Enlisting the help of various research institutions across the nation, IKEMOTO et al. have been pooling cultures of clinical isolates of respiratory tract infections and mapping out the correlations between patient backgrounds and the causative bacteria and the changes in the sensitivity spectra of the bacteria to various antibacterial and antibiotic agents annually since 1981. The following is a report of the 1986 findings.
During the period from September, 1986 to March, 1987, 558 cases of respiratory infections were reported at 17 institutions across the nation and a total of 657 apparent causative strains were isolated from sputum samples. Of these strains, 75 strains of Staphylococcus aureus, 108 of Streptococcus pneumoniae, 150 of Haemophilus influenzae, 107 of Pseudomonas aeruginosa (non-mucoid production type), 21 of P. aeruginosa (mucoid production type), 32 of Klebsiella pneumoniae, 8 of Escherichia coli, and 55 of Branhamella catarrhalis were subjected to MIC determination of various antibacterial and antibiotic agents to map drug sensitivities. In addition, diagnoses, age distributions by diagnoses, frequencies of infectious diseases, types of isolated bacteria, and usage statuses of the antibacterial and antibiotic agents the times of at isolation were also investigated.
MIC determinations were carried out to investigate susceptibilities of causative organisms of respiratory tract infections to various antibacterial and antibiotic agents. From the 558 cases of respiratory tract infections, 657 strains were detected at concentrations not less than 10^4-6/ml and identified to be the causative organisms. Of these strains, 603 could be used for MIC determination. An overwhelming majority of major causative bacteria, inclusive of H. influenzae and S. pneumoniae, showed sensitivity patterns similar to the sensitivity patterns found a year earlier. P. aeruginosa alone, however, showed some increase in its susceptibility to penicillin and cephem antibiotics.
Regarding patient backgrounds, the age distribution was heavily biased towards the higher end of the scale, which patients with ages of 50 or higher accounting for 77.9%, compared to 73.5% in 1985. When the patients were classified by diagnoses, chronic bronchitis, bacterial pneumonia and bronchiectasis accounted for the majority of the infections: 28.7%, 23.3%, and 19.0%, respectively. The percentages of chronic bronchitis and bacterial pneumonia 28.7% and 23.3%, respectively, were somewhat higher in 1986 than in 1985. The disease which was comparatively frequent in all age groups was bronchiectasis, which accounted for 44.0% in patients with ages 29 years or lower, 18.4% in patients between 30 years and 69 years, and 16.7% in patients with ages 70 years or higher. The next most frequent infection was bacterial pneumonia. Another finding of note was that whereas acute bronchitis was more frequent among young patients, chronic bronchitis was more prevalent in elderly patients.
When classified by diagnoses, H. influenzae, P. aeruginosa, and S. pneumoniae were frequent in most infectious diseases. Of 355 strains isolated from patients with chronic bronchitis, diffuse panbronchiolitis or bronchiectasis, P. aeruginosa, H. influenzae, and S. pneumoniae accounted for 27.9%, 26.2%, and 18.0%, respectively. However, the incidence of H. influenzae has been declining yearly, while the frequencies of P. aeruginosa and S. pneumoniae are increasing. It is noteworthy that the includence of B. catarrhalis is on a slow but steady increase.
In cases of bacterial pneumonia, the frequency of S. aureus was comparatively high.

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In vitro antibacterial activity of 30 β-lactam antibiotics including 2 β-lactamase inhibitors (clavulanic acid and sulbactam) against 2 major pathogenic Nocardia, i. e. Nocardia asteroides group and Nocardia brasiliensis was studied.
Among the antibiotics tested, a newly developed carbapenem antibiotic, imipenem (IPM), was found to be the most active, followed by oxacephem group antibiotic flomoxef (FMOX). IPM exhibited activity against only N. asteroides group (N. asteroides, Nocardia farcinica and Nocardia nova). On the other hand, FMOX showed activity against all pathogenic Nocardia tested.
A 2-to 30-fold decreases in minimum inhibitory concentration (MIC) for N. asteroides, N. brasiliensis and N. farcinica was noted when antibiotics and β-lactamase inhibitors were combined compared to antibiotics alone.
Further combination and enzymatic studies indicated that all pathogenic Nocardia possess β-lactamase except for a half of the strains of N. nova.
These species specific sensitivity patterns of pathogenic Nocardia are discussed in this paper with references to their taxonomic positions.

