Novel multidrug-resistant sublineages of Staphylococcus aureus clonal complex 22 discovered in India

Abstract

Staphylococcus aureus is a major pathogen in India causing community and nosocomial infections, but little is known about its molecular epidemiology and mechanisms of resistance in hospital settings. Here, we use whole-genome sequencing (WGS) to characterize 478 S. aureus clinical isolates (393 methicillin-resistant Staphylococcus aureus (MRSA) and 85 methicilin-sensitive Staphylococcus aureus (MSSA) collected from 17 sentinel sites across India between 2014 and 2019. Sequencing results confirmed that sequence type 22 (ST22) (142 isolates, 29.7%), ST239 (74 isolates, 15.48%), and ST772 (67 isolates, 14%) were the most common clones. An in-depth analysis of 175 clonal complex (CC) 22 Indian isolates identified two novel ST22 MRSA lineages, both Panton-Valentine leukocidin+, both resistant to fluoroquinolones and aminoglycosides, and one harboring the the gene for toxic shock syndrome toxin 1 (tst). A temporal analysis of 1797 CC22 global isolates from 14 different studies showed that the two Indian ST22 lineages shared a common ancestor in 1984 (95% highest posterior density [HPD]: 1982-1986), as well as evidence of transmission to other parts of the world. Moreover, the study also gives a comprehensive view of ST2371, a sublineage of CC22, as a new emerging lineage in India and describes it in relationship with the other Indian ST22 isolates. In addition, the retrospective identification of a putative outbreak of multidrug-resistant (MDR) ST239 from a single hospital in Bangalore that persisted over a period of 3 years highlights the need for the implementation of routine surveillance and simple infection prevention and control measures to reduce these outbreaks. To our knowledge, this is the first WGS study that characterized CC22 in India and showed that the Indian clones are distinct from the EMRSA-15 clone. Thus, with the improved resolution afforded by WGS, this study substantially contributed to our understanding of the global population of MRSA. IMPORTANCE The study conducted in India between 2014 and 2019 presents novel insights into the prevalence of MRSA in the region. Previous studies have characterized two dominant clones of MRSA in India, ST772 and ST239, using whole-genome sequencing. However, this study is the first to describe the third dominant clone, ST22, using the same approach. The ST22 Indian isolates were analyzed in-depth, leading to the discovery of two new sublineages of hospital-acquired Staphylococcus aureus in India, both carrying antimicrobial resistance genes and mutations, which limit treatment options for patients. One of the newly characterized sublineages, second Indian cluster, carries the tsst-1 virulence gene, increasing the risk of severe infections. The geographic spread of the two novel lineages, both within India and internationally, could pose a global public health threat. The study also sheds light on ST2371 in India, a single-locus variant of ST22. The identification of a putative outbreak of MDR ST239 in a single hospital in Bangalore emphasizes the need for routine surveillance and simple infection prevention and control measures to reduce these outbreaks. Overall, this study significantly contributes to our understanding of the global population of MRSA, thanks to the improved resolution afforded by WGS.

Keywords: India; ST22; ST239; ST772; Staphylococcus aureus; WGS; outbreak.

FIG 1

(A) Geographic and temporal distributions and main virulence factors of the STs identified in 478 Indian isolates. The map shows the locations of 17 collecting hospitals across 14 different cities. The markers are proportional in size to the number of contributing hospitals in a particular town. For example, Bengaluru, Karnataka, has three contributing hospitals, i.e., hospitals 12, 14, and 6, and hospitals 16 and 4 in Tiruchirappalli, Tamil Nadu. The pie charts show the proportion of the STs identified at each location. (B) Phylogeny of the 478 Indian S. aureus isolates. The midpoint-rooted phylogenetic tree was inferred from 97,120 informative sites obtained after mapping the genomes to the complete genome of strain Staphylococcus aureus MSSA476 (strain GCF_000011525.1 (ST1) and masking regions of recombination and mobile genetic elements. Tree nodes are colored according to their sequence types and are indicated on the map. Scale bars represent the number of single-nucleotide polymorphisms per variable site. Tree nodes are colored according to major STs present in the isolate collection. (C) The timeline shows the distribution of samples across the period from February 2014 to December 2019. The bars are colored by ST and are proportional to the number of isolates collected per month. This view is available at https://microreact.org/project/2xDvKQhriNveJ4kiVYsmSQ-s-aureus-wgs-study#i6az-ghru-tree.

FIG 2

(A) The timeline shows the distribution of ST239 samples across the outbreak period from March 2014 to October 2016. The bars are colored year-wise. This view is available at https://microreact.org/project/2xDvKQhriNveJ4kiVYsmSQ-s-aureus-wgs-study#ffxiv-st239-outbreak. (B) Phylogenetic tree, with linked genotypic data of outbreak isolates at one sentinel site in Bangalore. The maximum-likelihood phylogenetic tree of 43 closely related ST239 isolates was generated using S. aureus ST239 T0131 GCF_000204665.1_ASM20466v1 as a reference genome, identified based on similarity using BactInspector (46). The central cluster of 33 isolates collected from hospital 12 in Bangalore, between 2014 and 2016, is highlighted, together with information regarding the isolates’ SCCmec type, spa type, and AMR genetic determinants. The nodes are colored as per the year of collection. The interactive view is available at https://microreact.org/project/2xDvKQhriNveJ4kiVYsmSQ-s-aureus-wgs-study#yxiv-st239-outbreak-with-genome-t0131-as-a-reference.

FIG 3

(A) Indian CC22 strains in a global context. Phylogenetic tree of the ST22 global collection. The midpoint-rooted phylogenetic tree was obtained from 44,703 SNPs after mapping the genomes to the complete genome of strain EMRSA_15_GCF_000695215 and masking regions of recombination and mobile genetic elements. Tree tips are colored by country of origin as described in the legend. Scale bars represent the number of single-nucleotide polymorphisms per variable site. The interactive views are available at https://microreact.org/project/2xDvKQhriNveJ4kiVYsmSQ-s-aureus-wgs-study#olhe-cc22-global-view-rectangular-tree https://microreact.org/project/2xDvKQhriNveJ4kiVYsmSQ-s-aureus-wgs-study#6w7r-cc22-global-view-radial-tree. (B) Bayesian temporal tree of 1621 ST22 Indian and global isolates with estimated ancestral dates. Tree nodes from major ST22 lineages have been collapsed for clarity. The tree is annotated with the SCCmec types, the presence/absence of the PVL and tsst-1 virulence genes, and the predicted resistance to quinolones and aminoglycosides. The MRCA of IND-1 and IND-2 is marked with blue on the timeline. The data used to generate this tree are available at https://microreact.org/project/2xDvKQhriNveJ4kiVYsmSQ-s-aureus-wgs-study#6w7r-cc22-global-view-radial-tree.