Gene Mutations Responsible for Overexpression of AmpC -Lactamase in Some Clinical Isolates of Enterobacter cloacae
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微生物临床杂志 2005年第6期
Department of Environmental Infectious Diseases, Graduate School of Medical Sciences
Department of Microbiology, School of Medicine, Kitasato University, 1-15-1 Kitasato, Sagamihara, Kanagawa
Department of Central Laboratory, School of Medicine, Teikyo University, 2-11-1, Kaga, Itabashi-ku, Tokyo, Japan
ABSTRACT
AmpC regulatory genes in 21 ceftazidime-resistant clinical isolates of Enterobacter cloacae (MICs of 16 μg/ml) were characterized. All isolates exhibited AmpC overproduction due to AmpD mutation. Additionally, we found two AmpR mutants among the isolates. This is the first report of chromosomal ampR mutation in clinical isolates of E. cloacae.
TEXT
Resistance of members of the family Enterobacteriaceae (e.g., Enterobacter cloacae and Serratia marcescens) to expanded-spectrum cephalosporins is a serious clinical problem (14). The resistance of these bacteria is principally due to constitutive overexpression of chromosomal ampC (which encodes AmpC, a class C -lactamase) (5, 33). The ampC gene is located on the chromosome of many gram-negative bacilli and is inducible in the presence of -lactam antibiotics (9).
The mechanism of AmpC induction is closely linked to a cell wall recycling system (8, 12). The cell walls of gram-negative bacilli must be reconstituted during growth. Escherichia coli (and presumably E. cloacae) degrades 40 to 50% of the cell wall peptidoglycans during each generation, and 90% of the degradation products are recycled (28). Peptidoglycan degradation products (anhydromuropeptides) released into the periplasm are transported into the cytoplasm by AmpG, a transmembrane permease (6, 16, 22). Subsequently, these anhydromuropeptides are hydrolyzed by AmpD, which is a cytoplasmic N-acetyl-anhydromuramyl-L-alanine amidase that specifically hydrolyzes several anhydromuropeptides such as (GlcNAc-)anhMurNAc tripeptide and GlcNAc-anhMurNAc tetrapeptide (11). This action of AmpD prepares anhydromuropeptides for further recycling. In the presence of -lactams, however, anhydromuropeptides increase in both the periplasm and cytoplasm. Cytoplasmic anhydromuropeptides that have avoided hydrolysis by AmpD bind to AmpR, a transcriptional regulator of ampC (9, 19, 21). In the absence of an activating ligand, AmpR represses ampC expression with UDP-MurNAc pentapeptide, but cell wall degradation products [(GlcNAc-)anhMurNAc tripeptides] may be an AmpR-activating ligand that causes the activation of ampC expression. In AmpD-defective bacterial strains, unhydrolyzed anhydromuropeptides accumulate in the cytoplasm (7, 13), and this causes activation of AmpR, which results in the semiconstitutive or constitutive expression of ampC. The role of AmpD in the induction of AmpC in gram-negative bacilli has been studied in detail (15, 18, 20).
AmpD-dependent constitutive expression of AmpC occurs in vitro at a frequency of 10–5 to 10–7 (19, 29). In addition, we previously reported that AmpR-dependent constitutive overproduction of AmpC occurred at a frequency of 10–6 in AmpD-defective strains (17). However, the isolation rate of AmpD mutants among cephalosporin-resistant clinical strains has not been determined. In addition, there have been no reports describing the isolation of chromosomal AmpR mutants from clinical isolates.
Twenty-one ceftazidime-resistant strains of E. cloacae KU6323 to KU6332, KU6334, KU6336 to KU6341, and KU6343 to KU6346 were isolated during a 1-year period in 1999 from patients attending a university-affiliated hospital in Tokyo, Japan. KU4892, KU4894, and KU1917 were positive-control strains (blaTEM-1, blaTOHO-1, and blaIMP-1, respectively) and were created by introduction of plasmids pKU507, pMTY010, and pMS361, respectively (10, 34). E. cloacae KU3262 (wild-type AmpD) was a clinical isolate and used for complementation assay of ampR (17). The MICs were determined by the agar dilution method according to CLSI (formerly NCCLS) guidelines (25). -Lactamase activity was detected by a colorimetric assay using cephalothin as a substrate (17, 27). For inhibition of class A -lactamase, clavulanic acid (at a final concentration of 5 μg/ml) was added to either substrate solution or extracted enzyme solution. Results were analyzed by Student's t test, and values were expressed as the means ± standard deviations (SDs). A P of <0.001 was defined as indicating statistical significance.
