Evaluation of CTX-M steady-state mRNA, mRNA half-life and protein production in various STs ofEscherichia coli

Abstract

ObjectivesHigh 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.MethodsDisc 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.ResultsmRNA 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.ConclusionsCTX-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.

Inhibitory Effects of the Spore-forming Bacillus spp. on the Expression Levels of eae, luxS, flu, and ctxM Genes in E. coli Isolates

“…Spore-forming Bacillus spp. are one of these bacteria with high levels of tolerance to harsh conditions that are currently being used in dry probiotic products due to their ability to survive in conditions of high or low temperatures, aridity, and high oxygen levels [28].…”

OmpC regulation differs between ST131 and non-ST131 Escherichia coli clinical isolates and involves differential expression of the small RNA MicC

“…Previous data from our laboratory showed that ST did not impact CTX-M protein levels, but perhaps the presence of a particular CTX-M could impact porin production. 20 The overall trend for ompC expression was highest for ST131 isolates (Figure 1) with a range of expression from 457-to 6483-fold compared with XQ13 (ST68) regardless of whether CTX-M-14 or CTX-M-15 was produced. The trend for OmpC mRNA levels in non-ST131 isolates was lower and ranged from no difference compared with XQ13 to 638-fold.…”

“…Ten CTX-M-14-producing and 10 CTX-M-15-producing E. coli clinical isolates of various STs were collected from urine. 20 These isolates were collected from varying geographical regions to ensure that the data represented a wide distribution of CTX-M-producing isolates and not a local clonal outbreak (Table 1). The K-12 derivative WT strain BW25113 (BW) and its single-gene knockouts JW2203-1 (DompC), JW0912-1 (DompF) and JW4130-1 (Dhfq) were obtained from the Keio collection.…”

“…RNA isolation and expression analyses were carried out as previously described. 20 Relative mRNA expression was calculated using the 2 #DDCt method using the clinical isolate XQ13 as the comparator. Because the level of OmpC and OmpF protein production was equivalent to the laboratory strain BW25113, we chose to use strain XQ13 as our comparator as this strain represents WT levels of protein production within a clinical isolate.…”

Model-driven promoter strength prediction based on a fine-tuned synthetic promoter library inEscherichia coli

“…Total RNA was extracted using the Ultrapure RNA Kit (Novoprotein, Shanghai, China) and reverse transcribed using the SuperRT One-Step RT-PCR Kit (Novoprotein, Beijing, China). Real time quantitative PCR (qPCR) using a SuperRT One-Step RT-PCR Kit (Novoprotein, Beijing, China) was performed and analyzed according to the protocol 57,58 .…”