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Antimicrobial activities (MICs) of cefotetan (CTT) against 575 strains of 16 spp. of Gram-negative bacilli isolated in 1988 were determined to investigate distribution of MICs in comparison with those of cefmetazole (CMZ), cefoxitin (CFX), latamoxef (LMOX) and cefazolin (CEZ). The change in frequencies of incidents of cephem-resistant strains in the latter half of the 1980 were also investigated.
1. Distribution of MIC of CTT varied with test strains. No or very few MICs were at or higher than 12.5μg/ml at an inoculation of 10⁶cfu/ml, thus rates of CTT-resistant strains were low among Escherichia coli, Citrobacter diversus, Klebsiella spp., Proteus spp., Providencia spp., and Haemophilus influenzae. High rates of resistance to CTT were observed, however, among Citrobacter freundii, Enterobacter spp., Serratia marcescens, Morganella morganii, and Bacteroides fragilis.
2. Most CTT-resistant strains of C. freundii, Enterobacter spp. and S. marcescens were also resistant to LMOX. These resistant strains were considered to be multi-resistant strains to antibiotics including oxime type cephalosporins and monobactams.
3. Cephem-resistant E. coli was confirmed to be resistant to 22% of CEZ, 14% of CFX, 10% of CMZ and 2% of CTT tested. The incidence of cephem-resistant E. coli unconditionally showed an increasing tendency.
4. The incidence of resistant strains against cephamycins including CTT is discussed with regard to the mechanism of resistance against all β-lactam antibiotics, and the problem of the appearance of resistant strains is close and inseparable from social background.

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In vitro activities of imipenem/cilastatin (IPM/CS) against 413 clinical isolates were studied through the evaluation of MICs and the results of disk susceptibility tests. The MICs were determined using the agar dilution method at an inoculum level of 10⁶CFU/ml.
The MIC₈₀s of imipenem (IPM) against Staphylococcus aureus and Staphylococcus epidermidis were 25 and 1.56μg/ml, respectively, showing a bimodal MIC distribution. However, the distribution of MICs against other bacteria studied was of monomodal pattern. Group A Streptococcus, Enterococcus faecalis were inhibited by IPM at dose levels less than 0.025 and 6.25μg/ml, respectively. IPM inhibited Escherichia coli at 0.20μg/ml, Klebsiella pneumoniae at 0.39μg/ml, Pseudomonas aeruginosa at 3.13μg/ml except one strain showed a MIC of 25μg/ml, Serratia spp. at 3.13μg/ml except one with MIC>100μg/ml, Citrobacter freundii at 0.78μg/ml and Enterobacter spp. at 0.39μg/ml. Indole (-) Proteus and indole (+) Proteus were inhibited by this drug at levels of 3.13 and 1.56μg/ml, respectively.
The reliability of the IPM disk diffusion susceptibility test in quantitative estimation of antimicrobial activities was well demonstrated using commercialized 8mm diameter Showa disks containing 30μg antibiotic and also disks containing 1-30μg prepared in this laboratory. For the interpretation of the Showa disk susceptibility test, a 4 category system was used. In the 4 category system for Showa IPM disk the following classification of inhibitory zone diameters has been proposed; (+++) MIC≤3μg/ml,(++) MIC>3-15μg/ml,(+) MIC>15-60μg/ml,(-) MIC>60μg/ml. The results of the test using Showa 30μg disk against various clinical isolates were accurately classified into the 4 groups, showing false positive 8 out of 304 strains (2.6%) and false negative 1 of 304 strains (0.3%). With Showa 30μg disks subclassification of strains with MIC less than 3μg/ml cannot be achieved. In this study, however, the differentiation of strains with MICs less than 1μg/ml was made with disks containing 5-10μg, which afforded to set MIC break points at 1 and 3μg/ml.
According to current concepts on pharmacokinetics for antibiotics including the penetration of drugs into tissues and inflammatory fluids, serum protein binding of drugs appears to be one of the important determinants of drug distribution in the body. Only free, unbound drug molecules can readily pass through capillary pores into tissue fluids except in the hepato-billiary system. In addition, serum protein-bound drugs show decreased antimicrobial activities compared to unbound, free molecules. Therefore, levels of free, unbound antibiotics in plasma must be considered with respect to clinical efficacy. Since serum protein binding of IPM is less than 2%, total pharmacokinetic blood levels can be used for the assessment of clinical efficacy of this drug.
Based on antimicrobial activities and pharmacokinetic data obtained through the recommended modest dosage schedule (0.25-0.5g twice a day) for IPM, we, propose the application of MIC break points of 1 and 3μg/ml using disks containing 5-10μg for the disk susceptibility test. This system appears to be more useful than a 4 category system with disks containing 30μg in evaluating antimicrobial activities with respect to clinical efficacy, particularly for the assessment of blood concentration of the drug many folds greater than in vitro MICs.

