Biosynthetic Incorporation of Site-Specific Isotopes in ß-Lactam Antibiotics Enables Biophysical Studies.

A biophysical understanding of the mechanistic, chemical, and physical origins underlying antibiotic action and resistance is vital to the discovery of novel therapeutics and the development of strategies to combat the growing emergence of antibiotic resistance. The site-specific introduction of stable-isotope labels into chemically complex natural products is particularly important for techniques such as NMR, IR, mass spectrometry, imaging, and kinetic isotope effects. Toward this goal, we developed a biosynthetic strategy for the site-specific incorporation of 13C labels into the canonical ß-lactam carbonyl of penicillin G and cefotaxime, the latter via cephalosporin C. This was achieved through sulfur-replacement with 1-13C-l-cysteine, resulting in high isotope incorporations and milligram-scale yields. Using 13C NMR and isotope-edited IR difference spectroscopy, we illustrate how these molecules can be used to interrogate interactions with their protein targets, e.g., TEM-1 ß-lactamase. This method provides a feasible route to isotopically labeled penicillin and cephalosporin precursors for future biophysical studies.

A method to detect important residues using protein binding site comparison.

As the number of protein sequences with unknown function increases, assigning accurate function to unknown protein becomes increasingly an important issue. Protein function is often encoded in a small number of residues located in binding pocket, and there have been many attempts to predict the function using the binding site. Here, we developed a binding site comparison method which can easily identify spatially matched residues between binding sites. Using clique detection algorithm, the new method finds the matched residues of maximum size, and then these matched residues are scored in a way similar to sequence alignment scoring. In addition, the significance of matched score is estimated from the empirical random score distribution. Results of benchmark test suggest that the method successfully detects functionally related binding sites. Furthermore, conserved residues and subfamily-specific residues in the functional family can be identified. In addition, we investigated systematic relationship between binding sites and functions using the binding site comparison method. Result showed that proteins with similar binding site largely perform similar function.

Effect of penicillin resistance of Streptococcus pneumoniae on the presentation, prognosis, and treatment of pneumococcal endocarditis in adults.

We performed a clinical study of pneumococcal endocarditis (PE) in adults at 15 major Spanish hospitals during a 21-year period (1978-1998). During this time, 63 patients had PE due to Streptococcus pneumoniae diagnosed. Of the 63 isolates recovered from these patients, 24 (38%) and 6 (10%) showed resistance to penicillin (minimum inhibitory concentration [MIC], 0.1-4 microg/mL) and cefotaxime (MIC, 1 microg/mL), respectively. Twenty-two (35%) of the patients died. Left-side heart failure, but not penicillin resistance, was independently associated with a higher risk of death (odds ratio, 1.33; 95% confidence interval, 1.04-1.71; P=.026). Patients without meningitis who had PE due to penicillin-resistant S. pneumoniae could be treated with high-dose penicillin or a third-generation cephalosporin if the MIC for penicillin was < or =1 microg/mL. For patients with concurrent meningitis, high doses of cefotaxime could be used if the MIC for cefotaxime was < or =1 microg/mL. Early recognition of heart failure and surgery may help to decrease mortality.

Penicillin-binding protein 2x of Streptococcus pneumoniae: enzymic activities and interactions with beta-lactams.

The high-molecular-mass penicillin-binding protein (PBP) 2x, one of the primary targets of beta-lactam antibiotics in Streptococcus pneumoniae, has been produced as a soluble form and purified in large amounts. It has been shown to catalyse hydrolysis and transfer reactions with different ester and thiolester substrates and its catalytic behaviour was often similar to that of the soluble DD-peptidase from Streptomyces R61. This provided an easy method to monitor the activity of the PBP. For the first time, a reliable kinetic study of the interaction between a lethal target and beta-lactam antibiotics has been performed. Characteristic kinetic parameters were obtained with different beta-lactam compounds. These results not only validated the mechanism established with non-essential extracellular enzymes, but will also constitute the basis for comparative studies of the low-affinity variants from penicillin-resistant strains.

Penetration of cefotaxime and desacetylcefotaxime into brain abscesses in humans.

