Unravelling the Roles of Multidrug-resistant Plasmids in Bacteria

理解多重耐藥性質粒在細菌中扮演的角色

Student thesis: Doctoral Thesis

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Author(s)

Detail(s)

Awarding Institution
Supervisors/Advisors
  • Chi Kong Terrence LAU (Supervisor)
  • Yung-fu Chang (External person) (Supervisor)
  • Yung Fu CHANG (External person) (External Co-Supervisor)
Award date13 Jan 2020

Abstract

Antibiotic resistance has become one of the most emerging threats to global public health. Millions of lives are endangered due to the rapid development of drug resistance. Noticeably, the extended-spectrum beta-lactamases (ESBLs) and the carbapenemases are the two most concerning enzymes nowadays due to their ability to destroy beta-lactam and carbapenem antibiotics, which belong to the Model List of Essential Medicines (EML) of the World Health Organization (WHO). Plasmids, as efficient self-replicating genetic vehicles, play a particularly important role in the dissemination of drug-resistant genes. However, previous studies regarding multidrug-resistant (MDR) plasmids were mainly focused on resistance only, whereas the complete functional role of these genetic entities was often omitted. Plasmid and bacteria have co-evolved for so long that the interaction between plasmids and hosts could be far more complicated than previously thought. 

To explore the functional roles of major MDR plasmids, six MDR plasmids (four ESBLs carrying plasmids and two carbapenemases carrying plasmids) of different incompatibility (Inc) groups from clinical pathogen isolates in a Hong Kong hospital were studied. The six MDR plasmids used in this study were pCTXM123_C0996 (ESBLs, IncI1), pCTXM64_C0967 (ESBLs, IncI2), pHK01 (ESBLs, IncF2), pNDM-HK (carbapenemases, IncL/M), pNDM-HN380 (carbapenemases, IncX3) and pJIE143 (ESBLs, IncX4). To reveal the details of gene regulatory network mediated by MDR plasmids, RNA-sequencing was applied to E. coli J53 transconjugants of the six MDR plasmids in non-stressed and stressed conditions, including iron deprivation, oxidative stress and stationary phase. The transcriptome data were normalised in the R software and further analysed by the Sample Level Enrichment Analysis (SLEA). Several MDR plasmid-induced pathways that are closely related to adaption and survival in novel environments, such as biofilm formation, stress-induced responses and various metabolisms, were identified. Furthermore, specific pathways that are regulated by individual MDR plasmids were also found, such as chemotaxis and the galactose salvage pathway. These results illustrated the diverse functional roles that MDR plasmids play in the transcriptome regulatory network.

To validate the results observed in RNA-seq and to investigate phenotypic traits mediated by MDR plasmids, physiology and small RNA (sRNA) studies of transconjugants of the six MDR plasmids were performed. Cell growth of transconjugants in the standard LB medium were measured and compared. Slower growth and longer doubling times in the exponential phase were observed across all transconjugants when compared to the isogenic E. coli J53, suggesting that the carriage of MDR plasmids imposed noticeable fitness costs on the bacterial host. The motility of all transconjugants was also found to be slower than J53, indicating that the presence of MDR plasmids influenced both bacterial flagellar synthesis and chemotaxis. In addition, the stability of plasmids (plasmid persistence) was examined by the plasmid stability assay. All MDR plasmids were found be retained in their transconjugants after 600 generations except for pNDM-HK which was lost soon after the 100th generation. The lack of HN-S protein in pNDM-HK might contribute to the reduced plasmid stability in the absence of selection pressure. Overall, the high persistence of MDR plasmids implies that, even in the presence of perceivable fitness costs, most MDR plasmids can be very stable in a bacterial host for a long time. In addition, plasmid-encoded sRNAs from MDR plasmids were also identified, and their functionalities were studied. Intriguingly, the sRNA IGR plas2 from pNDM-HN380 was found to act as a regulator of fucR, which controls fucose metabolism. Via the knockdown of IGR plas2 using an antisense decoy, the formation of biofilm was inhibited in the bacterial host. This sRNA demonstrates a potential way of utilising plasmid-encoded sRNA against infectious bacteria.

To provide a more comprehensive understanding of the functional roles of MDR plasmids, plasmid-induced bacterial persistence was also measured and investigated in this study. Bacterial persistence and MDR plasmids were previously commonly considered as two unrelated topics; however, they are both very important regarding antibiotic resistance, and both are major contributors to the recalcitrance of infections. In this study, we first reported that five MDR plasmids, except for pNDM-HN380, were found to positively affected the fluoroquinolone persistence level of their bacterial host by more than 200%. A plasmid-borne gene from pNDM-HK, mucA, was shown to be a functional homolog of the chromosomally-encoded SOS mutagenesis gene umuD, and when mucA was overexpressed, it increased fluoroquinolone persistence remarkably in both laboratory (>300%, *, P<0.05) and clinical (>300%, **, P<0.01) E. coli strains. Furthermore, genes involved in the SOS response pathway, including the master regulator recA, were found to be upregulated (*, P<0.05) in the transcriptome following the introduction of pNDM-HK. Our finding suggests that MDR plasmids can impose secondary antibiotic tolerance to bacterial hosts in addition to the plasmid-encoded drug-resistant genes, therefore becoming a potential severe threat to the effectiveness of current medicines. 

Here, a comprehensive study of the functional roles of major MDR plasmids was presented. The results demonstrated the similarity and versatility of different major MDR plasmids in moderating the cellular physiology of bacteria. The knowledge of how MDR plasmids facilitate beneficial traits like bacterial persistence would be very useful for the development of new strategies against infectious pathogens in the future.

    Research areas

  • drug resistance plasmid, RNA-Seq, persistence