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Background: MRSA (methicillin-resistant Staphylococcus aureus)-infective endocarditis (IE) is associated with high morbidity and mortality. This study aimed to assess data from patients with MRSA-IE across multiple facilities in Japan, with a specific focus on antimicrobial therapy and prognosis., Methods: This retrospective study enrolled patients with a confirmed diagnosis of IE attributed to MRSA, spanning the period from January 2015 to April 2019., Results: Sixty-four patients from 19 centers were included, with a median age of 67 years. The overall mortality rate was 28.1% at 30 days, with an in-hospital mortality of 45.3%. The most frequently chosen initial anti-MRSA agents were glycopeptide in 67.2% of cases. Daptomycin and linezolid were selected as initial therapy in 23.4% and 17.2% of cases, respectively. Approximately 40% of all patients underwent medication changes due to difficulty in controlling infection or drug-related side effects. Significant prognostic factors by multivariable analysis were DIC for 30-day mortality and surgical treatment for 30-day and in-hospital mortality. For vancomycin as initial monotherapy, there was a trend toward a worse prognosis for 30-day and in-hospital mortality (OR, 6.29; 95%CI, 1.00-39.65; p = 0.050, OR, 3.61; 95%CI, 0.93-14.00; p = 0.064). Regarding the choice of initial antibiotic therapy, statistical analysis did not show significant differences in prognosis., Conclusion: Glycopeptide and daptomycin were the preferred antibiotics for the initial therapy of MRSA-IE. Antimicrobial regimens were changed for various reasons. Prognosis was not significantly affected by choice of antibiotic therapy (glycopeptide, daptomycin, linezolid), but further studies are needed to determine which antimicrobials are optimal as first-line agents., Competing Interests: Declaration of Competing interest Tokimatsu I. received scholarship donations from Shionogi Pharmaceuticals, Inc. and Daiichi Sankyo Co. Ltd. Ukimura A. received scholarship donations from Shionogi Pharmaceuticals, Inc. Hiroshige Mikamo received speaker honoraria from MSD K.K., FUJIFILM Toyama Chemical Co., Ltd., Miyarisan Pharmaceutical Co., Daiichi Sankyo Co., Ltd., Pfizer Japan Inc., Sanofi K.K., Sumitomo Pharma Co., Ltd., Kyorin Pharmaceutical Co., Ltd., Shionogi & Co., Ltd. Japan, Kowa Co. Ltd., Gilead Sciences K.K., GSK Group of Companies, Saraya Co. Ltd., Tsumura & Co. Japan, Nippon Becton Dickinson Company, Ltd., and FUKOKU Co., Ltd., grant supports from Asai Kasei Pharma Co., Shionogi & Co., Ltd., Sumitomo Pharma Co., Ltd., and FUKOKU Co., Ltd. Mitsutake K, Shinya N, Seki M, Ohara T, Uemura K, Fukunaga M, Sakai J, Nagao M, Sata M, Hamada Y, Kawasuji H, Yamamoto Y, Nakamatsu M, Koizumi Y, Aoyagi T, Sawai T, Tanaka T, Izumikawa K, Takayama Y, Nakamura K, Kanemitsu K, Nakajima K, and Akine D have no conflict of interest., (Copyright © 2024 Japanese Society of Chemotherapy, Japanese Association for Infectious Diseases, and Japanese Society for Infection Prevention and Control. Published by Elsevier Ltd. All rights reserved.)

Background: Infective endocarditis (IE) caused by MRSA (methicillin-resistant Staphylococcus aureus) is associated with a high mortality rate. This study aimed to elucidate the characteristics of patients with MRSA-IE in Japan and identify the factors associated with prognosis., Methods: This retrospective study included patients with a confirmed diagnosis of IE caused by MRSA, between January 2015 and April 2019., Results: A total of 65 patients from 19 centers were included, with a mean age of 67 years and 26 % were female. Fifty percent of the patients with IE were had nosocomial infections and 25 % had prosthetic valve involvement. The most common comorbidities were hemodialysis (20 %) and diabetes (20 %). Congestive heart failure was present in 86 % of patients (NYHA class I, II: 48 %; III, IV: 38 %). The 30-day and in-hospital mortality rates were 29 % and 46 %, respectively. Multi-organ failure was the primary cause of death, accounting for 43 % of all causes of death. Prognostic factors for in-hospital mortality were age, disseminated intravascular coagulation, daptomycin and/or linezolid as initial antibiotic therapy, and surgery. Surgical treatment was associated with a lower mortality rate (odds ratio [OR], 0.026; 95 % confidence interval [CI], 0.002-0.382; p = 0.008 for 30-day mortality and OR, 0.130; 95 % CI; 0.029-0.584; p = 0.008 for in-hospital mortality)., Conclusion: Mortality due to MRSA-IE remains high. Surgical treatment is a significant prognostic predictor of MRSA-IE., Competing Interests: Declaration of competing interest Tokimatsu I. received scholarship donations from Shionogi Pharmaceuticals, Inc. and Daiichi Sankyo Co., Ltd. Ukimura A. received scholarship donations from Shionogi Pharmaceuticals Inc. Hiroshige Mikamo received speaker honoraria from MSD K.K., FUJIFILM Toyama Chemical Co., Ltd., Miyarisan Pharmaceutical Co., Daiichi Sankyo Co., Ltd., Pfizer Japan Inc., Sanofi K.K., Sumitomo Pharma Co., Ltd., Kyorin Pharmaceutical Co., Ltd., Shionogi & Co., Japan. Ltd., Kowa Co. Ltd., Gilead Sciences K.K., the GSK Group of Companies, Saraya Co. Ltd., and Tsumura and Co. Japan, Nippon Becton Dickinson Company, Ltd., and FUKOKU Co., Ltd., and grant support from Asai Kasei Pharma Co., Shionogi & Co., Ltd., Sumitomo Pharma Co., Ltd., and FUKOKU Co., Ltd. Mitsutake K, Shinya N, Seki M, Ohara T, Uemura K, Fukunaga M, Sakai J, Nagao M, Sata M, Hamada Y, Kawasuji H, Yamamoto Y, Nakamatsu M, Koizumi Y, Aoyagi T, Sawai T, Tanaka T, Izumikawa K, Takayama Y, Nakamura K, Kanemitsu K, Nakajima K, and Akine D have no conflict of interest., (Copyright © 2024 Japanese Society of Chemotherapy, Japanese Association for Infectious Diseases, and Japanese Society for Infection Prevention and Control. Published by Elsevier Ltd. All rights reserved.)

