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We evaluated the antibacterial activity of faropenem against penicillin-susceptible Streptococcus pneumoniae (PSSP) and penicillin-resistant S. pneumoniae (PRSP). It was shown that the minimum inhibitory concentrations against 90% of the clinically isolated strains (MIC90) of faropenem, penicillin G, cefaclor, cefcapene, and cefditoren against PSSP were 0.032, 0.063, 2, 0.25, and 0.125 micrograms/ml, respectively. While those against PRSP were 0.5, 2, > 128, 1, and 1 micrograms/ml, respectively. Furthermore, we evaluated the bactericidal activity, at the level of 1/4, 1, and 4 MIC, of faropenem and the above four reference antibacterial agents against PSSP and PRSP. Against PSSP No. 127, a sensitive strain to both penicillin G and cefcapene, faropenem showed almost the same bactericidal activity as those of reference agents. Against PSSP No. 108, a penicillin-susceptible and cephem-resistant strain, and PRSP No. 57, a resistant strain to both of penicillin and cephem, faropenem of 1 MIC showed bactericidal activity, but reference agents needed 4 MIC to show bactericidal activity.

Two hundred and thirteen clinical strains of coagulase-negative staphylococci isolated in Japan between 1980 and 1997 were analyzed for glycopeptide susceptibility by determining MIC using both Mueller-Hinton agar (MHA) and Brain Heart Infusion agar (BHIA) plates. Of 37 Staphylococcus epidermidis strains isolated between 1980 and 1981, all were susceptible to vancomycin and teicoplanin on both MHA and BHIA. However, of 122 isolates of Staphylococcus epidermidis isolated between 1994 and 1997, 1 (0.8%) was intermediate to vancomycin on MHA and 39 (32%) were intermediate on BHIA, while 3 (2.5%) and 27 (22.1%) were intermediate or resistant to teicoplanin on MHA and BHIA, respectively. It was demonstrated that the susceptibilities of the strains in 1990s to vancomycin and teicoplanin were significantly decreased compared with those in 1980s. Population analysis was performed with six strains each of Staphylococcus epidermidis and Staphylococcus haemolyticus (three with vancomycin MIC > or = 8 micrograms/ml and three with vancomycin MIC < or = 4 micrograms/ml using BHIA). The population curves of the Staphylococcus epidermidis strains showed a homogeneous pattern of susceptibility. Whereas, those for two Staphylococcus haemolyticus strains (vancomycin MIC = 8 micrograms/ml using BHIA) showed a typical heterogeneous pattern. Vancomycin-resistant mutants (MIC > or = 32 micrograms/ml) were obtained with a high frequency of 10(-4)-(-5) from the strains by one-step selection with 16 micrograms/ml of vancomycin.

We have developed a new method of detecting clinical S. aureus strains possessing heterogeneous resistance to vancomycin (hetero-VRSA). The method exploits characteristic antagonism between vancomycin and beta-lactam antibiotics against the strain Mu3, a representative hetero-VRSA. The method comprises of the following procedures: 1) overnight culture of bacteria is streaked on brain heart infusion agar containing 4 micrograms/ml of vancomycin, 2) paper discs impregnated with any one of beta-lactam antibiotics are placed onto the plate, and 3) the plate is incubated at 37 degrees C and the cell growth was observed after 24 h and 48 h of incubation. The Mu3 cells were grown only around the beta-lactam discs due to the antagonism between vancomycin and beta-lactam against Mu3 cells. A total of 321 MRSA clinical strains isolated in 1995 and 1996 from 12 medical facilities in Japan were tested with this method. A total of 39 strains (12.1%; 24 h) and 67 strains (20.96%; 48 h) grew around the beta-lactam discs. All the 10 strains (representing 10 facilities), tested with the analysis of resistant subpopulations to vancomycin, showed similar heterogeneous patterns as observed with Mu3. The method was considered as a convenient and rapid substitute for the population analysis, the authentic method for the detection of hetero-VRSA strains.

The mechanism of resistance was studied with vancomycin-resistant Staphylococcus aureus (VRSA) strain Mu50. It was demonstrated that the incorporation of 14C-N-acetylglucosamine into the cell wall of Mu50 was not suppressed in the presence of 8 microliters/ml of vacomycin, whereas it was completely suppressed in vancomycin-susceptible strains FDA209P and H-1. Increased binding of vancomycin to the wall of Mu50 was observed compared to the control strains: 1.7 x 10(16) (Mu50), 6.1 x 10(15) (209P), and 6.7 x 10(15) (H-1) vancomycin molecules/mg cell wall, respectively. Remarkable proportion of the cell-wall component muropeptides were non-amidated in the cell wall of Mu50. In concordance with this phenomena, peptidoglycan cross-linkage decreased strikingly in the Mu50 strain. Free D-Ala-D-Ala residues at the end of muropeptides in the pre-existing cell wall generated by decreased cross-linkage seems to account for increased vancomycin binding. The increase of vancomycin-resistance level is presumably caused by sequestration of vancomycin molecules from primary target point on cell membrane. It was considered that at least two phenotypic changes are required for the vancomycin resistance in the Mu50 strain. First, as we have described previously, is the activated cell wall synthesis, and second, the reduction of cross-linkage of peptidoglycan by production of non-amidated muropeptide precursors.

The mechanism of vancomycin resistance in methicillin-resistant Staphylococcus aureus (MRSA) Mu50 was investigated. More than 3 times increase of the incorporation of 14C-GluNAc into the cell wall of Mu50 was observed compared to those of vancomycin-susceptible strains FDA209P, H-1, LR5P1. The amount of cytoplasmic murein-monomer precursor increased more than 3 times in Mu50 compared to those of control strains. There was an increased production of PBP1, PBP2, and PBP2', which were 1.51, 17.2, and 7.06 times greater, respectively, in Mu50 than those in H-1, and 2.38, 4.46, and 1.96 times greater respectively, than those in LR5P1. By transmission electromicrograph, it was shown that the cell wall of Mu50 was twice thicker than that of LR5P1. Increase of tightly-bound vancomycin to the cell wall fraction was observed in Mu50 when compared to those in FDA209P and H-1 strains. From these results, the increase of the vancomycin targets, free D-Ala-D-Ala residues in the cell wall, in number, due to the activated cell wall synthesis, and/or decrease of the cross-linkage of the cell wall was suggested to be the mechanism of vancomycin resistance in the Mu50 strain.

We evaluated a clinical strain of methicillin-resistant Staphylococcus aureus (MRSA), Mu50, for vancomycin susceptibility. Mu50 was isolated from a patient with infection of a surgical incision site which resisted vancomycin therapy. Mu50 showed a decrease in susceptibility to vancomycin, but this strain did not carry vanA or vanB or vanC genes as judged from PCR amplification. MICs of vancomycin against Mu50 and vancomycin-susceptible S. aureus FDA209P, S. aureus ATCC-29213, and MRSA H-1 were 8, 1, 1, and 1 microgram/ml, respectively by agar dilution and macro-broth dilution methods according to NCCLS. MIC values with agar dilution method using MHA + 20% horse serum, HIA, and BHIA agreed with the MIC values with micro- and macro-broth dilution method. Population analysis revealed that vancomycin concentration required for inhibition of ca. 10(7) cells of Mu50, S. aureus FDA209P, S. aureus ATCC29213, and MRSA H-1 were 36, 2, 2, and 2 micrograms/ml, respectively. These results showed that the activity of vancomycin against Mu50 was at least 8-fold decreased compared to that against S. aureus FDA209P, S. aureus ATCC29213, and MRSA H-1.