Combing multiple site-directed mutagenesis of penicillin G acylase from Achromobacter xylosoxidans PX02 with improved catalytic properties for cefamandole synthesis.

Penicillin G acylase (PGA) was an important biocatalyst for enzymatic production of second-generation cephalosporin. PGA from Achromobacter xylosoxidans PX02 (AxPGA) showed relatively lower identity to EcPGA (54.9% in α subunit and 51.7% in ß subunit), which could synthesize cefamandole in the kinetically controlled N-acylation (kcNa). Semi-rational design of AxPGA and "small and smart" mutant libraries were developed with minimal screening to improve cefamandole production. A triple mutant αR141A/αF142I/ßF24G by combining the mutational sites (ßF24, αR141, and αF142) from different subunits of AxPGA showed better performance in cefamandole production, with 4.2-fold of improvement in the (kcat/Km)AD value for activated acyl donor (R)-Methyl mandelate. Meanwhile, the (kcat/Km)Ps value for cefamandole by mutant αR141A/αF142I/ßF24G was sharply dropped by 25.5 times, indicating its highly synthetic activity and extremely low hydrolysis of cefamandole. Strikingly, the triple mutant αR141A/αF142I/ßF24G could form cefamandole with a yield of 85% at an economical substrate ratio (acyl donor/nucleophile) of 1.3:1 (82% at 1.1:1), which advanced the greener and more sustainable process of cefamandole production than the wild type. Furtherly, the improved synthetic ability and lower hydrolysis of cefamandole by mutant were rationalized using molecular docking.

Study on the interaction characteristics of cefamandole with bovine serum albumin by spectroscopic technique.

The interaction of cefamandole with bovine serum albumin (BSA) was studied by fluorescence quenching in combination with UV-Vis spectroscopic method under near physiological conditions. The fluorescence quenching rate constants and binding constants for BSA-cefamandole system were determined at different temperatures. The fluorescence quenching of BSA by cefamandole is due to static quenching and energy transfer. The results of thermodynamic parameters, ΔH (-268.0 kJ mol(-1)), ΔS (-810.0 J mol(-1) K(-1)) and ΔG (-26.62 to -8.52 kJ mol(-1)), indicated that van der Waals interaction and hydrogen bonding played a major role for cefamandole-BSA association. The competitive experiments demonstrated that the primary binding site of cefamandole on BSA was located at site III in sub-domain IIIA of BSA. The distance between cefamandole and a tryptophane unit was estimated to be 1.18 nm based on the Förster resonance energy transfer theory. The binding constant (KA) of BSA-cefamandole at 298 K was 2.239×10(4) L mol(-1). Circular dichroism spectra, synchronous fluorescence and three-dimensional fluorescence studies showed that the presence of cefamandole could change the conformation of BSA during the binding process.

Some ligands enhance the efflux of other ligands by the Escherichia coli multidrug pump AcrB.

By measuring quantitatively the active efflux of cephalosporins by the RND (resistance-nodulation-division) family efflux pump AcrB in intact cells of Escherichia coli, we found that the simultaneous presence of another substrate, such as chloramphenicol, benzene, cyclohexane, or Arg ß-naphthilamide, significantly enhanced the extrusion of cephalosporins. The stimulation occurred also in a strain expressing the covalently linked trimer of AcrB, and thus cannot be ascribed to the enhanced assembly of the trimer from AcrB monomers. When Val139 of AcrB was changed into Phe, the stimulation by benzene was found to occur at much lower concentration of the solvent. A plausible explanation of these observations is that the AcrB pump is constructed to pump out very rapidly the solvent or chloramphenicol molecules, and thus the efflux of cephalosporins, which presumably bind to a different subsite within the large binding pocket of AcrB, can become facilitated. Computer simulations of ligand binding to AcrB, both by docking and by molecular dynamics simulations, produced results supporting and extending this hypothesis. Benzene and the cephalosporin nitrocefin can bind simultaneously to the distal binding pocket of AcrB, both in the wild type and in the V139F variant. Interestingly, while the binding position and strength of benzene are almost unaffected by the presence of nitrocefin, this latter substrate is significantly displaced toward the exit gate in both wild type and mutant transporter in the presence of benzene. Additionally, the cephalosporin efflux may be enhanced by the binding of solvents (sometimes to the cephalosporin-free protomer), which could accelerate AcrB conformational changes necessary for substrate extrusion.

Structures of the Michaelis complex (1.2 Å) and the covalent acyl intermediate (2.0 Å) of cefamandole bound in the active sites of the Mycobacterium tuberculosis ß-lactamase K73A and E166A mutants.

