Rapid Detection and Antibiotic Susceptibility of Genital Mycoplasma Isolated from Male with Urethritis and Prostatitis , Iraq

1Biology Department, College of Science-University of Babylon, Hilla-Iraq. 2Biology Department, College of Science for Women-University of Babylon, Hilla-Iraq. 3Clinical Science Department, College of PharmacyUniversity of Babylon, Hilla-Iraq. 4Urology Center, Hilla Teaching Hospital, Hilla-Iraq. 5Biology Department, College of Science-University of Babylon, Hilla-Iraq. 6College of Agriculture-University of Al-Qasim Green, , Hilla-Iraq.

Ureaplasma urealyticum and Mycoplasma hominis are most important genital mycoplasma responsible for many genitourinary tract infections.Nongonococcal urethritis (NGU) and bacterial prostatitis are most clinically significant infections that affect a wide spectrum of male age groups.The incorrect or inappropriate diagnosis and susceptibility testing leads to unsuitable medication and as a results delay of recovery and bad squeal like infertility.During a period of sex months, twenty two expressed prostate secretion (EPS) samples and eighteen urethral swabs were collected from patients with age (44.55±21.22years).All samples were inoculated to strip and after 24 hours at 37° C the results recorded.The results of detection of genital mycoplasma revealed that only 3 (16.7%) of expressed prostate secretion samples give positive results for both Ureaplasma urealyticum and Mycoplasma hominis while 10 (45.5%) urethral swab were positive for both Ureaplasma urealyticum and Mycoplasma hominis.Common age group of prostatitis infected with genital mycoplasma is (41-60 years) while urethritis was detected among all three groups.The results of antibiotic susceptibility revealed that all isolates were fully sensitive (100%) to all antibiotics incorporated within the well except for erythromycin, roxithromycin, tetracyclin and levofloxacin in which one isolate (7.7%) was exhibit resistance in both Ureaplasma urealyticum and Mycoplasma hominis.The current study conclude the genital mycoplasmas (Ureaplasma urealyticum and Mycoplasma hominis) are predominant among non gonococcal urethritis and the macrolides still the choice for treatment while prescription of quinolones and tetracyclin must be under control due to emergence of resistance isolates.Keywords: U. urealyticum, M. hominis, Prostatitis, EPS.
Mycoplasmas are cell wall lacking, small bacteria that not effected by cell wall synthesis inhibiting antibiotics.Clinically significant Mycoplasmas includes Ureaplasma urealyticum, Mycoplasma pneumoniae, Mycoplasma genitalium, and Mycoplasma hominis 1 .As a causative agents of non-gonococcal, con-chlamydial urethritis and prostatitis Ureaplasma urealyticum and Mycoplasma hominis were recovered from many males and females undergoing sexually transmitted disease with variable percentage 2,3 .
Inflammation of urethra called urethritis and it is may be due to bacterial, viral and trichomonal infection.Bacteria urethritis is most common and be on two form: gonococcal and nongonococcal urethritis.Nongonococcal urethritis is emerge due to bacterial infection other than those caused by gonococci.Commonly bacterial causes of nongonococcal urethritis comprises: CHYPERLINK "https://en.wikipedia.org/wiki/Chlamydia_trachomatis".HYPERLINK "https://en.wikipedia.org/wiki/Chlamydia_trachomatis" trachomatis, U. urealyticum and M. hominis 4,5 .Bacterial prostatitis is the infection of prostate gland and regards hard-treated urologic illness that need 2-12 weeks of antibiotics treatment to be cured effectively.Genitourinary mycoplasmas (U. urealyticum and M. hominis) can cause prostatitis and may implicated in the prostate cancer development 6,7 .
Both of U. urealyticum and M. hominis were associated with sexually transmitted diseases and have the ability to destroy genitourinary epithelial cells 8,9 .Three species have been isolated from the surface of the genitourinary tract mucosa: M. hominis, U. urealyticum and recently discovered Mycoplasma genitalium 10,11,12 .M. hominis colonizes the genito-urinary tract and behave as an opportunistic pathogen, causing urethritis, cervicitis, pelvic inflammatory disease or chorioamnionitis.U. urealyticum regarded the common pathogen among men with urethritis and its complications 13,14 .
Abuse and misuse of antibiotics leads to emerging of antibiotics resistance which push a risk for human health.Due to that they are targeting cell wall, the â-lactam antibiotics and vancomycin are not active against Mycoplasmas and their susceptibility be conferred to only agents that inhibit protein synthesis, including aminoglycosides, macrolides, tetracycline chloramphenicol.They are also sensitive to that anti-topoisomerases antibiotics like fluoroquinolones 15 .Spreading of resistance to tetracycline and erythromycin leading to the use of clindamycin, fluoroquinolones or other macrolides following the failure of treatment with tetracycline or erythromycin 16,17,18 .
The current study aimed to detect genital mycoplasma (U. urealyticum and M. hominis) and their antibiotic susceptibility among nongonococcal urethritis and prostatitis patients in Hilla city-Iraq.

