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Antimicrobial Agents and Chemotherapy, September 1998, p. 2352-2358, Vol. 42, No. 9
0066-4804/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Salmonella enteritidis: AmpC Plasmid-Mediated Inducible beta -Lactamase (DHA-1) with an ampR Gene from Morganella morganii

Guilene Barnaud,1 Guillaume Arlet,1,* Charlotte Verdet,1 Olivier Gaillot,2 Philippe H. Lagrange,1 and Alain Philippon1

Service de Microbiologie, Hôpital Saint-Louis,1 and Service de Microbiologie, Hôpital Boucicaut,2 Paris, France

Received 15 January 1998/Returned for modification 10 April 1998/Accepted 12 June 1998

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

DHA-1, a plasmid-mediated cephalosporinase from a single clinical Salmonella enteritidis isolate, conferred resistance to oxyimino-cephalosporins (cefotaxime and ceftazidime) and cephamycins (cefoxitin and moxalactam), and this resistance was transferable to Escherichia coli HB101. An antagonism was observed between cefoxitin and aztreonam by the diffusion method. Transformation of the transconjugant E. coli strain with plasmid pNH5 carrying the ampD gene (whose product decreases the level of expression of ampC) resulted in an eightfold decrease in the MIC of cefoxitin. A clone with the same AmpC susceptibility pattern with antagonism was obtained, clone E. coli JM101(pSAL2-ind), and its nucleotide sequence was determined. It contained an open reading frame with 98.7% DNA sequence identity with the ampC gene of Morganella morganii. DNA sequence analysis also identified a gene upstream of ampC whose sequence was 97% identical to the partial sequence of the ampR gene (435 bp) from M. morganii. The gene encoded a protein with an amino-terminal DNA-binding domain typical of transcriptional activators of the LysR family. Moreover, the intercistronic region between the ampC and ampR genes was 98% identical to the corresponding region from M. morganii DNA. AmpR was shown to be functional by enzyme induction and a gel mobility-shift assay. An ampG gene was also detected in a Southern blot of DNA from the S. enteritidis isolate. These findings suggest that this inducible plasmid-mediated AmpC type beta -lactamase, DHA-1, probably originated from M. morganii.

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

The principal mechanism of resistance to broad-spectrum cephalosporins such as cefotaxime and ceftazidime in Salmonella strains involved the production of extended-spectrum class A beta -lactamases such as SHV-2, TEM-3, TEM-25, and TEM-27 (derived from TEM-or SHV-type enzymes) (12, 20, 38, 41) and CTX-M-2 and PER-2 (3, 4). This mechanism has also been reported in other members of the family Enterobacteriaceae, especially in Klebsiella pneumoniae, a species which, like Salmonella species, lacks the chromosomal ampC gene. These enzymes are not active against cephamycins such as cefoxitin, and susceptibility to oxyimino-cephalosporins can be restored by clavulanic acid.

An additional form of beta -lactam resistance has recently been observed. Plasmid-mediated AmpC type beta -lactamases have been reported in clinical strains of K. pneumoniae, Escherichia coli, Enterobacter aerogenes, and Salmonella spp. in nine countries. These enzymes include MIR-1, CMY-1, CMY-2, MOX-1, FOX-1, FOX-2, FOX-3, LAT-1, LAT-2, BIL-1, and ACT-1 (5-7, 11, 18, 19, 24, 29, 36, 39, 40, 45). These beta -lactamases have biochemical properties typical of AmpC beta -lactamases and result in a derepressed phenotype of resistance to beta -lactams. Combined with impermeability, as for K. pneumoniae strains producing ACT-1 (11), they can confer resistance to imipenem. Some have DNA and amino acid sequences very similar to those of chromosome-mediated AmpC beta -lactamases of Citrobacter freundii (CMY-2, LAT-1, LAT-2, BIL-1) (5, 15, 18, 29, 46) or Enterobacter cloacae (MIR-1, ACT-1) (11, 39), whereas the phylogenies of the other enzymes are unclear (6, 7, 19, 25, 36).

