Prevalence and genetic diversity of azole-resistant Malassezia pachydermatis isolates from canine otitis and dermatitis: A 2-year study.

Despite previous reports on the emergence of Malassezia pachydermatis strains with decreased susceptibility to azoles, there is limited information on the actual prevalence and genetic diversity of azole-resistant isolates of this yeast species. We assessed the prevalence of azole resistance in M. pachydermatis isolates from cases of dog otitis or skin disease attended in a veterinary teaching hospital during a 2-year period and analyzed the ERG11 (encoding a lanosterol 14-α demethylase, the primary target of azoles) and whole genome sequence diversity of a group of isolates that displayed reduced azole susceptibility. Susceptibility testing of 89 M. pachydermatis isolates from 54 clinical episodes (1-6 isolates/episode) revealed low minimum inhibitory concentrations (MICs) to most azoles and other antifungals, but 11 isolates from six different episodes (i.e., 12.4% of isolates and 11.1% of episodes) had decreased susceptibility to multiple azoles (fluconazole, itraconazole, ketoconazole, posaconazole, ravuconazole, and/or voriconazole). ERG11 sequencing of these 11 azole-resistant isolates identified eight DNA sequence profiles, most of which contained amino acid substitutions also found in some azole-susceptible isolates. Analysis of whole genome sequencing (WGS) results revealed that the azole-resistant isolates from the same episode of otitis, or even different episodes affecting the same animal, were more genetically related to each other than to isolates from other dogs. In conclusion, our results confirmed the remarkable ERG11 sequence variability in M. pachydermatis isolates of animal origin observed in previous studies and demonstrated the value of WGS for disentangling the epidemiology of this yeast species.

We analyzed the prevalence and diversity of azole-resistant Malassezia pachydermatis isolates in a veterinary hospital. A low prevalence of multi-azole resistance (c.10% of isolates and cases) was found. Whole genome and ERG11 sequencing of resistant isolates revealed remarkable genetic diversity.

Phylogenomic analysis of the genus Rosenbergiella and description of Rosenbergiella gaditana sp. nov., Rosenbergiella metrosideri sp. nov., Rosenbergiella epipactidis subsp. epipactidis subsp. nov., Rosenbergiella epipactidis subsp. californiensis subsp. nov., Rosenbergiella epipactidis subsp. japonicus subsp. nov., Rosenbergiella nectarea subsp. nectarea subsp. nov. and Rosenbergiella nectarea subsp. apis subsp. nov., isolated from floral nectar and insects.

The genus Rosenbergiella is one of the most frequent bacterial inhabitants of flowers and a usual member of the insect microbiota worldwide. To date, there is only one publicly available Rosenbergiella genome, corresponding to the type strain of Rosenbergiella nectarea (8N4T), which precludes a detailed analysis of intra-genus phylogenetic relationships. In this study, we obtained draft genomes of the type strains of the other Rosenbergiella species validly published to date (R. australiborealis, R. collisarenosi and R. epipactidis) and 23 additional isolates of flower and insect origin. Isolate S61T, retrieved from the nectar of an Antirrhinum sp. flower collected in southern Spain, displayed low average nucleotide identity (ANI) and in silico DNA-DNA hybridization (isDDH) values when compared with other Rosenbergiella members (≤86.5 and ≤29.8 %, respectively). Similarly, isolate JB07T, which was obtained from the floral nectar of Metrosideros polymorpha plants in Hawaii (USA) had ≤95.7 % ANI and ≤64.1 % isDDH with other Rosenbergiella isolates. Therefore, our results support the description of two new Rosenbergiella species for which we propose the names Rosenbergiella gaditana sp. nov. (type strain: S61T=NCCB 100789T=DSM 111181T) and Rosenbergiella metrosideri sp. nov. (JB07T=NCCB 100888T=LMG 32616T). Additionally, some R. epipactidis and R. nectarea isolates showed isDDH values<79 % with other conspecific isolates, which suggests that these species include subspecies for which we propose the names Rosenbergiella epipactidis subsp. epipactidis subsp. nov. (S256T=CECT 8502T=LMG 27956T), Rosenbergiella epipactidis subsp. californiensis subsp. nov. (FR72T=NCCB 100898T=LMG 32786T), Rosenbergiella epipactidis subsp. japonicus subsp. nov. (K24T=NCCB 100924T=LMG 32785T), Rosenbergiella nectarea subsp. nectarea subsp. nov. (8N4T = DSM 24150T = LMG 26121T) and Rosenbergiella nectarea subsp. apis subsp. nov. (B1AT=NCCB 100810T= DSM 111763T), respectively. Finally, we present the first phylogenomic analysis of the genus Rosenbergiella and update the formal description of the species R. australiborealis, R. collisarenosi, R. epipactidis and R. nectarea based on new genomic and phenotypic information.

