STUDY ON BACTERIAL AND MYCOTIC INFECTION OF THE MIDDLE EAR

Fathy Mohamed E. Serry 1 , Ashraf Ahmed Kadry Yosusef 1 and Fatma Ahmed N. M. H. Elmeselhy 2 . 1. Professor of Microbiology & Immunology, Faculty of Pharmacy, Zagazig University, Egypt. 2. Pharmacist, Zagazig Fever Hospital, Ministry of Health, Egypt. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History

Chronic otitis media (COM) is a permanent abnormality on the tympanic membrane following a long standing middle ear infection emanating from previous AOM or negative pressure to the middle ear. Chronic suppurative otitis media (CSOM) is the most severe form of OM and represents the most important cause of moderate conductive hearing loss in many developing countries (Chirwa et al., 2015).
A number of factors may contribute to the development of ear infections. Most experts consider Eustachian tube dysfunction to be the primary cause for both acute and chronic ear infections. Obstruction at the Eustachian tube isthmus (i.e., the narrowest portion) results in accumulation of middle ear secretions; secondary bacterial or viral infection of the effusion causes suppuration and features of AOM (Rovers et al., 2004).

The Aims of the work:-
This study aimed at identification of bacteria and fungi that cause otitis media, determination of susceptibility of isolates to antimicrobial agents & investigation of the possible mechanisms of antimicrobial resistance.

Materials And Methods:-
This study was carried out in the Microbiology and Immunology Department, Faculty of Pharmacy, Zagazig University on 85 patients suffering from ear discharge secondary to otitis media who were attending the out-patient clinic of ENT Department, Zagazig University Hospitals during the period from February 2013 to February 2016. All patients included in this study were not using local and/or systemic antibiotics for one week before obtaining the samples.
Ear swabs specimens were obtained, after cleaning the external auditory canal of cerumen with sterile saline and 70% alcohol swab. Specimens were handled according to the approved microbiological procedures as described by Koneman et al (1997), transported to laboratory, examined and cultured within one hour of collection.
Ear swabs were streaked directly on a) nonselective media; Nutrient agar and Blood agar, and b) selective media; Chocolate agar, MacConkey agar, Cetrimide agar, Mannitol salt agar and Sabouraud's dextrose agar (SDA) with chloramphenicol and gentamicin then incubated at 37°C for 24 hours except of SDA plates were incubated at 30°C and the cultures paraffin sealed plates were held for 4 weeks and were examined twice weekly for growth. The isolated bacterial colonies and Candida were microscopically examined after Gram staining (Collee et al., 1996). Mould cultures were mounted into lactophenol cotton blue stain and examined microscopically (Davise, 2011). Slide culture was prepared according to Riddel, (1950) for mould identification. Candida isolated colonies were presumptively identified according to their characters on SDA (Rippon, 1982

Results:-
During this study, a total of 120 isolates were obtained from 85patients. Single isolate (58.82%) were obtained from 50 specimens and double organisms (41.18%) were obtained from 35 specimens. samples were either mixed bacterial combination 27 (80%) or combination of bacteria and fungi 6 (20%).

Figure (1):-The distributions of types and percentages of isolates
The bacterial isolates were tested for their susceptibility to the different antimicrobial chemotherapeutic agents by disk diffusion method; the diameters of inhibition zones were measured and interpreted according to CLSI (2012) guidelines (table 1).  1420

Figure (2):-D-test results for Staphylococcus aureus
Phenotypic detection of β-lactamases done for Klebsiella pneumoniae isolate and Haemophilus influenzae isolates. These phenotypic confirmatory tests showed one positive Klebsiella pneumoniae isolate that was EBLs producter and showed four Haemophilus influenzae isolates that were βlactamase producer (carried out by using Cefinase disks).
Effect of efflux pump inhibitor carbonyl cyanide 3-chlorophenyl hydrazone (CCCP) on susceptibility of Pseudomonas aeruginosa isolates to antimicrobial agents piperacillin, gentamicin, amikacin, ciprofloxacin, levofloxacin, aztreonam and cefepime revealed that resistance of Pseudomonas aeruginosa isolates to these antimicrobial agents may be due to efflux activity.

