Although high-throughput marker gene studies provide valuable insight into the diversity and relative abundance of taxa in microbial communities, they do not provide direct measures of their functional capacity. Recently, scientists have shown a general desire to predict functional profiles of microbial communities based on phylogenetic identification inferred from marker genes, and recent tools have been developed to link the two. However, to date, no large-scale examination has quantified the correlation between the marker gene based taxonomic identity and protein coding gene conservation. Here we utilize 4872 representative prokaryotic genomes from NCBI to investigate the relationship between marker gene identity and shared protein coding gene content. Even at 99–100% marker gene identity, genomes share on average less than 75% of their protein coding gene content. This occurs regardless of the marker gene(s) used: V4 region of the 16S rRNA, complete 16S rRNA, or single copy orthologs through a multi-locus sequence analysis. An important aspect related to this observation is the intra-organism variation of 16S copies from a single genome. Although the majority of 16S copies were found to have high sequence similarity (> 99%), several genomes contained copies that were highly diverged (< 97% identity). This is the largest comparison between marker gene similarity and shared protein coding gene content to date. The study highlights the limitations of inferring a microbial community’s functions based on marker gene phylogeny. The data presented expands upon the results of previous studies that examined one or few bacterial species and supports the hypothesis that 16S rRNA and other marker genes cannot be directly used to fully predict the functional potential of a bacterial community.

Twelve wild North American porcupines (Erethizon dorsatum) out of a total of 44 of this species examined in an 8-year period were diagnosed with dermatopathies while being cared for at two wildlife rehabilitation clinics. Biopsy and necropsy were performed on seven and five animals, respectively. Atypical dermatophytosis was diagnosed in all cases. Lesions consisted of diffuse severe epidermal hyperkeratosis and mild hyperplasia with mild lymphoplasmacytic dermatitis and no folliculitis. Dermatophytes were noted histologically as hyphae and spores in hair shafts, and follicular and epidermal keratin. Trichophyton sp. was grown in 5/6 animals where culture was performed, with a molecular diagnosis of Arthroderma benhamiae/Trichophyton mentagrophytes in these five cases. Metagenomic analysis of formalin-fixed paraffin-embedded tissue samples from three cases identified fungi from 17 orders in phyla Basidiomycota and Ascomycota. Alteration of therapy from ketaconazole, which was unsuccessful in four out of five early cases, to terbinafine or nitraconazole led to the resolution of disease and recovery to release in four subsequent animals. In all, six animals were euthanized or died due to dermatopathy, no cases resolved spontaneously, and six cases were resolved with therapy. The work we present demonstrates an atypical lesion and anatomical distribution due to dermatophytosis in a series of free-ranging wild porcupines and the successful development of novel techniques for extracting and sequencing nucleic acids from fungus in archival formalin-fixed paraffin-embedded animal tissue.

Twelve wild North American Porcupines (Erethizon dorsatum) were diagnosed with dermatopathies while being cared for at two wildlife rehabilitation clinics. Biopsy and necropsy were performed on 7 and 5 animals respectively. Atypical dermatophytosis was diagnosed in all cases. Lesions consisted of diffuse severe epidermal hyperkeratosis and mild hyperplasia, with mild lymphoplasmacytic dermatitis, and no folliculitis. Dermatophytes were noted histologically as hyphae and spores in hair shafts, and follicular and epidermal keratin. Trichophyton sp. was grown in 5/6 animals where culture was performed, with molecular diagnosis of Arthroderma benhamiae / Trichophyton mentagrophytes in these 5 cases. Metagenomic analysis of formalin-fixed paraffin-embedded tissue samples from 3 cases identified fungi from 17 orders in phyla Basidiomycota and Ascomycota. Alteration of therapy from ketaconazole, which was unsuccessful in 4 of 5 early cases, to terbinafine or nitraconazole lead to resolution of disease and recovery to release in four subsequent animals. In all, 6 animals were euthanized or died due to dermatopathy, no cases resolved spontaneously, and 6 cases resolved with therapy. The work we present demonstrates an atypical lesion and anatomical distribution due to dermatophytosis in a series of free-ranging wild porcupines and successful development of novel techniques for extraction and sequencing nucleic acids from fungus in archival formalin-fixed paraffin-embedded animal tissue.