The genome of Mycobacterium tuberculosis (TB) contains a gene that encodes a highly active ß-lactamase, BlaC, that imparts TB with resistance to ß-lactam chemotherapy. The structure of covalent BlaC-ß-lactam complexes suggests that active site residues K73 and E166 are essential for acylation and deacylation, respectively. We have prepared the K73A and E166A mutant forms of BlaC and have determined the structures of the Michaelis complex of cefamandole and the covalently bound acyl intermediate of cefamandole at resolutions of 1.2 and 2.0 Å, respectively. These structures provide insight into the details of the catalytic mechanism.

Kinetic behavior of the major multidrug efflux pump AcrB of Escherichia coli.

Multidrug efflux transporters, especially those that belong to the resistance-nodulation-division (RND) family, often show very broad substrate specificity and play a major role both in the intrinsic antibiotic resistance and, with increased levels of expression, in the elevated resistance of Gram-negative bacteria. However, it has not been possible to determine the kinetic behavior of these important pumps so far. This is partly because these pumps form a tripartite complex traversing both the cytoplasmic and outer membranes, with an outer membrane channel and a periplasmic adaptor protein, and it is uncertain if the behavior of an isolated component protein reflects that of the protein in this multiprotein complex. Here we use intact cells of Escherichia coli containing the intact multiprotein complex AcrB-AcrA-TolC, and measure the kinetic constants for various cephalosporins, by assessing the periplasmic concentration of the drug from their rate of hydrolysis by periplasmic beta-lactamase and the rate of efflux as the difference between the influx rate and the hydrolysis rate. Nitrocefin efflux showed a K(m) of about 5 microM with little sign of cooperativity. For other compounds (cephalothin, cefamandole, and cephaloridine) that showed lower affinity to the pump, however, kinetics showed strong positive cooperativity, which is consistent with the rotating catalysis model of this trimeric pump. For the very hydrophilic cefazolin there was little sign of efflux.

Modulation of penicillin acylase properties via immobilization techniques: one-pot chemoenzymatic synthesis of Cephamandole from Cephalosporin C.

The modulation of penicillin G acylase (PGA) properties via immobilization techniques has been performed studying the acylation of 7-aminocephalosporanic acid with R-mandelic acid methyl ester. PGA from Escherichia coli, immobilized onto agarose activated with glycidol (glyoxyl-agarose), has been used for the design of a novel one-pot synthesis of Cephamandole in aqueous medium and without isolation of intermediates, through three consecutive biotransformations catalyzed by D-amino acid oxidase, glutaryl acylase and PGA.

Pharmacokinetics of antibiotic prophylaxis in major orthopedic surgery and blood-saving techniques.

The pharmacokinetics of cefuroxime, cefotiam, cefamandole, and ampicillin/sulbactam were randomly measured in 40 patients undergoing major orthopedic surgery associated with high blood and volume turnover and intraoperative blood salvage. Serum and bone concentrations and the pharmacokinetics occurring in the context of these procedures were measured. No changes in elimination half-life relative to a normal population occurred with cefuroxime, cefotiam, and ampicillin. Serum and tissue concentrations were slightly lower with cefamandole and sulbactam, but reapplication of the initial dose was required with all antibiotics 4 hours after the first application.

Construction of a modified penicillin-binding protein 2a from methicillin-resistant Staphylococcus aureus and purification by immobilized metal affinity chromatography.

The mecA-27r gene, which encodes PBP2a-27r, was modified by site-specific mutagenesis, resulting in replacement of the N-terminal membrane anchor with a short chelating peptide (CP-PBP2a-27r). CP-PBP2a-27r retained the same binding affinity for beta-lactam antibiotics as the wild-type enzyme. Approximately 95% pure CP-PBP2a-27r was recovered in a single step by use of chelating-peptide-immobilized metal ion affinity chromatography.

Site-directed mutagenesis of the mecA gene from a methicillin-resistant strain of Staphylococcus aureus.

The mecA-27r gene from Staphylococcus aureus 27r encodes penicillin-binding protein 2a (PBP2a-27r), which causes this strain to be methicillin resistant. Removal or replacement of the N-terminal transmembrane domain had no effect on binding of penicillin, but removal of portions of the putative transglycosylase domain (144, 245, or 341 amino acids after the transmembrane region) destroyed penicillin-binding activity. The SXXK, SXN, and KSG motifs, present in all penicillin-interacting enzymes, were found in the expected linear spatial arrangement within the putative transpeptidase region of PBP2a-27r. Alterations of amino acids in all three of these motifs resulted in elimination of penicillin-binding activity, confirming their roles in the interaction with penicillin.