Sugar Concentration, Nitrogen Availability, and Phylogenetic Factors Determine the Ability of Acinetobacter spp. and Rosenbergiella spp. to Grow in Floral Nectar.

The floral nectar of angiosperms harbors a variety of microorganisms that depend predominantly on animal visitors for their dispersal. Although some members of the genus Acinetobacter and all currently known species of Rosenbergiella are thought to be adapted to thrive in nectar, there is limited information about the response of these bacteria to variation in the chemical characteristics of floral nectar. We investigated the growth performance of a diverse collection of Acinetobacter (n = 43) and Rosenbergiella (n = 45) isolates obtained from floral nectar and the digestive tract of flower-visiting bees in a set of 12 artificial nectars differing in sugar content (15% w/v or 50% w/v), nitrogen content (3.48/1.67 ppm or 348/167 ppm of total nitrogen/amino nitrogen), and sugar composition (only sucrose, 1/3 sucrose + 1/3 glucose + 1/3 fructose, or 1/2 glucose + 1/2 fructose). Growth was only observed in four of the 12 artificial nectars. Those containing elevated sugar concentration (50% w/v) and low nitrogen content (3.48/1.67 ppm) were limiting for bacterial growth. Furthermore, phylogenetic analyses revealed that the ability of the bacteria to grow in different types of nectar is highly conserved between closely related isolates and genotypes, but this conservatism rapidly vanishes deeper in phylogeny. Overall, these results demonstrate that the ability of Acinetobacter spp. and Rosenbergiella spp. to grow in floral nectar largely depends on nectar chemistry and bacterial phylogeny.

In vitro activity of fidaxomicin and combinations of fidaxomicin with other antibiotics against Clostridium perfringens strains isolated from dogs and cats.

BACKGROUND: Previous studies have demonstrated that fidaxomicin, a macrocyclic lactone antibiotic used to treat recurrent Clostridioides difficile-associated diarrhea, also displays potent in vitro bactericidal activity against Clostridium perfringens strains isolated from humans. However, to date, there is no data on the susceptibility to fidaxomicin of C. perfringens strains of animal origin. On the other hand, although combination therapy has become popular in human and veterinary medicine, limited data are available on the effects of antibiotic combinations on C. perfringens. We studied the in vitro response of 21 C. perfringens strains obtained from dogs and cats to fidaxomicin and combinations of fidaxomicin with six other antibiotics. RESULTS: When tested by an agar dilution method, fidaxomicin minimum inhibitory concentrations (MICs) ranged between 0.004 and 0.032 µg/ml. Moreover, the results of Etest-based combination assays revealed that the incorporation of fidaxomicin into the test medium at a concentration equivalent to half the MIC significantly increased the susceptibility of isolates to metronidazole and erythromycin in 71.4% and 61.9% of the strains, respectively, and the susceptibility to clindamycin, imipenem, levofloxacin, and vancomycin in 42.9-52.4% of the strains. In contrast, » × MIC concentrations of fidaxomicin did not have any effect on levofloxacin and vancomycin MICs and only enhanced the effects of clindamycin, erythromycin, imipenem, and metronidazole in ≤ 23.8% of the tested strains. CONCLUSIONS: The results of this study demonstrate that fidaxomicin is highly effective against C. perfringens strains of canine and feline origin. Although fidaxomicin is currently considered a critically important antimicrobial that has not yet been licensed for veterinary use, we consider that the results reported in this paper provide useful baseline data to track the possible emergence of fidaxomicin resistant strains of C. perfringens in the veterinary setting.

