Analysis of the effects of -42 and -32 ampC promoter mutations in clinical isolates of Escherichia coli hyperproducing AmpC

Abstract

Escherichia coli usually produces only very small amounts of a constitutive AmpC beta-lactamase, but clinical strains overproducing this enzyme have been isolated. Three different ampC promoters of E. coli clinical strains were cloned upstream of the chloramphenicol acetyltransferase (CAT) gene in the pKK232-8 reporter plasmid and their relative strengths were compared by two different methods. The strength of the promoters from AmpC hyperproducers was 70- to 120-fold higher than those from a low-level AmpC producer. One of the strong promoters, which differs from strain K12 at bases -88, -82, -42, -18, -1 and +58, was mutated to abolish the -42 mutation. This change resulted in a 43-fold decrease in CAT concentration. In another promoter, with eight different mutations at positions -88, -82, -32, -18, -1, +5, +24 and +58, the -32T-->A transversion, which created perfect homology with the -35 consensus sequence, was reverted; this led to a 13-fold decrease in CAT concentration. The -42 and -32 mutations play an important role in E. coli resistance to beta-lactams by increasing ampC transcription.

ChromosomalampCmutations in cefpodoxime-resistant, ESBL-negative uropathogenicEscherichia coli

“…Strains with chromosomal ampC mutations can produce enzymes in higher amounts and are said to hyper-produce the AmpC enzyme, leading to clinical resistance and treatment failures. 3 The common DNA sequence seen in numerous E. coli promoters is a -35 box (TTGACA) separated from a -10 box (TATAAT) by 17bp. 2 The normal ampC promoter sequence, however, includes single nucleotide difference in each of these hexameric boxes, together with a spacer difference of 16bp ( Figure 1).…”

“…2 The normal ampC promoter sequence, however, includes single nucleotide difference in each of these hexameric boxes, together with a spacer difference of 16bp ( Figure 1). These small differences are sufficient to affect the function of the promoter and decrease the normal AmpC enzyme production to its 3 constitutive low level. 4 Mutations in the ampC promoter region can arise, and include transitions and insertions in the -35 or -10 boxes, which create a region more closely related to the standard E. coli promoter sequence, and thus a stronger promoter.…”

Role of ISAba1 and ISAba125 in governing the expression of bla ADC in clinically relevant Acinetobacter baumannii strains resistant to cephalosporins

“…This confers resistance to oxyimino-and 7-alphamethoxy-cephalosporins and monobactams (Sanders, 1987). Class C b-lactamases produced by Gram-negative bacteria can hydrolyse many b-lactam antibiotics including the cephamycins and oxyimino-cephalosporins (Philippon et al, 2002;Sanders, 1987;Caroff et al, 2000). These enzymes can also hydrolyse monobactams, such as aztreonam, to a lesser extent (Bush et al, 1995;Mammeri & Nordmann, 2007).…”

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

“…normally produced constitutively in small amounts (Caroff et al 2000). Although bla AmpC genes can be acquired on plasmids from other species, chromosomal mutations in the ampC promoter region are a more common cause of increased enzyme production (Lewis et al 2015).…”

“…A PCR protocol of 95 o C for 5 mins, followed by 35 cycles of 95 o C for 10s and 60 o C for 30s was used, with melting curve analysis at the end. A 271bp region of the ampC gene, including the promoter region, was amplified using previously published primers (0.1 umol/L) (Caroff et al 2000) and the same PCR protocol. PCR products were sent to an external company (Eurofins Genomics, Germany) for sequencing, using the forward amplification primer.…”