Abstract: Mycoplasma hominis is normally found in the urinary tract of human and its role in Urinary Tract Infection (UTI) has been proved. This bacterium causes inflammatory responses and accumulation of leucocytes in urethra. In spite of the presence of the bacteria, the urine culture might be negative. Mycoplasma hominis can be transferred sexually and causes human infertility. The present study was conducted to detect and identify Mycoplasma hominis by molecular methods in urine samples of the patients with UTI, who were referred to our hospital. A total number of 864 urine samples from the patients with UTI were subjected to this study. After routine culture, urine analysis were performed on the samples. The DNA was extracted from the sediments of the urine samples, using phenol and chloroform method. Polymerase Chain Reaction (PCR) was conducted on the extracted DNA to detect the 16S rRNA of Mycoplasma hominis, with the primers; RNAH1 and RNAH2. Based on the results of PCR tests, out of 100 pyuria positive samples, 9 and 1% were infected with Mycoplasma sp. and Mycoplasma hominis, respectively. The sequencing of amplified product of 16S-rRNA revealed a single nucleotide substitution (269 T A), compared with the reference gene of this species.
INTRODUCTION
Urinary Tract Infection (UTI) caused by bacteria is frequently encountered in the outpatient settings in the USA, accounting for 8.6 million visits in 2007 (Hooton et al., 2013). UTIs are among the most common infectious diseases of human and the most common nosocomial infections in the developed world (Ulett et al., 2013). It has been estimated that 40-50% of women and 5% of men will develop UTI in their lifetime and that UTI accounts for more than 1 million hospitalizations and $1.6 billion of medical expenses each year in the USA (Ulett et al., 2013). Acute uncomplicated lower UTI, also known as cystitis, is characterized by burning sensation on urination and frequent urination without fever or flank pain (Trestioreanu et al., 2010). Most UTIs in women are acute uncomplicated cystitis caused by Escherichia coli (86%), Staphylococcus aprophyticus (4%), Klebsiella spp. (3%), Proteus spp. (3%), Enterobacter spp. (1.4%), Citrobacter spp. (0.8%), or Enterococcus spp. (0.5%) (Colgan and Williams, 2011). It has been reported that 7.6% of the UTI cases are caused by both Mycoplasma and Ureaplasma (Latthe et al., 2008). Mycoplasma hominis is generally a genitourinary (GU) pathogen (Henao-Martinez et al., 2012). Mycoplasma are the smallest-living, atypical bacteria that possess a very small genome and are characterized by their strict dependence on the host for their nutrients (Al-Daghistani and Abdel-Dayem, 2010). The genome of this bacterium is composed of 580,000 base pairs and contains 482 genes (Larsen and Hwang, 2010). The role of mycoplasmas in the etiopathogenesis of the urogenital system is still a subject of controversy (Bayraktar et al., 2010). The importance of mycoplasmas is obscured by the presence of Mycoplasma hominis and Ureaplasmaurea lyticum in many asymptomatic persons from whom these bacteria could be isolated. The prevalence of Mycoplasma infectious has been reported to vary from 10-40% in the male seminal fluid and has been shown to have a role in varicose infertile men with higher rate of as then ozoospermia (Al-Daghistani and Abdel-Dayem, 2010). Mycoplasmas have been associated with various pathological conditions and intrauterine infections, including pyelonephritis, pelvic inflammatory disease, chorioamnionitis, endometritis and postpartum fever, leading to significant complications, such as; preterm birth, low birth, spontaneous abortion, stillbirth, premature birth, infertility and perinatal mortality (Bayraktar et al., 2010). Mycoplasma hominis, a commensal bacterium of the GU tract, is a rare cause of mediastinitis, deep sterna wound infection and blood-culture-negative endocarditis (Myers et al., 2010). Genital mycoplasmas represent a group of microorganisms that are commonly found in the GU tract of pregnant and non-pregnant women. They are most commonly present in the genital tract of sexually active women (Bayraktar et al., 2010). However, generations of subclinical genital infection or nongonococcal urethritis have been detected in 25% of infertile men (Al-Daghistani and Abdel-Dayem, 2010). The Mycoplasma hominis colonization values range between 20 and 30% around the world (Bayraktar et al., 2010). Mycoplasma hominis is a fastidious slow-growing organism, which may not be readily identified through routine culture protocols and whose growth might further be inhibited by sodium polyanethole used in some culture media. These factors are the major limitations to the identification of Mycoplasma hominis using standard culture media and explain the delay in correct diagnosis and treatment in patients (Myers et al., 2010). Molecular methods have revolutionized our understanding of these bacteria and the simultaneous use of culture and molecular detection methods, such as PCR, offers great sensitivity in the detection of mycoplasmas (Larsen and Hwang, 2010).
