Microevolutionary Analysis of Methicillin Resistant Staphylococcus aureus in a Cystic Fibrosis Patient from Argentina Using Next Generation Sequencing

HAIM MS; ZAHEER R; BHARAT A; KNOX N; DI GREGORIO S; DI CONZA J; GALANTERNIK L; GOLDING G; GRAHAM M; VAN DOMSELAAR G; CARDONA ST; MOLLERACH M

Nueva Orleans

Congreso; ASM Microbe 2017; 2017

American Society for Microbiology

TRABAJO ACEPTADO PARA SU PRESENTACIONBackground: Staphylococcus aureus is commonly observed in the airways of cystic fibrosis (CF) patients. Chronic colonization allows this pathogen to adapt over time to cope with changing selection pressures. We have previously shown that the main clones related to community acquired methicillin- resistant S. aureus (CA-MRSA) infections in Argentina, ST5 and ST30, are also frequently isolated from the sputum of CF patients but usually display multi-drug antimicrobial resistance (AMR). We used next generation sequencing to examine the emergence of AMR and study the microevolution of S. aureus in the lungs of a pediatric CF patient.Methods: the genomes of 8 sequential isolates belonging to ST5 obtained over 40 months from the airways of a persistently colonized CF patient were sequenced on the Illumina MiSeq platform. Isolates were isogenic by pulsed field gel electrophoresis but showed differences in the AMR profiles over time. Sequence reads were de novo assembled using SPAdes. Coding sequences on contigs were annotated using PROKKA. Reads were mapped to the N315 reference genome and single nucleotide variants (SNVs) identified using SNVPhyl (doi: https://doi.org/10.1101/092940). SNVs were annotated with SnpEff. A maximum likelihood tree was constructed with PhyML based on total SNV variations among CF isolates using a core genome of 97.8%.Results: the assembled draft genomes yielded the following average results: total genome length of 2733670 bp; GC content of 32.71%; 20 contigs > 1kb in length and N50 of 456448 bp. PROKKA annotated an average of 2511 coding sequences. An average of 26 SNVs were identified between the first isolate and the following ones some of which were stable and related to genes influencing antibiotic resistance: 23S rRNA, rpsJ, mepA, gyrA and gyrB. Moreover, preliminary analysis of mobile genetic elements showed the acquisition of Tn4001 and the mobilization of IS256. Phylogenomic analysis revealed selection of a subpopulation with a greater number of mutations.Conclusions: our comparative analysis sheds light on significant genomic changes occurring in the long-term colonization of CF airways. These within-patient microevolutionary changes may be the result of numerous selective pressures in CF airways and responsible for the adaptation of bacteria to this particular ecological niche. Moreover, this study describes tigecycline-related resistance mutations in ST5 clinical isolates. To our knowledge, this is the first report of reduced susceptibility to tigecycline in clinical isolates in Argentina.