Int J Pharm Pharm Sci, Vol 17, Issue 1, 12-20Original Article

WAR IN THE MIDDLE EAR: MICROBIOLOGY OF CHRONIC SUPPURATIVE OTITIS MEDIA WITH SPECIAL REFERENCE TO ANAEROBES AND ITS ANTIMICROBIAL SUSCEPTIBILITY PATTERN: OPTIMIZING ANTIMICROBIAL THERAPY IN A TERTIARY CARE HOSPITAL OF RURAL INDIA

PRIYA BHAT, UPASANA BHUMBLA*, JASLEEN KAUR

Department of Microbiology, Adesh Institute of Medical Sciences and Research, Bathinda
*Corresponding author: Upasana Bhumbla; *Email: ucupasana124@gmail.com

Received: 17 Sep 2024, Revised and Accepted: 02 Dec 2024

ABSTRACT

Objective: To isolate etiological organisms of Chronic Suppurative Otitis Media (CSOM), study the antimicrobial susceptibility pattern, study the risk factors and associated co-morbid conditions with CSOM, and detect biofilm production in isolated bacteria.

Methods: An ear discharge specimen was obtained from the diseased ear of the patient, using three separate sterilized swabs. One of the swabs was used for Gram staining and Aerobic culture. Antibiotic susceptibility testing was done by the Kirby-Bauer standard disc diffusion method all the g-negative isolates were screened for Extended-Spectrum Beta-Lactamase (ESBL) production and Amp C ß-lactamase production. Methicillin-Resistant Staphylococcus Aureus (MRSA) screening was done using a Cefoxitin disc. Biofilm production was done using the Tube method and Microtiter plate method. Anaerobic culture was inoculated into Robertson’s Cooked Meat (RCM) broth. Potassium hydroxide (KOH) mount was done using 10% KOH for the presence of budding yeast cells, fungal hyphae, and spores. Tubes showing positive cultures were examined by LactoPhenol Cotton Blue (LPCB) mount. Final Candida speciation and Antifungal susceptibility testing was done using VITEK-2.

Results: A total of 500 patients were included in the study, with 62.8% being males. The most common organism isolated was Pseudomonas aeruginosa. Fungi accounted for 30 isolates (5%) and anaerobes were 5 isolates (1%). Gram-negative bacteria were found to be most sensitive to Piperacillin-tazobactam (96%) while the highest resistance was noted to Amoxicillin-clavulanate (65%). 100% of Staphylococcus aureus isolates were sensitive to Vancomycin, Linezolid, and Teichoplanin.13% of S. aureus were found to be Cefoxitin resistant (MRSA). The highest resistance in S. aureus isolates was noted to Ciprofloxacin (65%).13% of isolates of Gram-negative bacteria had ESBL production while Amp C β-Lactamase production was noted in 9% of isolates. Out of 186 isolates tested for biofilm production, 149 isolates (80%) showed biofilm production. A total of 30 fungal isolates were obtained in culture. Aspergillus spp. Accounted for 66.6% of fungal isolates while Candida spp. accounted for 33.3% of fungal isolates. The sensitivity of Candida isolates was tested to Fluconazole, Itraconazole, Voriconazole, and Flucytosine and they were found to be 100% sensitive to all the antifungals tested.

Conclusion: CSOM often has polymicrobial pathology and creates a war in the middle ear environment, augmented by mixed aerobic and anaerobic organisms and synergism between aerobic and anaerobic organisms. A high rate of Clindamycin resistance was seen in anaerobe which is noteworthy because this is a frequently utilized antimicrobial for treating anaerobic infections.

Keywords: CSOM, Anaerobic profile, Biofilm, Demographic factors, Antibiotic resistance

© 2025 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open access article under the CC BY license (https://creativecommons.org/licenses/by/4.0/)
DOI: https://dx.doi.org/10.22159/ijpps.2025v17i1.52692 Journal homepage: https://innovareacademics.in/journals/index.php/ijpps

INTRODUCTION

CSOM is defined as chronic inflammation of the middle ear and mastoid cavity that presents with recurrent ear discharge of more than three months duration through a perforated tympanic membrane. It is a massive health problem in developing countries and India is one of the countries with the highest CSOM prevalence (>4%) where urgent attention is needed. According to a survey, about 65-330 million people suffer from ear infections worldwide and 60% of them will develop significant hearing impairment [1].