Background: Nursing- and healthcare-associated pneumonia (NHCAP) constitutes most of the pneumonia in elderly patients including aspiration pneumonia in Japan. Lascufloxacin (LSFX) possesses broad antibacterial activity against respiratory pathogens, such as Streptococcus spp. And anaerobes inside the oral cavity. However, the efficacy and safety of LSFX in NHCAP treatment remains unknown. We aimed to evaluate the efficacy and safety of LSFX tablets in the treatment of patients with NHCAP., Methods: In this single-arm, open-label, uncontrolled study, LSFX was administered to patients with NHCAP at 24 facilities. The study participants were orally administered 75 mg LSFX once daily for 7 days. The primary endpoint was the clinical efficacy at the time of test of cure (TOC). The secondary endpoints included clinical efficacy at the time of end of treatment (EOT), early clinical efficacy, microbiological efficacy, and safety analysis., Result: During the study period, 75 patients provided written informed consent to participate and were included. Finally, 56 and 71 patients were eligible for clinical efficacy and safety analyses, respectively. The median age of the patients was significantly high at 86 years. All patients were classified as having moderate disease severity using the A-DROP scoring system. LSFX tablets demonstrated high efficacy rates of 78.6 % at TOC and 89.3 % at EOT. The risk factors for resistant bacteria or aspiration pneumonia did not affect clinical efficacy. No severe adverse events associated with the study drugs were observed., Conclusion: Oral LSFX is an acceptable treatment option for moderate NHCAP in elderly patients who can take oral medications., Competing Interests: Declaration of competing interest Hiroshi Mukae received lecture honoraria and research grants from KYORIN Pharmaceutical Co., Ltd. Outside of the submitted work., (Copyright © 2024 Japanese Society of Chemotherapy, Japanese Association for Infectious Diseases, and Japanese Society for Infection Prevention and Control. Published by Elsevier Ltd. All rights reserved.)

We report on the findings of the first antimicrobial susceptibility surveillance study in Japan of isolates recovered from odontogenic infections. Of the 38 facilities where patients representing the 4 groups of odontogenic infections were seen, 102 samples were collected from cases of periodontitis (group 1), 6 samples from pericoronitis (group 2), 84 samples from jaw inflammation (group 3) and 54 samples from phlegmon of the jaw bone area (group 4) for a total of 246 samples. The positivity rates of bacterial growth on culture were 85.3%, 100%, 84% and 88.9%, respectively, for groups 1, 2, 3 and 4. Streptococcus spp. isolation rates according to odontogenic infection group were 22% (group 1), 17.7% (group 3) and 20.7% (group 4). Anaerobic isolation rates were 66.9% (group 1), 71.8% (group 3) and 68.2% (group 4). Drug susceptibility tests were performed on 726 strains excluding 121 strains that were undergrown. The breakdown of the strains subjected to testing was 186 Streptococcus spp., 179 anaerobic gram-positive cocci, 246 Prevotella spp., 27 Porphyromonas spp., and 88 Fusobacterium spp. The isolates were tested against 30 antimicrobial agents. Sensitivities to penicillins and cephems were good except for Prevotella spp. The low sensitivities of Prevotella spp is due to β-lactamase production. Prevotella strains resistant to macrolides, quinolones, and clindamycin were found. No strains resistant to carbapenems or penems were found among all strains tested. No anaerobic bacterial strain was resistant to metronidazole. Antimicrobial susceptibility testing performed on the S. anginosus group and anaerobic bacteria, which are the major pathogens associated with odontogenic infections, showed low MIC 90 values to the penicillins which are the first-line antimicrobial agents for odontogenic infections; however, for Prevotella spp., penicillins combined with β-lactamase inhibitor showed low MIC 90 values., Competing Interests: Declaration of competing interest Tetsuya Matsumoto received speaker honoraria from MSD K.K., and Pfizer Japan Inc., Hiroshi Kiyota received scholarship donations from Taisho Toyama Pharmaceutical Co., Ltd., Fujifilm Toyama Chemical Co., Ltd., Daiichi Sankyo Co., Ltd., Astellas Pharma Inc., Taiho Pharmaceutical Co., Ltd., and Sanofi K.K., Kazuhiro Tateda received speaker honoraria from Pfizer Japan Inc., MSD K.K., Sumitomo Dainippon Pharma Co., Ltd., Meiji Seika Pharma Co., Ltd., and Taisho Toyama Pharmaceutical Co., Ltd., and grant support from Hitachi, Ltd., Nissui Pharmaceutical Co., Ltd., BD Japan Co., Ltd., Denka Co., Ltd., Kaneka Co., and Shimadzu Co., received scholarship donations from Fujifilm Toyama Chemical Co., Ltd., Meiji Seika Pharma Co., Ltd., Daiichi Sankyo Co., Ltd., Pfizer Japan Inc., Sumitomo Dainippon Pharma Co., Ltd., Shionogi & Co., Ltd., and Asahi Kasei Pharma Co., Hiromitsu Kishimoto received speaker honoraria from Daiichi Sankyo Co., Ltd., (Copyright © 2020 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

