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Microbiology of Middle Ear Infections: Do You Hear What I Hear?
Clinical Microbiology N e w s l e t
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Vol. 38, No. 11 June 1, 2016 www.cmnewsletter.com I n T hi s
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87 Microbiology of Middle Ear Infections: Do You Hear What I Hear?
Stay Current... Stay Informed.
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Microbiology of Middle Ear Infections: Do You Hear What I Hear? Donna M. Wolk, MHA, Ph.D., D(ABMM),1,2,3 1Department of Laboratory Medicine, Geisinger Health Systems, Danville, Pennsylvania, 2Weis Center for Research, Danville, Pennsylvania, 3Wilkes University, Wilkes-Barre, Pennsylvania
Abstract Infectious conditions of the middle ear are a common and significant cause of morbidity and sometimes even mortality, especially in young children and elderly individuals. Pathogens and harmless commensal bacteria, viruses, and fungi co-inhabit the auditory canal and form intricate ecological networks, collectively known as a microbiome. Few studies that describe the normal flora of the middle ear have been published, and controversy exists about the roles of several possible pathogens. This review describes current literature examining otitis media and the roles various microbes play in the pathogenesis of middle ear infections. The review also highlights evolving research in middle ear microbiome studies, which begs the question, “Whose hearing could be damaged by what we don’t yet know about middle ear infections?”
Background and History
Corresponding author: Donna M. Wolk, Department of Laboratory Medicine, Geisinger Health Systems, 100 N. Academy Ave., Danville, PA 17822-1930. Tel.: 570-2716388. Fax: 570-214-9792. E-mail: [email protected].
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Middle ear effusion in otitis media (OM) was at one time considered sterile, but in the 1950s, bacteria were cultured from middle ear effusions [1]. Since then, the presence of Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis has been associated with OM, including acute and chronic infections, as well as those with effusions. These mono-pathogen infections are often called the “one-organism, one-disease” infectious disease framework, in which highly common bacterial infections, including OM and pneumonia, were thought to be caused by a limited number of pathogens. Scientists are now aware of the potential role of the nasopharyngeal lymphoid tissue as a reservoir of both middle ear and tonsil disease is supported by a wide range of clinical and microbiological studies [2-6], and it is known that healthy children also harbor a diverse number of bacterial species in the adenoid microbiota [3,4,7,8]. Complicating the microbiologic scenario further, the common pathogens associated with ear infections are also frequently observed commensal
residents of the upper respiratory tract. Together with harmless commensal bacteria, viruses, and fungi, they form intricate ecological networks collectively known as the microbiome. Similar to the intestinal microbiome, the normal respiratory microbiome is thought to be beneficial to the host by priming the immune system and providing colonization resistance, while an imbalanced biome might predispose to bacterial overgrowth and development of respiratory infections. The current literature suggests niche-specific upper respiratory tract human microbiota profiles, hosts, and changes occurring within these profiles exist, and that changes are associated with respiratory infections [9]. Biofilms detected in a wide range of infections of the ear, nose, and throat [6,10] comprise a community of sessile organisms embedded in an adherent matrix of extracellular polymeric substances. They are three-dimensional aggregates of bacteria that are highly resistant to both immune-mediated killing and antimicrobial agents, characteristics that both enhance survival of the bacteria and decrease the sensitivity of
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Figure 1. Middle ear anatomy and otitis media. Shown is a schematic representation of the ear under normal and CSOM conditions. Under normal conditions, the middle ear cavity is clear and empty. In contrast, the middle ear becomes red and inflamed with the presence of fluid under CSOM conditions. The red color denotes inflammation, while yellow indicates fluid during CSOM.
culture-based detection. Biofilm formation by multiple mucosal pathogens, notably Pseudomonas spp., is implicated in a variety of otorhinolaryngologic diseases, such as OM with effusion (OME), chronic serous OM (SOM), and recurrent adenotonsillitis [11-13]. Traditional culture techniques are known to be less sensitive for detecting bacteria that reside in biofilms and bacterial communities like those of the middle ear [14,15].
Disease Conditions of the Middle Ear To understand the progression of middle ear infections, certain definitions of the various conditions are relevant. OM is a generic term to describe an inflammation of the middle ear without linkage to a specific etiology but often associated with infection. The middle ear anatomy related to OM is depicted in Fig. 1. OM is very common; studies show that around 80% of children have experienced at least one episode by their third birthday [16]. OM describes any one of a continuum of diseases — acute OM (AOM), recurrent AOM (RAOM), OME, chronic otitis media (COM), and chronic OME (COME). Despite the more granular classifications, OM is generally classified into two broad types, acute and chronic. AOM is characterized by the rapid onset of signs of inflammation, specifically bulging and possible perforation of the tympanic membrane, fullness, and erythema, as well as symptoms associated with
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inflammation, such as otalgia, irritability, and fever [16]. COM is the term used to describe a variety physical symptoms and findings caused by infection and inflammation that result in long-term damage to the middle ear. Findings include chronic or recurrent inflammation, drainage, or fluid buildup, and can progress to bone erosion, cholesteatoma (keratinizing squamous epithelial cells in the middle ear and/or mastoid process), perforation or retraction of the eardrum, or even spread to the meninges. Despite appropriate therapy, AOM can progress to chronic suppurative OM (CSOM), associated with persistent drainage, ear drum perforation, and purulent discharge. When examined by otoscope, the middle ear looks red and inflamed, with purulent discharge. AOM is one of the most common chronic infectious diseases worldwide, especially affecting children; hearing impairment is one of the most common sequelae and can have a negative impact on a child’s speech development, education, and behavior [16]. In CSOM, the effusion prevents the middle ear ossicle (the bones of the middle ear) from properly relaying sound vibrations from the eardrum to the oval window of the inner ear, causing hearing loss. In addition, the associated inflammatory mediators can penetrate into the inner ear through the round window. This can cause the loss of hair cells in the cochlea, leading to sensorineural hearing loss.
