Difference in pathway of Escherichia coli outer membrane permeation between penicillins and cephalosporins

The antibacterial activities of tosufloxacin and other quinolones against and apparent uptakes of tosufloxacin and other quinolones by outer membrane mutants of Escherichia coli, Proteus mirabilis, and Salmonella typhimurium were studied. The hydrophobicity of tosufloxacin was nearly equal to that of ofloxacin or lower than those of sparfloxacin and nalidixic acid. OmpF-and OmpC-deficient E. coli and 40-kDa porin-deficient P. mirabilis mutants were twofold more susceptible to tosufloxacin and sparfloxacin but two-to fourfold less susceptible to other quinolones than their parent strains. In S. typhimurium lipopolysaccharide-deficient (rough) mutants, the differences in susceptibility to tosufloxacin were similar to those to sparfloxacin and nalidixic acid. The apparent uptake of tosufloxacin by intact cells was increased in porin-deficient mutants compared with that by their parent strain. These results suggest that the permeation route of tosufloxacin across the outer membrane is different from that of other fluoroquinolones and that tosufloxacin may permeate mainly through the nonporin pathway, presumably phospholipid bilayers. However, this characteristic is independent of the hydrophobicity of the molecule.

Section: Resultsmentioning

confidence: 89%

In vitro antibacterial activities of tosufloxacin against and uptake of tosufloxacin by outer membrane mutants of Escherichia coli, Proteus mirabilis, and Salmonella typhimurium

Mitsuyama

Itoh

Takahata

et al. 1992

“…Escherichia coli K-12 strains YA21 (22) and C600 (1) were used for all experiments except for the propagation of bacteriophage M13mpl9bla-I and M13mpl9bla-II, when E. coli JM105 was used. Plasmids pBR322AP1 and pBR322AP3 were made from pBR322 (4).…”

Section: Methodsmentioning

confidence: 99%

Lincomycin increases the half-life of beta-lactamase mRNA

Matsushita

Okabe

Hayashi

et al. 1989

Escherichia coli K-12 strains isolates carrying plasmid pBR322 were grown in the presence of subinhibitory concentrations of lincomycin, which stimulated ,3-lactamase synthesis about 2.5-fold, and the effects of the drug on the synthesis and degradation of bla mRNA were studied. The bla mRNA levels determined by 1-min pulse-labeling with [3H]uridine were significantly higher in a lincomycin-containing culture than in the control culture, indicating that stimulation of f-lactamase synthesis is caused by an increase in the amount of bla mRNA. The enhancing effect of lincomycin was observed in strains harboring pBR322AP1 and pBR322AP3, which lacked the P1 or P3 promoter, respectively, as well as in the strain harboring pBR322. Si nuclease analysis showed that the half-life of bla mRNA increased about 2.7-fold when lincomycin was present. These results indicate that the increase in jI-lactamase synthesis caused by lincomycin is due to an increase in the stability of bla mRNA rather than activation of its synthesis.Lincomycin stimulates production of some bacterial toxins at subinhibitory concentrations. It has been suggested that it increases enterotoxin production by Clostridium difficile (8) and causes an antibiotic-associated disease, pseudomembranous colitis (2, 6). It can also increase the production of cholera toxin (12, 13) and heat-labile enterotoxin (LT) (12,26). This phenomenon has been used in the detection of LT (9). The mechanism of this stimulatory effect of lincomycin is not well understood. We have found that TEM P-lactamase also increases in the presence of lincomycin (20). This enzynme is a monomeric secretory protein, in contrast to cholera toxin and LT, which consist of two different subunits that are encoded in polycistronic operons (23,24). Therefore, it may be easier to analyze the lincomycin effect on P-lactamase synthesis, and this information may help to elucidate the mechanism of enhancement of toxin production by lincomycin. In previous reports (19,20) we have shown that lincomycin stimulates the rate of synthesis of 3-lactamase and that stimulation involves neither an increase in copy number of the bla gene nor activation of secretion. We suggested that stimulation could occur at the level of either transcription or translation. In the present report we show that an increase in the half-life of bla mRNA is responsible for the stimulation of 1-lactamase synthesis by lincomycin. We also describe a possible mechanism for the stabilization of bla mRNA and relate it to the effects of lincomycin on toxin production. MATERIALS AND METHODSBacterial strains, plasmids, and bacteriophages. Escherichia coli K-12 strains YA21 (22) and C600 (1) were used for all experiments except for the propagation of bacteriophage M13mpl9bla-I and M13mpl9bla-II, when E. coli JM105 was used. Plasmids pBR322AP1 and pBR322AP3 were made from pBR322 (4). To delete the ,B-lactamase promoter P1, pBR322 was digested with EcoRI and BamHI. The ends were filled in with the Klenow fragment of DNA polymerase I and ligated to ci...