A plasmid-mediated AmpC beta -lactamase, DHA-1, which confers resistance to cephamycins and oxyimino-cephalosporins and which is characterized by a probable inducible production, was identified in 1992 in Saudi Arabia in a clinical isolate of Salmonella enteritidis (17). In the present study, we analyzed the nucleotide sequences of the contiguous ampC and ampR genes and the phylogeny and regulation of the corresponding AmpC enzyme.

    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Bacterial strains, plasmids, and media. The bacterial strains and plasmids used in this study are listed in Table 1. S. enteritidis KF92 encoding DHA-1 (17) was isolated in November 1992 from the stool samples from a patient with lung carcinoma hospitalized at Dhahran Hospital, Dhahran, Saudi Arabia.

                              
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  		TABLE 1.   Bacterial strains and plasmids used in this study

E. coli K-12 strain HB101 (10) was used for conjugation experiments, and E. coli JM101 was used for cloning and transformation. The clinical strains of S. enteritidis, K. pneumoniae, and Klebsiella oxytoca used in DNA-DNA hybridization experiments were isolated at Saint-Louis Hospital (Paris, France).

Plasmid vector pACYC184 (13) was used for cloning and was prepared by the alkaline lysis method (9). Drigalski agar (Sanofi Diagnostics Pasteur, Marnes-la-Coquette, France) containing amoxicillin (20 µg/ml) and chloramphenicol (30 µg/ml) was used for cloning.

Antibiotic susceptibility testing. MICs were determined by the agar dilution method with Mueller-Hinton agar (Sanofi Diagnostics Pasteur). Inocula of 104 to 105 CFU per spot were delivered with a multipoint inoculator. The resistance phenotype was determined by the disk diffusion method with Mueller-Hinton agar (14).

Synergy. A double-disk synergy test was performed with various cephalosporins: cefotetan, ceftazidime, cefoxitin, and cefotaxime. Disks of antibiotics were placed on agar plates inoculated with the transconjugant E. coli HB101 or with the clone to be tested. A disk of inhibitor was placed on the agar surface 30 mm (center to center) from the central disk. Two inhibitors were tested: clavulanate, a specific inhibitor of penicillinase (10 µg per disk), and RO48-1220, which is specific for cephalosporinase (20 µg per disk) (42) and which was obtained from Hoffmann-La Roche SA (Basel, Switzerland). The plates were examined after overnight incubation at 37°C.

beta -Lactamase preparation and induction. Crude cell extracts were prepared by sonication of cells grown overnight in Trypticase soy broth (Sanofi Diagnostics Pasteur). Cells of E. coli JM101 containing pSAL2-ind were induced by dilution in fresh broth containing imipenem at 0.25, 0.5, 1, or 2 µg/ml and were incubated for 3 h before harvesting. One unit of beta -lactamase activity is the enzyme activity that hydrolyzes 1 µM cephaloridine per min and is expressed per milligram of protein.

beta -Lactamase assays. Microacidimetric assays of beta -lactamase activity in cell-free extracts were performed as described previously with 100 µM cephalothin as the substrate (27). Protein concentrations were determined by using the Bio-Rad protein assay (Bio-Rad Laboratories, Ivry sur Seine, France) with bovine serum albumin as the standard.

IEF. Analytical isoelectric focusing (IEF) of crude extracts was performed in a polyacrylamide gel (pH 3.5 to 9.5) (37) that was subjected to electrophoresis for 18 h at 15°C. beta -Lactamase activity was detected with the chromogenic compound nitrocefin. TEM-1 (pIP1100; pI 5.4), TEM-3 (pCFF04; pI 6.3), OXA-1 (RGN238; pI 7.4), and SHV-4 (pUD21; pI 7.8) were used as pI markers.

Transformation with pNH5. The E. coli transconjugants producing DHA-1, MIR-1, and BIL-1 were transformed with plasmid pNH5 carrying the ampD gene by the standard CaCl2 technique (35). The plasmid carried a kanamycin resistance gene, and kanamycin was used at a concentration of 50 µg/ml to select transformants on Drigalski agar.

Enzymes. Restriction endonucleases and T4 DNA ligase were used according to the recommendations of the manufacturer (Boehringer Mannheim Biochemicals, France S.A. Meylan, France).

