PCR Development for Analysis of Some Type II Toxin-Antitoxin Systems, relJK, mazEF3, and vapBC3 Genes, in Mycobacterium tuberculosis and Mycobacterium bovis.

Toxin-Antitoxin (TA) systems are some small genetic modules in bacteria that play significant roles in resistance and tolerance development to antibiotics. Whole genome sequencing (WGS) is an effective method to analyze TA systems in pathogenic Mycobacteria. However, this study aimed to use a simple and inexpensive PCR-Sequencing approach to investigate the type II TA system. Using data from the WGS of Mycobacterium tuberculosis (M. tuberculosis) strain H37Rv and Mycobacterium bovis (M. bovis) strain BCG, primers specific to the relJK, mazEF3, and vapBC3 gene families were designed by Primer3 software. Following that, a total of 90 isolates were examined using the newly developed PCR assay, consisting of 64 M. tuberculosis and 26 M. bovis isolates, encompassing both 45 rifampin-sensitive and 45 rifampin-resistant strains. Finally, 28 isolates (including 14 rifampin-resistant isolates) were sent for sequencing, and their sequences were aligned and compared to the mentioned reference sequences. The amplicons size of mazEF3, relJK, and vapBC3 genes were 825, 875, and 934 bp, respectively. Furthermore, all tested isolates showed the specific amplicons for these TA families. To evaluate the specificity of the primers, PCR was performed on S. aureus and E.coli isolates. None of the examined samples had the desired amplicons. Therefore, the primers had acceptable specificity. The results indicated that the developed PCR-Sequencing approach can be used to effectively investigate certain types of TA systems. Considering high costs of WGS and difficulty in interpreting its results, such a simple and inexpensive method is beneficial in the evaluation of TA systems in Mycobacteria.

Evaluation of Mutations Related to Streptomycin Resistance in Mycobacterium tuberculosis Clinical Isolates.

Drug resistance to streptomycin in the clinical isolates of Mycobacterium tuberculosis (MTB) needs special consideration. It can mostly be caused by mutations in four genes with the names rpsL, rrs, gidB, and whiB7. The main objective of this study was the evaluation of the type and frequency of mutations in these mentioned genes using the PCR-sequencing method. This study was performed on 15 streptomycin-resistant and five streptomycin-sensitive isolates. Among resistant isolates, 11 samples contained mutations in codon 43 of the rpsL gene, which caused the lysine to be converted to arginine. Additionally, all of the isolates had mutations in the gidB. Missense mutations in codons 92 and 20 of this gene result in the amino acids Glutamic acid or Arginine being changed to Aspartic acid or Proline, respectively. No mutations in the rrs or whiB7 were found in any of the samples. Simultaneous mutations of rpsL and gidB were found in 10 isolates, the majority of which were Beijing strain. The results showed that the mutations of rpsL and gidB genes are mostly responsible for the streptomycin resistance in the evaluated MTB isolates. Furthermore, the discovery of dual mutations in Beijing strains highlights the strain's considerable potential for developing Tuberculosis drug resistance.

The Role of Genital Mycoplasmas in Preterm Labor.

Background: Growth of Mycoplasma in genital tract can cause problems such as infertility, pelvic inflammatory disease (PID), and preterm labor. This study was designed to evaluate the role of these bacteria in preterm labor among individuals in Gorgan city which is located in north of Iran. Methods: The study included 100 women with complaints of pain in preterm labor before 37 weeks of pregnancy (case group) and 100 women with term labor (control group) who were referred to Shahid Sayyad Shirazi Teaching Hospital in Gorgan city, north of Iran. Vaginal swabs, collected from all of these women, were evaluated for genital Mycoplasma sp. by molecular method using specific primers with polymerization chain reaction (PCR). The comparison of results was done by conducting X 2 and p<0.05 was considered significant. Results: Genital Mycoplasma was detected in 78 cases (39%) of 200 vaginal samples. Genital Mycoplasma colonization rates in the preterm and term samples were 60% and 18%, respectively, with relative risk of 2.05 (1.78-2.37) (p=0.001). The proportion of Ureaplasma parvum (44% and 15%), Ureaplasma urealyticum (11%, 3%), and Mycoplasma homins (5%, 0%) was significantly higher in women with preterm birth (PTB) than term labor. No cases of Mycoplasma genitalum were detected in this study. Conclusion: There is a significant relationship between presence of genital Mycoplasma in vaginal secretion and the risk of preterm labor.

Occurrence and risk factors of nontuberculous mycobacteria in tuberculosis-suspected patients in the north of Iran.

BACKGROUND AND OBJECTIVES: Some Nontuberculous Mycobacteria (NTM) can occasionally infect the human population and cause infections having symptoms similar to tuberculosis (TB). This study tried to provide updated data about the frequency and diversity of NTM species. MATERIALS AND METHODS: Suspicious samples of Mycobacterium tuberculosis (MTB) with both positive results in Ziehl-Neelsen (ZN) staining and Löwenstein-Jensen medium culturing were evaluated during January 2016 and December 2018 in Gorgan, Iran. After determination of MTB isolates by the growth rate, pigmentation status, the niacin test, and the insertion sequence 6110 (IS6110) PCR assay, other unknown isolates (presumably NTM) were detected by the 16S rDNA sequencing method and drawing the phylogenetic tree. Based on the patients' demographic information, their risk factors were also assessed. RESULTS: Among 226 culture-positive samples, obtained from 2994 individuals with suspected symptoms of TB, the analyses found 12 (5.3%) NTM and three Mycobacterium caprae isolates. Mycobacterium simiae (6/12) was the most prevalent NTM species. The average nucleotide similarity value was 98.2% ± 3.7. In comparison to patients with MTB (211 confirmed cases), other mycobacterium infections were more common in patients over 65 years old (Odd ratio (95% convenience interval): 2.96 (0.69 - 12.59), P = 0.14). CONCLUSION: Although the NTM species has a small portion in TB suspected patients, their prevalence has increased, mainly in elderly patients. Moreover, M. simiae was the most prevalent NTM species in our region. Therefore, identification of common species in each region is recommended and clinicians should pay more attention to them in each region.

