Next-generation DNA sequencing (NGS) has progressed enormously over the past decade, transforming genomic analysis and opening up many new opportunities for applications in clinical microbiology ...laboratories. The impact of NGS on microbiology has been revolutionary, with new microbial genomic sequences being generated daily, leading to the development of large databases of genomes and gene sequences. The ability to analyze microbial communities without culturing organisms has created the ever-growing field of metagenomics and microbiome analysis and has generated significant new insights into the relation between host and microbe. The medical literature contains many examples of how this new technology can be used for infectious disease diagnostics and pathogen analysis. The implementation of NGS in medical practice has been a slow process due to various challenges such as clinical trials, lack of applicable regulatory guidelines, and the adaptation of the technology to the clinical environment. In April 2015, the American Academy of Microbiology (AAM) convened a colloquium to begin to define these issues, and in this document, we present some of the concepts that were generated from these discussions.
High-resolution melting (HRM) analysis can be a diagnostic tool to evaluate the presence of resistance genes with the added bonus of discriminating sequence modifications. A real-time, multiplex PCR ...assay using HRM was designed for the detection of plasmid-mediated ampC genes. The specificity and sensitivity of this assay were 96% and 100%, respectively.
A novel metallo-β-lactamase gene, blaIMP-27, was identified in unrelated Proteus mirabilis isolates from two geographically distinct locations in the United States. Both isolates harbor blaIMP-27 as ...part of the first gene cassette in a class 2 integron. Antimicrobial susceptibility testing indicated susceptibility to aztreonam, piperacillin-tazobactam, and ceftazidime but resistance to ertapenem. However, hydrolysis assays indicated that ceftazidime was a substrate for IMP-27.
High levels of β-lactamase production can impact treatment with a β-lactam/β-lactamase inhibitor combination. Goals of this study were to: (i) compare the mRNA and protein levels of CTX-M-15- and ...CTX-M-14-producing Escherichia coli from 18 different STs and 10 different phylotypes; (ii) evaluate the mRNA half-lives and establish a role for chromosomal- and/or plasmid-encoded factors; and (iii) evaluate the zones of inhibition for piperacillin/tazobactam and ceftolozane/tazobactam.
Disc diffusion was used to establish zone size. RNA analysis was accomplished using real-time RT-PCR and CTX-M protein levels were evaluated by immunoblotting. Clinical isolates, transformants and transconjugants were used to evaluate mRNA half-lives.
mRNA levels of CTX-M-15 were up to 165-fold higher compared with CTX-M-14. CTX-M-15 protein levels were 2-48-fold less than their respective transcript levels, while CTX-M-14 protein production was comparable to the observed transcript levels. Nineteen of 25 E. coli (76%) had extended CTX-M-15 mRNA half-lives of 5-15 min and 16 (100%) CTX-M-14 isolates had mRNA half-lives of <2-3 min. Transformants had mRNA half-lives of <2 min for both CTX-M-type transcripts, while transconjugant mRNA half-lives corresponded to the half-life of the donor. Ceftolozane/tazobactam zone sizes were ≥19 mm, while piperacillin/tazobactam zone sizes were ≥17 mm.
CTX-M-15 mRNA and protein production did not correlate. Neither E. coli ST nor phylotype influenced the variability observed for CTX-M-15 mRNA or protein produced. mRNA half-life is controlled by a plasmid-encoded factor and may influence mRNA transcript levels, but not protein levels.
Cloning is a basic molecular biology procedure that has many applications in biomedical and industrial research. However, cloning protocols can be challenging and screening transformants by PCR ...amplification on conventional heat block thermal cyclers can require over 4 hours. A faster alternative is to use the rapid cycling capabilities of the Streck Philisa(R) Thermal Cycler to screen transformants for the cloned gene of interest. Here we use the Philisa(R) Thermal Cycler to evaluate potential clones containing the b-lactamase gene, blaCTX-M-15, ligated into the cloning vector, pMP220.
Abstract A previously designed end-point multiplex PCR assay and singleplex assays used to detect β-lactamase genes were evaluated using rapid PCR amplification methodology. Amplification times were ...16–18 minutes with an overall detection time of 1.5 hours. Rapid PCR amplifications could decrease the time required to identify resistance mechanisms in Gram-negative organisms.
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-14CTX-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-Mmediated 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-14CTX-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 IS Ecp1 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 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-14CTX-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 plasmidencoded 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-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
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