Article Figures & Data
Figures
- Fig. 1.
PBP profiles of AMP-susceptible and BLNAR H. influenzae strains (A) and Rd strains transformed with theftsI gene encoding PBP 3 (B). Membrane fractions were labeled with [3H]PEN for 10 min at 30°C and were then subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis and fluorography (I). PBPs were named according to the proposal of Parr and Bryan (19), our data on PBP affinity, and morphologic observations. AMP MICs are as follows: for strain MT053 (lane 1) and MT196 (lane 2), 0.125 μg/ml; for strain MT066 (lane 3), 2 μg/ml; for strain ME870 (lane 4), 4 μg/ml; for strain MT040 (lane 5), 2 μg/ml; for strain ME1133 (lane 6), 4 μg/ml; for strain ME587 (lane 7), 1 μg/ml; for strain KK01 (lane 8), 1 μg/ml; for strain ME495 (lane 9), 1 μg/ml. (B) lane 1, recipient Rd strain of H. influenzae; lane 2, transformed strain Rd3A/ME870-11; lane 3, transformed strain Rd3A/ME870-12; lane 4, BLNAR strain ME870.
- Fig. 2.
Fluorograms of PBPs of H. influenzae TK422 pretreated with AMP (A), CTX (B), and FRM (C). A 30-μl aliquot of each membrane fraction was incubated with 3 μl of each of the nonradioactive β-lactams diluted to various concentrations for 10 min and then further incubated with 3 μl of [3H]PEN for another 10 min. PBPs were visualized by autoradiography after exposure of the dried gels to X-OMAT film for 20 days at −80°C. The numbers identifying the lanes are concentration (in micrograms per milliliter).
- Fig. 3.
Deduced amino acid sequence of part of PBP 3 from the strains listed in Table 1. Only amino acids that differed from the Rd amino acid sequence are shown. Boxes represent the conserved amino acid motifs of Ser-Thr-Val-Lys (STVK), Ser-Ser-Asn (SSN), and Lys-Thr-Gly (KTG).
- Fig. 4.
Deduced amino acid sequence of part of PBP 4s from the strains listed in Table 1. Only amino acids that differed from the Rd amino acid sequence are shown. Boxes indicate conserved amino acid sequences of Ser-Thr-Gln-Lys (STQK), Ser-Asp-Asn (SDN), and Lys-Thr-Gly (KTG).
Tables
- Table 1.
Serotype, β-lactamase production, PBP affinity, and susceptibility to β-lactam antibiotics of the H. influenzae strains useed in this study
Assigned group Strain Sourcea Serotype β-Lactamase PBP affinity MIC (μg/ml) PBP 3A PBP 3B PBP 4 AMP CEC CTX CRO CPD CDR CDN CPN FRM MEM Control MT196 Na NSb − Nc N N 0.125 0.5 0.008 0.004 0.016 0.25 0.008 0.008 0.25 0.031 MT053 Na NS − N N N 0.25 1 0.008 0.004 0.031 0.125 0.008 0.008 0.25 0.031 TK422 Na NS − N N N 0.5 2 0.031 0.008 0.063 0.5 0.016 0.016 0.5 0.063 TC01 CSF b + NDd ND ND 8 2 0.016 0.004 0.063 0.5 0.016 0.016 0.5 0.031 AK01 CSF b + ND ND ND 8 2 0.016 0.008 0.063 0.5 0.008 0.016 0.25 0.031 I ME2014 Ot NS − De N N 1 32 0.063 0.016 0.25 2 0.031 0.063 2 0.031 ME1547 Na NS − D N N 1 64 0.063 0.008 0.5 4 0.031 0.125 1 0.063 ME692 Na NS − D N N 1 