Samples
Twenty two expressed prostate secretion (EPS) samples and eighteen urethral swabs were collected from patients with age (44.55±21.22years) during a period of 6 months.All samples were transfereed directly to transport media supplied with kit 19,20 .

Mycoplasma IES Plus Kit Principle
It is Dehydrated culture medium based assay for the screening, indicative enumeration and the identification of UU (U. urealyticum) and MH (M.hominis) in genitourinary tract of human.In the case of positive screening result, Mycoplasma IES Plus provides an extra strip for further susceptibility testing, which is partially based on CLSI (Clinical and Laboratory Standards Institute) recommendations.The mixed medium is prepared by mixing the freeze-dried powder and the diluent.After Mycoplasma has been cultivated, urease in UU will breakdown the urea and release NH3.Release of NH3 also occur by MH via breakdown of arginine by arginase leading to increases the pH of the liquid medium, the result is judged according to the color change of the indicator.The strip contains 11 antibiotics.Changing the color to red meaning growth of UU, MH or both although presence of antibiotics which reveal resistance to the specific antibiotic in the well 21 .

Mycoplasma IES Plus Kit Component
The kit components includes: 1-10 strips (with 30 wells have dehydrated culture medium and another supplied with antibiotics with different concentration).

Strip Inoculation
The sample transfereed directly to the transport medium, mixed well and transferred completely to the freeze-dried powder, and shake to mix completely.After the sample is inoculated, 0.5 ml of the inoculated medium incubated at 36-38 ! for 24 hours and if the color of the culture medium does not turn to red or peachblow, the sample could be deemed to be negative and no further operations are needed.When the color of the culture medium turns to red or peachblow, a 100 ìl of the culture medium will dispensed to the wells of strip, the strip was shacked to dissolve the coated materials and then one drop of the mineral oil will added to prevent evaporation during incubation.The cover the strip placed and the strip will incubated at 36-38 °C for 24 hours.The yellow color indicate negative results while peachblow to red indicate positive results.

RESULTS AND DISCUSSIONS
The results of detection of genital mycoplasma revealed that only 3 (16.7%) of   3).The positive and negative results for both Ureaplasma urealyticum and Mycoplasma hominis were showed in figure ( 2) and ( 3) respectively.Many studies stated that the coexistence of Ureaplasma urealyticum and Mycoplasma hominis was common and the affected age group 30-50 years.M. hominis is found as a colonizer of the genito-urinary tract in sexually active, and is also associated with bacterial vaginosis 22,23,24,25 .
U. urealyticum and M. hominis are prominent pathogens among men with urethritis and its complications and occur more frequently in the semen of prostatitis patients 11,26 .
lacking of cell wall, target of betalactam antibiotics and vancomycin, the most common and appropriate medication for urogenital infections caused by mycoplasmas depends upon tetracyclines, macrolides, and quinolones [15,27] .The results of antibiotic susceptibility revealed that all isolates were fully sensitive (100%) to all antibiotics incorporated within the well except for erythromycin, roxithromycin, tetracyclin and levofloxacin in which one isolate (7.7%) was exhibit resistance in both Ureaplasma urealyticum and Mycoplasma hominis.Our data was highly similar with those gathered from many studies which found that all genital mycoplasma were fully sensitive to doxycycline, josamycin, ofloxacin and pristinamycin 27,28,29,30,31 .Resistance to erythromycin were recorder for both of Ureaplasma urealyticum and Mycoplasma hominis isolated from genital samples of sexually active individuals.Resistance to erythromycin among mycoplasmsas is naturally occur while resistance to tetracyclin and quinolones can be attributed to antibiotic targets mutation and it may be related to higher pathogenicity 15,32,33 .
The current study conclude that the genital mycoplasmas (Ureaplasma urealyticum and Mycoplasma hominis) are common among non gonococcal urethritis and the macrolides still the choice for treatment while prescription of quinolones and tetracyclin must be under control due to emergence of resistance isolates.

Fig. 2 .
Fig. 2. Show the positive results of culture for both U. urealyticum and M. hominis

Fig. 3 .
Fig. 3. Show the negative results of culture for both U Ureaplasma urealyticum and Mycoplasma hominis

Table 1 .
Show the wells for identification and enumeration

Fig. 1. Show the Mycoplasma IES Plus strip Susceptibility tests (wells no. 6 to 30)
These wells are used to test the susceptibility of the strain with 11 antibiotics (table2).

Table 2 .
Show the wells for antibiotic susceptibility

Table 3 .
Distribution of genital mycoplasma among different age groups Ureaplasma urealyticum and Mycoplasma hominis while 10 (45.5%) urethral swab were positive for both.The common age group of prostatitis infected with genital mycoplasma is (41-60 years) while urethritis was detected among all three groups (table