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From our study on cefuzonam (CZON), a new antibiotic agent, with regard to pulmonary-tissue uptake and use in prophylaxis of postoperative infections in 20 patients undergoing thoracotomy, we were able to reach the following conclusions:
1. Following intravenous infusion over 1 hour of 1g of CZON immediate preoperatively, the mean serum concentration at 1 hour after commencement of the infusion reached at 41.5 /ml (serum peak value), then gradually decreased with a half-life in the β-phase of 1.20 hours.
2. The mean CZON concentration in normal pulmonary tissue at 1 hour after commencement of the infusion was 12.7μg/g with a half-life in the β-phase of 1.28 hours. Although the number of cases examined was small at only 4, the mean pulmonary tissue level in obstructive pneumonitic lesions was 13.7μg/g, and an average half-life in the A-phase was 1.65 hours.
3. Mean bronchiolar CZON concentrations at 2, 4, and 6 hours after commencement of the infusion were 14.5, 3.1, and 2.8μg/g, respectively, with a half-life in the β-phase of 3.53 hours.
4. Among the patients undergone thoracotomy with use of CZON, no postoperative infections occurred nor severe adverse reaction was observed.
CZON attained good transfer in the pulmonary tissues, particularly in the bronchiolar tissue, hence it appears useful as a prophylactic against postoperative infections in respiratory disease.

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Cefotaxime (CTX) was intravenously administered in a dose of 1g to patients just prior to lung surgery. Lung tissue specimens were collected at 1, 2 and 3 hours after the CTX administration, and the concentration of CTX in each specimen was determined. At the same time, the concentration of CTX in the serum was also measured. The results are summarized below.
1. Determination of the CTX concentration in the lung tissue using bioassay showed values of 3.78±1.93μg/g at 1 hour after CTX administration, 1.91±0.92μg/g at 2 hours, and 1.19± 1.04μg/g at 3 hours.
2. Determination of the CTX concentration in the serum using bioassay showed values of 36.9±14.4μg/ml at 30 minutes after CTX administration, 22.5±10.5μg/ml at 1 hour, 12.8±6.32 g/ml at 2 hours, 8.52±5.54μg/ml at 3 hours, and 3.98±3.19μg/ml at 6 hours. The serum half-life was calculated to be 1.82 hours.
3. The CTX concentration of 3.78±1.93μg/g in the lung tissue at 1 hour after CTX administration was more than 10 times higher than the MIC₈₀ values for Streptococcus pneumoniae (MIC₈₀≤0.025μg/ml) and Klebsiella pneumoniae (0.05μg/ml), 2 of the principal causative organisms of respiratory tract infections. The pattern of change in concentrations of CTX in the serum of these surgical patients was concluded to be similar to the pattern in healthy adult subjects.
On the basis of the results summarized above, it appears that CTX is a useful antimicrobial agent for application in the field of respiratory surgery

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Acute toxicity studies of miporamicin and its trace ingredients, degradations and metabolites were conducted in mice and rats.
1. Following oral administration of miporamicin (MPM), none died among mice or rats even at the highest dosage levels. Therefore, its LD₅₀ values were estimated to be >2,500mg/kg for mice and >2,000mg/kg for rats. The LD₅₀ value of MPM was the highest by oral route, followed, in order, by subcutaneous route and intravenous route. There was no difference in this respect between sexes of animals studied.
2. No signs of abnormalities were observed among mice or rats following oral administration of MPM. In animals dosed with MPM by subcutaneous route, such inflammatory reactions as swelling, subcutaneouse hyperemia and hemorrhage, and loss of hair incrustation at the site of injection were noted. Animals among those given MPM by intravenous injection developed postdosing depression of motor activity, respiratory depression or arrest, tremor and convulsion.
3. Deaths from administration of MPM were estimated to be due to paralysis of respiratory function inasmuch as fatally affected animals exhibited respiratory depression and cyanosis and, subsequently, respiratory arrest was followed by cardiac arrest.
4. Trace ingredients, metabolites and degradation products of MPM proved to be essentially the same as MPM in acute toxicities.