Since clinical trials comparing the efficacies of different antibiotic regimens for treatment of brain abscesses are difficult to perform, the choice of antibiotics must rely on the antibacterial spectrum and the ability of the drug to penetrate into the abscess fluid. The aim of this investigation was to study the ability of cefotaxime and its active metabolite desacetylcefotaxime to penetrate into brain abscesses. Eight patients were given 3 g of cefotaxime intravenously every 8 h. Abscess fluid samples, obtained at surgery at various times after dosing, and blood samples were analyzed for their concentrations of cefotaxime and desacetylcefotaxime by using a newly developed microbiological assay. The brain abscess concentrations of cefotaxime and desacetylcefotaxime were 1.9 +/- 1.7 and 4.0 +/- 2.2 mg/liter, respectively. Simultaneous concentrations in plasma were 2.0 +/- 1.0 and 3.9 +/- 1.8 mg/liter, respectively. With increasing time following cefotaxime dosing there was a significant increase in the abscess:plasma concentration ratio of desacetylcefotaxime. Since both cefotaxime and desacetylcefotaxime penetrate well into the brain abscess, reaching concentrations above the MIC for probable bacteria except gram-negative anaerobes, it is concluded that cefotaxime in combination with metronidazole may be used as an alternative in the treatment of brain abscesses.

Penetration of cefotaxime into respiratory secretions.

The objective of this study was to quantitate cefotaxime and its active metabolite, desacetyl cefotaxime, in the distal airways and to compare these levels to concentrations in plasma. Respiratory secretions were obtained from the subsegmental level in 17 adult patients undergoing fiber-optic bronchoscopy within 2 h after receiving four doses of cefotaxime (2 g intravenously every 6 h). In 11 patients, cefotaxime levels measured by high-pressure liquid chromatography in bronchial secretions were below detectable limits (less than 0.5 mg/liter); however, levels of desacetyl cefotaxime exceeded 1.5 mg/liter in 9 of these 11 patients (range, 1.6 to 10 mg/liter). Concentrations of desacetyl cefotaxime in lung secretions (6.9 +/- 0.85 [standard error] mg/liter) was 77% of mean levels of desacetyl cefotaxime in plasma (8.9 +/- 1.26 mg/liter). In summary, concentrations of desacetyl cefotaxime in bronchial secretions are markedly higher than those of cefotaxime.

Distribution of cefodizime to exudate in the croton oil-induced rat granuloma pouch and its therapeutic effects on experimental infections in the pouch.

Cefodizime was compared with cefotaxime (CTX) in regard to its distribution to an inflammatory site (exudate in croton oil-induced granuloma pouch) of rats and its therapeutic effects on experimental infections in such pouches after intravenous injection. Cefodizime levels in rat serum and pouch exudate were higher than those of CTX, and the former compound disappeared from the serum and pouch exudate far more slowly than the latter. In the tests for therapeutic effects, cefodizime showed almost the same degree of inhibitory activity as CTX against growth in pouch exudate of Escherichia coli Ec-7, Proteus mirabilis Pm-428, and Serratia marcescens Sm-390 for which the minimum inhibitory concentrations (MICs) of cefodizime were equal to or greater than the CTX values, and such activity of cefodizime lasted for a longer period than that of CTX. These results suggest that the above pharmacokinetic features of cefodizime compensate for its MICs against organisms displaying lower values for CTX.

A comparison of cefotaxime versus cefamandole in prophylaxis for surgical treatment of the biliary tract.

