Introduction: Staphylococcus aureus is a commensal Gram-positive organism present on the skin and mucosal surface with the capability of surviving on dry surfaces due to its thick peptidoglycan layer [1]. Risk factors of S. aureus infections include external devices, history of surgery, and extensive antibiotic use [2]. Patients at risk of S. aureus infections include neonates, children with poor sanitation, women during menstruation, and patients with intravascular catheters. This microorganism could mainly lead to the bacteremia, endocarditis, osteomyelitis, pneumonia, skin and soft tissue infections [3]. Although S. aureus is generally considered as an opportunistic pathogen, some clones may be more capable of causing invasive disease due to the presence of certain virulence factors facilitating access to normally sterile sites [4]. Recently, S. aureus has exhibited great resistance against multiple antimicrobial agents, which is of great concern. Methicillin-resistant S. aureus (MRSA) strains; including hospital-acquired MRSA (HA-MRSA), community-acquired MRSA (CA-MRSA), and livestock-associated MRSA (LA-
MRSA) strains; are resistant to all β-lactam
antibiotics through acquiring mobile genetic elements called staphylococcal cassette chromosome mec (SCCmec) [5-7]. In addition to acquiring antibacterial resistance, differences in staphylococcal pathogenicity, depending on different geographical regions and epigenetics, necessitate the investigation of the genomic structure, polymorphism, and phylogenetic relationships between different
S. aureus clinical isolates. ..references?Multi-locus sequence analysis (MLSA) is a powerful high-resolution method which provides data on genetic changes in housekeeping genes and could be served as a valid technique for the study of epidemiological relationships [8]. In fact, MLSA is able to compare the primary DNA sequences of multiple conserved protein-coding loci in order to assess the diversity and relationship between different isolates and to determine the sources and evolutionary alterations of different taxa.
Objectives: This study aimed to investigate the genetic diversity of S. aureus clinical strains isolated from different body sites. In this study, a MLSA protocol was developed
based on genes coding for β-subunit
bacterial RNA polymerase (rpoB) and heat-shock protein 70 (hsp70) as well as 16S rRNA gene. These genes are essential for bacteria, and their polymorphism is highly important
in determining bacterial genetic behaviors corresponding to different environmental factors
Methods: growth conditions&Isolation of Genomic DNA&Polymerase Chain Reaction (PCR) and Sequence data analyses&Sequencing&Findings Genetic variations by MLSA&Phylogenetic analysis of the strains using the Neighbor Joining method andPhylogenetic analysis of the strains
using the Maximum Parsimony method.
Results: Phylogenetic trees constructed based on 16s rRNA gene using the neighbor joining method showed that the Strain 14 was different from the Strains 23, 49, and 42 (>99% difference) as well as the Strains 5 and 6 (>73%). Moreover, Strain 48 showed a sequence different from the Strains 6, 14, 15, 19, 22, 23, 42, and 49 (>99% difference). Finally, the Strain 50 was different in sequence from the Strains 1, 2, 3, 4, 5, 6, 11, 16, 20, 25, 30, 33, 37, 38, 39, 40, 46, 48, and 49 (Fig. S1A). Phylogenetic trees based on hsp70 gene showed that the Strains 5, 30, 43, and 49 were different from other strains in sequence (>99% difference). However, other strains were highly correlated, and the sequence similarity of the strains could be considered as an indicative of common ancestors (Fig. S2A). Based on the rpoB gene sequences, the Strain 11 showed a high correlation to the reference strain and Strains 2, 3, 5, 11, 24, 26, 37, 39, and 43, while it was different from other strains (>99% difference). Moreover, the Strain 15 was highly correlated with other strains, except for 19, 25, 29, 35, 41, 45, and 49 (>99% difference). Phylogenetic trees constructed based on the concatenation of 16s rRNA, rpoB, and hsp70 genes sequences by maximum parsimony and neighbor-joining methods showed that the Strain 5 was different from the Strains 6, 15, 19, 30, 32, 42, 46, 47, 48, and 40 with a bootstrap value of 99%. Moreover, the Strain 11 showed a phylogenetic difference with the Strains 1, 2, 3, 4, 5, 6, 15, 19, 30, 32, 42, 46, 47, 48, and 49 with a bootstrap value of 99%. However, other strains were highly similar in sequence and showed a close relationship to one other. Notably, the Strain 24 only showed similar genetic sequence to the reference strain and Strain 7 and showed 99% difference with other strains (Fig. S4A). According to the results of phylogenetic analysis based on 16s rRNA gene using maximum parsimony method, all S. aureus isolates showed similar genetic sequences and were not phylogenetically different and were presented in a monophyletic manner. However, it must be noted that the Strains 13 and 39 showed a trivial difference with other isolates with a bootstrap value of 63% (Fig. S1B). Molecular analysis based on
hsp70 gene indicated that the Strains 2, 5, 27, and 30 were different from other strains with a bootstrap value of 62%, while the Strains 1 and 24 were different from other strains with a bootstrap value of 61% (Fig. 12B). Finally, Phylogenetic analysis based on
rpoB gene using the maximum parsimony method revealed the differentiation of the
Strains 5 and 11 with 99% bootstrap value as well as the differentiation of the Strain 33 with 53% bootstrap value compared to the other strains (Fig. S3B). Phylogenetic analysis based on 16srRNA, rpoB, and hsp70 genes in concatenation indicated that the Strains 5 and 11, 1 and 24, and 37 and 43 were different from other strains with the respective bootstrap values of 99, 58, and 51%.
Conclusion: today, understanding the phylogeny of bacteria based on housekeeping genes is of great importance. In this study, the polymorphisms were determined, and a MLSA technique was developed for 50 S. aureus clinical isolates based on 16S rRNA,
hsp70, and rpoB genes. The MLSA method was clearly capable of discriminating between the S. aureus genotypes. Overall, there was a high genetic diversity in the three studied MLSA loci among the 50 S. aureus clinical isolates compared to the reference strain of S. aureus NCTC 8325. The use of multi-locus sequence analysis and the study of polymorphisms in S. aureus clinical isolates are proposed for infection control and surveillance.