Otitis media (OM) or middle ear (ME) infection/inflammation remains a major public health problem worldwide, with an estimated 60% of hearing-impaired children under 5 years old having OM as the cause of hearing loss (GBD 2019 Hearing Loss Collaborators, 2021). Risk factors for OM include young age, lack of breastfeeding, allergies, upper respiratory infection, second-hand smoke exposure, low social status, daycare attendance, multiple siblings, and family history (Zhang et al., 2014; Brennan-Jones et al., 2015). OM may persist as recurrent acute (RA)OM or chronic (C)OM, for which treatment includes antibiotics and surgery. In the United States, annual health care use due to office visits, antibiotics, and surgeries for OM in children is estimated to cost >$5 billion (Suaya et al., 2018). Antibiotics are prescribed for 67% of children with OM (Hersh et al., 2016), inappropriately in 10-33% of cases, causing concern for antibiotic resistance (Fleming-Dutra et al., 2016; DeMuri et al., 2017). Globally, age-standardized rates for years lived with disability due to acute OM are still increasing (GBD 2019 Diseases and Injuries Collaborators, 2020). In children, OM can cause not only permanent hearing loss but also impairments in speech perception, auditory processing or phonological awareness while reading, thereby affecting academic performance (O'Niel et al., 2015; Khavarghazalani et al., 2016; Cai and McPherson, 2017; le Clercq et al., 2017; Carroll and Breadmore, 2018). If persisting to adulthood, COM, even with only mild-to-moderate hearing loss, is associated with poorer quality of life and mental health, particularly if revision surgeries were performed (Bakir et al., 2013). After tympanoplasty surgery, the 10-year recurrence rate of COM is 15-26% (Nardone et al., 2012).

Cholesteatoma is a non-cancerous cyst-like lesion in the ME that either develops congenitally (rare) or, more commonly, is acquired in COM. Cholesteatoma was estimated to develop in 10-24% of OM cases (O'Connor et al., 2009; Rosito et al., 2017). In a tertiary hospital setting in Colorado, out of 67,661 children seen over 10 years, 36.5% had OM and 12.0% were diagnosed with COM; of those with COM, 5.1% of children presented with cholesteatoma (unpublished data from COMPASS database). Prevalence is higher in adults: of 1,006 adults with OM, 24.4% had COM; of those with COM, 39.2% had cholesteatoma. These data show that while COM and cholesteatoma are prevalent in lower income countries such as the Philippines (Carrillo et al., 2007; Santos-Cortez et al., 2007), these disease entities are also significant health issues in high-income countries including the United States.

Cholesteatoma has a propensity for insidious growth and erosion of the ossicles or temporal bone that houses neural structures and may lead to disabling complications such as hearing loss, facial nerve palsy, vertigo, or intracranial extension. Surgery is required for treatment, with high rates of recurrence even after >10 years (Kuo et al., 2012; Nardone et al., 2012). Histologically, cholesteatoma is filled by keratin debris and lined by keratinized squamous epithelium (matrix), which is similar to the outer epidermal layer of the tympanic membrane (TM) but with Langerhans cells and basal cells (Lim and Saunders, 1972). Between the matrix and ME mucosa, there is a layer of loose connective tissue (i.e., perimatrix) containing collagen fibers and fibrocytes, typically with inflammation between the perimatrix and ME mucosa (Lim and Saunders, 1972). Under the operating microscope, these tissues can be differentiated well: the cholesteatoma is the encapsulated collection of keratin debris within the ME space, while the hyperplastic, severely inflamed mucosa that is in contact with the cholesteatoma appears as granulation tissue that is distinct from normal-looking but infected ME mucosa (Saunders et al., 2011). Fifty years after its histology was resolved, there is still no consensus on how a cholesteatoma forms, except that the process is probably a hybrid of theories (Maniu et al., 2014; Kuo et al., 2015): [a] desquamated keratin accumulates in a retraction pocket formed by negative ME pressure; [b] squamous epithelium of the TM migrates to the ME; [c] ME mucosa transforms into keratinizing epithelium; [d] keratin-filled microcysts form within the basal layer of the TM and invade subepithelial tissue. Better understanding of cholesteatoma pathology will aid in implementation of standard classifications of lesions to guide management and predict surgical outcomes (James et al., 2019). It was suggested that an exaggerated inflammatory response causes cholesteatoma growth, proliferation, and bony erosion (Maniu et al., 2014; Kuo et al., 2015). Biofilm formation and aberrant gene expression are also associated with cholesteatomas (Chole and Faddis, 2002; Maniu et al., 2014; Kuo et al., 2015; Baschal et al., 2019).

