Otitis external infection is a common clinical feature observed as an acute or chronic disease [1]. Most studies reported that otitis externa is frequently caused by Pseudomonas aeruginosa, followed by Staphylococcus epidermidis and Staphylococcus aureus. Almost 50% percent of otitis external cases caused by a single organism and others caused by two organisms include a variety of bacteria spp. and they are less frequently caused due to fungal agents such as Aspergillus spp. and Candida spp [1-4].

Otitis external infection caused by Staphylococcus spp. can easily be treated with a single antibiotic, whereas, infection due to P. aeruginosa requires a combination of topical antibiotics, such as aminoglycosides, polymyxin B, or fluoroquinolones with steroid preparations. However, treatment with systemic antibiotics is rarely needed for acute otitis external infection [1-5].

More recent studies from Jordan and other Middle Eastern Arab countries have shown that P. aeruginosa isolates from clinical specimens were commonly Multidrug Resistant (MDR) to many frequently used antibiotics in clinical medicines such as amikacin, aztreonam, gentamicin, piperacillin-tazobactam and ciprofloxacin, while they were mostly susceptible to imipenem, ceftazidime and colistin B [6-9]. Many studies have recommended treating patients with colistin B, gentamicin or ciprofloxacin topical preparations as the first-line treatment of otitis externa [2, 5, 10].

The prevalence of Extended-Spectrum β-Lactamases (ESBLs), carbapenemases (KPC), and Metallo-Beta-Lactamases (MBLs), especially VIM, IMP among P. aeruginosa has increased in recent years over the world including the Middle Eastern countries [6, 7, 11, 12].

P. aeruginosa carried a variety of virulence factors which allow the organism to develop biofilm and adhere to infected tissue surfaces, increase its survival rate and induce damage in the infected host. The most important of these virulence factors are exopolysaccharide called alginate lyase enzyme, Las B elastase, exotoxin A, Exoenzyme S and U [6, 11, 12].

This study investigated the spectrum of microbial causative agents of otitis external infections among Jordanian patients, with emphasis on P. aeruginosa and its association with antimicrobial susceptibility pattern and virulence genes

This prospective convenience sampling study was conducted on patients presented to ENT Clinics of The Jordan University Hospital (JUH), and who were diagnosed with otitis external infections over the period from January 2017 through September 2017. A total of 128 patients were included, and specific biographical data of each patient was recorded on special forms as part of routine clinical investigation. The form included age, gender, name, and duration of hospitalization, disease history, clinical diagnosis, and general treatment with antibiotics if they were consumed was at the time of sampling the specimens.

This study was first approved by the Schools of Medicine and Graduate Studies, The University of Jordan, Amman, Jordan. The ethical approval was obtained from the Institutional Review Board (IRB) and The ethical committee of the Jordan University Hospital (2/2017, 10/1/2017), and signed consent was obtained from each examined patient.

Two clinical samples were collected from the discharge and external auditory canals of each patient with symptoms of otitis external infection using one cotton swab carried in transport medium, and the second was a wet swab immersed in physiological saline. The first swab was cultured on blood, chocolate agar, MacConkey agar and Sabouraud dextrose agar (Oxoid, England). The second swab was used for wet preparation and Gram-stain. All positive bacterial cultures were first identified using Gram-stain, oxidase and catalase tests, secondly, they were identified according to conventional methods described by Baily & Scott's/Diagnostic Microbiology¹³ and few isolates were identified using ViteK 2 System (Biomeriex, France). All culture plates were incubated for 2 days at 37°C.

Fungal positive cultures were identified by macroscopic and microscopic examination and suspected Candida isolates by using further CandidaChrom agar (Oxoid, England) and germ tube test [13]. All fungal culture plates were incubated additionally for 7 days at room temperature (20-24 °C). First identified P. aeruginosa isolates were subcultured on Cetrimide Pseudomonas Selective Agar Base (Merck, Germany), incubated for 24-48 hours at 37 °C, and examined for the presence of blood hemolysis, fluorescent-colored colonies with yellow or blue-green pigmentation. All P. aeruginosa isolates were inoculated and stored in cryogenic tubes containing brain-heart infusion broth with 20% glycerol at -75 ºC for further investigation and were confirmed as P.aeruginosa using specific primers and PCR.

The demographic characteristics and clinical features of 128 examined patients are presented in Table 1. The spectrum of microorganisms isolates from ear samples was 105/128 (82%) as shown in Table 2. The list included all bacterial isolates 68/82 (82.9%) which revealed significant growth based on the number of detected bacterial colonies (≥10) in culture plates. Fungal isolates accounted for 14/82 (17.1%), and mixed cultures 17/82 (20.7%) were defined as one significant bacterial type isolate (≥10), and the second with few colonies less than 10 of any organism.

