Isolation and Characterization of Bacteria Causing Acute Suppurative Otitis Media in Egypt

1Department of Microbiology and Immunology, Faculty of Pharmacy, Modern University for Technology & Information, Cairo, Egypt. 2Department of Otolaryngology, Faculty of Medicine, Cairo University, Cairo, Egypt. 3Department of Microbiology and Immunology, Faculty of Medicine, Assuit University, Assuit, Egypt. 4Department of Microbial Genetics, Faculty of Biotechnology, Misr University for Science and Technology, 6th of October, Egypt. 5 Universität Ulm Medizinische Fakultät, Ulm, Deutschland.

Acute suppurative otitis media is inflammation of the mucous membrane lining of the middle ear cleft (consisting of the eustachian tube, tympanic cavity, mastoid antrum and mastoid air cells) produced by pus-forming organism 1 .It is a disease of multiple etiologies and is well known for its recurrence.ASOM often starts in infancy, and is among the top common childhood illnesses.It mainly affects children 2 .Infections usually results from bacterial and fungal causes, and in some cases secondary to other viral infections like upper respiratory tract infections (URTI) 3 .It may result in serious complications as mastoiditis, meningitis or intracranial abscess 4 .
In the pre-antibiotic era of the early 1900s, b-haemolytic group A Streptococcus pyogenes (S. pyogenes, group A Streptococcus, GAS) was the most common frequently isolated pathogen from people with acute otitis media (AOM), especially when AOM was a complication of acute tonsillitis or scarlet fever 5,6 .However, since the 1950s, it has been rapidly replaced by S. pneumoniae, H.influenzae, and M.catarrhalis 5,7 , which are nowadays the main bacterial pathogens followed by other less frequent pathogens as S.aureus, and more rarely, anaerobic Gram negative bacilli such as Escherichia coli or Pseudomonas aeruginosa 8,9 .With the advent of antibiotics, the incidence of complications had decreased.However, due to unnecessary and improper use of antibiotics, resistance by bacteria, and increase in susceptibility of individuals, complications still occur.Prevention of the disease by risk factor modification is attractive because it diminishes the costs and the side-effects of antimicrobial therapy and surgery 10 .The study of the microorganisms commonly associated with ASOM is very pertinent for the clinician to plan a general outline of treatment.Despite the frequent diagnosis of ASOM in our hospitals, little information exists in the literature of this condition.Therefore, the present study was carried out to report the isolation and characterization of causative bacteria involved in ASOM.

Subjects
The study population consisted of 76 outpatients who visited Kasr Al Aini Teaching Hospital, Cairo, Egypt outpatient clinic from February 2009 -August 2009, and were diagnosed with ASOM according to the results of their medical history and physical examinations.The population included 45 males, and 31 females.

Collection of Samples
Middle-ear aspirates for cultures were collected aseptically.The external auditory canal was thoroughly cleaned with 70% alcohol first and was allowed to act for 30-40 seconds to achieve sterile area.Then the contents of the ME were aspirated into the syringe.All clinical samples were transported to the Bacteriology laboratory at Faculty of Biotechnology, Misr University for Science and Technology, 6 th of October City, Egypt.

Isolation and identification of bacteria
Collected samples were inoculated in blood agar, nutrient agar, chocolate agar, and MacConkey agar plates.All plates were incubated aerobically at 37°C for 24 hours.Then all samples were subjected to Gram stain and biochemical reactions.Isolates were identified by colony morphology, gram staining reaction, catalase test, coagulase test, mannitol fermentation, blood hemolysis, and optochin sensitivity as per standard procedures 11 .

RESULTS
Seventy six patients were examined by the attending otorhinolaryngologists during the period of the study.They were diagnosed as having ASOM based on the criteria employed (Table 1); 74 (97.36 per cent) of these presented with unilateral discharging ears, while only 2 (2.63 per cent) presented with bilateral discharging ears (Table 2).Of these patients, 54 (71.05 per cent) presented with infected left ear, while 20 (26.31 per cent) presented with infected right ear.Forty five patients (59.21 per cent) were males and 31 (40.78 per cent) were females.Their age ranged from 5 to 80 years old.The incidence in relation to their sex and age is shown in Table 3. Thirty patients (39.47 per cent) presented in winter, while 10 patients (13.15 per cent) presented in summer (Table 4).
Table 5 shows that the total number of cultures was 78, all of which were monomicrobial; no polymicrobial cultures.Out of the total 78 cultures, S.aureus accounted for 66 cultures (78.37 per cent), while S. pneumoniae accounted for 12 cultures (13.79 per cent) as shown in Table 6.