CSOM is classically divided into) a tubo-tympanic type affecting the middle ear muco-periosteum and 2) an attico-antral type which is an active squamous disease with a growth of squamous epithelium into the middle ear cleft [2]. Recently CSOM has been modified into broader types like active mucosal, inactive mucosal, active squamous, inactive squamous and healed dimeric, tympanosclerosis, etc [3]. Unsafe CSOM is characterized by an attic cholesteatoma or a posterior-superior cholesteatoma with a history of scanty and foul-smelling ear discharge which is at times blood-stained. Cholesteatoma is a keratinizing stratified squamous epithelium accumulating within the middle ear or other pneumatised portions of the temporal bone and may get infected involving the entire middle ear cleft and can cause bone resorption possibly due to the activity of collagenase [4].

Gram-negative bacteria like Pseudomonas aeruginosa continue to be the most isolated organisms from cases of CSOM with maximum sensitivity for Piperacillin-tazobactam followed by Meropenem, Piperacillin while among the g-positive organisms, Staphylococcus aureus and remains the maximally isolated organism with maximum sensitivity to Vancomycin, Linezolid, however, the prevalence of ESBL, Carbapenemases, Amp C ß-lactamase, Metallo β-Lactamase (MBL), MRSA, Vancomycin-Resistant Staphylococcus Aureus (VRSA) is rising in this era of emerging multidrug resistance [5]. Although facultative anaerobes constitute the major chunk of causative organisms, anaerobes cannot be neglected. Peptostreptococcus, Bacteroides species, and Fusobacterium are the predominant anaerobic bacterial isolations from cases of CSOM. Though the anaerobes can live in complete harmony with the host and exhibit a mutually beneficial relationship with aerobic commensal, they can still become pathogenic and create a war-like environment in the middle ear when the host's defense mechanism gets impaired. There is a high tendency to see the anaerobic infection in samples showing polymicrobial appearance in Gram stain from clinical samples and this can be more virulent than those involving a single organism. Synergism between aerobic and anaerobic organisms has been recognized. Most clinical laboratories neither identify obligate anaerobes nor perform antimicrobial susceptibility studies as it is time-consuming and involves various technical skills and hence is overlooked. The frequency of antimicrobial resistance in anaerobic organisms has been increasing globally at the same pace as in aerobes and facultative anaerobes in the past few decades [6, 14].

Evaluation of the microbiological agents and their antibiotic sensitivity pattern is helpful in the initiation of specific therapy and thus minimizing its complications like skull base osteomyelitis, meningitis, etc, and the emergence of resistant strains. Due to the high incidence, long period of morbidity of CSOM, and the repeated occurrences of otorrhea during that period, patients are often prescribed empiric antibiotics in outpatient clinics resulting in bacterial resistance and ototoxicity with both topical and systemic antibiotics. Also, it is necessary that anaerobic susceptibility testing be performed and awareness of the emerging resistance among the anaerobic pathogens must be created to provide appropriate antimicrobial therapy for better patient care.

We conducted this study with the following aims and objectives:

1. To isolate, and identify aerobic bacteria from chronic suppurative Otitis media cases and to study antibiotic susceptibility patterns of isolated aerobes and facultative anaerobes.

2. To isolate, and identify anaerobic bacteria from chronic suppurative Otitis media cases and to study antibiotic susceptibility patterns of isolated obligate anaerobes.

3. To isolate, identify fungi causing CSOM and to study the antifungal susceptibility pattern of isolated candida species.

MATERIALS AND METHODS

This prospective study was conducted in the Department of Microbiology at a tertiary care teaching hospital. A total of 500 clinically diagnosed cases of CSOM attending ENT OPD, and being admitted in ENT wards were included in the study. Ethical clearance was obtained from the Institutional Ethical Committee (letter number: AIMSR/2023/IEC/59, dated: 15th September 2023) before the study. A written consent was also obtained from the patients before they participated in the study. Subjects were explained the purpose of the study and the procedures involved.

The sample size was calculated using the Daniel formula, n = 4 pq/d2 and depending upon previous years' statistics.

Statistical analysis

The data was analyzed by using Statistical Package for Social Sciences (SPSS) version 21.0 and the prevalence of organisms was determined and expressed in percentage.