Cloning of ampC and ampR genes. Plasmid pSAL1 DNA was isolated from the E. coli HB101 transconjugant by the alkaline lysis method of Birnboim and Doly (9). It was partially digested with Sau3A and ligated into the BamHI site of pACYC184. Recombinant plasmid was introduced into E. coli JM101 by the standard CaCl2 technique. Transformants were selected on the basis of resistance to amoxicillin (20 µg/ml) and chloramphenicol (30 µg/ml) and were further characterized by analysis of their antibiotic susceptibility patterns and determination of their pIs. The size of the insert in the recombinant plasmid was estimated by restriction enzyme digestion and electrophoresis in 1 to 3% agarose gels.

Sequencing. Double-stranded DNA was sequenced by the procedure of Sanger et al. (43) by using fluorescent dye-labeled dideoxynucleotides, thermal cycling with Taq polymerase, and an ABI 373A DNA sequencer (Applied Biosystems, Foster City, Calif.).

DNA and amino acid sequence analyses. Sequence analyses and comparison with other known sequences were performed with the BLAST (1) and FAST programs at the NCBI. The Clustal W program was used to produce multiple alignments of the predicted amino acid sequence of DHA-1 with the sequences of other proteins in the protein data bank.

Preparation of protein extracts. Protein extracts were prepared as described by Keegan et al. (28). E. coli JM101 cells containing the putative recombinant plasmid pSAL2-ind which confers inducible expression of the beta -lactamase gene were grown in 250 ml of Trypticase soy broth. The cells were collected by centrifugation, washed once in 150 mM NaCl, and suspended in 2 ml of buffer A (25 mM HEPES [N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid; pH 7.5], 5 mM MgCl2, 0.1 mM EDTA, 5 mM 2-mercaptoethanol, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride). This mixture was subjected to 10 cycles of sonication on ice, and cellular debris was removed by centrifugation at 48,000 × g for 30 min. The protein concentration was determined as described above.

Gel mobility-shift assays. Gel mobility-shift assays were performed as described previously (16). A 173-bp DNA fragment containing the 110-bp ampR-ampC intercistronic region was prepared by PCR with the oligonucleotides 5'-GGAAATCAGTGTTGCAGA and 3'-GGGTTAAGGGGGAGATAA as primers and pSAL1 as the template. Binding reactions were carried out in 10 µl of buffer B (25 mM HEPES [pH 7.5], 0.1 mM EDTA, 5 mM dithiothreitol, 10% glycerol, 50 mM KCl) containing 2 pM 173-bp DNA fragment, 2 µg of poly(dI-dC) (Pharmacia Biotech, Saclay, France), and various amounts of protein extracts (2.6 to 13 µg). The mixture was incubated at 25°C for 15 min and was subjected to electrophoresis in a 3% low-melting-point agarose gel in Tris-borate-EDTA buffer. After migration, the DNA was transferred from the agarose gel to a Hybond N+ membrane (Amersham France SA, Les Ulis, France) by the Southern alkaline transfer method (35). The membrane was probed with the fluorescein-labeled 173-bp DNA fragment. The DNA labeling and detection kit was used as recommended by the manufacturer (Amersham Life Science, Les Ulis, France).

Detection of ampG in total S. enteritidis KF92 DNA. An ampG probe (852 bp) was prepared by PCR with E. coli HB101 DNA as the template and the oligonucleotide primers 5'-GCTCGCCACGCAAATCCTG-3' and 5'-GACATAAACTCGCCCTACA-3' (positions 252 to 270 and 1104 to 1086, respectively [the numbering is as for the nucleotide sequence of E. coli JRG582 ampG]) (34). Before using this probe, the PCR product (ampG) was analyzed by PCR-restriction fragment length polymorphism analysis with Sau3A, HpaII, and HhaI. The restriction endonuclease patterns obtained were consistent with the patterns generated with DNA software (DNA Strider, version 1.2). Total KF92 DNA was digested with EcoRI, subjected to electrophoresis, and transferred to a Hybond N+ membrane as described above. The membrane was then probed with the fluorescein-labeled DNA probe. The DNA labeling and detection kit was used according to the recommendations of the manufacturer (Amersham Life Science).