Fiscal considerations prompted comparison of cefotaxime (a third generation cephalosporin) with cefamandole (a second generation cephalosporin) for prophylaxis in the surgical treatment of the biliary tract. One hundred and eight patients who underwent an operation upon the biliary tract received three 1 gram doses of cefotaxime (54 patients) or cefamandole (54 patients) at induction of anesthesia and then one and three hours later. The study was prospective, blinded and randomized. The groups (cefotaxime versus cefamandole) were statistically comparable for age, sex, diagnosis, type and duration of operation and positive cultures. The most prevalent bacteria isolated from qualitative aerobic and anaerobic cultures of bile and the wall of the gallbladder were Escherichia coli, Streptococcus and Klebsiella. The incidence of bactibilia in patients with one of these conditions was: 75 per cent for cancer; 69 per cent for patients more than 60 years old; 33 per cent for jaundice; 58 per cent for pancreatitis; 60 per cent for exploration of the common bile duct, and 22 per cent for acute cholecystitis. Microbiologic agar diffusion assays of tissue from the wall of the gallbladder, subcutaneous fat and rectus muscle and samples of bile and serum obtained 30 minutes after the second dose of antibiotic showed a statistically significant greater concentration of cefamandole in the wall of the gallbladder. Otherwise there was no difference between the concentration of cefamandole and cefotaxime. The groups showed no statistical difference for temperature of more than or equal to 38 degrees C. on two consecutive measurements, postoperative wound and urinary infections, postoperative hospital stay and days in the intensive care unit and incidence of readmission within a month. Prophylactic use of cefotaxime in a three dose regimen provided no advantage in prophylaxis compared with cefamandole.

[Pharmacokinetic and clinical studies on cefotaxime in plastic and reconstructive surgery].

Pharmacokinetic and clinical studies on cefotaxime (CTX) in plastic and reconstructive surgery field were carried out. The results obtained are summarized as follows. Two grams of CTX was administered intravenously by single bolus injection to each of 4 patients with burn blisters and mean levels of CTX concentration in blister exudates were investigated. Thirty minutes after administration, the mean level of CTX in the exudates was 3.90 micrograms/ml and it reached a peak of 9.81 micrograms/ml in 2 hours. The clinical efficacy rate for 30 patients with burn infections and postoperative infections was 73.3%, and the efficacy rate for 29 patients in prophylactic use was 72.4%. A side effect (eruption) and abnormal laboratory findings were observed in each one case out of 59. From the above results, CTX may be considered to be useful in plastic and reconstructive surgical treatments.

[Prophylactic effects of cefmenoxime against postoperative infections after thoracotomy. Studies of cefmenoxime transfer from serum to pleural fluid and of clinical effects of cefmenoxime].

Cefmenoxime (CMX) at a dose of 1 g was administered intravenously to each of 10 patients undergoing thoracotomy, and concentrations of CMX in the serum and pleural fluid were measured. Serum concentration of CMX reached its peak of 43.71 micrograms/ml at 1 hour and decreased to 4.15 micrograms/ml at 3 hours after the administration. The concentration of CMX in the pleural fluid reached its peak of 7.61 micrograms/ml at 3 hours and decreased slowly 5.26 micrograms/ml at 7 hours after the administration. A clinical study with 21 patients was performed to evaluate the effect of CMX as a prophylactic antimicrobial agent in thoracotomy. Patients received intravenous administration of 4 g/day of CMX for 7-10 days following operations. Each patient was evaluated daily for fever, sign of allergic reaction, and wound infection and other symptoms. No apparent infection occurred in those clinical patients except 1 patient with a suspected infection, and 1 case of allergic reaction as exanthema was observed during this study. Prophylactic effect of CMX against postoperative infection after thoracotomy was good.

The intravitreal penetration of cefotaxime in man following systemic and subconjunctival administrations.

Of 30 patients scheduled to undergo elective vitreal surgery, five received a single dose of 1000 mg of cefotaxime intramuscularly and 25 received 100 mg subconjuctivally. Specimens of serum and vitreous were collected from 30 minutes to five hours after drug administration, and were assayed for cetotaxime concentration by bioassay and high pressure liquid chromatography. In the five patients who received the intramuscular dose, no cefotaxime or its major metabolite could be detected in the vitreous. In the 25 patients who received the subconjunctival dose, cefotaxime or its metabolite could be only detected in patients with aphakia or in those who previously underwent vitreal surgery. Cefotaxime in intramuscular administration should not be relied upon as appropriate prophylaxis for vitreal surgery. Cefotaxime in subconjunctival administration reaches therapeutic levels in the vitreous only in previously operated eyes.

[A study of prostatic tissue levels of latamoxef, cefoperazone and cefotaxime].

