pathogens Article Characterization of Oral Microbiota in Cats: Novel Insights on the Potential Role of Fungi in Feline Chronic Gingivostomatitis Janina A. Krumbeck 1, Alexander M. Reiter 2, James C. Pohl 3, Shuiquan Tang 1, Young J. Kim 3, Annika Linde 3, Aishani Prem 1 and Tonatiuh Melgarejo 3,* 1 MiDOG LLC, Irvine, CA 92614, USA; [email protected] (J.A.K.); [email protected] (S.T.); [email protected] (A.P.) 2 School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA; [email protected] 3 College of Veterinary Medicine, Western University of Health Sciences, Pomona, CA 91766, USA; [email protected] (J.C.P.); [email protected] (Y.J.K.); [email protected] (A.L.) * Correspondence: [email protected]; Tel.: +1-(909)-706-3829 Abstract: Previous studies have suggested the involvement of viral and bacterial components in the initiation and progression of feline chronic gingivostomatitis (FCGS), but the role of fungi remains entirely unknown. This pilot study aimed to investigate the bacteriome and mycobiome in feline oral health and disease. Physical exams, including oral health assessment, of privately owned, clinically healthy (CH) cats (n = 14) and cats affected by FCGS (n = 14) were performed. Using a sterile swab, oral tissue surfaces of CH and FCGS cats were sampled and submitted for 16S rRNA and ITS-2 next-generation DNA sequencing. A high number of fungal species (n = 186) was detected, Citation: Krumbeck, J.A.; Reiter, with Malassezia restricta, Malassezia arunalokei, Cladosporium penidielloides/salinae, and Aspergillaceae A.M.; Pohl, J.C.; Tang, S.; Kim, Y.J.; sp. being significantly enriched in FCGS samples, and Saccharomyces cerevisiae in CH samples. The Linde, A.; Prem, A.; Melgarejo, T. bacteriome was significantly distinct between groups, and significant inter-kingdom interactions Characterization of Oral Microbiota were documented. Bergeyella zoohelcum was identified as a potential biomarker of a healthy feline in Cats: Novel Insights on the Potential Role of Fungi in Feline oral microbiome. These data suggest that fungi might play a role in the etiology and pathogenesis Chronic Gingivostomatitis. Pathogens of FCGS, and that oral health should not simply be regarded as the absence of microbial infections. 2021, 10, 904. https://doi.org/ Instead, it may be viewed as the biological interactions between bacterial and fungal populations 10.3390/pathogens10070904 that coexist to preserve a complex equilibrium in the microenvironment of the mouth. Additional investigations are needed to improve our understanding of the feline oral ecosystem and the potential Academic Editor: Valentina interactions between viruses, bacteria, and fungi in FCGS. Virginia Ebani Keywords: next-generation DNA sequencing; oral microbiota; microbiome; mycobiota; 16S rRNA; Received: 19 June 2021 fungal ITS; feline stomatitis Accepted: 14 July 2021 Published: 17 July 2021 Publisher’s Note: MDPI stays neutral 1. Introduction with regard to jurisdictional claims in published maps and institutional affil- The prevalence of feline chronic gingivostomatitis (FCGS) is approximately 0.7–12% [1]. iations. Affected cats usually present with oral inflammation, pain, anorexia and dysphagia. Feline calicivirus (FCV) and feline herpesvirus-1 (FHV-1) have been implicated in the develop- ment of the disease, but it has also been hypothesized that cats with FCGS may show an aberrant reaction to plaque bacteria that leads to oral inflammation beyond gingivitis and periodontitis [2]. However, the exact cause of FCGS remains unknown, and there is no gold Copyright: © 2021 by the authors. standard in terms of treatment besides partial and full-mouth tooth extraction and exten- Licensee MDPI, Basel, Switzerland. This article is an open access article sive medical management. Consequently, FCGS can be a challenging disease, not only for distributed under the terms and clinicians but also for clients [3]. Current therapeutic approaches include professional den- conditions of the Creative Commons tal cleaning, tooth extractions (partial or full mouth), and extensive medical (antimicrobial, Attribution (CC BY) license (https:// anti-inflammatory, immunosuppressive, immunomodulatory) management. Antibiotic creativecommons.org/licenses/by/ therapy is included in most management plans, but bacterial culture and sensitivity testing 4.0/). are not typically performed. Empirical treatment of FCGS with amoxicillin, amoxicillin Pathogens 2021, 10, 904. https://doi.org/10.3390/pathogens10070904 https://www.mdpi.com/journal/pathogens Pathogens 2021, 10, 904 2 of 15 clavulanate, clindamycin or metronidazole without proper diagnostic information can promote bacterial resistance and thus become ineffective. To better understand the biological role of