OXA-162, a novel variant of OXA-48 displays extended hydrolytic activity towards imipenem, meropenem and doripenem

Figures & data

Figure 1.  (A) Schematic map of the construct on the recombinant plasmid pMSH-3-(OXA-162). The coding regions are shown as arrow-head boxes indicating the direction of open reading frame. The insertion sequence IS1R was shown as gray-shaded box. (B) The left insertion sequence IS1999 is disrupted by IS1R between the dedicated promoter signal sequences while leaving the -10 signal intact.

Table 1.  Kinetic parameters for OXA-48 and OXA-162.

	OXA-48			OXA-162
	kcat	KM	kcat/KM	kcat	KM	kcat/KM
	(s^−1)	(µM)	(M^−1· s^−1)	(s^−1)	(µM)	(M^−1· s^−1)
Ampicillin	1349	572.7	2.36E+06	2665	710.90	3.75E+06
Cefepime	7.87	2514.5	3.13E+03	5.18	610.20	8.49E+03
Ceftazidime	N.H.^a			N.H.
Ertapenem	0.112	2.4	4.67E+04	0.74	49.10	1.51E+04
Doripenem	N.H.^b			1.38	38.90	3.55E+04
Imipenem	22.48	28.3	7.94E+05	40.72	29.10	1.40E+06
Meropenem	0.112	5.5	2.04E+04	0.92	3.40	2.71E+05

^aCeftazidime hydrolysis could not be detected with increasing enzyme concentrations.

^bDoripenem hydrolysis could not be detected with increasing concentrations of OXA-48.

Table 2.  Minimum inhibitory concentration (MIC) of OXA-162 producing K. pneumoniae strain KIRK1, its transformant, OXA-162 producing clone and OXA-48 producing clone.

	K. pneumoniae KIRK1 (mg/L)	E. coli DH10B pKRU-1 (mg/L)	E. coli DH10B pMSH-3 (OXA-162) (mg/L)	E. coli DH10B pVT-1 (OXA-48) (mg/L)	DH10B (mg/L)
Ampicillin	≥512	≥512	≥512	≥512	4
Piperacillin	≥512	128	≥512	64	1
Pip/Tazo	256	64	≥512	128	1
Cefepime	4	0.25	0.5	<0.0625	<0.0625
Ceftazidime	32	0.25	0.5	0.13	0.13
Ertapenem	8	1	4	0.5	<0.0156
Doripenem	2	0.25	0.5	0.06	<0.0156
Imipenem	4	1	2	0.5	<0.25
Meropenem	1	0.25	0.25	0.25	<0.0156

Figure 2.  Examination of the active site regions of OXA-48 and modeled OXA-162. (A) OXA-48 homodimer (active site entry regions were pointed out with arrow heads and active site residues were colored). (B) Depiction of OXA-48 entry site. Dark orange colored amino acid (Thr213) is where the mutation occurred. Asp101 was located across the mutated amino acid. The measured distance between Thr213 and Asp101 was 9.12 Å. This was predicted to be the putative antibiotics entry site. (C) Depiction of the modeled OXA-162 entry site. Dark orange colored amino acid is the mutated amino acid (Ala188). Asp77 was located across the mutated amino acid. The measured distance between Ala188 and Asp77 was 10.07 Å which indicated the presence of 1 Å widening of the predicted entry site. Note that the corresponding amino acids for Ala188 and Asp77 in OXA-48 were Thr213 and Asp101, respectively.