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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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, blaDHA-1 and blaDHA-2, have been described, destroying the generalization.1,3
In this report we demonstrate that the plasmid-encoded ampC gene, blaACT-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 blaACT-1 by transcript analysis, using primer extension analysis.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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).
|
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).
|
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 blaACT-1/ampR intergenic region is AF362955.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 blaACT-1 structural gene and the intergenic region for the two genes. The blaACT-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 blaACT-1 and ampR overlapped by 330 bp. The data indicated that the arrangement of blaACT-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 blaACT-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 blaACT-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 blaACT-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 blaACT-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 blaACT-1 (Figure 1
). The mutation improves the match to the -10 consensus sequence of E. coli.
blaACT-1 mRNA expression studies
The presence of the ampR gene and the AmpR binding site next to blaACT-1 indicated that blaACT-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 blaACT-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 blaACT-1 mRNA level was increased five-fold (Figure 2). To allow comparison of the levels of blaACT-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, blaACT-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 blaACT-1. The primary start site for blaACT-1 transcription is the guanosine 50 bases upstream from the ATG start codon (Figure 2
).
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Notes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
2 . Hanson, N. D. & Sanders, C. C. (1999). Regulation of inducible AmpC beta-lactamase expression among Enterobacteriaceae. Current Pharmaceutical Design 5, 88194.[ISI][Medline]
3
.
Fortineau, N., Poirel, L. & Nordmann, P. (2001). Plasmidmediated and inducible cephalosporinase DHA-2 from Klebsiella pneumoniae. Journal of Antimicrobial Chemotherapy 47, 20710.
4 . Bradford, P. A., Urban, C., Mariano, N., Projan, S. J., Rahal, J. J. & Bush, K. (1997). Imipenem resistance in Klebsiella pneumoniae is associated with the combination of ACT-1, a plasmid-mediated AmpC beta-lactamase, and the loss of an outer membrane protein. Antimicrobial Agents and Chemotherapy 41, 5639.[Abstract]
5
.
Hanson, N. D., Thomson, K. S., Moland, E. S., Sanders, C. C., Berthold, G. & Penn, R. G. (1999). Molecular characterization of a multiply resistant Klebsiella pneumoniae encoding ESBLs and a plasmid-mediated AmpC. Journal of Antimicrobial Chemotherapy 44, 37780.
6 . Chen, Y. C., Shipley, G. L., Ball, T. K. & Benedik, M. J. (1992). Regulatory mutants and transcriptional control of the Serratia marcescens extracellular nuclease gene. Molecular Microbiology 6, 64351.[ISI][Medline]
7 . Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A. et al. (1989). Short Protocols in Molecular Biology. Vol. 1. John Wiley & Sons, New York.
8 . Sanders, C. C. (1989). Beta-lactamase stability and in vitro activity of oral cephalosporins against strains possessing wellcharacterized mechanisms of resistance. Antimicrobial Agents and Chemotherapy 33, 13137.[ISI][Medline]
9 . Honore, N., Nicolas, M. H. & Cole, S. T. (1986). Inducible cephalosporinase production in clinical isolates of Enterobacter cloacae is controlled by a regulatory gene that has been deleted from Escherichia coli. EMBO Journal 5, 370914.[Abstract]
10 . Chow, J. W., Fine, M. J., Shlaes, D. M., Quinn, J. P., Hooper, D. C., Johnson, M. P. et al. (1991). Enterobacter bacteremia: Clinical features and emergence of antibiotic resistance during therapy. Annals of Internal Medicine 115, 58590.[ISI][Medline]
Received 5 July 2001; returned 5 November 2001; revised 22 November 2001; accepted 6 December 2001