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 ~  Abstract
 ~ Introduction
 ~ Methodology
 ~ Results
 ~ Discussion
 ~ Conclusion
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  Table of Contents  
Year : 2019  |  Volume : 37  |  Issue : 3  |  Page : 442-445

Determination of Biofilm-Forming Capacity of Otopathogens Isolated from Discharging Ears in Children with Chronic Otitis Media

1 Department of ENT, Christian Medical College, Vellore, Tamil Nadu, India
2 Department of Clinical Microbiology, Christian Medical College, Vellore, Tamil Nadu, India

Date of Submission14-Nov-2019
Date of Decision25-Nov-2019
Date of Acceptance24-Dec-2019
Date of Web Publication29-Jan-2020

Correspondence Address:
Dr. P Naina
Department of ENT, Christian Medical College, Vellore - 632 004, Tamil Nadu
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/ijmm.IJMM_19_404

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 ~ Abstract 

Chronic otitis media is a common disease of the developing world with persistent ear discharge, leading to major complications. This study describes the microorganisms isolated from the middle ear and nasopharynx of children with chronically discharging ears. Middle ear and nasopharyngeal swabs from 89 children were studied, and the microorganisms isolated were assessed for biofilm-forming ability. Methicillin-susceptible Staphylococcus aureus was common in the nasopharynx, while the middle ear showed predominantly pseudomonas and Methicillin-resistant S. aureus. Pseudomonas aeruginosa showed strong biofilm formation, whereas Escherichia coli, Proteus sp. and Providentia sp. were weak biofilm producers. S. aureus isolates were negative for biofilm formation.

Keywords: Chronic otitis media, ear discharge, nasopharynx, otopathogens, Pseudomonas aeruginosa, Staphylococcus aureus

How to cite this article:
Ralte Z, Naina P, Amladi A, John M, Anndan S, Varghese AM. Determination of Biofilm-Forming Capacity of Otopathogens Isolated from Discharging Ears in Children with Chronic Otitis Media. Indian J Med Microbiol 2019;37:442-5

How to cite this URL:
Ralte Z, Naina P, Amladi A, John M, Anndan S, Varghese AM. Determination of Biofilm-Forming Capacity of Otopathogens Isolated from Discharging Ears in Children with Chronic Otitis Media. Indian J Med Microbiol [serial online] 2019 [cited 2020 Oct 25];37:442-5. Available from:

 ~ Introduction Top

Chronic otitis media (COM), a common problem among children worldwide, has a high prevalence of 7%–8% in developing countries like India.[1],[2] It is associated with long-term sequel such as hearing loss, affecting the overall development of a child. Various clinical and microbiological risk factors, including the role of nasopharyngeal microorganisms, have been implicated in the etiopathogenesis of COM.[2],[3] Microbiological factors, especially the role of biofilms, have been studied widely to explain the persistence of ear discharge despite appropriate antibiotics.[4],[5],[6],[7],[8] Keeping in mind the paucity of literature on this aspect from India, this prospective study was conducted to describe the profile of microorganisms in the nasopharynx and middle ear of children with chronically discharging ears. The study also analysed the biofilm-forming capacity of these organisms.

 ~ Methodology Top

This study was conducted in Christian Medical College, Vellore, a tertiary care centre in Southern India after approval from the Institutional Review Board (IRB No 9348) between 2015 and 2017. The inclusion criterion was children aged below 14 years with ear discharge secondary to COM of at least 12 weeks' duration being planned for tympanoplasty or mastoidectomy. They were excluded if they were immunocompromised or had any craniofacial anomalies. After obtaining informed consent, a thorough history was asked and detailed clinical examination was performed and COM was categorised as mucosal and squamous based on the otoscopic findings.[9]

Specimen collection and processing

Using proper aseptic precautions, a middle ear swab was collected using a sterile flocked swab. If inflamed mucosa was seen, a cupped forceps was used to take a tissue sample from the middle ear mucosa which was placed in saline containing sterile tube and transported immediately to the laboratory for culture. Concurrently, a nasopharyngeal swab was also taken in a similar manner. The tissue samples were ground, centrifuged and inoculated onto blood agar, chocolate agar, MacConkey agar and thioglycolate broth, incubated at 37°C overnight and at studied at 24 and 48 h. Gram-stain smears from the specimen were evaluated. Any growth on the culture media was further subjected to biochemical tests and identified up to species level (Practical Medical Microbiology, Mackie and Macartney).[10]


