The multidrug-resistant Staphylococcus capitis NRCS-A clone is responsible for sepsis in preterm infants in neonatal intensive care units (NICUs) worldwide. Here, to retrace the spread of this clone and to identify drivers of its specific success, we investigated a representative collection of 250 S. capitis isolates from adults and newborns. Bayesian analyses confirmed the spread of the NRCS-A clone and enabled us to date its emergence in the late 1960s and its expansion during the 1980s, coinciding with the establishment of NICUs and the increasing use of vancomycin in these units, respectively. This dynamic was accompanied by the acquisition of mutations in antimicrobial resistance- and bacteriocin-encoding genes. Furthermore, combined statistical tools and a genome-wide association study convergently point to vancomycin resistance as a major driver of NRCS-A success. We also identified another S. capitis subclade (alpha clade) that emerged independently, showing parallel evolution towards NICU specialization and non-susceptibility to vancomycin, indicating convergent evolution in NICU-associated pathogens. These findings illustrate how the broad use of antibiotics can repeatedly lead initially commensal drug-susceptible bacteria to evolve into multidrug-resistant clones that are able to successfully spread worldwide and become pathogenic for highly vulnerable patients.
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We thank M. Stegger and his team for insightful exchanges during the manuscript drafting and C. Allix-Béguec, C. Gaudin, M. Mairey and S. Duthoy for their help in genome sequencing. This project was supported by the European Society of Clinical Microbiology and Infectious Diseases study group (Project P307-14), the Fondation pour la Recherche Médicale (project ING20160435683) and the European Union Patho-Ngen-Trace (project FP7-278864).
The authors declare no competing interests.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Analysis was based on 55 genomes: all non-NRCS-A strains were included, however the clone NRCS-A was undersampled to avoid a statistical bias in favor of mutational changes. Dark blue horizontal bars indicate recombination events detected by the analysis.
a, NRCS-A isolates within an MSTREE based on the whole genome sequencing data. Each strain is represented by a circle or a fraction of a circle, colors correspond to different host types. Numbers indicate the mutational steps between the strains. b, Same data as above but represented in an MDS plot. c, Within NRCS-A diversity as assessed by mean pairwise SNP distances (N=197). d, Graphical chart representing the fraction of strains obtained from newborns in the basal, Proto-outbreak 1 and 2 and Outbreak strains.
Extended Data Fig. 3 Genome scan analysis of NRCS-A strains for detecting SNPs involved in local adaptation.
a, Plot of the first 2 principal components (PC). The 197 NRCS-A strains are represented by points and colorized according to their phylogenic origin (Proto-outbreak 1 and 2 in blue, and Outbreak in red). PC 2 is the one separating the basal proto-outbreak 1 and 2 strains from the outbreak strains. b, Manhattan plot representing the 3,658 SNPs and values obtained after performing Mahalanobis distances. The SNPs are colorized according to the PC to which they correlate most (PC1 = black, PC2 = red, PC3 = green and PC4 = blue).
Respectively 32 and 17 SNPs were specifically identified in Outbreak strains among NRCS-A strains (n=197) or in clade Alpha strains among Basal strains (n=53). Those SNPs were identified using PCADAPT.
a, Positions on the tertiary protein structure of outbreak specific non-synonymous mutations detected via PCADAPT and involved in antibiotic resistance (tigecycline and vancomycin). b, Positions on the tertiary protein structure of alpha-clone specific non-synonymous mutations for a set of two genes involved in cell wall synthesis. Visualization and predictions were executed by PHYRE2 software (http://www.sbg.bio.ic.ac.uk/phyre2).
Phenotypic data of S. capitis isolates (n=250) were obtained from agar dilution and biomarkers of antibiotic resistance were detected using GENEFINDER. Comparison between groups of isolates was performed using two-sided Fisher exact test.
Extended Data Fig. 7 Phenotypic assays comparing a subset of representative isolates of each of the four subgroups identified by the phylogeographical analysis (Outbreak, Proto-outbreak 1, Proto-outbreak 2 and ‘other isolates’).
In all 6 graphs, center values represent means. a, Culture supernatants cytotoxicity assay using THP1 cells, adjusted on a positive control (Triton) of 12 representative S. capitis isolates (two independent experiments in triplicate for each strains). b, Survival of strains (n=12) after 24 hours of persistence in desiccation conditions (two independent experiments in triplicate for each strains). c, Comparison of the doubling time of bacterial growth during the exponential phase in standard conditions (BHI) of 24 representative S. capitis isolates (three independent experiments in triplicate for each strains) and d, Under oxidative stress (ethanol-supplemented medium to a final concentration of 6.5%) (n=24 strains, three independent experiments in triplicate for each strains). e, Quantification of biofilm production of 24 representative S. capitis isolates using crystal violet method (expressed as optic densitometry at 590nm) (three independent experiments in triplicate for each strains). f, Phagocytosis index of monocytes and granulocytes from cord blood for a subset of 5 representative isolates of “Outbreak” and “Basal” isolates (four independent experiments). Of note, results of phagocytosis of neutrophils and activated neutrophils are not represented here because they were similar to those with granulocytes.
Here are represented genes with a -log10 (HMP) > 7.5 on either axis, and/or > 5 on both axes, thus considered significant.
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Wirth, T., Bergot, M., Rasigade, J. et al. Niche specialization and spread of Staphylococcus capitis involved in neonatal sepsis. Nat Microbiol 5, 735–745 (2020). https://doi.org/10.1038/s41564-020-0676-2