Isolation and Identification of Pathogenic Bacteria Causing Otitis Media in Misan Governorate

Sciences, Maysan, Iraq. Abstract This study examined 150 ear swab samples from patients with otitis media who consulted at Al-Sadr Teaching Hospital from January to April 2021 in Misan, Iraq. The participants were aged 14–50 years, among which the infection rate was highest in participants aged 14–22 years and lowest in those aged 40–50 years. Subsequently, bacterial isolates were identified based on their morphology in various culture media and using biochemical tests. Six bacterial species were identified, namely Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli, Streptococcus pneumoniae, Klebsiella pneumoniae, and Staphylococcus epidermidis . Staphylococcus aureus had the highest infection rate (30%), whereas Staphylococcus epidermidis had the lowest infection rate (8.55%). When the sensitivity of each isolate to antibiotics was determined, Escherichia coli was the most sensitive to trimethoprim (TMP), whereas Pseudomonas aeruginosa was the most resistant to 75% of

infections in children. If the upper respiratory infection is caused by a bacterium, this may travel to the middle ear. If the upper respiratory infection is viral, such as a cold, viruses may be attracted to the habitat of the microorganism and invade the middle ear as a serious infection. Fluid accumulates behind the eardrum because of the illness. [22][23][24] Prompt, fast, and successful ear infection treatments can greatly reduce the short-and longterm consequences of ear infection. Furthermore, they can enhance the lives of patients. Thus, it is critical to have the most up-to-date data on the etiology of ear infection and associated antibiotic susceptibility pattern. [25][26][27] Therefore, this study aimed to isolate and identify the most common bacteria causing OM in patients from the Misan governorate, Iraq, and exploring bacterial sensitivity to most commonly used antibiotics.

MATeRIAlS And MeTHOdS Sample Collection
A total of 150 patients diagnosed with OM by specialists at (name of a clinic) in Misan governorate, Iraq from January to April 2021 was included in this study. The patients were classified according to age, ranging from 14 to 50 years. Swab sample from an irritated area was collected using a sterile cotton swab. Samples were collected on patients who had not taken antibiotics for at least one week prior to swab collection.

Collection and Culturing of ear discharge
Firstly, the outer ear was cleaned using regular saline. Then, a sample of the discharge was collected. Swabs were subsequently inoculated onto MacConkey and blood agar plates, which were incubated aerobically at 37°C for 24-48 hours, and mannitol salt agar plates, which were incubated anaerobically at 37°C for 24-48 hours. 28

Identification of Bacteria
Bacterial colonies were initially identified based on their phenotype and cultural characteristics. Identification of the isolated bacteria was performed according to standard microbiological methods, including cultural characteristics, Gram staining; biochemical reactions, such as catalase, indole urea media, oxidase; diagnostic tests, such as motility, hemolysis, and the triple sugar iron (TSI); and the VITEK 2 Compact. 28,29

Measurement of Bacterial Sensitivity to Antibiotics
To evaluate the antibiotic susceptibility of bacteria isolated from patients with OM, we performed the procedures developed by. 31 Briefly, Muller-Hinton agar was used for the experiment, except for S. pneumoniae isolates, which were grown on 5% blood agar medium. Samples were incubated at 37°C for 24 hours, and then the diameters of growth inhibition zones were measured. 33 Antibiotic discs were chosen based on the frequency of prescription in the study region and the guidelines of the Clinical Laboratory Standards Institute (CLSI) and the European Association on Antibiotic Susceptibility (EUCAST). 32,41 The following antibiotics ( Oxoid, UK) were used: amoxicillin (AMC, 30µg) plus clavulanic acid, penicillin G (P-10 U), amoxicillin (AX, 25µg), and trimethoprim (TMP, 10µg).

Antibiotic Sensitivity Test
The following antibiotics were used in this study: amoxicillin plus clavulinic acid (AmC 30 g), penicillin G (P-10 U), amoxicillin (AX-25 g), and trimethoprim (TMP 10 g). In general, all bacteria isolated in this study showed high sensitivity to amoxicillin (100%) and a relatively high sensitivity to trimethoprim (83%). Furthermore, 67% of the isolated bacteria were resistant to penicillin, whereas 50% of the isolates were resistant to amoxicillin Table 4.
Overall, Gram-positive isolates were highly sensitive to the antimicrobials used, ranging from as low as 7.5 mm for Streptococcus pneumoniae sensitive to amoxicillin and as high as 12.5 mm for Staphylococcus epidermidis for amoxicillin and trimethoprim. Streptococcus pneumoniae was observed to be resistant to penicillin. For Gram-negative isolates, majority showed high resistance to almost all tested antibiotics. Pseudomonas aeruginosa were resistant to all tested antibiotics, except amoxicillin (12.5 mm), followed by Escherichia coli that reacted as 50% resistant to amoxicillin and penicillin, while showed 50 % sensitivity for amoxicillin (13.5mm) and for trimethoprim with 15 mm inhibition zone measure (Fig. 1).