A total of 28 (22%) of P. aeruginosa isolates were recovered and confirmed by biochemical tests and PCR. A yellow-green pigment (pyoverdin) was found in 16/28 (57%), while the production of the blue-green pigment (pyocyanin) was observed in 12/28 (43%). All P. aeruginosa isolates have shown complete hemolytic activity on both human and sheep blood agar plates after 48 hrs of incubation at 37ºC.

The antimicrobial susceptibility patterns of the 28 P. aeruginosa isolates are shown in Table 3. Ceftazidime, ciprofloxacin, imipenem and azetreonam were highly to moderately susceptible in the range of (82-68%), respectively, while the antimicrobial drugs; gentamicin and amikacin indicated high rates of resistance in the range of (72-57%), respectively. Only one isolate was resistant to colistin. Minimum inhibitory concentration ranges (MIC₅₀ and MIC₉₀) for 4 tested antibiotics are presented in Table 3. The most common virulence genes detected among P. aeruginosa isolates were algD and lasB (100%), followed by toxA gene (82%), exoU (72%), pilB (54%), exoS(25%) (Table 4). While 16/28(57%) of P. aeruginosa isolates were positive for potential KPC genes, but all were negative for the presence of potential VIM-2 and IMP-15 genes (Table 4).

The present study showed a wide spectrum of 19 bacteria and fungi species recovered from ear samples of patients with otitis externa. The two most common organisms were P. aeruginosa and S. coagulase-negative, each accounted for 22% of isolates, whereas, fungal isolates were presented only by 17%. The majority of the otitis externa cases are caused by single bacterial pathogen as shown in Table 2. Additionally, it has been found that 17.9% of the samples were negative for any growth. The reason for such negative cultures is most probably due to the treatment of patients with antibiotics before taking their ear samples (Table 1).

A five-year retrospective study from New Zealand (2007-2011), with recorded data of 347 patients with otitis externa from Wellington Hospital, showed that P. aeruginosa was the most common organism (46.5%), while S. aureus was the second most common (31.9%) [3]. Most studies published during the last 10-years from various countries reported that microbial otitis externa is frequently caused by P. aeruginosa and less frequently by other Gram-negative bacteria and S. aureus and fungi [3, 5, 9, 20].

Two old Jordanian studies have reported the spectrum of organisms isolated from a general ear infection as follows. The first study has investigated patients with otitis externa in the South of Jordan, and it has reported that positive culture results accounted for the following: P. aeruginosa (39%), Aspergillus spp (27%), Candida albicans (18%), S. aureus (18%), and no growth was detected in 8.5% [21]. The second study has investigated all ear infections in the Jordanian city Al-Zarqa and indicated that P. aeruginosa was found in 41.7%, followed by Aspergillus species (19.4%), Candida albicans (10.6%), S. aureus (16.1%) and Proteus mirabilis (2.8%) [22]. Both studies have shown higher percentages of positive fungi isolates than this study.

The present study shows that almost all patients with positive P. aeruginosa isolates had ear discharge (82.1%) and were already treated with antibiotics (75%) (Table 1). The study also found MDR P. aeruginosa which accounted for 39% of the isolates including resistant to antipseudomonal drugs, while a recent study in Jordan by Al dawodeyah et al. [6] showed that MDR P. aeruginosa isolates accounted for 52.5% of all isolates from respiratory tract samples of hospitalized patients, and all were susceptible to colistin. While the present study has indicated that one out of 28 P. aeruginosa isolates was resistant to colistin (3%) (Table 3). The detection of one P. aeruginosa isolate resistant to colistin is alarming, since this finding has not been previously reported in all recent studies carried out either in Jordan or other Arab neighboring countries among P. aeruginosa clinical isolates [6-8, 20, 23, 24].

Generally, the studies from this region demonstrated that P. aeruginosa isolates from the ear and other human infections were still moderately susceptible to antipseudomonal drugs such as piperacillin, piperacillin–tazobactam, ticarcillin-clavulanate and ceftazidime and imipenem [6, 8, 21, 24]. Recently, studies found that MDR P. aeruginosa clones can be associated with MBLs genes, mostly VIM and IMP types, which can be acquired by either chromosomal mutations or horizontal gene transfer. [25-26]. Therefore, studies investigated the incidence of MBLs and BlaKPC genes in clinical isolates of P. aeruginosa. It is highly important to select a proper treatment and to avoid the development of chronic cases.

The present study found that all 28 P. aeruginosa carried many potential virulence factors, including hemolysis and pigments as well as lasB, algD, toxA, exoU, and pil B genes in the range (100-54%), respectively (Table 4), and this result is similar to the recently reported study about respiratory isolates in Jordan [6]. Other studies found high prevalence of toxA, exoU, and pil B genes in P. aeruginosa clinical isolates from other parts of the body [11, 27]. However, this study has not found any significant relationship (P=0.631) between MDR P. aeruginosa isolates and the presence of potential virulence genes.

In conclusion, this study has demonstrated that MDR P. aeruginosa isolates associated with certain virulence factors are important causative agents of otitis externa in Jordanian patients.