N-formimidoyl Thienamycin Research Articles

A metallo-\u03b2-lactamase enzyme for internal detoxification of the antibiotic thienamycin

Thienamycin, the first representative of carbapenem antibiotics was discovered in the mid-1970s from soil microorganism, Streptomyces cattleya, during the race to discover inhibitors of bacterial peptidoglycan synthesis. Chemically modified into imipenem (N-formimidoyl thienamycin), now one of the most clinically important antibiotics, thienamycin is encoded by a thienamycin gene cluster composed of 22 genes (thnA to thnV) from S. cattleya NRRL 8057 genome. Interestingly, the role of all thn-genes has been experimentally demonstrated in the thienamycin biosynthesis, except thnS, despite its annotation as putative β-lactamase. Here, we expressed thnS gene and investigated its activities against various substrates. Our analyses revealed that ThnS belonged to the superfamily of metallo-β-lactamase fold proteins. Compared to known β-lactamases such as OXA-48 and NDM-1, ThnS exhibited a lower affinity and less efficiency toward penicillin G and cefotaxime, while imipenem is more actively hydrolysed. Moreover, like most MBL fold enzymes, additional enzymatic activities of ThnS were detected such as hydrolysis of ascorbic acid, single strand DNA, and ribosomal RNA. ThnS appears as a MBL enzyme with multiple activities including a specialised β-lactamase activity toward imipenem. Thus, like toxin/antitoxin systems, the role of thnS gene within the thienamycin gene cluster appears as an antidote against the produced thienamycin.

Mechanisms of action involved in the chemoattractant activity of three β-lactamic antibiotics upon human neutrophils

The effects produced by three β-lactamic antibiotics ( N-formimidoyl thienamycin or imipenem, cefmetazole and cefoxitin) in vitro on protein carboxylmethylation, cAMP and cGMP levels in human polymorphonuclear neutrophils were studied. These antibiotics (50 μg/mL) exhibited chemoattractant activity for phagocytic cells and produced a fast (0.5 min) and significant stimulation of protein carboxylmethylation. They also increased intraphagocytic cGMP levels although no changes in cAMP levels were observed. Since the involvement of the above-mentioned mechanisms in leukotaxis have been established, the stimulation of neutrophil chemotaxis by the three antibiotics studied could possibly be mediated by one or more of these mechanisns.

Phagocytic function and antibody-dependent cellular cytotoxicity of human neutrophils in the presence of N-formimidoyl thienamycin

The efficacy of an antibiotic in the treatment of bacterial infections depends upon the interactions of the drug, bacteria and phagocytes. We have studied "in vitro" the effect of N-formimidoyl thienamycin (Imipenem), a novel beta-lactamic antibiotic, on the phagocytic function and antibody-dependent cellular cytotoxicity of human neutrophil leukocytes. The incubation of these cells with 50 micrograms/ml of Imipenem similar to the therapeutic levels reached in plasma results in an increase of their adherence capacity to nylon fiber and to substrate, induced mobility or chemotaxis, opsonization, phagocytosis of Candida albicans (with serum, with decomplementarized serum and without serum) and latex beads, candidicidal power and the capacity of NBT reduction. Imipenem at this dose also presents chemoattractant power for neutrophils and enhances the antibody-dependent cellular cytotoxicity (ADCC).

Imipenem/cilastatin, an alternative treatment of pseudomonas infection in cystic fibrosis.

Imipenem, a new N-formimidoyl thienamycin was given together with cilastatin to 20 patients with cystic fibrosis and pulmonary infection due to Pseudomonas aeruginosa. The antibiotic was given in short-term infusions for 9-14 days (mean 11.5) in a dose of 45-60 mg/kg body weight/day. Good clinical results were obtained in all patients with significant improvement of clinical score, pulse rate, vital capacity and FEV1.0 (P less than 0.001). Blood PO2 increased and WBC decreased significantly. A slight increase in the minimum inhibitory concentration was noted during treatment but all strains examined were fully susceptible at follow-up one month later. The peak serum concentration was significantly increased in patients receiving the high dose of imipenem, but the sputum concentration was low in all patients and there was no difference in clinical or bacteriological outcome. The plasma and urinary clearance increased with body weight and was inversely correlated to clinical score. Imipenem/cilastatin appears a good alternative for the treatment of pulmonary infections caused by P. aeruginosa in cystic fibrosis.

