Outer membrane permeation of Bacteroides fragilis by cephalosporins

Abstract

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...

The Ferrous Iron-Responsive BqsRS Two-Component System Activates Genes That Promote Cationic Stress Tolerance

“…S4C ). Negatively charged stressors tested included cefsulodin (1 negative charge) ( 51 ), potassium chromate (2 negative charges), and sodium arsenate (1 or 2 negative charges; pK a , 7.03). For both chromate and arsenate, the MIC was the same for both strains regardless of the Fe(II) concentration.…”

Mechanism of action of cephalosporins and resistance caused by decreased affinity for penicillin-binding proteins in Bacteroides fragilis

“…B. fragilis G-242 produced a large amount of P-lactamase. In a previous paper, we reported that the outer membrane permeations by the five cephalosporins in B. fragilis G-242 were nearly equal (32). There are no data concerning the affinity for PBPs in B. fragilis G-242 because…”

The Medically Important Bacteroides spp. in Health and Disease

“…These differences may account for some cases of b-lactam resistance. In two studies, cefoxitin resistance of B. fragilis and B. thetaiotaomicron strains appeared to involve decreased affinity of the PBPs for the drug and a similar study of a cephalothin-resistant B. fragilis strain showed that both reduced permeation and PBP affinity were responsible for resistance (Piddock and Wise, 1986;Yotsuji et al, 1988b;Wexler and Halebian, 1990).…”

“…The Bacteroides spp. appear to have at least three high mol wt PBPs (58,000-90,000 Da) and several smaller sized PBPs as estimated by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE; Piddock and Wise, 1986;Piddock and Wise, 1987;Yotsuji et al, 1988b;Wexler and Halebian, 1990). The high mol wt PBPs have been correlated with similar proteins in E. coli, and each Bacteroides spp.…”