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Antimicrobial Agents and Chemotherapy, November 2008, p. 3922-3927, Vol. 52, No. 11
0066-4804/08/$08.00+0     doi:10.1128/AAC.00341-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Role of ampD Homologs in Overproduction of AmpC in Clinical Isolates of Pseudomonas aeruginosa

Amber J. Schmidtke and Nancy D. Hanson^*

Department of Medical Microbiology and Immunology and Center for Research in Anti-Infectives and Biotechnology, Creighton University School of Medicine, Omaha, Nebraska

Received 11 March 2008/ Returned for modification 16 May 2008/ Accepted 29 August 2008

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
AmpD indirectly regulates the production of AmpC β-lactamase via the cell wall recycling pathway. Recent publications have demonstrated the presence of multiple ampD genes in Pseudomonas aeruginosa and Escherichia coli. In the prototype P. aeruginosa strain, PAO1, the three ampD genes (ampD, ampDh2, and ampDh3) contribute to a stepwise regulation of ampC β-lactamase and help explain the partial versus full derepression of ampC. In the present study, the roles of the three ampD homologs in nine clinical P. aeruginosa isolates with either partial or full derepression of ampC were evaluated. In eight of nine isolates, decreased RNA expression of the ampD genes was not associated with an increase in ampC expression. Sequence analyses revealed that every derepressed isolate carried mutations in ampD, and in two fully derepressed strains, only ampD was mutated. Furthermore, every ampDh2 gene was of the wild type, and in some fully derepressed isolates, ampDh3 was also of the wild type. Mutations in ampD and ampDh3 were tested for their effect on function by using a plasmid model system, and the observed mutations resulted in nonfunctional AmpD proteins. Therefore, although the sequential deletion of the ampD homologs of P. aeruginosa can explain partial and full derepression in PAO1, the same model does not explain the overproduction of AmpC observed in these clinical isolates. Overall, the findings of the present study indicate that there is still an unknown factor(s) that contributes to ampC regulation in P. aeruginosa.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
In Pseudomonas aeruginosa, the overproduction of AmpC β-lactamases, known phenotypically as derepression, can result in resistance to nearly all β-lactam antibiotics except the carbapenems. Historically, it was understood that the regulation of AmpC production required three proteins: AmpG, a plasma membrane-bound permease; AmpD, a cytosolic peptidoglycan-recycling amidase; and AmpR, the transcriptional regulator of AmpC (6). Derepression has been associated previously with defects within the ampD structural gene (6, 19) or decreased ampD expression (18). Derepression represents the inability of AmpR to keep ampC expression at constitutively low wild-type levels. Full derepression is the noninducible, high-level expression of ampC. Partial derepression occurs when an organism constitutively expresses ampC at higher levels than those in a wild-type strain but the gene can still be induced. There have been several publications indicating the absence of mutations in AmpG, AmpD, AmpR, and the ampC-ampR intergenic region in derepressed isolates of P. aeruginosa (1, 2, 10, 23).

A recent study presented data which demonstrated a mechanism for the levels of derepression observed in P. aeruginosa (7). In 2006, Juan and colleagues (7) identified two AmpD homologs in P. aeruginosa, AmpDh2 and AmpDh3. Using a series of ampD knockout clones, Juan et al. were able to show that together, the three AmpD proteins contribute to the stepwise upregulation of ampC in the wild-type strain PAO1. The loss of the ampDh3 homolog gene in combination with the deletion of ampD (hereinafter referred to as ampD1) resulted in increased basal, although still inducible, expression of ampC compared to that in a strain with a single ampD1 deletion. The loss of ampC inducibility (full derepression) was achieved only in a triple ampD mutant. The deletion of ampD1 had the single greatest impact in terms of increased β-lactam MICs and high-level constitutive ampC expression. The loss of ampDh3 together with ampD1 increased β-lactam MICs and AmpC production more than the deletion of ampDh2 together with ampD1. In summary, the effects of the different ampD genes on AmpC overproduction were as follows: ampD1 > ampDh3 > ampDh2.

The goal of this study was to examine the relative expression levels of the different ampD genes in PAO1 and determine whether sequence abnormalities and/or altered expression of the ampD genes could explain derepression in a panel of clinical isolates.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Strains and susceptibility. P. aeruginosa strain PAO1 was used as the wild-type comparator strain. Nine clinical isolates of P. aeruginosa strains were examined, including four cystic fibrosis isolates (PA22, PA24, PA113, and PA367) and four non-cystic fibrosis clinical isolates (GB57, GB61, TX291, and TX292). A mutant, 164CD (5), derived from a clinical isolate by selection using ceftazidime, was also characterized. In the model system, the ampD-deficient Escherichia coli strain JRG582 (11) was used to test whether mutations in an ampD gene could complement the ampD-deficient phenotype (18). MICs of piperacillin, piperacillin-tazobactam, ceftazidime, cefepime, and aztreonam were determined by agar dilution, Etest (AB Biodisk, Sweden), and/or broth microdilution according to CLSI recommendations (3).