Nucleotide sequence accession number. The EMBL accession number for the nucleotide sequence reported here is Y16410.

    RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Inducible cephalosporinase activity. An antagonism between cefoxitin or imipenem and other beta -lactams such as cefotaxime and aztreonam was originally observed by the diffusion method for both strains of S. enteritidis and the transconjugant E. coli HB101. Therefore, we hypothesized that the enzyme would be inducible, and to test this, we introduced by transformation pNH5 which carries the ampD gene into this transconjugant (31). The ampD product decreases the level of ampC expression. The MIC of cefoxitin for the transformant as eight times lower, and those of cefotaxime, moxalactam, and ticarcillin were half of those for the controls (Table 2). The transconjugants E. coli J53-2 and C600 producing the AmpC plasmid-mediated beta -lactamases BIL-1 (J53-2) and MIR-1 (C600) were also transformed, but no decrease in the MICs of cefoxitin, cefotaxime, moxalactam, or ticarcillin was observed (Table 2).

                              
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  		TABLE 2.   MICs of beta -lactams for S. enteritidis KF92, its transconjugant, its transformant, E. coli JM101, E. coli HB101, E. coli J53-2 producing BIL-1, E. coli C600 producing MIR-1, and the transformants with pNH5a

We investigated whether enzyme synthesis could be induced. Cephalosporinase activity was measured in extracts prepared from transconjugant E. coli HB101 in the absence of imipenem and after induction with various concentrations of imipenem. The activity was dependent on the concentration of imipenem. It was 8 times higher in the presence of 0.25 µg of imipenem per ml (one-quarter the MIC) than in the absence of imipenem and reached the maximum (11 times higher) at 0.5 µg of imipenem per ml (one-half the MIC), suggesting the involvement of an ampR-like gene.

Cloning of the ampC and ampR genes and their expression in an E. coli recipient. We selected several E. coli transformants that had beta -lactam susceptibility patterns (Table 2) similar to that of the E. coli HB101 transconjugant that produces cephalosporinase: resistance to amoxicillin, ticarcillin, cephalothin, cefoxitin, cefotaxime, and the amoxicillin-clavulanate combination. The patterns of susceptibility to other beta -lactams such as mecillinam, cefepime, and imipenem were the same as that for the parent strain.

Synergy between RO48-1220 and the various cephalosporins, particularly cefotetan, was observed against these transformants, suggesting that an AmpC-type beta -lactamase was produced (Fig. 1). IEF analysis of cell extracts from the transformants indicated that they mediated a beta -lactamase with a pI of 7.8 which cofocused with the cephalosporinase of the wild-type strain S. enteritidis KF92 (data not shown).


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  		FIG. 1.   Double-disk synergy test with the transformant E. coli JM101(pSAL2-ind). (A) Synergy test with 10 µg of clavulanate (disk 6); (B) synergy test with 20 µg of RO48-1220 (disk 7). Disks: 1, cefotaxime; 2, ceftazidime; 3, moxalactam; 4, cefotetan; 5, aztreonam.

One transformant was found to produce an inducible enzyme (antagonism between cefoxitin and cefotaxime). It harbored a recombinant plasmid (pSAL2-ind) with an insert of about 4.9 kb.

Nucleotide sequence. The 4.9-kb DNA insert of pSAL2-ind was sequenced. Two long open reading frames (ORFs) were found (Fig. 2). The first was 1.137 kb long and mediated a putative protein of 378 amino acids. This ORF had an ATG start codon at position 987 and a stop codon at position 2124 (Fig. 2). Database searches with this ORF identified similarities with several chromosome- and plasmid-mediated class C beta -lactamases (Fig. 3). The product of this ORF was most similar to the chromosome-mediated AmpC beta -lactamase of M. morganii (98.7% sequence identity) (2). The product of the ORF had 53 to 58% sequence identity with AmpC beta -lactamases of C. freundii, E. cloacae, Yersinia enterocolitica, and E. coli, and five plasmid-mediated enzymes (CMY-2, ACT-1, LAT-1, BIL-1, and LAT-2). There was more divergence from the AmpC beta -lactamases of Serratia marcescens, Pseudomonas aeruginosa, and Aeromonas sobria and from FOX-1 and MOX-1 (37 to 45% sequence identity). There were several conserved serine beta -lactamase motifs: the SXSK motif from the active site of the serine beta -lactamase, the typical class C motif YXN, and the KTG domain.


