1 Department of Medical Microbiology and Immunology; 2 Center for Research in Anti-Infectives and Biotechnology, Creighton University School of Medicine, 2500 California Plaza, Omaha, NE 68178, USA
Received 5 August 2002; returned 13 December 2002; revised 11 February 2003; accepted 14 February 2003
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Abstract |
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Keywords: AmpC expression, resistance, plasmid-encoded, derepressed, copy number
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Introduction |
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The discovery of the AmpC ß-lactamase, CMY-1, in 1989 was the first published report of a plasmid-encoded AmpC ß-lactamase.15 Since then, over 20 plasmid-encoded AmpC ß-lactamases have been reported in several genera of bacteria (genetic backgrounds) including Salmonella spp., E. coli, Proteus mirabilis and Klebsiella pneumoniae.1518 Most of these plasmid-encoded genes lack the ampR gene and therefore do not display an inducible AmpC phenotype. How- ever, since 1998, three inducible plasmid-encoded AmpC ß-lactamases have been reported. Two of these genes, blaDHA-1 and blaDHA-2 are of M. morganii origin, whereas the most recently discovered gene, blaACT-1, is of Enterobacter origin.1821 These ampC genes are linked to an ampR gene that is transcribed divergently from ampC. This genetic organization is identical to that observed for inducible chromosomal ampC genes.
Mutations associated with AmpD lead to constitutive high-level expression (derepression) of the chromosomal ampC gene in clinical isolates resulting in high-level expression of the AmpC ß-lactamase.10 Although the general mechanism for high-level expression of chromosomal ampC genes is understood, the same cannot be said for the plasmid-encoded ampC genes. Several factors could contribute to the high-level expression of these genes. These factors include gene copy number, the genetic background of the organism and the genetic organization or context of the genetic locus from which the genes are expressed. It has been suggested that two factors may contribute to high-level expression from plasmid-encoded ampC genes in the absence of AmpR. These factors include expression from high-copy number plasmids22 and the absence of regulation by AmpR, in which ampC gene expression has been observed to increase 2.5- to 5.8-fold.4,12 However, the contributions of increases in ampC transcription and high copy number in the high-level expression of plasmid-encoded ampC genes have not been documented. The data presented in this paper correlate the overall gene expression from four ampC genes of Enterobacter origin in clinical isolates, with gene copy number, genetic organization and genetic background. These genes include the non-inducible plasmid-encoded ampC gene blaMIR-1, the inducible plasmid-encoded ampC gene blaACT-1, both expressed in K. pneumoniae isolates, and two chromosomal ampC genes expressed in Enterobacter cloacae, one inducible and one exhibiting high-level constitutive expression (derepressed).
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Materials and methods |
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The clinical isolates and bacterial strains used in this study are listed in Table .
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Total RNA was isolated from MuellerHinton broth (MHB) (Oxoid, UK) using modified Trizol (Invitrogen, Carlsbad, CA, USA). Briefly, 6 h 15 mL cultures were diluted 1:20 (100 mL total volume) in fresh pre-warmed MHB and allowed to grow to an OD600 of 0.65 at 37°C with shaking at 100 rpm. These cells represent the early log phase of growth. Cells were collected in pre-chilled centrifuge tubes and the cell pellet was suspended in 1 mL of modified Trizol solution. Induction assays using cefoxitin and primer extension analysis were carried out as previously described.20 Fifty micrograms of total RNA was used for each primer extension reaction for ampC transcripts whereas 1 µg was used for the 16S rRNA reactions. The primers for primer extension analysis are listed in Table .
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Hydrolysis assays for each of the four clinical isolates were carried out as described previously using 100 µM cefalothin as substrate.20,23 Enzyme was recovered from the same cultures used for RNA isolation.
MIC determinations
The susceptibility profiles were determined by agar dilution according to National Committee for Clinical Laboratory Standards (NCCLS) methods.24 The antimicrobial agents were obtained from different sources. Imipenem was obtained from Merck (Rahway, NJ, USA); cefotaxime and piperacillin were obtained from Sigma Chemical Co. (St Louis, MO, USA); ceftazidime was obtained from GlaxoSmithKline (Collegeville, PA, USA); cefpodoxime was from Pharmacia and Upjohn (Kalamazoo, MI, USA); cefepime and aztreonam were from Bristol-Myers Squibb (Princeton, NJ, USA); cefixime was from Wyeth-Ayerst Lederle (Bound Brook, NJ, USA).
