Download groups, blaOXY-1 and blaOXY-2. Klebsiella oxytoca are divided into

Chromosomal beta-lactamase genes of
Klebsiella oxytoca are divided into two main
groups, blaOXY-1 and blaOXY-2.
B Fournier, P H Roy, P H Lagrange and A Philippon
Antimicrob. Agents Chemother. 1996, 40(2):454.
These include:
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ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Feb. 1996, p. 454–459
0066-4804/96/$04.0010
Copyright q 1996, American Society for Microbiology
Vol. 40, No. 2
Chromosomal b-Lactamase Genes of Klebsiella oxytoca Are
Divided into Two Main Groups, blaOXY-1 and blaOXY-2
BÉNÉDICTE FOURNIER,1,2* PAUL H. ROY,2,3 PHILIPPE H. LAGRANGE,1
AND
ALAIN PHILIPPON1
Laboratoire de Microbiologie, Hôpital Saint-Louis, Université Paris VII, 75010 Paris, France,1 and Laboratoire et
service d’Infectiologie, Centre Hospitalier de L’Université Laval,2 and Département de Biochimie,
Faculté des Sciences et de Génie, Université Laval,3 Sainte-Foy Québec, Canada
Received 3 July 1995/Returned for modification 22 October 1995/Accepted 11 December 1995
There are a variety of mechanisms for resistance to b-lactams. One of the most common is production of b-lactamases,
enzymes that hydrolyze b-lactams. b-Lactamases are classified
into four classes based on substrate affinity and amino acid
sequence. Two of these classes are much more common than
the others; classes A and C. Class A includes various plasmidmediated b-lactamases (TEM-1, SHV-1), the plasmid-mediated extended-spectrum b-lactamases derived from TEM or
SHV, and some chromosomally encoded b-lactamases, such as
that produced by Klebsiella pneumoniae (2). These enzymes
preferentially inactivate penicillins (e.g., amoxicillin and ticarcillin) and are thus penicillinases. Class C contains chromosomally encoded b-lactamases from bacteria including Escherichia coli (21) and Enterobacter cloacae (22). These enzymes
inactivate penicillins but are more active against cephalosporins.
A b-lactamase gene from Klebsiella oxytoca (blaOXY-1) has
been sequenced (3). This b-lactamase belongs to class A (3).
However, it was included in the functional group of extendedspectrum b-lactamases (3, 7, 8) because of its ability to hydrolyze not only penicillins but also cephalosporins, including cefuroxime and ceftriaxone as well as aztreonam. This enzyme has
been investigated to identify amino acids involved in interactions
with its various substrates. For example, the threonine residue at
position 140 may be involved in cefotaxime recognition (38).
We cloned and sequenced the b-lactamase gene of the wildtype K. oxytoca SL911. The sequence of this new gene
(blaOXY-2) was used to design and synthesize a DNA probe. A
colony hybridization study of K. oxytoca strains of various origins revealed two hybridization groups, OXY-1 and OXY-2.
By analysis of isoelectric points, we confirmed that there were
two groups of b-lactamases.
MATERIALS AND METHODS
Bacterial strains and plasmids. The strains and plasmids used in this study are
listed in Table 1.
Strain SL911 was identified with Biotype 99-carbon source strips (bioMérieux)
to confirm its identification as K. oxytoca as opposed to Klebsiella planticola,
which is also indole positive.
K. oxytoca strains were recovered in hospitals in various countries (France,
Germany, Spain, Switzerland, United Kingdom, and the United States). They
were identified with the API 20E system (bioMérieux) and by two carbon substrate assimilation tests (histamine and ethanolamine) as described previously
(31). Clinical strains other than K. oxytoca are listed in Table 2. They were
isolated at St.-Louis Hospital (Paris, France) between 1991 and 1993. They were
biotyped with the API 20E system, and the b-lactamase type was deduced from
the resistance pattern (28) (disk diffusion method with Mueller-Hinton agar).
Plasmid-mediated b-lactamases were previously characterized by determination
of isoelectric points and by hybridization with intragenic probes specific for
TEM, SHV, and CARB b-lactamases (4).
Determination of susceptibility pattern. MICs of K. oxytoca SL911 were determined by an agar dilution method as previously described (15). The resistance
phenotype of each K. oxytoca strain was also determined by the disk diffusion
method with Mueller-Hinton agar (11). The inhibition zone diameters and corresponding MIC breakpoints used for assessing susceptibility, respectively, were
as follows: for cefuroxime, $22 mm and #8 mg/ml; and for aztreonam, $23 mm
and #4 mg/ml (11).