Contrasting effects of nectar yeasts on the reproduction of Mediterranean plant species.

PREMISE: Yeasts are often present in floral nectar and can influence plant fitness directly (independently of pollinators) or indirectly by influencing pollinator visitation and behavior. However, few studies have assessed the effect of nectar yeasts on plant reproductive success or compared effects across different plant species, limiting our understanding of the relative impact of direct vs. indirect effects. METHODS: We inoculated the nectar of six plant species in the field with the cosmopolitan yeast Metschnikowia reukaufii to analyze the direct and indirect effects on female reproductive success over 2 years. The pollinator assemblage for each species was recorded during both flowering years. RESULTS: Direct yeast effects on female fecundity were statistically nonsignificant for all plant species. There were significant indirect, pollinator-mediated effects on fruit production and seed mass for the two species pollinated almost exclusively by bumblebees or hawkmoths, with the direction of the effects differing for the quantity- and quality-related fitness components. There were no consistent effects of the yeast on maternal fecundity for any of the species with diverse pollinator assemblages. CONCLUSIONS: Effects of M. reukaufii on plant reproduction ranged from negative to neutral or positive depending on the plant species. The among-species variation in the indirect effects of nectar yeasts on plant pollination could reflect variation in the pollinator community, the specific microbes colonizing the nectar, and the order of microbial infection (priority effects), determining potential species interactions. Elucidating the nature of these multitrophic plant-pollinator-microbe interactions is important to understand complex processes underlying plant pollination.

Acinetobacter pollinis sp. nov., Acinetobacter baretiae sp. nov. and Acinetobacter rathckeae sp. nov., isolated from floral nectar and honey bees.

A detailed evaluation of eight bacterial isolates from floral nectar and animal visitors to flowers shows evidence that they represent three novel species in the genus Acinetobacter. Phylogenomic analysis shows the closest relatives of these new isolates are Acinetobacter apis, Acinetobacter boissieri and Acinetobacter nectaris, previously described species associated with floral nectar and bees, but high genome-wide sequence divergence defines these isolates as novel species. Pairwise comparisons of the average nucleotide identity of the new isolates compared to known species is extremely low (<83 %), thus confirming that these samples are representative of three novel Acinetobacter species, for which the names Acinetobacter pollinis sp. nov., Acinetobacter baretiae sp. nov. and Acinetobacter rathckeae sp. nov. are proposed. The respective type strains are SCC477T (=TSD-214T=LMG 31655T), B10AT (=TSD-213T=LMG 31702T) and EC24T (=TSD-215T=LMG 31703T=DSM 111781T).

Susceptibility testing of Prototheca bovis isolates from cases of bovine mastitis using the CLSI reference broth microdilution method and the Sensititre YeastOne colorimetric panel.