RNA analysis using real-time RT-PCR. RNA was isolated from mid-logarithmic-phase cultures of P. aeruginosa by using TRIzol Max solution (Invitrogen, Carlsbad, CA) as described previously (18). Eight micrograms of total RNA was DNase treated as described previously (18). Reverse transcriptase PCR (RT-PCR) was performed using the QuantiTect SYBR green RT-PCR kit as instructed by the manufacturer (Qiagen, Valencia, CA) with 250 ng of template RNA for each reaction. For the evaluation of the expression of ampD homolog genes, each strain was analyzed using five reactions: a no-RT control and the amplification of ampD1, ampDh2, ampDh3, and sodB (an endogenous control gene). Amplification was performed as described previously (18) using an annealing temperature of 53°C. The relative quantification of ampD and ampC expression in the nine clinical strains was calculated as described previously (13) using expression in PAO1 as the calibrator. For the determination of the ratio of expression levels within PAO1, a modification of the calculation described by Livak and Schmittgen (13) was employed such that the sodB threshold cycle (C_T) was used to normalize the average C_T value for each ampD gene and the ampD1 expression level was used as the calibrator (set to 1) against which ampDh2 and ampDh3 expression levels were compared.

The induction of ampC expression was evaluated by real-time RT-PCR. Briefly, cells were grown to mid-logarithmic phase, at which time imipenem (Merck Research Laboratories, Rahway, NJ) was added at a concentration of one-fourth the MIC for each strain and cells were incubated at 37°C for 20 min (22). Relative ampC expression was quantified by real-time RT-PCR as described above using primers listed in Table 1. The coefficient of variation (the standard deviation divided by the mean) among results from different experiments was <10%.

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TABLE 1. Primers used in this study

In order to compare the levels of expression of the three ampD genes within PAO1, great care was taken to ensure similar amplification efficiencies for all three genes. Therefore, primers were designed to have similar (and often identical) nucleotide lengths, G+C contents, theoretical melting temperatures, and target amplicon lengths (Table 1). The amplification efficiency was experimentally verified with template DNA by real-time PCR. DNA was isolated from a 1.5-ml aliquot of 8-h PAO1 broth cultures by using the DNeasy blood and tissue kit (Qiagen, Valencia, CA). Each amplification reaction mixture included 250 ng of DNA. The QuantiTect SYBR green RT-PCR kit (Qiagen, Valencia, CA), minus RT, was used with an ABI Prism 7000 thermal cycler (Applied Biosystems, Foster City, CA) set to parameters described previously (18). The annealing temperature of 53°C provided the best amplification efficiency for all three genes. The coefficient of variation for the amplification efficiencies was <10%.

DNA isolation, amplification, purification, and sequencing. DNA was isolated as described previously (14). The DNA template was amplified by PCR using primers listed in Table 1. Amplified products were verified on a 1% agarose gel and prepared for sequencing using the Microcon YM-50 filter column (Millipore, Bedford, MA). Sequencing was performed by automated cycle sequencing using an ABI Prism 3100-Avant genetic analyzer. Sequence analyses were performed using the Vector NTI Advance version 10.3 software program (Invitrogen, Carlsbad, CA). P. aeruginosa sequences were compared to the PAO1 genome sequence (GenBank accession number NC_002516) and E. coli sequences were compared to the K-12 genome sequence (GenBank accession number NC_000913).

Cloning of ampD genes and determination of AmpD function. ampD mutations observed in the clinical isolates of P. aeruginosa were evaluated for their effects on AmpD function by using a previously described plasmid-based AmpD model system (18). Briefly, the AmpD model system uses a plasmid containing the inducible plasmid-carried ampC gene bla_ACT-1, which acts as the indicator for AmpD function. An AmpD-deficient strain of E. coli (JRG582) is transformed with this inducible genetic system, which results in a ceftazidime resistance phenotype for the strain. To test the significance of nucleotide variations of ampD genes and the corresponding amino acid variations identified during sequence analyses of clinical isolates (in this case, strains of P. aeruginosa), the ampD test genes were amplified by PCR, cloned into pCR2.1 (Invitrogen, Carlsbad, CA), and subcloned into the XbaI and HindIII sites of pACYC184, the vector used in the model system (16). The ampD-deficient E. coli strain JRG582 (11) was transformed with the resulting constructs. The clones containing the ampD test genes were evaluated for the ability to complement the ampD-deficient E. coli strain by ceftazidime susceptibility testing using disk diffusion.

