Crystal Structures of Penicillin-Binding Protein D2 from Listeria monocytogenes and Structural Basis for Antibiotic Specificity.

ß-Lactam antibiotics that inhibit penicillin-binding proteins (PBPs) have been widely used in the treatment of bacterial infections. However, the molecular basis underlying the different inhibitory potencies of ß-lactams against specific PBPs is not fully understood. Here, we present the crystal structures of penicillin-binding protein D2 (PBPD2) from Listeria monocytogenes, a Gram-positive foodborne bacterial pathogen that causes listeriosis in humans. The acylated structures in complex with four antibiotics (penicillin G, ampicillin, cefotaxime, and cefuroxime) revealed that the ß-lactam core structures were recognized by a common set of residues; however, the R1 side chains of each antibiotic participate in different interactions with PBPD2. In addition, the structural complementarities between the side chains of ß-lactams and the enzyme were found to be highly correlated with the relative reactivities of penam or cephem antibiotics against PBPD2. Our study provides the structural basis for the inhibition of PBPD2 by clinically important ß-lactam antibiotics that are commonly used in listeriosis treatment. Our findings imply that the modification of ß-lactam side chains based on structural complementarity could be useful for the development of potent inhibitors against ß-lactam-resistant PBPs.

QbD-Oriented Development and Characterization of Effervescent Floating-Bioadhesive Tablets of Cefuroxime Axetil.

The objective of the present studies was systematic development of floating-bioadhesive gastroretentive tablets of cefuroxime axetil employing rational blend of hydrophilic polymers for attaining controlled release drug delivery. As per the QbD-based approach, the patient-centric target product profile and quality attributes of tablet were earmarked, and preliminary studies were conducted for screening the suitability of type of polymers, polymer ratio, granulation technique, and granulation time for formulation of tablets. A face-centered cubic design (FCCD) was employed for optimization of the critical material attributes, i.e., concentration of release controlling polymers, PEO 303 and HPMC K100 LV CR, and evaluating in vitro buoyancy, drug release, and ex vivo mucoadhesion strength. The optimized formulation was embarked upon through numerical optimization, which yield excellent floatation characteristic with drug release control (i.e., T 60% > 6 h) and bioadhesion strength. Drug-excipient compatibility studies through FTIR and P-XRD revealed the absence of any interaction between the drug and polymers. In vivo evaluation of the gastroretentive characteristics through X-ray imaging and in vivo pharmacokinetic studies in rabbits revealed significant extension in the rate of drug absorption (i.e., T max, K a, and MRT) from the optimized tablet formulation as compared to the marketed formulation. Successful establishment of various levels of in vitro/in vivo correlations (IVIVC) substantiated high degree of prognostic ability of in vitro dissolution conditions in predicting the in vivo performance. In a nutshell, the studies demonstrate successful development of the once-a-day gastroretentive formulations of cefuroxime axetil with controlled drug release profile and improved compliance.

Molecular mechanism of Hfq-dependent sRNA1039 and sRNA1600 regulating antibiotic resistance and virulence in Shigella sonnei.

Bacillary dysentery caused by Shigella spp. is a significant concern for human health. Small non-coding RNA (sRNA) plays a crucial role in regulating antibiotic resistance and virulence in Shigella spp. However, the specific mechanisms behind this phenomenon are still not fully understood. This study discovered two sRNAs (sRNA1039 and sRNA1600) that may be involved in bacterial resistance and virulence. By constructing deletion mutants (WT/ΔSR1039 and WT/ΔSR1600), this study found that the WT/ΔSR1039 mutants caused a two-fold increase in sensitivity to ampicillin, gentamicin and cefuroxime, and the WT/ΔSR1600 mutants caused a two-fold increase in sensitivity to cefuroxime. Furthermore, the WT/ΔSR1600 mutants caused a decrease in the adhesion and invasion of bacteria to HeLa cells (P<0.01), and changed the oxidative stress level of bacteria to reduce their survival rate (P<0.001). Subsequently, this study explored the molecular mechanisms by which sRNA1039 and sRNA1600 regulate antibiotic resistance and virulence. The deletion of sRNA1039 accelerated the degradation of target gene cfa mRNA and reduced its expression, thereby regulating the expression of pore protein gene ompD indirectly and negatively to increase bacterial sensitivity to ampicillin, gentamicin and cefuroxime. The inactivation of sRNA1600 reduced the formation of persister cells to reduce resistance to cefuroxime, and reduced the expression of type-III-secretion-system-related genes to reduce bacterial virulence by reducing the expression of target gene tomB. These results provide new insights into Hfq-sRNA-mRNA regulation of the resistance and virulence network of Shigella sonnei, which could potentially promote the development of more effective treatment strategies.

