The patterns of antimicrobial susceptibility of 972 isolates of Serratia marcescens obtained from 5 hospitals during 1980-1983 were studied by using an agar dilution method. Of these strains, 572 (58.8%) were isolated from urine, 199 (20.5%) from sputum, 83 (8.5%) from pus and 118 (12.1%) from other clinical specimens. Antimicrobial agents used were kanamycin (KM), gentamicin (GM), tobramycin (TOB), dibekacin (DKB), sisomicin (SISO), butirosin (BT), amikacin (AMK), paromomycin (PRM), lividomycin (LVDM), ribostamycin (RSM) and neomycin (NM).
Among these 972 strains, 878 (90.3%) were resistant to one or more AGs. There was a little difference in the resistance rates in the isolates by specimens, i. e., 94. 8% of the isolates from urine, 85.5% from pus, 83.4% from sputum and 83.9% from other specimens were resistant to at least one of the antibiotics.
Of 878 resistant strains, the strains with RSM-resistance (88.2%) were isolated most frequently, then followed by those with DKB (57.7%)-. KM (56.4%)-, TOB (55.8%)-, BT (50.4%)-, SISO (47.6%)-, AMK (32.8%)-, GM (32.6%)-, NM (19.4%)-, PRM (18.8%)- and LVDM (16. 3%)- resistance. There was little difference by year except 1981 in the resistance rates of the isolates resistant to AGs, other than PRM, LVDM, NM and GM. The rates of those resistant to PRM, LVDM and NM decreased obviously year by year. On the other hand, the rate of those resistant to GM increased.
Among 878 resistant isolates, 189 (21.5%) carried transferrable R plasmid mediating AGs-resistance. The rate of strains carrying R plasmid in isolates in each specimen was as follows: 29.5% from urine, 18.3% from pus, 5.4% from sputum and 7.1% from other clinical specimens. During the 4 years, the rate in the isolates from urine decreased apparently. The rates in the isolates resistant to DKB, KM, TOB and SISO ranged from 46% to 58%, in 1980, and decreased to 16-18% in 1983. The rate in those resistant to GM ranged from 86% to 94% in 1980-1981, and decreased to 22% in 1983. On the other hand, the rates in those resistant to AMK and BT were very low, ranging from 3% to 6% in 1981-1983.
The AGs modifying enzymes in the transconjugants were presumed from their resistant patterns. AAD (2'') was most prevalent and was detected in the isolates of all 5 hospitals every year. APH (3') I and AAC (6') IV were also detected in those of 5 hospitals, whereas APH (3') II was detected in 3 hospitals.

