Integrating genomics and transcriptomics to understand the virulence and biofilm forming mechanism of selected vancomycin-resistant Enterococcus faecium / Lim Shu Yong

Lim, Shu Yong (2018) Integrating genomics and transcriptomics to understand the virulence and biofilm forming mechanism of selected vancomycin-resistant Enterococcus faecium / Lim Shu Yong. Masters thesis, University of Malaya.

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Abstract

Vancomycin-resistant enterococcus (VRE) is an emerging nosocomial pathogen which causes outbreaks in hospitals worldwide. It is, therefore, important to understand the virulence and how this organism persists as a nosocomial pathogen. Whole genome sequencing (WGS) provides a wealth of information to elucidate the genetic relationship, virulence potential, and resistance factors of VRE. However, such genomic studies are lacking in Malaysia. Moreover, since enterococci are often recovered from difficult-to-treat biofilm-mediated infections, a detailed study on the biofilm formed by VRE is useful to better understand its pathogenicity. The objectives of this study are to perform comparative genomics analysis on the four local vancomycin-resistant Enterococcus faecium (VREfm) and to elucidate the transcriptomic profile of biofilm cells in respect to the planktonic cells. Four VREfm strains were isolated from two fatal cases of nosocomial infections in a tertiary hospital in Kuala Lumpur. One of these strains (VRE2) was isolated from an index case (patient X), whereas the other three (VREr5, VREr6, VREr7) were isolated from different body sites of another patient (patient Y) at around one-week interval. WGS and comparative genomics analyses revealed that the four strains have different sequence types (STs), ST80 and ST203. Subsequent phylogenomic study showed that VREr5 was more closely related to VRE2, but was distantly related to VREr6 and VREr7 derived from the same host. Moreover, the genomic contents of VREr5 was also more similar to VRE2. The genomic data and clinical records suggested that patient Y was most probably infected by multiple strains of different clones. Alternatively, the strain that infected patient Y (VREr5) could have derived from the same clone from patient X (VRE2), given their high genomic similarity. The four local strains were multidrug resistant. All of them carried the vanA genotype and showed indistinguishable Tn1546 structure. Virulence profiling revealed that these strains harbored a total of 13 virulence genes mainly associated with adherence and biofilm formation. The transcriptomic analysis focused on the initial stage of biofilm formation to examine genes that are involved during the transition from planktonic to biofilm cells. Differential gene expression analysis revealed that the up-regulated genes in biofilm cells involved mainly in adherence, plasmid replication, and carbohydrate metabolism. Genes that have been reported to negatively regulate biofilm formation, such as the quorum sensing systems, fsr and luxS, and a transcriptional regulator gene spx, were highly down-regulated. The unique bee homolog of VREr5 was found to be down-regulated, implying a negative association of this locus to biofilm formation in VREr5. The results obtained from the gene expression study clearly reflected the attachment stage of biofilm development, including the preparation to enter the maturation stage. In conclusion, this study has contributed to the understanding of the genetic basis and diversity of local clinical strains which can be helpful to control the spread of VRE. This study also provides insight into the molecular mechanism of biofilm formation in VRE which might be useful in the development of new drugs.

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