A total of 62 Prototheca bovis isolates from cases of bovine mastitis were tested for susceptibility to different antifungal compounds by the Clinical and Laboratory Standards Institute (CLSI) reference microdilution method and a commercial colorimetric microdilution panel (Sensititre YeastOne). All isolates displayed low susceptibility to echinocandins (MICs > 8 µg/ml for anidulafungin, caspofungin, and micafungin), flucytosine (MIC > 64 µg/ml), and the azoles enilconazole and fluconazole (MICs > 4 and > 64 µg/ml, respectively). Moreover, 45.2, 32.3, and 1.6% of isolates had MICs > 4 µg/ml for ketoconazole, terbinafine, and voriconazole, respectively, when tested by the CLSI method. In contrast, all isolates were more susceptible to the polyene compounds amphotericin B and nystatin, and itraconazole, posaconazole, and ravuconazole (MICs ≤ 2 µg/ml, in all cases). Comparison of the results obtained in the CLSI and Sensititre methods showed excellent essential agreement (EA) for azoles (98.4% for itraconazole and posaconazole, and 100% for voriconazole) and moderate EA for amphotericin B (72.6%), when MICs were read after 48 and 24 h of incubation, respectively. In contrast, much lower EA values were obtained in some cases when the MICs for both techniques were determined after 48 h of incubation (e.g., 9.7% for amphotericin B and 69.4% for posaconazole). Therefore, the CLSI broth microdilution method and the Sensititre YeastOne panel can be used indistinctly for susceptibility testing of P. bovis isolates against azoles but not against amphotericin B until further optimization of the test conditions. LAY SUMMARY: The antifungal susceptibility of Prototheca bovis isolates was analyzed. All tested isolates displayed low susceptibility to echinocandins, flucytosine, and some azoles. Excellent agreement of the results of two different test methods was obtained for azoles, but not for the polyene amphotericin B.

Nitrogen Assimilation Varies Among Clades of Nectar- and Insect-Associated Acinetobacters.

Floral nectar is commonly colonized by yeasts and bacteria, whose growth largely depends on their capacity to assimilate nutrient resources, withstand high osmotic pressures, and cope with unbalanced carbon-to-nitrogen ratios. Although the basis of the ecological success of these microbes in the harsh environment of nectar is still poorly understood, it is reasonable to assume that they are efficient nitrogen scavengers that can consume a wide range of nitrogen sources in nectar. Furthermore, it can be hypothesized that phylogenetically closely related strains have more similar phenotypic characteristics than distant relatives. We tested these hypotheses by investigating the growth performance on different nitrogen-rich substrates of a collection of 82 acinetobacters isolated from nectar and honeybees, representing members of five species (Acinetobacter nectaris, A. boissieri, A. apis, and the recently described taxa A. bareti and A. pollinis). We also analyzed possible links between growth performance and phylogenetic affiliation of the isolates, while taking into account their geographical origin. Results demonstrated that the studied isolates could utilize a wide variety of nitrogen sources, including common metabolic by-products of yeasts (e.g., ammonium and urea), and that phylogenetic relatedness was associated with the variation in nitrogen assimilation among the studied acinetobacters. Finally, nutrient source and the origin (sample type and country) of isolates also predicted the ability of the acinetobacters to assimilate nitrogen-rich compounds. Overall, these results demonstrate inter-clade variation in the potential of the acinetobacters as nitrogen scavengers and suggest that nutritional dependences might influence interactions between bacteria and yeasts in floral nectar.

In vitro activity of amphotericin B-azole combinations against Malassezia pachydermatis strains.

Combination therapy has become popular in clinical practice, but limited data on the effects of combinations of antifungal agents is still available for most fungal pathogens. We studied the in vitro response of 30 genetically diverse clinical strains of the basidiomycetous lipophilic yeast Malassezia pachydermatis obtained from cases of canine otitis to several amphotericin B (AMB)-azole combinations. Broth microdilution checkerboard tests revealed that AMB antagonized the effects of itraconazole (ITC) and voriconazole (VRC) in 50% and 6.7% of the strains, respectively, but did not interact with fluconazole or posaconazole (fractional inhibitory concentration index (FICI) values were <4 in all cases). Subsequent Etest-based assays performed for a subset of strains did not confirm the antagonism between AMB and ITC or AMB and VRC. In summary, the results of this study suggest that antagonistic combination effects between AMB and azoles might occur when tested against M. pachydermatis. Nevertheless, as observed for other fungi, different in vitro analyses yielded contrasting results, and the response to AMB-azole combinations was compound- and strain-dependant.

Phylogenetic signal in phenotypic traits related to carbon source assimilation and chemical sensitivity in Acinetobacter species.