Mortality due to complications of CSOM is typically higher than in other types of OM, as intra-cranial complications, like brain abscess and meningitis, are the most common causes of death in CSOM patients [16]. In contrast, OME is characterized by a non-purulent effusion of the middle ear that may be either mucoid or serous. Typical symptoms of OME do not involve the classic pain and fever symptoms, but aural fullness is common, as are downstream complications, usually involving hearing loss. In children, hearing loss is generally mild and is often detected only with an audiogram. Finally, SOM is a specific type of OME caused by transudate formation. A transudate is a watery and clear fluid that has passed through a membrane or has been extruded from a tissue and is characterized by high fluidity and a low content of protein, cells, or solid matter derived from cells. Transudate forms as a result of a rapid decrease in middle ear pressure relative to the atmospheric pressure. Transudate is different from exudate, a fluid with a high content of protein and cellular debris that has escaped from blood vessels and has been deposited in or on tissues, usually as a result of inflammation.
Middle Ear Microbiology In a study of healthy humans, Stroman et al. sought to isolate and characterize bacteria and fungi from the normal healthy ear [17]. External auditory canal and cerumen (i.e., ear wax) specimens were collected from 164 healthy subjects. Winter and summer collections were obtained from 20 subjects (17 adults and 3 children) to compare seasonal colonization. For recovery of bacteria, the cerumen samples were emulsified in a 70% glycerol solution containing 3.5% NaHCO. Specimens were subcultured to thioglycollate broth and three solid media. Species level identification was obtained by combining phenotypic and genotypic data [17]. The overall distribution of microbes from the ear canal and cerumen by Gram morphology was as follows: Gram-positive organisms, 288 (93%) and 288 (92%); Gram-negative organisms, 14 (4.5%) and 3 (1.0%); and fungal isolates, 8 (2.5%) and 23 (7.0%). In this study, 17 ear canal and 16 cerumen specimens resulted in no growth. One hundred forty-eight cerumen specimens yielded 314 organisms, including 23 fungi. One hundred forty-seven ear canal specimens yielded 310 organisms, including 7 fungi. Of 291 bacteria isolated from cerumen, 99% were Gram-positive. Of 302 bacteria isolated from the canal, 96% were Gram-positive. Staphylococci represented 63% of both the cerumen bacteria and the ear canal bacteria. Corynebacteria and related species represented 22% of the bacteria in cerumen and 19% in the ear canal. Turicella otitidis was the primary coryneform isolated from both the canal and the cerumen. Streptococcus-like bacteria were 10% from the cerumen and 7% from the ear canal. In both cerumen and ear canal, Alloiococcus otitis represented more than 95% of the streptococcus-like bacteria. Prior to this study, the Centers for Disease Control and Prevention coryneform group ANF-1 bacteria were described as Corynebacterium afermentans, and group ANF-1-like bacteria were described as
T. otitidis. Over a 1.5-year period, 10 strains of a previously undescribed Gram-positive rod-shaped organism that was not partially acid fast and resembled ANF-1-like bacteria were isolated from different pediatric patients with ear infections. These previously undescribed coryneform bacteria exhibited a distinct colony morphology and consistency, had a carbon source utilization pattern distinct from the carbon source utilization patterns of C. afermentans and T. otitidis, had a cell wall based on meso-diaminopimelic acid, contained mycolic acids, and had DNA G+C contents of 68 to 74 mol%. A 16S rRNA gene sequence analysis revealed that these clinical isolates are members of the genus Corynebacterium and that they are distinct from C. afermentans and T. otitidis. On the basis of phenotypic and phylogenetic evidence, a new species, Corynebacterium auris was proposed for these Centers for Disease Control and Prevention coryneform group ANF-1-like bacteria. The type strain is strain DSM 44122 (CCUG 33426). Stroman and colleagues found that T. otitidis and A. otitidis were present at a much higher frequency than previously described, supporting evidence that they may not be normal flora [17], yet the controversy over pathogenicity remains [18-21]. Corynebacterium auris, previously reported only in children, was isolated from healthy adults. Fifteen Gram-negative organisms were isolated from the ear canal and cerumen, including four Pseudomonas aeruginosa strains [17]. Pathogens isolated in acute ear infections
AOM is predominantly caused by S. pneumoniae, Staphylococcus aureus, H. influenzae, and M. catarrhalis, with 91% of cases attributed to P. aeruginosa and S. aureus [22]. AOM is relatively well studied and other pathogens include Staphylococcus epidermidis, Mycobacterium otiditis, Mycobacterium alconae, Staphylococcus capitis, Staphylococcus haemolyticus, Aspergillus spp,, and Candida spp. in order of prevalence [22]. Despite the common nature of AOM, in a relatively large proportion of cases (29%), no pathogen was identified; viral pathogens have been implicated [22]. Pathogens isolated in chronic ear infections
Although the pathogenesis of AOM is well studied, very limited information is available to describe CSOM. P. aeruginosa and S. aureus are the most predominant aerobic causes of CSOM, followed by Proteus vulgaris and Klebsiella pneumoniae [23-25]. However, other studies reported S. aureus as the most predominant pathogen, followed by P. aeruginosa [16, 26]. The differences in the various studies could be due to the differences in the patient populations studied and to geographical variation. There are only a few studies available to help in understanding the pathogenesis and mechanisms of CSOM, and they are summarized in a recent review [16]. With the emergence of antibiotic resistance, as well as the ototoxicity of antibiotics and the potential risks of surgery, there is an urgent need to develop effective therapeutic strategies against CSOM and to understand the role of host immunity and how the bacteria evade these potent immune responses [27]. Both P. aeruginosa and S. aureus can enter the middle ear through the external auditory canal. P. aeruginosa thrives in the ear environment and is difficult to eradicate, damaging tissues, interfering with normal body defenses, and inactivating antibiotics by various
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enzymes and toxins [16]. Bacteroides spp., Clostridium spp., Peptococcus spp., Peptostreptococcus spp., Prevetolla melaninogenica, and Fusobacterium spp. are anaerobic pathogens associated with CSOM, but there is disagreement as to which of these pathogens could be normal microbial flora in the middle ear instead of pathogenic agents. Since limited studies are available to describe the normal microflora of the middle ear in a wide variety of subjects, controversy exists, and more work must be done to better characterize the normal microbiome of the middle ear, which will help in differentiating normal ear flora from the pathogens that cause CSOM. CSOM can also be characterized by co-infections with more than one type of bacterial and viral pathogen; fungi have also been identified in cultures from patients with CSOM [16]. Of note, the presence of fungi can be due to treatment with antibiotic ear drops, which causes suppression of bacterial flora and the subsequent emergence of fungal flora [16], isolated in greater numbers when the climate is humid. This condition could increase the incidence of fungal superinfection, and as with other types of microbiome imbalance; even the less virulent fungi become more opportunistic under these conditions. Recurrent CSOM is due to one or a combination of several factors. They include treatment with oral antibiotics alone, treatment with non-antibiotic drops, non-compliance with treatment, infection with resistant bacteria, and the presence of cholesteatoma. Disease can also be particularly recalcitrant and recurrent in patients with a distorted ear anatomy or who are prone to infections [16]. SOM pathogens by age and disease duration
