Prediction of the effects of inoculum size on the antimicrobial action of trovafloxacin and ciprofloxacin against Staphylococcus aureus and Escherichia coli in an in vitro dynamic model.
(49/79677)
The effect of inoculum size (N0) on antimicrobial action has not been extensively studied in in vitro dynamic models. To investigate this effect and its predictability, killing and regrowth kinetics of Staphylococcus aureus and Escherichia coli exposed to monoexponentially decreasing concentrations of trovafloxacin (as a single dose) and ciprofloxacin (two doses at a 12-h interval) were compared at N0 = 10(6) and 10(9) CFU/ml (S. aureus) and at N0 = 10(6), 10(7), and 10(9) CFU/ml (E. coli). A series of pharmacokinetic profiles of trovafloxacin and ciprofloxacin with respective half-lives of 9.2 and 4 h were simulated at different ratios of area under the concentration-time curve (AUC) to MIC (in [micrograms x hours/milliliter]/[micrograms/milliliter]): 58 to 466 with trovafloxacin and 116 to 932 with ciprofloxacin for S. aureus and 58 to 233 and 116 to 466 for E. coli, respectively. Although the effect of N0 was more pronounced for E. coli than for S. aureus, only a minor increase in minimum numbers of surviving bacteria and an almost negligible delay in their regrowth were associated with an increase of the N0 for both organisms. The N0-induced reductions of the intensity of the antimicrobial effect (IE, area between control growth and the killing-regrowth curves) were also relatively small. However, the N0 effect could not be eliminated either by simple shifting of the time-kill curves obtained at higher N0s by the difference between the higher and lowest N0 or by operating with IEs determined within the N0-adopted upper limits of bacterial numbers (IE's). By using multivariate correlation and regression analyses, linear relationships between IE and log AUC/MIC and log N0 related to the respective mean values [(log AUC/MIC)average and (log N0)average] were established for both trovafloxacin and ciprofloxacin against each of the strains (r2 = 0.97 to 0.99). The antimicrobial effect may be accurately predicted at a given AUC/MIC of trovafloxacin or ciprofloxacin and at a given N0 based on the relationship IE = a + b [(log AUC/MIC)/(log AUC/MIC)average] - c [(log N0)/(log N0)average]. Moreover, the relative impacts of AUC/MIC and N0 on IE may be evaluated. Since the c/b ratios for trovafloxacin and ciprofloxacin against E. coli were much lower (0.3 to 0.4) than that for ampicillin-sulbactam as examined previously (1.9), the inoculum effect with the quinolones may be much less pronounced than with the beta-lactams. The described approach to the analysis of the inoculum effect in in vitro dynamic models might be useful in studies with other antibiotic classes. (+info)
In vitro activities of cephalosporins and quinolones against Escherichia coli strains isolated from diarrheic dairy calves.
(50/79677)
The in vitro activities of several cephalosporins and quinolones against 195 strains of Escherichia coli isolated from diary calves affected by neonatal diarrhea were determined. One hundred thirty-seven of these strains produced one or more potential virulence factors (F5, F41, F17, cytotoxic necrotizing factor, verotoxin, and the eae gene), but the remaining 58 strains did not produce any of these factors. From 11 to 18% of the E. coli strains were resistant to cephalothin, nalidixic acid, enoxacin, and enrofloxacin. However, cefuroxime, cefotaxime, and cefquinome were highly effective against the E. coli isolates tested. Some significant differences (P < 0.05) in resistance to quinolones between the strains producing potential virulence factors and nonfimbriated, nontoxigenic, eae-negative strains were found. Thus, eae-positive, necrotoxigenic, and verotoxigenic (except for nalidixic acid) E. coli strains were significantly more sensitive to nalidixic acid, enoxacin, and enrofloxacin than nonfimbriated, nontoxigenic, eae-negative strains. Moreover, eae-positive strains were significantly more sensitive to enoxacin and enrofloxacin than F5-positive strains. Thus, the result of this study suggest that the bovine E. coli strains that produce some potential virulence factors are more sensitive to quinolones than those that do not express these factors. (+info)
Influences of urinary pH on ciprofloxacin pharmacokinetics in humans and antimicrobial activity in vitro versus those of sparfloxacin.
