INTRODUCTION

Infertility constitutes a grave emotional and social problem in societies where great importance is attached to having children ¹ Male infertility refers to the inability to achieve spontaneous pregnancy in a fertile female in one year. ² A decline in the male fertility may be attributed to many factors, which include testicular failure, urogenital abnormalities, immunologic problems, varicocele, genetic abnormalities, endocrine disturbances, systemic diseases, cancer, genital tract infections, environmental agents, distorted lifestyle and gonadotoxic factor exposure. ²Infection is a powerful mechanism that can lead to sperm damage, deformity

and eventually, male infertility. Genital tract infection and inflammation have been associated to 8-35% of male infertility cases. ³ Spermatozoa subsequently can be affected by infections at different points in their development and maturation. Acute and chronic infections can compromise spermatogenesis, resulting in quantitative and qualitative reductions.^4,5Infections of the male genitourinary tract represent a significant health care problem and account for almost 15% of cases of male infertility.⁶Among bacterial species that interact with spermatozoa are well known causative pathogens of genitourinary tract infection such as Escherichia coli, Ureaplasma urealyticum, Mycoplasma hominis and Chlamydia trachomatis.^6,7,8 E. coli probably represents the most frequently isolated microorganism in genitourinary infections. E. coli rapidly adheres to human spermatozoa in vitro, resulting in agglutination of spermatozoa.⁹ Agglutination of spermatozoa was produced only by live pathogenic E. coli whereas killed bacteria failed to do so.¹⁰The presence of antimicrobial components in blood and other bodily fluids, leukocytes and tissues has been known since the end of the 1800s and many were reported in the first half of the 1900s.¹¹ Attempts to improve the efficacy of available antibiotics, particularly the older and cheaper ones have been suggested.¹² Medicinal plants continue to play a central role in the healthcare systems of large proportions of the world’s population, particularly in developing countries, where herbal medicine has a long and uninterrupted history of use.¹³This raises the prospects of obtaining novel chemotherapeutic compounds if this vastly untapped resource could be adequately explored. The prospect of obtaining drugs from plants has been demonstrated by some notable examples of important pharmaceuticals derived from plant precursors.¹⁴The rich chemical diversity in plants has also been reported to be a promising source of antibacterial compounds raising hopes of obtaining novel antibiotics that can aid the fight against resistant infection.¹⁵This study was conducted to evaluate antibacterial activity of Punica granatum as therapeutic agents against multidrug resistance bacterial species contaminate semen and its association with sperm quality and semen characteristics of infertile patients visiting International Islamic Center for Population Studies and Research (IICPSR), Al-Azhar University, Cairo, Egypt.

 PATIENT AND METHODS

Collection of samples:seminal fluid samples were collected from 100 males attending the fertility clinic at the International Islamic Center for Population Studies and Research (IICPSR), Al-Azhar University, Cairo, Egypt. The samples were collected from patients during the period of May 2016 to October 2017. Upon collection, samples were transferred to the laboratory in a temperature around 37^oC. Semen samples are collected by masturbation after a 2 to 7 days of abstinence.^16,17The container must be clean, sterile and wide mouthed to minimize collection error. The semen specimen should be maintained at room or body temperature and examined within one hour of collection.¹⁷

Examination of samples: all semen samples included in this study were examined for physical appearance, volume, viscosity, total motility, and sperm count.

Physical examination: semen volume is by drawing the entire ejaculate up into a sterile (and warmed) graduated glass pipette.¹⁸While the color in a normal semen sample has a homogenous, gray opalescent appearance.¹⁹The semen odor may be affected by some foods or drugs excreted in semen. It may be offensive in some cases of genital infection or uriniferous in cases of semen contamination by urine.¹⁷

Microscopic examination:microscopic examination includes evaluation of sperm concentration, motility and morphology.   

Isolation and identification of bacterial species in semen samples: the seminal fluid samples were cultured in aseptic condition, within 1 h of collection on different types of enrichment and selective media. Bacteria were isolated by agar streaking method onto surface plates of MacConkey agar, mannitol salt agar and blood agar media. Then the plates were incubated aerobically and anaerobically at 37◦C for 24h. After incubation, separate bacterial colonies were picked up and subjected to purification processes by inoculating on different selective media. After purification, different colonies that appear from different samples were selected for the primary identification based on morphological and biochemical parameters ²⁴

Automated identification of bacterial isolates by using Biomerieux Vitek 2 System: automated highly identification system for microorganisms and reproducible results as shown in multiple independent studies. With its colorimetric reagent cards, and associated hardware and software advances, VITEK 2 offers a state-of-the-art technology platform for phenotypic identification methods.

