Comparative study of biofilm formation in tubotympanic and atticoantral ear disease

INTRODUCTION

Biofilms are organized bacterial communities that may be homogeneous or heterogeneous. Biofilm is a vicious layer that adheres to the inflamed tissue surfaces like mucosal lining or osteitic bone and they play a significant role in the pathogenesis of chronic rhinosinusitis, chronic tonsillitis, chronic suppurative otitis media and develop device related infections. Despite this clinical importance, only a limited number of studies have been done to examine the presence of biofilms in tissues from patients with different types of middle ear infections, COM of tubotympanic type or cholesteatomas.¹

Etiology of COM is multifactorial. Bacterial infection is a major factor in inflammation that lasts longer in COM. Bacteria that are involved in the etiogenesis of COM like

P. aeruginosa, S. aureus, gram positive and negative strains have the ability to a secrete polysaccharide exopolymer which form a glycocalyx matrix. This matrix along with the bacterial colony together forms the biofilm.² Bacterial biofilms have some defense abilities towards an unfavorable environment like resistance to antibiotics. Bacterial biofilm in COM can provide signals that result in increased exudation of fluid from epithelial surface, turnover rate of epithelial cells and keratin production. This accelerates the formation of cholesteatoma and/or local inflammation in COM, resulting in more complications and difficulty in treatment.^2-4 Management aimed at surgical debridement and cleaning up the pathologic tissue is an effective standard treatment.⁵

The aim of our study was to determine the formation of biofilm in COM and to compare the pattern of biofilm formation between tubotympanic and atticoantral COM.

METHODS

This was a prospective cross sectional study done in the patients of COM presenting for treatment to the department of otorhinolaryngology, institute of medical sciences, Banaras Hindu university, Uttar Pradesh between December 2018 to March 2020. Sample size taken was a total of 60 patients of COM (divided into 2 groups: group 1 had 30 patients of atticoantral disease and group 2 had 30 patients of tubotympanic disease.

Inclusion criteria

Patients of active COM aged between 10‐65 years irrespective of gender, socio-economic status who consented to participate in the study were included.

Exclusion criteria

Patients with pregnancy, malignancy, tuberculosis were excluded.

Pus sample was collected with sterile swab from the external auditory canal in tubotympanic COM cases. In atticoantral COM cases tissue from periphery of cholesteatoma site was collected intra-operatively in sterile vials containing phosphate buffered solution (pH 7.2).

All samples were sent to the microbiology department of the institute immediately after collection.

Isolation and characterization of aerobic bacteria

The collected samples were directly inoculated onto MacConkey and blood agar media. The plates were kept for overnight incubation at 37°C. Post incubation their colony morphology and cultural characteristics were observed post-incubation. Further phenotypic identification was done through gram staining and biochemical reaction. The pure isolated colonies were stocked in peptone medium and stored at room temperature for further processing.

Detection of biofilm formation

To assess biofilm formation, stocked bacterial isolates were subcultured onto MacConkey and blood agar media and the plates were kept for overnight incubation at 37°C. The isolated pure colonies were suspended into normal saline and the suspension was adjusted according to 0.5 McFarland ̴1.5×108 CFU/ml. In 96 well microtiter plate, 190 μl Luria Bertani broth media and 10 μl of a bacterial suspension was added. The plate was overnight incubated at 37°C. Following day, the wells were rinsed with sterile phosphate buffer saline and air dried. Each well was stained with 100 μl of 0.1% crystal violet. Excess stain in the well was rinsed with 95% ethanol followed by 20 min incubation at room temperature. The optical density at 578 nm (OD578) of each well was then measured using LisaScan® EM micro-titter plate reader. For the determination of biofilm formation OD value of standardized low, moderate and high biofilm producing S. aureus strains were taken a reference, while well containing only media were used as negative control. Reference strain OD578=low biofilms 0.040, moderate biofilms 0.060, high biofilms 0.091.

Statistical analysis was performed using statistical package for the social sciences (SPSS), version 23.0. IBM Corp., NY. The data was analyzed using Chi square test and Fisher’s exact test.

RESULTS

The following information were collected after going through clinical examination, surgical intervention, bacterial culture and biofilm formation. The following observations were made.

In our study, we observed that, in cases of atticoantral type of COM, most predominant bacteria found were P. aeruginosa and S. aureus equally as both amounted to 26.7%, followed by M. catarrhalis amounting to 16.7%, P. mirabilis amounting to 10% and Enterococcus to 3.3% and 10% cases had no bacterial growth (Figure 1).

In cases of tubotympanic type of COM, most predominant bacteria found was S. aureus amounting to 33.3%, followed by M. catarrhalis (13.3%), P. aeruginosa and P. mirabalis both amounting to 10% and 30% cases had no bacterial growth (Table 1).

Since p=0.308 there is no significant correlation between bacterial growth in our study groups.

In our study, we observed that, in cases with atticoantral type, 83.3% patients presented with biofilm formation whereas in cases with tubotympanic type, 60% patients presented with biofilm formation (Figure 2).

Since p=0.044, hence there was a significant correlation between the two groups of COM.

DISCUSSION

In our study, we observed that, in cases of atticoantral type of COM, the most predominant bacteria found were P. aeruginosa and S. aureus both amounting to 26.7%, followed by M. catarrhalis amounting to 16.7%, P. mirabilis amounting to 10% and Enterococcus to 3.3%, and 10% cases had no bacterial growth.

In our study, we observed that, in cases of tubotympanic type of COM, the most predominant bacteria found were S. aureus amounting to 33.3% , followed by M. catarrhalis amounting to 13.3%, and then P. aeruginosa and P. mirabalis both amounting to 10% and 30% cases had no bacterial growth. Since p=0.308 there is no significant difference between bacterial growth in our study group.