(51/79677)
The impact of acidification and alkalinization of urine on the pharmacokinetics of ciprofloxacin was investigated after single 200-mg oral doses were administered to nine healthy male volunteers. In addition, the effect of human urine on the MICs of ciprofloxacin and sparfloxacin against some common urinary tract pathogens such as Escherichia coli and Pseudomonas aeruginosa was investigated. Acidic and alkaline conditions were achieved by repeated oral doses of ammonium chloride or sodium bicarbonate, respectively. Plasma ciprofloxacin levels in all subjects were adequately described in terms of two-compartment model kinetics with first-order absorption. Acidification and alkalinization treatments had no effect on ciprofloxacin absorption, distribution, or elimination. The total amount of unchanged ciprofloxacin excreted over 24 h under acidic conditions was 88.4 +/- 14.5 mg (mean +/- standard deviation) (44.2% of the oral dose) and 82.4 +/- 16.5 mg (41.2% of the oral dose) under alkaline conditions, while the total amount of unchanged drug excreted over 24 h in volunteers receiving neither sodium bicarbonate nor ammonium chloride was 90.53 +/- 9.8 mg (45.2% of the oral dose). The mean renal clearance of ciprofloxacin was 16.78 +/- 2.67, 16.08 +/- 3.2, and 16.31 +/- 2.67 liters/h with acidification, alkalinization, and control, respectively. Renal clearance and concentrations of ciprofloxacin in urine were not correlated with urinary pH. The antibacterial activity of ciprofloxacin and sparfloxacin against E. coli NIHJ JC-2 and P. aeruginosa ATCC 27853 was affected by human urine and in particular by its pH. The activities of both quinolones against E. coli NIHJ JC-2 were lower at lower urinary pH and rather uniform, while in the case of P. aeruginosa ATCC 27853 ciprofloxacin was more active than sparfloxacin. (+info)
Structure-function studies of Ser-289 in the class C beta-lactamase from Enterobacter cloacae P99.
(52/79677)
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)
Molecular and biochemical characterization of VEB-1, a novel class A extended-spectrum beta-lactamase encoded by an Escherichia coli integron gene.
(53/79677)
A clinical isolate, Escherichia coli MG-1, isolated from a 4-month-old Vietnamese orphan child, produced a beta-lactamase conferring resistance to extended-spectrum cephalosporins and aztreonam. In a disk diffusion test, a typical synergistic effect between ceftazidime or aztreonam and clavulanic acid was observed along with an unusual synergy between cefoxitin and cefuroxime. The gene for VEB-1 (Vietnamese extended-spectrum beta-lactamase) was cloned and expressed in E. coli JM109. The recombinant plasmid pRLT1 produced a beta-lactamase with a pI of 5.35 and conferred high-level resistance to extended-spectrum (or oxyimino) cephalosporins and to aztreonam. Vmax values for extended-spectrum cephalosporins were uncommonly high, while the affinity of the enzyme for ceftazidime and aztreonam was relatively low. blaVEB-1 showed significant homology at the DNA level with only blaPER-1 and blaPER-2. Analysis of the deduced protein sequence showed that VEB-1 is a class A penicillinase having very low levels of homology with any other known beta-lactamases. The highest percentage of amino acid identity was 38% with PER-1 or PER-2, two uncommon class A extended-spectrum enzymes. Exploration of the genetic environment of blaVEB-1 revealed the presence of gene cassette features, i.e., (i) a 59-base element associated with blaVEB-1; (ii) a second 59-base element just upstream of blaVEB-1, likely belonging to the aacA1-orfG gene cassette; (iii) two core sites (GTTRRRY) on both sides of blaVEB-1; and (iv) a second antibiotic resistance gene 3' of blaVEB-1, aadB. blaVEB-1 may therefore be the first class A extended-spectrum beta-lactamase that is part of a gene cassette, which itself is likely to be located on a class 1 integron, as sulfamide resistance may indicate. Furthermore, blaVEB-1 is encoded on a large (> 100-kb) transferable plasmid found in a Klebsiella pneumoniae MG-2 isolated at the same time from the same patient, indicating a horizontal gene transfer. (+info)
Clindamycin suppresses endotoxin released by ceftazidime-treated Escherichia coli O55:B5 and subsequent production of tumor necrosis factor alpha and interleukin-1 beta.
