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Antimicrobial Agents and Chemotherapy, September 2000, p. 2549-2553, Vol. 44, No. 9
Servicio de Microbiología, Hospital
Ramón y Cajal, 28034 Madrid,1 and
Servicio de Microbiología, Hospital Insular, Las
Palmas de Gran Canaria,2 Spain
Received 8 October 1999/Returned for modification 28 February
2000/Accepted 9 June 2000
A clinical strain of Escherichia coli (Ec GCE)
displayed resistance to cefoxitin, cefotetan, cefotaxime, and
ceftazidime. Susceptibility was not restored by the addition of
clavulanic acid. Two Plasmid-mediated class A
Plasmid-mediated class C An E. coli strain (Ec GCE) that is resistant to a broad
spectrum of Susceptibility to
0066-4804/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Molecular Characterization of FOX-4, a New
AmpC-Type Plasmid-Mediated
-Lactamase from an Escherichia
coli Strain Isolated in Spain

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ABSTRACT
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Abstract
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References
-lactamases with apparent pIs of 5.4 and 6.4 were identified; the
-lactamase with a pI of 6.4 was transferred by
conjugation and associated with a 40-kb plasmid. Analysis of the
nucleotide sequence showed a new ampC
-lactamase gene
that is closely related to those encoding the FOX-3, FOX-2, and FOX-1
-lactamases but whose product has four novel amino acid mutations,
at positions 11 (M
T), 43 (A
E), 233 (V
A), and 280 (Y
H). This
first cephamycinase from Spain was named FOX-4.
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TEXT
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Abstract
Text
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-lactamases derived from the commonly found TEM and SHV enzymes have
been extensively reported (9, 10). They confer resistance to
extended-spectrum cephalosporins and aztreonam, but cephamycins are
unaffected. The enzymes remain susceptible to inhibition with the
addition of clavulanic acid.
-lactamases have been reported in the last
decade for Klebsiella pneumoniae, Escherichia
coli, Salmonella spp., and Proteus spp.
(2, 3, 4, 7, 14, 24). These
-lactamases show sequence
similarities to AmpC
-lactamases (1) of
Enterobacter cloacae (ACT-1 and MIR-1), Citrobacter
freundii (CMY-2, BIL-1, LAT-2, and LAT-1), and Pseudomonas
aeruginosa (CMY-1, FOX-1, and MOX-1) and confer a similar
resistance pattern to that of AmpC chromosomally mediated enzymes
(8).
-lactam antibiotics, including cephamycins, was isolated recently at Hospital Insular in Las Palmas de Gran Canaria (Canary Islands, Spain) from a patient suffering from a urinary tract infection
who was previously treated with cloxacillin, aztreonam, and cefotaxime
for an abdominal surgical infection. Our results showed that this
strain harbored a new plasmid-encoded cephamycin-hydrolyzing
-lactamase, the first plasmid-mediated cephamycinase isolated in Spain.
-lactams.
Antibiotic susceptibility
patterns of the Ec GCE clinical strain, as well as its transconjugant
and transformant, are shown in Table 1.
MICs were determined by the agar dilution method as recommended by the
National Committee for Clinical Laboratory Standards (18).
Antibiotics were provided as powders by the corresponding
manufacturers.
TABLE 1.
MICs of
-lactams for the Ec GCE clinical strain,
E. coli MC4100(pGC1), E. coli TG1, and
E. coli TG1(pGC-2)
-lactamase. In
addition, high levels of resistance to cefoxitin and cefotetan were
observed, together with a moderate level of resistance to moxalactam.
Higher MICs were obtained with the E. coli TG1 transformant harboring the pGC-2 plasmid, probably due to more copies of the ampC gene.
Isoelectric focusing. Isoelectric focusing was performed in polyacrylamide gels containing Ampholine with a pH range of 3.5 to 9.5, as previously described (17). The clinical isolate produced two enzymes, one with a pI of 5.4 (TEM-1 type) and one with a pI of 6.4 (AmpC type).
Conjugation experiments.
In this study the E. coli
MC4100 strain, with nalidixic acid and kanamycin resistances for
markers, was used in the conjugation experiments and E. coli
TG1 was used as the host for cloning the ampC gene. Plasmid
pBGS18
(23), with a kanamycin resistance
marker, was used for cloning the
-lactamase gene.
-lactamase with a pI of 6.4, was transferred by
conjugation into E. coli MC4100, with kanamycin (256 µg/ml), nalidixic acid (50 µg/ml), and ceftazidime (16 µg/ml) as
selector antibiotics. A few transconjugants grew which harbored an
identical plasmid of approximately 40 kb, named pGC-1. The other
plasmid from the Ec GCE clinical strain carried the
blaTEM-1 gene and would probably be transferred
if an appropriate antibiotic (i.e., ampicillin) was used for selection.
