Detection of a novel mutation G511T in the 530 loop in 16S rRNA in multi drugs resistant Mycobacterium tuberculosis isolated from Sudanese patients

Abstract

Background: Tuberculosis (TB) is a bacterial disease considered as a global public health emergency by the World Health Organization (WHO) since 1993. In Sudan, MDR-TB represents a growing threat and one of the most important challenges that faced national tuberculosis program to establish a comprehensive multidrug-resistant tuberculosis management system.Objective: To characterize the diversity and frequency of mutations in Sudanese MDR-TB strains isolated from Wad Madani, Al-Gadarif and Khartoum using 16S rRNA and phylogeny approach.Material and Methods: A total of 60 MDR-TB isolates from Wad-Madani, Al-Gadarif and Khartoum were tested with molecular LPA (Genotype MTBDR plus) and GeneXpert MTB/RIF assay and Spoligotyping to confirm their resistance to RIF and INH. Sequencing and phylogenetic analysis was carried out using in silico tools.Result: This study revealed the circulation of different Sudanese MDR-TB strains isolated from Wad Madani and Al-Gadarif belonging to two distinct common ancestors. Two isolates from Wad Madani (isolate3 and isolate11) found in one main group which characterized by a novel mutation G511T in the 530 loop.Conclusion: The recurrence of C217A mutation in Wad Madani (isolate11) indicates the spread of this mutation in Sudanese MDR-TB strains and the diversity of this inheritance leading to generate new G511T novel mutation. So, understanding the molecular characterization of resistance mechanisms in MD-TB can facilitate the early detection of resistance, the choice of appropriate treatment and ultimately the management of MD-TB transmission. Bioinformatics approaches provide helpful tools for analyzing molecular mechanisms of resistance in pathogens.

Molecular Phylogenetic Analysis of16S rRNASequences Identified Two lineages ofH. pyloriStrains Detected from Different Regions in Sudan Suggestive of Differential Evolution

“…The PCR reaction mixtures contained 2.5U of i-Taq TM DNA polymerase (5U/µl), 2.5mM of each deoxynucleoside tri-phosphates (dNTPs), 1X of PCR reaction buffer (10X),1X of gel loading buffer and 1µl of DNA template. The temperature cycle for the PCR was carried out using a method described previously (41,42). To detect the DNA, 3µl of each PCR products was loaded onto 2% agarose gels stained with 3µl ethidium bromide (10mg/ml) and subjected to electrophoresis in 1x Tris EDTA Buffer (TEB buffer) (89mM of Tris base, 89mM Boric acid and 2mM EDTA dissolved in 1Litter H 2 O) for 30 min at 120V and 50mA.…”

First insights into molecular basis identification of 16 s ribosomal RNA gene of Staphylococcus aureus isolated from Sudan

“…This mixture was added to the PCR master mix (GoTaq, Promega, USA) following the manufacturing guide. Then, run with a thermal cycler (SensoQuest, Germany) as follows: 30 cycles were performed in a thermocycler, each cycle has three steps of denaturation (95 °C for 1 min), annealing (54 °C for 1 min), extension (72° C for 3 min) and final extension time of 72 °C for 5 min [ 8 ]. Amplified products were analyzed by conventional electrophoresis, Bands were determined using an Imagemaster VDS image analysis system (SCIE-PIAS VISION U.K) [ 17 ].…”

“…Few studies have been published in Sudan on 16S rRNA gene sequencing. As described by Hassan et al [ 8 ], who showed the significance of microbial identification and phylogenetic markers for Staphylococci species from Sudanese isolates used for taxonomy. Prior research also been explored in Sudan by Merghani et al, suggested that PCR assay with primers targeted to the 16S rRNA gene sequence offered a useful method for the identification of bacteria to the species level and differentiated one species from others [ 9 , 10 ].…”

First insights into Molecular basis Identification of 16s Ribosomal RNA Gene of Staphylococcus aureus Isolated from Sudan

“…This mixture was added to the PCR master mix (GoTaq, Promega, USA) following the manufacturing guide. Then, run with a thermal cycler (SensoQuest, Germany) as follows: 30 cycles were performed in a thermocycler, each cycle has three steps of denaturation (95°C for 1 min), annealing (54°C for 1 min), extension (72° C for 3 min) and nal extension time of 72°C for 5 min [7]. Ampli ed products were analyzed by conventional electrophoresis, Bands were determined using an Imagemaster VDS image analysis system (SCIE-PIAS VISION U.K) [15].…”

“…Few studies have been published in Sudan on 16S rRNA gene sequencing. As described by Hassan et al, who showed the signi cance of microbial identi cation and phylogenetic markers for Staphylococci species from Sudanese isolates used for taxonomy [7]. Prior research also been explored in Sudan by Merghani et al, suggests that PCR assay with primers targeted to the 16S rRNA gene sequence offered a useful method for the identi cation of bacteria to the species level and differentiated one species from others [8,9].…”