Journal of Antimicrobial Chemotherapy

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Journal of Antimicrobial Chemotherapy (2002) 49, 557-560
© 2002 The British Society for Antimicrobial Chemotherapy

Brief report

The ACT-1 plasmid-encoded AmpC ß-lactamase is inducible: detection in a complex ß-lactamase background

Mark D. Reisbig^a,b and Nancy D. Hanson^a,b,*

^a Department of Medical Microbiology and Immunology and ^b Center for Research in Anti-Infectives and Biotechnology, Creighton University School of Medicine, Omaha, NE, USA

	Abstract

Top Abstract Introduction Materials and methods Results Discussion Acknowledgements References

 
The purpose of this study was to identify the genetic organization and inducibility of bla_ACT-1 in a clinical isolate of Klebsiella pneumoniae possessing at least five different ß-lactamases. The genetic organization of the bla_ACT-1/ampR region is identical to those of inducible chromosomal ampC genes. RNA analysis using primer extension demonstrated a five-fold increase in bla_ACT-1 transcript production on exposure to cefoxitin. These findings are significant because induction was detected in a complicated ß-lactamase background. In addition, this report is the first to describe an inducible plasmid-encoded AmpC ß-lactamase of Enterobacter cloacae origin.

	Introduction

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High-level expression of AmpC ß-lactamases by Gram-negative organisms can result in organisms that are resistant to almost all ß-lactam drugs.¹ Overexpression of inducible ampC genes occurs in at least three different ways: (i) induction of ampC in the presence of inducing ß-lactam antibiotics and the appropriate AmpR, a DNA binding protein that regulates ampC expression; (ii) perturbations of the induction pathway in the absence of ß-lactam drug (derepression); or (iii) expression from plasmid genes. Perturbations in the induction pathway and plasmid-mediated expression result in greatly increased, constitutive AmpC expression.²

It has been suggested that ampC genes carried on plasmids were originally chromosomal genes.¹ Several plasmid-encoded enzymes, probably originating from Citrobacter freundii, Enterobacter cloacae, Morganella morganii or Hafnia alvei, have been described.¹ Until recently, plasmid-encoded ampC genes were thought to be non-inducible due to lack of a functional AmpR or absence of an AmpR binding site. However, two inducible plasmid-encoded ampC genes of M. morganii origin, bla_DHA-1 and bla_DHA-2, have been described, destroying the generalization_.^1,3

In this report we demonstrate that the plasmid-encoded ampC gene, bla_ACT-1, discovered in a Klebsiella pneumoniae isolate expressing at least five different ß-lactamases, is inducible.^4,5 This is the first inducible plasmid-encoded ampC gene of Enterobacter origin to be described. The production of several ß-lactamases by the K. pneumoniae isolate made detection of ACT-1 induction by standard phenotypic methodology and ß-lactamase hydrolysis assays technically difficult and unreliable. Therefore, we measured induction of bla_ACT-1 by transcript analysis, using primer extension analysis.

	Materials and methods

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Bacterial strains

K. pneumoniae 225 is a clinical isolate.⁵ The strain was authenticated as K. pneumoniae by 16S rRNA gene analysis (Midi Labs, Newark, DE, USA).

Polymerase chain reaction (PCR) and DNA sequencing

DNA template was prepared from K. pneumoniae 225 as described.⁵ The primers used for PCR and sequencing are listed in the legend to Figure 1. PCR amplifications were carried out and products visualized as described previously,⁵ using 2 mM MgCl₂, 0.5 µM primer and 2 µL (1/250 volume) of the total template in a final reaction volume of 50 µL. PCR products were sequenced directly after gel purification using 1.5% agarose in Tris-acetate EDTA and extraction using a Qiagen gel extraction kit (Qiagen, Valencia, CA, USA).⁵ Sequence data were collected by generating overlapping sequences and sequencing the PCR products at least twice, on separate occasions. Sequence analyses were performed on line using the BLAST program (www.ncbi.nlm.nih.gov).

View larger version (39K): [in this window] [in a new window]   Figure 1. . Structural organization of the plasmid-mediated ampC ß-lactamase gene, blaACT-1. (a) blaACT-1, the intergenic region and the 5' portion of the ampR structural gene were amplified with primers 1F (5'-CGAACGAATCATTATTCAGCACCG-3')9 and 1R (5'-CGGCAATGTTTACTACACAGCG-3')4 resulting in a 1518 bp amplicon (lane 1); ampR and the intergenic region were amplified using primers 2F (5'-CCGTAATAGCGAGTCAAGGG-3')9 and 2R (5'-CTGACGGTCGTCACGTTGATTGC-3')9 (lane 2); primers 1R and 2R were used to amplify the entire 2263 bp blaACT-1/ampR fragment (lane 3). The 100 bp and 1 kb ladders are from Gibco BRL (Rockville, MD, USA). (b) Sequence of the overlapping blaACT-1 and ampR divergent promoter regions. The AmpR binding site is shaded. The -10 and -35 promoter elements are indicated in bold. The C to T transition in the blaACT-1 -10 promoter element is indicated in bold type. The bent arrow indicates the start site of transcription for blaACT-1 as determined by primer extension analysis (Figure 2). The ATG start codons are enclosed in boxes.

