Structure-function studies of Ser-289 in the class C beta-lactamase from Enterobacter cloacae P99. (1/96)

Site-directed mutagenesis of Ser-289 of the class C beta-lactamase from Enterobacter cloacae P99 was performed to investigate the role of this residue in beta-lactam hydrolysis. This amino acid lies near the active site of the enzyme, where it can interact with the C-3 substituent of cephalosporins. Kinetic analysis of six mutant beta-lactamases with five cephalosporins showed that Ser-289 can be substituted by amino acids with nonpolar or polar uncharged side chains without altering the catalytic efficiency of the enzyme. These data suggest that Ser-289 is not essential in the binding or hydrolytic mechanism of AmpC beta-lactamase. However, replacement by Lys or Arg decreased by two- to threefold the kcat of four of the five beta-lactams tested, particularly cefoperazone, cephaloridine, and cephalothin. Three-dimensional models of the mutant beta-lactamases revealed that the length and positive charge of the side chain of Lys and Arg could create an electrostatic linkage to the C-4 carboxylic acid group of the dihydrothiazine ring of the acyl intermediate which could slow the deacylation step or hinder release of the product.  (+info)

Bacteriologic efficacies of oral azithromycin and oral cefaclor in treatment of acute otitis media in infants and young children. (2/96)

A prospective, open-label, randomized study was conducted in order to determine the bacteriologic efficacies of cefaclor and azithromycin in acute otitis media (AOM). Tympanocentesis was performed on entry into the study and 3 to 4 days after initiation of treatment. Bacteriologic failure after 3 to 4 days of treatment with both drugs occurred in a high proportion of culture-positive patients, especially in those in whom AOM was caused by Haemophilus influenzae (16 of 33 [53%] of those treated with azithromycin and 13 of 34 [52%] of those treated with cefaclor). Although a clear correlation of the persistence of the pathogen with increased MICs of the respective drugs could be demonstrated for Streptococcus pneumoniae, no such correlation was found for H. influenzae. It is proposed that susceptibility breakpoints for H. influenzae should be considerably lower than the current ones for both cefaclor and azithromycin for AOM caused by H. influenzae.  (+info)

Macrolide antibiotics inhibit nitric oxide generation by rat pulmonary alveolar macrophages. (3/96)

There is evidence that macrolide antibiotics are effective in the treatment of chronic airway inflammatory diseases, probably through actions other than their antibacterial properties. In order to determine whether macrolides affect the nitric oxide-generating system in the respiratory tract, rat pulmonary alveolar macrophages (PAMs) were studied in vitro. The release of NO was assessed by direct measurement with a specific amperometric sensor for this molecule, and the expression of type II NO synthase (NOS) messenger ribonucleic acid (mRNA) was determined by Northern blotting. Incubation of PAMs with lipopolysaccharide from Escherichia coli and recombinant human interferon-gamma caused release of NO, which was accompanied by induction of type II NOS mRNA. The release of NO was reduced by coincubation of cells with the macrolides erythromycin, clarithromycin and josamycin in a concentration-dependent manner, the maximal inhibition being 73+/-10, 81+/-6 and 84+/-9%, respectively, but was not altered by amoxycillin or cefaclor. These macrolides likewise inhibited the induction of type II NOS mRNA, whereas no inhibitory effects were observed with amoxycillin or cefaclor. These results suggest that macrolide antibiotics specifically inhibit type II NO synthase gene expression and consequently reduce NO production by rat pulmonary alveolar macrophages, which might result in attenuation of airway inflammation.  (+info)

Presence of ROB-1 beta-lactamase correlates with cefaclor resistance among recent isolates of Haemophilus influenzae. (4/96)

beta-Lactamase production in Canadian isolates of Haemophilus influenzae has remained relatively constant (25-35%) over the last decade despite increasing cefaclor resistance (MIC >/= 32 mg/L). TEM (294/324, 90.7%) and ROB-1 (30/324, 9.3%) prevalence rates among 324 isolates of H. influenzae obtained from across Canada in 1997-1998 were similar (P > 0.05) to previously published reports. However, 66. 7% (26/39) of cefaclor-resistant isolates were ROB-1-positive (P < 0. 001) and the remaining four ROB-1-positive isolates were cefaclor-intermediate (MIC 16 mg/L). Susceptibilities to loracarbef (P < 0.001) and cefprozil were also reduced in the presence of ROB-1 while the activities of cefuroxime, cefotaxime, cefixime and imipenem were similar in both TEM- and ROB-1-positive solates.  (+info)

Cefaclor, a cephalosporin antibiotic, delays gastric emptying rate by a CCK-A receptor-mediated mechanism in the rat. (5/96)

Studies in vitro suggest that cephalosporin antibiotics release the gut hormone cholecystokinin. Cholecystokinin is known to inhibit gastric emptying. Here we examine the effects of cefaclor on gastric emptying and intestinal motility. Male Sprague-Dawley rats were fitted with gastric cannulas. Following a 3-week recovery, the rate of gastric emptying of saline, peptone (4.5%) or cefaclor was determined after instillation into the gastric cannula, while intestinal transit was measured by using the propagation of arabic gum + charcoal mixture given intraduodenally. Gastric emptying of saline was significantly delayed by the addition of cefaclor (3, 10, 30 or 100 mM). The CCK-A antagonist SR-27897B (1 mg kg(-1), i.p.) reversed the delay induced by 10 mM cefaclor, whereas the CCK-B antagonist CI-988 (1 mg kg(-1), i.p.) had no significant effect. In capsaicin-treated rats, 10 mM cefaclor emptied more rapidly than in vehicle-treated animals. Thirty-minute intestinal transit was increased at 30 and 100 mM of cefaclor, while the gastric acid secretion following cefaclor instillation was no different than the group which received saline. The cephalosporin antibiotic cefaclor appears to be a potent stimulant of CCK release from gut endocrine cells, resembling the effects of peptone. Cefaclor delays gastric emptying via capsaicin-sensitive afferent pathways, which involve CCK-A receptor interaction.  (+info)