Kinetic study on the inhibition of type Ib penicillinase by imipenem and thienamycin.

The mechanism of inhibition of type Ib penicillinase by imipenem (N-formimidoyl thienamycin) and thienamycin was studied. Imipenem and thienamycin. were found to inhibit the penicillinase in a progressive manner, but they differed from one another in the maximum level of enzyme inhibition, even with excess inhibitor. The maximum inhibitions by imipenem and thienamycin amounted to about 50% and 80%, respectively. To elucidate the mechanism of the equilibrium phenomenon, the rate constants in the proposed scheme were estimated. Computerassisted simulation using the rate constants revealed that there is an upper limit to the extents of inactivation by imipenem and thienamycin.

Cellular uptake and cell-associated activity of third generation cephalosporins.

The ability of the third generation cephalosporins to penetrate human polymorphonuclear leukocytes (PMNs) and their antibacterial activity against cell-associated Staphylococcus aureus (SA) and Haemophilus influenzae, type b (Hib) were studied. Utilizing radioactive uptake experiments, the cellular to extracellular concentration ratios were determined to be less than one for all cephalosporins at 10 and 120 min: cefotaxime (0.08 +/- 0.02, 0.34 +/- 0.08), ceftizoxime (0.21 +/- 0.11, 0.52 +/- 0.18), ceftriaxone (0.12 +/- 0.04, 0.38 +/- 0.23), and N-formimidoyl thienamycin (0.18 +/- 0.09, 0.33 +/- 0.14). Third generation cephalosporins were similar to penicillin in their exclusion from PMNs. The killing of cell-associated SA and Hib were evaluated in a preopsonized cell-associated bacterial assay with radiolabelled SA/Hib (cfu/cpm) comparing activity of PMNs + antibiotics to the PMN cell control (no antibiotics) at 0.5, 2, and 4 h. PMNs alone killed less than or equal to 0.5 log SA/Hib over 4 h. Clindamycin killed significantly more SA (p less than 0.01) than all other antibiotics; nafcillin killed significantly fewer SA (p less than 0.05) than all other antibiotics. Although each third generation cephalosporin showed good activity against cell-associated Hib, chloramphenicol had a significantly greater effect (p less than 0.05). N-formimidoyl thienamycin demonstrated good activity only after the concentration was increased in vitro to 8 micrograms/ml. Although cellular penetration of antibiotics may be important in the eradication of cell-associated pathogens, the overall cellular activity would appear to be multifactorial.

Penetration of N-formimidoyl thienamycin into extravascular fluids in rabbits.

The penetration of N-formimidoyl thienamycin into extravascular fluids was studied in rabbits. Two different models were used: subcutaneously implanted tissue chambers and fibrin clots. The antibiotic was given intramuscularly as a single dose of 10 mg/kg. The degree of penetration into these compartments was determined by comparing the area under the curve for the specific fluid with the area under the curve for plasma expressed in percent. The percentage of penetration of N-formimidoyl thienamycin into tissue chamber fluid (76.24%) was similar to that into fibrin clots (75.15%).

