The Open Microbiology Journal




ISSN: 1874-2858 ― Volume 14, 2020
RESEARCH ARTICLE

Otitis External Infections Among Jordanian Patients with Emphasis on Pathogenic Characteristics of Pseudomonas aeruginosa Isolates



Lubna Y. ALjaafreha1, Mohmmed Tawalbeh2, Asem A. Shehabi1, *
1 Department of Pathology-Microbiology, School of Medicine, The Jordan University, Queen Rania str. 100, Amman, Jordan
2 Department of Special Surgery/Division of Eye-Nose-Throat, The Jordan University Hospital, Queen Rania str. 100, Amman, Jordan

Abstract

Introduction:

Otitis external infection is an inflammation of the ear canal frequently caused by Pseudomonas aeruginosa, followed by Staphylococcus epidermis and Staphylococcus auerus.

Objective:

This study investigated the spectrum of bacterial and fungal agents that cause otitis external infection in Jordanian patients with an emphasis on important antimicrobial resistance genes and putative virulence factors of P. aeruginosa isolates using molecular PCR methods.

Methods:

A total of 128 ear swab samples were obtained from outpatients with otitis external infection of Ear-Nose-Throat Clinic (ENT) from the Jordan University Hospital (JUH). All samples were cultured for bacteria and fungi and their growth was identified by macroscopic and microscopic examination as well as recommended biochemical tests.

Results:

Positive growth of bacteria and fungi were found in 105/128 (82%) of the examined cases. A total of 28 (22%) of the recovered organisms from ear samples were P. aeruginosa. A total of 11/28 (39%) of P. aeruginosa isolates were Multidrug-Resistant (MDR) which are resistant to three or more antibiotic classes. Both blaIMP-15 and VIM genes were not detected, while KPC genes were found in 57% among all isolates. The rates of the potential virulence genes found among 28 P. aeruginosa isolates were as follows: lasB, algD, toxA, exoU PilB and exoS at 100%, 100%, 82%, 72%, 54% and 25%, respectively. All isolates produced beta hemolysis on both human and sheep blood agar and showed either the pigment pyoverdin (57.1%) or pyocyanin (42.8%).

Conclusion:

Accurate identification of the causative agent of otitis external infection and its susceptibility to antibiotics especially P.aeruginosa is highly important for successful treatment. No significant relationship has been found between MDR P. aeruginosa and the presence of virulence genes.

Keywords: Otitis externa, Jordanian patients, Causative organisms, P. Aeruginosa, Antimcicrobial resistanc, Virulence factors.


Article Information


Identifiers and Pagination:

Year: 2019
Volume: 13
First Page: 292
Last Page: 296
Publisher Id: TOMICROJ-13-292
DOI: 10.2174/1874285801913010292

Article History:

Received Date: 10/07/2019
Revision Received Date: 25/10/2019
Acceptance Date: 27/10/2019
Electronic publication date: 11/12/2019
Collection year: 2019

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© 2019 ALjaafreha et al.

open-access license: This is an open access article distributed under the terms of the Creative Commons Attribution 4.0 International Public License (CC-BY 4.0), a copy of which is available at: (https://creativecommons.org/licenses/by/4.0/legalcode). This license permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.


* Address correspondence to this author at Department of Pathology-Microbiology, School of Medicine, The Jordan University, Queen Rania str. 100, Amman, Jordan; Tel: +962795800618; E-mails: asashehabi2@gmail.com and ashehabi@ju.edu.jo





1. INTRODUCTION

Otitis external infection is a common clinical feature observed as an acute or chronic disease [1Wipperman J. Otitis externa. Prim Care 2014; 41(1): 1-9.
[http://dx.doi.org/10.1016/j.pop.2013.10.001] [PMID: 24439876]
]. 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 [1Wipperman J. Otitis externa. Prim Care 2014; 41(1): 1-9.
[http://dx.doi.org/10.1016/j.pop.2013.10.001] [PMID: 24439876]
-4Lee H, Kim J, Nguyen V. Ear infections: Otitis externa and otitis media. Prim Care 2013; 40(3): 671-86.
[http://dx.doi.org/10.1016/j.pop.2013.05.005] [PMID: 23958363]
].

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 [1Wipperman J. Otitis externa. Prim Care 2014; 41(1): 1-9.
[http://dx.doi.org/10.1016/j.pop.2013.10.001] [PMID: 24439876]
-5Schaefer P, Baugh RF. Acute otitis externa: An update. Am Fam Physician 2012; 86(11): 1055-61.
[PMID: 23198673]
].