The presence of the blaTEM-1 gene in the Ec GCE
strain was demonstrated by PCR assay with specific
blaTEM primers C1
(5'-GGGAATTCTCGGGGAAATGTGCGCGGAAC) and C2
(5'-GGGATCCGAGTAAACTTGGTCTGACAG).
Cloning experiments and nucleotide sequencing.
Plasmid DNA was
isolated by the alkaline lysis method (21) from the
ceftazidime-resistant transconjugant that produced a single
-lactamase with a pI of 6.4. Plasmid DNA was digested with
HindIII, and the resulting fragments were ligated to the plasmid pBGS18; which was previously digested with the same restriction enzyme. The ligation mixture was introduced into E. coli TG1 by transformation with CaCl2, and
transformants were detected on Luria-Bertani agar plates supplemented
with ceftazidime (4 µg/ml) and kanamycin (50 µg/ml). The resultant
plasmid carrying the bla gene was named pGC-2. The molecular
size of the insert was estimated by using restriction enzymes and
electrophoresis on 1% agarose gels.
-lactamase activity band from the E. coli transformant cofocused with the
-lactamase activity band from the Ec GCE clinical strain. Nucleotide sequencing of the 3.3 kb insert (Fig.
1) revealed, in
addition to a new bla gene, the presence
of ORF341 and a conserved integron sequence previously
identified in the In7 integron (2, 19). This new
bla gene was 1,149 bp long (Fig. 1), initiated with an ATG
codon, and ended with a TGA codon (382 amino acids long). The
initiation codon was preceded by a Shine-Dalgarno ribosome-binding sequence, GAGAA, and putative
10 and
35 promoter regions,
TTTATA and TTCGGAT, respectively. EMBL and SwissProt database
searches for this open reading frame revealed similarities with several class C chromosome- and plasmid-mediated
-lactamases. The new protein had the greatest homology with FOX-2, FOX-3, and FOX-1
-lactamases (97.4, 96.9, and 95.3% homology), AER14 and CEPS
-lactamases (20, 25) from Aeromonas sobria (76 and 74% homology), and to a lesser extent, CMY-1 (5), MOX-1
(13), and AmpC from P. aeruginosa (73 to 55%
homology) (15) and AmpC (6) from Acinetobacter baumannii (41.6% homology).
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-lactamases. In FOX-4, there are four
novel mutations, at positions 11 (M
T), 43 (A
E), 233 (V
A), and
280 (Y
H). The other FOX-4 amino acid changes are found in positions
previously described for FOX-2 and FOX-3
-lactamases, although in
some positions the amino acid changes were different from those
previously reported.
|
Determination of the
-lactamase kinetic constants
Km, Vmax, and
hydrolysis rate.
The substrate profile of the
-lactamase was
determined with enzyme that was partially purified by G100 Sephadex
(Pharmacia Fine Chemicals AB, Uppsala, Sweden), as previously described
(6). Initial hydrolysis rates were monitored
spectrophotometrically (UVIKON-930) at 25°C in 0.05 M phosphate
buffer (pH 7.4) (14). Kinetic parameters were determined in
duplicate experiments based on the initial steady-state rates at
different substrate concentrations (Lineweaver-Burk transformation).
-lactamase (14). The hydrolysis rates for moxalactam and
imipenem were very low, thus preventing the determination of reliable
Km and Vmax values.
|
Comparative studies.
In recent years, ampC genes
have been found in conjugative plasmids, mostly among K. pneumoniae and E. coli strains but also in
Klebsiella oxytoca, Salmonella enterica serovar
Enteritidis, and Proteus mirabilis (2, 3, 4, 7, 14,
24). The nucleotide and deduced amino acid sequences of the
-lactamase characterized here had very close homology with FOX-1,
FOX-2, and FOX-3
-lactamases (4, 14, 16). Moreover, the
deduced peptide sequence of blaFOX-4 contained
the common conserved motifs found in serine
-lactamases
(11): the SXSK motif of the active site of AmpC, the typical
class C motif YXN, and the KTG domain. In addition, kinetic experiments
performed with the semipurified enzyme revealed typical
cephalosporinase properties, especially a high hydrolytic efficiency of
the enzyme for cefoxitin and cefotaxime. These biochemical results
correlated well with the MICs of
-lactams for the clinical strain
and its transconjugant and transformant. A high cefoxitin hydrolytic
efficiency was also observed with the FOX-1
-lactamase.