Brook et al, correlated the microbiology of SOM in children with the duration of the condition and the patient’s age [28]. Aspirates of serous ear fluids from 114 children were examined for aerobic and anaerobic bacteria. Bacterial growth was noted in 47 patients (41%). Aerobic organisms only were recovered in 27 aspirates (57% of the culture-positive aspirates), anaerobic bacteria only in 7 (15%), and mixed aerobic and anaerobic bacteria in 13 (28%). A total of 83 bacterial isolates were recovered, accounting for 1.8 isolates per specimen (1.2 aerobes and 0.6 anaerobe). There were a total of 57 aerobic isolates, including H. influenzae (15 isolates), S. pneumoniae (13), and Staphylococcus spp. (12). Twenty-six anaerobes were recovered, including anaerobic gram-positive cocci (10), Prevotella spp. (8), and Propionibacterium acnes (4). The rate of positive cultures (20 of 36; 56%) was higher in patients younger than 2 years of age than in those older than 2 years of age (27 of 78; 35%). S. pneumoniae and H. influenzae were more often isolated in children younger than 2 years of age and those with effusion for 3 to 5 months, whereas anaerobes were recovered more often in those older than 2 years of age and those with effusion for 6 to 13 months. These data illustrate the effects of the length of effusion and age on the recovery of aerobic and anaerobic bacteria in SOM [28].
The Microbiome Several studies have addressed aspects of the middle ear microbiome [29-31]. In a sentinel study published in 2011, the role of the microbiome in otitis media and community analysis of ear and
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ear-related biomes showed the presence of distinct populations with some significant overlap [31]. Using pyrosequencing analysis of the 16S rRNA genes, Liu et al sought to better assess the polymicrobial middle ear, adenoid, and tonsillar microbiota [31]. They characterized complex microbial communities and combined genetic data with an ecological infectious disease framework, one in which bacterial community features, such as co-occurrence patterns or interactions between diverse bacterial types, are considered in the study of pathogenesis. In this study, remnant otologic, adenoid, and tonsil surgical specimens from a single pediatric patient with chronic SOM, adenotonsillar hypertrophy, and obstructive sleep apnea were examined. DNA was purified from each clinical specimen, the V3-V4 regions of the conserved bacterial 16S rRNA gene were amplified, and pyrosequencing was performed on the 454 Life Sciences GS FLX platform (Roche Diagnostics Corp, Branford, CT). The sequences were analyzed to determine the bacterial source based on the taxonomic designation (e.g., genus), and the resulting distribution of genus descriptions was used to evaluate the richness (i.e., the total number of unique bacterial types found), diversity (i.e., the collective measurement of both the abundance and number of unique bacterial types found), and overall composition of the adenoid, tonsil, and middle ear microbiota. The data were taxonomically classified to generate an abundance-based matrix, upon which comparative ecological analysis of the microbiota was performed [31]. In total, 1,042 sequences were obtained from the 3 samples. During taxonomic classification, more than 90% of the sequences were classified at a 95% bootstrap confidence level at the phylum, class, order, and family levels; however, this rate decreased to below 90% at the genus level, and therefore, subsequent comparative analysis of the microbiota was performed only at the family level. In this earliest of middle ear microbiome studies, a total of 17 unique bacterial families were detected, with 9 from the middle ear, 9 from the tonsil, and 12 from the adenoid specimens, respectively. The body sites in this study had lower richness and diversity than is found in most other human body sites. Pseudomonadaceae was the most commonly identified family in the middle ear microbiota (82.7% relative abundance), and Streptococcaceae was most common in the tonsil microbiota (69.2%). Overlap between the middle ear and the tonsil microbiota was minimal. In contrast, multiple bacteria, including Pseudomonadaceae, Streptococcaceae, Fusobacteriaceae, and Pasteurellaceae, dominated the adenoid microbiota, which encompassed bacteria detected from both the middle ear and tonsil, suggesting that the adenoid may be a source site for both the middle ear and tonsil microbiota [31]. The authors used the Shannon diversity index to show that the greatest diversity was found in the adenoid at 1.84, followed by 0.87 in the tonsil and 0.68 in the middle ear. A heat map visualization (Fig. 2) shows that the multiorganism-dominated adenoid microbiota overlapped with those of both the middle ear and the tonsil. Hierarchal clustering further indicated that the adenoid microbiota was more closely related to the bacterial composition of the tonsil than to that of the middle ear. Analysis of the microbial composition by genus again found that Pseudomonas was the
A
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Nocardiaceae Prevotellaceae Flavorbacteriaceae Bacillales family XI Carnobacteriaceae Enterococcaceae Streptococcaceae Clostridiales family Fusobacteriaceae Phlyobacteriacea Comamonadaceae Oxalobacteracea Neisseriaceae Enterobacteriaceae Pasturellaceae Moraxellaceae Pseudomonadaceae Xanthomonaceae
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Nocardiaceae Prevotellaceae Flavorbacteriaceae Bacillales family XI Carnobacteriaceae Enterococcaceae Streptococcaceae Clostridiales family Fusobacteriaceae Phlyobacteriacea Comamonadaceae Oxalobacteracea Neisseriaceae Enterobacteriaceae Pasturellaceae Moraxellaceae Pseudomonadaceae Xanthomonaceae
214 187 160 134 107 80 54 27 0
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Figure 2. Heat map visualization of the family level microbiota. (A) Comparison of the microbiota composition in the adenoid, tonsil, and middle ear specimens from a chronic SOM case showing that the adenoid microbiota is dominated by multiple bacterial groups, including Pseudomonadaceae, Streptococcaceae, and Fusobacteriaceae, in contrast to the single-bacterial-group-dominated microbiota in the middle ear and the tonsil. As shown by the color legend, red and purple represent the most abundant bacterial types, whereas yellow and gray signify the rarer types. The numbers by the color legend correspond to the number of sequences from a given bacterial group in each sample. (B) Visualization with hierarchal-clustering results using the family level microbiota suggests that while the adenoid microbiota is composed of bacterial families found in both the tonsil and the middle ear, the tonsil and adenoid microbiota are more closely related, with the middle ear microbiota as an outgroup. This finding likely reflects the closer proximity and greater exposure of the tonsil to the adenoid and the enclosed nature of the middle ear [31]. Used with permission from Arch Otolaryngol Head Neck Surg. 2011; 137(7):664-668. doi: 10.1001/archoto.2011.116.
