Ambler Class A Extended-Spectrum β-Lactamases in Pseudomonas aeruginosa: Novel Developments and Clinical Impact

DOI: 10.1128/AAC.47.8.2385-2392.2003

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FIG. 1.
Dendrogram obtained for class A ESBLs identified in P. aeruginosa by parsimony analysis. The alignments used for construction of the tree were carried out with the ClustalW program, followed by minor adjustments to fit the class A β-lactamase scheme (1). Branch lengths are drawn to scale and are proportional to the number of amino acid changes. The number of changes is indicated above each branch. The distance along the vertical axis has no significance.
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FIG. 2.
Double-disk synergy test performed with imipenem (IPM)- and ceftazidime (CAZ)-containing disks and a GES-1-producing P. aeruginosa isolate without (A) or with (B) cloxacillin (200 μg/ml)-containing Mueller-Hinton agar plates. No synergy was visible with a cefsulodin (CFS)-containing disk.

TABLE 1.

Ambler class A extended-spectrum β-lactamases known in P. aeruginosa

β-Lactamase	Genetic support^a	Country of first isolation	Yr of first isolation	Other countries of isolation	Reference(s)
VEB-1	C, P, I	France	1998	Thailand, India,^b China^b	8, 17, 32, 51
VEB-1a	C, I	Kuwait	1999		43
VEB-1b	C, I	Kuwait	1999		43
VEB-2	C, I	Thailand	1999		17
PER-1	C	France	1991	Turkey, Italy, Belgium	10, 12, 24, 34, 53, 54
SHV-2a	C, P	France	1995	Thailand, Poland^b	8, 31
SHV-5	P	Thailand	1994-1996	Greece^b	8
SHV-12	C	Thailand	1994-1996		8
TEM-4	P, C	France	1996		42
TEM-21	C	France	1997		13
TEM-24	P	France	1998		25
TEM-42	P	France	1992		30
GES-1	C, I	France	1999		14
GES-2	P, I	South Africa	2000		44, 45
IBC-2	C, I	Greece	1998		27

TABLE 3.

Comparative kinetic parameters for ESBLs found in P. aeruginosa^a

Antibiotic	GES-1			GES-2			PER-1			VEB-1			TEM-4			SHV-2a
Antibiotic	k_cat (s⁻¹)	K_m (μM)	k_cat/ K_m	k_cat (s⁻¹)	K_m (μM)	k_cat/ K_m	k_cat (s⁻¹)	K_m (μM)	k_cat/ K_m	V_max	K_m (μM)	V_max/ K_m	V_max	K_m (μM)	V_max/ K_m	V_max	K_m (μM)	V_max/ K_m
Benzylpenicillin	2.8	40	70	0.4	4	96	7.2	31.3	230	100	2.8	100	100	NA	NA	33	17	1.9
Amoxicillin	13	200	65	0.7	25.8	26	NA	NA	NA	110	6.0	50	50	NA	NA	NA	NA	NA
Ampicillin	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	47	28	1.7
Ticarcillin	0.3	400	0.7	0.06	13.3	4.5	NA	NA	NA	8	1	22	NA	NA	NA	NA	NA	NA
Piperacillin	8	900	13	0.3	22.8	23	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA
Cephalothin	179	3,400	52	0.3	3	112	12.4	46	269	700	6.0	325	NA	NA	NA	NA	NA	NA
Cephaloridin	53	2,000	26	0.5	7.7	65	13	237	22	2,300	12	533	232	NA	NA	100	30	3.2
Cefoxitin	0.9	30	33	NH	NH	NH	NA	NA	NA	NA	NA	NA	< 1	NA	NA	NA	NA	NA
Cefuroxime	NA	NA	NA	NA	NA	NA	NA	NA	NA	2,000	24	230	NA	NA	NA	NA	NA	NA
Ceftazidime	380	2,000	188	ND	>3,000	ND	70	3,519	19.9	NA	NA	NA	10	NA	NA	NA	NA	NA
Cefotaxime	68	4,600	15	2.2	890	2.5	43	652	66	4,300	38	314	300	NA	NA	7	26	0.2
Cefepime	2.8	1,800	1.6	1.1	1900	0.6	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA
Cefpirome	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	20	151	0.14
Imipenem	0.003	45	0.07	0.004	0.45	NA	NA	NA	NA	NA	NA	NA	<1	NA	NA	NA	NA	NA
Meropenem	NH	NH	NH	NH	NH	NH	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA
Aztreonem	NH	NH	NH	NH	NH	NH	43	442	97.3	400	500	2	<1	NA	NA	NA	NA	NA

↵ a Data were adapted from references 5, 36, 40, 41, and 45. k_cat values are available only for GES-1 and GES-2. V_max values are relative to that of benzylpenicillin and, for VEB-1, TEM-4 and SHV2a, relative to that of cephaloridine, which were set equal to 100. Abbreviations: NA, data not available; NH, not hydrolyzed (the initial rate of hydrolysis is reported to be lower than 0.001 μM⁻¹ s⁻¹); ND, not determinable due to K_m values that are too high.

TABLE 4.

MICs of β-lactams for several representative class A ESBL-producing P. aeruginosa isolates^a

β-Lactam^b	MIC (μg/ml) for the following extended-spectrum β-lactamase (isolate denomination):
β-Lactam^b	VEB-1 (JES-1)	PER-1 (RNL-1)	GES-1 (695)	GES-2 (GW-1)	SHV2a (RP-1)	TEM-4 (Stel)	IBC-2 (555)
Ticarcillin	>512	512	>512	>512	>512	>512	>256
Ticarcillin + CLA	256	256	64	>512	64	32	>256
Piperacillin	128	32	512	128	256	32	>256
Piperacillin + TZB	NA^c	NA	64	128	16	8	>256
Ceftazidime	512	128	32	32	32	8	>256
Ceftazidime + CLA	128	4	32	16	8	4	NA
Cefotaxime	NA	64	NA	128	>512	128	>256
Cefepime	128	NA	16	32	NA	8	NA
Imipenem	32	0.5	1	16	2	4	>128
Meropenem	8	NA	NA	16	NA	1	>128
Aztrenom	>256	256	4	16	32	16	32

↵ a Data were adapted from references 14, 27, 30 to 32, 34, 42, and 45.
↵ b CLA, clavulanic acid at a fixed concentration of 2 μg/ml; TZB, tazobactam at a fixed concentration of 4 μg/ml.
↵ c NA, data not available.

TABLE 5.

Primers used for detection of genes encoding class A ESBLs in P. aeruginosa

Primer name	Sequence (5′ to 3′)	Gene detected	Reference(s)
VEB-1A	CGACTTCCATTTCCCGATGC	bla _VEB	43
VEB-1B	GGACTCTGCAACAAATACGC
PER-A	ATGAATGTCATTATAAAAGC	bla _PER	10, 45
PER-B	AATTTGGGCTTAGGGCAGAA
GES-1A	ATGCGCTTCATTCACGCAC	bla _GES	45
GES-1B	CTATTTGTCCGTGCTCAGG
TEM-A	GAGTATTCAACATTTCCGTGTC	bla _TEM	45
TEM-B	TAATCAGTGAGGCACCTATCTC
SWSHV-A	AAGATCCACTATCGCCAGCAG	bla _SHV	31
SWSHV-B	ATTCAGTTCCGTTTCCCAGCGG