Copy number determination
Comparative PCR was used to obtain a ratio of band intensities between a single copy chromosomal gene, in this case ampD, and the gene of interest, ampC. ampD is a single copy gene, as verified by BLAST analysis of the E. coli and K. pneumoniae genomes.25 PCRs were carried out as previously described.26 Primers used for copy number determinations are indicated in Table . Ten-fold serial dilutions, ranging from 100 to 105, of total DNA prepared from each isolate served as templates for PCR. The PCR products were separated on a 0.8% agarose gel, stained with Vistra Green (Amersham Pharmacia, Piscataway, NJ, USA), and visualized with a Storm Molecular Imager (Molecular Dynamics Inc., Sunny, CA, USA). Quantification of the bands was carried out with ImageQuant software (Molecular Dynamics Inc.). A ratio was determined by comparison of band intensities between the PCR products of the target gene, ampC and the single copy gene, ampD. These ratios were determined between band intensities obtained from PCR products amplified from the same dilution of template (normally the 103 dilution), which was within the linear range of the PCR cycle. The initial copy number was verified by diluting the PCR product from the diluted template (103 dilution) by the calculated ampC to ampD ratio. The diluted PCR product and the undiluted ampD PCR product from the original diluted (103) template were visualized as described above. Initial copy number was verified when the peak intensities for each band were equivalent between the diluted target ampC gene product and the undiluted single copy ampD product (see text for further explanation).
Cloning and transformation
blaACT-1 and blaMIR-1 were amplified by PCR as described above using Platinum Taq DNA polymerase (Invitrogen, Carlsbad, CA, USA). Amplicons were visualized on a 0.8% agarose gel with Crystal Violet staining. DNA was extracted from the gel using a SNAP column (Invitrogen, Carlsbad, CA, USA) and ligated into the pCR-TOPO-XL cloning vector and transformed into E. coli Top10 competent cells (Invitrogen). The amplicons were subcloned into pACYC184 as an EcoRI fragment.27 The resulting plasmids were isolated and electroporated into E. coli Top10 (Invitrogen), E. coli SNO302 and E. coli JRG582 (Tables and
). The plasmid DNA from these clones was sequenced as described previously using the primers in Table
to verify the insert sequences.20
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Results |
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Primer extension and hydrolysis data confirmed the induction phenotypes of E. cloacae 55, K. pneumoniae 225, K. pneumoniae 96D and E. cloacae V204. These four isolates represent ampC ß-lactamase gene expression of Enterobacter origin in different genetic backgrounds (Table ). In addition, primer extension analysis was carried out to determine the relative amounts of steady-state ampC RNA expressed from each clinical isolate tested. As determined previously by primer extension analysis, blaACT-1 was induced five-fold over constitutive levels when RNA expression was analysed 15 min post-induction, whereas ß-lactamase hydrolysis assays indicated a 1.3-fold induction (Figure 1).20 ß-Lactamase hydrolysis assays showed that wild-type (WT) E. cloacae AmpC was also induced, whereas both MIR-1 and E. cloacae V204 (derepressed mutant) AmpC were uninducible (Figure 1). These induction phenotypes were also reflected at the RNA level (data not shown). However, when basal (non-induced) level RNA expression between these four strains was examined, there was a 33-, 95- and 75-fold difference between WTampC and blaACT-1, blaMIR-1 and DRampC, respectively (Figure 2a and b).