Cloning. Extraction of chromosomal DNA from K. oxytoca strains and transformation of plasmid DNA were performed as described previously (15).
Chromosomal DNA from K. oxytoca SL911 was completely digested with
EcoRI and ligated into the EcoRI site in pBGS18. The ligated DNA was introduced into E. coli JM101 by transformation. The transformants were selected on
Mueller-Hinton agar supplemented with ticarcillin (13 mg/ml) and kanamycin
(50 mg/ml). A recombinant plasmid DNA (pBOF-15) contained about 5.2 kb of
genomic K. oxytoca DNA (Table 1). pBOF-15 DNA was digested with PstI and
introduced into pBGS18. The newly created recombinant plasmid (pBOF-151)
contained a 1.1-kb EcoRI-PstI genomic DNA fragment of K. oxytoca (Table 1)
and carried the intact gene.
Genomic DNA from K. oxytoca was completely digested by PstI and ligated
into the polylinker site of the plasmid pBGS18. The newly created plasmid
(pBOF-16) contained a 1.5-kb PstI genomic fragment (Table 1). This DNA
fragment contained about 400 bp, upstream of the EcoRI site, of pBOF-151.
Sequencing and protein analysis. The b-lactamase gene and its 59-flanking
region were sequenced with the T7 sequencing kit (Pharmacia) from both
strands.
The nucleotide and peptide sequences were analyzed with Genetics Computer
Group software on a Sun computer. The DNA and protein sequences of other
b-lactamases were from the EMBL and Swiss-Prot databases. The Genetics
* Corresponding author. Mailing address: Laboratoire et service
d’Infectiologie, Centre Hospitalier de L’Université Laval, 2705 Blvd.
Laurier, Sainte-Foy Québec G1V4G2, Canada. Phone: (1) 418 654
2705. Fax: (1) 418 654 2715.
454
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The chromosomally encoded b-lactamase gene (blaOXY-2) of the wild-type Klebsiella oxytoca SL911 was cloned
and sequenced. Its nucleotide sequence similarity with the previously sequenced K. oxytoca b-lactamase gene
(blaOXY-1) (Y. Arakawa, M. Ohta, N. Kido, M. Mori, H. Ito, T. Komatsu, Y. Fujii, and N. Kato, Antimicrob.
Agents Chemother. 33:63–70, 1989) is 87.3%, and its amino acid similarity is 89.7%. This group of K. oxytoca
b-lactamases is related to chromosomal b-lactamases of Citrobacter diversus, Proteus vulgaris, and Yersinia
enterocolitica and to the plasmid-mediated extended-spectrum b-lactamases MEN-1 and Toho-1. By colony
hybridization with 86 strains susceptible and resistant to aztreonam, isolated in six countries, K. oxytoca
b-lactamase genes hybridized with either a specific blaOXY-1 DNA probe (668 bp) or a blaOXY-2 DNA probe (723
bp). Thus, b-lactamase genes could be divided into two groups: blaOXY-1 (47% of the strains) and blaOXY-2 (53%
of the strains). A study of isoelectric points confirmed the great variability reported in the literature. However,
the two b-lactamase groups were each represented by four different pIs: for OXY-2, 5.2, 5.7, 6.4, and 6.8, with
the 5.2 form representing 59% of all OXY-2 enzymes, and for OXY-1, 7.1, 7.5, 8.2, and 8.8, with the 7.5 form
representing 88% of all OXY-1 enzymes.
b-LACTAMASE GENES OF KLEBSIELLA OXYTOCA
VOL. 40, 1996
455
TABLE 1. Strains and plasmids used for cloning the b-lactamase gene of SL911
Genotype or relevant characteristicsa
Strain or plasmid
Strains
E. coli JM101
K. oxytoca SL911
Plasmids
pBGS18
pBOF-15
pBOF-151
pBOF-16
supE thi D(lac-proAB) F9[traD36 proAB1 lacIq lacZDM15]
Clinical isolate (1991)
46
Saint-Louis Hospital
Cloning vector, Kmr
5.2-kb EcoRI chromosomal fragment from SL911 that encodes blaOXY-2
cloned in pBGS18, Kmr Apr
1.1-kb EcoRI-PstI fragment from pBOF-15 subcloned in pBGS18, Kmr Apr
1.5-kb PstI chromosomal fragment from SL911 that encodes blaOXY-2 cloned
in pBGS18 (contains 400 bp of DNA upstream of the EcoRI site), Kmr Apr
42
This work
This work
This work
Km, kanamycin; Ap, ampicillin.
