Comparative genomics of sequential Staphylococcus aureus isolates recovered from respiratory samples of cystic fibrosis patients from Argentina.

HAIM MS; ZAHEER R; BHARAT A; GOLDING G; GALANTERNIK L; GRAHAM M; VAN DOMSELAAR G; MOLLERACH M; CARDONA S

Rosario

Congreso; XXIII Congreso Latinoamericano de Microbiología - XIV Congreso Argentino de Microbiología.; 2016

Asociación Argentina de Microbiología

Background: Cystic fibrosis (CF) airways offer a favourable environment for the colonization and proliferation of a large variety of microbes.Staphylococcus aureus is an early and commonly observed pathogen in CF airways. Previous results from our laboratory have shown that the prevalent clones of community acquired methicillin‐resistant S. aureus (CA‐MRSA) infections in Argentina are frequently isolated from respiratory samples of CF patients. Of great concern is that unlike typical CA‐MRSA lineages, most of the CA‐MRSA isolates recovered from CF respiratory samples are resistant to multiple antibiotics. The aim of this study is to gain insight into the emergence of antimicrobial resistance (AMR) and adaptation of S. aureus in CF patients´ lungs using Next Generation Sequencing (NGS).Materials and methods: Eleven sequential S. aureus isolates belonging to sequence types ST5 (n=8) and ST30 (n=3) recovered from respiratory samples of two paediatric CF patients over a period of 40 and 10 months respectively, and two non‐CF CA‐MRSA isolates from Argentina representing each ST were selected for NGS of the genome. Isolates belonging to the same ST were isogenic by PFGE but showed differences in the AMR profile over time. Using paired‐end libraries, sequence reads (300 bp average read length) were generated on an Illumina MiSeq platform and de novo assembled using the SPAdes assembler (version 1.2). Reads were mapped to the MRSA252 reference genome using Smalt (version 1.0.1) and single nucleotide polymorphisms (SNPs) were called with Freebayes (version 0.4.1) and Mpileup (version 2.0). A maximum likelihood tree was then constructed with PHyML (version 3.1) based on total SNP variations using a 90.4% core genome.Results: The mean estimated genome coverage was 103x. The assembled draft genomes yielded an average total genome length of 2755008 bp; a mean GC content of 32.71%; an average of 20 (ST5) and 30 (ST30) contigs > 1kb in length; a mean contig length of 123947 bp; and N50 of 397639 bp. Consistent with PFGE and MLST data, CF MRSA clustered into one clade of ST5 and one clade of ST30 with an average of only 21 and 6 intra‐clade SNPs respectively. Moreover, comparisons between CF and non‐CF CA‐MRSA ST5 and ST30 from Argentina revealed 278 and 97 SNPs respectively between each group. Preliminary SNP analysis results reveal that emergence of linezolid resistance between the isolates might be due to a 23S rRNA mutation.Conclusions: NGS is a very useful tool in tracking the microevolution of pathogens within the host. The substantial microevolutionary change between CF and non‐CF strains as revealed by SNP analysis might be the result of numerous selective pressures to which S. aureus is subjected to in CF airways. Further comparative genomics studies between these isolates are underway to advance our understanding of AMR mechanisms and host‐adaptations of S. aureus.