Antibiotic-Resistant Bacteria Are Major Threats of Otitis Media in Wollo Area, Northeastern Ethiopia: A Ten-Year Retrospective Analysis

Antibiotic resistance is an increasingly serious threat to human health that needs an urgent action. The aim of this study was to determine the prevalence and antibiotic susceptibility profiles of bacteria isolated from patient ear discharges suspected of otitis media. A retrospective analysis was performed using culture and antibiotic susceptibility test results of 1225 patients who visited Dessie Regional Health Research Laboratory from 2001 to 2011. Results showed a strong association (P < 0.001) between age and the risk of acquiring middle ear infection. The predominant bacterial isolates were Proteus spp. (28.8%), Staphylococcus aureus (23.7%), and Pseudomonas spp. (17.2%). Most of the isolated bacteria showed high resistance to ampicillin (88.5%), ceftriaxone (84.5%), amoxicillin (81.9%), and tetracycline (74.5%). About 72.5% of Proteus spp. and 62.2% of Pseudomonas spp. have developed resistance to one and more antibiotics used to treat them. This retrospective study also revealed the overall antibiotic resistance rate of bacterial isolates was increased nearly twofold (P = 0.001) over the last decade. Relatively, ciprofloxacin and gentamicin were the most effective antibiotics against all the isolates. In conclusion, antibiotic-resistant bacteria are alarmingly increasing in Wollo area, northeastern Ethiopia, and becoming a major public health problem in the management of patients with middle ear infection.


Introduction
Ear infection is a common clinical problem throughout the world and the major cause of preventable hearing loss in the developing world [1,2]. Microbial agents can infect the middle and external parts of the ear and may involve the skin, cartilage, periosteum, ear canal, and tympanic and mastoid cavities [3]. Ear infection can be classified as acute suppurative otitis media (ASOM), chronic suppurative otitis media (CSOM), or otitis externa (OE) [4]. Its chronic form is a serious problem in all age groups with less chance of recovery. In certain cases this condition can lead to serious life-threatening complications, such as hearing impairment, brain abscesses, or meningitis, mostly in childhood and late in life [2,4]. According to the World Health Organization (WHO) estimates of 2015 over 5% of the world's populations (328 million adults and 32 million children) have disabling hearing loss. The highest prevalence is found in the Asia-Pacific, South Asia, and Sub-Saharan African regions. Half of all cases of hearing loss are avoidable through primary prevention while many can be treated. A leading cause of hearing loss in younger ages, particularly in low-and middle-income countries, is untreated ear infections, often with discharge from the ear. Vaccine-preventable infectious diseases such as rubella, meningitis, measles, or mumps can also lead to hearing loss [2,4].
The causative agents of ear infection might be bacterial, viral, or fungal. However, the major causative agents of ear infection are bacterial isolates such as Pseudomonas aeruginosa, Escherichia coli, S. aureus, Streptococcus pyogenes, 2 International Journal of Microbiology Proteus mirabilis, Klebsiella spp., or mixed bacterial infection [5]. The microbiological profiles of ear infection are well documented in developed world. However, so far few studies have been conducted in most developing countries [6][7][8][9][10]. Moreover, the signs and symptoms of earache may often mislead the etiology of the infection, which makes it very difficult for the clinician to relate the disease to the exact etiology. Hence, the physician may advocate antibiotic therapy irrespective of the etiology of the disease. This may lead to unwanted economic loss, stress to the patient if the ear infection is due to the virus or fungi, and foremost antibiotic resistance [11]. For these reasons, it is very important to study the microbiological profiles of ear infection and their extent of antibiotic resistance in those developing countries for the proper management of patients with ear infections. Moreover, antibiotic resistance is a growing global problem listed among major threats to human health by the World Health Organization [10].
Ethiopia is among the least developing countries and there are only a handful of studies reported so far that showed the prevalence and antibiotic resistance profile of bacterial isolates associated with ear infections [12][13][14][15][16][17][18][19]. Therefore, in this study, we showed a ten-year retrospective analysis of culture and antibiotic susceptibility test results of middle ear discharge from patients suspected of having a middle ear infection and referred to Dessie Regional Health Research Laboratory (DRHRL), located in Dessie town, northeastern Ethiopia.