only genus found at all 3 sites, constituting 97.1% of the Pseudomonadaceae sequences. Anaerobe identification was most common in the adenoid, with Fusobacterium spp. and Prevotella spp. found at greater abundance in adenoid tissue than in either tonsil or middle ear specimens. Fusobacterium spp. were also uniquely abundant in the adenoid [31]. The complex bacterial communities identified encompassed a wide range of bacterial types, including many that have not been previously reported in culture-based studies. For example, in addition to Haemophilus spp. and Streptococcus spp., known to be associated with otitis media and adenotonsillar disease, other, previously unreported bacterial families included Comamonadaceae, Oxalobacteraceae, Clostridiales family XI, and Xanthomonadaceae. These families are fastidious and difficult to culture using traditional methods, but they can be detected and characterized using open, non-targeted, molecular-based methods, such as those in the study. In addition, rare organisms within a polymicrobial community that would otherwise be masked by dominant bacteria in a traditional culture-based assay were identified. Genetic analysis of the bacterial 16S rRNA gene revealed diverse bacterial communities in one set of pediatric middle ear, tonsil, and adenoid specimens, with much greater microbial diversity present at these anatomic sites than was previously detected. Among the 3 sites, the adenoid microbiota was the most complex and demonstrated the greatest overlap with the tonsil and the middle ear microbiota, suggesting
that nasopharyngeal tissue may serve as a bacterial reservoir for both middle ear and tonsillar disease. For newly identified members of the bacterial communities, the clinical roles remain unknown, and future studies must define their roles in pathogenesis. Although this study does not provide direct evidence of biofilm formation in the tissue specimens collected under sterile field operating room conditions, it does report increased detection of bacterial genera with known biofilm-forming capabilities, such as Pseudomonas. Finally, because this study is restricted to a sample from a single patient, the finding cannot be generalized until it is reproduced in a larger study population.
Limitations to Molecular Sequence Analysis of Microbiomes Of note, although molecular methods are highly sensitive for identification of bacteria within human tissues, detection of bacterial 16S rRNA does not necessarily equate with bacterial viability and activity; these aspects would be better supported by detection of bacterial transcripts or proteins in tissues. Reagent contamination is also an important challenge in studying small surgical specimens because the bacterial DNA carried through reagent synthesis can overwhelm the true bacterial content of the specimens. Contamination during specimen collection may also be a problem. For example, Pseudomonas spp. are known to colonize the external auditory canal, and thus, middle ear specimens are theoretically susceptible to unintentional contamination by the organism; to
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minimize the potential for cross-contamination, specimens should be collected in the operating room using sterile techniques.
Antimicrobial Penetration in the Middle Ear There is concern as to whether orally administered antibiotics reach concentrations in the middle ear that are sufficient to eradicate planktonic and biofilm bacteria [32]. Although systemic antibiotics are frequently used to treat OM, their effectiveness depends on whether an adequate antibiotic concentration is achieved in the middle ear, especially for biofilm infections such as OME, where high antibiotic concentrations are typically required for effective treatment. Investigators performed a literature search of studies that measured concentrations of antibiotics in the plasma and in the middle ear after oral administration of antimicrobials. These levels were compared to the MICs provided by the European Committee for Antimicrobial Susceptibility Testing (EUCAST) to determine if antibiotic doses reached sufficient levels to inhibit planktonic bacteria. The middle ear concentrations were then calculated as a multiple of the MIC to determine if the concentrations reached biofilm eradication concentrations (typically up to 1,000 × MIC). The results showed that the highest antibiotic levels reached were 8.3 × MIC against S. aureus, 33.2 × MIC against M. catarrhalis, 31.2 × MIC against H. influenzae, and 46.2 × MIC against S. pneumoniae. Macrolide antibiotics reached higher levels in the middle ear than in plasma. While orally administered antibiotics reach levels above the MIC in the middle ear, they do not reach levels that would typically be required to eradicate biofilms [32].
Summary Infections of the middle ear are a common but complicated condition, and the middle ear microbiome has yet to be fully described in a wide variety of subjects. The current literature suggests nichespecific upper respiratory tract human microbiota profiles exist and that changes occurring within these profiles seem to be associated with infections. It is thought that biofilms play a large role in the detection and therapy of infections in the ear, nose, and throat. Traditional culture techniques are known to be less sensitive for detecting bacteria that reside in biofilms and bacterial communities, like those of the middle ear; therefore, molecular methods appear to be the best option for understanding the roles of various microbes in the pathogenesis of middle ear infections.