Inclusion criteria

  1. Patients diagnosed as CSOM after thorough clinical evaluation i. e. the patients having ear discharge of more than 3 mo.

  2. Patients who were not on antibiotic (both systemic and topical) treatment for a minimum of 24 h before sample collection.

Exclusion criteria

  1. Patients suffering from CSOM who were on systemic antibiotics in the past days of presentation.

  2. Patients who were on topical medications to the ear.

  3. Patients having ear discharge due to some traumatic or neoplastic condition.

  4. Patients having ear discharge arising from otitis externa.

Sample collection and processing

Ear discharge was obtained from the diseased ear of the patient, using three separate sterilized swabs before the instillation of any topical medication. One of the swabs was used for Gram staining and Aerobic culture and was plated on 5% sheep Blood Agar (BA), MacConkey’s Agar (MA), and Chocolate Agar (CA) and incubated at 37 °C for 48 h. The plates were examined for growth and cultural characteristics and the isolated colonies were subjected to a standard set of biochemical tests for identification [7, 8]. Antibiotic susceptibility testing was done by Kirby-Bauer standard disc diffusion method on Muller-Hinton agar according to clinical and laboratory standards institute (CLSI) guidelines (M100-S24). All the g-negative isolates were screened for ESBL production by reduced zone of inhibition around Ceftazidime (30μg) and confirmed by Combined disc synergy test and were also screened for AmpC ß-lactamase by reduced susceptibility to Cefoxitin (30μg) with zone diameter less than 18 mm and confirmation by Amp C disc test. Isolates of Staphylococcus aureus were screened for MRSA by standard disc diffusion using Cefoxitin (30μg) disc. Detection of inducible Clindamycin resistance was done using a Standard disk diffusion procedure with 15μg Erythromycin and 2μg Clindamycin disks spaced 15-26 mm apart and the formation of D zone. Biofilm production among Pseudomonas aeruginosa and Staphylococcus aureus isolates was tested by the Tube method and Microtiter plate method [9, 12].

The second swab was used for anaerobic culture and was inoculated in RCM broth and incubated at 37 °C for 48-72 h, cultures were then done from RCM on anaerobic blood agar. A Metronidazole disc (5 µg) was placed at the junction of the secondary and tertiary streaking areas. An anaerobic jar (Anaerobic GEN box) was used for anaerobic culture. The jars were closed and incubated at 37 °C for 72 h and thereafter, examined for the zone of inhibition around the Metronidazole disk. An Anaerobic Gas Pack (H2S gas pouch) obtained from from biomerieux, which consisted of a bag containing a paper sachet filled with a black colored powder packet for Oxygen absorbing and Carbon dioxide generating agent was used. Pseudomonas inoculated plates were also used as an anaerobic indicator. The plates were examined for growth, and the organisms were identified by Colony characters, Gram staining, and sensitivity pattern to metronidazole disc. Growth around the Metronidazole disc was subcultured and inoculum suspensions were prepared from isolated colonies on anaerobic blood agar until a turbidity of between 2 to 3 McFarland standards was reached by using a calibrated vitek-2 densichek instrument. Identification of obligate anaerobes was further done by an automated method using a VITEK-2 ANC card [10]. The document M11-A8 was used as a reference for performing anaerobic Antimicrobial Susceptibility Testing (AST). The agar dilution method was performed using agar incorporation of different concentrations of the antimicrobial agent into Brucella agar followed by the application of a standardized number of bacterial cells to the surface of the agar plate. Plates were read after 48 h of growth by visually comparing the growths of different strains in the series, and the Minimum Inhibitory Concentration (MIC) is designated the lowest antimicrobial concentration that inhibits growth. Antimicrobial susceptibility testing was performed against Metronidazole and Clindamycin (HI Media Labs). MIC values were compared with the CLSI Standard for interpretation of results [10, 26] (table 1).