The inoculum was prepared from the bacterial cultures grown overnight on blood agar plates and the inoculum was set at 0.5 MacFarland standards. 100 μl of inoculum was pipetted in each well of 96-well U-bottom plate, and the peg plate [Figure 1] was placed over it and incubated overnight at 37°C for 24 h. After incubation, the peg plate was removed from the 96-well U-bottom plates and gently submerged in a small tray of 0.9% saline and was gently agitated. This process was repeated twice to remove unattached cells and media components to reduce background staining. One hundred and twenty-five microlitre of a 0.1% solution of crystal violet was added to each well of the 96-well microtitre plate and peg plate was placed over it for 10–15 min. The peg plates were rinsed 3–4 times with water by submerging in a tray of water, shake out and blotted vigorously on a stack of paper towels to get rid of the excess cells and dye. The peg plate was turned upside down and dried for a few hours or overnight at room temperature. Then, 125 μL of 30% acetic acid in water was added to each well of the 96 well plates, and peg plate was placed over it for 10–15 min to solubilise the coefficient of variation (CV). Finally, the peg plate was removed, and the microtitre plate which has the solubilised CV was read at 595 nm in a spectrophotometer (Scientific Multiskan GO).
Figure 1: Crystal violet stained peg plate showing formation of biofilm

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The biofilm formation (BF) was calculated using the formula: BF = AB − CW, where AB represents the optical density at 595 nm (OD595 nm) of a well-containing stained attached bacteria and CW, the OD595 nm of the stained control wells-containing bacteria-free medium. The samples were classified into four different groups as follows: strong: ≥0.300; moderate: 0.22–0.299; weak: 0.100–0.199 and negative: ≤0.100.

 ~ Results Top

A total of 89 children (65 males and 24 females) were recruited. Among them, 50 children had squamous disease, whereas 39 had mucosal disease. The number of episodes of ear discharge was found to be 7.3/year on an average. The average duration of days from the last administration of topical antibiotics was 79 days.

The distribution of microorganisms isolated in cultures from the nasopharynx and ear of patients with mucosal and squamosal disease is shown in [Figure 2]. Among the organisms isolated from the nasopharynx, methicillin-susceptible Staphylococcus aureus (82.1%) predominated followed by Haemophilus sp (15.8%). The cultures from the nasopharynx showed a higher percentage of positivity as compared to the ear. The nasopharyngeal colonisation was correlated with the age of the child. However, no statistically significant correlation was noted across different age groups in our study (P = 0.949). In the cultures from the ear, the most common organism was Pseudomonas sp. (50%), followed by Methicillin-resistant S. aureus (43.1%). On the correlation of the ear culture with the duration of COM, no statistically significant correlation was noted (P = 0.649). A total of 11 out of 39 children with mucosal disease and 25 among 50 children with squamous disease had a positive culture from both the middle ear and the nasopharynx. Of these 36 children, 14 children had the same organism isolated from both the sites. The most common organism found both in the middle ear and nasopharynx was S. aureus (8, 57%). The age distribution of children with the same organism isolated from the ear and nasopharynx was analysed. It was found that, except in the early age group, S. aureus was the most common organism colonising the ear and the nasopharynx together.
Figure 2: Distribution of microorganisms isolated in cultures from the nasopharynx and ear of patients with mucosal and squamosal disease

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Biofilm formation

The isolates of the common organism found in the nasopharynx and ear were evaluated for BF capacity. Twenty isolates were selected (non-fermenting Gram-negative bacteria [NFGNB] [n = 5], Pseudomonas aeruginosa [n = 2], S. aureus [n = 10] and  Escherichia More Details coli and Proveidentia and Proteus sp. [n = 1 each]). Biofilm-forming potential of the isolates was calculated as per the standard cutoffs. Accordingly, P. aeruginosa showed strong BF. NFGNB showed strong-to-moderate BF, whereas E. coli, Proteus sp. and Providentia sp. formed weak biofilm. S. aureus isolates were negative for BF [Figure 3].
Figure 3: Depicting biofilm formation efficiency of the study isolates in three categories, weak, moderate and strong biofilm producers. (SA: Staphylococcus aureus, PR: Providencia spp, EC: Escherichia coli, PT: Proteus mirabilis, PS: Pseudomonas aeruginosa, NFGNB: Non-fermenting Gram-negative bacteria)