Comparison of the amino acid sequences of P. aeruginosa AmpDh2 and AmpDh3 and E. coli AmiD. An alignment of the amino acid sequences of P. aeruginosa AmpDh2 and AmpDh3 and E. coli AmiD was generated by the MAFFT (multiple alignment using fast Fourier transform) program (9) available at http://www.ebi.ac.uk/mafft/index.html. AmpDh2 and AmpDh3 amino acid sequences were examined for transmembrane helix domains by using the TMHMM version 2 prediction software available at http://www.cbs.dtu.dk/services/TMHMM/.

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Expression studies. The relative expression of each ampD transcript in six independent PAO1 cultures was evaluated. The data were averaged and used for calculations as described in Materials and Methods, with ampDh2 and ampDh3 expression compared to that of ampD1. The ratio of the expression levels of the three genes, that of ampD1 to that of ampDh2 to that of ampDh3, was 1 to 0.24 to 10.7. The coefficient of variation for the six cultures was <10% for each gene tested and ranged from 0.04 to 0.07.

Partial or full derepression of ampC RNA expression in the nine clinical isolates in the absence and presence of imipenem was evaluated. As indicated in Table 2, all of the strains exhibited elevations in basal ampC expression compared to that in PAO1, ranging from a 65-fold increase for PA367 to a 5,853-fold increase for GB57. All of the cystic fibrosis isolates, PA22, PA24, PA113, and PA367, were inducible and therefore had partially derepressed AmpC production. Of the non-cystic fibrosis isolates, strains GB57, TX292, and 164CD were fully derepressed, with a 2-fold change in ampC expression when exposed to imipenem. However, ampC RNA expression in strains GB61 and TX291 increased by 4-fold in the presence of imipenem. Technically, these strains are considered to be partially derepressed.

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TABLE 2. Susceptibility phenotypes and associated ampC and ampD RNA expression for P. aeruginosa strains

The relative levels of expression of ampD RNA in the nine clinical strains were analyzed to determine whether decreased expression correlated with ampC overproduction. For most strains, no variation (2-fold) was observed in the expression of the ampD homologs. However, strains TX291 and TX292 expressed ampDh2 at 5.2- and 4.0-fold-higher levels than that of the expression of PAO1 ampDh2, respectively (Table 2). In addition, a threefold decrease in ampDh3 expression in GB61 was observed.

Sequence analysis and evaluation of AmpD function using the E. coli plasmid model system. ampD1 and ampDh3 from the wild-type P. aeruginosa strain PAO1 were able to complement the ampD-deficient E. coli strain, as indicated by a change in the ceftazidime zone diameter from 6 mm to 17 and 18 mm, respectively (Table 3). In contrast to the ampD1 and ampDh3 genes, PAO1 ampDh2 failed to complement the ampD-deficient clone, resulting in no change in the ceftazidime zone diameter.

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TABLE 3. Evaluation of ampD gene homologs from P. aeruginosa using the E. coli AmpD plasmid model system

The three ampD homolog genes in nine clinical isolates were sequenced to identify mutations which may contribute to ampC overproduction (Table 3). Regardless of whether ampC was fully derepressed or partially derepressed (Tables 2 and 3), all of the clinical isolates encoded AmpD1 mutations. The most frequently observed mutation in AmpD1 was a glycine-to-alanine change corresponding to codon 148, which occurred in five of the nine isolates. Three of the isolates, one cystic fibrosis isolate and two non-cystic fibrosis isolates, contained an 11- to 12-nucleotide deletion at position 5064907 (relative to the PAO1 genome sequence [GenBank accession number NC_002516]) that resulted in either a frameshift mutation with the absence of a stop codon (strains TX291 and TX292) or a four-codon deletion (strain PA24). All nine strains encoded wild-type AmpDh2 and did not require the evaluation of ampDh2 in the model. Six of the nine strains (67%) exhibited amino acid substitutions in AmpDh3. By using the PAO1 ampD1 and ampDh3 clones as controls, the ampD1 and ampDh3 mutations observed in the clinical isolates were evaluated for their effects on AmpD function.