Table 1: Clinical laboratory standards Institute (CLSI) interpretive values for anaerobic bacteria

Antibiotic Susceptible (µg/ml) Intermediate (µg/ml) Resistant (µg/ml)
Metronidazole <8 16 >32
Clindamycin <2 4 >8

The third swab was used for mycological culture and was inoculated onto 2 slopes of Sabouraud dextrose agar with pH 5.6 with antibiotics like Gentamicin to inhibit bacterial growth and incubated at 25 °C and 37 °C for 4-6 w. KOH mount was done using 10% KOH for the presence of budding yeast cells, fungal hyphae, and spores. Tubes showing positive cultures were examined for the rate of growth, texture, surface pigmentation, and pigmentation on the reverse. Microscopic examination was further done by LPCBmount and identified further based on the nature of hyphae (such as septate or aseptate, hyaline, narrow or wide), Conidiogenesis (origin, arrangement), Conidia (pigmented or hyaline, shape). A Germ Tube Test was done for presumptive identification of Candida species into Candida albicans and Non-albicansCandida. FinalCandida speciation and Antifungal susceptibility testing was done using VITEK-2 [11, 14].

RESULTS

A total of 500 patients were included in the study, with 62.8% of the patients being males while 37.2% of patients were females. The highest incidence of CSOM was found to be among patients in the age group of 20-29 y (32.8%) while the lowest incidence was found in patients ≥ 60 y of age (2.8%) (table 2). 400 patients (80%) were outpatients while 100 patients (20%) were inpatients.

Table 2: Age and sex-wise distribution of patients (n=500)

Age (y) Males Females Total Percentage p-value
<10 57 27 84 16.8% <0.0001
10-19 52 32 84 16.8% 0.0021
20-29 100 64 164 32.8% 0.0001
30-39 42 33 75 15% 0.1430
40-49 40 19 59 11.8% <0.0001
50-59 13 7 20 4% 0.0610
≥60 10 4 14 2.8% 0.0291
Total 314 186 500 100% <0.0001

The most common presenting complaint was ear discharge for more than 3 mo in 93% of cases followed by decreased hearing in 66% (fig. 1). Upper respiratory tract infection was noted in 33% of cases and allergic history was noted in 30% of cases. 26% of cases had a history of Modified Radical Mastoidectomy with tympanoplasty (table 3). Tonsillitis and Deviation of Nasal Septum (DNS) were noted in 20% of patients each. Tuberculosis and adenoids were seen in 4% and 2% of cases respectively (fig. 1, table 3).

Fig. 1: Distribution of presenting complaints

Table 3: Diseases associated with CSOM among patients

Associated features Number of cases Percentage (%)
URTI 165 33%
Allergy 150 30%
P/H OF modified radical mastoidectomy 130 26%
Tonsillitis 100 20%
DNS 100 20%
Tuberculosis 20 4%
Adenoids 10 2%

In this study, 550 isolates obtained 355 isolates were obtained in pure growth, 150 isolates (75 samples) were in mixed growth while about 45 isolates were commensals like diptheroids.310gram-negative isolates (56.4%) were obtained which outnumbered g-positive (aerobic and anaerobic) isolates160 (30%). The most common organism isolated was Pseudomonas aeruginosa 200 isolates (39%) followed by Staphylococcus aureus110 isolates (21%). Fungi accounted for 30 isolates (5%) and anaerobes were 5 isolates (1%). In mixed culture growth, the most common isolates were P. aeruginosa and K. pneumoniae (2.8%) followed by P. aeruginosa and E. coli (2.6%). Attico-antral type of CSOM was found in 150 cases (30%) while 350 cases (70%) were diagnosed with a tubo-tympanic type of CSOM (table 4,5,6 fig. 3-5).

Table 4: Culture results from patients with ear discharge (n=500)

Type of organism Total cases Total isolates Percentage
Pure growth 355 355 71%
Mixed growth 75 150(75*2) 15%
No growth 25 0 5%
Commensals (Diptheroids) 45 45 9%
Total 500 550 100%

Fig. 3: Distribution of isolates (n=500)

Fig. 4: Type of CSOM cases (n=500)

Table 5: Organisms isolated from CSOM cases (n=475)

Type of organism Total cases Percentage of cases Total isolates (Pure and mixed)
Pseudomonas aeruginosa 182 39% 200
Staphylococcus aureus 96 21% 110
Coagulase-Negative Staphylococci (CONS) 39 8% 50
Acinetobacter baumannii 9 6% 35
Klebsiella spp. 20 4% 25
E. Coli 15 3% 20
Proteus spp. 10 2% 15
Enterobacter spp. 5 1% 10
Citrobacter spp. 5 1% 5
Candida Spp. 8 2% 10
Aspergillus Spp. 16 3% 20
Anaerobes (Peptostreptococcus) 5 1% 5
Commensals(Diptheroids) 45 9% 45
Total 475 100 % 550