Computer Group programs FASTA and TFASTA were used for b-lactamase
sequence similarity searches (36). Multiple alignment of deduced peptide sequences was carried out with ClustalW software (European Molecular Biology
Laboratory, Heidelberg, Germany). This method uses the algorithm of Myers
and Miller (32), a memory-efficient variation of Gotoh’s algorithm (17). The
protein weight matrix used was that of Henikoff (Blosum series). To show the
relatedness of these b-lactamases, an alignment with truncated proteins, carried
out with ClustalW, was constructed. The C- and N-terminal portions were deleted so that compared sequences extended from residues 25 to 285 (ABL [class
A b-lactamase] consensus numbering scheme [1]), because the leader peptides of
the b-lactamases MEN-1 and D488 are unknown. A phylogenetic tree was
derived from this alignment by using the ClustalW software with Kimura’s
correction (26) and the neighbor-joining method of Saitou and Nei (39) (this tree
is unrooted).
Primer extension analysis. SL9111 is an overproducing mutant of SL911
produced as previously described (15). It was used because it contains very large
amounts of blaOXY mRNA. Total cellular RNA extraction by the hot-phenol
procedure and primer extension analysis were performed as described previously
(16). The primer P used for primer extension analysis was 59-d[GCA TCG GTA
CTG GCC CA]-39 (position 403 in the coordinates in Fig. 1 corresponds to the
first [59] base of the oligonucleotide).
Preparation of DNA probes. DNA probes were prepared by PCR. Primers C,
TABLE 2. Gram-negative rod-shaped bacteria and b-lactamases used for hybridization tests
Strain no.
1
2
3–5
6
7, 8
9–11
12, 13
14–23
24, 25
26, 27
28, 29
30, 31
32, 33
34, 35
36
37
38
39
40
41
42
43
44
45
46
47
48, 49
50
51, 52
53–55
56–58
59–61
a
Species
Klebsiella oxytoca SL781
Klebsiella oxytoca SL911
Klebsiella pneumoniae
Citrobacter amalonaticus VAN
Citrobacter diversus
Escherichia hermannii
Serratia marcescens
Enterobacter cloacae
Enterobacter aerogenes
Proteus vulgaris
Proteus rettgeri
Morganella morganii
Citrobacter freundii
Providencia stuartii
Pseudomonas aeruginosa PAO303
Escherichia coli K-12 J53-2
Escherichia coli K-12 J53-2
Escherichia coli K-12 J53-2
Escherichia coli K-12 J53-2
Pseudomonas aeruginosa Dalgleish
Pseudomonas aeruginosa PAO303
Escherichia coli K-12 J53-2
Escherichia coli K-12 J53-2
Escherichia coli K-12 J53-2
Escherichia coli K-12 J53-2
Escherichia coli E360
Proteus mirabilis
Shigella sonnei
Salmonella typhimurium
Yersinia enterocolitica
Klebsiella planticola
Klebsiella terrigena
Localization
of genetic
determinanta
Chr
Chr
Chr
Chr
Chr
Chr
Chr
Chr
Chr
Chr
Chr
Chr
Chr
Chr
pPIP1100
RP4
p453
pUD21
pMG19
pUD12
R151
RGN238
R46
R55
P
Chr
b-Lactamase:
Enzyme
OXY-1
OXY-2
TEM-1
TEM-2
SHV-1
SHV-4
PSE-4
CARB-4
OXA-10 (PSE-2)
OXA-1
OXA-2
OXA-3
MEN-1
Type(s)b
ESB
ESB
PHB
PHB
PHB
PHB
CPIC
CPIC
CPIC
CIC
CPIC
CPIC
CPIC
CPIC
CPIC
BSB
BSB
BSB
ESB
CaHB
CaHB
ClHB
ClHB
ClHB
ClHB
ESB
ND
ND
ND
CIC 1 PHB
PHB
ND
Chr, chromosomal; P, plasmid.