Mechanism analysis for the process-dependent driven mode of NaHCO3 in algal antibiotic removal: efficiency, degradation pathway and metabolic response.

This work provided a comprehensive perspective to investigate the performance of NaHCO3-driving effect and mechanism including the antibiotic removal, degradation pathway and metabolites analysis, and the algal physiological response during the removal process. Cefuroxime sodium was selected as the target antibiotic. Our results showed that NaHCO3 did not facilitate self-decomposition of the target antibiotic, while drove the improvement on the removal capacity of every algal cell, which then attributed to the total removal efficiency. After 24 h, there was an improvement on the removal rate of the target antibiotic (from 10.21% to 92.89%) when NaHCO3 was added. The degradation pathway of the target antibiotic was confirmed by the formation of three main products (M1, M2 and M3), and the degradation process, that from M1 to M2 and M2 to M3, was accelerated by the existence of NaHCO3. Besides, a 4-stage model illustrated the relationship between NaHCO3 and antibiotic removal process. Moreover, algal culture that supplemented with NaHCO3 demonstrated a better growth capacity. A large increase in the content of chlorophyll a and a moderate increase in the activity of two carbon metabolic enzymes (RuBisCO and CA) might be viewed as a positive response of the algae during the NaHCO3-driving process.

Characterization of a new metallo-beta-lactamase gene, bla(NDM-1), and a novel erythromycin esterase gene carried on a unique genetic structure in Klebsiella pneumoniae sequence type 14 from India.

A Swedish patient of Indian origin traveled to New Delhi, India, and acquired a urinary tract infection caused by a carbapenem-resistant Klebsiella pneumoniae strain that typed to the sequence type 14 complex. The isolate, Klebsiella pneumoniae 05-506, was shown to possess a metallo-beta-lactamase (MBL) but was negative for previously known MBL genes. Gene libraries and amplification of class 1 integrons revealed three resistance-conferring regions; the first contained bla(CMY-4) flanked by ISEcP1 and blc. The second region of 4.8 kb contained a complex class 1 integron with the gene cassettes arr-2, a new erythromycin esterase gene; ereC; aadA1; and cmlA7. An intact ISCR1 element was shown to be downstream from the qac/sul genes. The third region consisted of a new MBL gene, designated bla(NDM-1), flanked on one side by K. pneumoniae DNA and a truncated IS26 element on its other side. The last two regions lie adjacent to one another, and all three regions are found on a 180-kb region that is easily transferable to recipient strains and that confers resistance to all antibiotics except fluoroquinolones and colistin. NDM-1 shares very little identity with other MBLs, with the most similar MBLs being VIM-1/VIM-2, with which it has only 32.4% identity. As well as possessing unique residues near the active site, NDM-1 also has an additional insert between positions 162 and 166 not present in other MBLs. NDM-1 has a molecular mass of 28 kDa, is monomeric, and can hydrolyze all beta-lactams except aztreonam. Compared to VIM-2, NDM-1 displays tighter binding to most cephalosporins, in particular, cefuroxime, cefotaxime, and cephalothin (cefalotin), and also to the penicillins. NDM-1 does not bind to the carbapenems as tightly as IMP-1 or VIM-2 and turns over the carbapenems at a rate similar to that of VIM-2. In addition to K. pneumoniae 05-506, bla(NDM-1) was found on a 140-kb plasmid in an Escherichia coli strain isolated from the patient's feces, inferring the possibility of in vivo conjugation. The broad resistance carried on these plasmids is a further worrying development for India, which already has high levels of antibiotic resistance.

The biodegradation of cefuroxime, cefotaxime and cefpirome by the synthetic consortium with probiotic Bacillus clausii and investigation of their potential biodegradation pathways.

