Evaluating genotypic and phenotypic variability against polymyxins and a novel lipopeptide, octapeptin C4, in antibiotic-resistant Klebsiella pneumoniae

Abstract

Antibiotic resistance remains a longstanding global threat, resulting in minimal therapeutic options for severe bacterial infections and propelling modern medicine into a post-antibiotic era. This circumstance is driven by several variables including the inability for current methodologies to rapidly diagnose infections which has led to unnecessary or incorrect administration of antibiotics. The unregulated availability of antibiotics in several countries and the use in livestock as a growth factor has facilitated the increase in resistance. This has allowed bacteria to develop an abundance of resistance encoded on mobile elements which are readily transferred between bacteria. Furthermore, there is a lack of new antibiotics entering the clinic. These factors account for the global dissemination of multidrug resistance and the increasing reports of pandrug resistance whereby bacteria harbour resistance against all available antibiotics.The World Health Organisation (WHO) and Centers for Disease Control and Prevention (CDC) urge that new antibiotics are paramount for fighting these bacteria with Enterobacteriaceae declared as one of the highest priorities. Klebsiella pneumoniae, belonging to the Enterobacteriaceae family, is a community and hospital-acquired pathogen which frequently harbours multidrug resistance and leads to high mortality. In particular, the advent of carbapenem resistance, the prior last-line antibiotic for resistant K. pneumoniae, was defined as a critical threat by both the WHO and CDC. In order to combat these infections, the polymyxin class of antibiotics are utilised.My thesis explores the genetic mechanisms utilised by K. pneumoniae to develop resistance against polymyxins. Understanding these pathways gives further insight into drug infiltration processes and engagement with their targets. Establishing in vitro studies to monitor this resistance acquisition can further unravel antimicrobial mechanisms of known and novel compounds and their future viability in the clinic. Furthermore, unravelling these mechanisms may indicate alternative procedures for rapid detection of resistance. Chapter 1 provides an overview of the current literature on these aspects.Initially explored in research Chapter 2 is the genetic basis for resistance in extensively drug-resistant (XDR) K. pneumoniae clinical isolates sourced from Greece and Brazil. Whole genome sequencing (Illumina) and subsequent complementation assays revealed genomic variations contributing to and regulating resistance. Resistance was facilitated via chromosomal mutations rather than plasmid encoded resistance. Mutations were apparent in genes phoPQ, pmrAB and mgrB. Heteroresistance, where a subpopulation harbours resistance, was identified in these clinical isolates as well as heterogeneity of mutations. Interestingly, several mutations segregated with resistance conferring variants were detected which may represent partial suppressor mutations. This highlights the difficulties associated with detecting polymyxin resistance via DNA sequencing.Due to the various combinations of genomic variants and the difficulty determining which contribute to polymyxin resistance, the transcriptome for several of these isolates was interrogated in Chapter 3. Utilizing Oxford Nanopore Technologies (ONT) direct RNA sequencing, the pathway involved in polymyxin resistance was investigated along with the transcription of acquired antibiotic resistance genes in these strains. ONT MinION sequencing was performed on four of these clinical isolates where one isolate was polymyxin-susceptible as a reference. This portable device has the capacity to read long fragments of DNA or RNA and analyse data in real-time. Hence, the genome for these isolates was assembled and the majority of acquired resistance (≥75%) was detected on up to 5 plasmids. A real-time emulation revealed ≥70% of resistance genes were identified in 2 hours. Direct RNA sequencing was able to discern resistance, however, low level transcription was not detected as validated by qRT-PCR. Elevated expression of the pmrHFIJKLM operon, encoding the 4-amino-4-deoxy-arabinose modification to lipid A, was evident in polymyxin-resistant isolates.In Chapter 4 a novel antibiotic under early stage development to address the increasing resistance against polymyxins, octapeptin C4, was investigated. This antibiotic is structurally similar to polymyxins, however, retains activity against polymyxin-resistant isolates. To assess the potential liabilities of this new antibiotic in terms of its ability to induce resistance, a polymyxin-susceptible XDR K. pneumoniae isolate was subjected to increasing concentrations of octapeptin C4 over 20 days in vitro. In parallel, this isolate was exposed to polymyxin B and polymyxin E (colistin). Genomic alterations driven by polymyxin-induced resistance were comparable to that seen in the clinical isolates and produced high levels of resistance, although some novel variants in these genes were identified. In contrast, octapeptin C4 exposure resulted in a slow progression in resistance, vast differences in lipid A profiles and harboured variations in genes never associated with any other class of antibiotic.In conclusion, my PhD thesis has uncovered several new mutations regulating polymyxin resistance in K. pneumoniae which will aid the detection of resistance via sequencing. These findings further validate the potential for MinION sequencing to be ultilised as a rapid diagnostic, especially for bacteria harbouring high levels of resistance. Furthermore, the slow progression of resistance and mutations in an alternative pathway reveal octapeptins as a promising class of antibiotics to further explore for treating XDR infections.