Diversity of TEM Mutants in Proteus mirabilis

DOI: 10.1128/AAC.43.11.2671

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Fig. 1.
(A) Agarose (0.7%) electrophoresis of plasmid DNA from ESBL-producing E. coli transconjugants and from IRT-producing P. mirabilis. (B) Hybridization with TEM-specific DNA probes. Lanes: 1, plasmid RSa (39 kb); 2, plasmid TP114 (61 kb); 3, pCFF04 (TEM-3; 85 kb); 4, pCFF74 (TEM-24; 85 kb); 5, pCFF14 (TEM-5; 180 kb); 6, pCFF134 (TEM-3); 7, TrCF669 (TEM-66); 8, TrCF39 (TEM-3); 9, TrCF249 (TEM-24); 10, CF449 (TEM-44); 11, CF659 (TEM-65).

Table 1.

β-Lactam MICs for TEM-producingP. mirabilis strains

β-Lactam	MIC (μg/ml) for the following strains (enzymes):
β-Lactam	CF19 (TEM-1)	CF29 (TEM-2)	CF579 (TEM-57)
Amoxicillin	512	4,096	4,096
Amoxicillin + CA^a	4	16	16
Ticarcillin	128	512	512
Ticarcillin + CA	2	2	2
Piperacillin	8	256	256
Piperacillin + TAZ^b	0.5	1	1
Cephalothin	8	8	8

Table 2.

β-Lactam MICs for ESBL-producing P. mirabilis strains and their E. coli transconjugants

β-Lactams	MIC (μg/ml) for the following strains (enzymes):
β-Lactams	P. mirabilis CF249 (TEM-24)	E. coli TrCF249 (TEM-24)	P. mirabilis CF39 (TEM-3)	E. coliTrCF39 (TEM-3)	P. mirabilis CF669 (TEM-66)	E. coli TrCF669 (TEM-66)
Cefotaxime	0.12	1	2	4	8	4
Cefotaxime + CA^a	0.03	0.03	0.06	0.06	0.06	0.03
Ceftazidime	4	64	0.5	8	8	4
Ceftazidime + CA	0.12	0.25	0.12	0.25	0.12	0.12
Aztreonam	0.06	4	0.06	2	0.25	2
Aztreonam + CA	0.01	0.03	0.01	0.06	0.06	0.003

Table 3.

β-Lactam MICs for IRT-producingP. mirabilis strains

β-Lactam	MIC (μg/ml) for the following strains (enzymes):
β-Lactam	CF739 (TEM-73 [IRT-18])	CF749 (TEM-74 [IRT-19])	CF449 (TEM-44 [IRT-13])	CF659 (TEM-65 [IRT-16])
Amoxicillin	1,024	2,048	1,024	64
Amoxicillin + CA^a	1,024	512	512	64
Ticarcillin	8	32	16	1
Ticarcillin + CA	4	4	2	1
Piperacillin	16	16	8	2
Piperacillin + TAZ^b	4	1	0.5	0.5
Cephalothin	2	4	4	2

Table 4.

pIs and kinetic parameters inhibitors for TEM-type and IRT-type β-lactamases

Drug	Kinetic parameters for the following strains (enzymes; pIs):
	CF19 (TEM-1; 5.4)		CF29 (TEM-2; 5.6)		CF579 (TEM-57; 5.2)		CF739 (TEM-73 [IRT-18]; 5.2)		CF749 (TEM-74 [IRT-19]; 5.2)		CF449 (TEM-44 [IRT-13]; 5.4)		CF659 (TEM-65 [IRT-16]; 5.4)
	V_max^a	K_m (μM)	V_max	K_m (μM)	V_max	K_m (μM)	V_max	K_m (μM)	V_max	K_m (μM)	V_max	K_m (μM)	V_max	K_m (μM)
Benzylpenicillin	100	25	100	20	100	40	100	334	100	330	100	375	100	350
Amoxicillin	77	26	95	25	73	38	207	315	140	400	145	360	150	400
Ticarcillin	10	10	10	9	10	15	2	810	2	765	2.3	460	2	600
Cephalothin	10	250	25	500	17	900	1.5	1,000	1	300	2	312	2	400

↵a V _max values are given as a percentage of the V _max for benzylpenicillin, which was taken as 100%.

Table 5.

IC₅₀s of inhibitors for TEM-type and IRT-type β-lactamases

Table 6.

pI and kinetic parameters for ESBL-type β-lactamases

Drug	Kinetic parameters for the following strains (enzymes; pIs):
	CF249 (TEM-24; 6.5)		CF39 (TEM-3; 6.3)		CF669 (TEM-66; 6.0)
	V_max^a	K_m (μM)	V_max	K_m (μM)	V_max	K_m (μM)
Benzylpenicillin	100	5	100	4	100	5
Amoxicillin	62	43	72	5	70	5
Cephalothin	281	43	107	22	110	30
Cefotaxime	130	50	445	100	400	100
Ceftazidime	1,410	377	40	400	40	450

↵a V _max values are given as a percentage of the V _max for benzylpenicillin, which was taken as 100%.

Table 7.

Nucleotide and amino acid substitutions in thebla _TEM genes fromP. mirabilis strains

β-Lactamase and strain or enzyme	Mutation at the following nucleotide (substituted amino acid)^a:
β-Lactamase and strain or enzyme	32^b	226 (8)	263 (21)	317 (39)	346 (48)	436 (78)	477 (92)	512 (104)	604 (134)	682 (160)	692 (164)	911 (237)	914 (238)	917 (240)	925 (242)	929 (244)	990 (265)
TEM-like
TEM-1 (bla _TEM-1A)^c	`GCC-TA`	C (Phe)	C (Leu)	C (Gln)	A (Glu)	C (Gly)	G (Gly)	G (Glu)	G (Ala)	T (Thr)	C (Arg)	G (Ala)	G (Gly)	G (Glu)	G (Gly)	C (Arg)	C (Thr)
TEM-1 (bla _TEM-1B)^c	`GCC-TA`	T				T			T
TEM-2 (bla _TEM-2)^c	`GCT-TA`			A (Lys)	G	T				C					A
CF579 (TEM-57)	`GCTATA`				G	T	A (Asp)			C					A
ESBL
CF249 (TEM-24)	`GCT-TA`			A (Lys)	G	T		A (Lys)		C	A (Ser)	A (Thr)		A (Lys)	A
CF39 (TEM-3)	`GCT-TA`			A (Lys)	G	T		A (Lys)					A (Ser)		A
CF669 (TEM-66)	`GCT-TA`			A (Lys)	G	T	A (Asp)	A (Lys)					A (Ser)
IRT-like
CF449 (TEM-44 [IRT-13])	`GCT-TA`			A (Lys)	G	T				C					A	A (Ser)
CF659 (TEM-65 [IRT-16])	`GCT-TA`			A (Lys)	G					C					A	T (Cys)
CF739 (TEM-73 [IRT-18])	`GCT-TA`		T (Phe)	C	G	T				C					A	T (Cys)	T (Met)
CF749 (TEM-74 [IRT-19])	`GCT-TA`		T (Phe)	C	G	T				C					A	A (Ser)	T (Met)

↵a Nucleotide positions are from Sutcliffe (37), and amino acids positions are from Ambler et al. (1).
↵b The nucleotide at position 32 is given in boldface type.
↵c The sequences are from Chen and Clowes (12) and Goussard and Courvalin (15).