Review of the in vitro spectrum of activity of imipenem

Imipenem ( n-formimidoyl thienamycin, MK0787), a new carbapenem was found to have the widest antimicrobial activity of currently available beta-lactam drugs. Enterobacteriaceae had minimal inhibitory concentrations of imipenem of 8.0 μg/ml or less for 99.8 percent of clinical isolates. Only rare strains of Enterobacter species and Proteus mirabilis have higher imipenem minimal inhibitory concentration results. Hemophilus and Neisseria species were inhibited, but minimal inhibitory concentrations of imipenem were higher than those reported for third-generation cephalosporins. Only Pseudomonas maltophilia and Pseudomonas cepacia strains were imipenem resistant (MIC 50 greater than 32 μg/ml) among the commonly isolated non-enteric gram-negative bacilli. All anaerobes were found susceptible to imipenem with the exception of some strains of Clostridium difficile. Staphylococcus species and nonenterococcal streptococci were very susceptible to imipenem. Streptococcus faecalis had higher minimal inhibitory concentrations of imipenem (MIC 90 3.1 μg/ml) and S. faecium strains were frankly resistant. Methicillin-resistant S. aureus isolates had a MIC 90 of 27.2 μg imipenem/ml. Imipenem was generally bactericidal except for marked minimal inhibitory and minimal bactericidal concentration differences with enterococci, Listeria, methicillin-resistant staphylococci, and some P. aeruginosa strains. The minimal inhibitory and minimal bactericidal concentrations of imipenem were not significantly influenced by organism inoculum size, probably because of its beta-lactamase stability to nearly all commonly encountered bacterial enzymes. Imipenem was found to be an excellent inhibitor of beta-lactamases and a potent enzyme inducer. The induction characteristic seems responsible for the antagonistic interactions of imipenem with some enzyme-labile beta-lactams in combination. Imipenem had limited stability in some in vitro susceptibility test systems. The 10 μg disk test or dry-form broth microdilution systems were preferred, applying the interpretive criteria from the National Committee for Clinical Laboratory Standards (M2-A3). Imipenem-resistant strains were rarely found in clinical practice and bacteria resistant to newer beta-lactams and aminoglycosides were generally very susceptible to this new carbapenem.

Single-dose pharmacokinetics of imipenem-cilastatin in neonates.

The single-dose pharmacokinetics of imipenem (N-formimidoyl thienamycin), a beta-lactam antibiotic, used in combination with cilastatin, a renal dehydropeptidase I inhibitor, were evaluated in 10 neonates 1 to 8 days of age. The imipenem-cilastatin combination was given intravenously over a 15-min period at a dose of 15 or 25 mg/kg. Drug concentrations in serum, urine, and cerebrospinal fluid (when available) were determined by high-pressure liquid chromatography, and plasma disposition of the drugs was described by a two-compartment open model. The mean peak plasma levels of imipenem 30 min postinfusion were 55.4 and 27.2 micrograms/ml, and the mean t1/2 beta values were 2.1 and 1.8 h at doses of 25 and 15 mg/kg, respectively. The calculated volume of distribution was 0.41 liters/kg. In two patients from whom cerebrospinal fluid was obtained 1.5 h postinfusion, imipenem levels were 5.6 and 1.1 micrograms/ml at doses of 25 and 15 mg/kg, respectively, representing 10 and 4% of the 1-h serum levels. No side effects attributable to a single dose of imipenem-cilastatin were noted.

Occurrence and antimicrobial susceptibility of gram-negative nonfermentative bacilli in cystic fibrosis patients

Isolation of nonfermentative gram-negative bacilli (other than Pseudomonas aeruginosa ) from respiratory tract cultures of cystic fibrosis (CF) patients has increased in recent years. Species recovered include Pseudomonas cepacia, P. maltophilia, P. fluorescens/putida, P. alcaligenes, P. pseudoalcaligenes, P. stutzeri, Acinetobacter spp., Achromobacter xylosoxidans, Flavobacterium spp., and CDC groups IVe and Ve. Although colonization with most of these organisms is sporadic, P. cepacia (and to a lesser extent, P. maltophilia ) is usually isolated consistently, and can be associated with significant clinical deterioration. Occurrence of P. cepacia in CF respiratory tract cultures obtained close to the time of death rose nearly ten-fold from 1979 to 1982. Strains representing all nonfermentative gram-negative species encountered were assayed for susceptibility to 17 newer antimicrobial agents. Ceftazidime, n-formimidoyl thienamycin, and aztreonam were most active; cefsulodin, ceforanide, and ceftriaxone were not active against these isolates.

Imipenem ( N-formimidoyl thienamycin): In vitro antimicrobial activity and β-lactamase stability

In vitro studies with imipenem (N-formimidoyl thienamycin or MK0787) were performed with 8481 clinical isolates in three separate medical centers. More extensive comparative studies were also performed with 605 representative isolates, comparing imipenem to six other β-lactams. Although the newer β-lactams were often more active against susceptible species, imipenem demonstrated the broadest spectrum of antibacterial activity, with MIC 90s ≤ 4.0 μg/ml for all species tested except Pseudomonas maltophilia and P. cepacia . Imipenem was very active against all streptococci and staphylococci, in contrast to the third-generation cephalosporins. There was no evidence of cross-resistance between imipenem and the cephalosporins or penicillins. Resistance to hydrolysis by seven β-lactamase preparations was documented for imipenem, cefotaxime, and moxalactam. Like many other β-lactams, imipenem inhibited the Type I β-lactamase produced by Enterobacter cloacae . Other β-lactamases from gramnegative bacilli were also inhibited by high concentrations of imipenem.