To detect -lactamase genes, except for chromosomal ampC, PCR amplification was performed. Template DNA solution was prepared by the boiling preparation method (32). PCR was performed in a final volume of 100 μl containing 1x PCR buffer, 200 μM deoxynucleoside triphosphate, 0.2 μM of each primer, 2.5 U of HotStarTaq DNA polymerase (QIAGEN, Tokyo, Japan), and 5-μl aliquot of a template DNA solution. After initial incubation at 95°C for 15 min, DNA was amplified by 25 cycles, with 1 cycle consisting of 1 min at 95°C, 1 min at 55°C, and 1 min at 72°C. The final cycle included additional incubation at 72°C for 7 min. The primers used to amplify each bla gene were as follows: TEM#1 (5'-AAGCCATACCAAACGACGAG-3') and TEM#2 (5'-ATTGTTGCCGGGAAGCTAGA-3') for blaTEM (108 bp, a PCR product length), TOHO#1 (5'-TGGAAGCCCTGGAGAAAAGT-3') and TOHO#2 (5'-CTTATCGCTCTCGCTCTGTT-3') for blaTOHO (448 bp), and IMP#1 (5'-CATGGTTTGGTGGTTCTTGT-3') and IMP#2 (5'-ATAATTTGGCGGACTTTGGC-3') for blaIMP (164 bp). Amplified PCR products were confirmed by agarose gel electrophoresis.
Transformation of ampD mutants with wild-type ampD caused a decrease of AmpC expression (1). Therefore, complementation analysis of all isolates was performed using pKU420 (a plasmid containing wild-type ampD). pKU420 was constructed by blunt end cloning of a 994-bp PCR product into the SmaI site of pMW219 (Nippon Gene, Tokyo, Japan). The 994-bp PCR product, including the promoter and ampD-coding region of E. coli K-12 1037, was amplified by PCR using Pyrobest DNA polymerase (Takara, Kyoto, Japan) and the following pair of primers: ampD#1 (5'-ACGTCGGGTGTCAGGGTTAT-3')and ampD#2 (5'-GTGGCACGGGCATGGCTATCA-3').The PCR product was purified using a Qiaquick PCR purification kit (QIAGEN) and was subsequently ligated with SmaI-digested pMW219. E. coli K-12 XL1-Blue was used as the host strain for cloning. Transformation with the ligated plasmid was performed by electroporation as described previously (1). ampD clones were selected by incubation in LB agar containing 50 μg/ml of kanamycin, 100 μg/ml of 5-bromo-4-chloro-3-indolyl--D-galactopyranoside (X-Gal) (Takara), and 50 mM isopropyl--D-thiogalactopyranoside (IPTG) (Takara). The transcriptional orientation of ampD was opposite to the orientation of lacZ. Isolation of plasmids was performed using a QIAprep Spin Miniprep kit (QIAGEN). For the selection of clinical isolates transformed by pKU420, butirosin A (Sigma-Aldrich, St. Louis, Mo.) (at a final concentration of 50 μg/ml) was used.