The partially derepressed cystic fibrosis strains PA22 and PA367 carried identical mutations in ampD1 and ampDh3 (Table 3). Pulsed-field gel electrophoresis indicated that these strains were genetically related (unpublished data). The AmpD1 substitutions were a glycine-to-alanine change at position 148 and an aspartic acid-to-tyrosine change corresponding to codon 183. The AmpDh3 substitutions in strains PA22 and PA367 were an aspartic acid-to-glycine change at position 137 and an alanine-to-threonine change corresponding to codon 219. When tested in the model system, these mutations in ampDh3 resulted in a ceftazidime zone diameter indicating resistance (10 mm). PA24 and PA113, both partially derepressed cystic fibrosis strains, also carried ampD1 mutations as described above and ampDh3 mutations (Table 3). The ampDh3 genes in PA24 and PA113 carried a single mutation resulting in an Ala219Thr amino acid substitution.

Strain 164CD was a fully derepressed mutant derived from a clinical isolate by selection with ceftazidime (5) and carried unique mutations in both ampD1 and ampDh3 (Table 3). Together, the ampD1 mutations resulted in ceftazidime resistance in the ampD model system, with a zone diameter of 8 mm. The mutation observed in AmpDh3 was an isoleucine-to-threonine change at position 67, which also resulted in ceftazidime resistance (the zone diameter was 10 mm) when tested in the model system.

Involvement of E. coli amiD in ampDh2 complementation. Because PAO1 ampDh2 failed to complement the AmpD-deficient phenotype of E. coli strain JRG582 in the model system, it was possible that E. coli possessed an ampDh2 homolog. Recently, an AmpD homolog in E. coli, AmiD, has been identified (21). An alignment of the P. aeruginosa and E. coli AmpD homologs is presented in Fig. 1. AmiD of E. coli and AmpDh2 of P. aeruginosa share a lipobox motif sequence (L¹⁴AGC¹⁷) which is conserved in gram-negative bacteria (8, 21, 25). In addition, Lewenza et al. predicted that ampDh3 (gene PA0807) encodes a transmembrane helix in the N-terminal sequence (Fig. 1), suggesting that AmpDh3 may insert into the cytoplasmic membrane (12). Overall, E. coli AmiD shows 50% similarity to P. aeruginosa AmpDh2 and 47% similarity to P. aeruginosa AmpDh3. Given these similarities between ampDh2 of P. aeruginosa and amiD of E. coli, it is possible that within the AmpD model system, P. aeruginosa ampDh2 genes cannot be evaluated due to the presence of wild-type amiD. Sequences and levels of expression of amiD in E. coli JRG582 were examined, and no differences from those in the wild-type E. coli strain K-12 259 (corresponding to GenBank accession number D90770) were observed.

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FIG. 1. Alignment of E. coli (EC) AmiD and P. aeruginosa (PA) AmpDh2 and AmpDh3. Dashes indicate gaps, whereas asterisks, colons, and periods indicate identity, conserved substitution, and semiconserved substitution, respectively. The alignment was generated using the MAFFT program with the selection of a Clustal output. The lipobox sequence (L14AGC17) in E. coli AmiD and P. aeruginosa AmpDh2 is underlined. The transmembrane helix sequence in AmpDh3 is double underlined and was identified using the TMHMM version 2 prediction software (http://www.cbs.dtu.dk/services/TMHMM/).

Sequence analysis of ampR and the ampR-ampC intergenic region in GB57 and GB61. P. aeruginosa strains GB57 and GB61 had single amino acid substitutions in AmpD1, and both had wild-type AmpDh2 and AmpDh3 sequences. Therefore, the fully derepressed phenotype (Tables 2 and 3) of GB57 could not be explained through an AmpD mechanism. It was possible that mutations within ampR or the ampR-ampC intergenic region contributed to the high-level, noninducible RNA expression of ampC. Therefore, the ampR genes and the ampR-ampC intergenic regions in the fully derepressed strain GB57 and the partially derepressed strain GB61 were sequenced and compared. The ampR-ampC intergenic regions in GB61 and GB57 were both of the wild type, and the ampR sequence in GB61 was also of the wild type. However, mutations in the ampR structural gene of strain GB57 were observed and resulted in the following amino acid substitutions: Glu114Ala, Asp135Asn, Gly283Glu, and Met288Arg.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Derepressed phenotypes in P. aeruginosa in the absence of genetic mutations in the AmpC induction pathway have been observed previously (1, 2, 10). Juan et al. identified two ampD homologs in P. aeruginosa (ampDh2 and ampDh3) and demonstrated that the deletion of all three ampD genes in the laboratory strain PAO1 was required for a fully derepressed phenotype. In addition, they found that with respect to ampC expression and β-lactam MICs, the relative importance of the AmpD proteins was as follows: AmpD1 > AmpDh3 > AmpDh2 (7). In this study, an analysis of the RNA expression patterns of the three ampD genes in PAO1 relative to one another showed that ampDh3 was expressed at the highest level, followed by ampD1 and then ampDh2. These data, together with the data generated by Juan et al., suggest that (i) the role of ampDh2 in ampC regulation is minimal compared to those of ampD1 and ampDh3 and (ii) increased expression of ampDh3 relative to that of ampD1 and ampDh2 in PAO1 may reflect the importance of the AmpDh3 enzyme in cell wall metabolism. Further experimentation is required to substantiate these observations.