Efficacy of imipenem in experimental group B streptococcal bacteremia and meningitis.

We evaluated the activity of imipenem (N-formimidoyl thienamycin) against a type III group B streptococcal strain in vitro and in vivo. The minimal inhibitory and bactericidal concentrations of imipenem were 15 mg/l. In vivo studies using an infant rat model of group B streptococcal bacteremia and meningitis revealed that imipenem was highly effective. This was shown by (1) rapid bacterial clearance from the blood and cerebrospinal fluid; (2) rapid sterilization of the blood and cerebrospinal fluid; (3) prevention of the development of meningitis in bacteremic animals, and (4) reduction in mortality. Even 10 mg/kg of imipenem produced the mean serum bactericidal titers greater than 1:100 and cerebrospinal fluid bactericidal titers greater than or equal to 1:16 at 1-2 h after subcutaneous administration. These findings suggest that the activity of imipenem is bactericidal in vitro and in vivo and may be an effective regimen against group B streptococci in this experimental model of bacteremia and meningitis.

Primaxin in the treatment of acute bacterial pneumonia in adults.

Imipenem (N-Formimidoyl thienamycin) (MK-0787) is a new beta-lactam carbapenem antibiotic. When it is combined with the renal dipeptidase inhibitor cilastatin (MK-0791) the combination is known as primaxin. In this study 28 adult patients (24 males and 4 females) with acute bacterial pneumonia were treated with primaxin. Twenty-one patients were evaluable and 20 (95%) were clinically cured of their pneumonia. Bacteriological cures were demonstrated in 84% of the cases. One patient with a susceptible Pseudomonas aeruginosa failed. Major complications or toxic reactions included antibiotic associated diarrhoea in one patient; hypotension in one patient; increased grand mal seizures in one patient and elevated liver function studies in one patient. Results of this study suggest that primaxin will be useful in the treatment of a variety of serious Gram-positive and Gram-negative pneumonias. The true incidence of possible toxic reactions with this drug is not known at this time and awaits further experience.

The interaction of beta-lactam compounds with chromosomally mediated enzymes: relations to the molecular structure.

The interaction of 25 beta-lactam compounds with both chromosomally mediated beta-lactamases from Enterobacter cloacae and Citrobacter freundii was studied by enzyme kinetics. All the penams, cephems, and the penem Sch 29482 revealed 'competitive inhibition' of both enzymes. The penem required a preincubation period of approximately 5 min before reaching a state of equilibrium between the active and the inactive enzyme. Except for the recently developed compound HR 810, newer cephalosporins generally exhibited a high affinity for both enzymes. Consistent with earlier findings latamoxef, N-formimidoyl thienamycin, aztreonam and clavulanic acid showed a time dependent inhibition of the enzyme activity. These findings suggest a more complex reaction scheme including a second reversible complex. The last-mentioned compounds share one structural property: the modification or even the loss of the ring fused to the beta-lactam ring. Among these compounds, clavulanic acid exhibited the lowest affinity for the enzymes. It seems likely that the affinity of beta-lactam compounds is mainly influenced by the nature of the substituents.

Use of an in-vitro kinetic model to study antibiotic combinations.

A two compartment pharmacokinetic model was used to study combinations of piperacillin with N-formimidoyl thienamycin or amikacin, and azlocillin with netilmicin against strains of Pseudomonas aeruginosa. Antibiotic antagonism seen with in-vitro static tests of piperacillin and thienamycin did not occur with the kinetic model. Piperacillin plus amikacin showed enhanced activity, and azlocillin prevented bacterial regrowth seen with netilmicin alone during multiple dosing experiments at high bacterial inocula. This model is useful in the study of antibiotic combinations.

