Abstract
The prevalence of extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae is increasing rapidly. CTX-M type β-lactamases are the most prominent ESBL family worldwide and are produced mainly by E. coli. blaCTX-M-14 and blaCTX-M-15 genes are the dominant alleles circulating worldwide. The massive spread of CTX-M-producing organisms in both clinical and community settings have resulted in the CTX-M pandemic. However, the reasons for the rapid dissemination of this resistance mechanism remain unknown. It has been suggested that the success of CTX-M-15-producing E. coli is due to its association with the uropathogenic clone, sequence type 131 (ST131) that combines both virulence and multi-drug resistance mechanisms. The goal of my research was to understand the molecular mechanism(s) that contributes to CTX-M-mediated resistance with a focus on the two most dominant allotypes. Such studies could unveil potential targets for the development of new antibiotic therapies.|Initial steady-state expression studies demonstrated that CTX-M-15 mRNA was 8- to 165-fold higher than CTX-M-14 mRNA levels in E. coli strains isolated from human urine specimens from various geographical locations. Both CTX-M-14 and CTX-M-15 producers shared the same two promoters and transcriptional start sites and contained one copy of blaCTX-M-14 or blaCTX-M-15 on large clinical plasmids. Analysis of the upstream promoter regions using promoter deletion clones demonstrated that the proximal promoter elements within the non-coding region of ISEcp1 were responsible for the β-lactam resistant phenotype. Therefore, it was hypothesized that the genetic background of ST131 contributed to the upregulation of CTX-M-15 mRNA levels.|To this hypothesis, K12 transformants were constructed to evaluate the contribution of chromosomally-encoded factor(s) on the increased CTX-M-15 transcript levels VI observed. It was further hypothesized that CTX-M-14 and CTX-M-15 with the same K12 wild type E. coli background would have equivalent steady-state expression levels. The CTX-M-15 K12 transformant still showed an 11-fold increase in mRNA expression compared to the CTX-M-14 K12 transformant. These data indicated that the sequence type of the isolates was not a determining factor for the differential expression of these genes. Therefore, either an intrinsic structural feature was controlling transcription initiation of CTX-M-15 or a plasmid-encoded factor was causing differences in steady-state mRNA expression. Clones were created using heterologous promoters to drive expression of blaCTX-M-14/15 which still showed an upregulation of CTX-M-15. CTX-M chimeric clones were constructed through PCR to evaluate if the 5′ or 3′ halves of the CTX-M-15 gene contained an intrinsic structural element that affected transcription initiation. Expression of these constructs demonstrated that an element within the 5′ end of CTX-M-15 may control transcription initiation.|Additional studies that examined the stability of the CTX-M-14 and CTX-M-15 transcripts indicated that mRNA half-life also contributed to differential steady-state expression among these genes. The CTX-M-15 transcript produced by the majority of E. coli isolates had an extended half-life of 8-15 minutes that was controlled by a plasmid-encoded factor. Conjugation experiments involving three different E. coli hosts showed that the CTX-M harboring plasmid contained a factor that was also responsible for part of the differential expression among the CTX-M-14 and CTX-M-15 genes. However, the upregulation of CTX-M-15 mRNA levels did not correlate with CTX-M-15 β-lactamase production which is suggestive of either a post-transcriptional or translational regulation mechanism. Although some CTX-M-15 mRNA is translated into CTX-M-15 β-lactamase, the enzyme was not produced at a level to confer resistance to any of the β-lactam/β-VII lactamase inhibitor combinations evaluated including the new inhibitor, ceftolozane/tazobactam.|Collectively, my work has demonstrated the complexity associated with CTX-M β-lactamase expression in E. coli isolates collected from human urine samples. The data presented in this dissertation show that the regulation of CTX-M expression occurs at multiple levels including transcription inititation, mRNA half-life, and translation. This complex regulation could be a contributing factor for the successful spread of blaCTX-M-14 and blaCTX-M-15.