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Vol. 38, No. 11 June 1, 2016 www.cmnewsletter.com I n T hi s
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87 Microbiology of Middle Ear Infections: Do You Hear What I Hear?
Stay Current... Stay Informed.
t e r
Microbiology of Middle Ear Infections: Do You Hear What I Hear? Donna M. Wolk, MHA, Ph.D., D(ABMM),1,2,3 1Department of Laboratory Medicine, Geisinger Health Systems, Danville, Pennsylvania, 2Weis Center for Research, Danville, Pennsylvania, 3Wilkes University, Wilkes-Barre, Pennsylvania
Abstract Infectious conditions of the middle ear are a common and significant cause of morbidity and sometimes even mortality, especially in young children and elderly individuals. Pathogens and harmless commensal bacteria, viruses, and fungi co-inhabit the auditory canal and form intricate ecological networks, collectively known as a microbiome. Few studies that describe the normal flora of the middle ear have been published, and controversy exists about the roles of several possible pathogens. This review describes current literature examining otitis media and the roles various microbes play in the pathogenesis of middle ear infections. The review also highlights evolving research in middle ear microbiome studies, which begs the question, “Whose hearing could be damaged by what we don’t yet know about middle ear infections?”
Background and History
Corresponding author: Donna M. Wolk, Department of Laboratory Medicine, Geisinger Health Systems, 100 N. Academy Ave., Danville, PA 17822-1930. Tel.: 570-2716388. Fax: 570-214-9792. E-mail: [email protected].
CMN
Middle ear effusion in otitis media (OM) was at one time considered sterile, but in the 1950s, bacteria were cultured from middle ear effusions [1]. Since then, the presence of Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis has been associated with OM, including acute and chronic infections, as well as those with effusions. These mono-pathogen infections are often called the “one-organism, one-disease” infectious disease framework, in which highly common bacterial infections, including OM and pneumonia, were thought to be caused by a limited number of pathogens. Scientists are now aware of the potential role of the nasopharyngeal lymphoid tissue as a reservoir of both middle ear and tonsil disease is supported by a wide range of clinical and microbiological studies [2-6], and it is known that healthy children also harbor a diverse number of bacterial species in the adenoid microbiota [3,4,7,8]. Complicating the microbiologic scenario further, the common pathogens associated with ear infections are also frequently observed commensal
residents of the upper respiratory tract. Together with harmless commensal bacteria, viruses, and fungi, they form intricate ecological networks collectively known as the microbiome. Similar to the intestinal microbiome, the normal respiratory microbiome is thought to be beneficial to the host by priming the immune system and providing colonization resistance, while an imbalanced biome might predispose to bacterial overgrowth and development of respiratory infections. The current literature suggests niche-specific upper respiratory tract human microbiota profiles, hosts, and changes occurring within these profiles exist, and that changes are associated with respiratory infections [9]. Biofilms detected in a wide range of infections of the ear, nose, and throat [6,10] comprise a community of sessile organisms embedded in an adherent matrix of extracellular polymeric substances. They are three-dimensional aggregates of bacteria that are highly resistant to both immune-mediated killing and antimicrobial agents, characteristics that both enhance survival of the bacteria and decrease the sensitivity of
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Figure 1. Middle ear anatomy and otitis media. Shown is a schematic representation of the ear under normal and CSOM conditions. Under normal conditions, the middle ear cavity is clear and empty. In contrast, the middle ear becomes red and inflamed with the presence of fluid under CSOM conditions. The red color denotes inflammation, while yellow indicates fluid during CSOM.
culture-based detection. Biofilm formation by multiple mucosal pathogens, notably Pseudomonas spp., is implicated in a variety of otorhinolaryngologic diseases, such as OM with effusion (OME), chronic serous OM (SOM), and recurrent adenotonsillitis [11-13]. Traditional culture techniques are known to be less sensitive for detecting bacteria that reside in biofilms and bacterial communities like those of the middle ear [14,15].
Disease Conditions of the Middle Ear To understand the progression of middle ear infections, certain definitions of the various conditions are relevant. OM is a generic term to describe an inflammation of the middle ear without linkage to a specific etiology but often associated with infection. The middle ear anatomy related to OM is depicted in Fig. 1. OM is very common; studies show that around 80% of children have experienced at least one episode by their third birthday [16]. OM describes any one of a continuum of diseases — acute OM (AOM), recurrent AOM (RAOM), OME, chronic otitis media (COM), and chronic OME (COME). Despite the more granular classifications, OM is generally classified into two broad types, acute and chronic. AOM is characterized by the rapid onset of signs of inflammation, specifically bulging and possible perforation of the tympanic membrane, fullness, and erythema, as well as symptoms associated with
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inflammation, such as otalgia, irritability, and fever [16]. COM is the term used to describe a variety physical symptoms and findings caused by infection and inflammation that result in long-term damage to the middle ear. Findings include chronic or recurrent inflammation, drainage, or fluid buildup, and can progress to bone erosion, cholesteatoma (keratinizing squamous epithelial cells in the middle ear and/or mastoid process), perforation or retraction of the eardrum, or even spread to the meninges. Despite appropriate therapy, AOM can progress to chronic suppurative OM (CSOM), associated with persistent drainage, ear drum perforation, and purulent discharge. When examined by otoscope, the middle ear looks red and inflamed, with purulent discharge. AOM is one of the most common chronic infectious diseases worldwide, especially affecting children; hearing impairment is one of the most common sequelae and can have a negative impact on a child’s speech development, education, and behavior [16]. In CSOM, the effusion prevents the middle ear ossicle (the bones of the middle ear) from properly relaying sound vibrations from the eardrum to the oval window of the inner ear, causing hearing loss. In addition, the associated inflammatory mediators can penetrate into the inner ear through the round window. This can cause the loss of hair cells in the cochlea, leading to sensorineural hearing loss.