A common belief is that the phylogeny of bacteria may reflect molecular functions and phenotypic characteristics, pointing towards phylogenetic conservatism of traits. Here, we tested this hypothesis for a large set of Acinetobacter strains. Members of the genus Acinetobacter are widespread in nature, demonstrate a high metabolic diversity and are resistant to several environmental stressors. Notably, some species are known to cause opportunistic human infections. A total of 133 strains belonging to 33 species with validly published names, two genomic species and species of an as-yet unknown taxonomic status were analyzed using the GENIII technology of Biolog, which allows high-throughput phenotyping. We estimated the strength and significance of the phylogenetic signal of each trait across phylogenetic reconstructions based on partial RNA polymerase subunit B (rpoB) and core genome sequences. Secondly, we tested whether phylogenetic distance was a good predictor of trait differentiation by Mantel test analysis. And finally, evolutionary model fitting was used to determine if the data for each phenotypic character was consistent with a phylogenetic or an essentially random model of trait distribution. Our data revealed that some key phenotypic traits related to substrate assimilation and chemical sensitivity are linked to the phylogenetic placement of Acinetobacter species. The strongest phylogenetic signals found were for utilization of different carbon sources such as some organic acids, amino acids and sugars, thus suggesting that in the diversification of Acinetobacter carbon source assimilation has had a relevant role. Future work should be aimed to clarify how such traits have shaped the remarkable ability of this bacterial group to dominate in a wide variety of habitats.

Prevalence and characteristics of Clostridium perfringens and Clostridium difficile in dogs and cats attended in diverse veterinary clinics from the Madrid region.

Despite extensive research on the epidemiology of pathogenic clostridia in dogs and cats, most published studies focus on a selected animal population and/or a single veterinary medical centre. We assessed the burden of Clostridium perfringens and C. difficile shedding by small animals in 17 veterinary clinics located within the Madrid region (Spain) and differing in size, number and features of animals attended and other relevant characteristics. In addition, we studied the genetic diversity and antibiotic susceptibility of recovered isolates. Selective culture of all fecal specimens collected during a single week from dogs (n = 105) and cats (n = 37) attended in participating clinics yielded C. perfringens/C. difficile from 31%, 4.8% of the dogs, and 20%, 0% of the cats analyzed, respectively, and three dogs yielded both species. Furthermore, 17 animals (15 dogs and two cats) that yielded a positive culture for either species were recruited for a follow-up survey and C. perfringens was again obtained from nine dogs. Considerable differences in prevalence were observed among participating clinics for both clostridial species. C. perfringens isolates (n = 109) belonged to toxinotypes A (97.2%) and E (three isolates from one dog), whereas C. difficile isolates (n = 18) belonged to the toxigenic ribotypes 106 (33.3%) and 154 (16.7%), a 009-like ribotype (33.3%) and an unknown non-toxigenic ribotype (16.7%). Amplified fragment length polymorphism-based fingerprinting classified C. perfringens and C. difficile isolates into 105 and 15 genotypes, respectively, and tested isolates displayed in vitro resistance to benzylpenicillin (2.8%, 88.8%), clindamycin (0%, 16.7%), erythromycin (0.9%, 16.7%), imipenem (1.8%, 100%), levofloxacin (0.9%, 100%), linezolid (5.5%, 0%), metronidazole (4.6%, 0%) and/or tetracycline (7.3%, 0%). All animals from which multiple isolates were retrieved yielded ≥2 different genotypes and/or antimicrobial susceptibility profiles. Future studies should focus on the seasonal and geographical variations of prevalence and diversity patterns of clostridial species in small animals.

Isolation of Clostridium difficile from dogs with digestive disorders, including stable metronidazole-resistant strains.

The prevalence of Clostridium difficile in 107 dogs with diverse digestive disorders attended in a Spanish veterinary teaching hospital was assessed. The microorganism was isolated from 13 dogs (12.1%) of different disease groups. Isolates belonged to PCR ribotypes 078, 106, 154 and 430 (all of them toxigenic) and 110 (non-toxigenic), and were resistant to several antimicrobial drugs. Notably, seven isolates obtained from different dogs displayed stable resistance to metronidazole. The results of this study provide further evidence that dogs can act as a reservoir of C. difficile strains of epidemic ribotypes with resistance to multiple antibiotics.

