| Author: | Shenkutie, Abebe Mekuria |
| Title: | Molecular mechanisms of biofilm-specific antibiotic resistance in acinetobacter baumannii |
| Advisors: | Leung, Polly (HTI) |
| Degree: | Ph.D. |
| Year: | 2021 |
| Subject: | Drug resistance in microorganisms Biofilms Hong Kong Polytechnic University -- Dissertations |
| Department: | Department of Health Technology and Informatics |
| Pages: | xxi, 265 pages : color illustrations |
| Language: | English |
| Abstract: | Acinetobacter baumannii is a priority I antibiotic-resistant microbial pathogen. Although it has a remarkable capacity to acquire external genetic determinants of resistance, this is not the sole reason for failure to treat A. baumannii infections with antibiotics. Biofilm formation is an important strategy by which pathogens survive exposure to antibiotics. The emergence and spread of A. baumannii depends on both planktonic and biofilm-specific antibiotic resistance mechanisms. Antibiotic resistance mechanisms have been well studied in planktonic cultures of A. baumannii. However, limited in-depth studies have been conducted on the antibiotic resistance mechanisms associated with A. baumannii biofilms. To understand the connection between biofilm formation, antibiotic resistance and the mechanisms involved in the regulation of biofilm-specific antibiotic resistance, this thesis aims to (i) investigate the relationship between antibiotic susceptibility and the biofilm formation capability of clinical A. baumannii strains; (ii) compare the transcriptomes of A. baumannii biofilms to those of biofilm cells treated with sub-optimal concentrations of antibiotics; and (iii) investigate the regulatory role of small RNA00203 (sRNA00203) in biofilm formation and the development of biofilm-specific antibiotic resistance in A. baumannii. To understand the connection between biofilm formation capability and antibiotic susceptibility, 104 clinical A. baumannii strains were studied. We found that 59.6% of the strains were biofilm formers. We also observed that non-multidrug-resistant A. baumannii strains were strong biofilm formers. The antibiotic susceptibility of biofilm cells was evaluated in nine biofilm formers. The antibiotic concentrations required to eradicate the biofilm were 44–364 times higher than those required to kill planktonic bacteria. Of the nine strains tested, A. baumannii ST1894 developed the highest level of resistance to imipenem, ciprofloxacin and colistin. The reversibility test for antibiotic susceptibility showed that biofilm formation induced reversible antibiotic tolerance in the non-multidrug-resistant strains but resulted in a higher level of irreversible resistance in the extensively drug-resistant strain. To understand the mechanisms involved in the regulation of biofilm-specific antibiotic resistance, the hyper biofilm-forming strain A. baumannii ST1894 was subjected to global transcriptome profiling. Planktonic and biofilm cells of this strain were treated with colistin and imipenem at sub-inhibitory concentrations. The transcriptome profiles were obtained by RNA sequencing using an Illumina NovaSeq platform. We found that 1592 (51.8%) of the 3,075 transcribed genes were differentially expressed between the mature biofilm and planktonic cells; 106 and 368 biofilm-specific genes were differentially expressed in biofilm cells treated with sub-inhibitory concentrations of imipenem and colistin, respectively. The differentially expressed genes induced by imipenem and colistin in biofilm cells included genes that encoded outer membrane transport proteins, resistance–nodulation–cell division multidrug efflux systems, fimbrial proteins, homoserine lactone synthases and matrix synthesis proteins. The expression levels of metabolism-related genes, such as those coding for acinetobactin biosynthesis, DNA replication and translation and transport of D- and L-methionine, were significantly reduced in the biofilm cells. The effects of suboptimal concentrations of imipenem and colistin on biofilm-specific genes and their regulatory pathways may account for the reduced susceptibility of biofilm cells to antibiotics. When we investigated the regulatory role of sRNA00203, we identified 138 sRNAs as regulatory targets based on the transcriptome data of A. baumannii ST1894. Among the identified sRNAs, the gene encoding sRNA00203 was more strongly upregulated in the biofilm cells than in the planktonic cells. We found that deletion of this gene using suicide plasmid-mediated allelic exchange substantially impaired biofilm formation in A. baumannii ST1894. Deletion of sRNA00203 also increased the susceptibility of the biofilm cells to imipenem and ciprofloxacin. We showed that deletion of sRNA00203 significantly decreased the expression levels of genes that code for efflux pumps (novel00738), matrix synthesis (pgaB ), preprotein translocase subunit (secA) and the CRP transcriptional regulator. These findings demonstrate a clear role for sRNA00203 in the regulation of biofilm formation and development of antibiotic susceptibility in A. baumannii ST1894. The inhibition of sRNA00203 presents a new strategy for treating biofilm-specific infections by impairing biofilm production and increasing the antibiotic susceptibility of biofilm cells. In summary, this thesis demonstrates that non-multidrug-resistant A. baumannii strains are strong biofilm formers. The biofilm state of growth promoted reversible antibiotic tolerance in non-multidrug-resistant strains and higher levels of irreversible resistance in extensively drug-resistant strains. The transcriptome analysis demonstrated that the differential expression of genes associated with antibiotic resistance and metabolic quiescence could be responsible for the reduced susceptibility of biofilm cells to antibiotics. Furthermore, this thesis is the first of its kind to demonstrate the effect of sRNA00203 on the expression of genes associated with biofilm formation and the development of antibiotic resistance in clinical A. baumannii strains. |
| Rights: | All rights reserved |
| Access: | open access |
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