Affinity of cefotiam for the alternative penicillin binding protein PBP3SAL used by Salmonella inside host eukaryotic cells.

BACKGROUND: Following the invasion of eukaryotic cells, Salmonella enterica serovar Typhimurium replaces PBP2/PBP3, main targets of ß-lactam antibiotics, with PBP2SAL/PBP3SAL, two homologue peptidoglycan synthases absent in Escherichia coli. PBP3SAL promotes pathogen cell division in acidic environments independently of PBP3 and shows low affinity for ß-lactams that bind to PBP3 such as aztreonam, cefepime, cefotaxime, ceftazidime, ceftriaxone, cefuroxime and cefalotin. OBJECTIVES: To find compounds with high affinity for PBP3SAL to control Salmonella intracellular infections. METHODS: An S. Typhimurium ΔPBP3 mutant that divides using PBP3SAL and its parental wild-type strain, were exposed to a library of 1520 approved drugs in acidified (pH 4.6) nutrient-rich LB medium. Changes in optical density associated with cell filamentation, a read-out of blockage in cell division, were monitored. Compounds causing filamentation in the ΔPBP3 mutant but not in wild-type strain-the latter strain expressing both PBP3 and PBP3SAL in LB pH 4.6-were selected for further study. The bactericidal effect due to PBP3SAL inhibition was evaluated in vitro using a bacterial infection model of cultured fibroblasts. RESULTS: The cephalosporin cefotiam exhibited higher affinity for PBP3SAL than for PBP3 in bacteria growing in acidified LB pH 4.6 medium. Cefotiam also proved to be effective against intracellular Salmonella in a PBP3SAL-dependent manner. Conversely, cefuroxime, which has higher affinity for PBP3, showed decreased effectiveness in killing intracellular Salmonella. CONCLUSIONS: Antibiotics with affinity for PBP3SAL, like the cephalosporin cefotiam, have therapeutic value for treating Salmonella intracellular infections.

Molecular-weight-dependent, anionic-substrate-preferential transport of ß-lactam antibiotics via multidrug resistance-associated protein 4.

ß-Lactam antibiotics have cerebral and peripheral adverse effects. Multidrug resistance-associated protein 4 (MRP4) has been reported to transport several ß-lactam antibiotics, and its expression at the blood-brain barrier also serves to limit their distribution to the brain. Therefore, the purpose of this study was to clarify the structure-activity relationship of MRP4-mediated transport of ß-lactam antibiotics using MRP4-expressing Sf9 membrane vesicles. The transport activity was evaluated as MRP4-mediated transport per MRP4 protein [nL/(min·fmol MRP4 protein)] based on measurement of MRP4 protein expression by means of liquid chromatography-tandem mass spectrometry. Cefotiam showed the greatest MRP4-mediated transport activity [8.90 nL/(min·fmol MRP4 protein)] among the ß-lactam antibiotics examined in this study. Measurements of differential transport activity of MRP4 for various ß-lactam antibiotics indicated that (i) cephalosporins were transported via MRP4 at a greater rate than were penams, ß-lactamase inhibitors, penems, or monobactams; (ii) MRP4-mediated transport activity of anionic cephalosporins was greater than that of zwitterionic cephalosporins; and (iii) higher-molecular-weight anionic ß-lactam antibiotics showed greater MRP4-mediated transport activity than lower-molecular-weight ones, whereas zwitterionic ß-lactam antibiotics did not show molecular weight dependency of MRP4-mediated transport. These quantitative data should prove useful for understanding MRP-related adverse effects of ß-lactam antibiotics and their derivatives.

Cl- -dependent upregulation of human organic anion transporters: different effects on transport kinetics between hOAT1 and hOAT3.

Chloride ion has a stimulatory effect on the transport of organic anions across renal basolateral membranes. However, the exact mechanisms at molecular levels have been unclear as of yet. Human organic anion transporters hOAT1 and hOAT3 play important roles in renal basolateral membranes. In this study, the effects of Cl(-) on the activities of these transporters were evaluated by using HEK293 cells stably expressing hOAT1 or hOAT3 (HEK-hOAT1 or HEK-hOAT3). The uptake of p-[(14)C]aminohippurate by HEK-hOAT1 and [(3)H]estrone sulfate by HEK-hOAT3 was greater in the presence of Cl(-) than in the presence of SO(4)(2-) or gluconate. Additionally, the uptake of various compounds by HEK-hOAT1 and HEK-hOAT3 was significantly higher in the Cl(-)-containing medium than the gluconate-containing medium, suggesting that the influences of Cl(-) are not dependent on substrate and that Cl(-) directly stimulates the functions of hOAT1 and hOAT3. The substitution of gluconate with Cl(-) did not change the K(m) value for the uptake of p-[(14)C]aminohippurate by HEK-hOAT1 but caused an approximately threefold increase in the maximal uptake rate (V(max)) value. On the other hand, replacement of gluconate with Cl(-) decreased the K(m) value for the uptake of [(3)H]estrone sulfate and cefotiam by HEK-hOAT3 to about one-third, while it did not change the V(max) value. In summary, Cl(-) upregulates the activities of both hOAT1 and hOAT3, but its effects on transport kinetics differ between these transporters. It was suggested that Cl(-) participates in the trans-location process for hOAT1, and the substrate recognition process for hOAT3.