Cephalosporin residues in the environment are a great concern, but bioremediation options do exist. Bacillus clausii T reached a removal rate of 100% within 8 h when challenged with a mixture of cefuroxime (CFX), cefotaxime (CTX), and cefpirome (CPR). The co-culture of B. clausii T and B. clausii O/C displayed a higher removal efficiency for the mixture of CFX, CTX and CPR than a pure culture of B. clausii O/C. B. clausii T alleviated the biotoxicity of CFX and CPR. What's more, the biotoxicity of for CFX and CPR transformation products released by the co-culture of B. clausii T and B. clausii O/C was lower than that in pure cultures. Real-time PCR was applied to detect the changes in the expression levels of the relevant antibiotic-resistance genes of B. clausii T during CFX and CPR degradation. The results indicated that CFX and CPR enhanced the expression of the ß-lactamase gene bcl1. Hydrolysis, deacetylation and decarboxylation are likely the major mechanisms of CTX biodegradation by B. clausii. These results demonstrate that B. clausii T is a promising strain for the bioremediation of environmental contamination by CFX, CTX, and CPR.

TEM beta-lactamase mutants hydrolysing third-generation cephalosporins. A kinetic and molecular modelling analysis.

The catalytic properties of six "natural" mutants of the TEM-1 beta-lactamase have been studied in detail, with special emphasis on their activity versus third-generation cephalosporins. On the basis of the recently determined high-resolution structure of the wild-type enzyme, and of the substrates' structures optimized by the AMI quantum chemistry method, we have attempted to explain the influences of the mutations on the substrate profiles of the enzymes. Some of the kinetic results have thus received a satisfactory, semi-quantitative interpretation, especially in the case of single mutations. Analysis of the double mutants proved more hazardous. Extending the comparison to some other class A beta-lactamases showed that similar properties could result from different sequences, supplying an interesting example of convergent evolution within a generally diverging family.

The crystal structure of the penicillin-binding protein 2x from Streptococcus pneumoniae and its acyl-enzyme form: implication in drug resistance.

Penicillin-binding proteins (PBPs), the primary targets for beta-lactam antibiotics, are periplasmic membrane-attached proteins responsible for the construction and maintenance of the bacterial cell wall. Bacteria have developed several mechanisms of resistance, one of which is the mutation of the target enzymes to reduce their affinity for beta-lactam antibiotics. Here, we describe the structure of PBP2x from Streptococcus pneumoniae determined to 2.4 A. In addition, we also describe the PBP2x structure in complex with cefuroxime, a therapeutically relevant antibiotic, at 2.8 A. Surprisingly, two antibiotic molecules are observed: one as a covalent complex with the active-site serine residue, and a second one between the C-terminal and the transpeptidase domains. The structure of PBP2x reveals an active site similar to those of the class A beta-lactamases, albeit with an absence of unambiguous deacylation machinery. The structure highlights a few amino acid residues, namely Thr338, Thr550 and Gln552, which are directly related to the resistance phenomenon.

Purification and partial characterization of rat intestinal cefuroxime axetil esterase.

An esterase which hydrolyses the cephalosporin antibiotic, cefuroxime axetil has been isolated from rat intestinal washings and purified. Closely related cefuroxime esters were extremely poor substrates, but p-nitrophenyl acetate and alpha-naphthyl acetate were slowly hydrolysed by the purified enzyme. Analysis by gel filtration gave an Mr = 51,000 and on SDS-polyacrylamide gel electrophoresis the esterase resolved into two main bands of Mr = 31,500 and 26,800. Analytical isoelectric focusing resolved purified esterase into multiple forms active toward alpha-naphthyl acetate, the isoelectric points of which ranged from pH 4.5 to 6.3. The esterase bound specifically to Con A-Sepharose suggesting it could be a glycoprotein. Esterase activity was unaffected by the presence of dihydroxy bile salts (1-8 mM) and inhibition studies using organophosphates and eserine salicylate have classified the enzyme as a carboxylesterase.

Concentration of cefuroxime in middle ear effusion of children with acute otitis media.

BACKGROUND: Antibiotic concentrations in serum and middle ear effusion are important in determining therapeutic success in acute otitis media. For beta-lactams the most relevant pharmacokinetic index for clinical efficacy is the time for which serum concentrations exceed the minimum inhibitory concentration (MIC) of the pathogen, which should be at least 40 to 50% of the dosing interval. METHODS: In this open, single center study, the concentration of cefuroxime achieved in the serum and middle ear effusion of pediatric acute otitis media patients with purulent effusion was assessed between 2 and 5 h after a single oral dose of 15 mg/kg cefuroxime axetil suspension. RESULTS: Serum concentrations of cefuroxime ranged from 2.8 to 7.3 microg/ml and were consistent with the results of previous pharmacokinetic study. These results show that serum concentrations of cefuroxime remain above the MIC90 (2.0 microg/ml) for Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis for at least 5 h (42%) of the 12-h dosing interval. Cefuroxime was detected in 14 of 17 (82%) middle ear effusion samples and ranged from 0.2 to 3.6 microg/ml, indicating that cefuroxime penetrates well into the middle ear. CONCLUSIONS: Cefuroxime is well-absorbed and penetrates well into the middle ear after oral administration of cefuroxime axetil suspension.