Although, many studies on Mycoplasma hominis have been performed in Iran and around the world to determine the prevalence of this bacterium in clinical samples, no research has been conducted so far to study its molecular characteristics. Thus, the aim of the present study was to investigate and identify the characteristics of M. hominis in patients with UTI, who were referred to Shahid Beheshti Hospital in the second half of 2014 and reduce its hazardous staining prognoses.
MATERIALS AND METHODS
Mycoplasma isolation: In this descriptive study, 864 patients with UTI symptoms, who were referred to the Shahid Beheshti laboratory during the latter half of 2014, were included. The patients were provided with necessary instructions for appropriate urine sample collection and the mid-stream urine was collected in sterile tubes and transported to the Department of Microbiology Laboratory. Urinary analysis test was performed routinely with the samples and the presence of pyuria was determined.
DNA extraction: The DNA was extracted from the samples according to the procedures of Kojima et al. (1997). Based on the European Pharmacopoeia 2005, the negative and positive controls were PPLO broth medium and a standard strain of Mycoplasma hominis (PG21), respectively. A total of 500 μL of the samples were placed in a 1×5 μL Eppendorf tube and microcentrifuged at 13,000 rpm for 15 min. Then, 100 μL of the lysis buffer were added to 100 μL of the precipitate and the tube was placed in a 56°C bath for 4 h. Subsequently, 200 μL of saturated phenol were added to the tube and centrifuged at 13,000 rpm for 20 min. The supernatant was transferred to another tube and an equal volume of phenol/chloroform mixture (1:1) was added to it. After centrifuging at 13,000 rpm for 20 min, the aqueous phase was transferred to another tube and an equal volume of pure chloroform was added to it and again centrifuged at 13,000 rpm for 5 min. Subsequently, the supernatant was transferred to a new tube, mixed with 1/10 volume of sodium acetate (3 M), precipitated in a -20°C refrigerator with a twofold volume of cool and pure ethanol (20 min) and again centrifuged at 13,000 rpm for 15 min. Then, 200 μL of 70% ethanol were added and the tube was centrifuged at 13,000 rpm for 5 min. Finally, the DNA was dried and re-suspended in DDW at 4°C to be used for PCR (Kojima et al., 1997).
PCR: Based on the previous studies on Mycoplasma genus amplification, two primers (forward and reverse) (Stellrecht et al., 2004) were used and a 163-bp region of 16S rRNA gene was amplified. For Mycoplasma hominis, the primer sets RNAH1 and RNAH2 (Stellrecht et al., 2004), which amplify a 334 bp region of the lipoprotein gene were used. The primer sequences with their corresponding genes are shown in Table 1. The DNA amplification was performed in a total reaction volume of 25 μL containing 5 μL of DNA, 1 μL of each primer, 0.5 μL of dNTP mix (200 mM) (CinnaGen Inc.), 2 μL of MgCl2 (50 mM) (Cinnagen Inc.), 2.5 μL of PCR buffer (10×) (CinnaGen Inc.) and 0.25 μL of Taq DNA polymerase (5 U μL-1) (CinnaGen Inc.). The reaction mixture was thermocycled for 30 times beginning with an initial denaturation step at a minimum of 94°C. The temperature and time profiles of each cycle were as follows: The 94°C for 3 min (annealing) and 72°C for 1 min (extension). The PCRs were completed with a final extension step at 72°C for 10 min and the PCR products were stored at 4°C. The PCRs were performed using two programmable thermal cyclers (Primus and Master gradient). Positive and negative controls were included in all the tests. Each aliquot of every PCR product was mixed with 2 μL of loading buffer (6×). The PCR products and 100 bp DNA ladder were separated by electrophoresis on 1% agarose gel, stained with 0.5 μL mL-1 ethidium bromide (100 V for 1h) and viewed using a UV Transilluminator.