Mortality due to complications of CSOM is typically higher than in other types of OM, as intra-cranial complications, like brain abscess and meningitis, are the most common causes of death in CSOM patients [16]. In contrast, OME is characterized by a non-purulent effusion of the middle ear that may be either mucoid or serous. Typical symptoms of OME do not involve the classic pain and fever symptoms, but aural fullness is common, as are downstream complications, usually involving hearing loss. In children, hearing loss is generally mild and is often detected only with an audiogram. Finally, SOM is a specific type of OME caused by transudate formation. A transudate is a watery and clear fluid that has passed through a membrane or has been extruded from a tissue and is characterized by high fluidity and a low content of protein, cells, or solid matter derived from cells. Transudate forms as a result of a rapid decrease in middle ear pressure relative to the atmospheric pressure. Transudate is different from exudate, a fluid with a high content of protein and cellular debris that has escaped from blood vessels and has been deposited in or on tissues, usually as a result of inflammation.
Middle Ear Microbiology In a study of healthy humans, Stroman et al. sought to isolate and characterize bacteria and fungi from the normal healthy ear [17]. External auditory canal and cerumen (i.e., ear wax) specimens were collected from 164 healthy subjects. Winter and summer collections were obtained from 20 subjects (17 adults and 3 children) to compare seasonal colonization. For recovery of bacteria, the cerumen samples were emulsified in a 70% glycerol solution containing 3.5% NaHCO. Specimens were subcultured to thioglycollate broth and three solid media. Species level identification was obtained by combining phenotypic and genotypic data [17]. The overall distribution of microbes from the ear canal and cerumen by Gram morphology was as follows: Gram-positive organisms, 288 (93%) and 288 (92%); Gram-negative organisms, 14 (4.5%) and 3 (1.0%); and fungal isolates, 8 (2.5%) and 23 (7.0%). In this study, 17 ear canal and 16 cerumen specimens resulted in no growth. One hundred forty-eight cerumen specimens yielded 314 organisms, including 23 fungi. One hundred forty-seven ear canal specimens yielded 310 organisms, including 7 fungi. Of 291 bacteria isolated from cerumen, 99% were Gram-positive. Of 302 bacteria isolated from the canal, 96% were Gram-positive. Staphylococci represented 63% of both the cerumen bacteria and the ear canal bacteria. Corynebacteria and related species represented 22% of the bacteria in cerumen and 19% in the ear canal. Turicella otitidis was the primary coryneform isolated from both the canal and the cerumen. Streptococcus-like bacteria were 10% from the cerumen and 7% from the ear canal. In both cerumen and ear canal, Alloiococcus otitis represented more than 95% of the streptococcus-like bacteria. Prior to this study, the Centers for Disease Control and Prevention coryneform group ANF-1 bacteria were described as Corynebacterium afermentans, and group ANF-1-like bacteria were described as
T. otitidis. Over a 1.5-year period, 10 strains of a previously undescribed Gram-positive rod-shaped organism that was not partially acid fast and resembled ANF-1-like bacteria were isolated from different pediatric patients with ear infections. These previously undescribed coryneform bacteria exhibited a distinct colony morphology and consistency, had a carbon source utilization pattern distinct from the carbon source utilization patterns of C. afermentans and T. otitidis, had a cell wall based on meso-diaminopimelic acid, contained mycolic acids, and had DNA G+C contents of 68 to 74 mol%. A 16S rRNA gene sequence analysis revealed that these clinical isolates are members of the genus Corynebacterium and that they are distinct from C. afermentans and T. otitidis. On the basis of phenotypic and phylogenetic evidence, a new species, Corynebacterium auris was proposed for these Centers for Disease Control and Prevention coryneform group ANF-1-like bacteria. The type strain is strain DSM 44122 (CCUG 33426). Stroman and colleagues found that T. otitidis and A. otitidis were present at a much higher frequency than previously described, supporting evidence that they may not be normal flora [17], yet the controversy over pathogenicity remains [18-21]. Corynebacterium auris, previously reported only in children, was isolated from healthy adults. Fifteen Gram-negative organisms were isolated from the ear canal and cerumen, including four Pseudomonas aeruginosa strains [17]. Pathogens isolated in acute ear infections
AOM is predominantly caused by S. pneumoniae, Staphylococcus aureus, H. influenzae, and M. catarrhalis, with 91% of cases attributed to P. aeruginosa and S. aureus [22]. AOM is relatively well studied and other pathogens include Staphylococcus epidermidis, Mycobacterium otiditis, Mycobacterium alconae, Staphylococcus capitis, Staphylococcus haemolyticus, Aspergillus spp,, and Candida spp. in order of prevalence [22]. Despite the common nature of AOM, in a relatively large proportion of cases (29%), no pathogen was identified; viral pathogens have been implicated [22]. Pathogens isolated in chronic ear infections
Although the pathogenesis of AOM is well studied, very limited information is available to describe CSOM. P. aeruginosa and S. aureus are the most predominant aerobic causes of CSOM, followed by Proteus vulgaris and Klebsiella pneumoniae [23-25]. However, other studies reported S. aureus as the most predominant pathogen, followed by P. aeruginosa [16, 26]. The differences in the various studies could be due to the differences in the patient populations studied and to geographical variation. There are only a few studies available to help in understanding the pathogenesis and mechanisms of CSOM, and they are summarized in a recent review [16]. With the emergence of antibiotic resistance, as well as the ototoxicity of antibiotics and the potential risks of surgery, there is an urgent need to develop effective therapeutic strategies against CSOM and to understand the role of host immunity and how the bacteria evade these potent immune responses [27]. Both P. aeruginosa and S. aureus can enter the middle ear through the external auditory canal. P. aeruginosa thrives in the ear environment and is difficult to eradicate, damaging tissues, interfering with normal body defenses, and inactivating antibiotics by various
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enzymes and toxins [16]. Bacteroides spp., Clostridium spp., Peptococcus spp., Peptostreptococcus spp., Prevetolla melaninogenica, and Fusobacterium spp. are anaerobic pathogens associated with CSOM, but there is disagreement as to which of these pathogens could be normal microbial flora in the middle ear instead of pathogenic agents. Since limited studies are available to describe the normal microflora of the middle ear in a wide variety of subjects, controversy exists, and more work must be done to better characterize the normal microbiome of the middle ear, which will help in differentiating normal ear flora from the pathogens that cause CSOM. CSOM can also be characterized by co-infections with more than one type of bacterial and viral pathogen; fungi have also been identified in cultures from patients with CSOM [16]. Of note, the presence of fungi can be due to treatment with antibiotic ear drops, which causes suppression of bacterial flora and the subsequent emergence of fungal flora [16], isolated in greater numbers when the climate is humid. This condition could increase the incidence of fungal superinfection, and as with other types of microbiome imbalance; even the less virulent fungi become more opportunistic under these conditions. Recurrent CSOM is due to one or a combination of several factors. They include treatment with oral antibiotics alone, treatment with non-antibiotic drops, non-compliance with treatment, infection with resistant bacteria, and the presence of cholesteatoma. Disease can also be particularly recalcitrant and recurrent in patients with a distorted ear anatomy or who are prone to infections [16]. SOM pathogens by age and disease duration
