Multidrug-Resistant Methicillin-Resistant Staphylococcus aureus Associated with Bacteremia and Monocyte Evasion, Rio de Janeiro, Brazil

Abstract

We typed 600 methicillin-resistant Staphylococcus aureus (MRSA) isolates collected in 51 hospitals in the Rio de Janeiro, Brazil, metropolitan area during 2014-2017. We found that multiple new clonal complex (CC) 5 sequence types had replaced previously dominant MRSA lineages in hospitals. Whole-genome analysis of 208 isolates revealed an emerging sublineage of multidrug-resistant MRSA, sequence type 105, staphylococcal cassette chromosome mec II, spa t002, which we designated the Rio de Janeiro (RdJ) clone. Using molecular clock analysis, we hypothesized that this lineage began to expand in the Rio de Janeiro metropolitan area in 2009. Multivariate analysis supported an association between bloodstream infections and the CC5 lineage that includes the RdJ clone. Compared with other closely related isolates, representative isolates of the RdJ clone more effectively evaded immune function related to monocytic cells, as evidenced by decreased phagocytosis rate and increased numbers of viable unphagocytosed (free) bacteria after in vitro exposure to monocytes.

Keywords: Brazil; MRSA; MRSA and other staphylococci; Rio de Janeiro; Staphylococcus aureus; antimicrobial resistance; bacteremia; bacteria; bacterial infection; bloodstream infections; drug resistance; methicillin-resistant Staphylococcus aureus; molecular epidemiology; monocyte evasion; monocytes; multidrug-resistance; phagocytosis; phylogenetics.

Figure 1

Maximum-likelihood phylogenetic tree of 179 methicillin-resistant Staphylococcus aureus CC5 isolates from Rio de Janeiro, Brazil, 2014–2017 (red text) and 482 reference genomes (7). Red branches indicate the Rio de Janeiro clone of the lineage ST105(CC5)-SCCmecII-t002. Scale indicates substitutions per site. CC, clonal complex; SCC, staphylococcal chromosome cassette; ST, sequence type.

Figure 2

Distribution of 600 MRSA isolates by lineage (A), sample type (B), and patient age (C), Rio de Janeiro Brazil, 2014–2017. A) MRSA isolates by lineage (CC-SCCmec type) among 600 isolates. Labels indicate proportions. B) MRSA isolates by sample type. Labels indicate number of isolates. C) MRSA isolates by patient age (data available for 450 patients). Labels indicate number of isolates. BEC, Brazilian endemic clone; CC, clonal complex; EMRSA, epidemic methicillin-resistant Staphylococcus aureus; MRSA, methicillin-resistant Staphylococcus aureus; SCC, staphylococcal cassette chromosome.

Figure 3

Time-calibrated phylogenetic tree of methicillin-resistant Staphylococcus aureus ST105(CC5)-SCCmecII-t002 lineage, Rio de Janeiro, Brazil, 2014–2017. Chronogram constructed using Bayesian phylogenetic analysis of single-nucleotide polymorphisms from 73 genomes. Maximum clade credibility tree estimated using a strict clock rate of 1.1927 × 10^–6 substitutions/site/year (95% highest posterior density 1.5054–2.3351 × 10^–6). Node labels indicate 95% highest posterior density values of major clades. Asterisks (*) indicate posterior values >0.98. Scale indicates substitutions per site per year. CC, clonal complex; SCC, staphylococcal cassette chromosome; ST, sequence type.

Figure 4

Scatter plot representing gating strategy for identifying monocytes in the FSC-H versus SSC-H analysis of methicillin-resistant Staphylococcus aureus isolates, Rio de Janeiro, Brazil, 2014–2017. Representative flow cytometry chart shows the acquisition of THP-1 cells not exposed to methicillin-resistant Staphylococcus aureus. FSC-H, forward scatter height; SSC-H, side scatter height.

Figure 5

Histograms showing count versus green fluorescence intensity of THP-1 cells exposed or not to MRSA isolates, Rio de Janeiro, Brazil, 2014–2017. A) Acquisition of THP-1 cells not exposed to MRSA. B) Acquisition of THP-1 cells exposed to representative strains of 3 MRSA lineages. Blue indicates ST5-SCCmecIV-t002, strain CR14-026 (CC5-Basal lineage); green indicates ST5-SCCmecII-t539, strain CR15-071 (CC5-IIA lineage); and red indicates ST105-SCCmecII-t002, strain CD16–016 (CC5-IIB lineage). C) Acquisition of THP-1 cells exposed to representative strains of 3 MRSA lineages. Blue indicates ST5-SCCmecIV-t002, strain CHU15–056 (CC5-Basal lineage); green indicates ST5-SCCmecII-t539, strain CR14–016 (CC5-IIA lineage); and red indicates ST105-SCCmecII-t002 strain CD15–276 (CC5-IIB lineage). CC, clonal complex; FL1-H, forward light 1 height; MRSA, methicillin-resistant Staphylococcus aureus; SCC, staphylococcal cassette chromosome; ST, sequence type.

Figure 6

Scatter plots showing evasion of phagocytosis by MRSA isolates, Rio de Janeiro, Brazil, 2014–2017. A) Phagocytosis rates for representatives of the phylogenetic groups CC5-Basal, CC5-IIA, and CC5-IIB. Four independent experiments were conducted for each lineage using 1 fluorescence-activated single cell sorting determination for each experiment. Horizontal lines indicate means; whiskers indicate SDs. B) Viable count of unphagocytosed (free) bacteria after 30-min interaction with THP-1 monocytes. Six independent experiments were considered, with 2 replicates per lineage. Horizontal lines indicate means; whiskers indicate SDs. Statistical analyses conducted using 1-way analysis of variance and Tukey multiple comparison test. CC, clonal complex; NS, not significant; MRSA, methicillin-resistant Staphylococcus aureus; SCC, staphylococcal cassette chromosome; ST, sequence type.