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 [6Al Dawodeyah HY, Obeidat N, Abu-Qatouseh LF, Shehabi AA. Antimicrobial resistance and putative virulence genes of Pseudomonas aeruginosa isolates from patients with respiratory tract infection. Germs 2018; 8(1): 31-40.
[http://dx.doi.org/10.18683/germs.2018.1130] [PMID: 29564246]
-9Ninkovic G, Dullo V, Saunders NC. Microbiology of otitis externa in the secondary care in United Kingdom and antimicrobial sensitivity. Auris Nasus Larynx 2008; 35(4): 480-4.
[http://dx.doi.org/10.1016/j.anl.2007.09.013] [PMID: 18314283]
]. Many studies have recommended treating patients with colistin B, gentamicin or ciprofloxacin topical preparations as the first-line treatment of otitis externa [2Musa TS, Bemu AN, Grema US, Kirfi AM. Pattern of otitis externa in Kaduna Nigeria. Pan Afr Med J 2015; 21: 165.
[http://dx.doi.org/10.11604/pamj.2015.21.165.5577] [PMID: 26328002]
, 5Schaefer P, Baugh RF. Acute otitis externa: An update. Am Fam Physician 2012; 86(11): 1055-61.
[PMID: 23198673]
, 10Rostami S, Farajzadeh Sheikh A, Shoja S, et al. Investigating of four main carbapenem-resistance mechanisms in high-level carbapenem resistant Pseudomonas aeruginosa isolated from burn patients. J Chin Med Assoc 2018; 81(2): 127-32.
[http://dx.doi.org/10.1016/j.jcma.2017.08.016] [PMID: 29033110]
].

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 [6Al Dawodeyah HY, Obeidat N, Abu-Qatouseh LF, Shehabi AA. Antimicrobial resistance and putative virulence genes of Pseudomonas aeruginosa isolates from patients with respiratory tract infection. Germs 2018; 8(1): 31-40.
[http://dx.doi.org/10.18683/germs.2018.1130] [PMID: 29564246]
, 7Mansour SA, Eldaly O, Jiman-Fatani A, Mohamed ML, Ibrahim EM. Epidemiological characterization of P. aeruginosa isolates of intensive care units in Egypt and Saudi Arabia. East Mediterr Health J 2013; 19(1): 71-80.
[http://dx.doi.org/10.26719/2013.19.1.71] [PMID: 23520909]
, 11Nikbin VS, Aslani MM, Sharafi Z, Hashemipour M, Shahcheraghi F, Ebrahimipour GH. Molecular identification and detection of virulence genes among Pseudomonas aeruginosa isolated from different infectious origins. Iran J Microbiol 2012; 4(3): 118-23.
[PMID: 23066485]
, 12Bradbury RS, Roddam LF, Merritt A, Reid DW, Champion AC. Virulence gene distribution in clinical, nosocomial and environmental isolates of Pseudomonas aeruginosa. J Med Microbiol 2010; 59(Pt 8): 881-90.
[http://dx.doi.org/10.1099/jmm.0.018283-0] [PMID: 20430902]
].

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 [6Al Dawodeyah HY, Obeidat N, Abu-Qatouseh LF, Shehabi AA. Antimicrobial resistance and putative virulence genes of Pseudomonas aeruginosa isolates from patients with respiratory tract infection. Germs 2018; 8(1): 31-40.
[http://dx.doi.org/10.18683/germs.2018.1130] [PMID: 29564246]
, 11Nikbin VS, Aslani MM, Sharafi Z, Hashemipour M, Shahcheraghi F, Ebrahimipour GH. Molecular identification and detection of virulence genes among Pseudomonas aeruginosa isolated from different infectious origins. Iran J Microbiol 2012; 4(3): 118-23.
[PMID: 23066485]
, 12Bradbury RS, Roddam LF, Merritt A, Reid DW, Champion AC. Virulence gene distribution in clinical, nosocomial and environmental isolates of Pseudomonas aeruginosa. J Med Microbiol 2010; 59(Pt 8): 881-90.
[http://dx.doi.org/10.1099/jmm.0.018283-0] [PMID: 20430902]
].

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

2. METHODS

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 [13Baily & Scott's Diagnostic Microbiology. Editor, Patricia Tille, 14th Edition, 2014. ISBN: 9780323354820, Imprint: Mosby, St. Louis, Missouri ,USA. ]. 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.