Study Design, Site, and Data
Collection. This retrospective study was conducted from June 1 to August 31, 2012. During this period we reviewed culture and antibiotic susceptibility test results of middle ear discharges from 1225 patients suspected of middle ear bacterial infection and referred to DRHRL from September 2001 to September 2011 for antimicrobial sensitive test. DRHRL is located in Dessie town, South Wollo Zone, northeastern Ethiopia. This health research laboratory is the only regional referral laboratory found in the northeastern parts in Ethiopia serving populations of more than 4 million surrounding it [20]. Patient data were collected by the principal investigators using retrospective chart review data collection method based on the articulated aims of this study. Thus, all patient information registered in microbiology laboratory unit patient registration books from September 2001 to September 2011 were collected, including patient sociodemographic characteristics, isolated bacteria from middle ear discharge, and their antibiotic susceptibility test results categorized in each year of the study period. Of note, data with incomplete records or illegible handwriting were excluded from this study.

Laboratory Diagnostic Methods.
As the standard operational procedure of DRHRL microbiology unit shows, ear discharge specimens were taken aseptically using sterilized cotton swabs from each patient; then specimens were inoculated on MacConkey agar, blood agar, mannitol salt agar, and chocolate agar plates using sterilized 0.001 mL inoculation loop (all media from Oxoid Limited, UK). The plates of MacConkey, blood, and mannitol salt agar were placed in an aerobic incubator while the chocolate plate was incubated in a carbon-dioxide enriched atmosphere at 37 ∘ C for 24 hours. The swarming feature of Proteus spp. was managed by subculturing mixed colonies into MacConkey agar that has bile salt and by adding 5% of 90% ethanol. Bacterial isolation and identification were performed according to standard microbiological methods as described in the notable book of Monica Cheesbrough [21]. In brief, Gram-negative bacteria were identified by performing a series of biochemical tests, namely, triple sugar iron agar, indole, Simon's citrate agar, lysine iron agar, urea, mannitol, and motility. Gram-positive bacteria were identified based on their Gram reaction, novobiocin, catalase, and coagulase test results.

Statistical Analysis.
Data were checked for completeness, cleaned manually, entered, and analyzed using SPSS version 20 statistical software and Excel. The chi-square test ( 2 ) was used to measure the association between sex and age with susceptibility to middle ear infection and paired Student's -test was applied to compare antimicrobial resistance rates between the start and end of the study period. The value of < 0.05 was considered to show a statistically significant difference.

Ethical Considerations.
Ethical clearance was obtained from Ethical Review Committee of Wollo University. A formal supportive letter was also obtained from the Wollo University to the head of Dessie Regional Health Research Laboratory for cooperation and permission to get the tenyear data record. Patient privacy was protected by deidentification of records. Names of patients were replaced by initials. All data obtained in the course of the study were kept confidential and used only for this study.

Overall Antibiotic Susceptibility Profiles of Isolated Bacteria from Ear Discharge.
During the ten-year period 17 different antimicrobial agents were used to test the antibiotic susceptibility patterns of the pathogenic bacteria isolated from ear discharges. The overall susceptibility profiles of bacterial isolates are shown in Table 3. Out of the total antibiotics examined during the ten-year period, ampicillin had the highest overall resistance rate (88.5%) followed by ceftriaxone (84.5%), amoxicillin (81.9%), and tetracycline (74.5%). Conversely, majority of bacterial isolates were susceptible to ciprofloxacin, gentamicin, and nitrofurantoin with overall resistance rates of 6%, 17.2%, and 21.5%, respectively.

Species Specific Antibiotic Susceptibility Profile of Pathogenic Bacteria Isolated from Ear Discharges.
Species specific antibiotic susceptibility profiles are displayed in Table 4. Proteus spp., the most frequently isolated bacterium, showed high resistance rate (>74%) to each of the following antibiotics: amoxicillin, tetracycline, ampicillin, chloramphenicol, ceftriaxone, doxycycline, erythromycin, cefotaxime, and vancomycin. Likewise, S. aureus showed a resistance rate of (>52%) to each of the following antibiotics: amoxicillin, tetracycline, ampicillin, and cefotaxime. Pseudomonas spp., the third most common isolates, exhibited resistance rates of (>73%) to each of the following antibiotics: amoxicillin, tetracycline, TMP-SMX, ceftriaxone, doxycycline, erythromycin, and cefotaxime. E. coli had also showed resistance rates of more than 63% for each of the following antibiotics: amoxicillin, tetracycline, ampicillin, doxycycline, and erythromycin. In contrast, almost all the isolated pathogenic bacteria were susceptible to ciprofloxacin and gentamicin with resistance rates of 0%-10% and 2%-25%, respectively.

