NorM of Vibrio parahaemolyticus apparently is a new type of multidrug efflux protein, with no significant sequence similarity to any known transport proteins. Based on the following experimental results, we conclude that NorM is an Na ؉ -driven Na ؉ /drug antiporter. Drug resistance, especially multidrug resistance, is presently a serious problem in hospitals. Drug efflux from cells is one of the major mechanisms of drug resistance in both prokaryotes and eukaryotes (11,12,15,18,26). Many drug efflux systems are known to exist in the biological world, and these transporters can be divided into four families: the major facilitator (MF) family, the small multidrug resistance (SMR) family, the resistance nodulation cell division (RND) family, and the ATP binding cassette family (4,6,17). Membrane transporters of the MF family possess 12 to 14 transmembrane domains. Transporters of the SMR family are rather small and usually possess four transmembrane domains. Transporters of the RND family require multiple components to function effectively. An electrochemical potential of H ϩ across cell membranes seems to be the driving force for drug efflux by members of the MF, SMR, and RND families of transporters (13,18,28,29). ATP is utilized as the energy donor in members of the ATP binding cassette family of multidrug efflux pumps (3, 26).The electrochemical potential of H ϩ across cell membranes is established mainly by the respiratory chain in aerobic or facultative anaerobic bacteria. The electrochemical potential of H ϩ across the membrane is converted to that of Na ϩ by Na ϩ /H ϩ antiporters (25,27). Both of the electrochemical potentials of H ϩ and Na ϩ across cell membranes can be utilized to drive solute uptake in bacterial cells. Solutes are taken up into cells by an H ϩ /substrate symport mechanism or an Na ϩ / substrate symport mechanism (19). An electrochemical potential of H ϩ is also utilized to drive extrusion of substrate from cells. Most multidrug efflux pumps in bacteria are driven by H ϩ , which is a mechanism for H ϩ /drug antiport (18). However, no Na ϩ -driven extrusion system for drugs, i.e., no Na ϩ / drug antiporter, has been reported for bacterial cell membranes. Although an Na ϩ /Ca 2ϩ exchanger (16) and an Na ϩ / urea antiporter (9) have been reported for animal cells, no Na ϩ /drug antiporter has been reported for animal cells. Vibrio parahaemolyticus, a slightly halophilic marine bacterium, is one of the major causes of food poisoning in Japan and many other countries (14). This microorganism requires Na ϩ for its growth (2). Energy metabolism and energy coupling in membranes of this microorganism are unique (20). Cells of V. parahaemolyticus possess a primary respiratory Na ϩ pump (24) and Na ϩ -coupled membrane processes, such as an Na ϩ / solute symporter (21,22,24) and an Na ϩ -driven flagellar motor (1). We thought that Na ϩ /drug antiporters might exist in this marine organism.If an Na ϩ /drug antiporter were to exist, it would be anticipated that (i) Na ϩ would stimulate drug efflux fro...

An extract from Salvia officinalis (Sage) leaves showed antimicrobial activity against vancomycin-resistant enterococci (VRE). We isolated the effective compound and identified it as oleanolic acid, a triterpenoid. We also tested antimicrobial activity of similar triterpenoids, ursolic acid, uvaol, betulinic acid and betulin. We found that ursolic acid also showed antimicrobial activity against VRE. The minimum inhibitory concentrations (MICs) of oleanolic acid and ursolic acid were 8 and 4 m mg/ml, respectively. These two compounds also showed antimicrobial activity against Streptococcus pneumoniae and methicillin-resistant Staphylococcus aureus (MRSA). These compounds showed bactericidal activity against VRE at least for 48 h when added at concentrations that were two-times higher than their MICs.

Two new phenolic compounds, glicophenone (1) and glicoisoflavanone (2), were isolated from commercial licorice, and their structures were elucidated on the basis of spectroscopic data. Antibacterial assays of licorice phenolics for Staphylococcus aureus, including four strains of methicillin-resistant S. aureus (MRSA), and also for Escherichia coli K12 and Pseudomonas aeruginosa PAO1, were then examined. Two compounds among them, 8-(gamma,gamma-dimethylallyl)-wighteone (21) and 3'-(gamma,gamma-dimethylallyl)-kievitone (28), showed remarkable antibacterial effects [minimum inhibitory concentrations (MICs), 8 microg/ml on the MRSA strains and methicillin-sensitive S. aureus. Licochalcone A (14), gancaonin G (20), isoangustone A (24), glyasperins C (30) and D (31), glabridin, (32), licoricidin (33), glycycoumarin (34) and licocoumarone (40) showed antibacterial effects on the MRSA strains with MIC values of 16 microg/ml. Effects on the beta-lactam resistance of the MRSA strains were also examined, and licoricidin (33) noticeably decreased the resistance of the MRSA strains against oxacillin, as shown by the reduction in the MICs of oxacillin (lower than 1/128-1/1000 in the presence of 8 microg/ml of 33, and 1/8-1/32 in the presence of 4 microg/ml of 33). Mechanistic study suggested that 33 does not inhibit the formation of penicillin-binding protein 2' (PBP2'), but affects the enzymatic function of PBP2'.

Corilagin and tellimagrandin I are polyphenols isolated from the extract of Arctostaphylos uvaursi and Rosa canina L. (rose red), respectively. We have reported that corilagin and tellimagrandin I remarkably reduced the minimum inhibitory concentration (MIC) of β‐lactams in methicillin‐resistant Staphylococcus aureus (MRSA). In this study, we investigated the effect of corilagin and tellimagrandin I on the penicillin binding protein 2′ (2a) (PBP2′ (PBP2a)) which mainly confers the resistance to β‐lactam antibiotics in MRSA. These compounds when added to the culture medium were found to decrease production of the PBP2′ (PBP2a) slightly. Using BOCILLIN FL, a fluorescent‐labeled benzyl penicillin, we found that PBP2′ (PBP2a) in MRSA cells that were grown in medium containing corilagin or tellimagrandin I almost completely lost the ability to bind BOCILLIN FL. The binding activity of PBP2 and PBP3 were also reduced to some extent by these compounds. These results indicate that inactivation of PBPs, especially of PBP2′ (PBP2a), by corilagin or tellimagrandin I is the major reason for the remarkable reduction in the resistance level of β‐lactams in MRSA. Corilagin or tellimagrandin I suppressed the activity of β‐lactamase to some extent.

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Induction of the MexCD-OprJ multidrug efflux pump was investigated in wild-type Pseudomonas aeruginosa PAO1. MexCD-OprJ was induced by clinically important disinfectants such as benzalkonium chloride and chlorhexidine gluconate, and by some cytotoxic agents such as tetraphenylphosphonium chloride, ethidium bromide and rhodamine 6G. MexCD-OprJ was not induced by norfloxacin, tetracycline, chloramphenicol, streptomycin, erythromycin or carbenicillin, although they are substrates for the pump. Cells of PAO1 showed increased resistance to norfloxacin when grown in the presence of the inducers of the mexCD-oprJ operon mentioned above. These results indicate that MexCD-OprJ plays an important role in intrinsic multidrug resistance in wild-type P. aeruginosa in hospitals where disinfectants are used frequently.

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