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ABSTRACTThe activity of faropenem, a new oral penem, was tested against 579 strains of anaerobic bacteria by using the NCCLS-approved reference method. Drugs tested included amoxicillin-clavulanate, cefoxitin, clindamycin, faropenem, imipenem, and metronidazole. Of the 176 strains of Bacteroides fragilisgroup isolates tested, two isolates had faropenem MICs of 64 μg/ml and imipenem MICs of >32 μg/ml. Faropenem had an MIC of 16 μg/ml for an additional isolate of B. fragilis; this strain was sensitive to imipenem (MIC of 1 μg/ml). Both faropenem and imipenem had MICs of ≤4 μg/ml for all isolates of Bacteroides capillosus(10 isolates), Bacteroides splanchnicus(13 isolates), Bacteroides ureolyticus(11 isolates), Bilophila wadsworthia(11 isolates), Porphyromonasspecies (42 isolates), Prevotellaspecies (78 isolates), Campylobacterspecies (25 isolates), Sutterella wadsworthensis(11 isolates), Fusobacterium nucleatum(19 isolates), Fusobacterium mortiferum/varium(20 isolates), and other Fusobacteriumspecies (9 isolates). Faropenem and imipenem had MICs of 16 to 32 μg/ml for two strains of Clostridium difficile; the MICs for all other strains of Clostridiumtested (69 isolates) were ≤4 μg/ml. Faropenem had MICs of 8 and 16 μg/ml, respectively, for two strains of Peptostreptococcus anaerobius(MICs of imipenem were 2 μg/ml). MICs were ≤4 μg/ml for all other strains of gram-positive anaerobic cocci (53 isolates) and non-spore-forming gram-positive rods (28 isolates). Other results were as expected and reported in previous studies. No metronidazole resistance was seen in gram-negative anaerobes other than S. wadsworthensis(18% resistant); 63% of gram-positive non-spore-forming rods were resistant. Some degree of clindamycin resistance was seen in most of the groups tested.

Integration host factor (IHF) is a protein that binds to the H′ site of bacteriophage λ with sequence specificity. Genetic experiments implicated amino acid residue Glu44of the β-subunit of IHF in discrimination against substitution of A for T at position 44 of the TTR submotif of the binding site (Lee, E. C., Hales, L. M., Gumport, R. I., Gardner, J. F. (1992) EMBO J., 11, 305–313). We have extended this observation by generating all possible single-base substitutions at positions 43, 44, and 45 of the H′ site. IHF failed to bind these H′ site substitution mutants in vivo. The Kd(app) value for each H′ site substitution, except for H′45A mutant, was reduced >2000-fold relative to the wild-type site. Substitution of amino acid β-Glu44with alanine prevented IHF from discriminating against the H′44A variant but not the other H′ site substitution mutants. Further analysis with other substitutions at position β44 demonstrated that both oxygens of the wild-type glutamic acid are necessary for discrimination of AT at position 44. Because the β-Glu44residue does not contact the DNA, this residue probably enforces binding specificity indirectly through interaction with amino acids that themselves contact the DNA.

To analyze the role of amino acids in the steroid receptor DNA binding domain (DBD) recognition helix in binding of the receptor to the estrogen response element (ERE), we adapted the powerful P22 challenge phage selection system for use with a vertebrate protein. We used the progesterone receptor DNA binding domain and selected for mutants that gained the ability to bind to the ERE. We used a mutagenesis protocol based on degenerate oligonucleotides to create a large and diverse pool of mutants in which 10 nonconsensus amino acids in the DNA recognition helix of the progesterone receptor DNA binding domain were randomly mutated. After a single cycle of modified P22 challenge phage selection, 37 mutant proteins were identified, all of which lost the ability to bind to the progesterone response element. In gel mobility shift assays, approximately 70% of the genetically selected mutants bound to the consensus ERE with a >4-fold higher affinity than the naturally occurring estrogen receptor DBD. In the P-box region of the DNA recognition helix, the selected mutants contained the amino acids found in the wild-type estrogen receptor DBD, as well as other amino acid combinations seen in naturally occurring steroid/nuclear receptors that bind the aGGTCA half-site. We also obtained high affinity DBDs with Trp585as the first amino acid of the P-box, although this is not found in the known steroid/nuclear receptors. In the linker region between the two zinc fingers, G597R was by far the most common mutation. In transient transfections in mammalian cells using promoter interference assays, the mutants displayed enhanced affinity for the ERE. When linked to an activation domain, the transfected mutants activated transcription from ERE-containing reporter genes.