Tracking methicillin-resistant Staphylococcus aureus (MRSA) evolution to discover important biomarkers for rapid characterization of unique MRSA clones in hospital bloodstream infections

Methicillin-resistant Staphylococcus aureus (MRSA) is an important agent of hospital infections in which certain prevailing clones occur in epidemic waves and new clones replace previous ones. In this study, the authors used molecular and genomic techniques to reveal the entry of a new MRSA clone, more frequent in bloodstream infections (BSI), in Rio de Janeiro. This Rio de Janeiro clone (RdJ) belongs to clonal complex 5 (CC5) and proved to be more resistant to phagocytosis by human monocytes and neutrophils, suggesting a role in increasing the frequency of RdJ in BSI. Additionally, a biomarker was identified that allowed the development of a relatively rapid test for precisely determining the RdJ clone. Another finding was the increase in the MIC for the biocide Triclosan in 25% of the MRSA CC5 samples, a very high percentage compared to that seen in the genomes of international samples analyzed in the study (0.9%). Meanwhile, the study found that the loss of specific virulence genes may be important for the evolution of certain MRSA clones, thus contributing to their success as a pathogen in hospital settings. Such data challenge the paradigm that increasing the virulence arsenal would be the key to the evolution of a bacterial pathogen. Finally, genomic and molecular cloning studies revealed that a protein from the ArdA family of E. coli was able to increase gene acquisition by MRSA. The findings also indicate that anti-restriction genes in S. aureus act as a biomarker for hyper-resistance to antibiotics, present in the multidrug-resistant MRSA clone known as the Brazilian epidemic clone (BEC) and in strains that infect livestock, known as LA-MRSA, of the ST398 lineage, which are also hyper-resistant.

The project's main focus was the study of 600 MRSA samples previously obtained from 51 hospitals in Rio de Janeiro. About 40% of the samples had their genomes completely sequenced, and another 60 genomes were analyzed using DNA microarrays. Comparative genomic analyses associated with Bayesian phylogeny and pangenome matrix studies were performed to highlight potential genes associated with the predominance of MRSA clones prevalent in these hospitals. The analyses also focused on the search for specific biomarkers for the predominant RdJ clone. Once the target genes were revealed, in vitro, ex vivo, and in vivo studies were performed to unravel the molecular strategies used by hypervirulent and multidrug-resistant MRSA clones. The genomic studies were performed in partnership with researchers from the University of Pennsylvania and the Brazilian National Laboratory for Computational Science (LNCC), and the microarray analyses with the Université de Lyon.

MRSA strains belonging to clonal complex 5 (CC5)-SCCmecII were found to predominate in the target region, especially ST105-SCCmecII-t002, which showed more significant tropism for BSI when compared to strains of other MRSA clones occurring in Rio de Janeiro. Most of the MRSA samples analyzed were isolated from elderly patients. Whole genome sequencing revealed that strains of the ST105-SCCmecII-t002 lineage (clone RdJ) expanded locally, sharing a common ancestor with strains of the North American lineage ST5-SCCmecII. Phylogenetic analyses with Bayesian inference estimated that expansion of the RdJ clone in Rio de Janeiro started approximately in 2009 (95% HPD 2007-2010). Surprisingly, the ST105-SCCmecII-t002 strains showed higher resistance to phagocytosis by monocytes and neutrophils compared to strains from other related CC5 clones. These findings could explain the preponderance of clone ST105-SCCmecII-t002 in BSI. The studies also revealed a biomarker (an apomorphic point mutation in the aur gene, which was specific to the RdJ clone) that allowed the design of a 100% sensitive and specific molecular test without the need for complete genome sequencing. Another important finding was that 25% of CC5 samples were resistant to triclosan, carrying the fabI gene in a transposition element (TnSha1). Triclosan is an antiseptic widely used in cosmetics, toothpaste, and mouthwash, as well as in medical procedures and the control of hospital infections. Therefore, its extensive use in Brazil could explain the increase in MIC for this biocide. In addition to CC5, MRSA CC1 samples are found in the hospitals examined at a frequency of 13.2%, being the third most frequent clonal complex. Genomic analysis showed that the evolution of CC1 in Brazilian hospitals occurred due to the loss of cytotoxic and proapoptotic genes, causing a decrease in cytotoxicity in the analyzed bacteria.

Meanwhile, the RdJ clone increased the ability to survive within phagocytes, making it more apt to cause persistent infections, which are more challenging to overcome. Finally, the studies demonstrated that genes homologous to ardA (anti-restriction gene) are widely distributed in hyper-resistant MRSA strains. We proved, via molecular cloning, that the anti-restriction gene in MRSA increases the acquisition of exogenous genes. These studies thus revealed that such genes, called ardA-H1 and ardA-H2 in our study, act as biomarkers of antimicrobial hyper-resistance in MRSA and are widely disseminated in other species of gram-positive bacteria that infect humans and animals.

This study revealed that the prevalence of MRSA clones in the same hospital may vary according to the clinical source and the patient's age. Such clonal stratifications depend on specific mechanisms acquired during the evolution and dispersion of these clones. For example, the RdJ clone´s phagocytosis evasion is related to the prevalence of this clone in bloodstream infections. Through pangenome matrix analysis, a rapid method was designed to detect the RdJ clone without requiring DNA sequencing. The studies further demonstrated that the ardA-H1 and ardA-H2 genes were present in hyper-resistant strains of MRSA in humans and animals. ArdA-H1 also enhances the horizontal transfer of exogenous genes, including antimicrobial resistance genes. Studies like this could lead to the development of antimicrobials that can eliminate bacteria and prevent the spread of resistance among bacteria. Finally, the study demonstrated that the reductive evolution of the virulence repertoire was paramount to the evolutionary phenomenon for the establishment of MRSA CC1-SCCmecIV in hospitals in Rio de Janeiro, where it remains the third most frequent MRSA

The use of rapid methods for the detection of MRSA clones, such as the one developed for the RdJ clone, will facilitate studies on morbidity and mortality associated with specific clones, thus providing better guidance for physicians and hospital infection committees.

Given the predominance of CC5-SCCmecII in bloodstream infections, particularly this new strain, ST105-SCCmecII-t002, it is crucial to determine whether the presence of strains belonging to CC5-SCCmecII in these infections is predictive of increased case-fatality when compared to other MRSA clones. Furthermore, we intend to investigate whether this emerging clone is only found in Rio de Janeiro hospitals or if it is also widespread elsewhere in Brazil. Finally, studies are underway on the RdJ clone´s phagocytosis evasion. The research aims to reveal mechanisms involved in the clone´s phagocytosis evasion, both in bacteria and in phagocytes. These studies are essential because they can foster research on new immune system modulators to neutralize bacterial evasion.