Brook et al, correlated the microbiology of SOM in children with the duration of the condition and the patient’s age [28]. Aspirates of serous ear fluids from 114 children were examined for aerobic and anaerobic bacteria. Bacterial growth was noted in 47 patients (41%). Aerobic organisms only were recovered in 27 aspirates (57% of the culture-positive aspirates), anaerobic bacteria only in 7 (15%), and mixed aerobic and anaerobic bacteria in 13 (28%). A total of 83 bacterial isolates were recovered, accounting for 1.8 isolates per specimen (1.2 aerobes and 0.6 anaerobe). There were a total of 57 aerobic isolates, including H. influenzae (15 isolates), S. pneumoniae (13), and Staphylococcus spp. (12). Twenty-six anaerobes were recovered, including anaerobic gram-positive cocci (10), Prevotella spp. (8), and Propionibacterium acnes (4). The rate of positive cultures (20 of 36; 56%) was higher in patients younger than 2 years of age than in those older than 2 years of age (27 of 78; 35%). S. pneumoniae and H. influenzae were more often isolated in children younger than 2 years of age and those with effusion for 3 to 5 months, whereas anaerobes were recovered more often in those older than 2 years of age and those with effusion for 6 to 13 months. These data illustrate the effects of the length of effusion and age on the recovery of aerobic and anaerobic bacteria in SOM [28].
The Microbiome Several studies have addressed aspects of the middle ear microbiome [29-31]. In a sentinel study published in 2011, the role of the microbiome in otitis media and community analysis of ear and
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ear-related biomes showed the presence of distinct populations with some significant overlap [31]. Using pyrosequencing analysis of the 16S rRNA genes, Liu et al sought to better assess the polymicrobial middle ear, adenoid, and tonsillar microbiota [31]. They characterized complex microbial communities and combined genetic data with an ecological infectious disease framework, one in which bacterial community features, such as co-occurrence patterns or interactions between diverse bacterial types, are considered in the study of pathogenesis. In this study, remnant otologic, adenoid, and tonsil surgical specimens from a single pediatric patient with chronic SOM, adenotonsillar hypertrophy, and obstructive sleep apnea were examined. DNA was purified from each clinical specimen, the V3-V4 regions of the conserved bacterial 16S rRNA gene were amplified, and pyrosequencing was performed on the 454 Life Sciences GS FLX platform (Roche Diagnostics Corp, Branford, CT). The sequences were analyzed to determine the bacterial source based on the taxonomic designation (e.g., genus), and the resulting distribution of genus descriptions was used to evaluate the richness (i.e., the total number of unique bacterial types found), diversity (i.e., the collective measurement of both the abundance and number of unique bacterial types found), and overall composition of the adenoid, tonsil, and middle ear microbiota. The data were taxonomically classified to generate an abundance-based matrix, upon which comparative ecological analysis of the microbiota was performed [31]. In total, 1,042 sequences were obtained from the 3 samples. During taxonomic classification, more than 90% of the sequences were classified at a 95% bootstrap confidence level at the phylum, class, order, and family levels; however, this rate decreased to below 90% at the genus level, and therefore, subsequent comparative analysis of the microbiota was performed only at the family level. In this earliest of middle ear microbiome studies, a total of 17 unique bacterial families were detected, with 9 from the middle ear, 9 from the tonsil, and 12 from the adenoid specimens, respectively. The body sites in this study had lower richness and diversity than is found in most other human body sites. Pseudomonadaceae was the most commonly identified family in the middle ear microbiota (82.7% relative abundance), and Streptococcaceae was most common in the tonsil microbiota (69.2%). Overlap between the middle ear and the tonsil microbiota was minimal. In contrast, multiple bacteria, including Pseudomonadaceae, Streptococcaceae, Fusobacteriaceae, and Pasteurellaceae, dominated the adenoid microbiota, which encompassed bacteria detected from both the middle ear and tonsil, suggesting that the adenoid may be a source site for both the middle ear and tonsil microbiota [31]. The authors used the Shannon diversity index to show that the greatest diversity was found in the adenoid at 1.84, followed by 0.87 in the tonsil and 0.68 in the middle ear. A heat map visualization (Fig. 2) shows that the multiorganism-dominated adenoid microbiota overlapped with those of both the middle ear and the tonsil. Hierarchal clustering further indicated that the adenoid microbiota was more closely related to the bacterial composition of the tonsil than to that of the middle ear. Analysis of the microbial composition by genus again found that Pseudomonas was the
A
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Nocardiaceae Prevotellaceae Flavorbacteriaceae Bacillales family XI Carnobacteriaceae Enterococcaceae Streptococcaceae Clostridiales family Fusobacteriaceae Phlyobacteriacea Comamonadaceae Oxalobacteracea Neisseriaceae Enterobacteriaceae Pasturellaceae Moraxellaceae Pseudomonadaceae Xanthomonaceae
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Nocardiaceae Prevotellaceae Flavorbacteriaceae Bacillales family XI Carnobacteriaceae Enterococcaceae Streptococcaceae Clostridiales family Fusobacteriaceae Phlyobacteriacea Comamonadaceae Oxalobacteracea Neisseriaceae Enterobacteriaceae Pasturellaceae Moraxellaceae Pseudomonadaceae Xanthomonaceae
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Figure 2. Heat map visualization of the family level microbiota. (A) Comparison of the microbiota composition in the adenoid, tonsil, and middle ear specimens from a chronic SOM case showing that the adenoid microbiota is dominated by multiple bacterial groups, including Pseudomonadaceae, Streptococcaceae, and Fusobacteriaceae, in contrast to the single-bacterial-group-dominated microbiota in the middle ear and the tonsil. As shown by the color legend, red and purple represent the most abundant bacterial types, whereas yellow and gray signify the rarer types. The numbers by the color legend correspond to the number of sequences from a given bacterial group in each sample. (B) Visualization with hierarchal-clustering results using the family level microbiota suggests that while the adenoid microbiota is composed of bacterial families found in both the tonsil and the middle ear, the tonsil and adenoid microbiota are more closely related, with the middle ear microbiota as an outgroup. This finding likely reflects the closer proximity and greater exposure of the tonsil to the adenoid and the enclosed nature of the middle ear [31]. Used with permission from Arch Otolaryngol Head Neck Surg. 2011; 137(7):664-668. doi: 10.1001/archoto.2011.116.