2.1. Antimicrobial Susceptibility Using Disc Diffusion Method

Confirmed P. aeruginosa isolates were first examined for antimicrobial susceptibility using disc diffusion test according to the guidelines of the Clinical Laboratory and Standard Institute (CLSI, 2015) [14Clinical Laboratory and Standards Institute (CLSI). Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically; approved standard – tenth edition. CLSI document M07-A10 Villanova, PA, USA: CLSI,. 2016.]. Second, all MDR P. aeruginosa isolates have their minimum inhibitory concentrations (MICs) tested by E-test for ceftazidime, colistin, amikacin, azetronam, imipenem. The antimicrobial susceptibility results were interpreted in accordance with CLSI [14Clinical Laboratory and Standards Institute (CLSI). Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically; approved standard – tenth edition. CLSI document M07-A10 Villanova, PA, USA: CLSI,. 2016.]. P.aeruginosa ATCC 27853 was included as a control strain during all tests.

P. aeruginosa isolates stored in cryotubes at -70ºC were thawed at room temperature, and cultured on blood agar. After incubation at 37ºC for 24 hours, a few colonies were selected from the agar, inoculated into Mueller Hinton broth and incubated at 37ºC for 18 hours, using the Wizard genomic DNA Purification Kit, Promega (USA) according to the instructions of the manufacturer the bacterial DNA was extracted. The bacterial plasmid was extracted using the EZ-10 Spin Column Plasmid DNA Minipreps Bio Basic kit (Canada) according to the manufacturer’s instructions. Two PCR assays were performed; one is specific for the genus Pseudomonas, while the other is specific for P. aeruginosa, and two pairs of primers were used for each assay based on 16S ribosomal DNA (rDNA) sequence as reported by Spilker et al. [15Spilker T, Coenye T, Vandamme P, LiPuma JJ. PCR-based assay for differentiation of Pseudomonas aeruginosa from other Pseudomonas species recovered from cystic fibrosis patients. J Clin Microbiol 2004; 42(5): 2074-9.
[http://dx.doi.org/10.1128/JCM.42.5.2074-2079.2004] [PMID: 15131172]
]. Virulence genes (alg D, las B, tox A) were detected as described by Wolska et al. [16Wolska K, Szweda P. Genetic features of clinical Pseudomonas aeruginosa strains. Pol J Microbiol 2009; 58(3): 255-60.
[PMID: 19899619]
], whereas, virulence genes (exo S and exo U) were detected as reported by Mitov et al. [17Mitov I, Strateva T, Markova B. Prevalence of virulence genes among bulgarian nosocomial and cystic fibrosis isolates of Pseudomonas aeruginosa. Braz J Microbiol 2010; 41(3): 588-95.
[http://dx.doi.org/10.1590/S1517-83822010000300008] [PMID: 24031533]
].

BlaKPC genes among P. aeruginosa isolates were detected as reported by Akpaka et al. [18Akpaka PE, Swanston WH, Ihemere HN, et al. Emergence of KPC-producing Pseudomonas aeruginosa in Trinidad and Tobago. J Clin Microbiol 2009; 47(8): 2670-1.
[http://dx.doi.org/10.1128/JCM.00362-09] [PMID: 19494081]
], while the two MBLs genes (blaIMP and blaVIM) were detected as described by Pitout et al. [19Pitout JDD, Gregson DB, Poirel L, McClure J-A, Le P, Church DL. Detection of Pseudomonas aeruginosa producing metallo-β-lactamases in a large centralized laboratory. J Clin Microbiol 2005; 43(7): 3129-35.
[http://dx.doi.org/10.1128/JCM.43.7.3129-3135.2005] [PMID: 16000424]
].

2.2. Statistical Analysis

Data generated from the study were tabulated on Microsoft Excel sheet and uploaded to the Statistical Package for Social Sciences, version 20 (IBM Corp, Armonk, NY, USA). The frequencies and percentages were calculated for categorical data. Pearson’s chi-squared test or Fisher’s exact test were applied to determine potential factors associated with P. aeruginosa and to determine whether there are any statistical differences between the groups. The level of significance was set at a p-value of 0.05 to test the hypothesis without association. Fisher’s exact test replaces the chi-squared test when the minimum expected count is less than five patients.

3. RESULTS

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).

