RNomic Study of Antibiotic-resistant Bacteria: "Escherichia coli" and "Laribacter hongkongensis"

有關抗生素耐藥性細菌: 大腸桿菌及香港鷗桿菌的核糖核酸組學

Student thesis: Doctoral Thesis

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Award date9 Mar 2018

Abstract

Gram-negative bacteria carrying β-lactamases has been considered as a serious health problem in recent decades due to their multi-resistance to β-lactam antibiotics and efficient disseminations. Genes encoding β-lactamases are mainly disseminated by horizontal transfer via conjugation or transposition. Conjugation is an effective way to transfer genetic material, especially drug resistance plasmids among different species. Plasmids with designated incompatibility (Inc) groups possess specific replication and partition system. Among all replicons, IncF plasmid is the most prevalent for carrying blaCTX-M in human and animal isolates while IncM plasmid is commonly identified as carrier for clinically important β-lactamases including carbapenemases and AmpC-type β-lactamases. Carbapenemases exhibit broad spectrum of resistance to cephalosporins and carbapenems, which greatly restrict therapeutic options. On the contrary, AmpC-type β-lactamases are sensitive to carbapenems but are able to hydrolyze cephalosporins and β-lactamase inhibitors.

The main focus of drug-resistant bacteria in the past was mainly relied on genome or plasmid sequencing to identify possible resistance and dissemination mechanisms in clinical isolates. However, this approach was not comprehensive enough to explicate other complicated drug resistance pathways as well as their interacting network. Therefore, recent development of transcriptome profiling becomes a new approach for understanding bacteria that produced chromosomal or plasmid-encoded β-lactamases and their resistance mechanisms to different types of antibiotics. In addition, systematic transcriptome analysis provides more comprehensive data for future therapeutic development to combat the emergence of antimicrobial-resistant bacteria.

In this work, Escherichia coli (E. coli) transconjugants that bear pNDM-HK were analyzed by RNA-Sequencing. Plasmid pNDM-HK was the first carbapenemase NDM-1 carrying plasmid identified in Hong Kong. The carbapenem resistance was attributed to porin (ompC and ompF) loss in the transconjugant. Moreover, the porin loss was resulted from a nonsynonymous point mutation G183A that caused glycine to serine amino acid substitution in transcription factor ompR. Further investigation showed this mutant was defective in receiving phosphoryl group from cognate kinase EnvZ, therefore restricted expression of porin and further enhanced carbapenem resistance. In addition, gene moderation by ompR were also observed in bioB, csgD and dtpA. The result indicated that ompR is a master regulator which controls multiple pathways and mutation in ompR played an essential role in bacterial fitness as well as carbapenem resistance.

In order to further understand the influence of pNDM-HK to bacterial host, plasmid¬encoded small regulatory RNAs (sRNAs) were identified by sRNA sequencing. Small RNAs are non-coding RNAs with 50-250 nucleotides and provide post-transcriptional regulation in response to external stimuli. In this work, six novel sRNAs were identified from different regions of plasmid such as replication, stability and variable regions. Their regulatory targets and response to external stresses were examined. One of the sRNAs, NDM-sR3 was proved to be associated with bacterial host fitness in RNA chaperone (Hfq) dependent manner. In addition, sRNA NDM-sR2 was identified as counter-transcribed RNA (ctRNA) and it repressed the translation of replication protein A. Intriguingly, integrating NDM-sR2 into chromosome of transconjugants carrying pNDM-HK resulted in lethality. This result suggested a potential approach of sRNA to selectively inhibit the plasmid replication, in particular the widely disseminated plasmids such as IncM, IncI and IncF plasmids. The finding shed light on developing a specific therapeutic alternative to suppress dissemination of antibiotic resistant pathogens without causing any harms to other beneficial bacteria.

To further understand the drug resistance mechanism of other bacteria, expression profiles of Laribacter hongkongensis grown at freshwater and human body temperatures were compared. This bacterium encodes AmpC-type β-lactamases in the chromosome and survives as either fish or human pathogens. The expression levels of β-lactamases were similar between two temperatures but hyperexpression of efflux pumps and virulence factors were observed at freshwater temperature, indicating a potential risk on antibiotic resistance and pathogenicity. Moreover, expression level of metabolic genes and stress-related proteins were found to be switched according to host temperature. This result suggested temperature can activate different pathways in order to enhance the chance for survival at different environments. By understanding their adaption mechanisms in different hosts, treatment could be exploited to interfere bacterial infection.

To conclude, RNomic studies were carried out on antimicrobial resistant bacteria to reveal their drug resistance and physiological adaption mechanisms. Analyzing their transcriptome profile under various conditions and understanding their physiological changes in response to external stimuli will help to develop strategies to prevent and eliminate these drug-resistant bacteria. Moreover, these findings provided insight and essential molecular basis to the multidrug resistance mechanisms, which facilitate the therapeutic development in the future.