Although Cephem antibiotics are transferred to the prostatic fluid in relatively low levels, they are clinically effective for bacterial prostatitis. In the present study, the prostatic tissue concentration of Latamoxef (LMOX), Cefoperazone (CPZ) and Cefotaxime (CTX) were determined, and their penetration rates into the prostatic tissue were analyzed. Before the transurethral resection of the prostate (TUR-P), 2 g of LMOX (21 patients), 1 g of CPZ (15 patients) and 2 g of CTX (14 patients) were intravenously administered in a total of 50 patients with benign prostatic hypertrophy at our two Hospitals. The prostatic tissue was taken by TUR-P at 30 minutes and 60 minutes after the injection. The serum concentration and the prostatic tissue concentration of the three antibiotics were determined by the bioassay method. Additionally their serum concentration and the prostatic tissue concentration in the six cases of CTX-injected patients were also determined by high performance liquid chromatography (HPLC). The penetration rate into the prostatic tissue was obtained by the formula; the penetration rate = the prostatic tissue concentration/the serum concentration. The prostatic tissue concentrations determined by the bioassay method were, 36.9 +/- 10.6 micrograms/g at 30 minutes after the injection of 2 g of LMOX and 28.0 +/- 9.0 micrograms/g at 60 minutes, 31.0 +/- 8.3 micrograms/g at 30 minutes after the injection of 1 g of CPZ and 21.1 +/- 10.0 micrograms/g at 60 minutes, and 8.8 +/- 4.1 micrograms/g at 30 minutes after the injection of 2 g of CTX and 4.5 +/- 2.0 micrograms/g at 60 minutes (mean +/- S.D.). The penetration rates determined by the bioassay method were 36.2 +/- 10.9% at 30 minutes after the injection of LMOX and 34.8 +/- 15.3% at 60 minutes, 43.6 +/- 14.4% at 30 minutes after the injection of CPZ and 39.7 +/- 24.1% at 60 minutes, and 11.1 +/- 4.1% at 30 minutes after the injection of CTX and 9.6 +/- 3.8% at 60 minutes (mean +/- S.D.). The penetration rate of CPZ and LMOX were significantly higher than that of CTX (P less than 0.05, P less than 0.01). In 6 of the 14 CTX-injected patients, the serum and prostatic tissue concentrations of CTX and its metabolite, desacetyl-CTX, were also determined by HPLC. The penetration rate of CTX into the prostatic tissue was obtained by the formula; the penetration rate of CTX = the prostatic tissue concentration (CTX + Desacetyl-CTX)/the serum concentration (CTX + Desacetyl-CTX).(ABSTRACT TRUNCATED AT 400 WORDS)

[Concentrations of cefmenoxime in human bone marrow blood and osseous tissue].

One gram of cefmenoxime (CMX) was administered to each of 10 patients by a one shot intravenous injection at the beginning of surgery. At 30, 60, 90, 120 minutes after the administration, concentrations of CMX in venous blood, bone marrow blood and bone tissue were assayed by the agar-well method. CMX was smoothly transmigrated after the injection from venous blood to bone marrow blood and bone tissue, and effective concentrations higher than MIC80 values were maintained for several hours. CMX, therefore, appears to be a useful drug for the prophylaxis and the treatment of postoperative infections.

[Pharmacokinetics and clinical evaluation of cefotiam in pediatric surgery].

Pharmacokinetics and clinical studies on cefotiam (CTM) in pediatric surgery were performed and the results obtained are summarized below. In neonates without congenital heart disease (CHD), serum levels of CTM were rapidly increased following injection of CTM (20 mg/kg, one shot intravenous administration) and the peak values were found at 15 minutes. Six hours later, CTM almost disappeared from the blood. In the patient with CHD, however, CTM was retained in the serum and the urinary excretion was poor. The CTM was administrated prophylactically to prevent postoperative infections in 10 patients. Eight of 10 had no episodes during postoperative period. Wound infection, however, was found in 2 patients. No clinical and laboratory side effects were caused by CTM.

[Pharmacokinetic and clinical studies of cefotiam in the perinatal period].