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 ~ Discussion Top

COM is a common disorder in developing countries. Although the role of various epidemiological factors has been well studied, the role of microbiological factors such as colonisation by otopathogens (Streptococcus pneumoniae,  Moraxella More Details Catarrhalis and Haemophilus Influenza) and role of biofilms remains to be elucidated. In this study, S. aureus predominated in the nasopharynx followed by coagulase-negative staphylococci and Haemophilus parainfluenzae. S. aureus was also the most common organism colonising the ear and the nasopharynx together, the prevalence of which increased with the child's age in this study. Asymptomatic colonisation of the nasopharynx by S. pneumoniae and H. influenza can be a common occurrence in the younger age group.[11],[12] Children with otitis media are known to have a higher prevalence of otopathogens in the nose and nasopharynx, especially with more than one otopathogen.[13]S. aureus is another common otopathogen more frequently isolated in COM in middle ear discharge and less commonly in the nasopharynx.[13]

P. aeruginosa predominated in the middle ear cultures followed by S. aureus. With the loss of natural barriers of protection to the middle ear in COM, there is exposure to organisms from the surrounding, such as water entry into the ear facilitating the growth of P. aeruginosa. Pseudomonas is a sturdy organism with minimal growth requirement and thrives well in a humid environment surviving on exfoliated epithelium, such as in COM. It employs various strategies to survive and cause infection such as virulence mediators, resistance to quinolones and survival inside macrophages.[14],[15],[16]

The association between organisms in the ear and the nasopharynx in acute otitis media (AOM) and COM has been studied by many authors. While in AOM, this has been found to be significant, in COM no association has been determined.[13] This study also did not show any significant association. In COM, the middle ear profile consisted of mainly of Pseudomonas and Staph sp.

Recent studies have suggested the factors such as increased virulence of the pathogens, role of resistant organisms, reinfection with new organisms and biofilms formation for the persistence of disease.[9],[16] The role of biofilms in AOM is well described with biofilms found in the middle ear and nasopharynx tissue.[17] Dispersal of bacteria form a biofilm in the middle ear, serving as a bacterial reservoir may explain the recurrent and chronic nature of COM. The first evidence of bacterial mRNA in otitis media was by Rayner et al.[18] Presence of biofilm in the middle ear was first shown in an animal model and subsequently demonstrated described in the works of various authors.[6],[7],[8] Gu et al found bacterial biofilms in 92% and 85% of patients with COM and cholesteatoma, respectively.[5] In this study, P. aeruginosa, the most common pathogen in the ear, was also found to be a good biofilm producer. This explains why it overgrows other organisms and is found abundantly despite treatment.

The detection of biofilms is challenging for the microbiologists and the clinicians. Many methods have been successfully employed, but cost and availability limit their use in resource-poor countries. Most studies rely on methods for direct visualisation of biofilms which is an expensive and time-consuming process.[19] This study used a novel and inexpensive method. P. aeruginosa and NFGNB showed strong BF, whereas S. aureus did not show significant tendency for BF.

 ~ Conclusion Top

This study adds to the information on the common pathogens-forming biofilms in otitis media. Knowledge that a particular organism has a higher biofilm-forming capacity will help to sensitise the physician that factors such as biofilms may be at play and take appropriate measures. Future therapies for COM are more likely to be targeting the removal of biofilms by mechanical or enzymatic dispersal.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.