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To explore the action mechanism of tioconazole (TCZ), a novel topical imidazole-antimycotic, by which it inhibits the growth of or kills fungi, the biochemical effect of the drug on a sensitive parent strain of Candida albicans (TIMM 0144), as well as resistant mutant strains induced therefrom, was studied. The results are summarized as follows:
(1) All the syntheses of protein, RNA, DNA, cell wall homopolymers and total lipids that proceeded linearly in growing cells of C. albicans TIMM 0144 were instantly and completely arrested after exposure to fungicidal concentrations (40 and 80μg/ml) of TCZ. When the cells were treated with lower levels of the drug, the initial rate of biosynthesis was decreased to different extents from one cellular component to another. Biosynthesis of total lipids appeared to be most susceptible to the relatively low concentrations of TCZ and was significantly inhibited by 1.25 μg/ml of the drug at which it scarcely affected biosynthesis of any other major cellular components.
(2) TCZ markedly enhanced release of K⁺ and inorganic phosphate from cells of C. albicans TIMM 0144 into the ambient medium. The rate and extent of release of these intracellular materials was dependent on the drug concentrations in the medium; significant amounts of K⁺ and inorganic phosphate were released in the presence of ≥5μg/ml and 40μg/ml, respectively, of TCZ within 10 min after addition of the drug and an almost complete release of the materials was induced by 80μg/ml of the drug.
(3) When ≥5μg/ml of TCZ was added to C. albicans TIMM 0144 cell suspensions in deionized water, the pH value of the medium was rapidly increased. pH values of ≥0.1, ≥0.5 and ≥1.0 were attained within 1 min after addition of 20, 40 and 80μg/ml, respectively, of TCZ.
(4) Cellular lipids were extracted from cells of C. albicans TIMM 0144 grown with graded concentrations of TCZ for chromatographic analyses of sterol composition. Non-saponifiable lipids from cells treated with 0.08μg/ml or higher concentrations of TCZ were characterized by a marked decrease in the content of ergosterol, the major sterol of this fungus, and an excessive amount of a-methylated sterols as compared with those from untreated cells.
(5) Two mutant strains resistant to TCZ were induced by mutagenesis from C. albicans TIMM 0144 and their microbiological and biochemical responses to the drug were compared with those of the parent strain. Whereas IC₁₀₀ (MIC) values of TCZ for both mutant strains increased only by 2- to 4-fold, IC₅₀ values of TCZ for the strains increased by 100-fold or above, as compared with comparable values for the parent strain. However, the extent of K⁺ release induced by relatively high levels (40 and 80μg/ml) of TCZ from cells of the mutant strains was equal to or greater than that from cells of the parent strain. Both of the two mutant strains were shown to be defective in ergosterol biosynthesis.
(6) All the results lead us to the postulation that TCZ may have a dual mechanism of antifungal action; TCZ appear to cause inhibition of sterol biosynthesis without effect on the fungal cell membrane at sub-inhibitory drug levels, both inhibition of sterol biosynthesis and reversible membrane damage at fungistatic levels, and irreversible membrane damage at fungicidal levels.

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The mode of antifungal action of tolciclate, a new thiocarbamate antimycotic was studied using yeast-phase cells of a relatively sensitive fungus S. schenckii as the test organism. The results are summarized as follows:
(1) As monitored by optical density, dry weight and viable counts of fungal cultures, the extent of growth inhibition by tolciclate increased with increasing drug concentrations and a fungistatic or slight fungicidal activity was exerted by 1.25 to 80 μg/ml of the drug.
(2) Tolciclate inhibited synthesis of cell wall polysaccharides as well as lipids to greater extents than synthesis of the other cellular components in growing fungal cultures.(The extent of inhibition by the drug appeared to be comparable to this antifungal activity.)
(3) Tolciclate had no effect on the cell membrane function or the respiratory activity of S. schenckii cells.
(4) Tolciclate caused no significant inhibition of in vitro activities of DNA polymerases and RNA polymerases, both of which were prepared from S. cerevisiae.
(5) The in vitro activities of synthetases of three major cell wall polysaccharides prepared from S. schenckii were affected by tolciclate in a different fashion from synthetase to synthetase. The high concentrations of tolciclate substantially inhibited the chitin synthetase activity and, to a lesser extent, the mannan synthetase activity, but they were without effect on the β(1, 3)-glucan synthetase activity. The inhibition by tolciclate of the chitin synthetase was of competitive type and exhibited K_i value of 168 μM.
(6) Ergosterol synthesis in S. schenckii cells was potently inhibited by tolciclate even at concentrations lower than MIC. They induced significant reduction in ergosterol content and heavy accumulation of squalene, a biosynthetic precursor of sterol.
(7) All these results led us to the suggestion that inhibition by tolciclate of sterol synthesis through blocking the step of squalene epoxidation in a fungal sterol biosynthetic pathway may be primarily involved in the antifungal action of the drug.