Pseudomonas aeruginosa and Staphylococcus aureus are the most prevalent bacteria cultured from suppurative COM and cholesteatomatous OM (Ricciardiello et al., 2009). In previous bacterial profiling between different patients, the relative abundances of Alloiococcus, Haemophilus and Clostridiales were increased in cholesteatomas vs. healthy ME, non-cholesteatomatous OM, or tympanosclerotic plaques (Neeff et al., 2016; Kalcioglu et al., 2018). In another recent study of untreated cholesteatoma, no differences in bacterial or fungal profiles were found between cholesteatoma and ME mucosa (Weiss et al., 2019). In these previous microbiota studies, no biodiversity or relative taxa abundance estimates were significant, whether when comparing by case-control status or by sample-types, and when correcting for multiple testing [e.g. false discovery rate (FDR)] (Neeff et al., 2016; Minami et al., 2017; Kalcioglu et al., 2018; Weiss et al., 2019). On the other hand, in other studies up to 36% of cholesteatomas were reported as positive for human papillomavirus (HPV), a virus that can induce aggressive growth of tissue lesions such as papillomas or cancers (Bergmann et al., 1994; Franz et al., 2007).

In this study, we collected ME samples from Filipino patients undergoing tympanomastoidectomy for COM and submitted them for [1] broad-range bacterial 16S rRNA gene amplification and sequencing and [2] PCR screening for HPV and common viral otopathogens. Our goal was to determine if the microbiota of cholesteatoma tissue is different from other ME samples within the context of long-standing, insufficiently treated suppurative COM.

In this study, we examined the bacterial and viral microbiotas of cholesteatoma tissue and other ME specimens that were collected from Filipino patients undergoing tympanomastoidectomy surgery for long-standing COM. No viruses were identified in any ME sample, indicating that COM with or without cholesteatoma is primarily a bacterial disease. There was strong overlap in the bacteria found between sample-types whether from the same or across different patients ( Figure S1 ). Considering that multiple samples were taken from a limited field at the same ME surgery for each ear, the similarity in community composition is somewhat expected. Nonetheless, we found that the cholesteatoma tissues had moderately less biodiversity than did ME mucosal samples as indicated by richness and Shannon diversity indices ( Figure 2 ). Likewise, ME discharge had less alpha-diversity than ME mucosal samples, while granulation tissue had higher richness than cholesteatoma samples ( Figure 2 ).

Overall, our results indicate that factors such as quinolone use and cholesteatoma diagnosis had much more substantial effects on ME microbiota composition than did the particular sample-type collected ( Figures 1 , 3 , S4, S5 ). For example, granulation tissues and ME discharge from patients with cholesteatoma diagnosis differed substantially from those with suppurative COM without cholesteatoma ( Figure 3 ). Overall, these findings suggest that the microbiota of middle ears with cholesteatoma have a different profile than middle ears with suppurative COM only, which may necessitate a change in approach to treatment.

Differences in biodiversity also were documented across ages of patients with COM ( Figures 1 , 2 ), with greater diversity in patients >33 years old compared to teenagers ( Figure 1 ; Q1 vs Q3 and Q4). To place this finding within context, the Philippine General Hospital serves a large, indigent population from different regions of the country (Santos-Cortez et al., 2007), many of whom may not have had access to specialists in their locality. Thus, the differences by age bracket may, in fact, be reflective of the duration of ME infection, rather than chronological age per se. If the study participants did have some form of antibiotic or surgical treatment prior to sample collection, a large majority is assumed to have received sporadic treatment since the initial infection. By usual protocol, at initial consult, the patients are prescribed antibiotic otic drops (e.g., Ofloxacin, Neomycin-Polymixin B with or without steroids). However due to limited facilities and long waitlists, it may take months from the initial consult before surgery is performed. This situation is common among populations with limited access to otologic health care, particularly in lower-income countries or marginalized groups. Taken together, the differences that we observed across age quartiles are likely to reflect [1] long-standing, untreated or inadequately treated OM, and [2] recent antibiotic use on top of a potentially long history of antibiotic prescription (local or oral) and high likelihood of antibiotic resistance (Xu et al., 2020).

In addition to quinolone, other antibiotics may also have indirectly influenced our findings on ME bacterial profiles. Notably Pseudomonas, which is a common target of antibiotic otic drops, had greater relative abundance in older patients ( Figure S2 ). This finding may indicate antibiotic resistance and/or extensive formation of treatment-resistant biofilms due to long-standing infection. On the other hand, the most abundant taxa in cholesteatoma tissues were Corynebacterium, Porphyromonas, and Staphylococcus, with 11.5%, 8.3%, and 8.0% mean abundances ( Table S1 , Figure 1 ). Staphylococcus is often isolated from COM samples, but may be resistant to penicillin, macrolides or quinolones (Xu et al., 2020).

