Effects of the order of exposure to antimicrobials on the incidence of multidrug-resistant Pseudomonas aeruginosa.

Multidrug-resistant Pseudomonas aeruginosa (MDRP) is one of the most important pathogens in clinical practice. To clarify the mechanisms contributing to its emergence, we isolated MDRPs using the P. aeruginosa PAO1, the whole genome sequence of which has already been elucidated. Mutant strains resistant to carbapenems, aminoglycosides, and new quinolones, which are used to treat P. aeruginosa infections, were isolated; however, none met the criteria for MDRPs. Then, PAO1 strains were exposed to these antimicrobial agents in various orders and the appearance rate of MDRP varied depending on the order of exposure; MDRPs more frequently appeared when gentamicin was applied before ciprofloxacin, but were rarely isolated when ciprofloxacin was applied first. Exposure to ciprofloxacin followed by gentamicin increased the expression of MexCD-OprJ, an RND-type multidrug efflux pump, due to the NfxB mutation. In contrast, exposure to gentamicin followed by ciprofloxacin resulted in more mutations in DNA gyrase. These results suggest that the type of quinolone resistance mechanism is related to the frequency of MDRP and that the risk of MDRP incidence is highly dependent on the order of exposure to gentamicin and ciprofloxacin.

The role of RND-type efflux pumps in multidrug-resistant mutants of Klebsiella pneumoniae.

The emergence of multidrug-resistant Klebsiella pneumoniae is a worldwide problem. K. pneumoniae possesses numerous resistant genes in its genome. We isolated mutants resistant to various antimicrobials in vitro and investigated the importance of intrinsic genes in acquired resistance. The isolation frequency of the mutants was 10-7-10-9. Of the multidrug-resistant mutants, hyper-multidrug-resistant mutants (EB256-1, EB256-2, Nov1-8, Nov2-2, and OX128) were identified, and accelerated efflux activity of ethidium from the inside to the outside of the cells was observed in these mutants. Therefore, we hypothesized that the multidrug efflux pump, especially RND-type efflux pump, would be related to changes of the phenotype. We cloned all RND-type multidrug efflux pumps from the K. pneumoniae genome and characterized them. KexEF and KexC were powerful multidrug efflux pumps, in addition to AcrAB, KexD, OqxAB, and EefABC, which were reported previously. It was revealed that the expression of eefA was increased in EB256-1 and EB256-2: the expression of oqxA was increased in OX128; the expression of kexF was increased in Nov2-2. It was found that a region of 1,485 bp upstream of kexF, was deleted in the genome of Nov2-2. K. pneumoniae possesses more potent RND-multidrug efflux systems than E. coli. However, we revealed that most of them did not contribute to the drug resistance of our strain at basic levels of expression. On the other hand, it was also noted that the overexpression of these pumps could lead to multidrug resistance based on exposure to antimicrobial chemicals. We conclude that these pumps may have a role to maintain the intrinsic resistance of K. pneumoniae when they are overexpressed. The antimicrobial chemicals selected many resistant mutants at the same minimum inhibitory concentration (MIC) or a concentration slightly higher than the MIC. These results support the importance of using antibiotics at appropriate concentrations at clinical sites.

Comprehensive analysis of resistance-nodulation-cell division superfamily (RND) efflux pumps from Serratia marcescens, Db10.

We investigated the role of the resistance-nodulation-cell division superfamily (RND) efflux system on intrinsic multidrug resistance in Serratia marcescens. We identified eight putative RND efflux system genes in the S. marcescens Db10 genome that included the previously characterized systems, sdeXY, sdeAB, and sdeCDE. Six out of the eight genes conferred multidrug resistance on KAM32, a drug hypersensitive strain of Escherichia coli. Five out of the eight genes conferred resistance to benzalkonium, suggesting the importance of RND efflux systems in biocide resistance in S. marcescens. The energy-dependent efflux activities of five of the pumps were examined using a rhodamine 6 G efflux assay. When expressed in the tolC-deficient strain of E. coli, KAM43, none of the genes conferred resistance on E. coli. When hasF, encoding the S. marcescens TolC ortholog, was expressed in KAM43, all of the genes conferred resistance on E. coli, suggesting that HasF is a major outer membrane protein that is used by all RND efflux systems in this organism. We constructed a sdeXY deletion mutant from a derivative strain of the clinically isolated multidrug-resistant S. marcescens strain and found that the sdeXY deletion mutant was sensitive to a broad spectrum of antimicrobial agents.

