|Year : 2020 | Volume
| Issue : 2 | Page : 75-79
Biofilm-producing organisms and their antibiotic sensitivity in chronic otitis media – Mucosal disease
Rajeshwary Aroor1, Naina Narasimhadevara1, Pratibha Bhat2, Vadisha Bhat1, Marina Saldanha1, MK Goutham1
1 Department of Otorhinolaryngology (ENT), K. S. Hegde Medical Academy, Mangalore, Karnataka, India
2 Department of Microbiology, K. S. Hegde Medical Academy, Mangalore, Karnataka, India
|Date of Submission||05-Jan-2020|
|Date of Acceptance||17-Feb-2020|
|Date of Web Publication||17-Jul-2020|
Dr. Naina Narasimhadevara
Department of ENT, K. S. Hegde Medical Academy, Derelakatte, Mangalore - 575018, Karnataka
Source of Support: None, Conflict of Interest: None
Objectives: The objective of the study is to know the presence of biofilm-producing organisms in the middle ear mucosa in chronic otitis media (COM) mucosal disease and their antibiotic sensitivity. Materials and Methods: This was a prospective comparative study conducted on 100 patients who underwent tympanoplasty. They were divided into two groups: Group 1 included mucosal active disease (50) and Group 2 included mucosal inactive (50). During surgery, a biopsy from the middle ear mucosa was taken and sent for culture and sensitivity and tested for their biofilm-forming capability using tissue culture plate method. Results: In Group 1, 17 cultures were biofilm-producing organisms and 14 were non/weak biofilm-producing organisms. In Group 2, 14 were biofilm producing and 8 were non/weak biofilm producing. Piperacillin-tazobactam was the most sensitive drug for Pseudomonas aeruginosa, Klebsiella pneumoniae, and Staphylococcus epidermidis irrespective of their biofilm-producing capability. Linezolid had showed 100% sensitivity for Staphylococcus aureus. Ciprofloxacin was the most resistant drug irrespective of organisms and their biofilm-forming capability.Conclusion: P. aeruginosa and S. aureus are the common organisms present in the mucosa of the middle ear in COM. Piperacillin-tazobactam is the most sensitive drug for middle ear infection. The most resistant drug is ciprofloxacin.
Keywords: Biofilms, chronic otitis media, middle ear mucosa, mucosal disease
|How to cite this article:|
Aroor R, Narasimhadevara N, Bhat P, Bhat V, Saldanha M, Goutham M K. Biofilm-producing organisms and their antibiotic sensitivity in chronic otitis media – Mucosal disease. Indian J Otol 2020;26:75-9
|How to cite this URL:|
Aroor R, Narasimhadevara N, Bhat P, Bhat V, Saldanha M, Goutham M K. Biofilm-producing organisms and their antibiotic sensitivity in chronic otitis media – Mucosal disease. Indian J Otol [serial online] 2020 [cited 2020 Oct 31];26:75-9. Available from: https://www.indianjotol.org/text.asp?2020/26/2/75/289949
| Introduction|| |
Biofilms are an aggregate of bacteria attached in a self-produced matrix called extracellular polymeric substances that are associated with chronic infections. The increasing antibiotic resistance is believed to be due to biofilm formation. Pseudomonas aeruginosa, Proteus species, Staphylococcus aureus, K. pneumoniae, Diphtheroids, and Streptococcus are common bacteria in chronically discharging ears known for multidrug resistance due to overuse of antibiotics.
The presence of biofilms may be the reason for persisting ear discharge in active mucosal disease. This comparative study was done to know the presence of biofilm-producing organisms in the middle ear mucosa in active and inactive mucosal disease and their antibiotic sensitivity.
| Materials and Methods|| |
Ethical committee clearance was obtained and written informed consent was obtained from all the patients who were participating in the study. A prospective comparative study was done between October 2016 and September 2018 on patients of chronic otitis media (COM) mucosal disease undergoing tympanoplasty with or without cortical mastoidectomy.
The objectives of this study were to know the presence of biofilm-producing organisms in the middle ear mucosa in COM mucosal disease and to know their antibiotic sensitivity.