Ceftazidime MICs and -lactamase activities of clinical isolates of E. cloacae are shown in Table 1. The clinical isolates exhibited various levels of -lactamase activity, suggesting a diversity of genetic changes in ampD. After transformation with pKU420, all of the clinical isolates showed a dramatic decrease of -lactamase activity. This indicated that all clinical isolates resistant to ceftazidime harbor AmpD mutations. However, seven transformed strains (KU6331, KU6332, KU6334, KU6343, KU6344, KU6345, and KU6346) exhibited residual -lactamase activity comparable to that of KU3262, a ceftazidime-susceptible strain. blaTEM, blaTOHO, and blaIMP were detected in these strains, except for KU6334, KU6344, and KU6346 (Table 1). Additionally, -lactamase activity was inhibited by clavulanic acid in these strains, except for KU6334, KU6344, and KU6345. Since these -lactamases can hydrolyze cephalosporins, such as ceftazidime, residual -lactamase activity and the high MIC of ceftazidime in ampD transformants seem to be principally due to these -lactamases (10, 26). However, the -lactamase activities of KU6343 (2.97 U/mg protein) and KU6346 (0.39 U/mg protein) were only partially inhibited (to 1.43 and 0.21 U/mg protein, respectively). Thus, we confirmed the presence of residual AmpC activity in the KU6334, KU6343, KU6344, and KU6346 strains.
The DNA sequence of the ampD region was determined in six selected strains (KU6324, KU6325, KU6327, KU6328, KU6329, and KU6339). DNA sequencing was performed with an ABI PRISM 310 DNA sequencer (Applied Biosystems, Tokyo, Japan) using a BigDye Terminator v1.1 cycle sequencing kit (Applied Biosystems) according to the manufacturer's instructions. A homology search of the database was performed using the BLASTN program. All of the strains had mutations of AmpD compared with the wild-type sequence (GenBank accession no. Z14003). These amino acid substitutions were as follows: Ala-94 to Pro in strain KU6339; Asn-150 to Ile in KU6325; Leu-38 to Phe and Ala-60 to Val in KU6327 and KU6324; and Phe-63 to Tyr, Glu-96 to Gln, Ile-106 to Val, Arg-108 to Leu, and Ala-122 to Gly in KU6329 and KU6328. It has already been reported that not only mutations of the active-site core residues but also mutations of structurally independent residues can cause constitutive expression of AmpC (29). Therefore, we could not find any substitutions in the active-site core residues of AmpD, but each mutation seemed to cause overproduction of AmpC in the isolates.
Bartowsky et al. previously reported that a mutation of AmpR resulted in the semiconstitutive expression of AmpC (3, 4). In a previous study, we confirmed that introduction of a plasmid harboring mutant ampR into wild-type E. cloacae resulted in the constitutive expression of AmpC (17). Therefore, we used E. cloacae KU3262 with wild-type ampD to assess the effect of ampR mutation. The ampR region, including its promoter region and the partial ampC region, was amplified by PCR as described above. The primers were C#7 (5'-GCCTTGCCAAACGTGTAATAGTGC-3') and R#2 (5'-AAATCCATGTGCCAAGCGGTAAAT-3') for KU6334 and KU6343, while they were 6344C#1 (5'-GTTTGTGCAGTGACGGGTGTG-3') and 6344R#2 (5'-TCCTGACGGTGGTTACGCTGAT-3') for KU6344. Amplified PCR products (1,323 bp for KU6334 and KU6343 and 1,311 bp for KU6344) were purified and subsequently cloned using a PCR cloning kit (QIAGEN) according to the manufacturer's instructions. Although strain KU6346 exhibited residual AmpC activity, cloning of its ampR was not performed because there were no amino acid substitutions in the coding region. Transformation, clonal selection, and plasmid extraction were done as described above for ampD. The clonal ampR genes were subcloned into the BamHI and HindIII sites of pMW219.
The AmpC activity of E. cloacae KU3262 harboring these recombinant plasmids is shown in Table 2. Introduction of pKU430 and pKU431 into strain KU3262, significantly increased AmpC activity by 21-fold (1.56 U/mg protein) and 19-fold (1.35 U/mg protein) above the basal level (0.07 U/mg protein), respectively. Similarly, the MICs of ceftazidime also increased 16-fold (2 μg/ml) and 64-fold (8 μg/ml) above the basal value (0.25 μg/ml). In contrast, transformants of KU3262 harboring pKU432 did not exhibit any increase in the MIC of ceftazidime or AmpC activity. By comparison with wild-type ampR in MHN1 (GenBank accession no. X04730), amino acid substitutions were identified in all strains. The amino acid substitutions were Arg-86 to Ser in KU6334 and Thr-64 to Ile in KU6343. As for Arg-86, we have previously reported that introduction of a plasmid expressing this mutation of AmpR could increase AmpC activity (17). Additionally, the same phenomenon was observed by introducing pKU431 into KU3262. Considering these findings, mutations of residues 64 and 86 alter the conformational change of AmpR triggered by binding to anhydromuropeptides (the AmpR-activating ligand). On the other hand, the amino acids affecting constitutive AmpC expression (Arg-86, Gly-102, and Asp-135) were conserved in AmpR from KU6344, and introduction of pKU432 did not significantly induce expression of AmpC. Therefore, the residual AmpC activity of KU6344 appears to be related to unknown factors.