Oral antibiotic therapy for patients with acute exacerbations of chronic obstructive pulmonary disease (COPD) usually involves an aminopenicillin with clavulanic acid, a macrolide, or a quinolone. To date, however, the clinical efficacy and safety of the oral cephalosporin cefditoren pivoxil has not been evaluated in Japanese patients with acute exacerbations of COPD. We conducted a prospective, multicenter, single arm, interventional study from January 2013 to March 2017 to determine the efficacy and safety of oral administration of 200 mg cefditoren pivoxil three times daily for 7 days in a cohort of 29 eligible patients from 15 hospitals. The mean age (SD) of participants was 73.1 (8.1) years and 28 had a smoking history (the mean [SD] of smoking index, 1426.7 [931.7]). The primary efficacy endpoint was clinical response (cure rate) at test of cure, which was set at 5-10 days after treatment ceased. Of the 23 patients finally analyzed, cure was achieved in 15 (65.2%), while 8 (34.8%) remained uncured. Previous experience of acute exacerbations significantly affected the cure rate: none of the three patients who had at least two prior exacerbations were cured, while 15 of the 20 patients with one or fewer prior exacerbations were cured (p = 0.032). The microbiological eradication rate was 88.9% at test of cure. During treatment, mild pneumonia was reported as an adverse event in one patient (3.4%) but resolved within 10 days of onset. We conclude that cefditoren pivoxil represents a viable alternative for antibiotic therapy in patients with few prior exacerbations., (Copyright © 2019 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

Background: Parvimonas micra, a Gram-positive anaerobic coccus, is a rare pathogen for psoas abscess. We describe a case of a patient with iliopsoas abscess caused by P. micra., Case Presentation: An 81-year-old Asian man presented to our department with complaints of fever since the preceding day. Abdominal computed tomography revealed the presence of a low-density mass in the right iliopsoas muscle indicative of a psoas abscess. Computed tomography-guided percutaneous drainage of the psoas abscess was performed. Results of organism cultures of the abscess and blood were positive for P. micra. However, our patient had no known primary focus of infection. On the basis of these findings, a primary psoas abscess caused by P. micra was diagnosed, and treatment with ampicillin/sulbactam 1.5 g, administered intravenously every 8 h, was initiated. By day 7, the patient's white blood cell count normalized. By day 20, his C-reactive protein level was decreased to 0.35 mg/dl., Conclusion: Iliopsoas abscesses caused by anaerobic bacteria are relatively rare, and iliopsoas abscesses caused by P. micra are especially rare. Our patient's case revealed that P. micra can cause iliopsoas abscess. Therefore, clinicians should be aware of the possibility that P. micra may cause iliopsoas abscess.

Plant pathogenic bacteria cause huge yield losses in crops globally. Therefore, finding effective bactericides to these pathogens is an immediate challenge. In this study, we sought compounds that specifically inhibit the growth of Ralstonia solanacearum. As a result, we identified one promising compound, 1-(4-bromophenyl)-6-methoxy-2,3,4,9-tetrahydro-1H-β-carboline, which inhibited the growth of R. solanacearum (Rs1002) from a pilot library of 376 chemicals provided from RIKEN. We further obtained its structural analogues and assessed their ability to inhibit Rs1002 growth. Then we identified five compounds, named ralhibitins A to E, that specifically inhibit growth of Rs1002 at >5 μg/ml final concentration. The most effective compounds, ralhibitins A, C, and E completely inhibited the growth of Rs1002 at 1.25 μg/ml. In addition, ralhibitins A to E inhibited growth of Xanthomonas oryzae pv. oryzae but not the other bacteria tested at a final concentration of 10 μg/ml. Whereas, ralhibitin E, besides inhibiting R. solanacearum and X. oryzae pv. oryzae, completely inhibited the growth of X. campestris pv. campestris and the Gram-positive bacterium Clavibacter michiganensis subsp. michiganensis at 10 μg/ml. Growth inhibition by these compounds was stable at pH 6-9 and after autoclaving. Because Rs1002 grew in the culture medium in which ralhibitins were incubated with the ralhibitin-insensitive bacteria, the unaffected bacteria may be able to inactivate the inhibitory effect of ralhibitins. These results suggest that ralhibitins might be potential lead compounds for the specific control of phytopathogenic bacteria., (Copyright © 2018 Elsevier GmbH. All rights reserved.)