The patients with COM mucosal disease who underwent surgery for the same were divided into two groups. Group 1 consisted of 50 patients of active mucosal COM and patients with inactive mucosal COM were included in Group 2. Discharge from the middle ear was sent for culture and sensitivity using aseptic precautions under microscopic guidance in all mucosal active cases before the surgery. All the patients of both the groups underwent tympanoplasty with or without cortical mastoidectomy, during which biopsy from the middle ear mucosa was sent for culture and sensitivity. The isolates grown were subcultured. Tissue culture plate method was used to detect their biofilm-forming capability. Based on the optical density of the enzyme-linked immunoabsorbent assay (ELISA) plate, the biofilm-producing capability of the isolates was categorized into high, moderate, and non/weak biofilm-producing organism.
The institutional ethical committee has given ethical clearance prior to commencing the study. Our study does not involve any additional procedure or cost to the patient. Middle ear mucosal biopsy was taken intraoperatively during the surgery.
The optical density of the ELISA plate with the samples was read, and the values were categorized into non/weak, moderate, and high. Statistical analysis was done using”SPSS (Statistical Package for the Social Sciences) IBM SPSS Statistics for Windows, Version 23.0. (IBM Corp., Armonk, NY, USA).”
| Results|| |
Out of 50 mucosal active cases, 28 were female and 22 were male, whereas in the mucosal inactive group, 26 were male and 24 were female. Patients in both the groups were between the ages of 21 and 60 years with a mean age of 34 years.
Middle ear discharges were sent for culture preoperatively; P. aeruginosa was grown in seven samples and coagulase-negative Staphylococcus and S. aureus were grown inthree each.
In active mucosal disease, of 50 cultures from middle ear mucosal biopsies, 31 cultures showed bacterial growth and no organisms were grown in the remaining 19 cultures. Fourteen isolates were P. aeruginosa (45.2%), 10 isolates were S. aureus (32.3%), five isolates were Klebsiella pneumoniae (16.1%), and two isolates were Staphylococcus epidermidis (6.5%).
Among the 14 isolates of P. aeruginosa, five (42.9%) isolates had non/weak biofilm-producing capability and nine (57.1%) had moderate capability. Out of 10 isolates of S. aureus, six (60%) isolates had non/weak capability and four (40%) cultures showed moderate capability. K. pneumoniae was isolated in five of the cultures, of which three isolates had non/weak capability and two had moderate capability. S. epidermidis was isolated in two cultures, of which one had high and one had moderate biofilm-forming capability.
Of 50 samples in Group 2, 22 samples were culture positive and 28 samples were culture negative. S. aureus was isolated in 12 (54.5%) of the middle ear mucosa biopsies and P. aeruginosa (45.5%) was isolated in 10. Among the 10 isolates of P. aeruginosa, four isolates (40%) had non/weak, four had moderate (40%), and two had high (20%) biofilm-forming capability. S. aureus was isolated in 12 cultures, of which four isolates (33.3%) were non/weak, seven isolates (58.3%) were moderate, and one isolate (8.3%) had high biofilm-forming capability.
Antibiotic sensitivity of Pseudomonas aeruginosa
Nine of 14 samples of P. aeruginosa (64%) in Group 1 were sensitive to piperacillin-tazobactam and cefepime, followed by amoxicillin and meropenem (7 out of 14 [50%]). Ciprofloxacin (7 out of 14 [50%]) was the most resistant drug, followed by amoxicillin (five out of 14 [35.7%]). Among the biofilm-producing P. aeruginosa, eight were sensitive to piperacillin/tazobactam. In nonbiofilm-producing Pseudomonas aeruginosa, four of five isolates (80%) were sensitive to cefepime. Considering biofilm-producing organisms, ciprofloxacin was the most commonly resistant drug (5 out of 9) followed by ceftriaxone (four out of nine) In nonbiofilm-producing P. aeruginosa in active group, two of five were resistant to amoxicillin and ciprofloxacin.