Antibiotic sensitivity of bacterial isolates: the purified bacterial isolates were subjected to evaluation of antibiotic profile using different antibiotic groups in order to select the highest antibiotic resistant bacterial isolates. Antibiotic panel was Amoxicillin-Clavulanic acid 20/10μg/ml, Cefepime 30μg/ml, Azithromycin 15μg/ml, Clindamycin 2μg/ml, Levofloxacin 5μg/ml, Doxycycline 30μg/ml, Amikacin 30μg/ml, Erythromycin 15μg/ml, Chloramphenicol 30μg/ml, Cefaclor 30μg/ml, Aztreonam 30μg/ml, Ampicillin 15μg/ml, Cephradine 15μg/ml, Imipenem 10μg/ml, Cefotaxime 30μg/ml, Ceftriaxone 30μg/ml, Ampicillin-Sulbactam 10/10μg/ml, Piperacillin 75μg/ml, Cephalexin 75μg/ml, Cefetrizole-Sulbactam 75/30μg/ml antibiotic sensitivity profile was performed according to Kirby-Bauer disc-diffusion method on Mueller-Hinton agar. ^25,26

All strains were subculture on TSA plates and incubated over night at 37°C. The resulting culture was passage two additional times to ensure that the organism had an optimal growth and metabolic status.²⁷ Five to ten colonies of an overnight culture on TSA was used to inoculate 5ml of trypticase soy broth (TSB). Bacteria were then incubated four to five hours at 37 °C, with mild shaking to promote uniformity and growth optimization.

After incubation, the culture was adjusted to a turbidity equivalent to a 0.5 McFarland turbidity standard. Serial 1:10 dilutions in TSB was performed and 100µl of each dilution was spread over the surface of TSA plate and incubated at 37ºC overnight to determine the viable Colony Forming Unit (CFU) of each strain. The final concentration of bacteria in the agar plate would approximately 10⁸ CFU/ml. The plates were incubated at 37 ºC for 18-24h.

The experiment was carried out three times and the mean values are presented. The antimicrobial activity was evaluated by measuring the diameter of inhibition zone (mm) according to Vaghasiya and Chanda, 2010.²⁸

Collection of plant materials: the Punica granatummedicinal plant was used in this work collected from Faculty of Agriculture, Cairo University, during December 2017.

Preparation of the alcoholic extract: about 5g of air-dried plant powder were re-fluxed with 2.5 L of 70% Ethyl alcohol for 6 hours, and then filtered. The residue powder was then washed several times with hot alcohol. The combined filtrates were concentrated under reduced pressure at 50ºC, and then used for purification and identification

Gas chromatography–mass spectrometry (GC-MS) analysis: chemical composition of plant extract was separate, purified and identified by comparison of their retention times and mass spectra with those of WILEY 09 and NIST 11 mass spectral database.

Evaluation of antibacterial activity of purified Punica granatum extract:a loop full of bacterial isolate was inoculated in 25 ml of Muller Hinton broth in 150 ml conical flask and incubated at room temperature on a rotary shaker for 24hr in order to activate the test bacteria. The final cellular concentration was 1×10⁸ CFU/ml. The molten Mueller Hinton Agar was inoculated with 100μl of bacterial suspension and tilted well to ensure proper distribution in the plate.²⁶ Plant extracts were dissolved in 1% dimethylsulphoxide (DMSO) for antibacterial study. Extract concentration was adjusted to 20 mg/ml.²⁹ The well diffusion method was performed for the determination of antibacterial activity of plant extract against semen bacterial isolates. The well was loaded with 100µl of plant extract (well diameter: 6.0 mm). Then loaded plates were incubated at 4ºC for 2 h. to allow the diffusion of extracts into agar and then incubated at 37^◦C for 24h. The diameter of the inhibition zone was measured in millimeters.

RESULTS

Collection of samples and semen parameters:

One hundred samples of semen collected from men attending the fertility clinic of the International Islamic Center for Population Studies and Research (IICPSR), were examined routinely, for normal semen characteristics and bacterial contaminate. Among total cases, 20 cases (20%) showed at least one bacterial isolates and abnormalities range from 90 to 99% table (1)

    Table 1: Semen parameter of samples used to isolate bacterial species

Isolation of bacteria species from the collected semen samples:

Twenty bacterial isolates were isolated from the collecting samples by agar streaking method onto MacConkey agar, mannitol salt agar and blood agar media. A total of 20 bacterial isolates were identified. The overall prevalence of Gram-negative bacteria was 60% (12/20) and for Gram positive bacteria was 40% (8/20). The identification results were evident it, predominate bacterial isolates were found belong to E. coli (40%), Staphylococcus aureus (40%) Pseudomonas aeruginosa (20%) from total bacterial isolates as shown in figure (1).                                                                                                             

Antibiotics susceptibility of bacterial isolates:

Antibiotic sensitivity profile of bacterial isolates was performed in order to select the highest resistant bacteria against major of commercial antibiotic groups available at the market as showing in table (1).

Fig.1: Some bacterial species isolated from semen samples on MacConkey agar, blood agar media and Mannitol salt agar.

A total of 100 % of Escherichia coli. isolates were resistant to (clindamycin and piperacillin) and 87.5% to erythromycin, ampicillin   and cephalexin while Staphylococcus aureus and Pseudomonas aeruginosa isolates were resistance to amoxicillin/clavulanic acid, cefepime and ampicillin with ratio 100%  table (2).

Antibacterial activity of Punica granatum extracts against semen bacterial isolates:

Results showing that, the most potent extract from Punica granatum which give highest inhibition zones ranging from 18 to 36 mm against bacterial species isolated from semen samples table (3).

Table 3: Antibacterial activity of Punica granatum extracts against semen bacterial isolates.

Table 2: Antibiotics profile of semen bacterial isolates.