DISCUSSION
The study was carried out to determine the incidence of ASOM at Kasr Al Aini Teaching Hospital, Cairo, Egypt.The age ranged from 5 to 80 years.The criteria considered for diagnosing ASOM were those suggested in literature 12,13 .All the studied patients presented with perforated tympanic membrane, otalgia, and otorrhea.Forty eight patients (63.15 per cent) presented with nasal discharge, while 3 patients (3.94 per cent) presented with ear itching, and 4 patients (5.26 %) presented with hearing loss.These ratios are approximately similar to those obtained by Iseh and Adegbite [14]  who have found otorrhea and tympanic membrane perforation in all their studied subjects, but the nasal discharge and otalgia were found in 47.7 per cent and 35per cent of their studied population respectively.Ear itching was found in 3.4 per cent, and hearing loss was found in 2.5 per cent of the subjects.
In this study, unilateral infection was predominant (97.37 per cent), while bilateral infection was found in only 2.63 per cent, with a higher with left side incidence (71.05 per cent) compared to that of the right side (26.31 per cent).Olasunkanmi Oladapo 15 also has found that unilateral infection accounted for 85.6 per cent of his studied population while bilateral infection accounted for 14.4 per cent.On the contrary, Eugene Leibovitz et al 16 have found a predominance of bilateral infection over unilateral infection.
Our study showed that the maximum number of cases was found in the age group 5-10 years suggesting that the disease is highly incident in children.In a similar study, Iseh and Adegbite 14 reported that the majority of their studied subjects were within the age range 0-15 years, out of which 0-5 years accounted for 67.5 per cent.Children are more liable to ASOM because of being susceptible to upper respiratory tract infections, and having shorter, wider, more horizontal eustachian tube [17][18][19] .
The higher incidence of ASOM in males recorded in the study correlates with other incidences reported by various studies 14,20,21 , whereas Olasunkanmi Oladapo has found a slight predominance in females 15 .No rationale has been given for the inequality 1,22 , while Rakesh Kumar et al 23 suggested that the male predominance may be due to their more exposed way of life.
Among the studied cases, ASOM was more prevalent during winter (39.47 per cent), than in summer (13.15 per cent).This was also found by Eliza Royan et al. in a prospective cohort study 24 .Increased incidence of ASOM in winter is mainly attributed to repeated upper respiratory tract infection 25 .
In the present study, all cultures were monomicrobial indicating a 100 percent monomicrobial etiology and absence of polymicrobial etiology.On the contrary, chronic suppurative otitis media (CSOM) always show a considerable percentage of polymicrobial etiology.Upon isolating the organisms associated with CSOM in a tertiary care setup of Uttarakhand State, India, Rajat Prakash et al 26 have found that 57.84 per cent of samples showed monomicrobial growth whereas 33.33 per cent showed polymicrobial growth.
The organisms isolated were S.aureus and S. pneumoniae.S.aureus was frequently isolated (78.37 per cent) than S. pneumoniae (13.79 per cent).The predominance of S.aureus is similar to that found by Iseh and Adegbite who have found that S. aureus (46.2 per cent) was the commonest organism cultured in their study 14 .On the other hand, S. pneumoniae was the most cultured organism (82 per cent) by Katherine B Gibney et al 27 .In another study carried out in So Paulo, Brazil 28 , S. pneumoniae accounted for 16 per cent of the pathogens isolated from Brazilian children with acute otitis media, whereas S. aureus accounted for only 1 per cent.In Warsaw, Poland 29 , S. pneumoniae was the most frequently isolated pathogen (39.69 per cent) from the middle ear in children with acute otitis media treatment failure, while S.aureus was isolated from 16.03 per cent of the subjects.The difference in the distribution of bacteria may be due to the geographical variability 14 .

Table 1 .
Clinical and diagnostic profile of ASOM subjects

Table 2 .
Distribution of ASOM subjects according to Laterality &the side of the ear affected

Table 3 .
Incidence of ASOM among subjects in relation to their age and sex

Table 5 .
Incidence of Monomicrobial and polymicrobial cultures among ASOM subjects