Fecal pollution at coastal beaches in the Northeast, USA requires management efforts to address public health and economic concerns. Concentrations of fecal-borne bacteria are influenced by different fecal sources, environmental conditions, and ecosystem reservoirs, making their public health significance convoluted. In this study, we sought to delineate the influences of these factors on enterococci concentrations in southern Maine coastal recreational waters. Weekly water samples and water quality measurements were conducted at freshwater, estuarine, and marine beach sites from June through September 2016. Samples were analyzed for total and particle-associated enterococci concentrations, total suspended solids, and microbial source tracking markers for multiple sources. Water, soil, sediment, and marine sediment samples were also subjected to 16S rRNA sequencing and SourceTracker analysis to determine the influence from these environmental reservoirs on water sample microbial communities. Enterococci and particle-associated enterococci concentrations were elevated in freshwater, but suspended solids concentrations were relatively similar. Mammal fecal contamination was significantly elevated in the estuary, with human and bird fecal contaminant levels similar between sites. A partial least squares regression model indicated particle-associated enterococci and mammal marker concentrations had the most significant positive relationships with enterococci concentrations across marine, estuary, and freshwater environments. Freshwater microbial communities were significantly influenced by underlying sediment while estuarine/marine beach communities were influenced by freshwater, high tide height, and estuarine sediment. We found elevated enterococci levels are reflective of a combination of increased fecal source input, environmental sources, and environmental conditions, highlighting the need for encompassing MST approaches for managing water quality issues. IMPORTANCE: Enterococci have long been the federal standard in determining water quality at estuarine and marine environments. Although enterococci are highly abundant in the fecal tracts of many animals they are not exclusive to that environment and can persist and grow outside of fecal tracts. This presents a management problem for areas that are largely impaired by non-point source contamination, as fecal sources might not be the root cause of contamination. This study employed different microbial source tracking methods to delineate influences from fecal source input, environmental sources, and environmental conditions to determine which combination of variables are influencing enterococci concentrations in recreational waters at a historically impaired coastal town. Results showed that fecal source input, environmental sources and conditions all play a role in influencing enterococci concentrations. This highlights the need to include an encompassing microbial source tracking approach to assess the effects of all important variables on enterococci concentrations.

The identification of fungal pathogens in formalin-fixed paraffin-embedded (FFPE) tissues is an unmet need in human and animal medicine, and sequence-agnostic approaches are needed to identify emerging pathogens. Eleven FFPE biopsy specimens with etiologic diagnoses of fungal disease based on standard testing of paired fresh tissue samples were utilized here to evaluate metabarcoding approaches. The cases included tissues from three dogs, three cats, one box turtle, one goat, one common loon, and one gray tree frog. The diagnoses from the fresh tissues in these cases were Microsporum canis, Penicillium sp., Exophiala sp. (likely E. jeanselmei), Verticillium sp., Rhizopus sp., atypical Cryptococcus neoformans, Conidiobolus spp., Aspergillus fumigatus, Cryptococcus neoformans var grubii, Batrachochytrium dendrobatidis, Fusarium solani, Blastomyces dermatitidis, Coccidiodes immitis, and Histoplasma capsulatum. We compared the ITS1 and 28S D1 rRNA gene genetic markers in combination with several bioinformatic strategies to identify fungal pathogens in the FFPE tissue samples, with a success rate of 9/11. These methods could allow diagnosticians who receive only FFPE tissues and see fungal pathogens to speciate the pathogens and could be of value in retrospective studies wherein FFPE tissue is the only archived tissue. Furthermore, these techniques could be of use to researchers investigating polymicrobial communities where DNA preservation is suboptimal.