The authors have declared that no competing interests exist.
Bacteria of the genus Staphylococcus are pathogenic Gram-positive bacteria responsible for various infections, including skin suppuration, which can be severe or chronic. The objective of this study was to confirm Staphylococci strain’s identification isolated by bacteriological methods from biological products of CHU-B patients, by molecular methods based on the analysis of the gene coding for 16S rRNA. In total, 30 strains of Staphylococci were isolated including 8 (26.66%) community strains, 22 (73.33%) hospital strains. The products of the amplification of gene fragments encoding 16S rRNA from 10 strains of Staphylococci including 6 strains of
Bacteria are micro-organisms or unicellular living beings without a nucleus. Human body harbors several microorganisms, which can be commensal or pathogenic. As soon as they become pathogenic, they can be responsible for many infectious diseases
Traditional bacteriological methods, namely the study of morphological, cultural and biochemical characters, remain the reference for the identification of the Staphylococcus genus as for all other bacteria. On the other hand, molecular biology techniques are more advantageous for the detection of different bacteria
Staphylococci strains collected at the biomedical analysis laboratory of the Center Hospital and University Center of Brazzaville (CHU-B) using various biological fluids from hospitalized patients (hospital strains) and non-hospitalized patients (community strains) were used.Strains were isolated during the period of April 2020 to July 2020.
The isolation of the strains was carried out on nutrient agar associated with a selective Chapman agar
The identification of the strains was made on the basis of the morphological characters and the production of a catalase and a staphylocoagulase by the bacterial strain
Ten strains of Staphylococci identified by conventional bacteriological methods were the subject of a molecular analysis on the analysis of the gene coding for the 16S rRNA.
DNA extracts were isolated from
The DNA extracts were amplified by PCR (polymerase chain reaction) using specific primers for the 16S rRNA gene, the universal primers used are those designated by
The PCR reaction was used in a total volume of 50 microliters comprising 39.5 µL of PCR water (Nuclease-free water), 2 µL (20 µM) of each primer, 1 µL (10 µM) of dNTPs, 5 µL of buffer (10X), 2µl (20ng) of genomic DNA and 0.5µL (5Unit/µl) of Taq polymerase. The PCR reaction was carried out in a Biometra thermocycler under the following conditions: pre-denaturation 95°C at 5 min, followed by 30 cycles, each cycle comprising denaturation 95°C at 30 sec, hybridization 55°C, 30 sec and elongation 72° C, 1 min 30s and finally a final elongation 72°C, 5 min.
The PCR products were demonstrated by electrophoresis on a 1% agarose gel at 100 volts for 45 minutes with TBE buffer. The staining was carried out with a 1 μg/ml solution of ethidium bromide. The gel was visualized under a UV lamp, by fluorescence. The size of the sought gene was around 1500-1300 bp.
The PCR products resulting from the amplification of the gene encoding the 16S rRNA of the ten strains were purified using the NucleoFast 96 PCR plate (Macherey-Nagel EURL, France) and sequenced by the company Masrogen using the BigDye terminator chemistry on an ABI sequencer. 3730 (Applied Biosystems, Foster City, CA, USA). Baser DNA sequence assembler was used for sequence assembly.
The analysis of the sequences A2, A3, A4, A5, A6, A10 respectively sequences of the strains S2, S3, S4, S5, S6, S10, S. aureus) and A1, A7, A8 A10 of the S1, S7, S8, S10, SCN) was performed using the Local Baseline Alignment Search Tool (BLAST) available at the National Database, Center for Biotechnology Information (http://www. ncbi.nlm.nih.gov). The alignment of the nucleotide sequences was done on ClustalW. Molecular phylogenetic and evolutionary analyzes were performed using MEGA version 7
All strains isolated were Gram positive and catalase positive bacteria. The fresh state showed that all the stumps were cocci in immobile clusters.
Ten strains of clinical Staphylococci, including 6 S. aureus and 4 with Negative Coagulase by classical bacteriological methods, were the subject of a molecular analysis for confirmation on the analysis of the gene coding for 16S rRNA.
DNA extracts from each strain were amplified by PCR using 16S rRNA gene-specific primers.