 
Primer extension analysis

RNA was isolated from K. pneumoniae 225 cultures, treated or untreated with cefoxitin at an A₆₀₀ of 0.5 using x MIC of cefoxitin (128 mg/L) (Sigma Chemical Company, St Louis, MO, USA). Total RNA was extracted 15 min after addition of cefoxitin using hot phenol.⁶ Primers A and B (Figure 2) were annealed to 25 µg total RNA at 50°C and primer extension was performed using 100 U MuLV reverse transcriptase (Perkin-Elmer, Norwalk, CT, USA).⁷ Extension products were visualized by exposing the gel to a storage phosphor (Eastman Kodak Co., Rochester, NY, USA) for 2 days and scanning the image. mRNA was quantified using ImageQuant software (Molecular Dynamics Inc., Sunny, CA, USA).

View larger version (36K): [in this window] [in a new window]   Figure 2. . Primer extension analysis of blaACT-1 gene expression. Total RNA was isolated from K. pneumoniae 225 cells untreated or treated with 128 mg/L cefoxitin ( x MIC). The sequencing ladder was generated from the blaACT-1 sequence. The blaACT-1 transcript was mapped using 25 µg of total RNA and primer A (5'-GCCAATACCGAGCAGGAGGTG-3').4 The extension product is indicated with an arrow marked blaACT-1. The 16S rRNA-specific extension product is marked with an arrow and was obtained using 25 µg of total RNA using primer B (5'-CCAGACATTACTCACCCGTCC-3') (accession number Y17669). These data are representative of three independent experiments from which the average induction was five-fold.

 
ß-Lactamase assays

Crude cell extracts were obtained from a portion of cells from the same culture of K. pneumoniae 225 used to isolate RNA. Spectrophotometric hydrolysis assays were performed as described previously using cephalothin, 100 µM, as the substrate, both alone and in the presence of the ß-lactamase inhibitors clavulanic acid and cloxacillin. When an inhibitor was used, the ß-lactamase preparation (100 µL) was preincubated for 10 min at 37°C with the inhibitor (100 µL at 1000 µM).⁸

Nucleotide sequence accession number

The GenBank accession number for the K. pneumoniae 225 ampR gene and bla_ACT-1/ampR intergenic region is AF362955.

	Results

Top Abstract Introduction Materials and methods Results Discussion Acknowledgements References

 
Sequence analysis and genetic organization of plasmid-encoded bla_ACT-1

PCR amplification of the ampC gene of K. pneumoniae 225 using primers 1F and 1R (Figure 1) produced a 1518 bp fragment. The PCR fragment was sequenced, and revealed a partial 5' ampR sequence, the bla_ACT-1 structural gene and the intergenic region for the two genes. The bla_ACT-1 structural gene is 100% identical to the published nucleotide sequence.⁴ The presence of the entire ampR gene was verified by PCR using primers 2F and 2R (Figure 1). The 1075 bp fragment (Figure 1) was sequenced and revealed an ampR gene 83% similar to that of E. cloacae, encoding an AmpR with 90% identity to the E. cloacae AmpR.⁹ The sequences of the PCR products of bla_ACT-1 and ampR overlapped by 330 bp. The data indicated that the arrangement of bla_ACT-1 and ampR is the same as that for ampC and ampR in E. cloacae, where ampC is inducible. To confirm the genetic arrangement of the bla_ACT-1/ampR genes, PCR amplification was performed using primers 1R and 2R (Figure 1). The predicted size of the PCR product representing the entire ampR/ampC region is 2263 bp; the amplification product obtained was c. 2200 bp (Figure 1). These data, taken together, showed the organization of the bla_ACT-1/ampR genes in K. pneumoniae 225 to be identical to the chromosomal ampR/ampC region of E. cloacae.