Table 6: Micro-organisms isolated in pure culture (n=355)

Type of organism (pure culture) Total isolates (pure culture) Percentage (pure culture)
Pseudomonas aeruginosa 172 48.45%
Staphylococcus aureus 86 24.22%
CONS 35 9.87%
Acinetobacter baumannii 12 3.38%
Klebsiella Spp. 7 1.97%
E. Coli 7 1.97%
Proteus spp. 5 1.4%
Enterobacter Spp. 5 1.4%
Citrobacter Spp. 4 1.13%
Candida spp. 6 1.7%
Aspergillus Spp. 11 3.09%
Anaerobe (Peptostreptococcus) 5 1.4%
Total 355 100%

Fig. 5: Organisms seen in mixed culture (n=150)

Antibiotic susceptibility pattern in bacterial isolates

Gram-negative bacteria were found to be most sensitive to Piperacillin-tazobactam (96%) followed by Meropenem (94%) and Cefoperazone-Sulbactam (91%) while the highest resistance was noted to Amoxicillin-clavulanate (65%). Among Pseudomonas aeruginosa isolates, the highest sensitivity was noted for Piperacillin-tazobactam (97%) followed by Meropenem(95%), Cefoperazone (93%), and Piperacillin (93%) while only 68% isolates of P. aeruginosa were found to be sensitive to Ciprofloxacin.100% of Staphylococcus aureus isolates were found to be sensitive to Vancomycin, Linezolid, and Teicoplanin. 87% of S. aureus isolates were Cefoxitin sensitive (MSSA) and 13% of S. aureus isolates were found to be Cefoxitin resistant (MRSA). The highest resistance in S. aureus isolates was noted to Ciprofloxacin (65%) followed by Ampicillin (63%) and Amoxicillin-Clavulanate (54%). Among CONS isolates, 100% were sensitive to Linezolid and Vancomycin each, 94% were sensitive to Teichoplanin and 88% were sensitive to Amikacin while 92% isolates were resistant to Penicillin, 65% were resistant to Amoxicillin-Clavulanate and 58% were resistant to Ciprofloxacin. Among the isolates from unsafe CSOM, the highest sensitivity was noted for Piperacillin-Tazobactam (96%) while 95% of isolates from unsafe CSOM were resistant to Amoxicillin-clavulanate.13% isolates of Gram-negative bacteria had ESBL production while Amp C β-Lactamase production was noted in 9% isolates (fig. 6-8).

Biofilm production in pseudomonas aeruginosa and staphylococcus aureus

Out of 186 isolates tested for biofilm production, 149 isolates (80%) showed biofilm production. 50% of biofilm-producing isolates were moderately positive, 47% were weakly positive and 3% were strongly positive.

Out of the moderately positive and strongly positive biofilm producers, 90%(132) isolates were from unsafe CSOM out of which 107(80%) were Pseudomonas aeruginosa while 25 (20%) were Staphylococcus aureus. 87%(22) isolates of Staphylococcus aureus were MRSA while 13% (3) were MSSA. All these patients with unsafe CSOM had a history of recurrent CSOM and were on antibiotic therapy for many years (mean time on antibiotic treatment-8 y). 60 patients (84.5%) with unsafe CSOM with detection of moderately positive or strongly positive biofilm production had undergone Modified Radical Mastoidectomy (MRM) with type 2 tympanoplasty in the past.

Fig. 6: Antibiotic susceptibility pattern of g-negative bacteria (n=310)

Table 7: Antibiotic sensitivity pattern of anaerobic isolates obtained from CSOM cases (n=5)

Antibiotic Range noted(ug/ml) MIC50(ug/ml) MIC90(ug/ml) %Resistance
Metronidazole 0.25-4 0.5 2 0%
Clindamycin <0.25->128 16 >128 40%

Peptostreptococcus showed 100% sensitivity to Metronidazole while Clindamycin was sensitive in 40% of isolates. Table 7, fig. 9 depicts the MIC range.