Ceftazidime (CAZ), a cephem antibiotic for injection, was administered at dose of one gram q.i.d. to adult hospital-acquired pneimonia patients, and the clinical efficacy and blood concentration of CAZ was studied in five cases. The assessment of clinical efficacy was "efficacy" in all cases, and early improvement of examination value including body temperature, the value of CRP, and white blood cell counts were obtained. Abnormality of hepatic function as adverse effect was noted in two cases. However, the severity was mild and didn't affect the treatment in both cases. The maximum drug concentration immediately after the end of infusion was 72.1-176.5 microg/mL (median, 82.7 microg/mL) and the trough level was 5.1-72.1 microg/mL (median, 26.6 microg/mL), therefore the blood concentration was maintained a higher level than the MIC of bacteria estimated to be causative. From these results, administration method of ceftazidime one gram q.i.d. was considered to be a good way with increase of drug efficacy by maintaining highly blood concentration over prolonged period, and expected to be a good effect for hospital-acquired pneumonia.

We investigated the activities of linezolid, vancomycin, and teicoplanin in a murine model of hematogenous pulmonary infection with Staphylococcus aureus. Our results demonstrate that linezolid clearly reduced bacterial numbers in the methicillin-resistant S. aureus hematogenous infection model and significantly improved the survival rate of immunocompromised mice infected with vancomycin-insensitive S. aureus compared with vancomycin and teicoplanin. The pharmacokinetic profiles also reflected the effectiveness of linezolid.

Fourteen-membered macrolides (e.g. clarithromycin and erythromycin), but not 16-membered macrolides (e.g. josamycin), are effective in diffuse panbronchiolitis. However, there are no studies that have compared the effects of 14- and 16-membered macrolide antibiotics on biofilm formation. Treatment with high-dose clarithromycin (100 mg/kg) resulted in a significant decrease in the number of viable bacteria in an experimental murine model. Josamycin at a dose of up to 100 mg/kg had no effect on the number of viable bacteria in the lung. Our results may explain, at least in part, the clinical efficacy of 14-membered macrolide antibiotics in patients with chronic pneumonia caused by Pseudomonas aeruginosa.

Current Japanese clinical practice involves the usage of large amounts of new macrolides such as clarithromycin and roxithromycin for the treatment of diffuse panbronchiolitis, Helicobacter pylori and Mycobacterium avium complex infections. In this study, the phenotypes, genotypes, and macrolide resistance mechanisms of macrolide-inactivating Escherichia coli recovered in Japan from 1996 to 1997, were investigated. The isolation rate of erythromycin A highly-resistant E. coli (MIC > or = 1,600 microg/ml) in Japan slightly increased from 0.5% in 1986 to 1.2% in 1997. In six macrolide-resistant strains, recovered from the strains collected for this study during 1996 to 1997, the inactivation of macrolide could be detected with or without added ATP in the assay system. The appearance of erythromycin A-inactivating enzyme independent of ATP was novel from Japanese isolates, and the 1H NMR spectra of oleandomycin hydrolyzed by the three ATP-independent isolates were examined. It was clearly shown that the lactone ring at the position of C-13 was cleaved as 13-H signal in aglycon of oleandomycin upper shifted. These results suggested the first detection of macrolide-lactone ring-hydrolase from clinical isolates in Japan. These results suggested the first detection of an ATP-independent macrolide-hydrolyzing enzyme from Japanese clinical isolates. Substrate specificity of the macrolide-hydrolyzing enzyme was determined with twelve macrolides including the newer members of this group and it was found that not only erythromycin A but also the new macrolides, such as clarithromycin, roxithromycin, and azithromycin were inactivated. The NMR data, broad spectrum of activity, and independence of co-enzyme supported our naming of the enzyme as a macrolide esterase. PCR methodology was employed to detect an ereB-like gene from the 3 isolates producing macrolide esterase, and one of these was subsequently shown to contain both ereB-like and ermB-like genes. It was also clearly shown that the other three isolates, which inactivated macrolide in the presence of ATP, had an mphA-like gene.

