The esterase B from Sphingobium sp. SM42 has the new de-arenethiolase activity against cephalosporin antibiotics.

The esterase B (EstB) from Sphingobium sp. SM42, which was previously reported to be active towards dibutyl phthalate, can cleave some small aromatic ring side chains from cephalosporin derivatives. A new name, de-arenethiolase, has been proposed to represent this activity. We present the in vitro characterization of the activity of purified EstB toward cephalosporin substrates. Interestingly, EstB was highly active against cefoperazone and cefazolin resulting in 83 and 67% decreases in killing zone diameter, respectively. EstB also demonstrated a moderate activity towards ceftriaxone (18%) and cefotaxime (16%) while exhibiting no activity against cephalosporin C and cefixime. HPLC analysis indicated that EstB catalyzed the cleavage of the C-S bond found in cephalosporin derivatives to release the corresponding free aromatic ring side chains.

[Expression, purification and application of bla(TEM-116) extended-spectrum beta-lactamase].

To produce TEM-116 extended-spectrum beta-lactamase (ESBL) from recombinant bacteria in a cost-effective way, we purified and renatured the recombinant TEM-116 ESBL from the inclusion bodies by Ni(2+)-NTA affinity and gel filtration chromatography through subcloning the bla(TEM-116) into expression vector pET28a(+), transforming into Escherichia coli BL21(DE3) and inducing with IPTG. We characterized the purified protein that had the molecular weight of 30 kDa and specific activity of 476 IU/mg. The recombinant TEM-116 ESBL showed higher efficiency in eliminating penicillin and cephalosporin in vitro and in vivo. Specifically, the recombinant TEM-116 ESBL could eliminate 7000 mg penicillin G (PG) when used at 10.0 IU in 1 L fermentation medium. When used at 320.0 IU, it could also degrade a mix of PG, ampicillin and cefazolin each at 200 mg in 1 L of urine. In milk, 1.0-2.5 IU of the recombinant enzyme could remove 80 U/L of PG. The recombinant enzyme was fully active at the temperature ranged from 4 degrees C to 37 degrees C. Furthermore, the recombinant enzyme used at 2.0x10(4)-2.3x10(4) IU/(kg bw) (body weight) eliminated 8.0x10(4)-9.1x10(4) microg/(kg bw) PG in mouse models in vivo. The recombinant TEM-116 ESBL has the potential as a tool enzyme in food and environmental protection to eliminate harmful residues of antibiotics.

In vitro activities of tulathromycin and ceftiofur combined with other antimicrobial agents using bovine Pasteurella multocida and Mannheimia haemolytica isolates.

The purpose of this study was to determine the activities of two antibacterial agents used in the treatment of bovine respiratory infections-tulathromycin, a macrolide, and ceftiofur, a third-generation cephalosporin-alone, in combination with each other, and in combination with each of seven additional antibiotics (tilmicosin, florfenicol, enrofloxacin, danofloxacin, ampicillin, tetracycline, and penicillin G) against bovine Pasteurella multocida (n = 60) and Mannheimia haemolytica (n = 10) isolates for determination of synergy, antagonism, or indifference. Of 458 organism-drug combinations, 160 combinations of tulathromycin and 209 combinations of ceftiofur with eight antimicrobial drugs were indifferent. One combination was antagonistic (ceftiofur + florfenicol against one isolate of P. multocida). Time-kill studies showed loss of cidality for ceftiofur when combined with florfenicol at 1x the minimal inhibitory concentration. Overall, the in vitro data demonstrated that tulathromycin and ceftiofur, in combination with each other or seven other antimicrobial agents, primarily produce an indifferent response with no occurrences of synergism and rare occurrences of antagonism.

Inhibitory effect of zinc on the absorption of beta-lactam antibiotic ceftibuten via the peptide transporters in rats.