Corynebacterium is also commonly identified in COM but is typically viewed as a commensal, even potentially otoprotective in acute OM (Lappan et al., 2018; Jorissen et al., 2021). In contrast, Porphyromonas, Fusobacterium and Campylobacter ( Figure 1 , Figure 3 ) are less commonly isolated from patients with COM or cholesteatoma (Brook, 1995; Yusuf et al., 2015). Porphyromonas and Fusobacterium are more common in other anaerobic infections of the head and neck, such as within the oral cavity, oropharynx, and sinuses (Brook, 2011; Yusuf et al., 2015). However, recent 16S rRNA sequencing has identified these bacteria more often in COM cases, indicating the utility of sequencing towards greater understanding of the microbial communities in COM (Santos-Cortez et al., 2016; Johnston et al., 2019). Similar to anaerobic infections of the sinuses and head and neck tissues, the presence of Porphyromonas or Fusobacterium is likely due to seeding of necrotic or damaged tissue by anaerobic bacterial pathogens after failure of antibiotic treatment (Brook, 2011). These anaerobes may also facilitate biofilm formation (Wang et al., 2018). Identification of these bacteria in cholesteatoma samples that may not be reached by and/or are resistant to the usual otic drops suggest an important opportunity to administer intravenous or oral antibiotics with proper bacterial coverage (e.g., clindamycin, metronidazole, chloramphenicol) immediately after cholesteatoma removal. When the post-surgical ear dressing or packing is removed, instillation of antibiotic drops according to culture and sensitivity test results will help minimize risk of COM recurrence. Local information on antimicrobial susceptibility patterns, which may vary considerably within a few years (del Rosario et al., 1990; Abes et al., 1998; Ayson et al., 2006; Suzuki et al., 2015; Suzuki et al., 2020), will need to be updated regularly and is necessary to efficiently treat COM and cholesteatoma, particularly for the patients described here who lack regular access to health care. Also at the local level, the range of ototopical prescriptions might need to be expanded in order to include antibiotics with proper coverage of target bacteria for post-surgical treatment of cholesteatoma, e.g. chloramphenicol, clindamycin, bacitracin, gramicidin, sulphacetamide, or rifampicin otic drops (van Dongen et al., 2015; Brennan-Jones et al., 2020; de Jong et al., 2020).

There are several limitations of this study. The limited sample size allowed us to identify significant differences in alpha- and beta-diversity but may have been too limited to comprehensively identify differentially abundant taxa, particularly after FDR-correction. The differences in the surgical findings per patient also did not allow us to collect all four sample-types from each patient and reduced the number of samples for paired analyses. Because of restricted access to the ME and to the patients included in this study, all analyses are cross-sectional rather than longitudinal. Lastly, short-read 16S rRNA sequencing limits phylogenetic resolution and, therefore, we took the conservative approach of classifying sequences only to the genus-level. Furthermore, microorganisms such as archaea, fungi, and viruses were not targeted for broad-range sequencing. If feasible, given the low microbial biomasses observed in these specimens, future metagenomic sequence analyses will increase the phylogenetic depth and breadth of these studies. Bacterial species/strain identification will be more helpful in future patients whose ME samples may be submitted for culture and testing of antibiotic sensitivity for the taxa identified in this study.

While having a control population with no history of OM is desirable, we purposefully did not aspire to collect samples from healthy controls, (e.g., from patients undergoing cochlear implantation). There is ongoing debate whether the healthy ME is sterile (Jervis-Bardy et al., 2019; Lee et al., 2021). In our own pilot study of ME swabs from healthy ME of cochlear implant patients with no previous history of OM, no microbial DNA was isolated in 4 out of 5 patients. In one patient, DNA was amplified and submitted to sequencing; upon review of clinical history, this patient had a history of tympanomastoidectomy on the ear opposite to the sampling site, suggesting that the sampled ear was also seeded by previous ME infection, and thus not accurately described as a “healthy control”. Our data therefore support the sterile state of the healthy ME (Jervis-Bardy et al., 2019), precluding any comparison with the microbiota of our patients with COM.

To summarize, in this cohort of patients with long-standing COM, we found that age, cholesteatoma diagnosis, and quinolone use were significantly and independently associated with ME bacterial profiles. Biodiversity was moderately lower in cholesteatoma and ME discharge compared to ME mucosal tissues. Cholesteatoma also had less biodiversity than granulation tissue samples. The findings from this study will be useful in guiding surgical or medical treatment protocols for cholesteatoma and COM, especially in settings with limited health care resources. On a more practical level, these findings indicate that the details of patients’ medical histories and demographic factors are more critical for studies of ME microbiota than the choice or availability of particular types of ME specimens.

Demultiplexed 16S rRNA paired-end sequence data and associated metadata were deposited in the NCBI Sequence Read Archive under Bioproject ID PRJNA748418.

The study was approved by the University of the Philippines Manila Research Ethics Board (UPMREB 2015-238-01) and the Colorado Multiple Institutional Review Board (protocols 16-1525, 16-2673, and 17-1679). All adult participants and parents of minors provided informed consent.

Conceptualization – RS-C. Methodology and resources – DF, JM, KV, JS, KD, AM, EY, HD, RP, JL, JA, BG, KM, CR, GI, AC, S-LL, ET, NS, TY, CC, and RS-C. Formal analysis and investigation – DF, TB, JK, CR, S-LL, and RSC. Original draft preparation – DF, TB, and RS-C. Manuscript editing – all authors. All authors contributed to the article and approved the submitted version.

This work was funded by NIH-NIDCD grant R01 DC015004 (to RS-C).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.