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A 28-day oral dosage test of miporamicin (MPM), a new macrolide antibiotic, was performed to assess its toxicologic potential in groups of male and female rats receiving the compound in feed. Five graded dosage levels of 0, 3,200, 8,000, 20,000, and 50,000ppm were employed for treatment with MPM in feed and the treatment period was followed by a 28-day recovery phase observation period.
1. No deaths occurred throughout the course of the experiment. Animals receiving 50,000 ppm developed signs: ruffled hair coat and emaciation, which disappeared following withdrawal of the drug.
2. The MPM-50,000 group displayed depression of weight gain and decrease of feed and water intake during the treatment period. During the posttreatment recovery phase observation period the animals showed recovery in weight gain rate as well as in feed and water intake.
3. The achieved compound dosage was 273mg/kg/day in males and 288mg/kg/day in females in the MPM-3,200 group, 721 and 773mg/kg/day respectively in the MPM-8,000group, 1,738 and 1,856mg/kg/day in the MPM-20,000 group, and 3,405 and 3,611mg/kg/day in the MPM-50,000 group.
4. Hematological examinations revealed low values for RBC, WBC, hematocrit and hemoglobin concentration and decreased platelet counts in the MPM-50,000 group, which were considered to be due to the decreased feed intake. These changes disappeared or abated following withdrawal.
5. Of various serum biochemical parameters assessed, total protein, albumin, glucose and triglycerides showed lowered values in the MPM-50,000 group. All these changes were considered to be attributable to the decreased feed intake. During the ensuing recovery phase observation period, all these parameters showed restoration or abatement in parallel with the recovery in feed intake.
6. Urine analysis disclosed decrease of urine volume, lowered electrolyte concentration and elevation of urine osmolarity in the MPM-20,000 and the MPM-50,000 groups. These changes were considered to be secondary to cecal enlargement which is commonly seen with antibiotic medication, or to the decreased feed and water intake. Following drug withdrawal, all these changes disappeared with the recovery in feed and water intake and abatement of cecal hyperplasia.
7. At terminal necropsy, diminution of body fat and atrophy of the spleen and thymus that correlated with emaciation were noted in the MPM-50,000 group. Dose-related enlargement of the caecum was also noted in the treated groups. All these changes disappeared or abated following withdrawal.
8. Organ weight analysis showed low spleen and thymus weights in the MPM-50,000 group possibly attributable to the decreased feed intake, and a dose-related increase ofcecal weight. These changes disappeared or abated following withdrawal.
9. Histopathological examination revealed a mild degree of splenic atrophy, with tortuosity of the capsule and a slight decrease of perifollicular lymphocytes, in the MPM-50,000 group. In these animals, the thymus was also somewhat atrophic and the mesenteric lymph nodes showed a slight decrease of lymphocytes (T cells) in the paracortical regions. Increased fat cells were seen in the bone marrow of these rats. All these changes were due to the poor nutritive intake, and recovered by the end of recovery phase observation period.
10. Electron microscopic examination did not reveal any abnormalilies in the liver or the kidneys.
11. From the data obtained, the maximal non-effective level of MPM under the experimental conditions described was estimated to be 20,000ppm at which no malnutritive changes were observed.

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A six-month oral toxicity test of the new macrolide antibiotic miporamicin (MPM) was carried out in male and female rats receiving the compound in feed at concentrations of 1,280, 3,200, 8,000, or 20,000ppm. The animals were further observed for recovery for 2 months after the completion of the treatment period.
1. No death occurred at any dosage levels throughout the study period. The only notable signs observed were marginal blepharitis and aging-associated changes that were seen occasionally among the treated and control rats. There were no symptomatic changes of particular note.
2. Body weight, feed intake and water consumption data did not reveal any noticeable change.
3. The achieved test compound intake was 69mg/kg/day for males and 82mg/kg/day for females in the MPM-1,280 group, 176mg/kg/day for males and 207mg/kg/day for females in the MPM-3,200 group, 436mg/kg/day for males and 519mg/kg/day for females in the MPM-8,000 group, and 1,080mg/kg/day for males and 1,280mg/kg/day for females in the MPM-20,000 group.
4. No changes attributable to these treatments were noted in the hematological examination or serum biochemical tests.
5. Urinalysis disclosed mild changes with respect to urine volume, urinary electrolyte concentration and osmolarity. Animals recovered from all these changes during the recovery phase observation.
6. At gross pathologic examination a dose-related enlargement of the caecum was observed. The change disappeared or diminished following the 2-month recovery phase observation.
7. Organ weight analysis showed a dose-related increase of weight of the caecum. Animals recovered or abated in this respect during the 2-month recovery phase observation.
8. Histopathologic examination revealed no adverse toxicologic changes other than spontaneous or aging-associated ones.
9. No abnormalities were noted in the liver or in the kidneys as examined by electron microscopy.
10. The maximum non-effective level of MPM thus was estimated to be 20,000ppm at which no organic damage occurred.