Mycobacterium tuberculosis typing using Allele-specific oligonucleotide multiplex PCR (ASO-PCR) method.

Mycobacterium tuberculosis (M. tuberculosis) genotyping provides valuable information related to the origin and the evolution of the isolates. This study aimed to evaluate the applicability of single-nucleotide polymorphisms (SNPs) technique for lineages identification of M. tuberculosis and compare it with mycobacterial interspersed repetitive units-variable number of tandem repeats (MIRU-VNTR) method. The lineages of 162 clinically isolates were evaluated using six pair primers by Multiplex-PCR based on SNPs. Among 162 isolates, 70 (43.2%) isolates were lineage 4, following that 62 (38.3%) and 22 (13.6%) isolates were lineage 3 and 2, respectively. The method could not type 8 (4.9%) isolates. Moreover, we could identify 71 out of 79 unknown isolates resulted from the MIRU-VNTR method. The results showed that the SNP typing method has the potential to determine the lineages of M. tuberculosis as a rapid laboratory screening test.

New single nucleotide polymorphism G5508A in the SEPT12 gene may be associated with idiopathic male infertility in Iranian men.

BACKGROUND: Male infertility is a multifactorial disorder, which affects approximately 10% of couples at childbearing age with substantial clinical and social impact. Genetic factors are associated with the susceptibility to spermatogenic impairment in humans. Recently, SEPT12 is reported as a critical gene for spermatogenesis. This gene encodes a testis specific member of Septin proteins, a family of polymerizing GTP-binding proteins. SEPT12 in association with other Septins is an essential annulus component in mature sperm. So, it is hypothesized that genetic alterations of SEPT12 may be concerned in male infertility. OBJECTIVE: The objective of this research is exploration of new single nucleotide polymorphism G5508A in the SEPT12 gene association with idiopathic male infertility in Iranian men. MATERIALS AND METHODS: In this case control study, 67 infertile men and 100 normal controls were analyzed for genetic alterations in the active site coding region of SEPT12, using polymerase chain reaction sequencing technique. Fisher exact test was used for statistical analysis and p<0.05 was considered as statistically significant. RESULTS: Genotype analysis indicated that G5508A polymorphic SEPT12 alleles were distributed in three peaks of frequency in both control and diseases groups. Categorization of the alleles into (GG), (GA), (AA) types revealed a significant difference between infertile patients (azoospermic and asthenospermic) and normal controls (p=0.005). CONCLUSION: According to our finding we suggest that G5508A polymorphism in SEPT12 gene can affect spermatogenesis in men, the opinion needs more investigation in different populations.

Relationship between absence of annulus and asthenozoospermia in Iranian men.

PURPOSE: To find a relationship between absence of annulus and asthenozoospermia in Iranian men. METHODS: In the present study, semen samples from 100 asthenozoospermic and 20 normozospermic patients were analyzed for sperm concentration and motility. Spermatozoa were immunostained for the two septin subunits Sept4 and Sept7. The absence of the annulus structure was confirmed by transmission electron microscopy and western blot analysis for septin 4. DNA sequencing for all coding exons of SEPT12 was performed for a patient using peripheral blood sample. RESULTS: Specific antibodies for SEPT4 and SEPT7 consistently labeled the annuli in spermatozoa from all of the 20 normozospermic men, while in one of 100 patients with asthenozoospermia, 75% of sperms lacking septin 4 or septin 7 proteins at the annulus. It was shown that the structural defect in annulus formation is not caused by point mutation of SEPT12 gene. CONCLUSIONS: In conclusion, the results of this study demonstrated that the frequency of the absence of annulus in asthenozoospermic sample of Iranian population has a low frequency and could not be assume as a diagnostic marker for classifying asthenozoospermic patients.

Comparative expression analysis of Septin 14 in testes of infertile men with normal spermatogenesis and spermatogenic failure.

BACKGROUND: Septins are an evolutionary conserved group of GTP-binding and filament-forming proteins that have diverse cellular roles. An increasing body of data implicates the septin family in the pathogenesis of diverse states including cancers, neurodegeneration, and male infertility. OBJECTIVE: The objective of the study was to evaluate the expression pattern of Septin14 in testis tissue of men with and without spermatogenic failure. MATERIALS AND METHODS: The samples retrieved accessible random between infertile men who underwent diagnostic testicular biopsy in Royan institute. 10 infertile men with obstructive azoospermia and normal spermatogenesis and 20 infertile men with non-obstructive azoospermia were recruited for real-time reverse transcription (RT)-PCR analysis of the testicular tissue. Total RNA was extracted with trizol reagent. RESULTS: Comparison of the mRNA level of septin14 revealed that in tissues with partial (n=10) or complete spermatogenesis (n=10), the expression of septin 14 was significantly higher than sertoli cell only tissues. CONCLUSION: The testicular tissues of men with hypospermatogenesis, maturation arrest and sertoli cell only had lower levels of septin 14 transcripts than normal men. These data indicates that Septin 14 expression level is critical for human spermatogenesis.