The lipopolysaccharide (LPS)-compositions of 59 strains of Pseudomonas aeruginosa isolated from different clinical sources such as blood, urine, pus, sputum and feces were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the relationship between the LPS-compositions and gentamicin (GM)-susceptibilities of these strains was investigated. The 59 isolates tested were divided into three groups; a long-LPS chain (B-band LPS) group of 35 strains, a short-LPS chain (A-band LPS) group of 14 strains and an LPS-deficient group of 10 strains. The majority of the clinical isolates (12 of the 13 strains; 92%) from the blood specimens possessed the long-LPS chain. About 67% of the isolates from bot urine and feces possessed the long-lPS chain while a minority in both groups possessed the short chain. The LPS-deficient isolates were found in the sputum specimens at a considerably high rate (42%). Nineteen of the 35 isolates with the long-LPS chain (54%) were susceptible to GM, a poly-cationic antibiotic, and 12 isolates (34%) were resistant. Fourteen isolates with the short-LPS chain were divided into three groups, GM-resistant, GM-susceptible and intermediate. It was notable that 7 of the 10 LPSdeficient isolates were resistant to GM, 1 strain was moderatly resistant, and the remaining 2 strains were susceptible. The ionic binding of [3H] GM to the negative charge sites on the surface structures of P. aeruginosa differing in LPS-structures was the highest in the long-LPS strains followed in descending order by the shor-LPS strains and LPS-deficient strains. The [³H] GM-binding was also investigated with some LPS-deficient strains from P. aeruginosa PAC 1 R series. The [3H] GM-binding to these strains decreased with an increase in a lack of the repeated units of O-polysaccharide. On the other hand, PAC 605 strain, an LPS-mutant of the PAC 1 R of P. aeruginosa, was completely lacking in the repeated units of O-polysaccharide and also in some neutral sugar residues of the core-oligosaccharide region. This mutant strain was highly bound to [³H] GM, suggesting that the sites negatively charged in the deep core-oligosaccharide region and/or lipid A participated in the binding of [³H] GM. This manner of binding may be applied to P. aeruginosa No.45, a clinical isolate. P. aeruginosa PAC 1 R, PAC 605 and No.45 strains were each exposed to GM at a low concentration (20μg/ml) for 10 min. The viable cell counts of PAC 1 R strain decreased to about 70% of the initial count, whereas those of PAC 605 and No.45 strains markedly decreased to 3.6 and 11.0%, respectively, indicating that the vulnerability of both types of the LPSdeficient strains was enhanced by the bactericidal action of GM after a short incubation. The surface structure, such as LPSs, of clinical isolates of P. aeruginosa was changed by contact with various types of environmental factors including exposure to the antibiotics used, and changes in O-antigen structures and also in the susceptibility to poly-cationic antibiotics such as aminoglycosides, including GM, were observed. This type of drug resistance due to a decrease in the transport of antibiotics poses important problems in antibacterial chemotherapy and the development of new drugs which need to be resolved.

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The change in the sensitivity of clinical isolates to cefditoren (CDTR) was investigated by collecting samples in two surveys. In the first survey, 23 species including 805 strains which were isolated from clinical materials during the 3 month period from April to June 1995 were collected for measurement as test strains. Furthermore, 24 species including 803 strains collected during the one year period from July 1996 to June 1997, were used as the test strains in the second survey. The MIC distribution of CDTR in each test strain that was isolated at the time of the second survey demonstrated a tendency similar to that observed in the test strains isolated for the first survey. However, the MIC₈₀ values in Streptococcus pneumoniae, Proteus mirabilis, Providencia rettgeri and Morganella morganii were 4 to 8 times higher in the second survey, compared to the first survey. Of the strains collected during the second survey, CDTR demonstrated strong antibiotic action against 5 major species which cause respiratory. infections (methicillin susceptible Staphylococcus aureus, Streptococcus pyogenes, S. pneumoniae, Haemophilus influenzae, Moraxella catarrhalis). Furthermore, CDTR inhibited the growth of all the strains of H. influenzae, S. pyogenes, S. pneumoniae, S. aureus and M. catarrhalis at MICs of 0.05, 0.1, 0.39, 0.78 and 0.78μg/ml respectively. The above results coincide with those reported by each medical institution during the development stage of this drug.

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Multidrug-Resistant mutants of Pseudomonas aeruginosa, which are characterized as overproducing OprJ (nfxB-type), are known to emerge after treatment with norfloxacin (NFLX). We obtained P. aeruginosa R 2 of a nfxB-type drug-resistant strain from P. aeruginosa PAO 1 on NFLX (3.13μg/ml) containing agar plates. Differing from P. aeruginosa PAO 1, P. aeruginosa R 2 was highly resistant against cefepime (CFPM), cefzopran (CZOP), cefpirome (CPR), chloramphenicol (CP), tetracycline (TC) and quinolones. We studied the change in susceptibility of these strains against various β-lactams in the presence of fosfomycin. The synergystic effects could be observed on CFPM, CZOP, CPR, CAZ or NFLX in combination with fosfomycin. Although fosfomycin did not alter the amount of OprJ protein in P. aeruginosa R 2 it appeare to accumulate the amount of CFPM in the periplasm of P. aeruginosa R 2. Thus, we concluded that fosfomycin potentiated the bactericidal activities of β-lactams against multi-drugresistant P. aeruginosa not by reducing efflux proteins, but by increasing the drug concentration by some unknown mechanisms.