“…The permeation parameter C (12) and 80% of the overall Vintact in B. fragilis G-210, G-237, and G-242. On the contrary, the overall Vintact was between 2 and 15% of the overall vdis,upt of the same amount of cells.…”

mentioning

confidence: 99%

“…The permeation parameters of B. fragilis by cephalosporins were close to 10-5 cm3/min per ,ug of cell dry weight. When these values were compared with the permeation parameters of E. coli by the same compounds, it is clear that the outer membrane permeation of B. fragilis was about an order of magnitude lower than those of E. coli (12).…”

mentioning

confidence: 99%

See 1 more Smart Citation

Outer membrane permeation of Bacteroides fragilis by cephalosporins

Yotsuji

Mitsuyama

Hori

et al. 1988

Outer membrane permeation of Bacteroides fragilis by cephalosporins was examined by a previously described method. The permeation parameters of cephalosporins in B. fragilis were close to iO-5 cm3/min per ,ug of cell dry weight. These values were about an order of magnit4de lower than those in Escherichia coli. In B. fragilis, the permeation was not directly proportional to the hydrophilicity of cephalosporins, and the ion selectivity was weak.The mechanism of action of ,B-lactam antibiotics has been studied extensively in aerobic (4) and anaerobic (9) bacteria. Gram-negative bacteria, including Bacteroides fragilis, are covered by an outer membrane which acts as a permeability barrier for various toxic materials such as antibiotics and detergents (6). B. fragilis strains have been isolated from clinical specimens with an increasing frequency and are known to be moderately or highly resistant to penicillins and cephalosporins (2).In this study, we performed a detailed analysis of the outer membrape permeation of B. fragilis by cephalosporins.Bacterial strains. B. fragilis G-210, G-237, and G-242 were used in this study. These strains were isolated from clinical specimens and were highly resistant to 3-lactam antibiotics owing to ,-lactamase. B. fragilis G-210 and G-242 constitutively produce typical species-specific cephalosporinases of B. fragilis (14, 15). B. fragilis G-237 constitutively produces a novel cephalosporinase which shows a unique substrate profile in hydrolyzing cephalosporins, cephamycins, penicillins, and carbapenems (14). The ,B-lactamase produced in the periplasmic space was used for the measurement of outer membrane permeation by cephalosporins. Escherichia coli W3110 RGN823, which produces a TEM-type 3-lactamase, was also used in this study. B. fragilis and E. coli strains were stored in skim milk (10%) at -70°C.Antibiotics. The antibiotics used in this study were commercially available: cefamandole and cephaloridine, Shionogi Chemical Co., Ltd.; cefazolin and ceftezole, Fujisawa Pharmaceutical Co., Ltd.; cefoperazone, Toyama Chemical Co., Ltd.; cefsulodin, Takeda Chemical Industries, Ltd.; cephalothin, Torii Pharmaceutical Co., Ltd.Media. GAM broth and GAM agar (Nissui Pharmaceutical Co., Ltd.) were used to culture B. fragilis. Antibiotic medium 3 (Difco Laboratories, Detroit, Mich.) was used to culture E. coli.Reverse-phase thin-layer chromatography. The hydrophobic character of the cephalosporins was expressed as the Rf value, which was measured by reverse-phase thin-layer chromatography (TLC). The jI-Lactamase assay. ,B-Lactamase activity was assayed by a modification of the microiodometric method of Novick (8).Assay Qf outer membrane permeation by cephalosporins. The assay of outer membrane permeation by cephalosporins was carried out as described previously (11,16), except that the bacterial cells were grown in GAM broth for B. fragilis and on antibiotic medium 3 for E. coli. Cultures of 200 ml in the mid-logarithmic growth phase were harvested by centrifugation at 5,000 x g for 15 m...