Clinical isolates collected from clinical facilities across Japan in 1998 were tested against five aminoglycosides and three beta-lactams. The resistance of 50 strains each of methicillin sensitive Staphylococcus aureus, methicillin resistant Staphylococcus aureus (MRSA), Staphylococcus epidermidis, Escherichia coli, Citrobacter freundii, Klebsiella pneumoniae, Enterobacter sp., Serratia sp., Pseudomonas aeruginosa and Proteus sp. (P. mirabilis 25 strains and P. vulgaris 25 strains) to the aminoglycosides isepamicin (ISP), amikacin (AMK), gentamicin, tobramycin and dibekacin, and to the beta-lactams imipenem, ceftazidime and piperacillin (all three known to be effective against P. aeruginosa) were investigated using a micro liquid dilution method with the following results: 1. ISP was effective against all strains except for 14% of MRSA, 2% of Proteus sp., and 4% of P. aeruginosa. 2. Six strains of MRSA were resistant to all eight drugs; however, in these cases ISP exhibited a relatively low minimum inhibitory concentration (MIC) compared to the other compounds. 3. Four strains of MRSA were resistant to all drugs except ISP. MRSA was the only isolate to demonstrate a resistance to seven or more drugs. 4. Twenty-one strains of MRSA and 1 strain of P. aeruginosa were resistant to six drugs; however, all of these were susceptible to both ISP and AMK. 5. Against all strains tested, ISP generally exhibited a lower MIC compared to AMK. These results suggest that, even ten years after its entering the market, ISP is still an aminoglycoside having a high anti-bacterial activity against a wide range of clinical isolates.

Escherichia coli CU1, a clinical isolate recovered in Japan in 1997, was found to be highly-resistant to both 14-membered and 16-membered ring macrolide antibiotics. A crude extract prepared from strain CU1 inactivated 14-, 15- and 16-membered ring macrolides in the presence of ATP and the Rf value of inactivated oleandomycin was identical to that of oleandomycin 2'-phosphate. This suggested that strain CU1 produced the enzyme macrolide 2'-phosphotransferase [MPH(2')]. Substrate specificity of the crude enzyme from strain CU1 against 14-, 15- and 16-membered ring macrolides was basically similar to that of MPH(2')II from strain BM2506, differing in that the former more effectively inactivated roxithromycin and tylosin. Subsequent attempts were made to clone the novel mph gene encoding for MPH(2') in strain CU1. The mph gene carried by strain CU1 was located on nontransmissible plasmid DNA, designated pCU001. Its molecular weight, estimated by agarose electrophoresis, was approximately 57 kD. The DNA sequence of the cloned mph gene from the Japanese isolate CU1 was identical to that of mphB, which until now had only been recovered in France. The variance in the substrate specificity of MPH(2')II from each strain led us to speculate that other factors in the reaction affect the enzymatic inactivation activity.

Minimum inhibitory concentrations (MICs) of erythromycin, clarithromycin, roxithromycin, oleandomycin, triacetyloleandomycin, azithromycin, josamycin and midecamycin were investigated using 200 strains of methicillin-resistant Staphylococcus aureus (MRSA) clinically isolated in Japan during 1996. The results show that the MRSAs could be classified into five groups according to MIC patterns to various macrolides and that more than 88% of the strains used were highly-resistant to all macrolides tested. It was found that 9.0% of the strains examined showed a unique MIC pattern different to that of macrolide-lincosamide-streptogramin B antibiotic resistance type. This group was found to be highly resistant to 14-membered but susceptible to 16-membered macrolides. The resistance induction by erythromycin or oleandomycin was observed to increase for clarithromycin and roxithromycin resistances in a part of strains used. On the other hand, for azithromycin, such induction was not observed.

Antimicrobial susceptibility testing by the ATP-bioluminescence method has been noted for its speed; it provides susceptibility results within 2 to 5 h. However, several disagreements between the ATP method and standard methodology have been reported. The present paper describes a novel ATP method in a 3.5-h test which overcomes these deficiencies through the elimination of false-resistance discrepancies in tests on gram-negative bacteria with beta-lactam agents. In our test model using Pseudomonas aeruginosa and piperacillin, it was shown that ATP in filamentous cells accounted for the false resistance. We found that 0.5% 2-amino-2-methyl-1,3-propanediol (AMPD) extracted ATP from the filamentous cells without affecting normal cells and that 0.3 U of adenosine phosphate deaminase (APDase)/ml simultaneously digested the extracted ATP. We used the mixture of these reagents for the pretreatment of cells in a procedure we named filamentous cell treatment, prior to ATP measurements. This novel ATP method with the filamentous cell treatment eliminated false-resistance discrepancies in tests on P. aeruginosa with beta-lactam agents, including piperacillin, cefoperazone, aztreonam, imipenem-cilastatin, ceftazidime, and cefsulodin. Furthermore, this novel methodology produced results which agreed with those of the standard microdilution method in other tests on gram-negative and gram-positive bacteria, including P. aeruginosa, Escherichia coli, Staphylococcus aureus, and Enterococcus faecalis, for non-beta-lactam agents, such as fosfomycin, ofloxacin, minocycline, and aminoglycosides. MICs obtained by the novel ATP method were also in agreement with those obtained by the agar dilution method of susceptibility testing. From these results, it was shown that the novel ATP method could be used successfully to test the activities of antimicrobial agents with the elimination of the previously reported discrepancies.

