Whole-genome epidemiology, characterisation, and phylogenetic reconstruction of Staphylococcus aureus strains in a paediatric hospital

Abstract

Background: Staphylococcus aureus is an opportunistic pathogen and a leading cause of nosocomial infections. It can acquire resistance to all the antibiotics that entered the clinics to date, and the World Health Organization defined it as a high-priority pathogen for research and development of new antibiotics. A deeper understanding of the genetic variability of S. aureus in clinical settings would lead to a better comprehension of its pathogenic potential and improved strategies to contrast its virulence and resistance. However, the number of comprehensive studies addressing clinical cohorts of S. aureus infections by simultaneously looking at the epidemiology, phylogenetic reconstruction, genomic characterisation, and transmission pathways of infective clones is currently low, thus limiting global surveillance and epidemiological monitoring.

Methods: We applied whole-genome shotgun sequencing (WGS) to 184 S. aureus isolates from 135 patients treated in different operative units of an Italian paediatric hospital over a timespan of 3 years, including both methicillin-resistant S. aureus (MRSA) and methicillin-sensitive S. aureus (MSSA) from different infection types. We typed known and unknown clones from their genomes by multilocus sequence typing (MLST), Staphylococcal Cassette Chromosome mec (SCCmec), Staphylococcal protein A gene (spa), and Panton-Valentine Leukocidin (PVL), and we inferred their whole-genome phylogeny. We explored the prevalence of virulence and antibiotic resistance genes in our cohort, and the conservation of genes encoding vaccine candidates. We also performed a timed phylogenetic investigation for a potential outbreak of a newly emerging nosocomial clone.

Results: The phylogeny of the 135 single-patient S. aureus isolates showed a high level of diversity, including 80 different lineages, and co-presence of local, global, livestock-associated, and hypervirulent clones. Five of these clones do not have representative genomes in public databases. Variability in the epidemiology is mirrored by variability in the SCCmec cassettes, with some novel variants of the type IV cassette carrying extra antibiotic resistances. Virulence and resistance genes were unevenly distributed across different clones and infection types, with highly resistant and lowly virulent clones showing strong association with chronic diseases, and highly virulent strains only reported in acute infections. Antigens included in vaccine formulations undergoing clinical trials were conserved at different levels in our cohort, with only a few highly prevalent genes fully conserved, potentially explaining the difficulty of developing a vaccine against S. aureus. We also found a recently diverged ST1-SCCmecIV-t127 PVL- clone suspected to be hospital-specific, but time-resolved integrative phylogenetic analysis refuted this hypothesis and suggested that this quickly emerging lineage was acquired independently by patients.

Conclusions: Whole genome sequencing allowed us to study the epidemiology and genomic repertoire of S. aureus in a clinical setting and provided evidence of its often underestimated complexity. Some virulence factors and clones are specific of disease types, but the variability and dispensability of many antigens considered for vaccine development together with the quickly changing epidemiology of S. aureus makes it very challenging to develop full-coverage therapies and vaccines. Expanding WGS-based surveillance of S. aureus to many more hospitals would allow the identification of specific strains representing the main burden of infection and therefore reassessing the efforts for the discovery of new treatments and clinical practices.

Keywords: Bacterial pathogens; Microbial epidemiology; Microbial genomics; Staphylococcus aureus.

Fig. 1

Phylogenetic tree of the whole cohort. Phylogenetic tree based on the 1464 core genes (1,194,183 bases) of the 135 single-patient S. aureus isolates. STs are distinguished by means of numbers and background colours in the inner ring. Sample type, operative unit, PVL presence, and SCCmec type are colour-coded in the following rings. On the outermost ring, the number of virulence genes is reported as bar plot (total considered = 79)

Fig. 2

Whole-genome maximum likelihood phylogenetic trees of the four most relevant STs. All available reference genomes for ST22, ST121, and ST228 have been included. For ST5, 1478 reference genomes were available, but only 24 were included for the sake of clarity. The phylogenetic tree of ST1 and available reference genomes was also produced, but it is not reported here to avoid overlapping with Figure 5

Fig. 3

Overview of the SCCmecIV cassette variability in our cohort, compared with available reference cassettes for the recovered subtypes IVa, IVb, and IVc. Genes are marked as arrows in the direction of transcription. To avoid biases due to misassemble of the region of interest, only cassettes found on a single contig are reported. Annotated SCCmec are grouped together with the closest reference cassette subtype. Some genomes showed insertions of genes involved in resistance to trimethoprim (MR090) and to kanamycin and bleomycin (MF062)

Fig. 4

Presence/absence profile of 79 genes encoding for virulence factors (upper part of the heatmap) and 18 genes encoding for resistance (bottom part). Some virulence and resistance factors were more represented in specific STs (only STs found in > 1 samples are specifically mentioned), as in the case of gentamicin resistance that is more prevalent in the ST228 isolates. For a more detailed overview of the single genomes’ profiles, see Additional file 3: Table S2

Fig. 5

Bayesian timed tree of ST1 isolates, including reference genomes. Location and date of sample collection is reported for each isolate. For samples collected at Meyer’s Children Hospital (black circles), patient code is reported instead of location. The two North Dakota samples were collected from the same subject. “n/a” indicates that no information is available for location of sample collection. Numbers at selected nodes are posterior probabilities. Grey areas are the distributions of marginal posterior probabilities for the diversification of ST1 and the diversification of Mayer-specific clone