β-Lactamases of Kluyvera ascorbata, Probable Progenitors of Some Plasmid-Encoded CTX-M Types

Christel Humeniuk; Guillaume Arlet; Valerie Gautier; Patrick Grimont; Roger Labia; Alain Philippon

doi:10.1128/AAC.46.9.3045-3049.2002

DOI: 10.1128/AAC.46.9.3045-3049.2002

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FIG. 1.
Genetic environment of bla_KLUA-1 gene encoding the β-lactamase produced by K. ascorbata strain CIP 82.95T. The regions surrounding the chromosomal bla_KLUA-1 gene and the sequences adjacent to the plasmidic genes encoding β-lactamases CTX-M-2, CTX-M-4, CTX-M-5, CTX-M-6, CTX-M-7, and Toho-1 (2, 10, 11, 23) were compared. The RTR sequence and tnpA correspond to the right terminal repeat and transposase A, respectively, which are typical of ISEcp1.

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TABLE 1.

MICs of 12 β-lactams for the 12 clinical K. ascorbata isolates, recipient strain E. coli XL-1, and the E. coli XL-1 transformant producing KLUA-1

β-Lactam(s)^a	No. of isolates for which MIC (μg/ml) was:													MIC (μg/ml) for E. coli XL-1 recipient
β-Lactam(s)^a	<0.03	0.06	0.125	0.25	0.5	1	2	4	8	16	32	64	128	R+	R−
Amoxicillin						1	1	4	4	2				>128	4
Amoxicillin + CA			3^b	3	4	2								1	1
Ticarcillin										2	3	4	3	>128	0.5
Ticarcillin + CA			5^b	2	4	1								4	0.125
Piperacillin					1	4	4	3						128	1
Piperacillin + TZ			4^b	3	3	1	1							2	0.12
Cephalothin								2	7	3				>128	0.5
Cefuroxime							1	6	5					64	1
Cefoxitin				1	4	4	3							2	1
Cefotaxime	3	7	2											128	<0.03
Ceftriaxone	2	6	4											14	<0.03
Ceftazidime	6	5	1											4	0.06
Cefepime	9	2	1											4	<0.03
Aztreonam	4	6	2											8	0.12
Imipenem		1	6	5										0.25	<0.03

↵a CA, 2 μg of clavulanic acid per ml; TZ, 4 μg of tazobactam per ml.
↵b MIC, <0.125 μg/ml.
c R−, E. coli recipient; R+, E. coli with recombinant plasmid producing KLUA-1.

TABLE 2.

Kinetic constants (V_max, K_m) of β-lactamase produced by K. ascorbata strain CIP 82. 95T^a

Substrate	V_max (relative)	K_m (μM)	V_max/K_m (relative)
Benzylpenicillin	100	17.5	100
Ticarcillin	7.5	10	13
Cephalothin	1,100	141	136
Cefuroxime	80	35	40
Cefotaxime	110	375	5.1
Ceftazidime	<0.1	50	<0.04
Aztreonam	0.3	105	0.06
Cefoxitin	<0.1	110	<0.02

↵a V_max was expressed relative to the V_max for benzylpenicillin (which was set at 100%).

TABLE 3.

Genotypic characterization of β-lactamase contents of K. ascorbata strains and β-lactamase pIs

Strain	pI	Result of PCR with primer(s):			Gene	Genbank accession no.
Strain	pI	MA1-MA2 (208-751 bp; 543 bp)	MA3 (1-430 bp; 429 bp)	MA4 (461-883 bp; 422 bp)	Gene	Genbank accession no.
CIP 82.95T	8.0	+	+	+	bla_KLUA-1	AJ272538
IP 15.79	8.4	+	+	+	bla_KLUA-2	AJ251722
IP 5.79	8.0	+	+	+	bla_KLUA-3	AJ427461
IP 8.79	8.0	+	+	+	bla_KLUA-4	AJ427462
IP 12.79	8.0	+	+	+	bla_KLUA-5	AJ427463
SL Ka99	6.9	−	−	−
IP 2.89	6.9	−	−	−
IP 3.89	8.0	+	+	+	bla_KLUA-8	AJ427465
TN Ka01	8.4	+	+	+	bla_KLUA-9	AJ427466
IP 43.50	8.0	+	+	+	bla_KLUA-10	AJ427467
IP 4450.94	8.4	+	+	+	bla_KLUA-11	AJ427468
IP 684.74	8.4	+	+	+	bla_KLUA-12	AJ427469

TABLE 4.

Identities of amino acid sequences of K. ascorbata β-lactamases and CTX-M, Toho, UOE β-lactamases

Enzyme(s)	Range % identity (reference) with clusters including:
Enzyme(s)	KLUA-1, -2, -3, -4, -5, -8, -9, -10, -11, and -12 (this work)	CTX-M-1, -3, -10, -11, and -12 and UOE-1 (13, 18, 23^a)	CTX-M-2, -4, -5, -6, and -7 and Toho-1 (3, 10, 11, 23)	CTX-M-9, -13, -14, and -15 and UOE-2 (21^b)	Toho-2 (15)	CTX-M-8 (2)
KLUA-1, -2, -3, -4, -5, -8, -9, -10, -11, and -12	96-100	77-82	95-100	78-81	68-71	82-84
CTX-M-1, -3, -10, -11, and -12 and UOE-1		98-100	77-81	79-80	70-71	80-82
CTX-M-2, -4, -5, -6, and -7 and Toho-1			97-100	78-81	68-71	81-84
CTX-M-9, -13, -14, and -15 and UOE-2				99-100	87-88	85-86
Toho-2					100	75
CTX-M-8						100

↵a Genbank references numbers AY005110 for CTX-M-11 and AY013478 for UOE-1.
↵b Genbank reference numbers AF252621 for CTX-M-13, AF252622 for CTX-M-14, AF252623 for CTX-M-15, and AF311345 for UOE-1.