Novel mechanism of hydrolysis of therapeutic beta-lactams by Stenotrophomonas maltophilia L1 metallo-beta-lactamase. (6/96)

Stopped-flow tryptophan fluorescence under single turnover and pseudo-first-order conditions has been used to investigate the kinetic mechanism of beta-lactam hydrolysis by the Stenotrophomonas maltophilia L1 metallo-beta-lactamase. For the cephalosporin substrates nitrocefin and cefaclor and the carbapenem meropenem, a substantial quench of fluorescence is observed on association of substrate with enzyme. We have assigned this to a rearrangement event subsequent to formation of an initial collision complex. For the colorimetric compound nitrocefin, decay of this dark inter- mediate represents the overall rate-determining step for the reaction and is equivalent to decay of a previously observed state in which the beta-lactam amide bond has already been cleaved. For both cefaclor and meropenem, the rate-determining step for hydrolysis is loss of a second, less quenched state, in which, however, the beta-lactam amide bond remains intact. We suggest, therefore, that the mechanism of hydrolysis of nitrocefin by binuclear metallo-beta-lactamases may be atypical and that cleavage of the beta-lactam amide bond is the rate-determining step for breakdown of the majority of beta-lactam substrates by the L1 enzyme.  (+info)

E test for studying in vitro activity of seven antimicrobial agents against penicillin-susceptible and penicillin-resistant pneumococci. (7/96)

OBJECTIVE: To evaluate the comparative activity of seven oral antimicrobial agents against 100 strains of Streptococcus pneumoniae (S. pneumoniae). METHODS: Total 100 strains of S. pneumoniae were collected from general hospitals in Beijing from June 1996 to June 1997. E test method was used to detect the minimum inhibitory concentration (MIC) values of the following seven drugs against S. pneumoniae: penicillin, amoxicillin/clavulanate, cefaclor, cefuroxime, ceftriaxone, azithromycin and ofloxacin. The breakpoint of susceptibility categories was defined according to NCCLS. RESULTS: 74% of isolates were susceptible (S, MIC 0.06 mg/L) to penicillin, and 5% were penicillin resistant (R, MIC 2 mg/L). The intermediately resistant (I, MIC 0.1-1 mg/L) rate to penicillin was 21%. The total rates of R or I were 26% for penicillin, 6% for both amoxicillin/clavulanate and ceftriaxone, 9% for cefuroxime, 66% and 68% for azithromycin and ofloxacin respectively. Overall 94%, 94% and 91% of the pneumococcal isolates were susceptible to amoxicillin/clavulanate, ceftriaxone and cefuroxime respectively. S. pneumoniae was highly resistant to azithromycin tested as a representative of macrolides (MIC50 and MIC90 both > 256 mg/L, R rate 66%), and had a highly intermediately resistant rate to ofloxacin (I rate 62%). CONCLUSIONS: Our data suggest that in vitro activity of oral amoxicillin/clavulanate was as good as ceftriaxone and cefuroxime. It can be considered as an alternative compound in the treatment of community acquired pneumoniae and other respiratory tract infections caused by multiresistant S. pneumoniae.  (+info)

Haemophilus influenzae bla(ROB-1) mutations in hypermutagenic deltaampC Escherichia coli conferring resistance to cefotaxime and beta-lactamase inhibitors and increased susceptibility to cefaclor. (8/96)

The clinical use of cefaclor has been shown to enrich Haemophilus influenzae populations harboring cefaclor-hydrolyzing ROB-1 beta-lactamase. Such a selective process may lead to the increased use of extended-spectrum cephalosporins or beta-lactams plus beta-lactamase inhibitors and, eventually, resistance to these agents, which has not previously been observed in H. influenzae. In order to establish which bla(ROB-1) mutations, if any, could confer resistance to extended-spectrum cephalosporins and/or to beta-lactamase inhibitors, a plasmid harboring bla(ROB-1) was transformed into hypermutagenic strain Escherichia coli GB20 (DeltaampC mutS::Tn10), and this construct was used in place of H. influenzae bla(ROB-1). Strain GB20 with the cloned gene was submitted to serial passages in tubes containing broth with increasing concentrations of selected beta-lactams (cefotaxime or amoxicillin-clavulanate). Different mutations in the bla(ROB-1) gene were obtained during the passages in the presence of the different concentrations of the selective agents. Mutants resistant to extended-spectrum cephalosporins harbored either the Leu169-->Ser169 or the Arg164-->Trp164 substitution or the double amino acid change Arg164-->Trp164 and Ala237-->Thr237. ROB-1 mutants that were resistant to beta-lactams plus beta-lactamase inhibitors and that harbored the Arg244-->Cys244 or the Ser130-->Gly130 replacement were also obtained. The cefaclor-hydrolyzing efficiencies of the ROB-1 variants were strongly decreased in all mutants, suggesting that if bla(ROB-1) mutants were selected by cefaclor, this drug would prevent the further evolution of this beta-lactamase toward molecular forms able to resist extended-spectrum cephalosporins or beta-lactamase inhibitors.  (+info)

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