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In an attempt to obtain preclinical data which can explain the excellent therapeutic effectiveness of tioconazole (TCZ) for the topical treatment of dermatophytosis and other superficial mycoses, studies on the in vitro activity of this drug were carried out, mainly focussing on its fungicidal action toward several strains of pathogenic fungi, in comparison with miconazole nitrate (MCZ) and clotrimazole (CTZ) as reference drugs. They were followed by in vivo studies in which the topical effectiveness of cream preparations of TCZ, as well as the two reference drugs, in experimental Trichophyton mentagrophytes infection in guinea-pigs was tested. The estimation of efficacy of treatment with active formulations was made on the basis of the score of skin lesion, the rate of positive skin cultures and dermal contents of fungal chitin.
The results are summarized as follows:
(1) In growing cultures of Candida albicans strains, TCZ exerted a potent killing action at drug concentrations of 20 μg/ml or above. The extent and rapidity of this fungicidal effect of TCZ was greater than that of MCZ or CTZ if compared on the weight basis.
(2) When T. mentagrophytes conidia suspension was exposed to TCZ under the condition where no germination or subsequent outgrowth occurred, the imidazole significantly reduced viable counts recovered from the suspension at drug levels lower than those which were required for attaining a similar extent of lethal effect on growing cells of C. albicans. The fungicidal activity of TCZ toward T. mentagrophytes conidia was again superior to that of MCZ or CTZ.
(3) The in vitro antibiotic and cidal activity of TCZ against these susceptible fungi was only slightly affected by the pH value of testing media.
(4) The three parameters employed in the present in vivo studies denoting the severity of dermal infections were virtually comparable to each other.
(5) Both 0.2 and 1% creams of TCZ showed good therapeutic efficacy, although the former was less effective than the latter.
(6) Although no significant difference was observed in the therapeutic efficacy among the comparable cream preparations of TCZ, MCZ and CTZ when estimation was made at the end of 17-day experimental period of time, treatment with 1% cream of TCZ appeared to render the skin lesions to heal more rapidly than did treatment with comparable creams of MCZ or CTZ.

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To examine the effect of antibiotics on phagocytosis and bactericidal activity of human neutrophils in vitro, 17 antibiotics and 2 antibacterial agents were tested using peripheral neutrophils from normal subjects or patients with complicated pyelonephritis. The effect on phagocytosis was measured by counting engulfed cocci by microscopy, or those which had been labeled with ³H-Lysine with a liquid scintillation counter. Bactericidal activity was measured by QUIE's method. The effect of antibiotics on phagocytosis and bactericidal activity varied with the kind and concentration of antibiotic used. While many antibiotics suppressed phagocytosis and bactericidal activity below the normally clinically accepted maximum concentration levels in blood, CPIZ enhanced these activities markedly, in which the effect of antibiotics differed normal subjects and patients with pyelonephritis.

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In order to investigate the penetration of cefotaxime (CTX) into the skin, 1 gram of CTX was administered by one bolus intravenous injection to patients during operations for full-thickness skin grafts under general anesthesia. The CTX levels in the serum and the skin were determined at specified time intervals using bioassay and HPLC methods.
Thirty minutes after administration, the mean CTX level in the serum was 52.4 μg/ml by bioassay and 44.4 μg/ml by HPLC. In both methods, the curves for the declining levels were similar over the specified intervals of time, and in 4 hours, they reached 5.1 μg/ml and 2.8 μg/ml, respectively.
Meanwhile, the mean level of desacetyl-cefotaxime (D-CTX) in the serum was 5.4 μg/ml by HPLC 30 minutes after administration, equivalent to 12% of ahe CTX concentration at the corresponding point in time.
Thirty minutes after administration, the mean level of CTX in the skin was 7.8 μg/g by bioassay and 4.7 μg/g by HPLC. These values gradually decreased to 2.2μg/g and 1.2 μg/g after 4 hours.
The mean concentration of D-CTX in the skin reached a peak of 11.1 μg/g one hour after administration according to the HPLC, showing a gradual decline afterwards. The levels of D-CTX were higher than the levels of CTX in the skin.
The above results show that both CTX and D-CTX are maintained at an effective concentration in the skin for a long period. Therefore, good therapeutic effect against skin infection can be expected.