Enzyme type according to the classification of Bush et al. (8). Abbreviations: ESB, extended-spectrum b-lactamase inhibited by clavulanic acid (group 2be); PHB,
penicillin-hydrolyzing enzyme inhibited by clavulanic acid (group 2a); CPIC, cephalosporin-hydrolyzing b-lactamase poorly inhibited by clavulanate (group 1); CIC,
cephalosporinase inhibited by clavulanic acid (group 2e); BSB, broad-spectrum b-lactamase inhibited by clavulanic acid (group 2b); CaHB, carbenicillin-hydrolyzing
b-lactamase inhibited by clavulanic acid (group 2c); ClHB, cloxacillin-hydrolyzing b-lactamase (group 2d); ND, not detected.
b
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a
Source or reference
456
FOURNIER ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
RESULTS
MICs. The K. oxytoca strain SL911 was moderately resistant
to penicillins (MIC of amoxicillin, 64 mg/ml; MIC of ticarcillin,
64 mg/ml; and MIC of piperacillin, 4 mg/ml) and susceptible to
cephalosporins (cephalothin [2 mg/ml], cefuroxime and cefoxitin [1 mg/ml], cefotaxime and ceftriaxone [#0.0015 mg/ml],
and ceftazidime and moxalactam [#0.06 mg/ml]), aztreonam
(0.12 mg/ml), and imipenem (0.12 mg/ml).
FIG. 2. Localization of the blaOXY-2 transcription initiation site by primer
extension analysis. Lane 1, K. oxytoca SL9111. Lanes A, C, G, and T represent a
sequencing ladder of the coding strand of the strain SL911 with the same primer,
P. The sequences shown on the right correspond to the coding strands (capital
letters) and deduced noncoding strands (lowercase letters). The position of
transcript initiation is indicated by an arrow. The 210 sequence consensus of the
promoter is boxed.
Cloning and DNA sequencing. The complete nucleotide sequence of the K. oxytoca b-lactamase gene and the deduced
amino acid sequence are shown in Fig. 1. There was an 870nucleotide open reading frame encoding a protein of 290
amino acid residues. The initiation codon (ATG) was preceded
by a promoter, which was mapped by primer extension. The
transcriptional start point of the b-lactamase gene is a C 26
nucleotides upstream of the ATG translational start site and 6
nucleotides downstream of the 210 consensus sequence of the
promoter (Fig. 2). The nucleotide similarity between the cod-
FIG. 1. Nucleotide sequence of the blaOXY-2 region from the wild-type K.
oxytoca SL911 cloned in pBOF-151 and pBOF-16. The deduced polypeptide
sequence is indicated. The numbers at the end of each line refer to nucleotide
positions, and those in brackets are polypeptide positions. The stop codon is
indicated by asterisks, and the consensus sequences of the promoter are underlined once. The transcriptional start site is shown in boldface type and labeled
11. The ribosome-binding site is indicated by double underlining. The EcoRI
site is underlined with a dotted line.
FIG. 3. Alignment of the amino acid sequence of the b-lactamase from
SL911 with that of the b-lactamase from K. oxytoca E23004 (3). The asterisks
indicate the residues highly conserved in the consensus sequence of class A
b-lactamases (1). The numbering is that of Ambler et al. (1). Boxes I to VII are
conserved residues described by Joris et al. (23). The SDN loop is also boxed
(20). Vertical bars indicate amino acid identities between the two b-lactamases.
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59-d(GCG TAG CGC TGA TTA ACA CG)-39, and D, 59-d(CCT GCT GCG
GCT GGG TAA AA)-39, were used for the preparation of blaOXY-1 probe, and
primer L, 59-d(CAG ATC TCG AGA AGC GTT CA)-39 (position 421 [see Fig.
1]), and primer M, 59-d(ACC TCT TTG CGG TTT TTC GC)-39 (position 1144
[see Fig. 1]), were used for the preparation of the blaOXY-2 probe. PCR was
performed with 35 cycles of denaturation at 948C for 1 min, annealing at 608C
(for blaOXY-1) or 558C (for blaOXY-2) for 1 min, and extension at 728C for 1 min.
The PCR product was labeled with digoxigenin with the random-primed DNA
labeling kit (Boehringer Mannheim).