Fig. 7: Antibiotic sensitivity pattern of pseudomonas aeruginosa (n=200)

Fig. 8: Antibiotic sensitivity pattern of staphylococcus aureus (n=110)

MIC values of Metronidazole

MIC values of Clindamycin

Fig. 9: MIC values of metronidazole and clindamycin

Fungal isolates

A total of 30 fungal isolates were obtained in culture. Aspergillus Sp. Accounted for 66.6% of fungal isolates while Candida Sp. Accounted for 33.3% of fungal isolates. Among Aspergillus spp., Aspergillus Niger accounted for 43.3%, Aspergillus flavus 16.7% and Aspergillus fumigatus6.6%. Among Candida Sp., Candida albicans accounted for 26.7% and Candida tropicalis 6.7% (table 8).

Table 8: Distribution of fungal isolates (n=30)

Fungal isolate Number Percentage
Aspergillusniger 13 43.3%
Aspergillus flavus 5 16.7%
Aspergillus fumigatus 2 6.6%
Candida albicans 8 26.7%
Candida tropicalis 2 6.7%
Total 30 100%

Antifungal sensitivity pattern

The sensitivity of Candida isolates was tested via VITEK-2 to Fluconazole, Itraconazole, Voriconazole, and Flucytosine and they were found to be 100% sensitive to all the antifungals tested.

DISCUSSION

This prospective study, which was conducted on diagnosed cases of CSOM, included a total of 500 patients and 550isolates were obtained. CSOM was found to be more common among males than females with males accounting for 62.8% of cases. This may reflect the overall male predominance of infections due to behavioral differences between males and females.

Analysis of age distribution in our study showed peak incidence of CSOM in the third decade of life with 32.8% cases in the age group of 20-29 y which may be related to socioeconomic factors, lifestyle, and swimming in infected water. The findings of our study correlated with a study done byShreshtha et al. [15], Prakash et al. [6], however had reported peak incidence in the first decade of life. Children tend to have a higher predisposition to ear infections than adults because of the anatomy of the Eustachian tube. Other factors that predispose children to CSOM include attending childcare centers, having a mother who reported a history of purulent ear discharge, having smokers in the household, a high burden of upper respiratory tract infections, and racial factors as stated by Koch et al. [16].

The most common presenting complaint in our study was ear discharge for more than 3 mo (93% cases), followed by decreased hearing (66%). Some of the risk factors noted in our study were upper respiratory tract infection noted in 33% of cases and allergic history noted in 30% of cases. 26% of cases had a history of modified radical mastoidectomy with tympanoplasty.

In the present study, the majority of patients had tubo-tympanic type of CSOM (70%). This is in concordance with the previous study by Poorey et al. and Sharma et al. [17].

Analysis of the bacterial profile of the present study showed that Gram-negative bacilli (56.4%) outnumbered Gram-positive bacteria (30%) which is similar to the study by Malkappa et al. [18]. In contrast, Prakash et al. documented Staphylococcus aureus to be the predominant isolate comprising 48.69% of the total isolates followed by Pseudomonas aeruginosa [6]. These studies depict that the microbial profile varies between different regions, based on patient population and geographical distribution. Pseudomonas aeruginosa was the most isolated organism among cases of unsafe CSOM (51.3%) followed by Staphylococcus aureus (27.3%). Pseudomonas aeruginosa, the predominant cause of CSOM in the tropical region usually does not inhabit the upper respiratory tract, its presence in the middle ear cannot be ascribed to an invasion through the Eustachian tube and it is considered to gain access to the middle ear via a defect in tympanic membrane. Prakash et al. reported coliforms including Klebsiella pneumoniae and Escherichia coli in 9.42% and 7.33% of cases respectively [6]. Frequent isolation of coliforms like E. coli and Klebsiella indicates that individuals are at high risk of acquiring infection when they are exposed to water contaminated with fecal flora while bathing or in activities like swimming. Peptostreptococcus was the anaerobe isolated and accounted for 3.4% of the total isolates isolated in unsafe type of CSOM. Peptostreptococcus remain a significant anaerobic pathogen with reported rates of isolation ranging from 6-7%. However, a study from India found anaerobic etiology in 10.2% of cases. A review study found a minimum of 1.8% and a maximum of 22.4% isolation of anaerobes. The reason of this wide variation may be due to sampling error, prior administration of antibiotics, or differences in the timing of sampling during disease as stated by Aroop Mohanty et al. [27]. Anaerobes accounted for 29.41% of the isolates in a study conducted by Prakash et al. [6]. In the present study, fungi were isolated among 30 patients. Among the fungal isolates, Aspergillus species accounted for 66.7% of isolates and Candida species accounted for 33.3% of isolates. Among Aspergillus sp., Aspergillus niger was the most common isolate (43.3%) followed by Aspergillus flavus (16.7%) and Aspergillus fumigatus (6.7%). Candida albicans was isolated in 8 cases (26.7%) and Candida tropicalis in 2 cases (6.7%). In a study from Haryana, India, fungal etiology was found in 15% of cases, out of which 60% were Candida species 40% were Aspergillus species 74.2% of Aspergillus species, and 19.3% of Candida species among the fungal isolates were reported by Gupta et al. [19].