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Clinical and fundamental studies on a new cephalosporin, cefpiramide (CPM) were carried out and following results were obtained;
1. Clinical effects in total of 24 cases with complicated ulcer including burns were as follows; excellent response was seen in 1 case, good in 15 cases, fair in 6 cases, poor in 1 case and unknown in 1 case. Efficacy rate was 69.6%.
Especially, in 18 cases with burned infection, excellent response was seen in 1 case, good in 11 cases, fair in 4 cases, poor in 1 case, and unknown in 1 case. On the burned infection, efficacy rate was 70.6%.
A 100% or over 90% successful grafting rates were seen in all cases with skin graft.
2. Antibacterial activity of cefpiramide (CPM) were examined and compared with those of other cephalosporins, cefoperazone (CPZ), ceftizoxime (CZX), cefsulodin (CFS) against clinically isolated strains from burned infection site.
Strains were as follows: S. aureus (23 strains), S. epidermidis (5), S. faecalis (13), K. oxytoca (5), P. aeruginosa (27) and P. nzaltophilia (6).
The antibacterial activity of CPM against S. aureus, S. faecalis and P. maltophilia was recognized to be superior to CPZ.
CPM exhibited almost similar activity against P. aeruginosa with that of CSF and against S. epidermidis with CPZ, but showed somewhat less activity against K. oxytoca than that of CPZ and CZX.

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A double blind group comparative study was performed to ascertain efficacy and safety of cefpiramide (CPM) as compared with cefotiam (CTM) in treatment of postoperative wound infections (Trial A), postoperative infections in the abdominal cavity and dead space (Trial B).
Either of the drugs was administrated by intravenous drip infusion at 1 g twice a day, and the following results were obtained.
1. There was no significant difference in the patient characteristics between the CPM and the CTM groups in the Trial A. In the Trial B, however, in the type of bacterial infection, there was more cases of monobacterial infection in the CPM group than in the CTM group, even though there was no significant differences in the other backgrounds.
2. Clinical efficacy rates were 87%(62/71) in the CPM group and 77%(55/71) in the CTM group in Trial A, and 85%(47/55) in the CPM group and 58%(31/53) in the CTM group in Trial B. In Trial B, the statistical analysis showed significantly higher result in the CPM group than that in the CTM group by x²-test and U-test.
3. Final overall improvement rates were 79%(56/71) in the CPM group and 72%(51/71) in the CTM group in Trial A. In Trial B, they were 78%(43/55) and 58%(31/53) for the CPM and CTM group, respectively. In Trial B, this result was significantly higher in the CPM group than in the CTM group by x²-test.
4. As for bacteriological efficacy, the eradicated rates of clinical isolates were 53%(28/53) and 46%(26/57) in the CPM and CTM group in Trial A, respectively. In Trial B, the eradication rates were 59%(26/44) and 38%(10/32) in the CPM and the CTM group, respectively. The eradication rate in the CPM group was significantly higher than that in the CTM group by x²-test in Trial B.
5. Side effects were noted in 5 cases (3%) of CPM group and 3 cases (2%) of CTM group, and 7 of them were allergic except for a case of diarrhea. Abnormal laboratory findings were noted in 7 cases (5%) of CPM group and 16 cases (12%) of CTM group, and most of them were those as hepatic dysfunction. Significant differences were not observed between the two drugs, either in kind of side effect, or frequency of abnormal laboratory findings.
6. On clinical usefulness, there were no significant difference in the CPM and the CTM group in Trial A. In Trial B, the rate of usefulness in the CPM group was significantly higher than that in the CTM group by x²-test and U-test.
7. When clinical efficacy, final overall improvement, bacteriological effect and clinical usefulness were stratified by the type of bacterial infection in Trial B, only bacteriological effect of CPM (68%) in the monomicrobial infection was significantly superior to that of CTM (33%).
As a result, it was concluded that CPM was useful for the treatment of postoperative infections.

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