presence of ACME. Interesjng clusters are shaded and named A. -â H. ISOLATE COLLECTION. In total 1,453 community-â and hospital-âacquired ST8 isolates ...
Using genomics to unravel diversity and population structure of MRSA USA300 to an unprecedented depth Janina DORDEL1, Ma.hew TG HOLDEN1,2, Brian G SPRATT3, Anne-‐Catrin UHLEMANN4, Susan S HUANG5, Julian PARKHILL1
1 Pathogen Genomics, Sanger Ins4tute, Cambridge, UK 2 School of Medicine, University of St Andrews, St Andrews, UK 3 Department of Infec4ous Disease
Epidemiology, Imperial College London, London, UK 4 Department of Medicine, Division of Infec4ous Diseases, Columbia University Medical Center, New York, NY, USA 5 Division of Infec4ous Diseases and Health Policy Research Ins4tute, University of California Irvine School of Medicine, Irvine, CA, USA
INTRODUCTION Over the past two decades community-‐associated methicillin resistant Staphylococcus aureus (CA-‐MRSA) strains have drama-cally increased the global burden of S. aureus infec-ons. The pandemic sequence type ST8/USA300 is the dominant CA-‐MRSA clone in the United States, but its evolu-onary history and basis for biological success are incompletely understood. Here we present a detailed analysis of the distribu-on and diversity of mobile gene-c elements and their associated virulence and resistance factors to reveal how these shape the popula-on of USA300 and may have promoted its spread.
PHYLOGENY OF ST8 ISOLATE COLLECTION. In total 1,453 community-‐ and hospital-‐acquired ST8 isolates from Orange County, CA, Northern ManhaXan, NY, San Diego, TX, San Francisco, CA, and Houston, TX were collected between 2003 and 2011.
Geographical Origin Orange County, CA San Francisco, CA San Diego, TX Houston, TX
SCCmec IVa IVg
A IVb IVc MSSA
B
ACME
DISTRIBUTION OF PLASMIDS
➩ rep16 and rep21 most prevalent in USA300-‐like isolates indica-ng clonal propaga-on ➩ no significant geographical prevalence in USA300 ➩ sporadic acquisi-on and loss of all plasmids ➩ cluster A: lacks prevalent rep21 plasmid ➩ cluster H: acquisi-on of rep7 plasmid (pUSA02)
ogra
AC phic SCCm ME al o ec rigin
presence absence
PLASMID CLASSIFICATION. Plasmids were classified using conserved areas of the replica-on ini-a-ng genes (rep) (Lozano et al. 2012).
Ge
C D
H
E
F G
Fig. 1: Phylogene-c tree of 1,453 ST8 isolates based on 37,323 core genome SNPs, rooted by using the distantly related S. aureus isolate COL as an outgroup. The USA300 core clade is shaded in black, and the non-‐USA300 ST8 clades are in lighter shade. Colors of branches and the inner ring indicate the geographical origin of the isolates, then SCCmec type and presence of ACME. Interes-ng clusters are shaded and named A -‐ H.
➩ “bush” isolates distantly related (includes MSSA and USA500) ➩ geographical clustering The core clade: USA300-‐like isolates ➩ ~ 94 % of isolates cluster within a c l o s e l y r e l a t e d c o r e -‐ c l a d e represen-ng USA300-‐like isolates ➩ SCCmec IVa and ACME posi-ve ➩ limited geographical and HA/CA clustering ➩ clusters A -‐ H represent possible successful and more recent clusters (all linked to the acquisi-on of mobile gene-c elements)
Phylogenomics and phylogeography indicate a highly dynamic USA300-‐like MRSA popula4on even over very long distances.
DISTRIBUTION AND DIVERSITY OF PROPHAGES
Fig. 4: Phylogene-c tree as in Fig. 1. Colors in the rings represent presence and absence of 11 plasmid/rep gene types.
DRUG RESISTANCE IN SILICO ANTIBIOTICA RESISTANCE TESTING. Resistance against 16 an-bio-c families was tested by detec-on of 53 acquired resistance determinants and 12 SNPs in housekeeping genes.
