Diversity of Substitutions within or Adjacent to Conserved Amino Acid Motifs of Penicillin-Binding Protein 2X in Cephalosporin-Resistant Streptococcus pneumoniaeIsolates

Yasuko Asahi; Yasuo Takeuchi; Kimiko Ubukata

doi:10.1128/AAC.43.5.1252

DOI: 10.1128/AAC.43.5.1252

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Fig. 1.
Deduced amino acid sequences of part of PBP 2X from representative strains from each group. The sequence of PSSP R6 is shown on the top line. Numbering is based on published data on the R6 strain (15). Only amino acids differing from the R6 sequence are shown. Boxes represent conserved amino acid motifs.
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Fig. 2.
Stereoview of the transpeptidase domain of PBP 2X of the high-level CTX-resistant PRSP strain 31/KU5. The structure was constructed by using the crystallographic coordinates of PBP 2X of Pares et al. (PDB entry code 1 PMD) (21). Cefditoren of oral cephalosporin was shown in the long groove as a β-lactam model. Amino acids differing from the R6 sequence are indicated by colored circles (blue, substitutions within or adjacent to conserved motifs of STMK and KTG; green, substitutions adjacent to SSN and KSG in strains classified into groups I and II; yellow, general substitutions).

Tables

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Table 1.

Classification based on PBP 2X sequence differences and properties of S. pneumoniae strains

Group	Strain	Serotype	MIC (μg/ml)^a						Amino acid motif^b			PCR result^c
Group	Strain	Serotype	PC	CTX	CPDX	CDTR	CFDN	CCL	S T M K	A H S S N V	L K S G T	pbp1a	pbp2b
None	1/E9	15	0.031	0.031	0.125	0.016	0.125	1	––––	––––––	–––––	−	−
	2/HSB1	15	0.031	0.031	0.125	0.031	0.125	2	––––	V–––––	–––––	−	−
	3/YO42	3	0.031	0.063	0.125	0.031	0.125	1	––––	––––––	–––––	−	−
	4/HSC21	6	0.031	0.063	0.125	0.031	0.25	1	––––	––––––	–––––	−	−
	5/H48	19	0.031	0.063	0.125	0.031	0.25	1	––––	––––––	–––––	−	−
I	6/HSB5	3	0.008	0.125	0.5	0.031	0.25	2	––––	––––––	––––A	−	−
	7/T37	6	0.008	0.125	0.25	0.031	0.25	2	––––	––––––	––––A	−	−
	8/TJ24	9	0.016	0.125	0.25	0.031	0.25	1	––––	––––––	––––A	−	−
	9/E13	9	0.016	0.25	0.5	0.031	0.25	2	––––	––––––	––––A	−	−
II	14/TO22	19	0.031	0.063	0.25	0.063	0.125	0.5	––––	–L––––	V––––	−	−
	17/SU1	19	0.063	0.125	0.25	0.016	0.25	1	––––	–L––––	V––––	−	−
	21/H3	10	0.031	0.125	0.25	0.063	0.125	1	––––	–L––––	–––––	−	−
	16/MA37	23	0.063	0.125	0.25	0.125	0.25	1	––––	VL––––	–––––	−	−
	22/H23	14	0.031	0.063	0.25	0.063	0.25	1	––––	VL––––	–––––	−	−
III	11/KM99	3	0.031	0.125	0.5	0.063	0.25	0.5	–A––	––––––	–––––	−	−
	15/HSC9	3	0.031	0.125	0.5	0.125	0.25	1	–A––	––––––	–––––	−	−
	18/TJ41	3	0.031	0.125	0.5	0.063	0.25	2	–A––	––––––	–––––	−	−
	23/H69	3	0.031	0.25	0.25	0.063	0.25	1	–A––	––––––	–––––	−	−
	25/S19	3	0.031	0.125	0.5	0.125	0.25	1	–A––	––––––	–––––	−	−
IV	34/H31	6	1	0.5	1	0.5	4	32	–A––	––––––	V––––	+	+
	36/Z21	6	2	0.5	2	0.5	8	64	–A––	––––––	V––––	+	+
	37/H28	19	1	0.5	2	0.5	2	64	–A––	––––––	V––––	+	+
	38/KM90	19	2	0.5	2	0.5	4	64	–A––	––––––	V––––	+	+
	39/Z13	19	2	0.5	1	0.5	4	64	–A––	––––––	V––––	+	+
	40/H29	23	1	0.5	2	0.5	4	64	–A––	––––––	V––––	+	+
	41/B99	23	1	0.5	2	0.5	4	64	–A––	––––––	V––––	+	+
	42/H0	14	4	1	4	1	8	64	–A––	––––––	V––––	+	+
	27/S46	23	0.5	0.5	1	0.25	2	32	–A––	––––––	V––––	−	+
	1A22/HA5	6	0.25	0.5	1	0.25	2	16	–A––	––––––	V––––	−	+
V	20/SHA3	19	2	4	16	4	32	64	–A––	V–––––	V––––	+	+
	29/KK133	23	2	8	16	4	32	64	–AF–	––––––	V––––	+	+
	30/NG44	23	2	2	16	2	16	64	–AF–	––––––	V––––	+	+
	31/KU5	23	1	2	8	2	8	64	–AF–	––––––	V––––	+	+
	32/KU81	19	4	4	16	2	16	64	–AF–	––––––	V––––	+	+
	33/AK5	14	0.125	4	16	2	2	8	–AF–	––––––	V––––	+	−

↵a Susceptibilities to PC, CTX, cefpodoxime (CPDX), cefditoren (CDTR), cefdinir (CFDN), and cefaclor (CCL) were determined by previously described methods (1).
↵b Only amino acid residues differing from PBP 2X conserved motif sequences of the PSSP strain R6 are shown. Conserved amino acid motifs are underlined.
↵c Altered PBP genes were detected by PCR as described previously (29). +, altered; −, not altered.

Table 2.

Susceptibilities to β-lactam antibiotics of S. pneumoniae transformants obtained with pbp2x DNA

Strain^a	MIC (μg/ml)^b
Strain^a	PC	CTX	CPDX	CDTR	CFDN	CCL
Recipient, KK97	0.031	0.016	0.031	0.016	0.063	0.5
Transformant
KK97^2X34/H31	0.063	0.5	2.0	0.5	4.0	2.0
KK97^2X40/H29	0.063	0.5	2.0	0.5	4.0	2.0
KK97^2X29/KK133	0.063	1.0	2.0	0.5	8.0	2.0
KK97^2X32/KU81	0.063	1.0	4.0	0.5	8.0	2.0

↵a Nonencapsulated strain KK97 was transformed with PCR-amplified pbp2x from group IV and V strains as the donor DNA. Transformants were selected on plates containing 0.2 or 0.5 μg of CTX per ml. Example of transformant nomenclature, KK97^2X34/H31 corresponds to KK97 strain containing the pbp2x gene from strain 34/H31.
↵b For abbreviations of β-lactams, see Table1, footnote a .