Clinical study of cefuroxime in the treatment of lower respiratory tract infections.

Twenty-three hospital in-patients with severe lower respiratory tract infections were treated with cefuroxime sodium. The drug was given intramuscularly in a dose of either 750 mg or 1000 mg at 8-hourly intervals for 5 days. Of the 21 patients who could be assessed, the response to treatment was highly satisfactory and there were no treatment failures. Eight patients had failed to respond to a course of oral antibiotics before being seen. Most of the patients were elderly and all were very ill. The sputum became mucoid in all but 1 patient. There was no change in tests of liver or renal function. Cefuroxime appears to be an effective and well-tolerated drug for the treatment of patients with severe chest infections.

Levels of cefuroxime in bronchial secretion.

Levels of cefuroxime in bronchial secretion were measured in 49 patients requiring bronchoscopy for diagnostic purposes. Patients were randomly allocated to one of 4 groups which differed with respect to the size (750 mg or 1.5 g) and number (1 or 7) of intravenous doses of cefuroxime given approximately 1 hour prior to sampling. Levels of cefuroxime achieved after administration of all 4 dosages were sufficient to exceed the MIC values for the major chest pathogens. Penetration of cefuroxime into bronchial secretion was also found to be dose-related.

Cefuroxime: mechanisms of action, antimicrobial activity, pharmacokinetics, clinical applications, adverse reactions and therapeutic indications.

Cefuroxime is a second generation cephalosporin with a broad antimicrobial activity against both Gram-positive and Gram-negative organisms. It has excellent in vitro activity against staphylococcal strains, streptococcal strains (other than enterococci), N. gonorrhoeae, H. influenzae and N. meningitidis. It also has excellent in vitro activity against members of the Enterobacteriaceae with the exception of Serratia and indole-positive Proteus. Ps. aeruginosa and B. fragilis are resistant. Cefuroxime is relatively free of serious side effects. It is metabolically stable, and most of it is excreted unchanged in the urine. Three fourths of it are distributed in the extravascular compartment. Blood levels exceed the in vitro minimum inhibitory concentrations for many important gram negative pathogens. Clinical studies have shown cefuroxime to be effective therapy for infections of soft tissue, respiratory tract, urinary tract, genital tract (caused by N. gonorrhoeae) and the central nervous system. Superinfections with Ps. aeruginosa and enterococcal strains may present a problem. In spite of excellent diffusion into bone and joint tissues, the available clinical data are too limited to make a recommendation for its use in bone and joint infections.

The intraocular penetration of cefuroxime after parenteral administration.

Cefuroxime (Zinacef) is a new second generation cephalosporin which is resistant to beta-lactamases produced by both Gram-negative and Gram-positive organisms, with an antibacterial spectrum that includes practically all likely ocular pathogens except pseudomonas. It is shown to penetrate into the eye after intravenous (1.5g) or intramuscular (1 g) injection and achieve therapeutic concentrations, with absence of side effects. Cefuroxime is therefore a useful antibiotic for ophthalimic infections and should have a role in combination with gentamicin in intraocular infections, particularly those of unknown aetiology.

Pharmacokinetics of intravenous and intraperitoneal cefuroxime in patients undergoing peritoneal dialysis.

The pharmacokinetics of cefuroxime sodium, a parenteral beta-lactam antibiotic, were investigated in 9 patients during peritoneal dialysis. In 6 patients cefuroxime 500 mg was administered intravenously. Mean plasma levels of cefuroxime thereafter fell from 28.0 +/- 5.0 mg/l at 1 hr to 6.0 +/- 1.6 mg/l at 24 hr. Mean peak levels 4.6 +/- 1.9 mg/l in peritoneal effluent were found 7 hr after dosing and clearance of the drug by peritoneal dialysis averaged 4.7 ml/min. There was no evidence of net tubular secretion or of increased non-renal elimination. In 5 patients, the administration of cefuroxime, 100 mg/2 l dialyzate, in each cycle of dialysis maintained mean cefuroxime levels of 25.4 +/- 13 mg/l in the dialysis effluent. An average of 44% of the dose was not recovered in the effluent, and was presumably absorbed by the patient, and mean plasma levels of cefuroxime increased from 1.1 +/- 0.4 mg/l at 1 hr to 14.0 +/- 8.1 mg/l at 24 hr. If cefuroxime is used to treat peritoneal infections associated with peritoneal dialysis it should be given by both intraperitoneal and intravenous routes and followed up with parenteral therapy alone.