Multi-Antibiotic-Resistant Pathogenic Bacteria Isolated from Ear
Discharges. Almost all the isolated bacteria were found to be resistant to one and more of the commonly used antibiotics (Figure 1(a)). Among the total Proteus spp. isolated, the majority (72.5%) of the isolates have developed resistance to one and more antibiotics in clinical use. Similarly, about two-thirds of (62.2%) Pseudomonas spp. isolates were able to resist one and more antibiotics commonly used to treat them. About 34%-59.9% of the other bacterial isolates have also showed overall antibiotic resistance to one and more antibiotics used in clinics. Comparing the overall antibiotic resistance rate of bacterial isolates from start to end of the study periods (2001-2 versus 2010-11) the resistance rate to one and more of the antibiotics was increased nearly twofold (34% versus 66%, P = 0.001) (Figure 1(b)). spp.

Discussion
Ear infection is a more frequent treatable health care problem worldwide, yet if left untreated, it can cause a serious complication such as a speech development disorder, hearing loss, distress in patients and their family quality of life, and economic burden on the health care system [2]. The burden and prevalence of ear infection are more intense in developing countries due to the poor living standard and hygienic conditions along with lack of proper nutrition [5,19,23]. Thus, highlighting the etiologies of ear infection and their antibiotic susceptibility pattern will help to lessen the severe International Journal of Microbiology 5 Table 4: Antibiotic susceptibility profile of pathogenic bacteria isolated from ear discharges at Dessie Regional Health Research Laboratory from 2001 to 2011.

