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Antimicrobial Agents and Chemotherapy, November 2000, p. 3220-3223, Vol. 44, No. 11
0066-4804/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Heterogeneity of AmpC Cephalosporinases of
Hafnia alvei Clinical Isolates Expressing Inducible or
Constitutive Ceftazidime Resistance Phenotypes
Delphine
Girlich,
Thierry
Naas,
Samuel
Bellais,
Laurent
Poirel,
Amal
Karim, and
Patrice
Nordmann*
Service de Bactériologie-Virologie,
Hôpital de Bicêtre, Assistance Publique/Hôpitaux de
Paris, Faculté de Médecine Paris-Sud, 94275 Le
Kremlin-Bicêtre, France
Received 28 February 2000/Returned for modification 16 June
2000/Accepted 18 August 2000
 |
ABSTRACT |
Ten unrelated Hafnia alvei clinical isolates were
grouped according to either their low-level and inducible
cephalosporinase production or their high-level and constitutive
cephalosporinase production phenotype. Their AmpC sequences shared 85 to 100% amino acid identity. The immediate genetic environment of
ampC genes was conserved in H. alvei isolates
but was different from that found in other ampC-possessing
enterobacterial species.
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TEXT |
As with other
cephalosporinase-producing enterobacterial species (3, 15),
Hafnia alvei isolates may be grouped into two 
-lactamase
expression phenotypes, i.e., low-level and inducible cephalosporinase
production and ceftazidime susceptibility on the one hand and
high-level and constitutive cephalosporinase production and
ceftazidime resistance on the other hand (18). We have
previously shown (i) that an ampR gene is located upstream of the ampC gene and that its product acts as a repressor on
the basal level of AmpC biosynthesis and as an activator upon addition of a -lactam inducer and (ii) that transformation of an in
vitro-obtained ceftazidime-resistant H. alvei mutant with
ampD from Escherichia coli restores an inducible
phenotype (5). The chromosome-borne AmpC of H. alvei clinical isolate 1, ACC-2, shares 94% amino acid identity
with ACC-1, a plasmid-borne cephalosporinase from Klebsiella pneumoniae KUS (2, 5).
The aims of this study were to investigate the molecular heterogeneity
of the ampC genes present in several unrelated H. alvei clinical isolates expressing either of the two
cephalosporinase expression phenotypes and to analyze their
immediate genetic environment.
Bacterial strains, plasmid, and pulsed-field gel electrophoresis
(PFGE) analysis.
Ten H. alvei isolates were isolated
from biliary fluids (n = 3), a tracheobronchial
aspirate (n = 1), stool specimens (n = 3), blood cultures (n = 1), and urinary tract
infection specimens (n = 2) of patients hospitalized in
1997 and 1998 at the Hôpital de Bicêtre (Le
Kremlin-Bicêtre, France). These isolates, identified as described
previously (5), were chosen in order to exclude those (i)
isolated from patients hospitalized in the same department during the
same 2-month period and (ii) showing a -lactam resistance phenotype
consistent with that of a penicillinase as deduced from a routine
antibiogram. Plasmid DNA extractions (14) revealed that none
of the isolates harbored the plasmid.
Comparison of H. alvei genomic DNAs was performed by a PFGE
technique as reported previously (14). It showed that
H. alvei isolates were not clonally related (Fig.
1A). A PFGE gel of
SfiI-restricted DNAs of H. alvei isolates 1 to 10 followed by Southern hybridization using an intragenic probe of
blaACC-2 made of a PCR-amplified fragment
(primer A, 5'-GCGTAAAAAAATGCAGAACACC-3'; primer B,
5'-CACTTCCAACGAGCTCAGGATT-3') (5, 16) revealed
blaACC-2-like genes in each isolate and on
separate macrorestriction fragments (Fig. 1B).
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FIG. 1.
PFGE patterns of SfiI-restricted DNAs of 10 H. alvei isolates (A) and their Southern transfer and
hybridization with an internal probe of blaACC-2
from H. alvei 1 (B). Lanes 1 through 10, H. alvei
1 to 10, respectively; lane M, molecular size markers are in kilobase
pairs.
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Susceptibility testing and -lactamase assays.