Discussion:-
OM is a serious healthcare concern worldwide, not only because of the distress it causes to the patient and their family but also because of the substantial economic burden it imposes on the health care system (Ologe and Nwawolo, 2002 In the present study, the most common causative organisms were Gram negative bacteria (47.5%) then Gram positive bacteria (30.83%). This agrees with the study of Van Hasselt (2013).
In present study, the most frequently occurring organisms were Pseudomonas aeruginosa (20.3%) and In the present study, Haemophilus influenzae isolates represented low incidence (4.2%) of total isolates. Different result reported by Nwankwo and okeke (2014).
In our study, Escherichia coli and Klebsiella pneumoniae represented 6.7% and 3.3% of total isolates, respectively. Our results were comparable to that of In the present study, antibiotic susceptibility tests revealed that all Staphylococcus aureus isolates (100%) were susceptible to vancomycin. Our finding agrees with Abdelshafy (2015), showed that quinolones were the most effective antibiotic against Staphylococcus aureus.
In the present study, all Pseudomonas aeruginosa isolates (100%) sensitive to ceftazidime and 96% were sensitive to imipenem. The results agree with that of Ogbogu et al (2013). Pseudomonas aeruginosa showed also high sensitivity to cefepime (84%), followed by levofloxacin (64%). Cefotaxime was less active against most P. aeruginosa isolates with resistance rate of 56 %. This was in agreement with the study of Harvinder and Seth (2011).
In our study, Fungal isolates Candida albicans, Aspergillus niger and Aspergillus fumigatus were all fully susceptible to the two antifungal agents used amphotericin B and itraconazole (100% susceptibility rate). This was in agreement with the results Abd al-zaher (2004) stated that Aspergillus niger and Aspergillus fumigatus isolates had acceptably low MICs for amphotericin B and Itraconazole and Candida albicans isolates were fully susceptible to Itraconazole.

1423
In the current study, 100% of Staphylococcus aureus isolates that were resistant to erythromycin and intermediated to clindamycin had inducible clindamycin resistance (positive D-test). Yusuf et al (2014) found that out 72.8% of Staphylococcus aureus isolated that were resistant to erythromycin and susceptible to clindamycin, were positive to D-test.
In the present study, 3.7% of Enterobacteriaceae isolates (Escherichia coli, Proteus mirabilis and Klebsiella pneumoniae) were found to produce extended spectrum β-lactamases (ESBLs) that found in 25% of Klebsiella pneumoniae isolates. These results are different from that of Yusuf et al (2014).
In this study, Reduction in MICs (at least 2 fold) in presence of efflux inhibitor CCCP indicates the presence of proton gradient-efflux pump(s) in our Pseudomonas aeruginosa isolates. The highest rate for reduction in MIC was reported to aztreonam (2 to 3 fold reductions in MIC). Levofloxacin MICs decreased by 2 fold after addition of CCCP. Piperacillin, gentamicin, amikacin, ciprofloxacin and cefepime showed 2 to 3 fold reduction in MIC in presence of CCCP. These results are in agreement with that of Adabi et al (2015).
In the current study, the piperacillin resistant Pseudomonas aeruginosa isolates were tested for the possible role of efflux in such resistance by testing the activity of efflux pump through demonstrating the effect of efflux inhibitor (DNP) on ethidium bromide accumulation inside cells.

Conclusion:-
In our study Pseudomonas, Staphylococcus aureus, Streptococcus pneumoniae, Proteus mirabilis and Aspergillus species were the most important organisms associated with middle ear infection. For Gram negative bacteria, imipenem, amoxicillin-clavulanic acid, cefotaxime levofloxacin and amikacin appear to be the first line antibiotics to treat CSOM. However, imipenem, vancomycin, sulfamethoxazole/trimethoprim, levofloxacin, ciprofloxacin and clindamycin appear to be the first line antibiotics in the treatment of Gram positive bacteria.
The antibiotic susceptibility patterns must be continuously and periodically evaluated to decrease the risk of resistant strains.