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Although the relationship between β-lactam antibiotics and penicillin-binding proteins (PBPs) has been frequently investigated, their clinical benefits have not yet been elucidated. In the present study, the affinities of several, β-lactam antibiotics, having different fundamental structures, to PBPs of Escherichia coli were determined in order to investigate the effects of these antibiotics on MIC, bactericidal action, and morphological changes. The affinity of β-lactam antibiotics to PBPs was represented by the inhibitory ratio (Ir:%) of binding of [³H] benzylpenicillin to the PBPs of E. coli, using the method described previously by Spratt. Two-fold diluted, β-lactam antibiotics from 1/32 MIC to 32 MIC were added to membrane fractions of E. coli. The correlation between the concentrations of the antibiotics pre-added and Ir was the highest for PBP 1 B. The correlation coefficient was 0.4661 at an Jr of 10% and 0.7569 at an Jr of 90%, showing a concave linear pattern. The MIC seems difficult to explain in terms of the affinity of β-lactams to PBP 1 B alone. Thus. Jr of each β-lactam to each PBP at their MIC points was obtained in order to determine the correlation between Ir and MIC. The correlation coefficient was the highest with 0.8257 for PBP 1 B, and with 0.5 or higher for other PBPs, suggesting that MICs of β-lactams can be explained by the Irs for all PBPs at their MIC point. Multiple regression analysis was conducted using MIC as a criterion variable and Jr as an explanatory variable. The results indicated that MIC was significantly correlated with Irs to PBP 1 B plus PBP 4, Jr to PBP 3, and PBP 2. Similarly, discriminant analysis of bactericidal action and morphological changes was conducted by categorizing the results obtained after 2 hour cultivation with each 4 MIC of, β-lactams. Whereas bactericidal action increased as Ir to PBP 4 increased, it significantly decreased as Irs to PBP 2 plus PBP 3 increased. Significant filamentous formation and ovoid formation of the cells occurred in proportion to increases in Ir to PBP 3 and to PBP 2, respectively. On the other hand, significant bulge formation was seen with increases in both Ir to PBP 4 and to PBP 1 B. These results suggest that awareness of low-molecular weight PBP, which has not been evaluate as a target of β-lactams at this time, is required for the development of β-lactams.

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The growth of Bacteroides species in the environment of mixed infection was studied. The growth of Bacteroides species in a nutritionally limited in vivo environment with antibiotics is different from that under optimum conditions in the laboratory. In an attempt to mimic possible in vivo conditions, slow growing Bacteroides species were produced by limiting nutrition or in an sub-mic antibiotic (6.25μg/ml cefmetazole) environment. Clinical strains of Bacteroides species were used, and five strains of Bacteroides species (Bacteroides fragilis 2, Bacteroides caccae 2, Bacteroides eggerthii 1) were obtained; which grew slowly at 37.5°C in GAM broth under 100% CO₂. About 100 colony forming units per ml (CFU/ml) Bacteroides species were cultured alone or in the presence of 10⁶CFU/ml Pseudomonas aeruginosa at 37.5°C in GAM broth in an anaerobic environment. The numbers of viable cells were counted at various times of incubation. One slowly growing B. fragilis strain prepared by the limiting nutrition showed optimal growth without dormant or slow growing states in the presence of P. aeruginosa, and the number of viable cells after 30 hours was about 10⁹CFU/ml. Four Bacteroides strains prepared with cefmetazole exposure showed slow growth in the absence of P. aeruginosa. On the otherhand, they showed optimal growth in the presence of P. aeruginosa. All slow growing strains in this study almost recovered their optimal growth in the presence of P. aeruginosa. Therefore, the choice of antimicrobial agents should include at least agents with activity against both Bacteroides species and P. aeruginosa in treatment of polymicrobial aerobic/anaerobic bacterial infections.

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We determined the in vitro combination effects of vancomycin (VCM) plus flomoxef (FMOX), VCM plus cefpirome (CPR), and VCM plus imipenem (IPM) by the checkerboard method on 560 methicillin-resistant Staphylococcus aureus (MRSA), which were clinically isolated in 1995, 1996 and 1997, and compared their MICs and fractional inhibitory concentration (FIC) index. For these three years, the antibacterial activities of VCM alone showed high levels against the 560 MRSA with the same MIC₉₀ of 1μg/ml, and no strains detected of MIC with more than 4μg/ml. During the same period, each β-actam alone demonstrated the same antibacterial activities with the same MIC₉₀ of 128μ/ml, 64μg/ml and 64μg/ml to FMOX, CPR and IPM, respectively. Combinations of VCM plus FMOX and of VCM plus IPM displayed synergistic effects, an FIC index of≤0.5 against more than 90% of the strains, and additive effects, an FIC index from >0.5 to <2.0 against all other strains. The combination of VCM plus CPR led to additive effects against most strains. None of the combinations showed antagonistic effects, an FIC index of ≥2.0, against any of the strains. By comparing the bactericidal activities against MRSA, the combinations of 0.5μg/ml VCM plus 2, 4 and 8μg/ml FMOX showed higher activities than that of each antibiotic alone. These results suggest that combination therapies of VCM plus FMOX and of VCM plus IPM can be useful in the treatment of MRSA infection.

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