In Group 2, cefepime and amikacin (7 out of 10) were the most commonly sensitive drugs followed by piperacillin/tazobactam and ceftriaxone (6 out of 10). When considering biofilm-producing P. aeruginosa in inactive disease (six isolates), piperacillin/tazobactam and cefepime (5 out of 6) were most sensitive, followed by amikacin (4 out of 6). Among the nonbiofilm-producing P. aeruginosa in inactive disease, amikacin and ceftriaxone were more sensitive drugs (3 out of 4). P. aeruginosa in inactive group was most resistant to amoxicillin (50%) followed by ciprofloxacin (30%). In the biofilm-producing organisms (6 isolates), three of six were resistant to amoxicillin (50%) followed by ciprofloxacin (2 out of 6). In nonbiofilm-producing organisms (four isolates), two isolates were resistant to amoxicillin (50%) [Table 1].
Antibiotic sensitivity of Staphylococcus aureus:
In mucosal active, 10 isolates were showing S. aureus, of which four were biofilm producing and six were nonbiofilm producing.
All 10 isolates of S. aureus in mucosal active were sensitive to linezolid (100%) followed by clindamycin (90%) and gentamycin (80%). Ciprofloxacin was resistant for four isolates (40%) followed by gentamycin in two isolates (20%).
All the biofilm-producing S. aureus in mucosal active (four isolates) were sensitive to levofloxacin and linezolid (100%), followed by erythromycin, clindamycin, and gentamycin (3 out of 4 isolates). Two out of four were resistant to ciprofloxacin (50%). Among the nonbiofilm-producing S. aureus in mucosal active (six isolates), all were sensitive to clindamycin and linezolid (100%) followed by gentamycin (5 out of 6). Two out of the six isolates of nonbiofilm-producing S. aureus were found to be resistant to ciprofloxacin.
In the mucosal inactive group, levofloxacin was the most sensitive drug (10 out of 12) followed by gentamycin and linezolid (9 out of 10). The most resistant drugs in inactive disease for S. aureus were ciprofloxacin and gentamycin (3 out of 10). In biofilm-producing isolates of S. aureus in inactive group (eight isolates), seven of eight were sensitive to levofloxacin and gentamycin. The most resistant drug was found to be ciprofloxacin (2 out of 8). In the nonbiofilm-producing isolates of S. aureus in the inactive group (four isolates), three of four were sensitive to ciprofloxacin, levofloxacin, erythromycin, linezolid, oxacillin, and trimethoprim/sulfamethoxazole. Two of four were observed to be resistant to gentamycin [Table 2].
Antibiotic sensitivity of Klebsiella pneumoniae:
Of five isolates of K. pneumoniae which were isolated in only Group 1, two were biofilm producing and three were non-producing. All five were sensitive to piperacillin/tazobactam and cefoperazone, i.e., 100%, three of the five isolates were resistant to ciprofloxacin i.e., 60% [Table 3].
Antibiotic sensitivity of Staphylococcus epidermidis
S. epidermidis was isolated in two samples of which both were biofilm producing and were sensitive to cefepime and piperacillin/tazobactam and were resistant to ciprofloxacin.
Using Fisher's exact test, there was no significant difference between the both groups when comparing the high, moderate, or weak biofilms producing capability (P = 0.341).
Using Fisher's exact test, there was a statistically significant difference between the two groups when comparing the different types of organisms isolated, i.e., Group 1 – four organisms and Group 2 – two organisms (P = 0.029).
| Discussion|| |
COM is one of the most common ENT problems that patients present with persistent ear discharge despite adequate treatment. In recent years, it is believed that biofilm production is the cause for persistent infection and antibiotic resistance. Biofilm formation has become increasingly significant and plays an important role in many otorhinolaryngologic infections and results in persistent infection and difficulty in the eradication of the disease.
Biofilms are an aggregation of bacteria in a self-produced extracellular matrix of exopolysaccharides, proteins, and some micromolecules such as DNA. It can form on biotic and abiotic surfaces. These biofilms are highly resistant to the immune killing system and antibiotic therapy. They are also capable of shedding bacteria into the circulatory system causing bouts of systemic infection, which may recur despite antibiotic therapy.
Biofilms have been reported in otitis media with effusion, cholesteatomas,,,,, adenotonsillitis, tracheostomy tubes, and chronic rhinosinusitis (CRS). They have also been isolated on the surfaces of implants and prosthesis such as voice prosthesis, tympanostomy tubes, and cochlear implants.
In our study, 53 middle ear mucosal cultures were positive out of 100 COM including both active and inactive mucosal disease, we found that 24 isolates (45.28%) were P. aeruginosa, 22 isolates (41.51%) were S. aureus, five isolates were K. pneumoniae (9.43%), and two isolates (3.77%) were S. epidermidis.