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In contrast to the predicted 2.5- to 5.8-fold increase in the absence of ampR for plasmid-encoded ampC genes, differences of 33- (ampR present) and 95-fold increases (ampR absent) over chromosomal ampC expression were observed (Figure 2a and b). However, the constitutive level of RNA expression between the two plasmid-encoded ampC genes, blaACT-1 and blaMIR-1 was only three-fold. These data indicated a difference in the regulation of constitutive expression between the plasmid-encoded AmpC ß-lactamase genes compared with each other and WTampC. One explanation for the large increase in overall RNA expression from the plasmid-encoded genes could be gene copy number. Therefore, the copy number of each of these genes was determined by a comparative analysis using PCR. A ratio was determined between amplified products of the ampC gene in question and the single copy chromosomal gene, ampD (Figure 3). PCR amplification of each ampC gene and the respective ampD gene for each organism (K. pneumoniae or E. cloacae) was carried out using 10-fold serial dilutions of template DNA (100105) from each bacterial strain (Figure 3a and c). The band intensities of the ampC and ampD amplified products from the 103 template dilutions (Figure 3a and c) are represented by the graphs in Figure 3(b and d). The area under the curve for each peak was used to determine the ratio of amplified products of blaACT-1 to ampD (Figure 3b) and blaMIR-1 to ampD (Figure 3d). The amount of blaACT-1 and blaMIR-1 products with respect to the ampD genes revealed a ratio of 2 (Figure 3a and b) and 12 (Figure 3c and d), respectively.
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Similarly, comparing the band intensities for the 1:12 dilution of the blaMIR-1 103 PCR product and the undiluted 103 ampD product, the area under the curve was equivalent (Figure 3f). Therefore, the copy number of the inducible plasmid encoded blaACT-1 is 2, whereas the copy number of the non-inducible plasmid encoded blaMIR-1 is 12. This method was also used to verify the copy numbers of the chromosomal ampC genes from E. cloacae 55 and V204, which were both one (data not shown). As a control for the technique, blaACT-1 and blaMIR-1 were cloned into the vector pACYC184 which has a copy number of 10 to 12.27 The copy number of both blaACT-1 and blaMIR-1 cloned into pACYC184 was determined to be 11 for each gene (data not shown). These data indicate that the copy number protocol designed in this study is an accurate method for determining relative gene copy number.
Promoter sequence and expression comparisons
Primer extension data were used to map the transcriptional start sites for the mRNA transcript of each gene studied (Figures 2a and 4). As expected, the blaACT-1 mapped to the guanosine at position +1 (Figure 4), 50 bp upstream of the ATG start codon.20 The start site of transcription for the E. cloacae chromosomal ampC genes, WTampC and DRampC was a cytosine at the position identical to the blaACT-1 transcriptional start site. Surprisingly, the transcriptional start site of blaMIR-1 mapped to the cytosine at position 36 relative to the transcript start sites for blaACT-1 and chromosomal ampC genes (Figure 4), 89 bp upstream of the ATG start codon. Putative 10, TAaAtT, and 35, TTGAat, promoter elements having similarity to the E. coli 70 promoter consensus sequences 10, TATAAT and 35, TTGACA were identified. The spacing between these putative sites was 17 bp, the optimal distance between these two elements for maximal expression as described for E. coli.28 The relative level of expression from each promoter was determined by primer extension analysis using clones E. coli WTpACT-1 and E. coli WTpMIR-1 (Table
). These clones comprise the native promoter in addition to the structural gene cloned into pACYC184. RNA levels expressed from the same vector were only slightly increased (1.6-fold) for E. coli WTpMIR-1 when compared with RNA expression from E. coli WTpACT-1 (data not shown).
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Constitutive high-level expression (derepression) of the chromosomal ampC gene of E. cloacae V204 gave a 75-fold increase in expression and significant increases in ß-lactam MICs (Figure 2b and Table ). In contrast, the WT strain, E. cloacae 55, expressing an inducible chromosomal ampC gene does not exhibit high ß-lactam MICs. However, the susceptibility profile of an organism expressing an inducible plasmid-encoded ampC gene in an AmpD background is unknown. Therefore, ß-lactam MICs were determined for E. coli strains that were either AmpD+ or AmpD expressing either ACT-1 or MIR-1. Under these conditions, the ß-lactam MICs increased two- to 16-fold in AmpD E. coli expressing ACT-1 when compared with AmpD+ E. coli expressing ACT-1, whereas the ß-lactam MICs for E. coli
DpMIR-1 did not change from those for E. coli WTpMIR-1 for any drug tested (Table
). As indicated in Table
, the ß-lactam MICs observed for E. coli WTpACT-1 were within one dilution of those for both E. coli WTpMIR-1 and E. coli
DpMIR-1.