4. DISCUSSION

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%) [3Jayakar R, Sanders J, Jones E. A study of acute otitis externa at Wellington Hospital, 2007-2011. Australas Med J 2014; 7(10): 392-9.
[http://dx.doi.org/10.4066/AMJ.2014.2094] [PMID: 25379060]
]. 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 [3Jayakar R, Sanders J, Jones E. A study of acute otitis externa at Wellington Hospital, 2007-2011. Australas Med J 2014; 7(10): 392-9.
[http://dx.doi.org/10.4066/AMJ.2014.2094] [PMID: 25379060]
, 5Schaefer P, Baugh RF. Acute otitis externa: An update. Am Fam Physician 2012; 86(11): 1055-61.
[PMID: 23198673]
, 9Ninkovic G, Dullo V, Saunders NC. Microbiology of otitis externa in the secondary care in United Kingdom and antimicrobial sensitivity. Auris Nasus Larynx 2008; 35(4): 480-4.
[http://dx.doi.org/10.1016/j.anl.2007.09.013] [PMID: 18314283]
, 20Kiakojuri K, Mahdavi Omran S, Jalili B, et al. Bacterial otitis externa in patients attending an ENT Clinic in Babol, North of Iran. Jundishapur J Microbiol 2016; 9(2)e23093
[http://dx.doi.org/10.5812/jjm.23093] [PMID: 27127584]
].

Table 1
Demographic characteristics of 128 investigated patients.


Table 2
Types of organisms isolated from ear discharges of 128 patients.


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% [21Al-Asaaf SM, Farhan MJ. Otitis externa in a localized area at the South of Jordan. Saudi Med J 2000; 21(10): 928-30.
[PMID: 11369954]
]. 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%) [22Battikhi MN, Ammar SI. Otitis externa infection in Jordan. Clinical and microbiological features. Saudi Med J 2004; 25(9): 1199-203.
[PMID: 15448765]
]. Both studies have shown higher percentages of positive fungi isolates than this study.

Table 3
Antimicrobial susceptibility pattern and MICs of 28 P. aeruginosa isolates.


Table 4
Distribution of potential virulence genes, blaKPC and MBLs genes among 28 P. aeruginosa isolates.


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. [6Al Dawodeyah HY, Obeidat N, Abu-Qatouseh LF, Shehabi AA. Antimicrobial resistance and putative virulence genes of Pseudomonas aeruginosa isolates from patients with respiratory tract infection. Germs 2018; 8(1): 31-40.
[http://dx.doi.org/10.18683/germs.2018.1130] [PMID: 29564246]
] 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 [6Al Dawodeyah HY, Obeidat N, Abu-Qatouseh LF, Shehabi AA. Antimicrobial resistance and putative virulence genes of Pseudomonas aeruginosa isolates from patients with respiratory tract infection. Germs 2018; 8(1): 31-40.
[http://dx.doi.org/10.18683/germs.2018.1130] [PMID: 29564246]
-8Hamze M, Mallat H, Dabboussi F, Achkar M. Antibiotic susceptibility and serotyping of clinical Pseudomonas aeruginosa isolates in Northern Lebanon. IAJAA 2012; 4(2): 1-6., 20Kiakojuri K, Mahdavi Omran S, Jalili B, et al. Bacterial otitis externa in patients attending an ENT Clinic in Babol, North of Iran. Jundishapur J Microbiol 2016; 9(2)e23093
[http://dx.doi.org/10.5812/jjm.23093] [PMID: 27127584]
, 23El Mahallawy WM, Zafer H, Ghaith MM. D. & Abdel hamid R. Lab based surveillance of multidrug resistant Pseudomonas aeruginosa in cairo university hospitals, Egypt. J Microbiol Experiment 2015; 2(2): 1-5., 24Asem A. Shehabi, Aya M. Kamal. Pseudomonas aeruginosa, a common opportunistic pathogen in Jordan: A short review article. IAJAA 2019; 9(1): 1.].