Bioinformatics studies: After purification, the best PCR products were sent to MWG Institute, Germany, to determine and verify their sequences. The nucleotide sequences of the samples were compared using MEGA software v. 5.04 and the similarity matrix was illustrated in Excel. The phylogenetic tree of the samples was analyzed and constructed by Bootstrap 1000 software via neighbor-joining tree method. Subsequently, the obtained sequences were compared with other sequences of Mycoplasma hominis available in GenBank to ensure their distinctiveness. The similarity matrix developed in the present study was compared with others constructed in Iran and other regions of the world, which have been recorded in GenBank.
| Table 1: | Nucleotide sequences and primers used for identification of Mycoplasma hominis by PCR |
The phylogenetic tree for all the samples was constructed and all the sequences were compared with each other in accordance with the shortest sequence.
RESULTS
The results obtained from the electrophoresis of the PCR products demonstrated the existence of 163 and 334 base pair bonds. These unique bonds belonged to Mycoplasma and Mycoplasma hominis, respectively (Fig. 1 and 2). Out of the 864 urine samples cultured in the laboratory, 100 were pyuria positive and among them, 10 samples (10%) were infected with mycoplasmas and 1 sample (1%) was infected with Mycoplasma hominis. All of the obtained sequences were similar (Fig. 3), except for m.hom-Kashan, which was different from the source sequence, exhibiting substitution of T with A. The lack of difference between the sequences of the samples studied in the present study and the source sequence could be owing to the absolute coincidence of the samples with the source sequence of Mycoplasma hominis, which proved the lineages of this species.
| Fig. 1: | Specificity of the PCR detection assay using the primers 163 bp. M: Marker 100 bp, +: Positive control, -: Negative control and 1-8: Suspected sample |
| Fig. 2: | Specificity of the PCR detection assay using the primers RNAH1 and RNAH2. M: marker 100 bp; PC: positive control [Mycoplasma hominis (PG21)], NC: Negative control [PPLO broth media], 1: Positive Mycoplasma genus samples. The formation of 334 bp in positive specie |
| Table 2: | Similarities matrix of Mycoplasma hominis in this study |
| Fig. 3: | Comparison between sequence of Mycoplasma hominis isolates in this study and others similar sequences in gen bank |
The sequences of the sample (Table 2) that were related to Mycoplasma hominis sequences presented no deviation, whereas the genomic region of the 10 samples infected with mycoplasmas was correlated to that of the other species of Mycoplasma with relative similarity. These species were placed in different branches of the phylogenetic tree, which confirmed the resultant resolution of the arranged sequence. The similarity matrix (Fig. 3) revealed 100% similarity with Mycoplasma hominis and up to 95%similarity with the sequences of other mycoplasmas. The sequencing results were deposited in GenBank as m.hom-Kashan strains.
DISCUSSION
One of the pivotal outcomes of this study is the determination of the Mycoplasma hominis sequence. Furthermore, this study is the first to determine and analyze the sequence and phylogenetic characteristics of Mycoplasma hominis in Iran by using bioinformatics software. In a previous study, Kenri et al. (2012) reported that Mycoplasma pneumoniae strain 309, a type 2a (subtype 2 variant) strain, has variations in the P1 protein, which is responsible for the attachment of this bacterium to the host cells. Furthermore, Musatovova and Baseman (2009) identified common and distinct sequences among the Texas clinical strains of Mycoplasma genitalium and obtained 18 DNA sequence variants, which were compared with all the other available clinical sequences. A detailed analysis revealed silent mutations of six amino acid residues within the encoded region of the MgPaadhesin in numerous clinical strains. In addition, missense mutations of limited numbers of amino acids were observed. The alignment of putative amino acid sequences revealed the simultaneous occurrence of several mutations and the existence of identical or similar protein variants in strains from different locations (Musatovova and Baseman, 2009). Pereyre et al. (2002) investigated the mechanisms of intrinsic resistance of Mycoplasma hominis to 14 and 15 membered macrolides and compared them with those of M. pneumoniae, which is naturally susceptible to macrolides. In addition, two Mycoplasma hominis clinical isolates with acquired resistance to 16-membered macrolides were also examined for mutations in domains II and V of 23S rRNA and in ribosomal proteins L4 and L22. Overall, their study achieved exhaustive characterization of the intrinsic resistance of Mycoplasma hominis to 14 and 15 membered macrolides and provided the first description of Mycoplasma clinical isolates resistant to macrolide, lincosamide and streptogramin antibiotics and harboring a mutation at position 2611 in the 23S rRNA (Pereyre et al., 2002).