DisCUssiON
Middle ear inflammation associated with OM is a common childhood and adult disease. 34 This study enrolled 150 patients from the Misan governorate, Iraq, who were screened to determine the causative bacteria of OM and their susceptibility to several commonly used antibiotics. Additionally, we investigated the prevalence of OM across the examined age groups. The findings indicated that women (66%) had a higher prevalence of OM than men (44 %). This observation is consistent with 35,36 who reported that females (66.1%) were more affected by OM than males (33.9 %). Several studies have demonstrated an equal prevalence of AOM in men and women, while numerous previous studies have indicated an increased incidence in boys. This could be a reflection of the environmental conditions (in-depth, recommended study) associated with the onset of OM symptoms.  Age is a significant factor in determining the percentage of OM infections. In the present study, the highest infected age group was 14-22 (33.3 %), followed by 22-26 (33.3 %) (20%). A low distribution of OM (4.6 %) was discovered in adults aged 40-50. Children are evidently more susceptible to OM due to their susceptibility to upper respiratory tract infections. 37 reported that majority of their subjects were aged of 0-15 years, with 0-5 years accounting for 67.5%. This finding is consistent with the reasons children are more likely to contract OM than adults: children have a more vulnerable immune system and must fight off multiple infectious pathogens. Furthermore, the eustachian tube is a relatively small passageway. A child's ear is shorter and more angular than that of an adult, making it more susceptible to blockage. Additionally, infection with adenoids may contribute to pathogen transmission into the eustachian tube. 38 Other studies discovered that OM was the most prevalent disease in the first two years of children. 39,40 Majority of the bacteria isolated in this study were Gram-positive (50 %), and the most common isolate was Staphylococcus aureus (30 %). Pseudomonas aeruginosa represented the majority with 25.3 percent. Similar findings were observed by Roland and Storman, 42 who observed that Pseudomonas aeruginosa and Staphylococcus aureus were the most dominant bacterial pathogens in OM, and 43-46,55 who reported that Pseudomonas aeruginosa and Staphylococcus aureus were the most common bacteria among patients with chronic OM.
S. pneumoniae represented 16% of infections isolated from Brazilian participants with severe otitis media, whereas S. aureus was responsible for only 1%. 48 S. pneumoniae was the most frequently isolated pathogen from the middle ear of children with otitis media drug resistance with (39.69%), whereas S. aureus was recovered from 16.03 % of the participants. 47 Geographical heterogeneity may be responsible for the differences in the distribution of bacteria. 49 This study discovered that amoxicillin plus clavulanic acid (AmC 30 g) was 100% effective against most bacterial isolates from patients with OM, whereas trimethoprim TMP (10 g) was 83% effective. Amoxicillin is primarily bactericidal as a β-lactam antibiotic. It inhibits the third and final stages of bacterial cell wall synthesis by preferentially binding to a specific PBP found in the bacterial cell wall. As is the case with all β-lactam antibiotics, inhibition PBP-mediated cell wall synthesis results in cell lysis. 50,51 Clavulanic acid is a β-lactamase inhibitor with negligible antimicrobial activity, and it acts as an effective "suicide" suppressor of a range of infectious β-lactamases mediated by plasmids and chromosomes. 52 Pseudomonas aeruginosa exhibited the highest resistance to 75% of antibiotics tested but showed good sensitivity to amoxicillin. Escherichia coli was identified as the most susceptible isolate to trimethoprim (15 mm) and amoxicillin (13.5 mm). Antibiotic resistance is increasing worldwide as a result of antimicrobial misappropriation and overuse. 53 A number of pediatricians and infectious disease organizations have recently launched a program to limit antibiotic use for competitive reasons. 54,56 The Centers for Disease Prevention and U.S. Centers for Disease Control collaborated to review the prudent use of antimicrobial agents in infectious agents and recommend measures for potential pathogens, including those causing AOM. 57,59 These guidelines emphasize the importance of limiting the use of antimicrobial agents. If these guidelines are adopted, the selective pressure caused by widespread antiviral use may be alleviated. Antimicrobial resistance is a significant challenge to international public health, and such guidelines may help reduce the therapeutic failures caused by drug-resistant bacteria. 58

CONClUsiON
The findings of this study shed light on pathogenic bacteria that caused OM in patients from Misan, Iraq, and highlighted the phenomenon of antibiotic resistance, which has captured the interest of biological scientists, particularly microbiologists, in recent years. The most frequently isolated organism was Staphylococcus aureus, followed by Pseudomonas aeruginosa. Antibiotics, such as amoxicillin and trimethoprim, were the most efficient against most of the isolates. We firmly advocate for a nationwide antimicrobial surveillance to ensure appropriate antibiotic recommendations and rigorous adherence to antibiotic use policies and