Zinc is an essential metal ion for the body, and is widely used for nutritional and clinical purposes. Previously, we showed that zinc inhibits the transport of glycylsarcosine via the intestinal peptide transporter PEPT1 in the human intestinal cell line Caco-2. In this study, we examined the effect of zinc on the activity of peptide transporters in rats using the oral beta-lactam antibiotic ceftibuten as a model drug. The plasma ceftibuten concentration after intraintestinal administration was decreased in the presence of zinc. The maximum plasma concentration (C(max)) was significantly decreased and the time required to reach C(max) (T(max)) was prolonged by zinc coadministration. The plasma ceftibuten concentration after iron coadministration or two hours after zinc administration was not affected. The in situ loop technique revealed 50% inhibition of ceftibuten absorption by zinc. In conclusion, zinc inhibits the transport activity of PEPT1 in vivo as well in vitro.

Protective effect of serum thymic factor, FTS, on cephaloridine-induced nephrotoxicity in rats.

Serum thymic factor (FTS), a thymic peptide hormone, has been reported to increase superoxide disumutase (SOD) levels in senescence-accelerated mice. In the present study, we examined the effect of FTS on cephaloridine (CER)-induced nephrotoxicity in vivo and in vitro. We previously reported that CER led to extracellular signal-regulated protein kinase (ERK) activation in the rat kidney. So, we also investigated whether FTS has an effect on ERK activation induced by CER. Treatment of male Sprague-Dawley rats with intravenous CER (1.2 g/kg) for 24 h markedly increased BUN and plasma creatinine levels and urinary excretion of glucose and protein, decreased creatinine clearance and also led to marked pathological changes in the proximal tubules, as revealed by electron micrographs. An increase in phosphorylated ERK (pERK) was detected in the nuclear fraction prepared from the rat kidney cortex 24 h after CER injection. Pretreatment of rats with FTS (50 microg/kg, i.v.) attenuated the CER-induced renal dysfunction and pathological damage. FTS also suppressed CER-induced ERK activation in the kidney. In vitro treatment of the established cell line, LLC-PK1 cells, with FTS significantly ameliorated CER-induced cell injury, as measured by lactate dehydrogenase (LDH) leakage. Our results, taken together with our previous report that MEK inhibitors ameliorated CER-induced renal cell injury and ERK activation induced by CER, suggest that FTS participates in protection from CER-induced nephrotoxicity by suppressing ERK activation induced by CER.

Investigation of the mechanism of resistance to third-generation cephalosporins by class C beta-lactamases by using chemical complementation.

The widespread use of antibiotics to treat bacterial infections has led to the continuing challenge of antibiotic resistance. For beta-lactam antibiotics, the most common form of resistance is the expression of beta-lactamase enzymes, which inactivate the antibiotics by cleavage of the beta-lactam core. In this study, chemical complementation, which is a general method to link the formation or cleavage of a chemical bond to the transcription of a reporter gene in vivo, was employed in combination with combinatorial mutagenesis to study the mechanism by which the class C beta-lactamase P99 might evolve resistance to the commonly administered third-generation cephalosporin cefotaxime. The chemical complementation system was first shown to be able to distinguish between the wild-type (wt) class C beta-lactamase P99 and the clinically isolated extended-spectrum class C beta-lactamase GC1 in the presence of cefotaxime. The system was then employed to evaluate the activity of mutants of wt P99 towards cefotaxime. A number of single-point mutations at position 221 (Tyr in wt P99) were identified that conferred resistance towards inhibition by cefotaxime, with as much as a 2000-fold increase in k(cat) and a 100-fold increase in k(cat)/K(M) (k(cat)=the rate of catalysis; K(M)=the Michaelis constant), as compared to those of the wt enzyme. Finally, the chemical complementation system was employed in a high-throughput screen to identify a number of mutants of P99 that have multiple mutations around the substrate-binding pocket that increase resistance towards cefotaxime inhibition. The catalytic turnover of cefotaxime by the most active mutant identified was 5500 times higher than that of the wt P99. The resistant mutants suggest a mechanism by which a number of mutations can confer resistance by increasing the flexibility of the Omega loop and altering the positioning of residue 221. Thus, as illustrated in this study, chemical complementation has the potential to be used as a high-throughput screen to study a wide range of enzyme-drug interactions.