Sasaki et al. (2002) determined the complete genomic sequence of an intracellular bacterial pathogen, Mycoplasma penetrans HF 2 strain. The genome was found to contain the two component system, but lacked the essential cellular gene, uridinekinase. The relatively large genome of M. penetrans HF 2 among the Mycoplasma species may be owing to its rich core proteome and the presence of a number of paralog families corresponding to 25.4% of all coding sequences (CDSs). The largest paralog family was found to be the p35 family, which encodes surface lipoproteins, including the major antigen, P35. A total of 44 genes for p35 and p35 homologs were identified and 30 of them were noted to form one large cluster in the chromosome. The genetic tree of p35 paralogs suggested the occurrence of dynamic chromosomal rearrangement in paralog formation during evolution. Thus, M. penetrans HF 2 may have acquired diverse repertoires of antigenic-variation related genes to allow its persistent infection in humans (Sasaki et al., 2002). Jamalizadeh (2013) studied Mycoplasma hominis in infertile men referred to the Infertility Clinic of Kerman and genetically classified mycoplasmas into five separate lineages. It was pointed out that there was a rare similarity between the five lineages. In addition, two lineages presented a significant genetic similarity with those available in the GenBank of Japan, Denmark, Russia and the United Kingdom and were arranged into one ancestry. The other three lineages were arranged into ancestry independent of the whole lineages and those available in GenBank. These lineages could be recorded in GenBank as native Iranian ones. Although, a significant genetic similarity had been indicated between seven other lineages, there was no similarity between those available in GenBank and the five individual lineages (Jamalizadeh, 2013). In another study, Mohseni examined M. genitalium in infertile men referred to the Infertility Clinic of Kerman and classified this bacterium into four different ancestries, with rare similarity between them. In addition, there was a significant similarity between the four lineages and those available in GenBank of Denmark and the USA and were thus classified into one ancestry. One lineage was arranged into an ancestry independent of the rest of the lineages and those available in GenBank. This lineage could be recorded in GenBank as the native Iranian one. The other two lineages were placed in two ancestries; however, there was a rare similarity between them and the rest of the lineages as well as those available in GenBank (Mohseni, 2013).
In the study by Lathle et al. (2008) no significant difference was observed between the Mycoplasma hominis substance (10%) and the findings obtained by examining 1032 samples (with 7.6% of the cases positive for Mycoplasma and Ureaplasma) (Latthe et al., 2008). In addition, no significant difference between the obtained results and the findings reported by Gonzalez Pedraza et al. (2003) was noted, who examined 1507 patients with GU infection (2% Mycoplasma hominis). Latthe et al. (2008) reported that the highest rate of staining for E. coli in the urinary samples was 35%, similar to that mentioned for this bacterium (44.34%) by Gonzalez-Pedraza et al. (2003) with no significant difference between the results obtained in both the studies.
Maeda et al. (2004) reported that the rate of infection with Mycoplasma hominis in 153 patients with non gonococcal urethritis in Japan was 2.6%. Furthermore, Nassar et al. (2008) indicated that the rate of infection with Mycoplasma hominis in 2400 urinary samples (with 200 samples being pyuria positive) was 3%, similar to that observed by Baka et al. (2009) in 153 women with chronic GU infection (3.3%) in Greece. In addition, in a study carried out in Japan with 100 urinary samples, Takahashi et al. (2006) reported that the rate of infection with Mycoplasma hominis was 4%. Thus, it can be observed that there are no significant differences in these above-mentioned results.
CONCLUSION
In the present study, infection with Mycoplasma hominis in patients who are pyuria positive, but show negative routine culture was pointed out. Occasionally, the existence of mycoplasmas in the GU tract could be detected with no clinical symptoms. Thus, microbial screening for pyuria-positive patients is necessary, especially for teenagers, because GU infections resulting from the colonization of these bacteria could cause serious diseases, such as Pelvic Inflammatory Disease (PID) and infertility, if left untreated. The results obtained in the current study not only open a new window to treatments for the above-mentioned diseases but also make it possible to access the lineages of Mycoplasma hominis through identification of its molecular characteristics for future research on infectious factors correlated to human GU system.
ACKNOWLEDGMENTS
This research was supported by the Kashan University of Medical Sciences, Shahid Beheshti Hospital, Kashan, Iran and the Faculty of Specialized Veterinary Sciences, the Reference Mycoplasma Laboratory, Razi Vaccine and Serum Research Institute, Karaj, Iran.