Molecular Identification of Staphylococcus spp Isolated from Clinical samples

Abstract

The analysis of 16S rRNA gene sequences has been the technique generally used to study and confirm the identification and taxonomy of staphylococci. However, bacterial species cannot always be distinguished from each other using cultural methods. Thus, clinical samples were collected from 190 cases only 31 positive for staphylococcal infections with Urinary Tract Infection, Wounds, Burns, Otitis media, diarrhea infections, were applied for microbiological analysis which include: cultures on Manitol salt agar and HiCrome UTI Agar medium all the isolates gave positive golden yellow and identify as Staphylococcus spp. DNA was extracted from Staphylococcus spp and the 16srRNA gene were amplified by using specific primer, then sequencing of nucleic acid of genes was performed by machine is AB13730XL, Applied Biosystem, Macro gen company, the DNA sequencing results of flank sense of 16srRNA gene from 31 strains of Staphylococcus was confirm the identification into species level: Staphylococcus haemolyticus, Staphylococcus aureus, Staphylococcus epidermidis And Staphylococcus sciuri. Analysis of the sequences appeared that there two substitution(Transversion, Transition) in the Staphylococcus aureus strains with Sequence ID LC090540.1 location at Range of nucleotide from 4 to 636, 100% compatibility with NCBI while no substitution appeared in the Staphylococcus haemolyticus strains which have the sequence ID LN998078.1, 99% compatibility with NCBI also the sequence ID KR812401.1 which related to the strain Staphylococcus sciuri not appeared any substitution after sequencing analysis. Types of substitution detected in partial 16srRNA gene in Staphylococcus epidermidis strains 13 Transversion and 5 transition substitution location at range of nucleotide from 6 to 1026 have the Sequence ID KF575160.1 compared with data obtained from Gene Bank, these finding lead to conclusion, our assay allows rapid and confirm the detection to avoid possibility of misidentification of Staphylococcus species based on cultural analysis, the study aimed to propose the partial sequencing of the gene as an alternative molecular tool for the analysis of Staphylococcus species and for decreasing the possibility of misidentification. New submission of local Iraqi Staphylococcus clinical isolated during the current study show successfully record of four isolate Staphylococcus sciuri, Staphylococcus epidermidis, Staphylococcus aureus and Staphylococcus haemolyticus with GenBank accession number: KY938530.1 ,KY938529.1, ,KY938528.1, and GenBank: KY938527.1respectivelly.

Keywords

• Staphylococcus Spp, HiCrome UTI Agar, Manitol salt Agar, and Sequencing, Gen Bank.