Single-dose pharmacokinetics of imipenem in children

The single-dose pharmacokinetics of imipenem (N-formimidoyl thienamycin) was evaluated in 13 pediatric patients (mean age 5.2 +/- 3.5 years). Imipenem was administered in combination with cilastatin as either a 10 mg/kg or 25 mg/kg dose (not to exceed 500 mg) over 15 minutes. Plasma disposition in children was best described by a two-compartment open model. The distribution phase was rapid (t1/2 lambda 1 = 0.18 hours) and was followed by a monoexponential elimination phase (t1/2 lambda 2 = 1.2 hours). The calculated value for the apparent volume of distribution (0.66 L/kg) was similar to that of total body water. The total plasma clearance was rapid (0.36 L/hr/kg). Direct proportionality was exhibited between administered dose and either resultant plasma concentration or area under the plasma concentration versus time curve. Comparison of imipenem plasma pharmacokinetic data derived from these children with data reported from adult subjects revealed disparities for both the apparent volume of distribution and plasma clearance. Based on preliminary pharmacokinetic simulations using parameters generated from our study, a 25.0 mg/kg dose of imipenem administered every 6 hours appears adequate for initiation of therapy in children.

Postantibiotic effect of imipenem on Pseudomonas aeruginosa

Imipenem (formerly N-formimidoyl thienamycin) and ceftazidime were investigated for their postantibiotic effect on Pseudomonas aeruginosa. Four strains of P. aeruginosa in the logarithmic phase of growth were exposed for 1 and 2 h to concentrations of antibiotics achievable in human serum. Recovery periods of test cultures were evaluated after dilution or addition of beta-lactamase. A consistent postantibiotic effect against all strains was obtained with imipenem but not with ceftazidime. Although ceftazidime did not have a postantibiotic effect, it did suppress the growth of the organisms at concentrations equivalent to one-third of the MIC. The clinical implications of these effects need further evaluation.

In vitro antagonism between N-formimidoyl thienamycin and aztreonam, ticarcillin and ticarcillin/clavulanic acid

One-hundred eighty-seven bacterial strains were tested by the two-disk-agar diffusion method for the interaction between N-formimidoyl thienamycin and aztreonam, ticarcillin and ticarcillin-clavulanic acid. An antagonism between N-formimidoyl thienamycin and the other 3 beta-lactams was noted in half of the evaluable tests, especially against Pseudomonas, Escherichia coli and Morganella.

Epidemiology of colitis induced by Clostridium difficile in hamsters: application of a bacteriophage and bacteriocin typing system.

The epidemiology of colitis induced by Clostridium difficile in hamsters was studied with a new bacteriophage and bacteriocin typing system. Fatal enterocolitis was induced by administration of N-formimidoyl thienamycin. Environmental cultures were obtained repeatedly throughout the experiments. Thirteen percent of 90 healthy hamsters were already colonized with C difficile on arrival from the supplier. Mortality from enterocolitis after antibiotic administration was 75% and was not diminished by use of a laminar-flow facility. The same uncommon bacteriocin type (83/1309/2329) of toxigenic C difficile that colonized hamsters on arrival was recovered from the cecal contents of all hamsters dying with enterocolitis and from most environmental isolates. Previously uncolonized , antibiotic-treated hamsters placed into cages where animals had died from enterocolitis also developed enterocolitis with the same bacteriocin type (83/1309/2329), an outcome suggesting acquisition of C difficile from the environment.

The morphological response of Pseudomonas aeruginosa to azthreonam, cefoperazone, ceftazidime and N-formimidoyl thienamycin.

The morphological responses of Pseudomonas aeruginosa to N-formimidoyl thienamycin, azthreonam, ceftazidime and cefoperazone were studied by transmission electron microscopy. They were correlated with the results of viable counts and continuous turbidimetric monitoring. N-formimidoyl thienamycin initially caused the formation of abnormally shaped cells which developed into spheroplasts. The other antibiotics caused filamentation of the bacteria, which subsequently underwent lysis. The degree and mechanism of lysis varied between the antibiotics. Exposure to azthreonam also resulted in the deposition of electron-dense intracellular material. As judged by conventional minimum-inhibitory-concentration tests, all the agents exhibited similar activity against the strains of P. aeruginosa tested, with ceftazidime displaying the highest activity. However, N-formimidoyl thienamycin caused the most extensive morphological damage and resulted in the most rapid fall in viable count.