In the present study, we found that the resistance of E. cloacae clinical isolates to ceftazidime was principally due to AmpD-dependent constitutive expression of AmpC. On the other hand, we identified chromosomal AmpR mutants among clinical isolates. One of the problems in clinical practice is the spread of plasmid-mediated AmpC (30). In particular, strains expressing inducible plasmid-mediated AmpC with functional AmpR have been isolated (2, 23, 24, 31). Among those, blaCFE-1, a plasmid-encoded AmpC that we reported previously, seems to be involved in AmpR-dependent constitutive expression of AmpC (24). Our results suggest that chromosomal AmpR mutations may have the potential to confer not only high-level constitutive expression of chromosomal AmpC but also plasmid-mediated constitutive AmpC overproduction to clinical isolates.
Nucleotide sequence accession numbers. The nucleotide sequence data in this study will appear in the EMBL/GenBank/DDBJ nucleotide sequence databases. The accession numbers are as follows: AY789446 for ampR from E. cloacae KU6334, AY789447 for ampR from KU6343, and AY789448 for ampR from KU6344.
ACKNOWLEDGMENTS
This work was supported by grants (14370096) from the Japanese Ministry of Education, Culture, Sports, Science and Technology; the COE program of the Japanese Ministry of Education, Culture, Sports, Science and Technology; the Japanese Ministry of Health, Labor and Welfare (the Research Project of Emerging and Establishment of Rapid Identification Methods [H15-Sinko-9]); and the Charitable Trust Clinical Pathology Research Foundation of Japan.
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Department of Microbiology, School of Medicine, Kitasato University, 1-15-1 Kitasato, Sagamihara, Kanagawa
Department of Central Laboratory, School of Medicine, Teikyo University, 2-11-1, Kaga, Itabashi-ku, Tokyo, Japan
ABSTRACT
AmpC regulatory genes in 21 ceftazidime-resistant clinical isolates of Enterobacter cloacae (MICs of 16 μg/ml) were characterized. All isolates exhibited AmpC overproduction due to AmpD mutation. Additionally, we found two AmpR mutants among the isolates. This is the first report of chromosomal ampR mutation in clinical isolates of E. cloacae.
TEXT
Resistance of members of the family Enterobacteriaceae (e.g., Enterobacter cloacae and Serratia marcescens) to expanded-spectrum cephalosporins is a serious clinical problem (14). The resistance of these bacteria is principally due to constitutive overexpression of chromosomal ampC (which encodes AmpC, a class C -lactamase) (5, 33). The ampC gene is located on the chromosome of many gram-negative bacilli and is inducible in the presence of -lactam antibiotics (9).
