Introduction

Urethritis is one of the most common sexually transmitted diseases (STDs) that is also defined as urethral inflammation (1-4). Some common clinical signs, such as stinging or itching mucoid, mucopurulent, and/or purulent discharge, dysuria and penile irritation, and urethritis can often be asymptomatic. Urethritis may occur due to infectious or non-infectious causes, such as using condoms or spermicide and mechanical traumas (1,2,4).

N. gonorrhoeae is the agent of gonococcal urethritis (GU), which is still the most important agent in developing countries, whereas Chlamydia trachomatis, Ureaplasma urealyticum, Mycoplasma species, Trichomonas vaginalis, Herpes simplex virus, and Adenoviruses are the causes of non-GU (NGU) in developed countries. More commonly microorganisms than N. gonorrhoeae are detected (1,3-6).

Penicillins, tetracyclines, fluoroquinolones, and oral third-generation cephalosporins are recommended for N. gonorrhoeae therapy (7-10). Macrolides, tetracyclines, and quinolones are antibiotics of choice for the treatment of Chlamydia, Mycoplasma, and Ureaplasma due to the emergence of resistance, and antibiotic susceptibilities should also be tested (11,12).

This study aimed to report the distribution of Neisseria gonorrhoeae, Chlamydia trachomatis, Ureaplasma urealyticum, and Mycoplasma hominis and the antibiotic resistance rates in clinical specimens obtained from male patients with urethritis in a third-step hospital between 2010 and 2019.

Method

Patient Population and Study Design

Between 2010 and 2019, 748 male patients with urethritis having clinical complaints and admitted to İstanbul Yeni Yüzyıl  University, Medical Faculty Gaziosmanpaşa  Hospital were evaluated. Male patients were 20-74 (mean; 38) years old. Patients had clinical signs, such as urethral discharge and/or dysuria. Urethral discharge, swab, and ejaculation samples were collected after the clinical examination using sterilized cotton swabs and placed in transport agar (SEEDSWAB ƳNo2, Eiken Chemical Co. Ltd. Tokyo, Japan). All samples were dyed with Gram’s stain and all preparations were examined for the presence of polymorph nuclear leukocytes and microorganisms.

N. gonorrhoeae, M. hominis, and U. urealyticum were isolated and their antibiotic susceptibilities were also tested. The antibiotic testing panels are changed in diagnostic kits over 10 years; thus, not all isolated strains could be tested for the same groups of antibiotics. The immune chromatographic method was used to detect C. trachomatis antigen.

Isolation of N. gonorrhoeae and Antibiotic Susceptibility Testing

All samples were cultivated on the Thayer-Martine agar and incubated for 24-48 h at 35 °C in 5% CO₂ atmospheric conditions to isolate N. gonorrhoeae. The identification was performed using VITEK-2. The antimicrobial susceptibility testing was performed by disc diffusion method for cefuroxime, ceftriaxone, penicillin, levofloxacin, ciprofloxacin, and tetracycline according to the recommendations of Centers for Disease Control and Prevention (2005) and Clinical & Laboratory Standards Institute (2012) (13,14). During the antimicrobial susceptibility testing, our results were confirmed with the results of clinical N. gonorrhoeae strains that were performed in an accredited laboratory to validate the internal quality of our test conditions.

Isolation of M. hominis and U. urealyticum and Antibiotic Susceptibility Testing

A quantitative commercial test (Mycoplasma IES, Autobio) was used to identify and test the antibiotic susceptibility (roxithromycin, clarithromycin, erythromycin, ofloxacin, tetracycline, ciprofloxacin, clindamycin, and levofloxacin) of M. hominis and U. urealyticum. This test detects urease and arginase activities by releasing NH₃ to identify Ureaplasma and Mycoplasma, respectively. The positive results were determined with color changes in the wells that contain antibiotics, wherein changes mean that the bacterium is resistant. The tests were performed following the manufacturer’s recommendations.

Detection of C. trachomatis Antigen

A rapid chromatographic immunoassay (Ultimed) was used to detect C. trachomatis antigen. The test was performed according to the manufacturer’s recommendations.

Results

Distributions of Patients, Clinical Samples, and Detected Microorganisms

A total of 987 patients with prediagnosed urethritis were evaluated. To determine the prevalence and antibiotic resistance rates of causative microorganisms, 748 (75.8%) male patients were included in the present study, wherein 165 (22.05%) were positive for at least one investigated microorganism. Among 165 patients with urethritis, 28 (16.9%) have mixed infection (infected with more than one causative agent). A total of 194 different microorganisms were detected. Except for one urine and three ejaculate samples, the majority of the clinical samples were urethral swabs.