In order to test the hypothesis of Juan et al. (7), the role of the three ampD genes in AmpC overproduction in nine clinical isolates with either partially or fully derepressed ampC expression was examined. All of the ampD1 and ampDh3 genes tested in the model encoded nonfunctional proteins, as indicated by a phenotype of ceftazidime resistance. But the sequence for ampDh2 in all the isolates was of the wild type. This finding contradicts the requirement that all three ampD genes of P. aeruginosa PAO1 be deleted for the isolate to become fully derepressed (7). It was possible that ampDh2 expression was altered in the clinical isolates, since a decrease in ampD gene expression has been associated previously with elevated levels of ampC expression in the presence of a wild-type AmpD sequence (18). However, instead of decreased ampD expression, the partially and fully derepressed strains TX291 and TX292 had elevated levels of ampDh2 RNA expression. What role the elevated expression of ampDh2 may play in the fully derepressed phenotypes of these strains is unclear.

Strain GB57 had the highest levels of ampC expression and was fully derepressed yet had only a single amino acid substitution in AmpD1. However, mutations in the structural gene ampR were observed and have been observed previously in other derepressed clinical isolates of P. aeruginosa (1, 15, 20). The Asp135Asn AmpR substitution observed in GB57 has been correlated previously with a large increase (16,000-fold) in β-lactamase activity (1). Therefore, it is possible that the combination of the mutations observed in both the ampR and ampD1 genes was responsible for the 5,000-fold increase in basal-level ampC RNA expression and the fully derepressed phenotype.

Strains PA113 and GB61 highlight the need for further studies investigating alternative mechanisms in the ampC regulatory pathway. PA113 is a cystic fibrosis isolate which displayed a partially derepressed phenotype of ampC expression, with basal-level RNA expression 442-fold higher than that in PAO1. GB61 was also partially derepressed and expressed ampC RNA at a basal level 1,616 times higher than that in PAO1. The difference in the basal levels of expression between the two strains was fivefold. Although both strains had amino acid substitutions in AmpD1, the mutations in AmpDh3 differed. Strain PA113 had an amino acid substitution in AmpDh3, while GB61 had wild-type AmpDh3 but threefold-lower ampDh3 RNA levels. Both strains carried wild-type ampR and ampR-ampC intergenic regions. Although the effect of ampDh3 expression on ampC expression remains unknown, these data further indicate the complexity involved in the regulation of ampC gene expression.

The present study explored the impact of ampD homologs on ampC expression in clinical isolates and further substantiated the notion that the regulation of ampC β-lactamase in P. aeruginosa is exceedingly complex. These data support observations that additional mechanisms besides the known regulators of AmpC β-lactamase contribute to ampC regulation.

 
PAO1 was generously provided by Keith Poole. We thank Daniel Wolter for performing pulsed-field gel electrophoresis on the clinical isolates used in this study.

 
* Corresponding author. Mailing address: Department of Medical Microbiology and Immunology, Creighton University, 2500 California Plaza, Omaha, NE 68178. Phone: (402) 280-5837. Fax: (402) 280-1875. E-mail: ndhanson{at}creighton.edu

Published ahead of print on 8 September 2008.

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Antimicrobial Agents and Chemotherapy, November 2008, p. 3922-3927, Vol. 52, No. 11
0066-4804/08/$08.00+0     doi:10.1128/AAC.00341-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

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• Lister, P. D., Wolter, D. J., Hanson, N. D. (2009). Antibacterial-Resistant Pseudomonas aeruginosa: Clinical Impact and Complex Regulation of Chromosomally Encoded Resistance Mechanisms. Clin. Microbiol. Rev. 22: 582-610 [Abstract] [Full Text]  
• Yang, T.-C., Huang, Y.-W., Hu, R.-M., Huang, S.-C., Lin, Y.-T. (2009). AmpDI Is Involved in Expression of the Chromosomal L1 and L2 {beta}-Lactamases of Stenotrophomonas maltophilia. Antimicrob. Agents Chemother. 53: 2902-2907 [Abstract] [Full Text]  
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