Una síntesis versátil y eficiente de nuevas cefalosporinas «caballo de Troya» / An efficient and versatile synthesis of new Trojan-horse cephalosporins

Se describe un procedimiento útil para la síntesis de nuevas cefalosporinas de doble acción. Estas moléculas podrían representar una herramienta fascinante para el tratamiento de enfermedades infeccio- infecciosas sas bacterianas, ya que presentan una posible actividad inhibidora hacia las bacterias que Expresan la â-lactamasa. La principal ventaja de este enfoque sintético de tres pasos reside en su versatilidad, que permite la preparación sistemática de un amplio conjunto de nuevas moléculas

A useful synthesis of new dual-action cephalosporins is reported. These molecules could represent a fascinating tool for treatment of bacterial infectious diseases, since they display a possible inhibitor activity towards â-lactamase expressing bacteria. The major advantage of this 3-step synthetic approach lies in its versatility, which allows the systematic preparation of a wide pool of new molecules

Neutrophil elastase contributes to the development of ischemia-reperfusion-induced liver injury by decreasing endothelial production of prostacyclin in rats.

We previously reported that nitric oxide (NO) derived from endothelial NO synthase (NOS) increased endothelial prostacyclin (PGI(2)) production in rats subjected to hepatic ischemia-reperfusion (I/R). The present study was undertaken to determine whether neutrophil elastase (NE) decreases endothelial production of PGI(2), thereby contributing to the development of I/R-induced liver injury by decreasing hepatic tissue blood flow in rats. Hepatic tissue levels of 6-keto-PGF(1alpha), a stable metabolite of PGI(2), were transiently increased and peaked at 1 h after reperfusion, followed by a gradual decrease until 3 h after reperfusion. Sivelestat sodium hydrochloride and L-658,758, two NE inhibitors, reduced I/R-induced liver injury. These substances inhibited the decreases in hepatic tissue levels of 6-keto-PGF(1alpha) at 2 and 3 h after reperfusion but did not affect the levels at 1 h after reperfusion. These NE inhibitors significantly increased hepatic tissue blood flow from 1 to 3 h after reperfusion. Both hepatic I/R-induced increases in the accumulation of neutrophils and the microvascular permeability were inhibited by these two NE inhibitors. Protective effects induced by the two NE inhibitors were completely reversed by pretreatment with nitro-l-arginine methyl ester, an inhibitor of NOS, or indomethacin. Administration of iloprost, a stable derivative of PGI(2), produced effects similar to those induced by NE inhibitors. These observations strongly suggest that NE might play a critical role in the development of I/R-induced liver injury by decreasing endothelial production of NO and PGI(2), leading to a decrease in hepatic tissue blood flow resulting from inhibition of vasodilation and induction of activated neutrophil-induced microvascular injury.

Ceftriaxone induced lipid peroxidation and its inhibition with various antioxidants: Part II. Evaluation of glutathione and probucol as antioxidants.

Exploratory studies on drug induced lipid peroxidation in goat whole blood and its inhibition with antioxidants were carried out using sodium ceftriaxone (CTS) as the representative drug and glutathione and probucol as the representative antioxidants. The studies showed that CTS could induce lipid peroxidation to a significant extent. Lipid peroxidation is a toxicity mediating process, this finding may be correlated with the toxic potential of the drug. It was further found that glutathione and probucol caused significant suppression of CTS induced lipid peroxidation. The results suggest that glutathione and probucol merit further assessment to explore their potential to reduce drug induced lipid peroxidation and thus to increase therapeutic index of the drug by way of reducing toxicity that may be mediated through free radical mechanism.