The mechanism of AmpC induction is closely linked to a cell wall recycling system (8, 12). The cell walls of gram-negative bacilli must be reconstituted during growth. Escherichia coli (and presumably E. cloacae) degrades 40 to 50% of the cell wall peptidoglycans during each generation, and 90% of the degradation products are recycled (28). Peptidoglycan degradation products (anhydromuropeptides) released into the periplasm are transported into the cytoplasm by AmpG, a transmembrane permease (6, 16, 22). Subsequently, these anhydromuropeptides are hydrolyzed by AmpD, which is a cytoplasmic N-acetyl-anhydromuramyl-L-alanine amidase that specifically hydrolyzes several anhydromuropeptides such as (GlcNAc-)anhMurNAc tripeptide and GlcNAc-anhMurNAc tetrapeptide (11). This action of AmpD prepares anhydromuropeptides for further recycling. In the presence of -lactams, however, anhydromuropeptides increase in both the periplasm and cytoplasm. Cytoplasmic anhydromuropeptides that have avoided hydrolysis by AmpD bind to AmpR, a transcriptional regulator of ampC (9, 19, 21). In the absence of an activating ligand, AmpR represses ampC expression with UDP-MurNAc pentapeptide, but cell wall degradation products [(GlcNAc-)anhMurNAc tripeptides] may be an AmpR-activating ligand that causes the activation of ampC expression. In AmpD-defective bacterial strains, unhydrolyzed anhydromuropeptides accumulate in the cytoplasm (7, 13), and this causes activation of AmpR, which results in the semiconstitutive or constitutive expression of ampC. The role of AmpD in the induction of AmpC in gram-negative bacilli has been studied in detail (15, 18, 20).
AmpD-dependent constitutive expression of AmpC occurs in vitro at a frequency of 10–5 to 10–7 (19, 29). In addition, we previously reported that AmpR-dependent constitutive overproduction of AmpC occurred at a frequency of 10–6 in AmpD-defective strains (17). However, the isolation rate of AmpD mutants among cephalosporin-resistant clinical strains has not been determined. In addition, there have been no reports describing the isolation of chromosomal AmpR mutants from clinical isolates.
Twenty-one ceftazidime-resistant strains of E. cloacae KU6323 to KU6332, KU6334, KU6336 to KU6341, and KU6343 to KU6346 were isolated during a 1-year period in 1999 from patients attending a university-affiliated hospital in Tokyo, Japan. KU4892, KU4894, and KU1917 were positive-control strains (blaTEM-1, blaTOHO-1, and blaIMP-1, respectively) and were created by introduction of plasmids pKU507, pMTY010, and pMS361, respectively (10, 34). E. cloacae KU3262 (wild-type AmpD) was a clinical isolate and used for complementation assay of ampR (17). The MICs were determined by the agar dilution method according to CLSI (formerly NCCLS) guidelines (25). -Lactamase activity was detected by a colorimetric assay using cephalothin as a substrate (17, 27). For inhibition of class A -lactamase, clavulanic acid (at a final concentration of 5 μg/ml) was added to either substrate solution or extracted enzyme solution. Results were analyzed by Student's t test, and values were expressed as the means ± standard deviations (SDs). A P of <0.001 was defined as indicating statistical significance.
To detect -lactamase genes, except for chromosomal ampC, PCR amplification was performed. Template DNA solution was prepared by the boiling preparation method (32). PCR was performed in a final volume of 100 μl containing 1x PCR buffer, 200 μM deoxynucleoside triphosphate, 0.2 μM of each primer, 2.5 U of HotStarTaq DNA polymerase (QIAGEN, Tokyo, Japan), and 5-μl aliquot of a template DNA solution. After initial incubation at 95°C for 15 min, DNA was amplified by 25 cycles, with 1 cycle consisting of 1 min at 95°C, 1 min at 55°C, and 1 min at 72°C. The final cycle included additional incubation at 72°C for 7 min. The primers used to amplify each bla gene were as follows: TEM#1 (5'-AAGCCATACCAAACGACGAG-3') and TEM#2 (5'-ATTGTTGCCGGGAAGCTAGA-3') for blaTEM (108 bp, a PCR product length), TOHO#1 (5'-TGGAAGCCCTGGAGAAAAGT-3') and TOHO#2 (5'-CTTATCGCTCTCGCTCTGTT-3') for blaTOHO (448 bp), and IMP#1 (5'-CATGGTTTGGTGGTTCTTGT-3') and IMP#2 (5'-ATAATTTGGCGGACTTTGGC-3') for blaIMP (164 bp). Amplified PCR products were confirmed by agarose gel electrophoresis.