(54/79677)
Treatment of septicemia caused by Escherichia coli with ceftazidime (CAZ) may be associated with the development of septic shock due to the release of bacterial lipopolysaccharide. We examined the suppressive effect of clindamycin (CLDM) on CAZ-induced release of endotoxin by cultured E. coli and the subsequent production of inflammatory cytokines (tumor necrosis factor alpha [TNF-alpha] and interleukin-1 beta [IL-1 beta]). E. coli ATCC 12014 was incubated in inactivated horse serum with or without CLDM for 1, 4, or 18 h, followed by the addition of CAZ and collection of the culture supernatant at 0, 1, and 2 h. The concentration of endotoxin in each sample was measured by a chromogenic Limulus test. Another portion of the culture supernatant was added to THP-1 cell culture and incubated for 4 h, and the concentrations of TNF-alpha and IL-1 beta in the supernatant were measured by an enzyme-linked immunosorbent assay. In the control group (no CLDM), CAZ administration resulted in significant increases in endotoxin, TNF-alpha, and IL-1 beta concentrations. Pretreatment of E. coli with CLDM for 4 or 18 h before the addition of CAZ significantly suppressed the concentrations of endotoxin, TNF-alpha, and IL-1 beta in a time-dependent manner. In addition, CAZ treatment transformed E. coli from rodshaped bacteria to filament-like structures, as determined by electron microscopy, while pretreatment with CLDM prevented these morphological changes. Our in vitro studies showed that CAZ-induced release of large quantities of endotoxin by E. coli could be suppressed by prior administration of CLDM. (+info)
Use of an isogenic Escherichia coli panel to design tests for discrimination of beta-lactamase functional groups of Enterobacteriaceae.
(55/79677)
A study was designed to determine if an isogenic panel of Escherichia coli strains containing many different beta-lactamases could be used for the preliminary screening of a large number of beta-lactam agents to identify which might be most useful in the development of a definitive test for specific beta-lactamases found among the members of family Enterobacteriaceae. The susceptibilities of 46 strains, comprising the isogenic panel, to expanded-spectrum cephalosporins, cephamycins, and aztreonam were determined in the presence and absence of beta-lactamase inhibitors in broth microdilution tests. The results indicated that strains producing extended-spectrum beta-lactamases (ESBLs) could be distinguished from strains producing other Bush-Jacoby-Medeiros functional group 2 or group 1 beta-lactamases. For strains producing group 1 beta-lactamases, cefpodoxime and ceftazidime MICs were > or = 4 micrograms/ml and addition of clavulanate did not reduce the MICs more than fourfold. For strains producing group 2 enzymes other than ESBLs, cefpodoxime and ceftazidime MICs were < or = 2 micrograms/ml. With a single exception (ceftazidime for the strain producing SHV-3), among strains producing ESBLs, cefpodoxime and ceftazidime MICs were > or = 4 micrograms/ml and addition of clavulanate reduced the MICs by more than eightfold. Cephamycins could also be used to discriminate between strains producing group 1 beta-lactamases and ESBLs, since only the former required cefotetan concentrations as high as 8 micrograms/ml or cefoxitin concentrations of > 16 micrograms/ml for inhibition. Other cephalosporins provided some discrimination between the various beta-lactamase producers, although they were not as reliable as either cefpodoxime or ceftazidime. These results indicate the utility of an isogenic panel for identification of candidate drugs among many for further testing with clinical isolates of the family Enterobacteriaceae to determine the best agents for detection of specific beta-lactamases in this family. (+info)
Esterases in serum-containing growth media counteract chloramphenicol acetyltransferase activity in vitro.
(56/79677)
The spirochete Borrelia burgdorferi was unexpectedly found to be as susceptible to diacetyl chloramphenicol, the product of the enzyme chloramphenicol acetyltransferase, as it was to chloramphenicol itself. The susceptibilities of Escherichia coli and Bacillus subtilis, as well as that of B. burgdorferi, to diacetyl chloramphenicol were then assayed in different media. All three species were susceptible to diacetyl chloramphenicol when growth media were supplemented with rabbit serum or, to a lesser extent, human serum. Susceptibility of E. coli and B. subtilis to diacetyl chloramphenicol was not observed in the absence of serum, when horse serum was used, or when the rabbit or human serum was heated first. In the presence of 10% rabbit serum, a strain of E. coli bearing the chloramphenicol acetyltransferase (cat) gene had a fourfold-lower resistance to chloramphenicol than in the absence of serum. A plate bioassay for chloramphenicol activity showed the conversion by rabbit, mouse, and human sera but not bacterial cell extracts or heated serum of diacetyl chloramphenicol to an inhibitory compound. Deacetylation of acetyl chloramphenicol by serum components was demonstrated by using fluorescent substrates and thin-layer chromatography. These studies indicate that esterases of serum can convert diacetyl chloramphenicol back to an active antibiotic, and thus, in vitro findings may not accurately reflect the level of chloramphenicol resistance by cat-bearing bacteria in vivo. (+info)