In majority of the studies, P. aeruginosa was the most commonly recovered organism from the chronically draining ear. Various researchers over the past few decades have recovered pseudomonas from 48‐98% of patients with CSOM. Also some studies have shown that S. aureus is the second most common organism isolated from chronically diseased middle ears. Reported data estimate infection rates from 15‐30% of culture‐positive draining ears.

The remainder of infections is caused by a large variety of gram negative organisms. Klebsiella (10‐21%) and Proteus (10‐15%) species are slightly more common than other gram negative organisms. Sivarajan et al (1991), conducted a study on the bacteriological profile in 146 cases of CSOM and found that Pseudomonas was the highest followed by Staphylococcus and the Proteus.⁶

In our study, we found out that in cases with atticoantral type of COM, on bacterial culture, the most common organism were P. aeruginosa, S. aureus followed by M. catarrhalis whereas in cases with tubotympanic COM, most common organism was S. aureus. In cases with atticoantral type of COM 10% specimen culture were sterile whereas in cases with tubotympanic type of COM 30% cases were sterile.

In our study, we observed that, in cases with atticoantral type, 83.3% patients presented with biofilm formation whereas in cases with tubotympanic type, 60% patients presented with biofilm formation. Since p=0.044, hence there was a significant difference between the two groups of COM, with biofilm formation being more common in atticoantral type of COM.

In the study by Romling et al almost all bacteria can be found as part of a biofilm. Some bacteria like S. aureus, P. aeruginosa and E. coli are more likely to be found in a biofilm community than others.⁷

In the study by Gu et al (2014) bacterial biofilm were found in 85%, 92%, 16% of patient with middle ear cholesteatoma, COM and tympanic membrane perforation, respectively.¹

Artono et al conducted a study using scanning electron microscopy (SEM) with 33 patients and divided into 16 unsafe type and 17 patient safe type, the test result showed that in unsafe type CSOM 12 (75%) was positive with biofilm bacteria and 4 (25%) negative bacteria biofilm. Safe type CSOM showed 6 (35.35%) with positive biofilm bacteria and 11 (67.9%) negative biofilm bacteria.⁵

Lampikoski et al conducted a study using confocal scanning laser microscopy (CSLM) of the 17 chlesteatoma patient, mastoid mucosal biofilm formation could be seen in 14 cases (82%). In CSOM group, 5 (42%) of 12 patient showed mastoid mucosal biofilm. In control group (patient undergoing cochlear implantation, with no history of COM), 1 (9%) patient of 11 showed mastoid mucosal biofilm.⁸

Kaya et al (2013) showed 7 out of 10 CSOM patient had biofilm (70%), 6 of the 11 groups of non-suppurative COM showed biofilm (54.5%). Cholesteatoma group showed biofilm in 8 of 13 patient (61.5%).⁹

In our study, in cases with atticoantral type of COM biofilm formation was found to be significantly more common as compared to tubotympanic type of COM. A long-term follow up after surgical intervention could yield more information about relationship of biofilm and treatment outcome, which was the major limitation of this study.

CONCLUSION

In our study, we found out that in cases with atticoantral type of COM, on bacterial culture, the most common organism was P. aeruginosa and S. aureus followed by M. catarrhalis whereas in cases with tubotympanic COM, most common organism was S. aureus. We found that, in atticoantral type of COM biofilm formation was significantly more common as compared to that in tubotympanic type of COM. Therefore, we can conclude that there is a significant correlation in biofilm formation between atticoantral and tubotympanic type of COM. Biofilm formation in COM is even higher when atticoantral disease is present.

Funding: No funding sources

Conflict of interest: None declared

Ethical approval: The study was approved by the Institutional Ethics Committee

REFERENCES

1. 1. Gu X, Keyoumu Y, Long L, Zhang H. Detection of bacterial biofilms in different typs of chronic otitis media. Eur Arch Otorhinolaryngol. 2014;271(11):2877-83.
2. 2. Hall-Stoodley L, Costerton JW, Stoodley P. Bacterial biofilm: from natural envirnment to infectious disease. Nat Rev Microbiol. 2004;2(2):95-108.
3. 3. Drenkard E. Antimicrobial resistance of Pseudomonas aeruginosa biofilm. Microbes Infect. 2003;5(13):1213-9.
4. 4. Morris DP, Hagr A. Biofilm: why the sudden interest. J Otolaryngol. 2005;34(2):56-9.
5. 5. Artono A, Purnami N, Rahmawati R. Biofilm bacteria plays a role in CSOM pathogenesis and has significant correlation with unsafe type CSOM. FMI. 2015;51(4).
6. 6. Nandy A, Mallya PS, Sivarajan K. Chronic suppurative otitis media: a bacteriological study. Indian J Otolaryngol. 1991;43(3):136-8.
7. 7. Romling U, Balsalobre C. Biofilm infections, their resilience to therapy and innovative treatment strategies. J Intern Med. 2012;272(6):541-61.
8. 8. Lampikoski H, Aarnisalo AA, Jero J, Teemu J. Kinnari department of otorhinolaryngology head and neck surgery, Helsinki university central hospital, Helsinki, Finland. Otol Neurotol. 2012;33:785-8.
9. 9. Kaya E, Dag I, Incesulu A, Gurbuz MK, Acar M, Birdane L. Investigation of the presence of biofilm in chronic suppurative otitis media, nonsuppurative otitis media, and chronic otitis media with choleateatoma by scanning electron microscopy. Scient World J. 2013:1-6.