32 0.063 0.016 0.5 4 0.063 0.125 0.5 0.125 ME1503 Na NS − D N N 1 64 0.063 0.016 1 8 0.063 0.125 2 0.125 H17 Na NS − D N N 1 32 0.063 0.008 0.25 2 0.031 0.125 1 0.125 N130 Na NS − D N N 1 32 0.063 0.016 0.25 2 0.031 0.125 1 0.25 H2 Na NS − D N N 4 64 0.25 0.031 1 2 0.063 0.25 1 0.025 ME1495 Na NS + ND ND ND 64 64 0.125 0.016 0.5 4 0.063 0.125 1 0.031 H7 Na NS + ND ND ND 64 64 0.125 0.031 4 8 0.063 0.5 1 0.25 II ND01 CSF b − D N N 1 32 0.063 0.016 0.25 8 0.031 0.125 2 0.25 KK01 CSF b − D N N 1 64 0.063 0.031 0.25 4 0.031 0.125 0.5 0.125 ME570 Na NS − D D N 1 64 0.063 0.016 1 2 0.031 0.125 1 0.25 ME587 Na NS − D D N 1 64 0.063 0.008 0.25 4 0.031 0.063 1 0.25 ME495 Na NS − D N N 1 64 0.125 0.031 1 4 0.063 0.25 2 0.125 MT273 Na NS − D N N 2 32 0.063 0.016 0.5 2 0.031 0.125 8 0.5 MT160 Na NS − D N N 2 32 0.063 0.008 0.5 2 0.031 0.125 2 0.5 ME267 Ot NS − D D N 2 32 0.063 0.016 0.25 1 0.031 0.063 2 0.25 ME040 Ot NS − D N D 2 64 0.063 0.063 1 4 0.063 0.25 2 0.25 ME1210 Na NS − D D N 4 64 0.125 0.031 1 4 0.063 0.25 2 0.25 MT313 Na NS − D N N 4 64 0.125 0.031 1 4 0.063 0.25 2 0.25 ME1976 Na NS + ND ND ND 64 32 0.063 0.016 0.25 2 0.016 0.125 1 0.063 III MT066 Na NS − D D N 2 64 1 0.25 4 8 0.25 1 2 0.25 MT070 Na NS − D D N 2 64 1 0.25 4 8 0.125 1 4 0.125 ME1133 Na NS − D N D 4 64 1 0.25 8 16 0.25 2 2 0.25 ME870 Na NS − D D N 4 64 2 0.25 4 16 0.5 2 4 0.5 - Table 2.
Deduced amino acid substitutions identified in PBP 3s from 25 BLNAR strains and 5 AMP-susceptible strains of H. influenzae
Assigned group Strain Amino acid substitution Asp-350 Ser-357 Met-377 Ser-385 Leu-389 Arg-517 Asn-526 Control MT196 MT053 TK422 TC01 AK01 I ME2014 His ME1547 Asn Thr His ME692 Asn Asn His ME1503 Asn Asn His H17 Asn His N130 Asn Asn His H2 Asn Asn His ME1495 Asn His H7 Asn Asn IIe Thr His II ND01 Asn Asn Lys ME570 Asn Lys ME587 Asn Lys KK01 Asn Asn Lys ME495 Asn Asn Lys MT273 Asn Asn Lys MT160 Asn Asn Lys ME267 Asn Asn Lys MT040 Asn Asn Lys ME1210 Asn Asn Lys MT313 Asn Asn Lys ME1976 Asn Lys III MT066 Asn Asn IIe Thr Phe Lys MT070 Asn Asn Ile Thr Phe Lys ME1133 Asn Asn Ile Thr Phe Lys ME870 Asn Asn Ile Thr Phe Lys - Table 3.
Susceptibilities to β-lactam antibiotics of H. influenzae strains transformed with ftsI DNA fragment
Assigned group Straina MIC (μg/ml) AMP CEC CTX CDR CPD CDN CPN MEM Rd (ATCC51097) 0.25 1 0.008 0.25 0.063 0.008 0.016 0.031 I Rd3A/H17 1 16 0.063 1 0.25 0.063 0.063 0.125 II Rd3A/KK01 1 16 0.063 1 0.5 0.031 0.063 0.125 III Rd3A/ME870 2 16 0.5 2 2 0.125 1 0.25 ↵a H. influenzae Rd (ATCC 51097) (the recipient) was transformed with PCR-amplifiedftsI gene DNA fragments from group I, II, and III strains as the donors. Transformants were selected on plates containing 5 or 10 μg of CEC per ml. As an example of the transformant nomenclature, Rd3A/ME870 corresponds to Rd strain containing theftsI gene from strain ME870.