The MICs of
selected -lactams were determined and -lactamase assays were
performed as described previously (5). Cephalosporinase basal-level and induction experiments allowed the division of H. alvei isolates into two groups: those with low-level and inducible expression of cephalosporinase and those with high-level and
constitutive expression of cephalosporinase (Table
1). The first phenotype (H. alvei isolates 1 to 6) conferred susceptibility to
extended-spectrum cephalosporins and cefoxitin (Table 1) except in
H. alvei 6, for which the MICs of ceftazidime, cefotaxime,
and cefpirome were increased, which might be due to an additional
decrease in the permeability of the outer membrane (Table 1). The
second phenotype (H. alvei isolates 7 to 10) conferred
resistance or intermediate susceptibility to extended-spectrum
cephalosporins, including cefpirome, and susceptibility to cefoxitin
and cefepime. For H. alvei isolates that expressed a
constitutive phenotype, full susceptibility to cefoxitin and the
uncommon decreased susceptibility to cefpirome for an AmpC
cephalosporinase corresponded to the atypical biochemical properties
described for ACC-2 of H. alvei 1 (5).
Isoelectric focusing of cultures of H. alvei clinical
isolates (5) identified pIs ranging from 7.7 to 8.1 (Table
1), which lies close to the pI of 8 determined for ACC-2
(5).
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TABLE 1.
-Lactamase expression phenotypes, MICs of
selected -lactams, pIs, and sequences of -lactamases for 10 H. alvei clinical isolates
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H. alvei clinical isolates 7 and 8, which express a
constitutive phenotype, were transformed with plasmid pNH5, which
contains
the
ampD gene of
E. coli (
5).
Both recombinant strains exhibited
a low-level and inducible
cephalosporinase expression phenotype
(data not shown) associated with
a decrease in
-lactam MICs such
as those of ceftazidime, cefpirome,
and cefepime, being 1, 0.5,
and 0.03 µg/ml, respectively. Thus, the
role of an AmpD-like protein
in the regulation of the expression of
H. alvei cephalosporinase
was confirmed with clinical
isolates as shown with an in vitro-obtained
ceftazidime-resistant
H. alvei 1 mutant (
5).
Sequence analysis of ampCs and their genetic
environment.
Using a set of internal and external primers to the
blaACC-2 sequence (5) (primers A, B,
C [5'-TCTTTTGCATGCTGATTGGC-3'], D
[5'-CCGAGAAATCGGTGACTC-3'], E
[5'-AATCAGGCGGCGATAGCGGATAT-3'], and F
[5'-GCTTCAAGGTGTTCTGCATTT-3']), PCR amplification products were obtained by using genomic DNAs of H. alvei 2 to 10 as
templates. The products were sequenced and analyzed as described
previously (5). AmpC amino acid identities among H. alvei isolates ranged from 85 to 100%, allowing us to divide them
into three subgroups (Fig. 2): point
mutant derivatives of ACC-1, namely, ACC-1a (H. alvei 2, 6, and 10), ACC-1b (H. alvei 4), ACC-1c (H. alvei
7), and ACC-1d (H. alvei 9); ACC-2, previously identified
from H. alvei 1, which possesses 94% amino acid identity
with ACC-1 (5); and ACC-3 (H. alvei 3 and 8),
which possesses 87% amino acid identity with ACC-1, and a point mutant
derivative, ACC-3a (H. alvei 5). The amino acid identity
between ACC-2 and ACC-3 was 85%. The ACC-1-like enzymes clustered in
the same subgroup as that of enterobacterial cephalosporinase (data not
shown). As exemplified by ACC-1a found in H. alvei 2, 6, and
10, no correlation was established between the AmpC sequence and its
cephalosporinase phenotype. Moreover, relative activities of restricted
and extended-spectrum cephalosporins were similar for each H. alvei cephalosporinase, whatever the expression phenotype was
(data not shown).
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FIG. 2.
Amino acid sequence comparison of the chromosome-borne
AmpCs from H. alvei isolates with the plasmid-mediated
cephalosporinase ACC-1 from K. pneumoniae KUS (2)
and the previously identified chromosome-borne ACC-2 from H. alvei 1 (5). Dashes indicate identical amino acids.
ACC-1a was from H. alvei 2, 6, and 10; ACC-1b was from
H. alvei 4, ACC-1c was from H. alvei 7, ACC-1d
was from H. alvei 9, ACC-3 was from H. alvei 3 and 8, and ACC-3a was from H. alvei 5. Numbering is
according to that of the ACC-1 sequence (2), to which 4 amino acids have been added at its N terminus in order to match the
consensus sequence derived from the analysis of the cephalosporinase
sequences.