In vitro efficacy of six cephalosporins tested against Enterobacteriaceae isolated at 38 North American medical centres participating in the SENTRY Antimicrobial Surveillance Program, 1997-1998.

The SENTRY Antimicrobial Surveillance Program is an ongoing international collaboration that monitors the predominant bacterial and fungal pathogens and antimicrobial susceptibility patterns associated with community-acquired and nosocomial infections. SENTRY data on the current cephalosporin susceptibility patterns (1997-98) of North American isolates of clinically important Enterobacteriaceae were analyzed. Susceptibility to a selection of cephalosporins was assessed at a central laboratory using reference broth microdilution methods and interpretive criteria specified by the National Committee for Clinical Laboratory Standards. The third- and fourth-generation cephalosporins tested demonstrated excellent activity against Escherichia coli and Klebsiella pneumoniae, whereas some of the older agents maintained good efficacy. Extended spectrum beta-lactamases were detected in all regions of the United States and Canada (1.8-10.7%). Cefepime was the most active agent tested against pathogens with the potential for enzyme-mediated resistance due to Amp C. The third-generation agents maintained acceptable efficacy against Serratia marcescens, but were less effective against Citrobacter and Enterobacter species. The older cephalosporins were generally inadequate against these pathogens, in contrast to cefepime, which was the widest spectrum cephalosporin overall. Some significant regional variations in spectrum were detected.

Structure-activity relationships of carbapenems to the antagonism of the antipseudomonal activity of other beta-lactam agents and to the beta-lactamase inducibility in Pseudomonas aeruginosa: effects of 1beta-methyl group and C-2 side chain.

The antagonism of the antipseudomonal activity of ceftazidime by meropenem (1a) was much less than those by imipenem (2a) and panipenem (2b). To reveal the major structural features of carbapenem compounds responsible for the antagonism, we investigated the structure-activity relationships of carbapenems to their antagonism of the antipseudomonal activity of ceftazidime and to their beta-lactamase-inducibility in P. aeruginosa. The antagonistic effect of 1a was less than that of desmethyl-meropenem (1b). Two other meropenem-analogues (3, 4), with the highly basic C-2 side chain, showed greater antagonistic effects than that of 1a, which has a weakly basic C-2 side chain. The beta-lactamase-inducibility of 1a in P. aeruginosa was lower than those of 2a, 1b and 4. These results indicated that the antagonism of the antipseudomonal activity of ceftazidime by carbapenems was due to the induction of beta-lactamase in P. aeruginosa. As a result of the study on the structure-activity relationships, we clarified that the introduction of a 1beta-methyl group and/or the reduction of the basicity (cationic character) of the C-2 side chain in carbapenem skeleton decreased the antagonistic effect of carbapenems on the antipseudomonal activity of ceftazidime resulted mainly from the decreasing the beta-lactamase inducibility.

Bacteriologic and clinical applications of a new extended-spectrum parenteral cephalosporin.

Although third-generation cephalosporins have been considered the backbone of antibiotic therapy for the treatment of many kinds of serious infections, including those in hospitalized patients, lack of activity against some important pathogens still exists among currently available drugs. In addition, increasing accounts of antibiotic resistance, particularly in the hospital environment, are of deep concern and have thus led to the need for the development of newer antimicrobial agents. Cefepime is a now parenteral cephalosporin with an extended spectrum of antibacterial activity that includes both aerobic gram-negative and gram-positive bacteria. It is also active against many gram-negative organisms resistant to ceftriaxone and cefotaxime, as well as many strains of Enterobacter and Citrobacter resistant to ceftazidime. Cefepime appears to be less likely to select out resistant organisms, and it may be less likely to change hospital flora than currently available antimicrobials. Cefepime has been shown to be very well tolerated and effective in the treatment of a variety of infections including moderate-to-severe pneumonia (including cases associated with concurrent bacteremia), complicated and uncomplicated urinary tract infections (also including cases associated with concurrent bacteremia), and skin and skin-structure infections. Clinical response rates are > or = 75% for most infections and have been comparable to ceftazidime in comparative trials. In addition, pretreatment susceptibility testing indicates that >94% of organisms isolated in patients enrolled in clinical trials were susceptible to cefepime.