P. aeruginosa was the most common organism isolated from the discharge in actively discharging ear (preoperatively seven out of 50, 14%) and in the culture of the middle ear mucosa in actively discharging ear (14 out of 31). In discharging ear, we observed that out of 50 cases, only 13 samples showed organisms in cultures sent from the discharge, whereas middle ear mucosal culture showed growth in 31 out of 50 samples. K. pneumoniae and S. epidermidis were grown only in the middle ear mucosal culture. This indicates that the absence of discharge does not signify the absence of infection in the middle ear.
S. aureus was the second most common organism cultured from the middle ear mucosa in actively discharging ear (32%, 10 out of 31) after P. aeruginosa. However, in the middle ear mucosa of inactive mucosal disease, S. aureus was the predominant organism (12 out of 22, 54.5%) followed by P. aeruginosa (10 out of 22, 45.4%).
“Galli et al.” found biofilms in the middle ear in 14 out of 15 cholesteatoma cases, but bacterial cultures were negative in all the samples. Chronic exposure of the middle ear mucosa through the perforation of the tympanic membrane perforation is the cause for a positive bacterial culture of the middle ear mucosa in our study. Although the presence of biofilms in the middle ear was observed for decades, their antibiotic sensitivity was not observed in any of the study.
S. aureus was the most common biofilm-producing organism in CRS. In our study, P. aeruginosa was the most common biofilm-producing organism. We observed that biofilm-forming organisms are more seen in mucosal active disease when compared to mucosal inactive disease.
Antibiotic resistance is an increasing global threat. This is due to inadequate or overuse of antibiotic therapy. One of the causes for this could be due to biofilm formation. Pseudomonas aeruginosa in active mucosal disease was equally and most sensitive to piperacillin-tazobactam and cefepime. Ciprofloxacin followed by amoxicillin was the most common resistant drugs. In the inactive group, amoxicillin was the most resistant drug for P. aeruginosa irrespective of their capability to produce biofilms.
Linezolid showed 100% sensitivity for S. aureus in active mucosal disease, followed by clindamycin and gentamycin. In the inactive mucosal disease, levofloxacin was the most sensitive drug for S. aureus both in biofilm-producing and nonproducing organisms. Ciprofloxacin was the most resistant drug in biofilm-producing and nonbiofilm-producing S. aureus.
Piperacillin-tazobactam and cefoperazone showed 100% sensitivity for K. pneumoniae. Again, ciprofloxacin was the most resistant drug for K. pneumoniae. S. epidermidis was isolated in only two cases and both had biofilm-forming capability and showed 100 % sensitivity to piperacillin-tazobactam and cefepime. Both isolates were resistant to ciprofloxacin.
Similar to our study “Asati and Chaudhary” studied in burns wound, a total of 190 isolates were studied, of which 68.9% of isolates showed biofilm formation. They observed that biofilm-producing bacterial isolates showed a high level of resistance to all drugs that are commonly used, such as fluoroquinolones, cephalosporins, aminoglycosides, and tetracyclines. The most effective drug against Gram-positive bacteria was vancomycin and the most effective drug against Gram-negative bacteria was carbapenems. However, from our study, we can conclude that antibiotic resistance in middle ear infection is not dependent on their biofilm-forming capability.
No study was done on the capability of biofilm production and their antibiotic sensitivity in COM similar to this study. We believe that high resistance to ciprofloxacin is probably due to overuse of antibiotic ear drops.
| Conclusion|| |
P. aeruginosa and S. aureus are common organisms isolated in the mucosa of the middle ear in COM which are known to produce biofilms. We conclude that there is no significant difference between the biofilm-forming capability of bacteria in the middle ear mucosa in mucosal active and mucosal inactive diseases. Piperacillin-tazobactam is the most sensitive drug for P. aeruginosa and linezolid is the most sensitive drug for S. aureus irrespective of their capability to form biofilms. Ciprofloxacin is the most resistant drug irrespective of the organism and their biofilm-producing capability.
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Conflicts of interest
There are no conflicts of interest.
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[Table 1], [Table 2], [Table 3]