Hyperproduction of AmpC ß-lactamase in a clinical isolate of Escherichia coli associated with a 30 bp deletion in the attenuator region of ampC

Felipe Fernández-Cuenca1,*, Alvaro Pascual1 and Luis Martínez-Martínez2

1 Department of Microbiology, University Hospital Virgen Macarena and School of Medicine, University of Seville, Apdo. 914, 41009 Seville, Spain; 2 Service of Microbiology, University Hospital Marqués de Valdecilla, Santander, Spain


* Corresponding author. Tel: +34-954-552-862; Fax: +34-954-377-413; Email: felipefc{at}us.es

Keywords: E. coli , AmpC , mutations , antimicrobial resistance

Sir,

The expression of the chromosomally encoded AmpC ß-lactamase of Escherichia coli is not inducible, because of the absence of the regulatory gene ampR. The ampC gene of E. coli is regulated by a weak promoter and a transcriptional attenuator. Hyperproduction of AmpC can arise by gene amplification of ampC or mutations at either the promoter and/or the attenuator of ampC, which increase the transcription rate of ampC.1

Constitutive low-level expression of the AmpC ß-lactamase of E. coli does not contribute to a clinically relevant level of resistance to ß-lactams. However, hyperproduction of this enzyme causes resistance to penicillins, first- and second-generation cephalosporins, and cephamycins, and may decrease the activity of oxyimino-cephalosporins. As AmpC is not inhibited by clavulanic acid and similar inhibitors of class A ß-lactamases, strains hyperproducing this enzyme are also resistant to the combinations of penicillins with these ß-lactamase inhibitors.

In this study we describe a novel mutation in the attenuator of the ampC gene of E. coli 47/94, an AmpC-hyperproducing clinical strain isolated in 1994 from a urine sample of a patient at the Clinical Microbiology Department, University Hospital Virgen Macarena, Seville, Spain.2

Mutations in the promoter/attenuator region of the ampC gene were identified as described previously3 by DNA sequencing of the PCR products in both directions using the Big dye terminator v3.0 sequencing kit (Applied Biosystems, Foster City, CA, USA). Sequence analysis was performed on a 3700 DNA Analyzer (Applied Biosystems).

Five point mutations at positions –88 (C->T), –82 (A->G), –42 (C->T), –18 (G->A) and –1 (C->T) of the ampC promoter of E. coli 47/94 were observed (Figure 1). All these mutations have been described previously in clinical isolates of E. coli hyperproducing AmpC.36 The role of the mutation at –42 in the hyperproduction of AmpC has been studied in more detail than the mutations at –88, –82, –18 and –1. The C->T transition at position –42 creates a perfect TTGACA box upstream of the normal –35 box, generating a strong ampC promoter and modifying the transcription initiation site.



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Figure 1. Nucleotide sequence of the ampC promotor/attenuator of E. coli K-12 and E. coli 47/94. Point mutations in the ampC promoter are shown in bold. The DNA sequence of the attenuator of ampC containing the dyad symmetry region is shown in bold and is also underlined.

 
In addition to the these mutations, a deletion of 30 nucleotides between positions +16 and +45 containing the dyad symmetry region of the ampC attenuator was also observed in E. coli 47/94 (Figure 1). This deletion has not been reported previously, and is likely to cause high-level transcription of ampC by decreasing the stability of the hairpin loop of the attenuator. The MICs of cefotaxime and ceftazidime for E. coli 47/94 were higher (64 and 16 mg/L, respectively) than those reported for other E. coli isolates with similar point mutations in the ampC promoter but without this deletion.1,6 This suggests that the 30 bp deletion in the attenuator region of ampC may contribute to an increase in the level of resistance of E. coli 47/94 to oxyimino-cephalosporins. Additional studies are being undertaken to determine further the effect of this deletion on the ampC promoter of this isolate of E. coli.

In conclusion, the hyperproduction of AmpC in E. coli 47/94 was associated with point mutations in the ampC promoter and a deletion of 30 bp affecting the attenuator region of ampC.

References

1. Nelson EC, Elisha BG. Molecular basis of AmpC hyperproduction in clinical isolates of Escherichia coli. Antimicrob Agents Chemother 1999; 43: 957–9.[Abstract/Free Full Text]

2. Martínez-Martínez L, Conejo MC, Pascual A et al. Activities of imipenem and cephalosporins against clonally related strains of Escherichia coli hyperproducing chromosomal ß-lactamase and showing altered porin profiles. Antimicrob Agents Chemother 2000; 44: 2534–6.[Abstract/Free Full Text]

3. Caroff N, Espaze E, Gautreau D et al. Analysis of the effects of –42 and –32 ampC promoter mutations in clinical isolates of Escherichia coli hyperproducing AmpC. J Antimicrob Chemother 2000; 45: 783–8.[Abstract/Free Full Text]

4. Siu LK, Lu PL, Chen JY et al. High-level expression of AmpC ß-lactamase due to insertion of nucleotides between –10 and –35 promoter sequences in Escherichia coli clinical isolates: cases not responsive to extended-spectrum-cephalosporin treatment. Antimicrob Agents Chemother 2003; 47: 2138–44.[Abstract/Free Full Text]

5. Forward KR, Willey BM, Low DE et al. Molecular mechanisms of cefoxitin resistance in Escherichia coli from the Toronto area hospitals. Diagn Microbiol Infect Dis 2001; 41: 57–63.[CrossRef][ISI][Medline]

6. Clarke B, Hiltz M, Musgrave H et al. Cephamycin resistance in clinical isolates and laboratory-derived strains of Escherichia coli, Nova Scotia, Canada. Emerg Infect Dis 2003; 9: 1254–9.[ISI][Medline]





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