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Amino acid changes in
H. alvei cephalosporinases occurred
throughout the entire cephalosporinase sequence, as has been described
for cephalosporinases of
Citrobacter freundii,
Enterobacter cloacae,
and
Pseudomonas aeruginosa
(
4,
8,
17) (Fig.
2). However,
none of these amino acid
changes were located in the putative
active site of the
H. alvei cephalosporinase, unlike those identified
in the active
sites of some cephalosporinases of
E. cloacae,
C. freundii, and
Serratia marcescens that confer an
extended hydrolytic
profile (
7,
9,
10-13,
19).
The genetic variability of AmpC sequences in
H. alvei
corresponded to that found in
C. freundii; the amino acid
identity between
AmpCs of
C. freundii I113 and 0S60 was only
82% (
8). On the
other hand, the sequence identity of AmpCs
of three
E. cloacae isolates was higher, since they differed
only by point mutations
at eight positions (
4). In
P. aeruginosa, the sequence identity
of AmpCs is much higher than in
H. alvei (99.6 versus 85%) (
17).
An
ampR gene had been identified upstream of the
ampC gene in
H. alvei 1 (
5). A further
540 bp upstream of this
ampR gene,
part of an open reading
frame, the deduced protein of which shared
74% identity with the
glutathione reductase of
E. coli, was found
in
H. alvei 1 (
5,
6). Using several sets of primers (primer
1 [5'-GTACGCACAGTAGCAGGATC-3'], primer 2 [5'-GCTCTTCGCGCATTTGAAGC-3'],
primer 3 [5'-CCGCCAATTGCGAGATAGTC-3'], and primer 4 [5'-GGTGTTCTGCATTTTTTTACGC-3'])
(Fig.
3), PCR amplifications were attempted
using
H. alvei 2 to
10 DNAs as templates. Similar-sized PCR
fragments indicated that
ampR-like and glutathione
reductase-like genes were present in
each
H. alvei isolate
in the same positions relative to that of
the
ampC gene
(Fig.
3). This result indicated that the genetic
environment of
H. alvei ampC genes was conserved, as with other
enterobacterial
ampC genes (Fig.
3). However, the genetic
environments
of the
ampC genes differ from one
enterobacterial species to the
next (Fig.
3).
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FIG. 3.
Comparison of the sequences surrounding ampC
in several enterobacterial species. The positions and directions of the
fumarate operon (frdABCD), hybF (hydrogenase),
orf-1 (unknown function), gro (glutathione
reductase), ampC, and ampR genes are indicated
with arrows. The locations of the putative promoters (p) and the
primers used in this study (small arrows and numbers) are also
indicated.
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Conclusion.
This work further underlines the relationship
between the plasmid-borne cephalosporinase ACC-1 and the
chromosome-borne point mutant ACC-1 derivatives of several unrelated
H. alvei isolates. Similarly, 100% amino acid identity is
known for the plasmid-mediated cephalosporinase DHA-1 of
Salmonella enteritidis and the chromosome-borne cephalosporinase of Morganella morganii (1, 15).
The heterogeneity of AmpC sequences of H. alvei may explain
why several plasmid-mediated cephalosporinases are not just point
mutant derivatives of known chromosome-borne cephalosporinases and why
they appear to be distantly related or even unrelated to
chromosome-borne cephalosporinases.
Nucleotide sequence accession number.
The nucleotide sequences
data reported in this paper will appear in the GenBank and EMBL
nucleotide databases under the accession no. AF180953 to AF180961.
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ACKNOWLEDGMENTS |
This work was funded by the Ministère de l'Education
Nationale et de la Recherche, Université Paris XI, Faculté
de Médecine Paris Sud (UPRES, grant JE-2227), and the network Les
-lactamases: de l'observation clinique à la structure, Paris, France.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Service de
Bactériologie-Virologie, Hôpital de Bicêtre, 78 rue
du Général Leclerc, 94275 Le Kremlin-Bicêtre Cedex,
France. Phone: 33 1 45 21 36 32. Fax: 33 1 45 21 63 40. E-mail:
nordmann.patrice{at}bct.ap-hop-paris.fr.
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Antimicrobial Agents and Chemotherapy, November 2000, p. 3220-3223, Vol. 44, No. 11
0066-4804/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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