Regulation of virulence and β-lactamase gene expression in Staphylococcus aureus isolates: cooperation of two-component systems in bloodstream superbugs

Abstract

Background: Methicillin-resistant Staphylococcus aureus (MRSA)-bloodstream infections (BSI) are predominantly seen in the hospital or healthcare-associated host. Nevertheless, the interactions of virulence factor (VFs) regulators and β-lactam resistance in MRSA-BSI are unclear. This study aims to characterize the molecular relationship of two-component systems of VFs and the expression of the β-lactamase gene in MRSA-BSI isolates. In this study, 639 samples were collected from BSI and identified by phenotypic methods. We performed extensive molecular characterization, including SCCmec type, agr type, VFs gene profiles determinations, and MLST on isolates. Also, a quantitative real-time PCR (q-RT PCR) assay was developed for identifying the gene expressions.

Results: Ninety-one (91) S. aureus and 61 MRSA (67.0%) strains were detected in BSI samples. The presence of VFs and SCCmec genes in MRSA isolates were as follows: tst (31.4%), etA (18.0%), etB (8.19%), lukS-PVL (31.4%), lukF-PV (18.0%), lukE-lukD (16.3%), edin (3.2%), hla (16.3%), hlb (18.0%), hld (14.7%), hlg (22.9%), SCCmecI (16.3%), SCCmecII (22.9%), SCCmecIII (36.0%), SCCmecIV (21.3%), and SCCmecV (16.3%). Quantitative real-time PCR showed overexpression of mecRI and mecI in the toxigenic isolates. Moreover, RNAIII and sarA genes were the highest expressions of MRSA strains. The multi-locus sequence typing data confirmed a high prevalence of CC5, CC8, and CC30. However, ST30, ST22, and ST5 were the most prevalent in the resistant and toxigenic strains.

Conclusion: We demonstrated that although regulation of β-lactamase gene expressions is a significant contributor to resistance development, two-component systems also influence antibiotic resistance development in MRSA-BSI isolates. This indicates that resistant strains might have pathogenic potential. We also confirmed that some MLST types are more successful colonizers with a potential for MRSA-BSI.

Keywords: Antibiotic resistance; Methicillin-resistant Staphylococcus aureus; Virulence factors; β-Lactamase.

Fig. 1

Differential expression of virulence and β-lactamase regulatory genes in BSI isolates of S. aureus. a: Heatmap of virulence and β-lactamase regulatory genes expression patterns in all 91 S. aureus. Red represents up-regulation and blue down-regulation relative to control. b: The expression levels of β-lactamase regulatory genes in MDR, XDR, and PDR strains, discriminated based on p-value and log₂ (fold-change) at an α level of 0.05. c: The expression levels of β-lactamase regulatory genes in hemolysin producer, toxin producer, and non-virulent strains, discriminated based on p-value and log₂ (fold-change) at an α level of 0.05. d The expression levels of virulence regulatory genes in MDR, XDR, and PDR strains, discriminated based on p-value and log₂ (fold-change) at an α level of 0.05. e: The expression levels of virulence regulatory genes in hemolysin producer, toxin producer, and non-virulent strains, discriminated based on p-value and log₂ (fold-change) at an α level of 0.05. Error bars standard errors: 0.05. Student’s t-test and Tow-Way ANOVA test were performed for testing differences between groups. *: p < 0.05, **: p < 0.001, ***: p < 0.0001

Fig. 2

The multi-locus sequence typing (MLST) phylogenetic tree, unrooted, maximum likelihood, and circular-dendrogram clustering of S. aureus based on sequence type (ST) profiles. The circular-dendrogram was estimated by neighbor-joining using the k2 + G model, with MEGA version 6 and Figtree version 1.4.4