In the present study, Gram-negative bacteria showed the highest sensitivity to Piperacillin-tazobactam (96%) followed by Meropenem (94%) and Cefoperazone-sulbactam (91%). 70% of isolates of Gram-negative bacteria showed sensitivity to Ceftazidime whereas 83% showed sensitivity to Ceftazidime-clavulanate. 83% of isolates of Gram-negative bacteria were sensitive to Amikacin while 71%showed sensitivity to Ciprofloxacin. Antibiotic susceptibility pattern of P. aeruginosa revealed 97% of the isolates were sensitive to Piperacillin-tazobactam, 95% were sensitive to Meropenem, 93% isolates were sensitive to Piperacillin, 93% to Cefoperazone, 90% to Imipenem, 81% to Amikacin and 68% to Ciprofloxacin. The sensitivity pattern of Pseudomonas aeruginosa correlated with a study done by Lee et al. [20]. Aminoglycosides are bactericidal antibiotics that are frequently used because of their activity against Gram-negative bacteria. In the present study majority of the Gram-negative isolates including Pseudomonas aeruginosa isolates were found to be more sensitive to Amikacin than Gentamicin. Fluoroquinolones inhibit the bacterial DNA gyrase, or the topoisomerase II and they have a broad range of activity against Pseudomonas aeruginosa. In the present study, 71% of Gram-negative isolates and 68% of P. aeruginosa were found to be sensitive to Ciprofloxacin which may be due to the frequent use of topical and intravenous formulations of fluoroquinolones and is similar to the sensitivity reported by Kumar S et al. Ceftazidime is the most frequently prescribed third-generation cephalosporin that has an extended Gram-negative spectrum [4]. However, resistance to Ceftazidime is increasing, complicating the management of patients with such isolates. In the present study, 70% of isolates of Gram-negative bacteria were sensitive to Ceftazidime which is in concordance with the study conducted by Mahajan et al. who reported 84% sensitivity to Ceftazidime in Gram-negative bacteria [21]. Ceftazidime resistance is mainly mediated by the production of β-lactamases such as ESBL, MBL, and occasionally AmpC-β-lactamases. In the present study out of 310 isolates of Gram-negative bacteria, 13% were found to be positive for ESBL production, and 9% of the Gram-negative isolates were observed to be Amp C producers in our study. This is in concordance with the studies of Aggarwal et al. who documented ESBL production of about 21% [22]. ESBL-producing organisms are frequently resistant to other classes of antibiotics, including aminoglycosides and fluoroquinolones due to the coexistence of genes encoding drug resistance to other antibiotics on the plasmids which encode ESBL. The second most isolated organism in our study was Staphylococcus aureus comprising 110 (22%) of the isolates.87% of Staphylococcus aureus were found to be MSSA whereas 13% were found to be MRSA.100% isolates of Staphylococcus aureus were sensitive to Vancomycin, Linezolid, and Teicoplanin. 65% of Staphylococcus aureus isolates were resistant to Ciprofloxacin, 63% were resistant to Ampicillin and 54% were resistant to Amoxicillin-Clavulanate. This indicates a high level of resistance among Staphylococcus aureus for β-Lactams and Fluoroquinolones. In a study done by Prakash et al. out of 181 isolates, 48% of isolates were Staphylococcus aureus, and all were found to be methicillin-sensitive [6].

In our study, Escherichia coli showed 80% sensitivity to Amikacin and 20% sensitivity to ciprofloxacin. The sensitivity of Proteus mirabilis was good with Amikacin and Gentamicin. Klebsiella pneumoniae showed 85% sensitivity with Amikacin and 57% sensitivity with both Ciprofloxacin and Gentamicin. Among NFGNB isolates, 100% sensitivity was noted in Amikacin, Gentamycin, Ciprofloxacin, and Piperacillin-Tazobactam while 89% of isolates were sensitive to Piperacillin.

