Understanding persistence and emergence of antibiotic resistance during Staphylococcus aureus infection

Abstract

Persistent Staphylococcus aureus infection despite appropriate antibiotic therapy is a well-recognized, difficult to treat condition that is frequently reported in the medical literature. These persistent S. aureus infections may be associated with increased morbidity and mortality, and in some cases are associated with the in vivo evolution of enhanced antimicrobial resistance. However, the molecular basis for persistence is not well understood. This project aimed to characterise the molecular basis for persistence and enhanced antimicrobial resistance in a series of clinical S. aureus isolates obtained from a patient that had a methicillin-resistant S. aureus (MRSA) infection lasting over 115 days. The final clinical isolate (JKD6229) showed extensive phenotypic diversity compared to the first clinical isolate (JKD6210), including a small colony variant phenotype associated with significantly reduced growth rate, and acquired resistance to a number of antimicrobials including rifampicin and ciprofloxacin, and reduced susceptibility to last-line agents, vanconycin and linezolid. Detailed phenotypic and genomic characterisation of 47 clinical isolates obtained from 23 positive blood culture specimens and one spinal aspirate was undertaken. Whole genome sequencing was used to uncover molecular mechanisms for persistence and resistance, accompanied by detailed analysis of antimicrobial susceptibility profiles, analysis of isolate susceptibility to innate immune factors, microarray transcriptional analysis and genetic manipulation experiments to investigate the impact of specific mutations. Comparative genomic and single nucleotide polymorphism analysis demonstrated prominent DNA changes within the bacterial population, highlighted by both transient and conserved mutations associated with antimicrobial resistance or innate immune evasion. Mutations important for the adaptation of the organism during infection included a RelA F128Y mutation that led to an activated bacterial stringent response and partially explained the small colony variant phenotype and innate immune resistance; a codon insertion in a methyltransferase (RlmN) that altered ribosomal methylation and linezolid susceptibility; and a RpoB H481Y mutation that resulted in high level rifampicin resistance but also reduced susceptibility to human innate immune factors. This thesis has also uncovered a link between antibiotic resistance and resistance to innate immune factors caused by large chromosomal duplications. These duplications represent a previously unrecognized adaptive strategy employed by S.aureus to overcome antimicrobial exposure and host immune responses. The careful reconstruction of all the genetic changes that occurred over the course of a protracted multi-drug resistant S. aureus infection have revealed for the first time the extent to which the pathogen can adapt in the face of clinical interventions.