In vitro antimicrobial effect against Streptococcus pneumoniae of adding rifampin to penicillin, ceftriaxone, or 1-ofloxacin.

Adding rifampin to penicillin or l-ofloxacin diminished the rate at which these antibiotics killed 21 clinical isolates isolates of Streptococcus pneumoniae in vitro. A less pronounced inhibitory effect was observed when rifampin was added to ceftriaxone. Synergy was not observed for any bacterial isolate. The in vitro demonstration of indifference or antagonism using these antibiotic combinations argues against the empirical addition of rifampin to beta-lactams or fluoroquinolones in treating serious pneumococcal infections.

Mechanism and kinetics of transcellular transport of a new beta-lactam antibiotic loracarbef across an intestinal epithelial membrane model system (Caco-2).

Various processes involved in the transcellular transport (TT) of loracarbef (LOR) were studied in the Caco-2 cell monolayer, a cell culture model of the small intestinal epithelium. The results provide support for presence of two AP to BL peptide TT pathways in the intestinal epithelial cell monolayer (Caco-2). The H+ gradient-dependent pathway (Km = 0.789 mM, and Jmax = 163 pmol/min per cm2) is relatively "high affinity" and "low capacity" compared to H+ gradient-independent pathway (Km = 8.28 mM, and Jmax = 316 pmol/min per cm2). In addition, TT of LOR in the presence of a H+ gradient was inhibited 77% to 88% (p < 0.05) by 10 mM of cephalexin, enalapril, Gly-Pro and Phe-Pro, while TT of LOR in the absence of a H+ gradient was only inhibited 42% to 48% (p < 0.05) by 10 mM of Gly-Pro and Phe-Pro. Since AP uptake is H+ gradient-dependent and saturable while the BL efflux is mostly nonsaturable and not driven by a H+ gradient, these two transmembrane transport processes must be different, which could be the result of two different peptide carriers. In vivo, these two transport processes must have worked in concert to produce transcellular flux of loracarbef. To explain the differences between kinetic characteristics of AP uptake and TT transport, a cellular pharmacokinetic (PK) model was developed and the results indicate that the PK model appropriately described the kinetics of LOR TT. The use of this PK model may provide an additional advantage to the use of the cell culture model because kinetic parameters at both sides of the intestinal epithelial membrane may be obtained using the same preparation. Taken together, the Caco-2 model system represents an excellent model system for the study of carrier-mediated processes involved in the TT of peptides and peptide-like drugs.

A role for alanine in the ammonium regulation of cephalosporin biosynthesis in Streptomyces clavuligerus.

It is known that excess ammonium supply decreases cephalosporin production and represses cephalosporin synthases. We wondered whether an additional important effect could be inhibition of synthase action by alanine. We had previously shown that ammonium addition induced alanine dehydrogenase and increased intracellular alanine and that alanine could inhibit resting cell synthesis of cephalosporins. In the present work we confirm the alanine inhibition of antibiotic production by resting cells. We found L-alanine inhibited three of the four synthases tested: ACV synthetase, cyclase and expandase; the epimerase was not inhibited. These data suggest that interference in cephalosporin production by growth in ammonium salts involves synthase inhibition by intracellular alanine, in addition to the known role of ammonium in synthase repression.

Membrane potential-dependent and carrier-mediated transport of cefpiramide, a cephalosporin antibiotic, in canalicular rat liver plasma membrane vesicles.