Antifungal Susceptibility Testing of Ascomycetous Yeasts Isolated from Animals.

Recent studies suggest that antifungal resistance in yeast isolates of veterinary origin may be an underdiagnosed threat. We tested a collection of 92 ascomycetous yeast isolates that were obtained in Spain from birds, mammals and insects for antifungal susceptibility. MICs to amphotericin B and azoles were low, and no resistant isolates were detected. Despite these results, and given the potential role of animals as reservoirs of resistant strains, continuous monitoring of antifungal susceptibility in the veterinary setting is recommended.

Nectar yeasts of the Metschnikowia clade are highly susceptible to azole antifungals widely used in medicine and agriculture.

The widespread use of azole antifungals in medicine and agriculture and the resulting long-persistent residues could potentially affect beneficial fungi. However, there is very little information on the tolerance of non-target environmental fungi to azoles. In this study, we assessed the susceptibility of diverse plant- and insect-associated yeasts from the Metschnikowia clade, including several ecologically important species, to widely used medical and agricultural azoles (epoxiconazole, imazalil, ketoconazole and voriconazole). A total of 120 strains from six species were tested. Minimum inhibitory concentrations (MICs) were determined by the EUCAST broth microdilution procedure after some necessary modifications were made. The majority of species tested were highly susceptible to epoxiconazole, ketoconazole and voriconazole (>95% of strains showed MICs ≤ 0.125 mg l(-1)). Most strains were also very susceptible to imazalil, although MIC values were generally higher than for the other azoles. Furthermore, certain Metschnikowia reukaufii strains displayed a 'trailing' phenotype (i.e. showed reduced but persistent growth at antifungal concentrations above the MIC), but this characteristic was dependent on test conditions. It was concluded that exposure to azoles may pose a risk for ecologically relevant yeasts from the Metschnikowia clade, and thus could potentially impinge on the tripartite interaction linking these fungi with plants and their insect pollinators.

Genotyping and antifungal susceptibility testing of multiple Malassezia pachydermatis isolates from otitis and dermatitis cases in pets: is it really worth the effort?

A total of 216 colonies of Malassezia pachydermatis from 28 cases of fungal otitis or dermatitis in pets were genotyped by M13 fingerprinting and tested for antifungal susceptibility. A huge genetic diversity was found (157 M13 types in total), with all animals having a polyclonal pattern of infection (5.4 ± 1.5 genotypes/sample). Furthermore, analysis of molecular variance (AMOVA) revealed that most genetic diversity (44%) was found at the within sample level. In contrast, variability in antifungal susceptibility among isolates from the same sample was less important, with different M13 types displaying in most cases identical or very similar MIC results. Most isolates displayed high in vitro susceptibility to amphotericin B, terbinafine and all azoles tested except fluconazole, for which MIC values were always ≥4 µg/ml and a 26.9% of isolates displayed values ≥32 µg/ml. We conclude that although characterization of multiple yeast isolates results in a considerable increase in laboratory workload and expenses, it may help to get a better understanding of the epidemiology of M. pachydermatis in a given patient population.

Water Sources in a Zoological Park Harbor Genetically Diverse Strains of Clostridium Perfringens Type A with Decreased Susceptibility to Metronidazole.