Presence of an additional penicillin-binding protein in methicillin-resistant Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus hominis, and Staphylococcus simulans with a low affinity for methicillin, cephalothin, and cefamandole.

The presence of an additional penicillin-binding protein (PBP) was demonstrated in methicillin-resistant strains of Staphylococcus epidermidis, S. haemolyticus, S. hominis, and S. simulans. In these four species, the apparent molecular mass of this protein was analogous to that of PBP 2' of methicillin-resistant S. aureus SR 1550-9. It exhibited a low affinity for methicillin, cephalothin, and cefamandole; and its synthesis was methicillin inducible. Peptide mapping of this PBP from the four species yielded identical results that were analogous to those obtained with S. aureus SR 1550-9. These results suggest that this protein is similar to, if not the same as, PBP 2' of S. aureus and that it is involved in methicillin resistance in the four species studied.

Efficacy of prophylactic antibiotics in vascular surgery: an arterial wall microbiologic and pharmacokinetic perspective.

This prospective study examined microbiologic features of arterial tissue and pharmacokinetics and bioactivity of cefamandole and cefazolin in patients undergoing elective primary prosthetic aortoiliofemoral/infrainguinal reconstruction. Double-blind, randomized, perioperative prophylaxis (1 gm intravenously every 6 hours for nine doses) with cefamandole or cefazolin was administered to 47 patients. Specimens of blood serum, subcutaneous fat, thrombus, atheroma, and arterial wall were obtained for culture and minimal inhibitory concentration and drug level analysis by high-pressure liquid chromatography. The serum half-life (hr +/- SEM) was 1.43 +/- 0.36 for cefamandole and 2.22 +/- 0.40 for cefazolin. Over the first 2 hours of surgery and for all time intervals combined, the serum concentration of cefazolin was significantly higher (p less than 0.025) than cefamandole. Irrespective of sampling time, the tissue concentration of cefazolin was significantly greater (p less than 0.005) than cefamandole. Positive arterial tissue cultures were obtained in 12 of 29 patients (41.4%) from 23 of 116 (19.8%) arterial tissue specimens. Coagulase-negative Staphylococcus was the predominant isolate, 64 of 93 (68.8%). Twenty-five of the 51 coagulase-negative staphylocci tested (49%) were slime-producers. During surgery, the arterial tissue concentration of cefamandole fell below the geometric mean minimal inhibitory concentration against all organisms combined, and against S. aureus (with the highest minimal inhibitory concentration of the prevalent isolates), significantly more often than the concentration of cefazolin. The data show that a significant number of primary elective aortoiliofemoral/infrainguinal reconstructions are associated with positive arterial tissue cultures, which represent a potential source of graft infection.(ABSTRACT TRUNCATED AT 250 WORDS)

Pharmacokinetics of cefonicid in serum and its penetration into lung tissue, bronchial mucosa and pleura.

Cefonicid pharmacokinetics in serum and tissue penetration into the lung parenchyma, bronchial mucosa and pleura were studied in 39 patients undergoing lung excision for malignancy. Cefonicid concentrations in serum and tissues samples were assayed at different times after a single 1 g intramuscular administration. The concentrations observed were much higher than the reported minimal inhibitory concentrations for the microorganisms commonly responsible for bronchial and pulmonary infections and therapeutic concentrations were still detectable in the tissues 24 h after dosing. Kinetic findings demonstrated a similar half-life for cefonicid in tissues and in serum. These data provided a further kinetic explanation for the observed clinical efficacy of cefonicid with a single daily dose.

In vitro activity of cefotaxime against clinically significant pathogens.

The present in vitro antibacterial activities of cefotaxime and 8 other cephalosporins (cefoperazone, cefmenoxime, cefpiramide, latamoxef, cefamandole, cefmetazole, cefotiam and cephazolin) were evaluated simultaneously in 384 strains of Gram-positive cocci, 595 strains of Enterobacteriaceae, 240 strains of non-fermenters and 143 strains of anaerobes and miscellaneous organisms. The results were expressed as minimum inhibitory concentration (MIC) range, MIC50 and MIC90. Of the beta-lactams, cefotaxime and latamoxef exhibited the highest activity against a wide variety of Gram-positive and Gram-negative bacteria. MIC90 of cefotaxime, however, for species of Pseudomonas aeruginosa, Xanthomonas maltophilia, enterococci, Bacteroides sp. and Clostridium difficile were more than 100 mg/L. Cefpiramide and cefoperazone were generally less active than these 2 agents. All strains were tested for beta-lactamase production by the cefinase disc method and the relationship of susceptibility to beta-lactams was evaluated in each species. The need was demonstrated for periodic susceptibility testing to be performed to better guide empirical antimicrobial therapy.