The antibiotic susceptibilities of 43 strains of Escherichia coli O157:H7 identified in the summer of 1996 in Japan were investigated. Growth of 90% of O157 strains was inhibited at a concentration of < or = 0.5 micro/ml by several agents including fosfomycin with glucose-6-phosphate.

Among recent clinical isolates in Japan, strain CU264 was discovered which formed unusual colonies. This strain was identified as Rahnella aquatilis which is usually found in water. The antibiotic susceptibilities against tetracycline, carbenicillin, chloramphenicol, streptomycin, kanamycin, gentamicin, sulphonamide, neomycin, fosfomycin, rifampicin, norfloxacin and nalidixic acid, were investigated. The result demonstrated that the strain was highly resistant to fosfomycin only. It was further shown that this resistance was transmissible with low frequency to Serratia marcescens whereas it was not transmissible to Escherichia coli.

It is well known that fosfomycin (FOM) shows the combination effects with some other antibiotics. Suck effects have not been known against Klebsiella pneumoniae, however. In this report, combination effects of FOM with beta-lactam, aminoglycoside, and macrolide antibiotics were investigated against clinical isolates of both FOM-susceptible and resistant Klebsiella pneumoniae. FOM had synergistic activities with beta-lactams such as ampicillin (ABPC) and cefminox (CMNX), and macrolide antibiotics as erythromycin (EM) and midecamycin (MDM) against all strains tested. Among beta-lactams, penicillin V showed synergistic actions against FOM-susceptible strain of Tf341A and additive actions against FOM-resistant 3 strains with FOM. Pheneticillin was synergistic with FOM against FOM-highly susceptible strain Tf341A and the additive or nearly synergistic effects against other strains. FOM with amoxicillin was synergistic against Tf341A, and FOM-resistant strain of Tf170B and additive against other strains. While the activities of combination of FOM with kanamycin or dibekacin against FOM-susceptible 2 strains were additive, those with amikacin were synergistic. Five different aminoglycosides tested showed antagonistic activities with FOM against 3 FOM-resistant strains. From these results, FOM appears to be clinically useful in treating FOM-susceptible and resistant strains of Klebsiella pneumoniae in combination of 4 antibiotics such as ABPC, CMNX, EM, and MDM.

In a newly established murine model of chronic Pseudomonas aeruginosa respiratory infection mimicking diffuse panbronchiolitis (DPB), we investigated the effect of oral administration of clarithromycin on lymphocyte accumulation in the lung. Infection was produced by placement of a plastic tube precoated with P. aeruginosa in the bronchus. The number of bacteria on the tube was 6.25 +/- 0.22 log10 colony-forming units (cfu)/ml. Viable bacteria were constantly isolated at 10(5) to 10(6) cfu/specimen from the lungs for more than 1 yr. The histopathologic features resembled those of DPB consisting of massive accumulation of lymphocytes in the lung. The total number of pulmonary lymphocytes started to increase on Day 7, reaching a peak level within 12 d of intratracheal challenge. The number remained steady at that level for up to 120 d. There was also a steady fall in the CD4+/CD8+ ratio in the lungs, commencing on Day 7 and persisting to Day 120. A 10-d course of oral clarithromycin (10 mg/kg/d) from Day 7 resulted in a reduction of lymphocyte numbers to baseline level, although the dose did not influence the number of bacteria in the lungs. Treatment also increased the CD4+/CD8+ ratio to the baseline level from Day 7 to 17. Our results were similar to those detected in bronchoalveolar lavage fluid of patients with DPB and suggest that the therapeutic benefits of clarithromycin are due to its anti-inflammatory properties rather than antimicrobial effect.

Intact cells of aminoglycoside (AG) antibiotic-resistant Pseudomonas aeruginosa usually do not inactivate AG, even though they possess the AG-modifying enzyme. An assay method for determining the activity of inactivating enzyme in intact cells of streptomycin-resistant P. aeruginosa was previously reported. Although this assay method was applied to the determination of the activity of kanamycin (KM)-inactivating enzymes, it could not apply to some of the KM-resistant strains. A new simple assay method has now been investigated for determining the activity of KM-inactivating enzyme in intact cells of clinically isolated KM-resistant P. aeruginosa. The determination of AG-inactivating enzyme activity was attempted using lysozyme for release of the inactivating enzymes in washed cells, and both DNase and RNase were added to digestion of the nucleic acids released by bacteriolysis. This lysozyme-DNase-RNase (LDR) method has facilitated the confirmation of the presence of AG-inactivating enzyme in the strains used. In addition, the LDR technique was applicable to the determination of inactivating enzyme activity for various AGs other than KM. Since this simple assay method can determine any type of AG-inactivating enzyme activity of various P. aeruginosa strains, it may contribute significantly to the rapid selection of drugs in clinical use.

