Comparison of two RT-PCR methods for quantifyingampCspecific transcripts inEscherichia colistrains

Abstract

In Escherichia coli, beta-lactam resistance usually depends on beta-lactamase production. AmpC chromosomal cephalosporinase hyperproduction is generally due to mutations in the ampC gene promoter. In order to study ampC expression in E. coli clinical strains, we have compared two methods: conventional and real-time reverse transcription-polymerase chain reaction (RT-PCR). With both methods, ampC mRNA was found to be greatly increased in strains presenting -42 or -32 mutations in the ampC promoter, and moderately increased when a -11 mutation was present in the Pribnow box. Real-time RT-PCR represents a powerful tool combining amplification, fluorescent detection and analysis.

ampCgene expression in promoter mutants of cefoxitin-resistantEscherichia coliclinical isolates

“…Strains with promoter types with mutations only in the attenuator region or that did not contain mutations creating a consensus −35 box only expressed ampC 1–4‐fold, similar to strains with promoter types with mutations outside of functional elements (Table 1). This study supports previous studies that showed that it is the creation of a consensus −35 box, whether by a T→A transversion at position −32 or a C→T transition at position −42, that is the most dominant factor in strengthening the ampC promoter (Jaurin et al , 1982; Olsson et al , 1982, 1983; Caroff et al , 1999, 2000; Nelson & Elisha, 1999; Forward et al , 2001; Corvec et al , 2003). The results also support previous studies showing that optimal spacing between −35 and −10 boxes is also an important factor contributing to promoter strength (Jaurin et al , 1981; Siu et al , 2003).…”

“…A transversion at position À 32 or a C ! T transition at position À 42, that is the most dominant factor in strengthening the ampC promoter (Jaurin et al, 1982;Olsson et al, 1982Olsson et al, , 1983Caroff et al, 1999Caroff et al, , 2000Nelson & Elisha, 1999;Forward et al, 2001;Corvec et al, 2003). The results also support previous studies showing that optimal spacing between À 35 and À 10 boxes is also an important factor contributing to promoter strength (Jaurin et al, 1981;Siu et al, 2003).…”

Functional characterization of a putative β-lactamase gene in the genome of Zymomonas mobilis

“…The C t value of ZMO0103 transcript indicated that the ZMO0103 expression was greater when compared to the relative transcript quantities in antibiotic-susceptible bacteria like E. coli strain ATCC 25922 (Corvec et al 2003). The C t value of ZMO0103 transcript indicated that the ZMO0103 expression was greater when compared to the relative transcript quantities in antibiotic-susceptible bacteria like E. coli strain ATCC 25922 (Corvec et al 2003).…”

Differential expression of blaSHV related to susceptibility to ampicillin in Klebsiella pneumoniae

“…Total RNA was extracted with TRIzol (Invitrogen, Carlsbad, CA) as recommended, treated with RNase-free DNase (Promega) (1.5 U/g of RNA) for 35 min, purified with phenol and precipitated with 100% ethanol. cDNA synthesis was performed as described previously [6] using Moloney murine leukaemia virus reverse transcriptase (Promega). Transcriptional expression of bla SHV was measured by RT-PCR with primers SHV-C and SHV-D as described above.…”