Patients, who had undergone cesarean sections, and those who had experienced premature rupture of membranes, received cefotiam (CTM) and the clinical efficacy and the safety for mothers and fetuses were investigated. At the same time, pharmacokinetic analysis was done to study the maternal fetal transfer. Following results were observed. In cases of premature rupture of membranes, the maternal-fetal transfer ratio after intravenous administrations of CTM was 50.3% at a dosage of 1 g. Maternal and fetal serum concentrations of CTM were maintained higher levels than the MIC80 (0.78 micrograms/ml) against major pathogens excluding anaerobic of gynecologic-obstetric infections and were maintained up to 5.87 hours and 6.15 hours in mothers and fetuses, respectively. The CTM was administered once every 12 hours at a dosage of 1 g to 38 cases up to the 3rd or 4th day of puerperium after the rupture of membranes. Also, the CTM was administered up to times of delivery to another 20 cases, in one of which the fetus developed pneumonia. The maternal-fetal prophylactic effect was recognized in 98.3% (57/58) of cases. Forty-three cesarean section cases received CTM at a dosage level of 1 g by one-hour intravenous drip infusion in the following manner: after surgery to the 4th day, twice a day; from the 5th to the 7th day, once a day. Postoperative prophylactic effect against infection was achieved in all the cases. In 1 case, a slight transient elevation in the maternal GOT was observed. Neonatal jaundice with total bilirubin levels higher than 15.0 mg/dl was observed in 19 neonates (32.8%) in the group in which premature rupture of the membranes had occurred. However, the cause/effect relationship between CTM and the total bilirubin levels is unclear. The maternal-fetal transition of CTM was excellent, and the safety toward the fetus and neonate was high. When an antimicrobial activity and pharmacokinetics are considered, CTM will be a useful drug in the treatment of perinatal infections.

[Concentrations of cefmenoxime and cefotiam in the bile and gallbladder tissue following intravenous administration in patients with biliary tract diseases].

To test the effectiveness of cefmenoxime (CMX) and cefotiam (CTM) in patients with biliary tract diseases, concentrations of either antibiotic were measured after an intravenous bolus injection of 1.0 g of CMX or CTM, or simultaneous injection of both (1.0 g each). CMX or CTM was injected in 76 patients with biliary tract diseases (mostly cholelithiasis) prior to a cholecystectomy and concentrations of CMX or CTM were measured by the bioassay (agar well) method at 30 to 60 minutes after the injection. Average concentrations of both CMX and CTM in gallbladder bile and gallbladder tissue sufficiently exceeded the minimal inhibitory concentration (MIC) against main causative organisms of biliary tract infections. Concentrations of both antibiotics in gallbladder bile were significantly higher in patients with patent cystic ducts than with obstructed cystic ducts. Concentrations of both antibiotics in the gallbladder tissue reached at a similar high level regardless of the patency of the cystic ducts, but concentrations were lower in severely inflamed gallbladders. CMX and CTM were administered alternatively (cross-over fashion), or simultaneously (combined) to 13 patients with T-tube drainage or percutaneous transhepatic cholangio-drainage, and concentrations of both antibiotics in bile from the drainage tube were measured by high performance liquid chromatography at hourly intervals after the injection. Concentrations of both antibiotics were far greater than MICs against main attributable microorganisms in biliary tract infections. The concentration of CMX slightly exceeded that of CTM. Concentrations of both antibiotics were lower in bile of patients showing abnormally high serum GTP, A1-P, and total bilirubin levels than in bile of patients with normal values of these variables. It is speculated that the secretion of both antibiotics in the bile may decrease in cases with severe hepatic failure, but effective concentrations of both antibiotics in the gallbladder tissue should be maintained as long as the blood circulation in the gallbladder was maintained.

[Clinical study on the tissue distribution of cefmenoxime in the field of otolaryngology].

Concentrations of cefmenoxime (CMX) in serum and tissue were determined in surgical patients with chronic sinusitis, maxillary cyst or chronic tonsillitis. CMX was intravenously administered in a dose of 1 gram. Thirty minutes after the administration, patients were operated under local anesthesia. Concentrations of CMX in tissues were determined 1 hour after administration, and were 15.8 micrograms/g and 20.3 micrograms/g in mucous membrane of maxillary sinus and maxillary cyst. CMX was not detectable in tonsils of 8 patients out of 11 examined. An examination of MIC against various bacteria isolated from sinus pus and pharyngeal swab indicated that CMX was effective enough to produce a prophylactic effect against otolaryngo-infections.

