Rapid and real-time identification of fungi up to species level with long amplicon nanopore sequencing from clinical samples.

The availability of long-read technologies, like Oxford Nanopore Technologies, provides the opportunity to sequence longer fragments of the fungal ribosomal operon, up to 6 Kb (18S-ITS1-5.8S-ITS2-28S) and to improve the taxonomy assignment of the communities up to species level and in real-time. We assess the applicability for taxonomic assignment of amplicons targeting a 3.5 Kb region (V3 18S-ITS1-5.8S-ITS2-28S D2) and a 6 Kb region (V1 18S-ITS1-5.8S-ITS2-28S D12) with the What's in my pot (WIMP) classifier. We used the ZymoBIOMICSTM mock community and different microbiological fungal cultures as positive controls. Long amplicon sequencing correctly identified Saccharomyces cerevisiae and Cryptococcus neoformans from the mock community and Malassezia pachydermatis, Microsporum canis and Aspergillus fumigatus from the microbiological cultures. Besides, we identified Rhodotorula graminis in a culture mislabelled as Candida spp. We applied the same approach to external otitis in dogs. Malassezia was the dominant fungal genus in dogs' ear skin, whereas Ma. pachydermatis was the main species in the healthy sample. Conversely, we identified a higher representation of Ma. globosa and Ma. sympodialis in otitis affected samples. We demonstrate the suitability of long ribosomal amplicons to characterize the fungal community of complex samples, either healthy or with clinical signs of infection.

Effect of an anti-inflammatory pomegranate otic treatment on the clinical evolution and microbiota profile of dogs with otitis externa.

BACKGROUND: Canine otitis externa (OE) is a common disease characterised by inflammation of the epithelial tissue of the external ear canal. Secondary infections are frequent, and Malassezia pachydermatis and Staphylococcus pseudintermedius are routinely isolated and treated with antifungal and antibiotic compounds. HYPOTHESIS/OBJECTIVES: To analyse the otitis ear microbiome before and after a treatment with prednisolone plus pomegranate or antimicrobial drugs ANIMALS: 15 dogs with nonpurulent OE. METHODS AND MATERIALS: A 30 day, double-blinded, multicentre, randomized and controlled parallel-group (1:1) trial was conducted in 15 dogs with nonpurulent OE, following two different topical treatments (prednisolone plus pomegranate versus prednisolone plus antibiotic and antifungal drugs). On days (D)0, D15 and D30, serum and skin otic samples were collected, and clinical examination and microbiome analysis (bacteria and fungi) were performed. Results were compared with validated otitis clinical scores to assess the effectiveness of both treatments. RESULTS: Nine bacterial and four fungal families were detected during the three time-points tested. An increase in fungal diversity (Shannon index) and composition was the most significant change observed after both treatments. At treatment D15 and D30, the reduction in clinical signs was statistically significant in both treatment groups (P ≤ 0.05). Prednisolone plus pomegranate cleanser treatment was able to control the clinical signs of otitis as well as the bacterial and fungal overgrowth. CONCLUSIONS AND CLINICAL IMPORTANCE: Mild otitis cases associated with microbial overgrowth may be managed with topical antiseptic and anti-inflammatory agents without the need for antibiotic and/or antifungal compounds.

The microbiota of water buffalo milk during mastitis.

The aim of this study was to define the microbiota of water buffalo milk during sub-clinical and clinical mastitis, as compared to healthy status, by using high-throughput sequencing of the 16S rRNA gene. A total of 137 quarter samples were included in the experimental design: 27 samples derived from healthy, culture negative quarters, with a Somatic Cell Count (SCC) of less than 200,000 cells/ml; 27 samples from quarters with clinical mastitis; 83 samples were collected from quarters with subclinical mastitis, with a SCC number greater of 200,000 cells/ml and/or culture positive for udder pathogens, without clinical signs of mastitis. Bacterial DNA was purified and the 16S rRNA genes were individually amplified and sequenced. Significant differences were found in milk samples from healthy quarters and those with sub-clinical and clinical mastitis. The microbiota diversity of milk from healthy quarters was richer as compared to samples with sub-clinical mastitis, whose microbiota diversity was in turn richer as compared to those from clinical mastitis. The core microbiota of water buffalo milk, defined as the asset of microorganisms shared by all healthy milk samples, includes 15 genera, namely Micrococcus, Propionibacterium, 5-7N15, Solibacillus, Staphylococcus, Aerococcus, Facklamia, Trichococcus, Turicibacter, 02d06, SMB53, Clostridium, Acinetobacter, Psychrobacter and Pseudomonas. Only two genera (Acinetobacter and Pseudomonas) were present in all the samples from sub-clinical mastitis, and no genus was shared across all in clinical mastitis milk samples. The presence of mastitis was found to be related to the change in the relative abundance of genera, such as Psychrobacter, whose relative abundance decreased from 16.26% in the milk samples from healthy quarters to 3.2% in clinical mastitis. Other genera, such as SMB53 and Solibacillus, were decreased as well. Discriminant analysis presents the evidence that the microbial community of healthy and clinical mastitis could be discriminated on the background of their microbiota profiles.

Gastrointestinal microbial population of turkey (Meleagris gallopavo) affected by hemorrhagic enteritis virus.

Hemorrhagic enteritis (HE) is an acute viral disease that affects avian species, particularly turkeys, compromising their commercial production and having a negative effect on animal welfare. Turkey adenovirus 3 (TAdV-3), is the main causal agent of the disease. In this study, we considered 3 groups of turkeys to achieve 2 purposes: 1) A preliminary investigation on the microbiota content in the 4 parts of healthy turkey's intestine (group A), namely duodenum, jejunum, ileum, and ceca was done; 2) an investigation on the relationship between natural infections with TAdV-3 and the intestinal microbiota in the jejunum, where HE mostly develops, comparing group A with animals with molecular positivity for the virus and with clinical signs of HE (group B) and animals with molecular positivity for the virus but without clinical signs (group C). Massive sequencing of the hypervariable V1-V2 regions of 16S rRNA gene and QIIME 1.9.1 software analysis was performed, and operation taxonomic units (OTUs) were classified into 4 abundant phyla: Actinobacteria, Bacteroidetes, Firmicutes, and Proteobacteria. The microbial population of small intestine was distributed almost homogeneously in the healthy turkeys, and Firmicutes was the prevalent phylum (79.85% in duodenum, 89.57% in jejunum and 99.28% in ileum). As compared with small intestine, ceca microbial community was much more heterogeneous: Firmicutes (48.03%), Bacteroidetes (33.60%) and Proteobacteria (12.32%). In the natural infections of HEV, the main bacterial families were Bacteroidaceae (Bacteroidetes) and Peptostreptococcaceae (Firmicutes), uniquely detected in group B and C. Also Clostridiaceae (Firmicutes) was detected, uniquely in group B.