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Discussion |
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In addition to correlating ß-lactam MICs with ampC expression, this study aimed to determine what factors influence high-level expression of plasmid-encoded ampC genes of Enterobacter origin by correlating in vivo constitutive ampC expression levels with copy number and genetic characteristics. In studies using cloned ampC/ampR constructs expressed in E. coli, in the absence of AmpR, ampC expression was shown to increase 2.5- to 5.8-fold.4,12 Until recently, all plasmid-encoded ampC genes described in the literature were not associated with an ampR gene. Therefore, it was predicted that expression from a plasmid-encoded ampC gene, in the absence of AmpR, would increase 2.5- to 5.8-fold. However, using primer extension, this study demonstrated that blaMIR-1, a plasmid-encoded ampC gene expressed in the absence of AmpR, exhibited a 95-fold increase in expression relative to WTampC. These expression levels are 16- to 38-fold higher than the amount of expression previously reported using reporter gene assays in E. coli or cloning these genes into vectors such as pACYC184.12 These data indicate that the level of plasmid-encoded ampC expression in clinical isolates lacking AmpR is not due simply to a release from repression by AmpR. Furthermore, for constitutive blaACT-1 expression, a 33-fold increase in expression was observed (Figure 5). This level of expression was in contrast to what might be predicted based on the genetic arrangement of the plasmid and its correlation with chromosomal ampC genes.
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Contribution of copy number to overall gene expression was analysed in the four clinical isolates expressing ampC genes of Enterobacter origin by comparing the ratio of constitutive expression with gene copy number (Figure 5). The expression per copy number ratio for blaMIR-1 is 8, whereas for blaACT-1 it is 17. The similarities between the genetic context of blaACT-1 and the WTCampC would predict that the copy number for blaACT-1 would be the same as the relative fold increase in expression, i.e. 33-fold. This was not the case as the copy number of blaACT-1 is two, yielding an expression per copy number ratio of 17. These data indicate that copy number does not account for the difference observed in expression levels between blaACT-1 and WTCampC. When the promoter regions of blaACT-1 and WTampC were compared, only three mutations were observed. The most significant mutation occurred in the putative 10 promoter element of blaACT-1. This mutation was located at position 9 and resulted in a C to T transition, which increased the similarity of the blaACT-1 promoter to the E. coli 10 consensus sequence TATAAT (Figure 4).20 This same substitution in E. coli ampC promoters has been shown to increase expression seven- to 20-fold.32,33 Thus, these data indicate that the point mutation in the blaACT-1 promoter contributes to the increased expression observed for blaACT-1 transcription compared with wild-type expression.
The expression to copy number ratio of 8 for blaMIR-1 was surprising. Copy number has been implicated as the major mechanism to explain the increase in AmpC ß-lactamase activity when the gene is encoded by a plasmid.22 This hypothesis would predict that the overall expression of blaMIR-1 would be six-fold higher than blaACT-1. However, the difference in total RNA expression between the two genes was only three-fold. In addition, this same hypothesis would predict that the copy number difference observed between the two genes would be three-fold not six-fold. Furthermore, comparisons between promoter expression of blaMIR-1 and blaACT-1 in E. coli when the genes were encoded by the same vector indicated that the difference in expression levels was less than two-fold. These data support the expression to copy number ratio for blaMIR-1 being less than the blaACT-1 ratio and indicate that not all the plasmid copies of blaMIR-1 may participate equally in the overall expression. In addition to the expression to copy number ratio, the location of the start site of blaMIR-1 transcription was unique compared with other Enterobacter ampC genes. Mapping the blaMIR-1 start site to position 36, relative to the transcript start sites for blaACT-1 and chromosomal ampC genes, indicated that the putative 10 position correlates to TAAATT (Figure 4). This 10 element also has higher similarity to E. coli consensus than the 10 elements of the E. cloacae chromosomal ampC genes. These data indicate that ampC promoter mutations may be necessary to obtain high levels of ß-lactamase gene expression. In addition, the creation of new promoters upon insertion of ampC genes in the plasmid may be necessary to drive expression levels required for resistance.
In summary, the mechanism of plasmid-encoded AmpC ß-lactamase resistance is multifaceted and probably fluctuates depending on the genetic organization and background in which these genes are expressed. This will make predicting the ß-lactam MIC values for a particular plasmid-encoded AmpC ß-lactamase somewhat unpredictable and incredibly challenging.
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Acknowledgements |
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Footnotes |
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