 ~ References Top

Sophia A, Isaac R, Rebekah G, Brahmadathan K, Rupa V. Risk factors for otitis media among preschool, rural Indian children. Int J Pediatr Otorhinolaryngol 2010;74:677-83.  Back to cited text no. 1
Mukara KB, Lilford RJ, Tucci DL, Waiswa P. Prevalence of middle ear infections and associated risk factors in children under 5 years in Gasabo district of Kigali city, Rwanda. Int J Pediatr 2017;2017:1-8.  Back to cited text no. 2
Wang J, Chen B, Xu M, Wu J, Wang T, Zhao J, et al. Etiological factors associated with chronic suppurative otitis media in a population of Han adults in China. Acta Otolaryngol 2016;136:1024-8.  Back to cited text no. 3
Jensen RG, Johansen HK, Bjarnsholt T, Eickhardt-Sørensen SR, Homøe P. Recurrent otorrhea in chronic suppurative otitis media: Is biofilm the missing link? Eur Arch Otorhinolaryngol 2017;274:2741-7.  Back to cited text no. 4
Gu X, Keyoumu Y, Long L, Zhang H. Detection of bacterial biofilms in different types of chronic otitis media. Eur Arch Otorhinolaryngol 2014;271:2877-83.  Back to cited text no. 5
Ehrlich GD, Veeh R, Wang X, Costerton JW, Hayes JD, Hu FZ, et al. Mucosal biofilm formation on middle-ear mucosa in the chinchilla model of otitis media. JAMA 2002;287:1710-5.  Back to cited text no. 6
Dohar JE, Hebda PA, Veeh R, Awad M, Costerton JW, Hayes J, et al. Mucosal biofilm formation on middle-ear mucosa in a nonhuman primate model of chronic suppurative otitis media. Laryngoscope 2005;115:1469-72.  Back to cited text no. 7
Wessman M, Bjarnsholt T, Eickhardt-Sørensen SR, Johansen HK, Homøe P. Mucosal biofilm detection in chronic otitis media: A study of middle ear biopsies from Greenlandic patients. Eur Arch Otorhinolaryngol 2015;272:1079-85.  Back to cited text no. 8
Minami SB, Mutai H, Suzuki T, Horii A, Oishi N, Wasano K, et al. Microbiomes of the normal middle ear and ears with chronic otitis media. Laryngoscope 2017;127:E371-7.  Back to cited text no. 9
Gold HS, Moellering RC, Berman JD, King M, Edwards N, Omar MT, Jackman AL, Boyle FT, Harrap KR, Borst P, Quellette M. Mackie and McCartney, Practical Medical Microbiology. J Biol Sci 1996;4:1445-53.  Back to cited text no. 10
Kosikowska U, Korona-Głowniak I, Niedzielski A, Malm A. Nasopharyngeal and adenoid colonization by Haemophilus influenzae and Haemophilus parainfluenzae in children undergoing adenoidectomy and the ability of bacterial isolates to biofilm production. Medicine (Baltimore) 2015;94:e799.  Back to cited text no. 11
Raman R, Sankar J, Putlibai S, Raghavan V. Demographic profile of healthy children with nasopharyngeal colonisation of Streptococcus pneumoniae: A research paper. Indian J Med Microbiol 2017;35:607-9.  Back to cited text no. 12
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Coleman A, Wood A, Bialasiewicz S, Ware RS, Marsh RL, Cervin A. The unsolved problem of otitis media in indigenous populations: A systematic review of upper respiratory and middle ear microbiology in indigenous children with otitis media. Microbiome 2018;6:199.  Back to cited text no. 13
Mittal R, Parrish JM, Soni M, Mittal J, Mathee K. Microbial otitis media: Recent advancements in treatment, current challenges and opportunities. J Med Microbiol 2018;67:1417-25.  Back to cited text no. 14
Kim SH, Kim MG, Kim SS, Cha SH, Yeo SG. Change in detection rate of methicillin-resistant Staphylococcus aureus and Pseudomonas aeruginosa and their antibiotic sensitivities in patients with chronic suppurative otitis media. J Int Adv Otol 2015;11:151-6.  Back to cited text no. 15
Byrd MS, Pang B, Hong W, Waligora EA, Juneau RA, Armbruster CE, et al. Direct evaluation of Pseudomonas aeruginosa biofilm mediators in a chronic infection model. Infect Immun 2011;79:3087-95.  Back to cited text no. 16
Thornton RB, Rigby PJ, Wiertsema SP, Filion P, Langlands J, Coates HL, et al. Multi-species bacterial biofilm and intracellular infection in otitis media. BMC Pediatr 2011;11:94.  Back to cited text no. 17
Rayner MG, Zhang Y, Gorry MC, Chen Y, Post JC, Ehrlich GD. Evidence of bacterial metabolic activity in culture-negative otitis media with effusion. JAMA 1998;279:296-9.  Back to cited text no. 18
Vermee Q, Cohen R, Hays C, Varon E, Bonacorsi S, Bechet S, et al. Biofilm production by Haemophilus influenzae and Streptococcus pneumoniae isolated from the nasopharynx of children with acute otitis media. BMC Infect Dis 2019;19:44.  Back to cited text no. 19


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