Transformation of ampD mutants with wild-type ampD caused a decrease of AmpC expression (1). Therefore, complementation analysis of all isolates was performed using pKU420 (a plasmid containing wild-type ampD). pKU420 was constructed by blunt end cloning of a 994-bp PCR product into the SmaI site of pMW219 (Nippon Gene, Tokyo, Japan). The 994-bp PCR product, including the promoter and ampD-coding region of E. coli K-12 1037, was amplified by PCR using Pyrobest DNA polymerase (Takara, Kyoto, Japan) and the following pair of primers: ampD#1 (5'-ACGTCGGGTGTCAGGGTTAT-3')and ampD#2 (5'-GTGGCACGGGCATGGCTATCA-3').The PCR product was purified using a Qiaquick PCR purification kit (QIAGEN) and was subsequently ligated with SmaI-digested pMW219. E. coli K-12 XL1-Blue was used as the host strain for cloning. Transformation with the ligated plasmid was performed by electroporation as described previously (1). ampD clones were selected by incubation in LB agar containing 50 μg/ml of kanamycin, 100 μg/ml of 5-bromo-4-chloro-3-indolyl--D-galactopyranoside (X-Gal) (Takara), and 50 mM isopropyl--D-thiogalactopyranoside (IPTG) (Takara). The transcriptional orientation of ampD was opposite to the orientation of lacZ. Isolation of plasmids was performed using a QIAprep Spin Miniprep kit (QIAGEN). For the selection of clinical isolates transformed by pKU420, butirosin A (Sigma-Aldrich, St. Louis, Mo.) (at a final concentration of 50 μg/ml) was used.
Ceftazidime MICs and -lactamase activities of clinical isolates of E. cloacae are shown in Table 1. The clinical isolates exhibited various levels of -lactamase activity, suggesting a diversity of genetic changes in ampD. After transformation with pKU420, all of the clinical isolates showed a dramatic decrease of -lactamase activity. This indicated that all clinical isolates resistant to ceftazidime harbor AmpD mutations. However, seven transformed strains (KU6331, KU6332, KU6334, KU6343, KU6344, KU6345, and KU6346) exhibited residual -lactamase activity comparable to that of KU3262, a ceftazidime-susceptible strain. blaTEM, blaTOHO, and blaIMP were detected in these strains, except for KU6334, KU6344, and KU6346 (Table 1). Additionally, -lactamase activity was inhibited by clavulanic acid in these strains, except for KU6334, KU6344, and KU6345. Since these -lactamases can hydrolyze cephalosporins, such as ceftazidime, residual -lactamase activity and the high MIC of ceftazidime in ampD transformants seem to be principally due to these -lactamases (10, 26). However, the -lactamase activities of KU6343 (2.97 U/mg protein) and KU6346 (0.39 U/mg protein) were only partially inhibited (to 1.43 and 0.21 U/mg protein, respectively). Thus, we confirmed the presence of residual AmpC activity in the KU6334, KU6343, KU6344, and KU6346 strains.
The DNA sequence of the ampD region was determined in six selected strains (KU6324, KU6325, KU6327, KU6328, KU6329, and KU6339). DNA sequencing was performed with an ABI PRISM 310 DNA sequencer (Applied Biosystems, Tokyo, Japan) using a BigDye Terminator v1.1 cycle sequencing kit (Applied Biosystems) according to the manufacturer's instructions. A homology search of the database was performed using the BLASTN program. All of the strains had mutations of AmpD compared with the wild-type sequence (GenBank accession no. Z14003). These amino acid substitutions were as follows: Ala-94 to Pro in strain KU6339; Asn-150 to Ile in KU6325; Leu-38 to Phe and Ala-60 to Val in KU6327 and KU6324; and Phe-63 to Tyr, Glu-96 to Gln, Ile-106 to Val, Arg-108 to Leu, and Ala-122 to Gly in KU6329 and KU6328. It has already been reported that not only mutations of the active-site core residues but also mutations of structurally independent residues can cause constitutive expression of AmpC (29). Therefore, we could not find any substitutions in the active-site core residues of AmpD, but each mutation seemed to cause overproduction of AmpC in the isolates.
