Abstract
In recent years, Stenotrophomonas maltophilia has not only gained importance due to its increasing prevalence in human disease around the world but because of its intrinsic multidrug resistance, which makes this bacterium an interesting model to study mechanisms of drug resistance while informing rational drug design and changes to chemotherapy protocols.Here, we aimed to identify novel mechanisms of resistance to ceftazidime, amikacin, levofloxacin, and minocycline. Overexpression of the chromosomally encoded serine-β-lactamase (SBL), L2, and metallo- β-lactamase (MBL), L1, is known to confer resistance to β lactams, including ceftazidime. However, the mechanisms behind β-lactamase hyperproduction are not completely understood. Here, a new mechanism responsible for L1/L2 hyperproduction has been characterised: loss of function mutations in mpl which likely lead to accumulation of activator ligand for AmpR, the transcriptional regulator of L2 and L1. Additionally, for the first time a TonB energy dependent mechanism is proposed for ceftazidime uptake where the alteration of a proline-rich region, deactivating TonB, is responsible for ceftazidime resistance in non β lactamase hyper-producers.
Although reduced antimicrobial permeability in S. maltophilia due to upregulation of efflux pumps is well known, little is known about their regulation. Here evidence is provided to associate SmeYZ aminoglycoside efflux pump upregulation with ribosomal damage caused by mutations in the rplA gene, encoding a ribosomal subunit target of aminoglycosides and by ribosomal-acting agents such as gentamicin. Alterations in genes involved in lipid trafficking were found to be associated with SmeDEF efflux pump upregulation. Two novel ABC transporters were shown to be involved in levofloxacin resistance and reduced minocycline susceptibility; each is locally regulated by a two component system. Minocycline was found to be the most promising therapeutic agent and resistant mutants or clinical isolates could not be found.
In addition to characterise last-line antimicrobials, the potential of non-classical β-lactamase inhibitors to restore β-lactam activity was studied. It was found that the diazabicyclooctane avibactam and the new bicyclic boronate 2 have potential to combat β-lactam resistance in S. maltophilia when compared to the traditional β-lactam based inhibitor, clavulanic acid based on microbiological, kinetic and structural evidence. In cases where the non-traditional inhibitors failed to restore β-lactam antimicrobial activity (e.g. with ceftazidime) new combinations strategies (aztreonam/avibactam or aztreonam/bicyclic boronate 2) or new inhibitors (MBL inhibitors in combination with meropenem) showed significant promise. In addition, the sideromimic modification of γ lactam antibiotic, lactivicin (LTV-17) was found tp increase its antimicrobial activity 1000-fold against S. maltophilia. Although LTV-17 induces L1//L2 production, lactivicin is only slowly hydrolysed by β-lactamases. Therefore, LTV-17 antimicrobial activity is still strong against β-lactamase and efflux pump hyper-producing mutants, and extensively-drug resistant clinical isolates. Mutants with reduced LTV-17 susceptibility were identified, and were found to have the same loss of the TonB energy-transducer seen in ceftazidime resistant mutants. This work has given insight into the mechanisms of resistance in S. maltophilia to assist the optimisation and identification of possible candidates to combat this species in the clinic.
| Date of Award | 25 Sep 2018 |
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| Original language | English |
| Awarding Institution |
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| Supervisor | Jim Spencer (Supervisor) & Matthew B Avison (Supervisor) |