[Clinical studies on the transference of cephem-type antibiotics into bile and gallbladder tissues with special reference to cefotiam and cefmenoxime].

Cefotiam (CTM) and cefmenoxime (CMX) were studied for their serum concentrations and transference into bile in patients with PTCD or T-tube. One gram of CTM or either 1 g or 2 g of CMX was administered by an intravenous drip infusion for over 30 minutes. These drugs were also studied for their serum concentrations, bile concentrations, and tissue concentrations in the walls of the gallbladder of patients operated on for cholelithiasis. Intravenous drip infusion (over 30 minutes) was used to administer 1 g of CTM or 1 g or 2 g CMX immediately before surgery. Both CTM and CMX were readily transferred into bile. Their bile concentrations, however, varied greatly among patients, and extremely low levels were detected in some patients. A crossover analysis of concentrations of CMX in bile of patients given doses of 1 g and 2 g revealed a dose-response relationship. The crossover analysis of drug concentrations in bile of patients given CTM and CMX showed that CMX is transferred more readily to bile. The relationship between liver functions and drug transfer to bile was examined by plotting the total bilirubin level against drug concentrations in bile. The plots formed an exponential curve with a correlation coefficient (r) being -0.52 in cases when each subjects received 1 g of CTM and -0.72 in cases when each subjects received 1 g of CMX. A study of 3 patients given CMX at a dose of 1 g suggested that bile levels of CMX may be correlated to ICG. Concentrations of CTM and CMX in tissues of the gallbladder wall were fairly high, with unexpectedly small variance among patients. Even in patients with low bile concentrations of these drugs, drug levels in the tissues of the gallbladder wall were high. Drug levels in the noninflammatory tissues were higher than those in inflammatory lesions. The above findings suggest that CTM should be the antibiotic of choice for patients with ordinary biliary tract infections and after the surgery of the liver and biliary tract system, while CMX should be the antibiotic of choice for patients with severe biliary tract infections, and for compromised hosts after the surgery of the liver and biliary tract system.

[Augmentation by serrapeptase of tissue permeation by cefotiam].

Cefotiam (CTM) is a new cephalosporin with a broad spectrum of activity against both Gram-positive and Gram-negative microorganisms. Cephalosporins are widely used for prophylaxis of infections in patients undergoing thoracotomy. Augmentation by serrapeptase on tissue permeation of CTM was examined in 35 thoracotomy patients with lung cancer. The subjects were divided into two groups according to the method of the administration of CTM. Group I consisted of 17 subjects, each of whom received a single dose of 2 g of CTM alone by an instillation for 30 minutes. Group II consisted of 18 subjects, each of whom received a combination of CTM and serrapeptase; serrapeptase was given 2 tablets (10 mg) each time for three times/day until the day before surgery, and then CTM was administered by the same procedure. The following results were obtained: Individual difference was observed for the permeation of CTM into tissues. Pathologic differences also affected the permeation. Nevertheless, the CTM levels in pulmonary tissues reached about a half of those in the blood in both the single dose group and the combination group, hence sufficient concentrations exceeding MIC80 for main microorganisms that caused infections in the lung were obtained. The concentrations of CTM in inflammatory tissues have showed lower levels than those of normal tissues in both CTM single dose and the combination groups. Decrease of blood flow volume may have contributed to the reduction in levels of CTM in the inflammatory tissues. The ratio of the concentration of the drug in pulmonary tissues to that in the blood was 29.1 +/- 2.5% in the single dose group, and 44.2 +/- 6.0% in the combination group, the latter showing quite a significant increase (P less than 0.05). Combined administrations of CTM and serrapeptase deserves more trials in the case when surgical treatments of the lung are performed. An antiinflammatory effect of serrapeptase in the respiratory system is expected, and in addition, the combined use of CTM and serrapeptase should stimulate permeation of the antibiotic into tissues.