only genus found at all 3 sites, constituting 97.1% of the Pseudomonadaceae sequences. Anaerobe identification was most common in the adenoid, with Fusobacterium spp. and Prevotella spp. found at greater abundance in adenoid tissue than in either tonsil or middle ear specimens. Fusobacterium spp. were also uniquely abundant in the adenoid [31]. The complex bacterial communities identified encompassed a wide range of bacterial types, including many that have not been previously reported in culture-based studies. For example, in addition to Haemophilus spp. and Streptococcus spp., known to be associated with otitis media and adenotonsillar disease, other, previously unreported bacterial families included Comamonadaceae, Oxalobacteraceae, Clostridiales family XI, and Xanthomonadaceae. These families are fastidious and difficult to culture using traditional methods, but they can be detected and characterized using open, non-targeted, molecular-based methods, such as those in the study. In addition, rare organisms within a polymicrobial community that would otherwise be masked by dominant bacteria in a traditional culture-based assay were identified. Genetic analysis of the bacterial 16S rRNA gene revealed diverse bacterial communities in one set of pediatric middle ear, tonsil, and adenoid specimens, with much greater microbial diversity present at these anatomic sites than was previously detected. Among the 3 sites, the adenoid microbiota was the most complex and demonstrated the greatest overlap with the tonsil and the middle ear microbiota, suggesting
that nasopharyngeal tissue may serve as a bacterial reservoir for both middle ear and tonsillar disease. For newly identified members of the bacterial communities, the clinical roles remain unknown, and future studies must define their roles in pathogenesis. Although this study does not provide direct evidence of biofilm formation in the tissue specimens collected under sterile field operating room conditions, it does report increased detection of bacterial genera with known biofilm-forming capabilities, such as Pseudomonas. Finally, because this study is restricted to a sample from a single patient, the finding cannot be generalized until it is reproduced in a larger study population.
Limitations to Molecular Sequence Analysis of Microbiomes Of note, although molecular methods are highly sensitive for identification of bacteria within human tissues, detection of bacterial 16S rRNA does not necessarily equate with bacterial viability and activity; these aspects would be better supported by detection of bacterial transcripts or proteins in tissues. Reagent contamination is also an important challenge in studying small surgical specimens because the bacterial DNA carried through reagent synthesis can overwhelm the true bacterial content of the specimens. Contamination during specimen collection may also be a problem. For example, Pseudomonas spp. are known to colonize the external auditory canal, and thus, middle ear specimens are theoretically susceptible to unintentional contamination by the organism; to
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minimize the potential for cross-contamination, specimens should be collected in the operating room using sterile techniques.
Antimicrobial Penetration in the Middle Ear There is concern as to whether orally administered antibiotics reach concentrations in the middle ear that are sufficient to eradicate planktonic and biofilm bacteria [32]. Although systemic antibiotics are frequently used to treat OM, their effectiveness depends on whether an adequate antibiotic concentration is achieved in the middle ear, especially for biofilm infections such as OME, where high antibiotic concentrations are typically required for effective treatment. Investigators performed a literature search of studies that measured concentrations of antibiotics in the plasma and in the middle ear after oral administration of antimicrobials. These levels were compared to the MICs provided by the European Committee for Antimicrobial Susceptibility Testing (EUCAST) to determine if antibiotic doses reached sufficient levels to inhibit planktonic bacteria. The middle ear concentrations were then calculated as a multiple of the MIC to determine if the concentrations reached biofilm eradication concentrations (typically up to 1,000 × MIC). The results showed that the highest antibiotic levels reached were 8.3 × MIC against S. aureus, 33.2 × MIC against M. catarrhalis, 31.2 × MIC against H. influenzae, and 46.2 × MIC against S. pneumoniae. Macrolide antibiotics reached higher levels in the middle ear than in plasma. While orally administered antibiotics reach levels above the MIC in the middle ear, they do not reach levels that would typically be required to eradicate biofilms [32].
Summary Infections of the middle ear are a common but complicated condition, and the middle ear microbiome has yet to be fully described in a wide variety of subjects. The current literature suggests nichespecific upper respiratory tract human microbiota profiles exist and that changes occurring within these profiles seem to be associated with infections. It is thought that biofilms play a large role in the detection and therapy of infections in the ear, nose, and throat. Traditional culture techniques are known to be less sensitive for detecting bacteria that reside in biofilms and bacterial communities, like those of the middle ear; therefore, molecular methods appear to be the best option for understanding the roles of various microbes in the pathogenesis of middle ear infections.
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