Enterobacter cloacae outer membrane permeability to ceftizoxime (FK 749) and five other new cephalosporin derivatives.

The ability of ceftizoxime to penetrate the outer membrane was compared with those of five other new cephalosporins: cefotiam, cefuroxime, cefotaxime, cefmetazole and cefoxitin, using a clinical isolate of Enterobacter cloacae as a test strain. Estimation of permeability was performed by a method utilizing the inhibitory activities of the cephalosporins against beta-lactamase located in the periplasm. Of the cephalosporins tested, both ceftizoxime and cefmetazole gave remarkably high concentrations in the periplasm, several times higher than those of cefotaxime and cefoxitin and ten or more times higher than those of cefuroxime and cefotiam. The approximate permeability coefficient of ceftizoxime was also several times higher than those of cefotiam and cefmetazole and over ten times higher than those of cefoxitin, cefuroxime and cefotaxime.

Acute prostatitis: which antibiotic to use first.

Six antimicrobial agents were administered to 48 patients (divided in 6 groups) who underwent prostatectomy. Half of the patients received the antibiotic in a single dose one hour before the operation and the rest in divided doses 24 hours before the operation. The concentration levels in serum and in prostatic tissue were measured for each of the antibiotics and for each mode of administration. The obtained ratios of prostatic tissue to serum concentrations and the relative antimicrobial activity to local pathogens of each agent indicate that the agent of choice for prostatic disease is netilmicin followed by aztreonam, cefuroxime and the ticarcillin-clavulanic acid combination.

An evaluation of the safety of cefuroxime axetil during six months oral administration to beagle dogs.

The effects of repeated oral administration of cefuroxime axetil were assessed in Beagles. The test material, an ester, is hydrolysed following absorption from the intestine to yield the therapeutically active moiety, cefuroxime, together with acetic acid and acetaldehyde; in this study cefuroxime and unhydrolyzed cefuroxime axetil were detected in the blood. Cefuroxime axetil was administered twice daily during 27 weeks by gavage of aqueous, suspensions, total daily doses were equivalent to 100, 400 or 1600 mg cefuroxime/kg/day. Apart from three cases of intercurrent illness, unrelated to treatment, the dogs remained in good health. Effects observed in the 1600 mg/kg group included vomiting and slight suppression of body weight gain. Minor variations in haematological measurements included rather low haemoglobin levels, packed cell volumes and erythrocyte counts. Slightly smaller numbers of neutrophils were thought to reflect reduced demand on normal defensive mechanisms due to continued antibiotic treatment. Prolongation of prothrombin time and activated partial thromboplastin time is attributed to disturbance of the intestinal microbial flora and reduced synthesis of vitamin K, on which the dog is highly dependent. Cefuroxime does not have the N-methylthiotetrazole side chain thought to be responsible for inhibition by other cephalosporins of the vitamin K-dependent step in the synthesis of clotting factors. Variations in plasma chemistry included rather low levels of plasma protein. Electrophoresis showed this to be a generalised reduction; only gamma globulins were proportionally decreased and this finding, like the low neutrophil counts, is attributed to the protective action of the antibiotic. Minor metabolic adjustments to the compound are reflected in plasma levels of cholesterol and triglycerides. This spectrum of findings was seen only to a very limited extent in the 400 mg/kg group; the 100 mg/kg group was, with very few exceptions, unaffected by the treatment. Macroscopic post mortem examination and microscopic examination of tissues revealed no treatment-related features indicative of toxicity. Cefuroxime axetil was thus shown to possess very little toxicity when administered repeatedly in large doses to Beagles. The lowest dose level in this study was ten times the proposed daily clinical dose in man.

The pharmacokinetics of cefuroxime lysine in the elderly.

Single doses of 750 mg of cefuroxime lysine were given to eight elderly patients. The mean plasma half life was 124 minutes and the mean time to peak was 82 minutes. When compared with previous studies in young healthy volunteers, while peak plasma concentrations are similar, there is prolongation of half life and delay of excretion of the drug in the elderly. In our group studied, no local discomfort was noted following injection.

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.