Analysis of the region upstream of bla_ACT-1 revealed -10 and -35 ampR promoter elements, a -35 ampC promoter element and an AmpR binding site, all of which are identical to sequences of the respective elements in E. cloacae associated with inducible chromosomal ampC genes (Figure 1b).⁹ The -10 bla_ACT-1 promoter element shows one mismatch from that in E. cloacae, a cytosine to thymine substitution 59 bases upstream of the ATG start codon of bla_ACT-1 (Figure 1). The mutation improves the match to the -10 consensus sequence of E. coli.

bla_ACT-1 mRNA expression studies

The presence of the ampR gene and the AmpR binding site next to bla_ACT-1 indicated that bla_ACT-1 expression is inducible. Traditionally, induction of ß-lactamases is examined by assaying ß-lactamase activity in the presence or absence of a good inducer, such as imipenem or cefoxitin. However, in organisms expressing several ß-lactamases, such as K. pneumoniae 225, demonstration of inducible ß-lactamase activity can be technically difficult. In spectrophotometric hydrolysis assays, other ß-lactamases may contribute to turnover of substrate, preventing accurate quantification of the activity of the enzyme of interest. In an attempt to measure ACT-1 ß-lactamase activity accurately, ß-lactamase assays were performed several times using cephalothin as the substrate, in the presence and absence of clavulanic acid. The results demonstrated only a 1.3-fold increase in enzyme activity in the induced preparation, which was considered not to be significant (data not shown).

To determine whether bla_ACT-1 expression in K. pneumoniae 225 is induced in the presence of cefoxitin, the fold increase in mRNA production was measured using primer extension analysis. On exposure to cefoxitin, the bla_ACT-1 mRNA level was increased five-fold (Figure 2). To allow comparison of the levels of bla_ACT-1 mRNA in untreated and treated cultures, the content of 16S rRNA in each preparation was determined using RNA isolated from the same cultures of K. pneumoniae 225. As indicated in Figure 2, the level of 16S rRNA did not change in cells exposed to cefoxitin. Accordingly, bla_ACT-1 mRNA levels were normalized to 16S rRNA levels for comparison (Figure 2). Primer extension was also used to map the start site of transcription for bla_ACT-1. The primary start site for bla_ACT-1 transcription is the guanosine 50 bases upstream from the ATG start codon (Figure 2).

	Discussion

Top Abstract Introduction Materials and methods Results Discussion Acknowledgements References

 
The increase in genetic complexity of resistance mechanisms found in Gram-negative organisms complicates identification of the mechanism of resistance to a particular antibiotic(s). Yet understanding these mechanisms in clinical isolates is necessary for a clear understanding of resistance. The standard method of measuring induction of ampC ß-lactamase is to assay for ß-lactamase activity with a standard ß-lactam drug.⁸ The assay results are relatively easy to interpret for clinical isolates when the number of ß-lactamases produced by the isolate is limited.⁸ However, as shown for K. pneumoniae 225, ß-lactamase assays are inadequate when several ß-lactamases, including ESBLs, are present. Therefore, new strategies are needed to examine expression of particular ß-lactamase genes in clinical isolates with complex ß-lactamase backgrounds. Primer extension analysis of mRNA is a technique that can specifically identify and quantify the amount of RNA transcribed from a given gene, allowing examination of gene expression in complex genetic backgrounds.

The discovery of inducible plasmid-mediated AmpC ß-lactamases requires a re-evaluation of the treatment options available for patients infected with pathogens expressing this resistance mechanism. An association between use of third-generation cephalosporins and emergence of resistance among organisms with inducible chromosomally encoded AmpC ß-lactamases has been established.¹⁰ The mutants overexpress the AmpC ß-lactamase and are known as derepressed mutants.² These mutants arise spontaneously and are thought to be present normally as minor subpopulations within the patient. Derepression of ampC expression is most commonly caused by mutations within the ampD gene.² This mechanism is easily detected in the clinical laboratory by identifying hyperexpression of the AmpC ß-lactamase by organisms that encode inducible chromosomal AmpC ß-lactamases, such as E. cloacae and C. freundii. Now, with the identification of inducible plasmid-encoded AmpC ß-lactamases new questions arise. First, what will be the consequence of an inducible plasmid-encoded ampC gene in an ampD mutant of Escherichia coli or K. pneumoniae and can this be recognized quickly in the clinical laboratory? Secondly, will treatment of patients be compromised when infected with these organisms? Future studies addressing these questions are required.

	Acknowledgements

Top Abstract Introduction Materials and methods Results Discussion Acknowledgements References

 
We thank Jennifer Black and Ellen Smith Moland for expert technical assistance. DNA sequencing was supported in part by Creighton University Core Facility and the technical expertise of Steve Kelly and by UNMC/ Eppley Cancer Center grant P30CA36727. This work was supported in part by the Center for Research in Anti-Infectives and Biotechnology, and the Health Future Foundation, Omaha, NE, USA.

	Notes

 
^* Correspondence address. Center for Research in Anti-Infectives and Biotechnology, Department of Microbiology and Immunology, Creighton University School of Medicine, 2500 California Plaza, Omaha, NE 68178, USA; Tel: +1-402-280-5837; Fax: +1-402-280-1875; E-mail: ndhanson{at}creighton.edu

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Received 5 July 2001; returned 5 November 2001; revised 22 November 2001; accepted 6 December 2001

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