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Experiments were conducted to assess the effects of miporamicin (MPM) on prenatal and postnatal development of fetuses and offsprings of rats receiving the compound at oral dosages of 40, 200, or 1,000mg/kg/day during the organogenesis stage of gestation. The drug treatment had no appreciable effect on maternal body weight during pregnancy or lactation period. The rats showed decreased food intake and increased water intake during gestation period in the groups given ≥200mg/kg/day, and increased food and water intakes during lactation period in the group given 1,000mg/kg/day. There was no macroscopic evidence of changes indicative of any effect of the treatment in the viscera of rat dams at terminal necropsy. Observation of the fetuses did not reveal any effect of the treatment with MPM with respect to the number of implantations, the number of living fetuses, the death rate of fetuses or incidence of external, visceral, or skeletal anomalies. Male fetal weights were low in the groups given ≥200mg/kg/day. Observation of the offspring post partum failed to disclose any abnormalities indicative of adverse effects of the treatment with respect to birth index, viability index, weaning index, postnatal external differentiation, body weight changes, external morphology, skeleton, viscera, organ weight, functional and behavioral tests, emotion, learning ability, or reproductive performance, or in respect of prenatal development of their fetuses (F₂). It is concluded from the results that no effect dose levels of MPM was 40mg/kg/day in rat dams, 40mg/kg/day also for their fetuses, and 1,000mg/kg/day for postnatal development of the offspring under the experimental conditions described.

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A teratogenicity study on miporamicin (MPM) was performed in New Zealand White rabbits. Following suitable dose range finding experiments, 60 mated rabbits were randomised into 4 treatment groups, each containing 15 animals. These animals were dosed once daily, by oral gavage, over days 6-18 inclusive of gestation, where the day of mating was designated as day 0. The dose levels applied were 0, 50, 100 and 200mg/kg/day.
A standard dose volume of 5ml dosing suspension per kilogram body weight was applied, the vehicle being an 0.5% solution of carboxymethylcellulose in distilled water.
There was an effect on maternal food consumption at 200mg/kg/day: cessation of consumption by over one quarter of the animals in the group.
Under the conditions of this study, MPM had no effect on the outcome of pregnancy at dose levels of up to 200mg/kg/day. There was generally no significant effect on either the mother or the conceptus at 100mg/kg/day.

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Experiments were performed to assess antigenicity and contact sensitivity of miporamicin (MPM) with the results leading to the conclusions as follows:
1. In the test to find out if an active systemic anaphylactic reaction would occur in guinea pigs, none of the animals developed any typical signs of anaphylactic reaction.
2. All the sera samples obtained from guinea pigs which had been given sensitizing, injections of MPM proved negative for homologous PCA reaction.
3. In a test to see if PCA reaction would occur in rats, all the sera samples obtained from mice which had been given sensitizing injections of MPM did not cause positive reactions.
4. MPM was negative for contact sensitivity tested in guinea pigs; thus, no evidence of contact sensitizing potential was observed.
Thus, MPM has proven to be not antigenic in guinea pigs or mice, nor to have any contact sensitivity in guinea pigs under the testing conditions employed

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Mutagenic activity of miporamicin (MPM), a new macrolide antibiotic for animal use, was examined using the reversion test with bacteria, the chromosomal aberration test with mammalian cells in culture and the micronucleus test with rodents.
In the reversion test, MPM exhibited severe growth inhibition effect on the test bacteria but caused no increase of revertant colonies over the baseline levels, alone or in combination with S 9 mixture.
In the chromosome aberration test, MPM induced a medium grade increase of chromosome aberrations at high concentrations, but induced no increase of polyploid cells over the control level.
In the micronucleus test, MPM had no effect on induction of micronucleated polychromatic erythrocyte even at the 1/2 LD₅₀ dose.
From these results, we concluded that MPM has no effect on the induction of point mutations but has a weak clastogenicity which is detectable only by in vitro tests.

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Local irritation effect of miporamicin (MPM), a new macrolide antibiotics for animal use, on skin and ocular tissues was determined in rabbits by the primary skin irritation test and ocular irritation test, respectively. In the former test, P. I. I. and inflammatory findings increased with increasing exposure time to MPM.
A short-time contact with MPM caused relatively little effect on ocular tissues. Contrarily, a long-time contact with MPM caused medium or high degree of inflammatory reactions on cornea, iris and conjunctiva tissues. The increased inflammation was naturally accompanied with increases of the DRAIZE score. All these results suggest that MPM per se has local irritation effect on both skin and ocular tissues.

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