-lactamase and all the FOX enzymes had the
greatest homology to the ampC genes from A. sobria; therefore, the ampC
-lactamase genes from
A. sobria may be ancestors of the FOX genes.
However, it is possible that other ampC genes, from
Enterobacteriaceae and other gram-negative rods, which have not yet been sequenced, may assume this role.
Recently, two new blaFOX genes have been
identified (N. D. Hanson, P. Coudron, E. S. Moland, and
C. C. Sanders, Abstr. 39th Intersci. Conf. Antimicrob. Agents
Chemother., abstr. 1458, 1999; G. Jacoby, J. Tran, and M. Nato, Abstr.
39th Intersci. Conf. Antimicrob. Agents Chemother., abstr. 1481, 1999).
Since we do not have enough experimental data to compare them, we named
our enzyme FOX-4.
In summary, we report here the molecular characterization of a novel
-lactamase, FOX-4, from an E. coli clinical strain. To
our knowledge, this is the first report of a cephamycinase in Spain.
Nucleotide sequence accession number. The nucleotide sequence of the blaFOX-4 gene has been given the EMBL database accession no. AJ277535.
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ACKNOWLEDGMENTS |
|---|
We thank L. De Rafael for his critical comments, Dolores Malpica for technical assistance, and Reyes Garcia for secretarial assistance.
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FOOTNOTES |
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* Corresponding author. Mailing address: Servicio de Microbiología, Hospital Ramón y Cajal, Carretera Colmenar Viejo km. 9.100, 28034 Madrid, Spain. Phone: (91) 3368082. Fax: (91) 3368809. E-mail: jmtzbeltran{at}hrc.insalud.es.
Present address: Department of Immunology, Mayo Clinic,
Rochester, MN 55905.
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REFERENCES |
|---|
|
|
|---|
| 1. |
Ambler, R. P.
1980.
The structure of -lactamases.
Philos. Trans. R. Soc. Lond. B
289:321-331 |
| 2. |
Barnaud, G.,
G. Arlet,
C. Verdet,
O. Gaillot,
P. H. Lagrange, and A. Philippon.
1998.
Salmonella enteritidis: AmpC plasmid-mediated inducible -lactamase (DHA-1) with an ampR gene from Morganella morganii.
Antimicrob. Agents Chemother.
42:2352-2358 |
| 3. |
Bauernfeind, A.,
I. Stemplinger,
R. Jungwirth, and H. Giamarellou.
1996.
Characterization of the plasmidic -lactamase CMY-2, which is responsible for cephamycin resistance.
Antimicrob. Agents Chemother.
40:221-224[Abstract].
|
| 4. |
Bauernfeind, A.,
S. Wagner,
R. Jungwirth,
I. Schneider, and D. Meyer.
1997.
A novel class C -lactamase (FOX-2) in Escherichia coli conferring resistance to cephamycins.
Antimicrob. Agents Chemother.
41:2041-2046[Abstract].
|
| 5. |
Bauernfeind, A.,
I. Stemplinger,
R. Jungwirth,
R. Wilhem, and Y. Chong.
1996.
Comparative characterization of the cephamycinase blaCMY-1 gene and its relationship with other -lactamase genes.
Antimicrob. Agents Chemother.
40:1926-1930[Abstract].
|
| 6. |
Bou, G., and J. Martínez-Beltrán.
2000.
Cloning, nucleotide sequencing, and analysis of the gene encoding an AmpC -lactamase in Acinetobacter baumannii.
Antimicrob. Agents Chemother.
44:428-432 |
| 7. |
Bradford, P. A.,
C. Urban,
N. Mariano,
S. J. Projan,
J. J. Rahal, and K. Bush.
1997.
Imipenem resistance in Klebsiella pneumoniae is associated with the combination of ACT-1, a plasmid-mediated AmpC -lactamase, and the loss of an outer membrane protein.
Antimicrob. Agents Chemother.
41:563-569[Abstract].
|
| 8. |
Bush, K.,
G. A. Jacoby, and A. A. Medeiros.
1995.
A functional classification scheme for -lactamases and its correlation with molecular structure.
Antimicrob. Agents Chemother.
39:1211-1233[Medline].
|
| 9. |
Chanal, C.,
D. Sirot,
J. P. Romaszko,
L. Bret, and J. Sirot.
1996.
Survey of prevalence of extended spectrum beta-lactamases among Enterobacteriaceae.