Pharmacokinetics of antibiotic prophylaxis in major orthopedic surgery and blood-saving techniques.

The pharmacokinetics of cefuroxime, cefotiam, cefamandole, and ampicillin/sulbactam were randomly measured in 40 patients undergoing major orthopedic surgery associated with high blood and volume turnover and intraoperative blood salvage. Serum and bone concentrations and the pharmacokinetics occurring in the context of these procedures were measured. No changes in elimination half-life relative to a normal population occurred with cefuroxime, cefotiam, and ampicillin. Serum and tissue concentrations were slightly lower with cefamandole and sulbactam, but reapplication of the initial dose was required with all antibiotics 4 hours after the first application.

Autolysis of methicillin-resistant Staphylococcus aureus is involved in synergism between imipenem and cefotiam.

Imipenem-induced autolysis and the activity of imipenem plus cefotiam were studied in 16 strains of methicillin-resistant Staphylococcus aureus (MRSA). The degree of imipenem-induced autolysis and the rate of synergistic action of imipenem plus cefotiam varied among strains and did not correlate with susceptibility to either imipenem or cefotiam. However, the degree of autolysis correlated well with susceptibility to the synergistic action of imipenem plus cefotiam. In methicillin-susceptible S. aureus strains, both imipenem-induced autolysis and the synergistic activity of the combined drugs were less than those observed in MRSA strains. Differences in the degree of autolysis were not due to differences in autolytic enzyme production. The autolysis of imipenem-pretreated MRSA was enhanced further by cefotiam, while treatment of cells in the reverse order did not enhance autolysis. These findings indicate that cell wall impairment in MRSA is caused by exposure to imipenem but not to cefotiam and that this difference in drug actions results in synergism between imipenem and cefotiam. The possible participation of penicillin-binding proteins PBP 2' and PBP4 in the observed effect is discussed.

Specificity of p-aminohippurate transport system in the OK kidney epithelial cell line.

Substrate specificity of the p-aminohippurate (PAH) transport system was investigated in the OK kidney epithelial cells. PAH uptake by OK cells from the basal side was inhibited by beta-lactam antibiotics such as benzylpenicillin (PCG) and cefazolin. The inhibition of PAH uptake by PCG was competitive and the Ki value was calculated as 108.8 microM. Transcellular transport of PCG across OK cell monolayers occurred unidirectionally from the basal to apical side, and transcellular transport and basolateral uptake were inhibited by PAH, probenecid and beta-lactam antibiotics. The basolateral uptake of cefazolin and cefotiam was also inhibited by PAH and probenecid. The basolateral uptake of PAH and PCG were not affected by aliphatic dicarboxylates with 3 or 4 carbon atoms, but were strongly inhibited by those with 5 or 6 carbon atoms. The inhibitory effect became weaker for a longer dicarboxylate with 7 carbon atoms, then increased again with increasing number of carbon atoms. Such a pattern of inhibition by dicarboxylates is essentially the same with that observed in rat renal proximal tubules in situ. These findings suggest that the PAH transport system in OK cells has a substrate specificity similar to that in rat renal proximal tubules, which is involved in the active secretion of various organic anions including drugs.

Interaction of cefpirome and a cephalosporinase from Citrobacter freundii GN7391.

The interaction of cefpirome and a cephalosporinase from Citrobacter freundii, including hydrolysis and inhibition, was studied in comparison with those of cefotiam, cefotaxime, and ceftazidime. Cefpirome was hydrolyzed by the enzyme more rapidly at Vmax than were cefotaxime and ceftazidime. However, the low affinity of the enzyme for cefpirome caused a reduction in the hydrolytic rate of cefpirome at a low drug concentration (0.1 microM). The high stability of cefpirome at a low concentration explains the high antimicrobial activity of the agent against cephalosporinase-producing bacteria.