E. coli
Citrobacter spp.  International Journal of Microbiology complication of the infection and guide the empirical antibiotic prescribed by the physicians, especially for developing countries [19,24]. On top of these issues, increased antimicrobial resistance is one of the greatest global public health challenges, which has been accelerated by overprescription of antibiotics worldwide. Infection with antibiotic-resistant bacteria may cause severe illness, increased mortality rates, and an increased risk of complications and admission to hospital and longer stay [24][25][26]. In light of these facts, this study revealed that Proteus spp., S. aureus, Pseudomonas spp., and E. coli were the most prevalent multi-antibiotic-resistant pathogenic bacteria isolated from suspected patient ear discharges with otitis media in Wollo area, the northeastern part of Ethiopia. This ten-year retrospective analysis revealed that gender has no influence on the risk of acquiring middle ear infection. Likewise, most investigators have reported no clear gender based difference exists in the risk of acquiring middle ear infection [23,27]. However, we found that age has a strong association with the risk of acquiring middle ear infection. Similar to our finding other previously reported data from Ethiopia and many other countries also showed age has significant influence on the risk of acquiring middle ear infections [18,27]. For example, children are highly vulnerable to frequent ear infection due to pathogenic bacteria colonization in the middle ear or upper respiratory tract [12,19,[28][29][30].
In this study, the main pathogenic bacterium associated with middle ear infection was Proteus spp. followed by S. aureus and Pseudomonas spp., respectively. Similarly, previously published articles from our study area and other parts of Ethiopia also reported Proteus spp. were the foremost bacteria associated with middle ear infection followed by the later ones [12][13][14][15][16][17][18][19]. Although it needs a further nationwide study, taking into account our study finding and others, it seems that Proteus spp. is the leading bacterial isolates associated with middle ear infection in Ethiopia. Conversely, several other published data pieces from Africa and elsewhere in the world reported Pseudomonas spp., mainly P. aeruginosa, is the primary pathogenic bacteria associated with middle ear infection [5,[31][32][33][34][35][36][37]. One possible explanation for this difference might be due to climate and geographical variations between Ethiopia and those countries. It is noteworthy that this study and many other previously reported data indicated S. aureus is the second common bacterial isolates that often associated with middle ear infection [15,18,32,34,37].
Ear infection is among the most common illnesses that leads to overprescription of antibiotic use, one of the reasons for the emergence of antibiotic resistant pathogenic bacteria [23,24]. In light of this evidence, our study revealed that most of the isolated pathogenic bacteria have become resistant to all the easily available antibiotics. In general, ampicillin, ceftriaxone, amoxicillin, and tetracycline had shown the highest antibiotic resistance rates to all bacterial pathogens isolated from middle ear discharge, respectively. Correspondingly, tetracycline, ampicillin, ceftriaxone, and erythromycin were the most clinically used antibiotics that showed a higher resistance rate to Proteus spp., which is in line with earlier reports from Ethiopia, Nigeria, and Egypt [17,18,38,39].
Likewise, amoxicillin and ampicillin showed higher resistance rates to S. aureus; similar high percentage of resistance was also reported from Ethiopia and elsewhere in the world [5,17,18,40,41]. Amoxicillin, TMP-SMX, and erythromycin also showed a high level of resistance to Pseudomonas spp.; these findings are consistent with other studies [17][18][19]42]. Among the total Proteus spp. and Pseudomonas spp. isolated during the ten-year period about 72.5% and 62.2% of the isolated bacteria have developed resistance to one and more antibiotics that were once in clinical use, respectively. Overall, more than half of the bacterial isolates of this study were characterized as multi-antibiotic-resistant pathogenic bacteria. The reason for this high degree of multiantibiotic resistance might be linked to indiscriminate use of antibiotics, including animal husbandry, self-medication, and poor infection prevention and control practices as indicated by the recent WHO antimicrobial resistance report and earlier studies [10,19,43,44].
Furthermore, this study revealed multi-antibioticresistant pathogenic bacteria have become increasing threats of otitis media over the last decade in Wollo area, northeastern Ethiopia. By comparing the start and end of the study periods (2001-2 versus 2010-11), the overall resistance rate of all bacterial isolates to one and more of the antibiotics was increased nearly twofold. This finding is higher than studies reported from other African countries and elsewhere in the world [45,46]. This increasing trend of multiantibiotic resistance rate against middle ear bacterial pathogens such as Proteus spp. and Pseudomonas spp. calls for more careful attention empirical treatment of middle ear infections in the study area.
Interestingly, our study revealed almost all the isolated pathogenic bacteria were considerably susceptible to ciprofloxacin and gentamicin. Particularly, ciprofloxacin was shown to be highly effective for the three leading pathogenic bacteria associated with middle ear infection in this study: Proteus spp., S. aureus, and Pseudomonas spp. Likewise, with a slight variation several other authors have shown a similar high efficiency of ciprofloxacin against these bacterial species [5,15,17,[47][48][49][50][51]. Thus, we propose that ciprofloxacin can be taken as a first-line optional treatment for middle ear infection in Wollo area, northeast Ethiopia, as it is available in oral, injection, and topical ear drops formulation [48,51]. This suggestion holds true, since the current first-line treatment for both acute and chronic otitis media in Ethiopia is amoxicillin [52], which already showed a high resistance rate (81.9%) for the majority of the bacterial isolates in this study and in other published data from different parts of Ethiopia [14][15][16][17][18][19]53]. Moreover, empiric therapy is advocated when the antibiotic resistance level is 10% or less [54]; however, our finding revealed that the resistance level of most alternative antibiotic treatments for middle ear infection was more than 10%. This implies most common antibiotics used in our study area are no longer appropriate for empiric management of otitis media.
As limitations, the result of this study may not completely represent all the antibiotics used in the clinical practice in the study area since only those who visited the health research laboratory were included in the study and a retrospective International Journal of Microbiology 7 study incomplete record and illegible handwriting eliminated some culture results and antibiotics from the study.

Conclusion
This study revealed that age and the risk of acquiring middle ear infection are strongly associated and Proteus spp., S. aureus, and Pseudomonas spp. were the three predominant bacteria isolates from patient ear discharges suspected of otitis media. Almost all the isolated bacteria showed a considerable level of resistance to more than one antibiotic that are commonly used in primary health care centers; particularly, majority of isolated bacteria were found to be highly resistant to ampicillin, ceftriaxone, amoxicillin, and tetracycline treatments. More than two-thirds of Proteus spp. and Pseudomonas spp. have developed resistance to one and more antibiotics used to treat them. A comparative analysis of the overall antibiotic-resistance rate between the start and end of the study periods revealed that the rate of bacterial isolates resistant to one and more of the antibiotics was increased nearly twofold over the last decade in Wollo area, northeastern Ethiopia. This study also indicated ciprofloxacin and gentamicin are effective against all the bacterial isolates and most were highly sensitive to ciprofloxacin. In general, the result of this study revealed that antibiotic-resistant bacteria are alarmingly increasing in Wollo area, the northeastern part of Ethiopia, and becoming a major public health problem in the management patients with middle ear infection. Therefore, we strongly recommend nationwide antimicrobial surveillance to make the right recommendation of alternative antibiotics along with strict adherence to antibiotic policy to reduce the spread of drug resistant microbes in the country.