A novel tetracycline derivative, DMG-DMDOT [9-(N,N-dimethylglycylamido)-6-demethyl-6-deoxytetracycline] , is one of the glycylcyclines which have a broad antibacterial spectrum, including many tetracyclineresistant bacteria (R.T. Testa, P.J. Petersen, N.V. Jacobus, P.-E. Sum, V.J. Lee, and F.P. Tally, Antimicrob. Agents Chemother. 37:2270-2277, 1993). The mechanism by which DMG-DMDOT overcomes efflux-based tetracycline resistance was investigated. Tetracycline-resistant Escherichia coli cells carrying an R plasmid encoding the tet(B) gene, which encodes the typical tetracycline efflux pump [TetA(B)] of gram-negative bacteria, were as susceptible to DMG-DMDOT as was the tetracycline-susceptible host. When mid-log-phase cells carrying the tet(B) gene were incubated with a subbactericidal concentration of DMG-DMDOT (0.5 micrograms/ml) for 2 h, a significant amount of the TetA(B) protein was detected in the cell membrane by Western blotting (immunoblotting) with an anti-carboxyl-terminal antibody, similar to the case in which tetracycline was used as the inducer, indicating that the tet repressor, TetR, can recognize DMG-DMDOT as an efficient inducer. Everted membrane vesicles prepared from cells producing the TetA(B) protein showed absolutely no transport activity for DMG-DMDOT. Furthermore, the presence of excess DMG-DMDOT had no effect on the tetracycline transport activity of the everted vesicles, indicating that DMG-DMDOT is not recognized as a substrate by the TetA(B) protein.

The class C beta-lactamase of Citrobacter freundii GN346 is a typical cephalosporinase comprising 361 amino acids, and substitution of the glutamic acid at position 219 in the enzyme by lysine was previously shown to broaden its substrate spectrum to oxyimino beta-lactams (K. Tsukamoto, R. Ohno, and T. Sawai, J. Bacteriol. 172:4348-4351, 1990). To clarify this spectrum extension from the kinetic point of view, the interactions of cefuroxime, ceftazidime, and aztreonam with the wild-type and mutant enzymes were analyzed. In addition to aztreonam, known as a progressive inhibitor of class C beta-lactamases, cefuroxime and ceftazidime were found to act as progressive inhibitors of the wild-type enzyme. On the other hand, only aztreonam showed weak progressive inhibition of the mutant enzyme. On the basis of kinetic parameters, a minimum scheme for interaction of the oxyimino beta-lactams with the wild-type enzyme was proposed, and the rate-limiting step of the hydrolysis of unfavorable substrates was indicated to be conversion of the stable acyl-enzyme intermediate to the unstable intermediate. In aztreonam hydrolysis by the mutant enzyme, the reaction rate at the rate-limiting step was 2,000 times that of the wild-type enzyme. These results indicate that the mutation at position 219 disturbs the stabilization of the stable intermediate.

The diffusion rates of nalidixic acid, ofloxacin and ofloxacin's two optically active isomers through OmpF channels were measured in proteoliposomes and compared with the rates of beta-lactams. The four quinolones showed high diffusion rates, exceeding that of cephaloridine and being comparable to imipenem. There was no significant difference in diffusion rate between nalidixic acid and ofloxacin, or between the two optically active isomers. The diffusion rates of enoxacin and norfloxacin were also estimated to be higher than many beta-lactams.

Two penicillins and 5 cephalosporins were evaluated for their ability to pass through the outer-membranes of Proteus morganii, Citrobacter freundii and Escherichia coli. Cefazolin, ceftezole and cephaloridine showed high permeability through the outer-membranes of these Gram-negative bacteria. Benzylpenicillin and cephalothin, on the contrary, showed low permeability. The outer-membrane permeability of ampicillin and cephalexin varied from species to species. C. freundii was found to have the highest barrier against both the penicillins and the cephalosporins, and E. coli appeared to have a low barrier against the cephalosporins. The hydrophobic character of the beta-lactam antibiotics, which was estimated by a reversed-phase thin-layer chromatography was closely related to the outer-membrane permeability. In general, the more hydrophilic antibiotic showed the higher outer-membrane permeability. However, cephaloridine, the most lipophilic compound among the antibiotics tested, showed good permeability.

Cefoxitin, N-formimidoyl thienamycin (MK0787), clavulanic acid, and penicillanic acid sulfone (CP45,899) were studied to determine their potency as suicide inhibitors of three very different kinds of beta-lactamases produced by Gram-negative bacteria: type Ib penicillinase (TEM-2 type), a typical cephalosporinase (the enzyme of Proteus morganii), and a cephalosporinase with broad substrate specificity (the enzyme of Proteus vulgaris). All these beta-lactams were confirmed to be quite stable to the three beta-lactamases. The absolute values of the turnover numbers (kcat) for these enzyme-catalyzed hydrolyses were determined, the values ranged from 0.25 minute-1 to 660 minute-1. All the beta-lactams studied, except cefoxitin, acted as suicide inhibitors of the typical cephalosporinase. Although cefoxitin did not exhibit such an effect, it was a powerful competitive inhibitor of this enzyme. Although the four beta-lactams acted as suicide inhibitors of the P. vulgaris cephalosporinase, the inactivated enzyme partially regained its activity after removing the effect of the free inhibitor. Type Ib penicillinase was also inactivated by the four beta-lactams, and the activity of the inactivated enzyme, except in the case of cefoxitin, was partially restored. The rate constants for enzyme inactivation or reactivation were calculated and are presented. The information obtained from this study suggests that the catalytic center of the P. vulgaris cephalosporinase is different not only from that of the penicillinase-type but also from that of the cephalosporinase-type beta-lactamases.