Hybridization tests. Membranes were inoculated with colonies and treated as
previously described (4). Hybridization and signal detection were performed
following the instructions of the manufacturer (Boehringer Mannheim), with
stringent washes of the filters after hybridization (twice for 15 min in 23 SSC–
0.1% SDS at room temperature and twice for 15 min in 0.13 SSC–0.1% SDS at
688C [13 SSC is 0.15 M NaCl plus 0.015 M sodium citrate]).
Two type strains were used to verify specificity of hybridization: SL781 (pI,
7.5), specific for blaOXY-1 (15), and SL911 (pI, 5.2), specific for blaOXY-2.
Determination of isoelectric points. Isoelectric points were determined by
analytical isoelectric focusing as previously reported (15).
Nucleotide sequence accession number. The nucleotide sequence data reported in this paper will appear in the EMBL-GenBank-DDBJ nucleotide sequence data libraries under accession no. Z49084.
VOL. 40, 1996
b-LACTAMASE GENES OF KLEBSIELLA OXYTOCA
457
FIG. 4. Phylogenetic tree for 28 class A b-lactamases. Branch lengths are to
scale and numbers along each branch are percentage divergences. The underlined percentages at the nodes refer to the number of times a grouping occurs in
100 sample trees (,50 indicates the uncertainty of nodes with bootstrap values
of less than 50%). b-Lactamase abbreviations (and references): OHIO-1 from
Enterobacteriaceae (41); SHV-1 from Klebsiella sp. (30); LEN-1 from K. pneumoniae (2); TEM-1 from E. coli (43); CARB-3 from Pseudomonas aeruginosa
(27); NMCA from E. cloacae (33); Sme-1 from S. marcescens (34); SL911 from
K. oxytoca (OXY-2 type); D488 from K. oxytoca (OXY-2 type) (38); E23004 from
K. oxytoca (OXY-1 type) (3); CDIV from C. diversus (37); MEN-1 from E. coli
(5); Toho-1 from E. coli (19); PVUL from P. vulgaris (44); YENT from Y.
enterocolitica (40); LENZ from L. enzymogenes (6); MFOR from M. fortuitum
(45); SLAV from S. lavendulae (14); SALB from S. albus (13); BMYC from
Bacillus mycoides (Swiss-Prot accession no. P28018); BCERI, b-lactamase I from
Bacillus cereus 569/H (29); BAMY from B. amyloliquefaciens (EMBL accession
no., Z35653); BLIP from Bacillus licheniformis (35); BCERIII, b-lactamase III
from B. cereus 569/H (18); NLACT from Nocardia lactamdurans (12); SBADI
from Streptomyces badius (14); ROB-1 from Haemophilus influenzae (25); and
PC-1 from S. aureus (9).
Downloaded from http://aac.asm.org/ on April 30, 2014 by PENN STATE UNIV
ing regions of the previously sequenced K. oxytoca b-lactamase
gene blaOXY-1 (3, 16) and this newly described b-lactamase
gene of K. oxytoca, blaOXY-2, was 87.3%. The amino acid similarity between the two deduced b-lactamases was 89.7% (Fig.
3). The seven canonical boxes typical of the class A b-lactamases
(23) and the SDN loop (20) were found in the b-lactamases of the
strain SL911 as well as in that of the strain E23004 (Fig. 3).
Similarity with other b-lactamases. The percentage similarities were determined for entire proteins. The OXY b-lactamases belong to class A as previously reported (3). The blaOXY
group includes blaOXY-1 types such as E23004 (3) or SL781
(16) and blaOXY-2 types such as D488 (38) or SL911. This
group is similar to chromosomal b-lactamases of Citrobacter
diversus, Proteus vulgaris, and Yersinia enterocolitica (similarities to the SL911 b-lactamase, 73, 67, and 57%, respectively)
and to the plasmid-mediated extended-spectrum b-lactamases
MEN-1 and Toho-1 (similarity of each to the SL911 b-lactamase, 73%). blaOXY enzymes are also similar to the chromosomal carbapenem-hydrolyzing b-lactamases Sme-1 from Serratia marcescens (47%) and NMC-A from E. cloacae (47%)
and to the chromosomal b-lactamases of Lysobacter enzymogenes (a nonpathogenic soil bacterium) (45%) and Bacillus
amyloliquefaciens (44%). Phylogenetic-tree analysis (Fig. 4)
suggested that the OXY b-lactamases belong to a subgroup
containing chromosomal b-lactamases of the family Enterobacteriaceae (C. diversus, P. vulgaris, Y. enterocolitica) and the
plasmid-mediated MEN-1 and Toho-1 from E. coli. This group
is close to another group of b-lactamases from L. enzymogenes,
Mycobacterium fortuitum, Streptomyces albus, and Streptomyces
lavendulae. It is more distantly related to the gram-negative
family of mainly plasmid-mediated b-lactamases of the SHV,
TEM, and CARB types and the chromosomal b-lactamase
from K. pneumoniae LEN-1.