Effect of protein binding on drug penetration into blister fluid.

The effect of protein binding on drug penetration into blister fluid was evaluated by using cefonicid, ceftizoxime, and cefotaxime. Drug concentrations in a chamber with a high surface area/volume ratio (i.e., paper disk) follow changes in serum more closely than do those in a chamber with a low surface area/volume ratio. Both the area under the concentration-time curve ratio and the concentration ratio (by the disk method) for cefonicid were statistically lower than the ratios for ceftizoxime and cefotaxime. The high degree of protein binding of cefonicid results in the availability of less drug for diffusion to blister fluid than with the low-protein-binding ceftizoxime and cefotaxime.

Microbiological and pharmacokinetic evaluation of cefonicid, a long-acting cephalosporin.

The in vitro antimicrobial activity of cefonicid has been tested against 27 recent clinical isolates of 7 different species (S. aureus, E. coli, K. pneumoniae, P. mirabilis, S. marcescens, E. cloacae and P. aeruginosa) using the MS-2 Research System, contrast phase microscopy and the colony forming unit assay. With the exception of P. aeruginosa, S. marcescens and E. cloacae, cefonicid showed excellent activity against the different bacterial species tested (i.e. S. aureus, E. coli, K. pneumoniae and P. mirabilis). Tissue penetration of cefonicid after a single i.m. or i.v. dose (1 or 2 g, respectively) was also studied using the suction blister method. In 14 adult subjects with normal renal and liver functions, cefonicid plasma half-life was 5.1 and 5.4 h following i.v. and i.m. administration. Drug concentrations achieved at peak in plasma and suction blister fluid were higher than the minimum inhibitory concentrations for most sensitive pathogens and remained above these values for 24 h. These data support the use of a single daily dose regimen of cefonicid, both i.v. and i.m., for the treatment of most common infections caused by sensitive pathogens in blood and tissues.

Cefamandole concentrations in the anterior chamber of the eye in rabbits and humans.

Cefamandole concentrations were determined in the aqueous humor of the anterior chamber of the eye in 10 rabbits and in 15 patients undergoing cataract extraction. The rabbits received 50 mg of cefamandole per kilogram of body weight in the ear vein and humans received 2 g of cefamandole by intravenous injection before operation. The levels recovered were several times higher than the minimum inhibitory concentrations (MICs) against the bacteria that are most often responsible for ocular infections.

The role of beta-lactamase and the permeability barrier on the activity of cephalosporins against members of the Bacteroides fragilis group.

The role of beta-lactamase and the permeability barrier on the activity of some beta-lactams against 53 strains of the Bacteroides fragilis group was investigated. Minimal inhibitory concentrations of cefamandole, cefoxitin, and cephalothin were determined with or without the addition of clavulanic acid and (or) ethylenediaminetetraacetate using an agar dilution technique. A significant increase of susceptibility with clavulanic acid indicated a role for beta-lactamase, and with ethylenediaminetetraacetate, a role for a permeability barrier. We found that both beta-lactamase and low permeability decreased the activity of the beta-lactams to some extent depending on the bacterial species and the antibiotic. The species-specific exception was B. distasonis which showed only a permeability barrier to all antibiotics tested.

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.

Peritoneal transport of cefonicid.

The pharmacokinetic characteristics of cefonicid, a highly protein-bound expanded-spectrum cephalosporin, were examined in six noninfected, clinically stable patients undergoing continuous ambulatory peritoneal dialysis. After a 1.0-g intravenous dose of cefonicid, the mean concentrations in serum were 105 +/- 25 and 35.6 +/- 14.4 micrograms/ml at 3 and 72 h, respectively. Despite a prolonged half-life in serum of 49.7 +/- 18 h, the penetration into peritoneal fluid was low. The average concentration in dialysate over the 72-h study period was 2.7 micrograms/ml. The serum clearance was 2.6 +/- 1.0 ml/min, and the distribution volume was 0.14 +/- 0.02 liter/kg. Dosage recommendations and clinical considerations for cefonicid use in continuous ambulatory peritoneal dialysis patients are discussed.

Penetration of cefonicid in skin suction blister fluid in healthy volunteers.

A new cephalosporin, cefonicid (1 g) was given intramuscularly to six healthy volunteers 12 h after induction of skin suction blister. Serum and blister fluid were collected during a 24 h period. Antibiotic assay was made by a microbiological method. The data were analysed by a one-compartment kinetic model. Despite the high protein binding, cefonicid penetrated in microbiologically effective concentration into blister fluid. Blister fluid concentrations were higher than the serum concentrations at 24 h.