Mutant strains lacking outer membrane protein(s) were isolated from Escherichia coli, Proteus mirabilis, and Enterobacter cloacae. The outer membrane protein(s) of P. mirabilis and E. cloacae corresponding to E. coli porin were identified on the basis of their function, namely, their ability to allow the permeation of glucose as demonstrated by [14C]glucose uptake by intact cells. P. mirabilis has only one outer membrane pore protein (molecular weight, 40,000), but E. cloacae has at least two such proteins (molecular weights, 37,000 and 39,000 to 40,000). When the bacteria lost these proteins or porin, the outer membrane permeation of cefazolin was found to be greatly reduced in these three species. Such a change in the outer membrane permeation closely correlated with a significant decrease in the bacterial susceptibility to cephalosporins, including cefoxitin. These results suggested that the main pathway for cephalosporin permeation is the pore made up of these proteins. The 39,000- to 40,000-molecular-weight protein in E. cloacae was also assumed to play an important role in the outer membrane permeation of tetracycline and chloramphenicol. On the other hand, the permeation route of penicillins was obscure. The susceptibility to penicillins, except in some cases, was little influenced by the absence of the proteins. Ampicillin was found to pass through the outer membrane via the same route as the cephalosporins, but the possibility that ampicillin and other penicillins possess another unknown route for outer membrane permeation could not be ruled out.

A novel carbapenem, SF2103A, is a strong inhibitor of various types of beta-lactamase. Equimolar concentrations of SF2103A completely inactivated the cephalosporinases of Proteus vulgaris and Citrobacter freundii and type Ib and type II penicillinases mediated by R plasmids in a progressive manner. The inactivation of the two penicillinases and P. vulgaris cephalosporinase was apparently irreversible; however, when the inactivated enzymes were separated from excess SF2103A by gel filtration, they showed very slow reactivation. The hydrolysis of SF2103A by these three beta-lactamases was below the limit of detection. It is concluded that SF2103A acts as a tight-binding competitive inhibitor for the penicillinases and P. vulgaris cephalosporinase. In contrast, the inactivation of C. freundii cephalosporinase by SF2103A was evidently reversible. The rate constant of reactivation of the enzyme was compatible with the turnover rate of the enzyme in the steady state of SF2103A hydrolysis. Thus, SF2103A simply acts as a poor substrate for C. freundii cephalosporinase.

The effect of the interaction of beta-lactamase production and outer membrane permeability on the bacterial resistance to beta-lactam antibiotics was investigated using the Escherichia coli K12 substrains, which have outer membrane mutation and produce TEM-type penicillinase. Experiments confirmed that the cefazolin concentrations in the periplasm of cells exposed to the MIC were restricted to 2.4-4.6 microM among the bacteria, even though the MICs for the bacterial strains were distributed from 13 microM to 1,726 microM. The drug concentrations in the periplasm could be calculated on the basis of parameters obtained experimentally, and the calculated values were in fair agreement with the experimental values. The effect of beta-lactamase activity (Vmax/Km) and the outer membrane barrier on the elevation of MICs was observed to be synergistic, and the contribution of beta-lactamase was more effectively expressed in the bacterial cells with higher outer membrane barrier.

Liposomes containing penicillinase or cephalosporinase were prepared from the phospholipids of Escherichia coli. After free beta-lactamase was inactivated by clavulanic acid or penicillanic acid sulfone followed by separation of inactivated enzyme and inhibitor from liposomes by gel filtration, the permeability of these liposomes to ampicillin, cefazolin and cephaloridine was estimated by measuring the hydrolysis of these antibiotics by the entrapped enzymes. The permeability parameter C (minute-1 microM lipid-1) of ampicillin, cefazolin and cephaloridine was calculated to be 2.35 X 10(-4), 0.33 X 10(-4) and 0.52 X 10(-4), respectively. The lipid bilayer permeability of these antibiotics was also measured by using the liposomes containing these antibiotics. About half of the initially entrapped ampicillin was released from the liposomes within 80 minutes, while no significant release of cefazolin and cephaloridine could be detected during the same period. These results clearly indicates that the lipid bilayer membrane is more permeable to ampicillin than cefazolin and cephaloridine, and they are consistent with the observations of Sawai et al., who showed that ampicillin was a more effective antibacterial drug than cefazolin and cephaloridine against the porin-deficient mutants.

A set of bacterial strains composed of nine bacterial groups, with each made up of three or four strains, was used to estimate the stability of beta-lactam antibiotics to nine types of beta-lactamases from Gram-negative bacteria. The strains in the same group produced the same kind of enzyme constitutively, but the enzyme activity achieved in the bacterial cell differed with different strains. The difference in antibacterial activity of each antibiotic on the strains of each group, easily measured by a simple plate technique, permits an estimation of its relative stability to each beta-lactamase. This method was applied to thirteen beta-lactam antibiotics including seven new ones.