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  		FIG. 2.   Nucleotide sequences of the ampC and ampR genes and of the intercistronic region from pSAL2-ind and the deduced amino acid sequence of DHA-1. Putative -35 and -10 regions of the promoters of the two genes are boxed.


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  		FIG. 3.   Schematic representation of multiple-sequence alignments of 25 class C beta -lactamases calculated with Clustal W. Amino acids sequences were directly obtained from the GenBank, EMBL, or SWISS-PROT database.

The second ORF containing 873 nucleotides (nt) was transcribed in the opposite orientation and was located 5' from the ampC structural gene (Fig. 2). It began with an ATG codon at nt 876 and ended with a stop codon at nt 3. By analogy with the ampC-ampR genes in members of the family Enterobacteriaceae which produce inducible AmpC beta -lactamases, this ORF may correspond to the regulatory gene ampR. The deduced sequence of the product of this ORF was very similar to the sequence of the transcriptional regulators of the LysR family, particularly to those of the AmpR proteins of the members of the family Enterobacteriaceae. The partial DNA sequence (435 bp) (2) of the corresponding gene from M. morganii was 97% identical to the sequence of this ORF. The deduced protein sequence of this ORF was only 60 to 62% identical to the sequences of the known AmpR proteins from C. freundii, E. cloacae, and Y. enterocolitica. The predicted sequence of 280 amino acids had the characteristics of a typical LysR transcriptional regulator protein (21), with a helix-turn-helix DNA-binding motif in the N-terminal region (data not shown), as previously observed in the ampR gene from M. morganii (2).

The 111-bp region between the ampR and ampC start codons contained putative overlapping promoters (Fig. 2). This region was 98% identical to the corresponding region of M. morganii DNA (2) and was smaller than the corresponding regions of C. freundii, E. cloacae, and Y. enterocolitica DNA (111 versus 140 bp) (23, 33, 44).

Binding of AmpR to DNA in the ampR-ampC intercistronic region. Because we have detected antagonism between antibiotics and positive induction of the gene, we tested whether the ampR gene was functional. To test the binding of AmpR to the 111-bp intercistronic region, we performed gel mobility-shift assays. The 173-bp fragment encompassing the intercistronic region was retarded when it was mixed with the cell extracts containing the AmpR protein prepared from pSAL2-ind (Fig. 4).


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  		FIG. 4.   Gel mobility-shift assay of the S. enteritidis ampR and ampC intercistronic region with cell protein extracts containing the AmpR regulator protein. The intercistronic sequence, an end-labeled 173-bp PCR product, was retarded with various concentrations of protein extracts from the transformant pSAL2-ind. Lane A, 2.6 µg; lane B, 5.2 µg; lane C, 7.8 µg; lane D, 10.4 µg; lane E, 13 µg; lane F, 0 µg. The position of the retarded complexes containing the intercistronic region is indicated by the arrow.

Detection of ampG in S. enteritidis KF92. The ampG gene is required for activation of the ampR gene in members of the family Enterobacteriaceae with an inducible cephalosporinase. We detected this gene in S. enteritidis chromosomal DNA by hybridization to an ampG probe from E. coli HB101. We found that S. enteritidis KF92 had in its genome an ampG gene which was involved in the inducible synthesis of this plasmid-mediated beta -lactamase (Fig. 5).


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  		FIG. 5.   Southern blot analysis of DNA from S. enteritidis KF92 probed with ampG from E. coli HB101. Lane A, clinical isolate of K. oxytoca; lane B, clinical isolate of K. pneumoniae; lane C, clinical isolate of S. enteritidis; lane D, S. enteritidis KF92; lane E, E. coli HB101.