Hybridization tests. A 668-bp intragenic DNA probe specific for the blaOXY-1 gene and a 723-bp intragenic DNA probe
specific for the blaOXY-2 gene were used for hybridization tests.
Eighty-six K. oxytoca strains from six countries (39 strains susceptible and 47 strains resistant to aztreonam) were examined
(Fig. 5A). Under high-stringency conditions, 40 strains (20
aztreonam-susceptible strains and 20 resistant strains) hybridized strongly with the blaOXY-1 probe and weakly with the
blaOXY-2 probe. Forty-five strains (18 aztreonam-susceptible
strains and 27 resistant strains) hybridized strongly with the
blaOXY-2 probe and weakly with the other probe. One strain
(no. 25) (Fig. 5A) hybridized strongly with both probes. However,
this strain was subsequently demonstrated to be a mixture of two
different strains. After separation and isolation of the two colonies, one strain hybridized with only the blaOXY-1 probe and the
other hybridized with only the blaOXY-2 probe (data not shown).
The two probes were tested for hybridization against a panel
of 59 gram-negative bacteria harboring different types of b-lactamase (Fig. 5B). The bacteria and b-lactamases are listed in
Table 2. No hybridization signal was observed with any of the
b-lactamase-producing strains tested. The two probes were
thus specific for the K. oxytoca genes.
Isoelectric points. The b-lactamase produced by K. oxytoca
458
ANTIMICROB. AGENTS CHEMOTHER.
FOURNIER ET AL.
DISCUSSION
SL911 had an isoelectric point of 5.2. The pIs of the enzymes
produced by each of 71 K. oxytoca strains (26 strains susceptible to aztreonam and 45 resistant strains; 34 blaOXY-1 type and
37 blaOXY-2 type) were determined. The different isoelectric
points are listed in Table 3.
The blaOXY-2 types gave four different pIs: 5.2, 5.7, 6.4, and
6.8, with 59% of OXY-2 enzymes having a pI of 5.2. The
blaOXY-1 type gave four different pIs: 7.1, 7.5, 8.2, and 8.8, with
88% of OXY-1 enzymes having a pI 7.5.
TABLE 3. Isoelectric points of b-lactamases from K. oxytoca
according to b-lactamase gene type and aztreonam resistance
phenotype
b-Lactamase
gene
pI
No. of strains with
phenotype
Susceptible
Resistant
Total no. of
strains (%)
blaOXY-2
5.2
5.7
6.4
6.8
8
2
2
0
14
5
2
4
22 (31)
7 (10)
4 (6)
4 (6)
blaOXY-1
7.1
7.5
8.2
8.8
0
13
0
1
1
17
2
0
1 (1)
30 (42)
2 (3)
1 (1)
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FIG. 5. Colony hybridization of various strains with probes specific for
blaOXY-1 and blaOXY-2 DNA. (1) Hybridization with blaOXY-1 probe; (2) Hybridization with blaOXY-2. (A) K. oxytoca strains. No. 1 to 42, clinical strains; no.
43, strain SL781 specific for blaOXY-1; no. 44 and 45, strains SL902 and SL911
specific for blaOXY-2; 46, E. coli. (B) Bacterium strains harboring various b-lactamases. No. 1, strain SL781 specific for blaOXY-1; no. 2, strain SL901 specific for
blaOXY-2; no. 3 to 61 are listed in Table 2.
We cloned and sequenced the b-lactamase gene from the
wild-type K. oxytoca SL911. This is the second type of b-lactamase gene identified in K. oxytoca (blaOXY-2). This gene is very
similar to the first type sequenced (blaOXY-1 [3]), with 87%
nucleotide similarity. The promoter 235 and 210 consensus
sequences are identical, but the DNA flanking these sequences
differs. Some clinical strains harboring the b-lactamase OXY-2
were aztreonam resistant (Table 3). A mutation in the 210
consensus sequence of the promoter of the blaOXY-1 gene has
been shown to lead to resistance to aztreonam and broadspectrum cephalosporins (16). The blaOXY-2 promoter could
therefore presumably acquire a similar mutation, and the
strain will probably present the same overproduction.
