Infections caused by Pseudomonas aeruginosa often are hard to treat; inappropriate chemotherapy readily selects multidrug-resistant P. aeruginosa. This organism can be exposed to a wide range of concentrations of antimicrobials during treatment; learning more about the responses of P. aeruginosa to antimicrobials is therefore important. We review here responses of the bacterium P. aeruginosa upon exposure to antimicrobials at levels below the inhibitory concentration. Carbapenems (e.g., imipenem) have been shown to induce the formation of thicker and more robust biofilms, while fluoroquinolones (e.g., ciprofloxacin) and aminoglycosides (e.g., tobramycin) have been shown to induce biofilm formation. Ciprofloxacin also has been demonstrated to enhance the frequency of mutation to carbapenem resistance. Conversely, although macrolides (e.g., azithromycin) typically are not effective against P. aeruginosa because of the pseudomonal outer-membrane impermeability and efflux, macrolides do lead to a reduction in virulence factor production. Similarly, tetracycline is not very effective against this organism, but is known to induce the type-III secretion system and consequently enhance cytotoxicity of P. aeruginosa in vivo. Of special note are the effects of antibacterials and disinfectants on pseudomonal efflux systems. Sub-inhibitory concentrations of protein synthesis inhibitors (aminoglycosides, tetracycline, chloramphenicol, etc.) induce the MexXY multidrug efflux system. This response is known to be mediated by interference with the translation of the leader peptide PA5471.1, with consequent effects on expression of the PA5471 gene product. Additionally, induction of the MexCD-OprJ multidrug efflux system is observed upon exposure to sub-inhibitory concentrations of disinfectants such as chlorhexidine and benzalkonium. This response is known to be dependent upon the AlgU stress response factor. Altogether, these biological responses of P. aeruginosa provide useful clues for the improvement and optimization of chemotherapy in order to appropriately treat pseudomonal infections while minimizing the emergence of resistance.

We found that cells of Vibrio parahaemolyticus possess an energy-dependent efflux system for norfloxacin. We cloned a gene for a putative norfloxacin efflux protein from the chromosomal DNA ofV. parahaemolyticus by using an Escherichia coli mutant lacking the major multidrug efflux system AcrAB as the host and sequenced the gene (norM). Cells of E. coli transformed with a plasmid carrying the norMgene showed elevated energy-dependent efflux of norfloxacin. The transformants showed elevated resistance not only to norfloxacin and ciprofloxacin but also to the structurally unrelated compounds ethidium, kanamycin, and streptomycin. These results suggest that this is a multidrug efflux system. The hydropathy pattern of the deduced amino acid sequence of NorM suggested the presence of 12 transmembrane domains. The deduced primary structure of NorM showed 57% identity and 88% similarity with that of a hypothetical E. coli membrane protein, YdhE. No reported drug efflux protein in the sequence databases showed significant sequence similarity with NorM. Thus, NorM seems to be a novel type of multidrug efflux protein. We cloned the ydhE gene from E. coli. Cells ofE. coli transformed with the cloned ydhE gene showed elevated resistance to norfloxacin, ciprofloxacin, acriflavine, and tetraphenylphosphonium ion, but not to ethidium, when MICs were measured. Thus, it seems that NorM and YdhE differ somehow in substrate specificity.

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...

The MexXY components of the MexXY-OprM multidrug efflux system of Pseudomonas aeruginosa are encoded by a MexZ repressor-regulated operon that is inducible by antibiotics that target the ribosome. Mutant strains disrupted in a gene, PA5471, were shown to be compromised for drug-inducible mexXY expression and, therefore, MexXY-OprM-mediated antimicrobial resistance. The PA5471 gene was inducible by the same ribosometargeting agents that induce mexXY expression. Moreover, vector-driven expression of cloned PA5471 was sufficient to promote mexXY expression and MexXY-mediated resistance in the absence of antibiotic exposure, consistent with PA5471 directly or indirectly activating mexXY expression following its own upregulation in response to antibiotics. The requirement for PA5471 for mexXY expression and antimicrobial resistance was, however, obviated in mutants lacking the MexZ repressor of mexXY expression, suggesting that PA5471 directly or indirectly modulates MexZ activity in effecting mexXY expression. While the recruitment of PA5471 and MexXY in response to ribosome disruption by antimicrobials is consistent with their genes playing a role in protecting cells from the adverse consequences of disrupting the translation process, reminiscent of transtranslation, these genes appear to operate independently in their contribution to resistance: mutants defective in trans-translation showed a much more modest (twofold) decrease in resistance to ribosome-targeting agents than those lacking PA5471 or MexXY, and this decrease was observed whether functional PA5471/MexXY was present or not.Multidrug efflux systems of the resistance-nodulation-division (RND) family contribute significantly to intrinsic and acquired resistance to antimicrobials in a number of gram-negative bacteria (43, 45). Despite their significance as determinants of antibiotic resistance, however, RND-type multidrug exporters also, in many instances, accommodate biocides (42, 45), organic solvents (48), detergents (43), including bile salts (9,18,46,60), toxic fatty acids/lipids (54), and in some instances, plant-derived antimicrobials (phytoalexins and isoflavonoids) (7, 39), metabolic inhibitors (52), organometallic compounds (tributyltin) (25), quorum-sensing effector molecules (13,26,40), and, possibly, virulence factors (21) in addition to antibiotics. Clearly, RND pumps can and do function as other than antibiotic exporters.Pseudomonas aeruginosa expresses several three-component RND-type multidrug efflux systems, among which four, MexAB-

Helicobacter cinaedi was first isolated from rectal cultures from homosexual men in 1984. In the 1980s to mid 1990s, the microorganism was mainly isolated from samples from homosexual men or immunocompromised patients; however, during the last two decades, H. cinaedi has been isolated from immunocompromised and from immunocompetent individuals worldwide. In Japan, the isolation of this microorganism was first reported in 2003. Since then, many cases have been reported in hospitals across the country. Despite many reports, the etiological properties and pathogenicity of H. cinaedi remain elusive; however, we are increasingly able to recognize some of the features and the clinical relevance of infection. In particular, a long incubation period is essential for detection in an automatic blood culture system and many of the recent isolates are resistant to both macrolides and quinolones. Furthermore, there is an association between infection and severe or chronic illnesses, such as meningitis or arteriosclerosis, in addition to mild diseases such as fever, abdominal pain, gastroenteritis, proctitis, diarrhea, erysipelas, cellulitis, arthritis, and bacteremia. In this review, we introduce the current knowledge and our latest findings relating to H. cinaedi.