Structures and Antibacterial Properties of Isorugosins H-J, Oligomeric Ellagitannins from Liquidambar formosana with Characteristic Bridging Groups between Sugar Moieties.

Three new ellagitannin oligomers, isorugosins H (1), I (2), and J (3), together with 11 known hydrolyzable tannins were isolated from an aqueous acetone extract of the fresh leaves of Liquidambar formosana. Their chemical structures were elucidated based on spectroscopic data and chemical conversion into known hydrolyzable tannins. The bridging mode of the valoneoyl groups between their sugar moieties has been identified only in this plant species. Additionally, the effects of the isorugosins isolated from this species on drug-resistant bacteria were evaluated and showed that isorugosin A (4) exhibited the most potent antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA). The isorugosins also had a suppressing effect on pigment formation in Pseudomonas aeruginosa. The isorugosin-protein complexes were analyzed using size-exclusion chromatography and polyacrylamide gel electrophoresis to clarify the relationship of their antibacterial properties with their protein interaction potency as hydrolyzable tannins. The results suggested that the antibacterial properties of hydrolyzable tannins are not simply a result of their binding activity to proteins, but are due to other factors such as the accessibility of polyphenolic acyl groups to bacterial membranes.

A Compound Inhibits Biofilm Formation of Staphylococcus aureus from Streptomyces.

Biofilm is one virulence factor of bacteria. It contributes not only to bacterial adherence to many kinds of infection-establishing surfaces, but also to bacterial resistance against antimicrobial agents and antiseptic agents. Thus, inhibitors of bacterial biofilm formation should be useful in the prevention of infections. We found that a culture of Streptomyces sp. strain MC11024 showed inhibitory activity on biofilm formation by Staphylococcus aureus and isolated streptorubin B as an inhibitor of this formation in S. aureus. The biofilm formation of methicillin resistant S. aureus (MRSA) N315 was reduced to less than 30% at 1 µg/mL of streptorubin B, and at this concentration cell growth was not affected. Our study suggests that streptorubin B has the potential to be a leading compound of anti-infectious agents of S. aureus.

Action mechanism of 6, 6'-dihydroxythiobinupharidine from Nuphar japonicum, which showed anti-MRSA and anti-VRE activities.

BACKGROUND: Multidrug-resistant bacteria, such as methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin resistant enterococci (VRE), cause serious infections at clinical sites, for which the development of new drugs is necessary. We screened candidates for new antibiotics and investigated its action mechanism. METHODS: An antimicrobial compound was isolated from an extract of Nuphar japonicum. Its chemical structure was determined by NMR, MS, and optical rotation. We measured its minimum inhibitory concentration (MIC) using the microdilution method. The effects of the compound on DNA gyrase and DNA topoisomerase IV were investigated with DNA supercoiling, decatenation, and cleavage assay. RESULTS: We isolated and identified 6,6'-dihydroxythiobinupharidine as the antimicrobial compound. The MIC of this compound was 1-4 µg/mL against various MRSA and VRE strains. We also demonstrated that this compound inhibited DNA topoisomerase IV (IC50 was 10-15 µM), but not DNA gyrase in S. aureus, both of which are known to be the targets of quinolone antibiotics and necessary for DNA replication. However, this compound only exhibited slight cross-resistance to norfloxacin-resistant S. aureus, which indicated that DTBN might inhibit other targets besides topoisomerase IV. These results suggest that 6,6'-dihydroxythiobinupharidine may be a potent candidate or seed for novel antibacterial agents. CONCLUSIONS: DTBN from N. japonicum showed anti-MRSA and anti-VRE activities. DTBN might be involved in the inhibition of DNA topoisomerase IV. GENERAL SIGNIFICANCE: DTBN might be useful as a seed compound. The information on the inhibition mechanism of DTBN will be useful for the modification of DTBN towards developing novel anti-MRSA and anti-VRE drug.

