Molecular Characterization of Cefoxitin-Resistant Escherichia coli from Canadian Hospitals

Abstract

A study designed to gain baseline information on strains of Escherichia coli displaying resistance to cefoxitin in Canada is described. A total of 29,323 E. coli isolates were screened at 12 participating hospital sites as part of an extended-spectrum beta-lactamase surveillance initiative. A total of 411 clinically significant, nonrepeat isolates displaying reduced susceptibilities to the NCCLS-recommended beta-lactams were submitted to a central laboratory over a 1-year period ending on 30 September 2000. Two hundred thirty-two isolates were identified as resistant to cefoxitin. All cefoxitin-resistant strains were subtyped by pulsed-field gel electrophoresis, and of these, 182 strains revealed a unique fingerprint and 1 strain was untypeable. PCR and sequence analysis of the ampC promoter region revealed 51 different promoter or attenuator variants and 14 wild-type promoters. Three promoter regions were interrupted by insertion elements, two contained IS10 elements, and one contained an IS911 variant. PCR and sequence analysis for the detection of acquired AmpC resistance (by the acquisition of ACT-1/MIR-1, CMY-2, or FOX) revealed that 25 strains contained CMY-2, including 7 of the strains found to have wild-type promoters. The considerable genetic variability in both the strain fingerprint and the promoter region suggests that AmpC-type resistance may emerge spontaneously by mutation of sensitive strains rather than by the spread of strains or plasmids in the hospital setting.Escherichia coli contains a chromosomal ampC gene which has a weak promoter as well as a transcriptional attenuator (13,14). Strains carrying the wild-type gene produce a low basal amount of AmpC and are susceptible to ampicillin. Analysis of cefoxitin-resistant strains has revealed that the ampC promoter and attenuator regions often carry mutations leading to the overproduction of the enzyme (3,4,5,7,10,14,20,21,22,29). These AmpC-overproducing strains are not only resistant to ampicillin but also usually have reduced susceptibilities to expanded-spectrum cephalosporins. Genetically, the most common mutations are those that create a strong promoter that more closely resembles the E. coli consensus promoter (12) and mutations that destabilize the attenuator.Plasmid-mediated ampC genes were first reported in 1988 (25, 31). The plasmid-mediated ampC genes are derived from inducible chromosomal genes that have become mobilized (11,24,25). The first genetically characterized plasmid-mediated AmpC was MIR-1, which was mobilized from an Enterobacter isolate (23). These plasmid-mediated genes are of special interest because their mobility allows them to emerge in one genus or species and spread to different organisms. The prevalence of plasmid-mediated AmpC-type resistance at the national level in most countries is unknown because studies have not examined the strains at the molecular level of detail required to elucidate the different mechanisms involved. A recent report from the United States, however, showed that among 752 Klebsiella spp. a...

ChromosomalampCmutations in cefpodoxime-resistant, ESBL-negative uropathogenicEscherichia coli

“…5 Whilst we found only three strains with the -42 mutation, other studies have reported a higher prevalence, 13,[17][18][19] with one study finding 100% of isolates with this mutation. 20 Eight of the 40 strains in our study had insertions of either one or two bases at position -13.…”

“…However, it is not clear whether this has a significant impact on enzyme function. 13 The geometric mean MIC for cefotaxime was 1.2mg/L for all strains, just above the recommended breakpoint of 1.0mg/L. 10 Those strains with the -32 mutation present had a lower cefotaxime MIC of 0.6mg/L, compared to 1.2mg/L for strains with other mutations.…”

“…The -42 (C→T) substitution creates a new -35 box, whereas the G→A mutation at -18 gives rise to a new -10 box region. These mutations are often reported together, 13 and result in two new regions 17bp apart at different locations. The increased distance between the two regions has been reported to affect promoter strength.…”

Multi-locus sequence typing of Escherichia coli isolates with acquired ampC genes and ampC promoter mutations

“…Other studies have found a higher prevalence for this particular mutation (Mulvey et al 2005;Jorgensen et al 2010;Mammeri et al 2010;Alonso et al 2016), with one reporting a prevalence of 100% (Bogaerts et al 2010). There were two strains (SW-007 and TR-032) that were found to have insertions (thymine) at position -20.…”

“…Unusually, the -32 mutation (T to A) was the most common (63%); whereas other studies have reported a higher prevalence for the -42 mutation (Mulvey et al 2005;Mammeri et al 2008;Bogaerts et al 2010;Jorgensen et al 2010;Alonso et al 2016). We therefore set out to investigate whether this was also the case in other parts of the South West of England.…”