PCR fragments were sequenced and assembled.
Codes | Max Score | E. Value | % de similarité | Accession numbers | Equivalent strains |
A1 | 1714 | 0.0 | 94,5% | OM281812.1. | Staphylococcus sp. strain PYCC 8255 16S ribosomal |
A2 | 1725 | 0.0 | 94 % | MW308320.1. | Staphylococcus aureus strain s18 16S ribosomal RNA gene |
A3 | 1720 | 0.0 | 94 % | MW308321.1. | Staphylococcus aureus strain s18 16S ribosomal RNA gene |
A4 | 1727 | 0.0 | 91,2% | OP067888.1. | Staphylococcus aureus strain s20 16S ribosomal RNA gene |
A5 | 1742 | 0.0 | 91,2% | MK809243.1. | Staphylococcus aureus strain RM_AST_SA012 Ribosomal RNA gene 16 |
A6 | 1705 | 0.0 | 92% | MK809241.1. | Staphylococcus aureus strain RM_AST_SA001 16S ribosomal RNA gene |
A7 | 1881 | 0.0 | 93,4% | KY218856.1 | Staphylococcus haemolyticus strain AP BFT16 16S ribosomal RNA gene |
A8 | 1882 | 0.0 | 92 ,4% | MK934564.1. | Staphylococcus haemolyticus strain AP BFT16 16S ribosomal RNA gene |
A9 | 1714 | 0.0 | 93,5% | GQ110719.1 | Uncultured bacteria clone nbw618g09c1 16S ribosomal RNA gene |
A10 | 1716 | 0.0 | 94,5 % | AF015929.1. | Staphylococcus aureus 16S ribosomal RNA gene |
Codes | Sequences of strains identified |
A1 | Staphylococcus sp. Strain BMGLG21A1 |
A2 | Staphylococcus aureus Strain BMGLG21A2 |
A3 | Staphylococcus aureus Strain BMGLG21A3 |
A4 | Staphylococcus aureus Strain BMGLG21A4 |
A5 | Staphylococcus aureus Strain BMGLG21A5 |
A6 | Staphylococcus aureus Strain BMGLG21A6 |
A7 | Staphylococcus haemolyticus Strain BMGLG21A7 |
A8 | Staphylococcus haemolyticus Strain BMGLG21A8 |
A9 | Uncultured bacteria Strain BMGLG21A9 |
A10 | Staphylococcus aureus Strain BMGLG21A10 |
The evolutionary history was deduced using the UPGMA method
Although classical bacteriological methods remain the reference for bacterial identification, we used the molecular method based on the analysis of the gene coding for 16S rRNA for the confirmation of the isolated staphylococcus strains and the different related species identified in this work. Analysis of the results by conventional bacteriological methods show a high percentage of
1% agarose gel electrophoresis of the strains' 16S rRNA gene PCR products showed a length of approximately 1500 bp. This size (1500Pb) has been endorsed by several studies which have shown the importance of the 16S rRNA gene in the classification and identification of the molecular genus and species of prokaryotes identical to the results obtained by,
The analysis of the multiple alignment of the sequences of the RNA16S gene corresponding to all the strains of staphylococci identified and the homologs in the database using the CLUSTAL W algorithm allowed the highlighting of the mutations (losses of nucleotides), These results could explain the low rate of the percentage of similarity observed between the identified sequences and the homologs in Gen Bank. The multiple alignment of the sequences also made it possible to highlight highly conserved regions between the sequences, reflecting the belonging of the identified strains to the same family, to the same genus. These results also show that the strains have undergone evolutionary changes over time, evolutionary changes that are confirmed by the established Phylogenetic tree.
This study made it possible to confirm the identification of Staphylococcus strains made by microbiological methods based on morphological and biochemical characters (coagulase test) by molecular biology techniques, more precisely by the amplification of the 16S rRNA gene from DNA extracts from ten strains. The amplification of the 16S RNA gene is a universal, precise and objective method, whose inter-operator variability is limited compared to conventional techniques, it has enabled us to broaden our knowledge of the bacterial world, more precisely of the genus staphylococcus both at the level of the species and of the genus by highlighting the existing phylogenetic interferences.