only 15% of infected nymphs moulted into adults. All biological parameters of treated groups differed significantly (p < 0.001) from those of control groups.ConclusionsThis study demonstrates that a suspension containing 107 conidia/ml of a native B. bassiana strain is highly virulent towards all life-cycle developmental stages of R. sanguineus s.l. and may be of potential interest as a biological control agent against these ticks.

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).

Laboratory evaluation of a native strain of Beauveria bassiana for controlling Dermanyssus gallinae (De Geer, 1778) (Acari: Dermanyssidae).

The poultry red mite, Dermanyssus gallinae (De Geer, 1778) (Acari: Dermanyssidae) is one of the most economically important ectoparasites of laying hens worldwide. Chemical control of this mite may result in environmental and food contamination, as well as the development of drug resistance. High virulence of Beauveria bassiana sensu lato strains isolated from naturally infected hosts or from their environment has been demonstrated toward many arthropod species, including ticks. However, a limited number of studies have assessed the use of B. bassiana for the control of D. gallinae s.l. and none of them have employed native strains. This study reports the pathogenicity of a native strain of B. bassiana (CD1123) against nymphs and adults of D. gallinae. Batches of nymph and adult mites (i.e., n=720 for each stage) for treated groups (TGs) were placed on paper soaked with a 0.1% tween 80 suspension of B. bassiana (CIS, 10(5), 10(7) and 10(9) conidia/ml), whilst 240 untreated control mites for each stage (CG) were exposed only to 0.1% tween 80. The mites in TG showed a higher mortality at all stages (p<0.01) when compared to CG, depending on the time of exposure and the conidial concentration. A 100% mortality rate was recorded using a CIS of 10(9) conidia/ml 12 days post infection (DPI) in adults and 14 DPI in nymphs. B. bassiana suspension containing 10(9) conidia/ml was highly virulent towards nymph and adult stages of D. gallinae, therefore representing a possible promising natural product to be used in alternative or in combination to other acaricidal compounds currently used for controlling the red mite.

Storage of Beauveria bassiana Conidia Suspension: A Study Exploring the Potential Effects on Conidial iability and Virulence against Dermanyssus gallinae De Geer, 1778 Acari: Dermanyssidae

Beauveria bassiana are usually formulated as conidia infection suspensions CIS for field and laboratory applications as biocontrol agent, but the factors affecting CIS shelf-life and virulence, are unexplored. With the aim of investigating the best storage conditions for extending the CIS shelf-life, the potential effects of storage at room 22 ± 1°C and refrigerated temperatures 4 ± 1°C, with and without light and agitation, on viability and virulence of B. bassiana CISs, was assessed. The viability of conidia was evaluated monthly, over a one year period, and the B. bassiana virulence against D. gallinae, five times in one year. B. bassiana had a significantly higher rate of survival at 4 ± 1°C compared to 22 ± 1°C, regardless of the other conditions of storage. The highest mortality rate of D. gallinae was registered for CIS stored at 4 ± 1°C, in dark and without agitation p<0.05. These results indicate that refrigerating CIS in the dark and without agitation seems to guarantee the best viability and performance of B. bassiana against D. gallinae. Data obtained in this study confirm B. bassiana as potential promising candidate for controlling red mites and provide evidences that appropriate storage condition extend CIS shelf-life for up to one year.