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 [6Al Dawodeyah HY, Obeidat N, Abu-Qatouseh LF, Shehabi AA. Antimicrobial resistance and putative virulence genes of Pseudomonas aeruginosa isolates from patients with respiratory tract infection. Germs 2018; 8(1): 31-40.
[http://dx.doi.org/10.18683/germs.2018.1130] [PMID: 29564246]
, 8Hamze M, Mallat H, Dabboussi F, Achkar M. Antibiotic susceptibility and serotyping of clinical Pseudomonas aeruginosa isolates in Northern Lebanon. IAJAA 2012; 4(2): 1-6., 21Al-Asaaf SM, Farhan MJ. Otitis externa in a localized area at the South of Jordan. Saudi Med J 2000; 21(10): 928-30.
[PMID: 11369954]
, 24Asem A. Shehabi, Aya M. Kamal. Pseudomonas aeruginosa, a common opportunistic pathogen in Jordan: A short review article. IAJAA 2019; 9(1): 1.]. 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. [25Voor In ’t Holt AF, Severin JA, Lesaffre EM, Vos MC. A systematic review and meta-analyses show that carbapenem use and medical devices are the leading risk factors for carbapenem-resistant Pseudomonas aeruginosa. Antimicrob Agents Chemother 2014; 58(5): 2626-37.
[http://dx.doi.org/10.1128/AAC.01758-13] [PMID: 24550343]
-26Rafiee R, Eftekhar F, Tabatabaei SA, Minaee Tehrani D. Prevalence of extended-spectrum and metallo β-Lactamase production in AmpC β-Lactamase producing Pseudomonas aeruginosa isolates from burns. Jundishapur J Microbiol 2014; 7(9)e16436
[http://dx.doi.org/10.5812/jjm.16436] [PMID: 25485066]
]. 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 [6Al Dawodeyah HY, Obeidat N, Abu-Qatouseh LF, Shehabi AA. Antimicrobial resistance and putative virulence genes of Pseudomonas aeruginosa isolates from patients with respiratory tract infection. Germs 2018; 8(1): 31-40.
[http://dx.doi.org/10.18683/germs.2018.1130] [PMID: 29564246]
]. Other studies found high prevalence of toxA, exoU, and pil B genes in P. aeruginosa clinical isolates from other parts of the body [11Nikbin VS, Aslani MM, Sharafi Z, Hashemipour M, Shahcheraghi F, Ebrahimipour GH. Molecular identification and detection of virulence genes among Pseudomonas aeruginosa isolated from different infectious origins. Iran J Microbiol 2012; 4(3): 118-23.
[PMID: 23066485]
, 27Shaver CM, Hauser AR. Relative contributions of Pseudomonas aeruginosa ExoU, ExoS, and ExoT to virulence in the lung. Infect Immun 2004; 72(12): 6969-77.
[http://dx.doi.org/10.1128/IAI.72.12.6969-6977.2004] [PMID: 15557619]
]. However, this study has not found any significant relationship (P=0.631) between MDR P. aeruginosa isolates and the presence of potential virulence genes.

CONCLUSION

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.

ETHICS APPROVAL AND CONSENT TO PARTICIPATE

The ethical approval was obtained from the Institutional Review Board (IRB) and the ethical Committee of the Jordan University Hospital, Jordan (2/2017, 10/1/2017).

HUMAN AND ANIMAL RIGHTS

Not Applicable.

CONSENT FOR PUBLICATION

Informed written consent was obtained from each participant.

AVAILABILITY OF DATA AND MATERIALS

The source of all clinical data of patients was obtained from their records at The Jordan University Hospital in Amman, and all sources of culture media, control bacterial strains, primers for virulence factors, potential resistance genes of ESBLs, KPC and MBLs are mentioned within text.

FUNDING

This work was supported by a grant from The Jordan University, Dean of Research (No. 2017-2016/96), Jordan.

CONFLICT OF INTEREST

The authors declare no conflict of interest, financial or otherwise.

ACKNOWLEDGEMENTS

Thanks for the administration of The Jordan University Hospital in Amman, for its permission to carry out this study.

REFERENCES

[1] Wipperman J. Otitis externa. Prim Care 2014; 41(1): 1-9.
[http://dx.doi.org/10.1016/j.pop.2013.10.001] [PMID: 24439876]
[2] Musa TS, Bemu AN, Grema US, Kirfi AM. Pattern of otitis externa in Kaduna Nigeria. Pan Afr Med J 2015; 21: 165.
[http://dx.doi.org/10.11604/pamj.2015.21.165.5577] [PMID: 26328002]
[3] Jayakar R, Sanders J, Jones E. A study of acute otitis externa at Wellington Hospital, 2007-2011. Australas Med J 2014; 7(10): 392-9.
[http://dx.doi.org/10.4066/AMJ.2014.2094] [PMID: 25379060]
[4] Lee H, Kim J, Nguyen V. Ear infections: Otitis externa and otitis media. Prim Care 2013; 40(3): 671-86.
[http://dx.doi.org/10.1016/j.pop.2013.05.005] [PMID: 23958363]
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