The distributions of positive patients for urethritis according to years were shown in Table 1; in years 2015 and 2017 the positivity was the highest. In 2011, only eight patients were found to be infected with the investigated microorganisms.

U. urealyticum (114 patients) was revealed as the most prevalent microorganism over a 10-year period, which was followed by N. gonorrhoeae (43 patients), M. hominis (24 patients), and C. trachomatis (10 patients) (Figure 1).

Antibiotic Susceptibility Results

All 42 N. gonorrhoeae strains isolated over the 10-year period were susceptible (92.3-100%) to all six tested antibiotics (Table 2).

The resistance rate of U. urealyticum strains to clindamycin was 89.6%, followed by ciprofloxacin (78.5%) and tetracycline (43.9%). Strains were also intermediate resistant to ofloxacin (47.5%) (Table 3).

All M. hominis strains were resistant to erythromycin, followed by clarithromycin (90%), clindamycin (70%), and ciprofloxacin (43.8%) (Table 4).

Discussion

Urethritis is the most common syndrome among STDs, which are globally important public health concerns (3,5,15,16). Early diagnosis and treatment were implicated. Increasing resistance rates in agents of STDs is also very important and can cause treatment failure (3,17,18).

N. gonorrhoeae and C. trachomatis remain as the most important causative agents of urethritis in male patients (2,4,5,8). Asymptomatic cases are frequent and important for infection transmission (19-21). The body of evidence suggests that N. gonorrhoeae has a great capacity to develop antibiotic resistance, and in many countries, resistance rates are very high to penicillin, macrolides, tetracycline, and quinolones; therefore, ceftriaxone (extended-spectrum cephalosporin) has become an important antibiotic (17,18,22). The World Health Organization and CDC have emphasized the importance of surveillance studies performed by culture and antibiotic susceptibility testing to prevent the dissemination of multidrug-resistant N. gonorrhoeae strains (18).

Previous studies from India, Kuwait, Zimbabwe, and the United States reported that the detection rates of N. gonorrhoeae and C. trachomatis from male patients range 23.9-81.9% and 12.4-31.4%, respectively according to different geographical regions (17,23-25).

A study reported from France analyzed 1,944 cases of urethritis in male patients during 10 years and 38% of patients were found to be infected with C. trachomatis while the isolation rate of N. gonorrhoeae was 32.5%. Additionally, 6.7% of the patients were found to be co-infected (5). Our study also includes strains that are isolated from the samples obtained in male patients in the last 10 years. Our isolation rates were 21.6% and 5.2% for N. gonorrhoeae and C. trachomatis, respectively, and the difference between isolation rates can be associated with the number of included patients and techniques used to detect microorganisms.

Many literature reports on antibiotic susceptibility were also investigated. As an example, Hamasuna et al. (26) reported that the majority of N. gonorrhoeae strains were resistant to ciprofloxacin in Japan. The prevalence of fluoroquinolone resistance rates of N. gonorrhoeae has been reported to reach almost 70% (26-28).

A study by Buder et al. (18) in Germany revealed that a total of 537 N. gonorrhoeae strains were isolated during an extended surveillance program in the whole country, wherein none of the strains were found to be resistant to ceftriaxone but with resistance rates to azithromycin and ciprofloxacin of 10.8% and 64.9%, respectively. Consistent with Buder et al. (18), Latif et al. (17) reported that all N. gonorrhoeae strains were susceptible to ceftriaxone and cefixime, but resistance to ciprofloxacin and kanamycin were 18.6% and 2%, respectively. Contrarily, many literatures reported the susceptibility rates of N. gonorrhoeae strains showed to be decreased to cefixime (29,30).

Yeshanew and Geremew (15) found in Ethiopia that, among 207,044 gonorrhoeae suspected patients, 20.8% were confirmed to be infected with N. gonorrhoeae. All isolated strains were resistant to tetracycline (100%) and penicillin (100%), and resistance rates to ciprofloxacin, ceftriaxone, and cefoxitin were 52%, 48%, and 44%, respectively (15).

Some studies revealed that N. gonorrhoeae strains are found resistant to various antibiotics, even to ceftriaxone (15,26-28); however, in our study, none of the strains were resistant to ceftriaxone and cefuroxime and even penicillin-resistance rates were found to be low (6.6%).