The K. oxytoca b-lactamases are most similar to chromosomal b-lactamases of gram-negative bacteria (C. diversus, P.
vulgaris, and Y. enterocolitica). This is consistent with recent
reports (8, 34). MEN-1 (5) and Toho-1 (19), two plasmidmediated extended-spectrum b-lactamases, are probably derived from the chromosomal b-lactamase of K. oxytoca. This
group is closely related to another group of b-lactamases carrying two recently identified b-lactamases (those produced by
L. enzymogenes and M. fortuitum) and two b-lactamases from
actinomycetes (S. albus and S. lavendulae). Seoane and Garcia
Lobo (40) described two main groups of class A b-lactamases:
the chromosomal branch, which contains Bacillus and Streptomyces enzymes as well as those of K. oxytoca, Y. enterocolitica,
and Staphylococcus aureus PC-1, and the transposon branch,
which contains TEM-, SHV-, and CARB-type enzymes and
LEN-1 enzymes. Each group contains b-lactamases characterized by amino acids conserved at particular positions. In the
transposon branch, all b-lactamases are plasmid-mediated except the LEN-1 enzyme of K. pneumoniae. The LEN-1 enzyme
could be the result of a recent integration of a plasmid-mediated b-lactamase gene (OHIO-1 or SHV-1 type) on the chromosome (40). This could explain the great difference between
the amino acid sequences of LEN-1 and K. oxytoca b-lactamases (only 37% identity with the b-lactamase produced by the
strain SL911) (3, 24, 38). The presence of MEN-1 and Toho-1
in the chromosomal group could be explained by the incorporation of the blaOXY gene into a transposon after the separation of the two branches during evolution (40).
Each K. oxytoca strain hybridized with either the blaOXY-1
probe or the blaOXY-2 probe. The pIs of K. oxytoca b-lactamases are particularly widely distributed (Table 3). We found all
the pIs reported in the literature (10). For each group of
b-lactamases, four isoelectric points were obtained; those for
OXY-1 ranged from 7.1 to 8.8, and those for OXY-2 ranged
from 5.2 to 6.8. The b-lactamase produced by K. oxytoca
E23004 has an isoelectric point of 7.4 (3) and belongs to the
OXY-1 group. b-Lactamases from strain D488, sequenced by
Reynaud et al. (38), and strain SL911 belong to the OXY-2
group and have isoelectric points of 6.3 and 5.2, respectively.
There are only three amino acid differences between the D488
b-lactamase and the SL911 b-lactamase: Asn and Asp, position
199; Val and Ala, position 225, and Asn and Asp, position 255
(Fig. 1). In each group, one form of b-lactamase is more
frequent than all the others combined. It is probable that only
a few amino acid substitutions determine the pI differences
observed for the other forms of b-lactamases.
In conclusion, b-lactamase genes in K. oxytoca are divided
into two main groups, blaOXY-1 and blaOXY-2. Each group of
b-lactamases is represented by at least four different forms
according to their isoelectric points.
b-LACTAMASE GENES OF KLEBSIELLA OXYTOCA
VOL. 40, 1996
ACKNOWLEDGMENTS
We thank P. C. Appelbaum (Hershey Medical Center, Hershey,
Pa.), F. Baquero (Hospital Ramon y Cajal, Madrid, Spain), A. Bauernfeind (Max von Pettenkofer-Institut, Munich, Germany), F. Goldstein
(Hôpital Saint-Joseph, Paris, France), V. Jarlier (Hôpital de la Pitié,
Paris, France), D. M. Livermore (London Hospital Medical College,
London, United Kingdom), F. C. Tenover (National Center for Infectious Diseases, CDC, Atlanta, Ga.), and R.L. Then (Hoffmann-La
Roche, Basel, Switzerland) for the gifts of K. oxytoca strains. We thank
P. A. D. Grimont (Institut Pasteur, Paris, France) for identifying the
strain SL911. We also acknowledge A. Gravel (Université Laval, Québec,
Canada) for helping one of us (B.F.) use programs for sequence analysis.
B.F. was supported by a grant from the Ministère de la Recherche
et de l’Espace.
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