the microbial populations on the devel- opment of FCGS, two scientific groups partially described the cat oral microbiome using culture-based methods [4,5]. These studies showed that the feline oral cavity is typi- cally polymicrobial with a preponderance of obligate anaerobic and facultative anaerobic bacteria, of which only a few bacterial strains are cultivatable using routine laboratory methods [6]. Molecular microbial tools, like next-generation DNA sequencing (NGS), have the advantage that they are culture-independent and more sensitive. NGS uses an untargeted sequencing approach and can identify and quantify bacteria and fungi present in a sample, including previously unknown microbes. This technique has been previously applied to study the feline oral cavity in health [7–11] and disease [12–17], showing that the bacterial profile in the mouth of cats is rich and highly diverse. In clinically healthy cats, Porphyromonas, Moraxella, and Fusobacterium are the most abundant genera [18], and Capnocytophaga canimorsus is the most prevalent species, followed by Xanthomonadaceae and Bergeyella spp. [10]. In cats affected by gingivitis, mild periodontitis, or other oral diseases, the key pathogens from the phyla Spirochaetes and Bacteroidetes [11] and the Peptostrepto- coccaceae family [13] were dominant. Another study showed that members of Pasteurella multocida subsp. multocida were significantly more prevalent in diseased cats compared to controls [5]. Besides bacteria, fungi are an important part of the mammal microbiome in various species [19] and infections [16,20]. To date, only two studies have investigated fungal oral microbiota, i.e., mycobiota, of healthy [9] or allergic cats [21]. To our knowledge, there is no study investigating the oral mycobiome of cats with FCGS. Understanding the role of oral microbiota in FCGS could provide critical insight into disease pathophysiology as well as outline new approaches for prevention and treatment. The aim of the present study was to investigate the bacterial and fungal microbiota of CH cats and cats with FCGS. It was hypothesized that the feline oral microbial composition and structure will differ between the two groups and that these differences and population dynamics may provide clinicians with a better understanding of the complex etiology of and potential therapeutic approaches to FCGS. 2. Results A total of 249 bacterial and 186 fungal genera were identified in this dataset. All samples harbored both bacteria and fungi, with only one exception of a CH sample (CH_5) that apparently had no fungal species present. A commonly used measurement tool for the microbial diversity within samples, i.e., how many different types of microbes are present in a sample, is the alpha-diversity (Figure1a–c). The alpha-diversity measurements for CH and FCGS samples showed that the number of observed bacterial species was significantly higher than the number of fungal species. There was a mean of 76 and 90 (rounded) bacterial species in the FCGS and CH samples, respectively. However, no significant differences were found with regards to the number of fungal species between FCGS and CH samples with a mean of 15 and 13 (rounded), respectively (Figure1a). The bacterial diversity was significantly different between the groups for both the number of observed species and the Shannon diversity index (a value that increases with biodiversity), showing a higher alpha-diversity in the CH samples (Figure1a,c). For the fungal profile, both the Shannon diversity index and the Simpson diversity index—which measures the relative abundance of species—were significantly higher in FCGS samples compared to CH samples (Figure1b,c). The beta-diversity—a measurement of microbial diversity between samples—showed that FCGS samples were not significantly distinct from the CH samples for the bacterial or fungal composition (Figure2a,b). Pathogens 2021, 10, 904 3 of 15 Pathogens 2021, 10, x 3 of 15 2.1. Analysis of the Bacterial Microbiota in CH and FCGS Cats TheThe bacterial bacterial composition composition profile profile was was analyzed analyzed in ineach each group group (Figure (Figure 3,3 ,top). top). In In healthyhealthy cats, cats, the the top top five five most most domina dominantnt bacterial bacterial species species on onaverage average were were PorphyromonasPorphyromonas gulaegulae (10.0%),(10.0%), MoraxellaMoraxella sp. (7.3%),sp. (7.3%), PorphyromonasPorphyromonas sp. (2.8%)sp. (2.8%) PorphyromonasPorphyromonas circumdentaria circumden- (2.6%),taria and(2.6%), Bacteroidales and Bacteroidales sp. (2.6%).sp. The (2.6%). top five The most top dominant five most bacterial dominant species bacterial based species on Porphyromonas gulae averagebased relative on average