In the present study, all 5 isolates of Peptostreptococcus showed 100% sensitivity to Metronidazole while Clindamycin was sensitive in 40% of isolates. However, few studies have shown varying rates of Metronidazole resistance. Padmaja et al. 25 reported resistance rates of 5.5% against Metronidazole among Peptostreptococcus spp. Clindamycin, a lincosamide antibiotic has a good spectrum of activity against most of the Gram-positive anaerobic organisms and our study showed higher rates of resistance of 40% to Clindamycin. There are reports of emerging resistance against Clindamycin among anaerobes as stated by Padmaja et al. and Brook et al. [23, 26].

In our study, we tested 186 isolates for biofilm production out of which 149 isolates (80%) showed biofilm production. 50% of biofilm-producing isolates were moderately positive, 47% were weakly positive and 3% were strongly positive. Out of the moderately positive and strongly positive biofilm producers,(132) 90% of isolates were from unsafe CSOM out of which 107(80%) were Pseudomonas aeruginosa while 25 (20%) were Staphylococcus aureus. 87% (22) isolates of Staphylococcus aureus were MRSA while 13% (3) were MSSA. Host immune responses to planktonic species have been relatively well characterized and how they modulate anti-bacterial effector mechanisms when organized in this protective milieu as stated by Hank et al. [24]. Biofilms act as non-selective physical barriers that obstruct antibiotic diffusion and hinder the cellular and humoral immune. They have a characteristic physiology and architecture that form the basis of biofilm resistance to many antibiotics and mechanisms of host defense as stated by Stewart et al. [12]. Bacteria may communicate with each other through diffusible molecules within the milieu of a biofilm through a mechanism known as quorum sensing as per Li and Tian. [25]An epidemiological knowledge of the prevalence of biofilm production in CSOM is necessary as biofilm production may account for the recurrence of CSOM after treatment, failure of surgery, and resistance to antibiotic therapy. All the patients with unsafe CSOM in our study had a history of recurrent CSOM and were on antibiotic therapy for many years (mean time on antibiotic treatment – 8 yrs.). 60 patients (84.5%) with unsafe CSOM with detection of moderately positive or strongly positive biofilm production had undergone MRM with type 2 tympanoplasty in the past.

The study had a few limitations. We could not perform the antifungal susceptibility testing for molds in our study. Also, the antibiotic sensitivity profile of anaerobes could not be evaluated more extensively.

CONCLUSION

CSOM is generally treated by oral medications with Fluoroquinolone antibiotic drops, the absence of oral formulations of Cephalosporins and Carbapenems which limited the use of these antibiotics in patients with CSOM otorrhea in a past era, resulted in less resistance however there has been a recent trend in increasing resistance to beta-lactams due to production of ESBL and Amp C beta-lactamases. Isolation rate of MRSA has also seen an upward trend. The emergence of multi-drug-resistant organisms may complicate patient management. The formation of biofilm by certain bacteria may explain recurrence and resistance patterns in unsafe CSOM and post-surgical procedures like modified radical mastoidectomy with tympanoplasty creating surfaces for biofilm to develop. CSOM often has polymicrobial pathology creating war in the middle ear environment, augmentation of pathology being created by mixed aerobic and anaerobic organisms, and synergism between aerobic and anaerobic organisms has already been recognized. A high rate of Clindamycin resistance was seen in anaerobes which is noteworthy because this is a frequently utilized antimicrobial for treating anaerobic infections. Periodic surveillance of the antimicrobial susceptibility profile of bacteria is the need of the hour to guide empiric antibiotic therapy and to formulate local antibiotic policy.

FUNDING

Nil

AUTHORS CONTRIBUTIONS

Dr Priya Bhat collected the data performed analysis of samples and prepared the manuscript. Dr Upasana Bhumbla supervised the methodology of the study and was involved in the analysis of the data. Dr Jasleen Kaur was involved in the preparation of the manuscript and data analysis. All the authors approved the final manuscript.

CONFLICT OF INTERESTS

The authors have no conflicts of interest regarding this investigation.

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