In Turkey, Balıkçı and Aydın (31) reported that 1,226 N. gonorrhoeae strains were found to be susceptible to ceftriaxone, cefuroxime, and cefoxitin. However, resistance rates of penicillin, tetracycline, and ciprofloxacin were 62%, 61%, and 33%, respectively (31). Another study from Turkey revealed that, among 78 N. gonorrhoeae strains, the resistance rate was 64% for penicillin and 75.7% for tetracycline, whereas doxycycline, azithromycin, and ciprofloxacin were found to be the most effective antibiotics (22). In the present study, resistance rates to penicillin (6.6%), tetracycline (7.7%), and ciprofloxacin (8.3%) were much lower as mentioned above.

U. urealyticum and M. hominis is well known to colonize the genital tract when people were born, colonization rates increase up to 60% of the population after puberty, and that they can be responsible for some clinical manifestations, such as NGU, cervicitis, and cystitis (20,32). Generally, the prevalence of U. urealyticum in patients with NGU is nearly 6-60%, and infections are associated with socioeconomic status and education levels of the population (17,31).

Authors from Switzerland, China, Cuba, Africa, and Serbia informed that isolation rates of Ureaplasma and Mycoplasma from patients with urethritis range 13-89% according to different geographical regions (34-38).

Various reports from Turkey showed that isolation rates of U. urealyticum and M. hominis reached up to 82% and 30%, respectively (33). Pelit et al. (16) reported that U. urealyticum was the most prevalent (32%) microorganism that is isolated from the urethral discharges of 140 male patients followed by M. hominis (10.7%), C. trachomatis (8.6%), and N. gonorrhoeae (8.6%). Our study revealed that the isolation rate of U. urealyticum (58.8%) was higher than N. gonorrhoeae (21.6%), thus our results appear to support previous studies.

Quinolone resistance of Ureaplasma (0-62.5%) and Mycoplasma (17.6-41.2%) strains is increasing (32,39-41). Resistance rates of Mycoplasma and Ureaplasma strains to doxycycline, roxitromycin, and tetracycline were reported as 0-8%, 0-30%, and 19-50%, respectively. Thus doxycycline, clarithromycin, and azithromycin are most recommended antibiotics (42).

Our results revealed that resistance rates of U. urealyticum strains to clindamycin, quinolones, and tetracycline were 89.6%, 47.5-78.5%, and 43.9%, respectively. Additionally, Ureaplasma strains were susceptible to erythromycin (90.1%), roxithromycin (85.3%), and clarithromycin (83.6%). Similar to U. urealyticum, resistance rates of M. hominis strains were high to clindamycin (70%) and ciprofloxacin (43.8%). Tetracycline was also effective to Mycoplasma strains (90%) but not to Ureaplasma strains (36.6%). Therefore, roxithromycin was the most effective antibiotic against both Ureaplasma and Mycoplasma strains, with resistance rates of 14.7% and 28.6%, respectively.

Study Limitations

Limitations of our study include insufficient demographical data, knowledge about recent antibiotic usage and serological test results (anti-HIV, anti-syphilis, etc.), and the number of sexual partners or unprotected sexual intercourse. We also failed to perform antibiotic susceptibility testing for C. trachomatis.

Additionally, our training hospital is a third-step hospital, and patients are mostly admitted after a prior antibiotic therapy and due to financial difficulties, all diagnostic procedures could not be performed to clearly define urethritis etiology. Therefore, our number of positive cases, such as N. gonorrhoeae, is lower than some of the reported studies. The low prevalence rates could be the reason for lower resistance rates compared with previous studies.

Conclusion

Our results proved that, among the four major STD agents, U. urealyticum was the most common for the last decade in symptomatic male patients with urethritis. Roxithromycin is found to be the most effective antibiotic for both Ureaplasma and Mycoplasma strains. As mentioned in previous studies, N. gonorrhoeae strains were susceptible to mostly used therapeutic agents.

Ethics

Ethics Committee Approval: Ethical approval has been obtained from İstanbul Yeni Yuzyıl University Medical Faculty Research Ethics Committee (meeting date: 2020/04-05).

Informed Consent: Retrospective study.

Authorship Contributions

Surgical and Medical Practices: D.B.T., Concept: D.B.T., Design: D.B.T., D.G., F.K.Y., Data Collection or Processing: D.B.T., D.G., F.K.Y., Analysis or Interpretation: D.G., F.K.Y., Literature Search: D.G., F.K.Y., Writing: D.G., F.K.Y.

Conflict of Interest: No conflict of interest was declared by the authors.

Financial Disclosure: The authors declared that this study received no financial support.