Staphylococcus aureus typing by digestion of protein A coding gene using Bsp143I

Background: Protein A is the virulence factors of Staphylococcus aureus rolling in its pathogenesis, and its gene is used for typing. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) with different enzymes has been used for this action. Objectives: In this study, we used Bsp143I enzyme for digestion of the gene, coding protein A (spa gene) in S. aureus. The bacteria were isolated from patients and healthy carriers in Gorgan, north of Iran. Patients and Methods: DNAs of 128 S. aureus subjects (53 from healthy carriers and 75 from patients) were extracted and amplified using specific primers of the spa gene. The product was digested by Bsp143I enzyme and its pattern was assessed by gel electrophoresis. Results: There were seven spa types among the tested S. aureus samples, among which six types differed in the repeated X region of the spa gene, but the seventh type had a deletion on one of BSP143I restriction sites. The frequency of spa types among isolated S. aureus samples as well as healthy carriers was six and five, respectively. S. aureus isolated from wounds showed the most diverse spa types (five) among clinical samples. Types 1, 2 and 4 were observed in all clinical samples, while only one case of type 3 was identified among patients, whereas this type constituted over 32% of the isolates among carriers. We found seven and four spa types among methicillin-resistant S. aureus (MRSA) and methicillin-sensitive S. aureus (MSSA) isolates, respectively. Conclusions: Our results showed that typing the spa gene using PCR-RFLP using Bsp143I was an acceptable method for typing S. aureus. Furthermore, this survey showed that the types in healthy carriers and MSSA were more variable than patient and MRSA isolates, respectively. We used the Bsp143I enzyme, which was not used in any previous studies on the spa gene. The results of this study suggested that we can use PCR-RFLP of spa gene by Bsp143I for molecular typing and sequencing of S. aureus, instead of relatively expensive methods. This method is relatively rapid and inexpensive, and can be accomplished in centers with conventional molecular facilities. © 2014, Ahvaz Jundishapur University of Medical Sciences

Reproducibility of 15-Loci MIRU-VNTR method in mycobacterium tuberculosis genotyping

Background: Mycobacterium tuberculosis genotyping is an essential step for understanding the epidemiology of tuberculosis. Mycobacterial-interspersed-repetitive-units (MIRU)-variable-number-of-tandem-repeats (VNTR) typing is an important method for this purpose. Objectives: The study aimed to determine the reproducibility of 15-loci MIRU-VNTR method. Methods: DNA extraction and 15-loci MIRU-VNTR were carried out for genotyping of 60 M. tuberculosis isolates collected from different clinical samples of 27 M. tuberculosis patients, each with two to four clinical isolates of M. tuberculosis. The similarity of M. tuberculosis isolates of the same patient was investigated by MIRU-VNTRplus. Results: The patterns of MIRU-VNTR were identical in 43 M. tuberculosis isolates collected from 20 tuberculosis patients, giving the repeatability of 71.6 for the test. In 12 isolates of M. tuberculosis belonging to five patients, the variable-number tandem repeat (VNTR) in only one locus was different. In three M. tuberculosis isolates of one patient, two different genotypes were identified. Conclusions: This study confirms the high reproducibility of 15-loci MIRU-VNTR, except for limited cases. © 2019, Author(s)

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 fre-quency and diversity of NTM species. Materials and Methods: Suspicious samples of Mycobacterium tuberculosis (MTB) with both positive results in Zie-hl-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 cas-es), 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. © 2021 The Authors. Published by Tehran University of Medical Sciences