The presence of Clostridium perfringens in water is generally regarded as an indicator of fecal contamination, and exposure to waterborne spores is considered a possible source of infection for animals. We assessed the presence and genetic diversity of C. perfringens in water sources in a zoological park located in Madrid (Spain). A total of 48 water samples from 24 different sources were analyzed, and recovered isolates were toxinotyped, genotyped by fluorophore-enhanced repetitive polymerase chain reaction (rep-PCR) fingerprinting and tested for antimicrobial susceptibility. C. perfringens was recovered from 43.8 % of water samples and 50 % of water sources analyzed. All isolates (n = 70) were type A and 42.9 % were ß2-toxigenic (i.e., cpb2+), but none contained the enterotoxin-encoding gene (cpe). Isolates belonged to 15 rep-PCR genotypes and most genetic diversity (88 %) was distributed among isolates obtained from the same sample. Most isolates displayed intermediate susceptibility (57.1 %; MIC = 16 µg ml-1) or resistance (5.7 %; MIC ≥ 32 µg ml-1) to metronidazole. No resistance to other antimicrobials was detected, although some isolates showed elevated MICs to erythromycin and/or linezolid. Finally, a marginally significant association between absence of cpb2 and decreased susceptibility to metronidazole (MIC ≥ 16 µg ml-1) was detected. In conclusion, our results reveal a high prevalence of C. perfringens type A in the studied water reservoirs, which constitutes a health risk for zoo animals. The elevated MICs to metronidazole observed for genetically diverse isolates is a cause of additional concern, but more work is required to clarify the significance of reduced metronidazole susceptibility in environmental strains.

In vitro amphotericin B susceptibility of Malassezia pachydermatis determined by the CLSI broth microdilution method and Etest using lipid-enriched media.

We determined the in vitro amphotericin B susceptibility of 60 Malassezia pachydermatis isolates by the CLSI broth microdilution method and the Etest using lipid-enriched media. All isolates were susceptible at MICs of ≤ 1 µg/ml, confirming the high activity of amphotericin B against this yeast species. Overall, the essential agreement between the tested methods was high (80% and 96.7% after 48 h and 72 h, respectively), and all discrepancies were regarded as nonsubstantial.

Floral nectar and honeydew microbial diversity and their role in biocontrol of insect pests and pollination.

Sugar-rich plant-related secretions, such as floral nectar and honeydew, that are commonly used as nutrient sources by insects and other animals, are also the ecological niche for diverse microbial communities. Recent research has highlighted the great potential of nectar and honeydew microbiomes in biological pest control and improved pollination, but the exploitation of these microbiomes requires a deep understanding of their community dynamics and plant-microbe-insect interactions. Additionally, the successful application of microbes in crop fields is conditioned by diverse ecological, legal, and ethical challenges that should be taken into account. In this article, we provide an overview of the nectar and honeydew microbiomes and discuss their potential applications in sustainable agricultural practices.

Analysis of the culturable gut yeast microbiota of dogs with digestive disorders.

Despite the increasing interest in studying the gut mycobiota of dogs, the association between fungal colonization and the development of digestive disorders in this species remains largely understudied. On the other hand, the high prevalence of antifungal-resistant yeasts detected in previous studies in samples from animals represents a major threat to public health. We analyzed the presence of culturable yeasts in 112 rectal swab samples obtained from dogs with digestive disorders attended in a veterinary teaching hospital. Our results revealed that Malassezia pachydermatis was frequently isolated from the studied dog population (33.9% of samples), and that the isolation of this yeast was significantly associated to the age of animals, but not to their sex, disease group, or the presence of vomits and/or diarrhea. In contrast, other yeast species were less prevalent (17.9% of samples in total), and their isolation was not significantly associated to any variable included in the analysis. Additionally, we observed that 97.5% of the studied M. pachydermatis isolates (n = 158, 1-6 per positive episode) displayed a minimum inhibitory concentration (MIC) value >4 µg/ml to nystatin, 31.6% had a MIC ≥32 µg/ml to fluconazole, and 27.2% had a MIC >4 µg/ml to amphotericin B. The antifungal susceptibility profiles of non-Malassezia (n = 43, 1-7 per episode) were more variable and included elevated MIC values for some antifungal-species combinations. These results confirm that the intestine of dogs is a reservoir of opportunistic pathogenic yeasts and suggest that the prevalence of M. pachydermatis colonization depends more on the age of animals than on any specific digestive disorder.