The biliary excretion mechanism of cefpiramide, a typical biliary excretion type cephalosporin antibiotic, was investigated by measuring the uptake by purified rat liver bile canalicular membrane vesicles. The initial uptake of cefpiramide showed significant temperature and concentration dependencies; the kinetic parameters were estimated to be 1.73 mM, 2.64 nmol/40 sec/mg protein and 0.17 nmol/40 sec/mg protein/mM for Michaelis constant, maximum uptake rate and nonsaturable uptake rate constant, respectively. An intravesicular positive membrane potential induced by valinomycin significantly enhanced the initial uptake of cefpiramide. Furthermore, by inducing an inside negative membrane potential, the initial efflux was significantly enhanced. Organic anions such as bile acids, bromosulfophthalein, bilirubin, probenecid, furosemide and 4,4'-diisothiocyanostilben-2,2'-disulfonic acid significantly reduced the uptake of cefpiramide, whereas amino acids, dipeptides, organic neutral compounds and cationic compounds had no effect. The inhibitory effects of taurocholic acid and bromosulfopthalein were especially competitive. Most of the beta-lactam antibiotics tested significantly reduced the uptake of cefpiramide in a competitive manner. Furthermore, benzylpenicillin showed a countertransport effect on the cefpiramide uptake. From these results it is suggested that neither organic cation nor dipeptide carrier systems participate in the uptake of beta-lactam antibiotics, and most of the derivatives are transported by recognition as organic anions in the bile canalicular membrane.

Estudio sobre cepas productoras de beta lactamasas plasmídicas de espectro extendido en Argentina / Plasmid mediated extended spectrum beta-lactamase strains isolated in Argentina

La inactivación de cefalosporinas de tercera generación (C3G) por beta lactamasas de enterobacterias era hasta hace poco responsabilidad exclusiva de cepas productoras de cefalosporinasas cromosómicas. Actualmente, se han descripto nuevas beta lactamasas plasmídicas, transferibles, de espectro ampliado (BLEA) que hidrolizan C3G. Para determinar la incidencia de enterobacterias productoras de BLEA se estudiaron las cepas aisladas de 824 pacientes de siete centros asistenciales de Buenos Aires. Se seleccionaron por: 1) obtención de un halo de <26mm, por el método de discos frente a cefotaxima (CTX) o ceftacidima (CAZ) y 2) aumento del halo a >30mm al emplear discos de CTX o CAZ adicionados de clavulanato (30:10). Reunieron estas condiciones 44 Klebsiella spp y 2 E. coli. Estas últimas resultaron ser simultaneamente hiperproductoras de beta lactamasa cromosónica y de TEM-1. Todas las cepas de Klebsiella fueron productoras de BLEA en base a: perfil de resistencia por CIM, punto isoeléctrico (pI) de las beta lactamasas, acción de inhibidores de beta lactamasas y transferencia de la resistencia a C3G. Las cepas productoras de BLEA, se aislaron en el 14,6 por ciento de pacientes internados en Unidades de Cuidados Intensivos, 3,6 por ciento en otras áreas y 1,2 por ciento de pacientes externos. Un 56,5 por ciento provinieron de infecciones urinarias, 17,3 por ciento de bacteriemias, 15,2 por ciento de infecciones respiratorias y 11 por ciento de otras localizaciones. Los fracasos terapéuticos con C3G predominaron entre las infecciones extraurinarias. De acuerdo a la CIM todas las cepas fueron resistentes a las penicilinas, excepto a temocilina; a las cefalosporinas de primera y segunda generación, excepto a la cefoxitina. Para las C3G todas presentaron CIM entre 50 y 100 veces más elevadas que las habituales en Klebsiella spp. no productoras de BLEA. Cefoperazona y CAZ fueron las más perjudicadas mientras que ceftizoxima y cefpiroma fueron las más estables. Aztreonama fue más afectada que carumonama. Todas fueron sensibles a imipenem y moxalactama y también a ciprofloxacina, excepto una cepa. Todas las cepas presentaron bandas a pI correspondientes a derivados SHV y todas, salvo una, a pI:5,4 correspondiente a TEM-1...