Bartowsky et al. previously reported that a mutation of AmpR resulted in the semiconstitutive expression of AmpC (3, 4). In a previous study, we confirmed that introduction of a plasmid harboring mutant ampR into wild-type E. cloacae resulted in the constitutive expression of AmpC (17). Therefore, we used E. cloacae KU3262 with wild-type ampD to assess the effect of ampR mutation. The ampR region, including its promoter region and the partial ampC region, was amplified by PCR as described above. The primers were C#7 (5'-GCCTTGCCAAACGTGTAATAGTGC-3') and R#2 (5'-AAATCCATGTGCCAAGCGGTAAAT-3') for KU6334 and KU6343, while they were 6344C#1 (5'-GTTTGTGCAGTGACGGGTGTG-3') and 6344R#2 (5'-TCCTGACGGTGGTTACGCTGAT-3') for KU6344. Amplified PCR products (1,323 bp for KU6334 and KU6343 and 1,311 bp for KU6344) were purified and subsequently cloned using a PCR cloning kit (QIAGEN) according to the manufacturer's instructions. Although strain KU6346 exhibited residual AmpC activity, cloning of its ampR was not performed because there were no amino acid substitutions in the coding region. Transformation, clonal selection, and plasmid extraction were done as described above for ampD. The clonal ampR genes were subcloned into the BamHI and HindIII sites of pMW219.
The AmpC activity of E. cloacae KU3262 harboring these recombinant plasmids is shown in Table 2. Introduction of pKU430 and pKU431 into strain KU3262, significantly increased AmpC activity by 21-fold (1.56 U/mg protein) and 19-fold (1.35 U/mg protein) above the basal level (0.07 U/mg protein), respectively. Similarly, the MICs of ceftazidime also increased 16-fold (2 μg/ml) and 64-fold (8 μg/ml) above the basal value (0.25 μg/ml). In contrast, transformants of KU3262 harboring pKU432 did not exhibit any increase in the MIC of ceftazidime or AmpC activity. By comparison with wild-type ampR in MHN1 (GenBank accession no. X04730), amino acid substitutions were identified in all strains. The amino acid substitutions were Arg-86 to Ser in KU6334 and Thr-64 to Ile in KU6343. As for Arg-86, we have previously reported that introduction of a plasmid expressing this mutation of AmpR could increase AmpC activity (17). Additionally, the same phenomenon was observed by introducing pKU431 into KU3262. Considering these findings, mutations of residues 64 and 86 alter the conformational change of AmpR triggered by binding to anhydromuropeptides (the AmpR-activating ligand). On the other hand, the amino acids affecting constitutive AmpC expression (Arg-86, Gly-102, and Asp-135) were conserved in AmpR from KU6344, and introduction of pKU432 did not significantly induce expression of AmpC. Therefore, the residual AmpC activity of KU6344 appears to be related to unknown factors.
In the present study, we found that the resistance of E. cloacae clinical isolates to ceftazidime was principally due to AmpD-dependent constitutive expression of AmpC. On the other hand, we identified chromosomal AmpR mutants among clinical isolates. One of the problems in clinical practice is the spread of plasmid-mediated AmpC (30). In particular, strains expressing inducible plasmid-mediated AmpC with functional AmpR have been isolated (2, 23, 24, 31). Among those, blaCFE-1, a plasmid-encoded AmpC that we reported previously, seems to be involved in AmpR-dependent constitutive expression of AmpC (24). Our results suggest that chromosomal AmpR mutations may have the potential to confer not only high-level constitutive expression of chromosomal AmpC but also plasmid-mediated constitutive AmpC overproduction to clinical isolates.
Nucleotide sequence accession numbers. The nucleotide sequence data in this study will appear in the EMBL/GenBank/DDBJ nucleotide sequence databases. The accession numbers are as follows: AY789446 for ampR from E. cloacae KU6334, AY789447 for ampR from KU6343, and AY789448 for ampR from KU6344.
ACKNOWLEDGMENTS
This work was supported by grants (14370096) from the Japanese Ministry of Education, Culture, Sports, Science and Technology; the COE program of the Japanese Ministry of Education, Culture, Sports, Science and Technology; the Japanese Ministry of Health, Labor and Welfare (the Research Project of Emerging and Establishment of Rapid Identification Methods [H15-Sinko-9]); and the Charitable Trust Clinical Pathology Research Foundation of Japan.
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