➩ drug resistance does not differ between geographical origin or CA/HA state ➩ sporadic acquisi-on of resistance determinants without further propaga-on for most an-bio-c classes (for excep-ons see box and text below)
Acquisi4on and subsequent clonal propaga4on of kanamycin, macrolide & fluoroquinolone resistance together with core genome changes may have promoted the success of the USA300-‐like isolates.
PROPHAGE CLASSIFICATION. Prophages were classified using conserved areas of the integrase genes (int) (Goerke et al. 2013). Absence and presence of the PVL-‐genes were detected using an in silico PCR approach.
➩ ➩ ➩ ➩ ➩ ➩ Fig. 2: Phylogene-c tree as in Fig. 1. Colors in the outer rings represent presence and absence of 7 prophage types.
Fig. 3A: Distribu-on of SNPs along the φSa2int haplotype 7 prophage (shaded in red) with surrounding core genome areas (shaded in grey). Asterisk highlights the posi-on of the PVL locus. B: Representa-on of the φSa2int genome. Prophage genes are colored according to their func-on.
no significant geographical prevalence sporadic acquisi-on and loss of all prophages cluster A: acquisi-on of φSa5int subcluster within B: acquisi-on of φSa1int subcluster within C: acquisi-on of φSa5int cluster F: acquisi-on of φSa1int
The PVL carrying prophage φSa2int ➩ diversity analysis reveal seven haplotypes which match the phylogene-c tree of the ST8 host ➩ only haplotype 7 carries the PVL-‐gene ➩ presence of haplotype 7 matches core clade and progenitor isolates ➩ high SNP density in φSa2int haplotype 7 compared to the core genome ➩ PVL locus shows lowest number of SNPs highligh-ng the selec-ve advantage of this virulence factor ➩ gene boundaries show lower SNP density indica-ng the transfer of whole genes from a prophage gene pool
TAKE HOME MESSAGES ABOUT USA300-‐LIKE ISOLATES ➩ highly dynamic popula-on without geographical structures ➩ MGEs provide USA300-‐like isolates with resistance and virulence factors, which may shape the popula-on and promote its spread ➩ no significant geographical correla-on between presence/absence or degree of diversifica-on of MGEs ➩ examples for possible successful clusters within the USA300-‐like clade show accumula-on and further diversifica-on of MGEs
Fig. 5: Phylogene-c tree as in Fig 1. Colors in the outer rings represent -‐from inside to outside-‐ the SCCmec type and determinants conferring resistance against an-bio-cs.
➩ kanamycin (aph3) and macrolide (msrA) resistance clusters in ST8 SCCmec-‐IVg and USA300-‐like core clade. Inferred through uptake of p18805-‐P03 (rep16, see plasmids) ➩ fluoroquinolone resistance clusters in ST8 SCCmec-‐IVg and a cluster within the USA300-‐ like core clade defined through different alleles in grlA and gyrA, respec-vely ➩ cluster F: addi-onal fluoroquinolone resistance conferring SNP in grlB ➩ subcluster within H: tetracycline resistance (tetK) through acquisi-on of pUSA02 (rep7, see plasmids)
PATHOGENICITY ISLAND 5 DETECTION OF SaPI5. SaPI5 was extracted from the whole genome alignment of mapped reads against USA300 FPR3757. A threshold of 85 % mapping coverage was used to detect the presence of SaPI5. Associated enterotoxins sek2 and seq2 genes were detected using an in silico PCR approach.
Fig. 6: Phylogene-c tree as in Fig 1. Colors in the inner ring represent the presence and diversity (inferred by mapping %) of SaPI5. Rings 2 and 3 show the presence of associated enterotoxins.
➩ presence of SaPI5 matches core clade and progenitor isolates ➩ sporadic loss of SaPI5 and enterotoxins sek2 and seq2 ➩ d egree o f ma p p i n g covera ge revea l s degrada-on and/or further diversifica-on from the reference for clusters A, F & G and a subcluster within B
Combina4on of mobile gene4c elements and core genome SNPs provide USA300 with selec4ve advantages and promotes its spread.