J. Antimicrob. Chemother.
38:127-132 |
| 10. |
De Champs, C.,
D. Sirot,
C. Chanal,
M. C. Poupart,
M. P. Dumas, and J. Sirot.
1991.
Concomitant dissemination of three extended-spectrum beta-lactamases among different Enterobacteriaceae isolated in a French hospital.
J. Antimicrob. Chemother.
27:441-457 |
| 11. |
Ghuysen, J. M.
1991.
Serine -lactamases and penicillin-binding proteins.
Annu. Rev. Microbiol.
45:37-67[CrossRef][Medline].
|
| 12. |
Higgins, D. G.,
A. J. Bleasby, and R. Fuchs.
1992.
Clustal V: improved software for multiple alignments.
CABIOS
8:189-191 |
| 13. |
Horii, T.,
Y. Arakawa,
M. Ohta,
S. Ichiyama,
R. Wacharotayankun, and N. Kato.
1993.
Plasmid-mediated AmpC-type -lactamase isolated from Klebsiella pneumoniae confers resistance to broad-spectrum -lactams, including moxalactam.
Antimicrob. Agents Chemother.
37:984-990 |
| 14. |
Leiza, M. G.,
J. C. Perez-Diaz,
J. Ayala,
J. M. Casellas,
J. Martinez-Beltran,
K. Bush, and F. Baquero.
1994.
Gene sequence and biochemical characterization of FOX-1 from Klebsiella pneumoniae, a new AmpC-type plasmid-mediated -lactamase with two molecular variants.
Antimicrob. Agents Chemother.
38:2150-2157 |
| 15. |
Lodge, J. M.,
S. D. Minchin,
L. Piddock, and J. W. Busby.
1990.
Cloning, sequencing and analysis of the structural gene and regulatory region of the Pseudomonas aeruginosa chromosomal ampC -lactamase.
Biochem. J.
272:627-631[Medline].
|
| 16. |
Marchese, A.,
G. Arlet,
G. C. Schito,
P. H. Lagrange, and A. Philippon.
1998.
Characterization of FOX-3, an AmpC-type plasmid-mediated -lactamase from an Italian isolate of Klebsiella oxytoca.
Antimicrob. Agents Chemother.
42:464-467 |
| 17. |
Matthew, M.,
A. M. Harris,
M. J. Marshall, and G. W. Ross.
1975.
The use of analytical isoelectric focusing for detection and identification of -lactamases.
J. Gen. Microbiol.
88:169-178[Medline].
|
| 18. | National Committee for Clinical Laboratory Standards. 1997. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically. Approved standard M7-A4. National Committee for Clinical Laboratory Standards, Wayne, Pa. |
| 19. |
Parsons, Y.,
R. M. Hall, and H. W. Stokes.
1991.
A new trimethoprim resistance gene, dhfrX, in the In7 integron of plasmid pDGO100.
Antimicrob. Agents Chemother.
35:2436-2439 |
| 20. |
Rasmussen, B. A.,
D. Keeney,
Y. Yang, and K. Bush.
1994.
Cloning and expression of a cloxacillin-hydrolyzing enzyme and a cephalosporinase from Aeromonas sobria AER 14M in Escherichia coli: requirement for an E. coli chromosomal mutation for efficient expression of the class D enzyme.
Antimicrob. Agents Chemother.
38:2078-2085 |
| 21. | Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. |
| 22. |
Sanger, F.,
S. Nicklen, and A. R. Coulson.
1977.
DNA sequencing with chain-terminating inhibitors.
Proc. Natl. Acad. Sci. USA
74:5463-5467 |
| 23. | Spratt, B. G., P. J. Hedge, T. S. Heesen, A. Edelman, and J. K. Broome-Smith. 1986. Kanamycin-resistant vectors that are analogues of plasmids pUC8, pUC9, pEMBL8, and pEMBL9. Gene 41:337-342[CrossRef][Medline]. |
| 24. |
Verdet, C.,
G. Arlet,
S. Ben-Redjeb,
A. Ben-Hassen,
P. H. Lagrange, and A. Philippon.
1998.
Characterisation of CMY-4, an AmpC-type plasmid-mediated -lactamase in a Tunisian clinical isolate of Proteus mirabilis.
FEMS Microbiol. Lett.
169:235-240[Medline].
|
| 25. |
Walsh, T.,
L. Hall,
A. P. MacGowan, and P. M. Bennett.
1995.
Sequence analysis of two chromosomally mediated inducible beta-lactamases from Aeromonas sobria, strain 163a, one a class D penicillinase, the other an AmpC cephalosporinase.
J. Antimicrob. Chemother.
36:41-52 |
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