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(2015)Antibiotic consumption and molecular epidemiology of ESBL-producing escherichia coli and klebsiella pneumoniae in a Lebanese hospital Doctoral thesis, University of Surrey.
Escherichia coli is an important nosocomial pathogen and extended-spectrum cephalosporin (ESC) resistance E. coli is recognized as the global drug-resistant threat. This study aims to determine the resistance mechanisms of ESC and genetic diversity of Malaysian E. coli strains using various phenotypic and genotypic approaches. One hundred ten non-repeat E. coli strains were isolated from the stools of paediatric patients in 2009 from a tertiary hospital in Malaysia. Twenty-one were categorized as extended spectrum beta-lactamase (ESBL)-producers using the double-disk synergy test and ESBL E-test. CTX-M-15 was the pre-dominant CTX-M variant (77%). Class 1 integron was the most common class of integrons found in the E. coli strains (76%); however, they lack gene cassette encoding ESBL genes. Using PFGE, three strains displayed DNA degradation (Dnd) phenotype and few CTX-M-15-positive strains with indistinguishable pulsotypes were identified. To better understand the dissemination of CTX-M genes, E. coli strains from clinical, zoonotic, food and environment samples isolated between 2002 - 2011 in Malaysia were screened for ESBL-production. Using multi-locus sequence typing (MLST), 6 out of 35 CTX-M positive E. coli strains belonged to the sequence type (ST) ST131, an E. coli clone notorious for the global dissemination of CTX-M genes. Insertional sequences IS26 and ISEcp1 were the most common genetic environments for CTX-M genes in the Malaysian E. coli strains. The degradation phenotype which was observed earlier in this thesis was overcome for 12 Dnd+ E. coli strains by adding thiourea into the PFGE run. The dnd operon, responsible for the phosphorothioation modification of DNA, was detected in all Dnd+ E. coli strains by PCR. Further genomic analysis of the genetic environment of dnd operons of 52 global E. coli genomes revealed a total of 7 types of dnd-encoding genomic islands, indicating substantial diversity in these regions. dnd operons were more often found in pathogenic E. coli, indicating a possible linkage of the dnd operons with E. coli pathogenicity. Two highly related E. coli strains EC302/04 and EC096/10 with single band difference in their pulsotypes (identified in the first part of this study) were investigated using whole genome sequencing (WGS). EC302/04 and EC096/10 that were isolated from tracheal aspirate and a stool specimen, respectively, shared the same sequence type ST349, and serotype O166:H5. EC302/04 harboured a 140,232 bp IncFII plasmid, pEC302/04, that was absent in EC096/10. Plasmid pEC302/04 is self-transferable and carried resistance genes (blaTEM-1; integron-encoded- sul1, cml, and aadA) and two iron-acquisition systems. Phylogenomic analysis with 38 global E. coli of various pathotypes revealed that ExPEC and intestinal strains may share similar phylogenetic signals. This may help in identifying commensal-like strains with extraintestinal virulence potential. Phylogenomic analysis also unveiled ExPEC with rare genotypes, indicating the importance of genomic approach in detecting the potential emergence of new ExPEC lineage(s). Besides providing comprehensive understanding on the resistance mechanisms of the clinically important CTX-M genes, this thesis also demonstrates the usefulness of whole genome sequence approach in investigating the resistance mechanisms and genetic diversity of specific region or whole genome of various types of E. coli strains. (500 words)
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Screening and confirmation of ESBL-producing phenotypes among the clinical isolates were performed according to the guidelines of the Clinical and Laboratory Standard Institute (CLSI, 2012).
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