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

We report here on the nucleotide sequence, phylogeny, and regulation of an inducible AmpC plasmid-mediated beta -lactamase. This enzyme, previously called DHA-1 (17), has a pI of 7.8 and confers on clinical strain S. enteritidis KF92 and on transconjugant E. coli HB101 a pattern of beta -lactam resistance similar to those of strains of members of the family Enterobacteriaceae that overproduce their chromosome-mediated cephalosporinase and strains with plasmid-mediated AmpC beta -lactamase, especially clinical isolates of K. pneumoniae. Recently, another plasmid-mediated class C beta -lactamase (CMY-2b) was identified in a single clinical isolate of Salmonella senftenberg in Algeria (29). The incidence of these enzymes has increased since 1989. In K. pneumoniae, E. coli, and Salmonella spp., they are easy to detect on the basis of the isolate's phenotype. Inhibitors such as RO48-1220 that are active at least against class C beta -lactamases have been described (42) and may make it easier to detect these enzymes by the disk synergy test with cephamycins such as cefotetan in particular. Detection of such beta -lactamases is more difficult in Enterobacter spp. and Citrobacter spp. because these species have inducible chromosome-mediated cephalosporinases, and mutations in the ampD gene lead to overproduction of the enzyme.

Some plasmid-mediated class C beta -lactamases probably originate from the chromosomal ampC genes of E. cloacae (MIR-1 and ACT-1) or C. freundii (BIL-1, CMY-2, LAT-1, and LAT-2), whereas the origins of the other enzymes are unknown (CMY-1 and MOX-1) or uncertain (FOX-1, FOX-2, and FOX-3). We demonstrated by sequencing that this AmpC type beta -lactamase (DHA-1) has a sequence very similar to that of the chromosome-mediated cephalosporinase of M. morganii. Unlike other plasmid-mediated AmpC-type beta -lactamases, DHA-1 was inducible, as demonstrated by transformation with the ampD gene and the results of an induction test with imipenem as the inducer. These results strongly suggest the involvement of an ampR-like gene in pSAL1.

The nucleotide sequence of the upstream region of the ampC gene was very similar to that of the ampR gene and the intercistronic ampC-ampR region of M. morganii (97 to 98% identity) (2). The protein sequence deduced from ampR gene was very similar to that of the transcriptional regulators of the LysR family with a helix-turn-helix motif in the N-terminal region. This protein appeared to be functional, as shown by the gel mobility-shift assay.

Induction of the chromosome-mediated cephalosporinase AmpC of some members of the family Enterobacteriaceae (C. freundii, E. cloacae) is under the control of three genes, ampR, ampD, and ampG (8, 30, 32). The ampC and ampR genes are species specific and are present only in members of the family Enterobacteriaceae with inducible beta -lactamases. The ampD and ampG genes are probably very common and highly conserved in all members of the family Enterobacteriaceae because they encode enzymes involved in cell wall metabolism. AmpG is a permease and AmpD is a cytosolic amidase (22, 26, 34). The presence of the ampD gene, which is not closely linked to the ampR-ampC region, negatively regulates ampC expression (32). A homologous gene was detected in chromosomal DNA of Salmonella spp. by hybridization with an intragenic ampD probe from E. cloacae (38a). The ampG gene is required for activation of ampC by AmpR (30). We demonstrated the presence of the ampG gene in S. enteritidis KF92, so this strain has all the genes required for the inducible synthesis of DHA-1.

Thus, DHA-1 is highly related to the chromosome-mediated beta -lactamase of M. morganii and is inducible. The gene encoding the class C enzyme may have migrated from the chromosome of M. morganii to a plasmid, with this gene migration being mediated by transposable elements. Analyses of the sequences flanking the ampC and ampR genes in pSAL1 are in progress in our laboratory.

    FOOTNOTES

* Corresponding author. Mailing address: Service de Microbiologie, Hôpital Saint-Louis, 1, Ave. Claude Vellefaux, 75475 Paris Cedex 10, France. Phone: 33 (0)1 42 49 94 87. Fax: 33 (0)1 42 49 92 00. E-mail: alainphilippon{at}chu-stlouis.fr.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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Antimicrobial Agents and Chemotherapy, September 1998, p. 2352-2358, Vol. 42, No. 9
0066-4804/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


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