Characterization of MATE-type multidrug efflux pumps from Klebsiella pneumoniae MGH78578.

We previously described the cloning of genes related to drug resistance from Klebsiella pneumoniae MGH78578. Of these, we identified a putative gene encoding a MATE-type multidrug efflux pump, and named it ketM. Escherichia coli KAM32 possessing ketM on a plasmid showed increased minimum inhibitory concentrations for norfloxacin, ciprofloxacin, cefotaxime, acriflavine, Hoechst 33342, and 4',6-diamidino-2-phenyl indole (DAPI). The active efflux of DAPI was observed in E. coli KAM32 possessing ketM on a plasmid. The expression of mRNA for ketM was observed in K. pneumoniae cells, and we subsequently disrupted ketM in K. pneumoniae ATCC10031. However, no significant changes were observed in drug resistance levels between the parental strain ATCC10031 and ketM disruptant, SKYM. Therefore, we concluded that KetM was a multidrug efflux pump, that did not significantly contribute to intrinsic resistance to antimicrobial chemicals in K. pneumoniae. MATE-type transporters are considered to be secondary transporters; therefore, we investigated the coupling cations of KetM. DAPI efflux by KetM was observed when lactate was added to produce a proton motive force, indicating that KetM effluxed substrates using a proton motive force. However, the weak efflux of DAPI by KetM was also noted when NaCl was added to the assay mixture without lactate. This result suggests that KetM may utilize proton and sodium motive forces.

Overexpression of vmeTUV encoding a multidrug efflux transporter of Vibrio parahaemolyticus causes bile acid resistance.

We isolated deoxycholate-resistant mutants from Vibrio parahaemolyticus RTM34, which lacks four multidrug efflux transporters belonging to the resistance nodulation cell division (RND) family. RTM34 showed sensitivity to many antimicrobial agents such as cholate and deoxycholate. Deoxycholate-resistant mutants from RTM34 have elevated resistance to not only deoxycholate, but also antibiotics, disinfectants, and dyes. RT-PCR analysis revealed that the expression of vmeV, which encodes an RND-type multidrug efflux transporter, was higher in deoxycholate-resistant mutants than in parental strain RTM34. VPA0806, designated as vdeR, was located upstream of the vmeTUV operon, was oriented in the opposite direction of this operon, and encoded a putative TetR family transcriptional regulator. We determined the nucleotide sequences of vdeR and the vmeT promoter region in the genomes of deoxycholate-resistant mutants. A point mutation was identified in vdeR of seven deoxycholate-resistant mutants and a deletion mutation was identified in vdeR of one deoxycholate-resistant mutant. Since most mutations cause a frame shift mutation and premature stop codon, the function of VdeR is thought to be lost in these mutants. Taken together, the results of the present study indicate that deoxycholate resistance in these mutants was due to the overexpression of vmeTUV caused by a loss in the repression by VdeR.

Suppression of stop codon UGA in acrB can contribute to antibiotic resistance in Klebsiella pneumoniae ATCC10031.

We previously reported that Klebsiella pneumoniae MGH78578 exhibited higher resistance against various antimicrobials than K. pneumoniae ATCC10031. In this study, we showed that the plasmid, pKPN5, in K. pneumoniae MGH78578 played an important role in resistance against aminoglycosides, ampicillin, tetracycline, and chloramphenicol, while genome-derived ß-lactamases and drug efflux pumps appeared to be more important in resistance to cloxacillin. acrAB, encoding a potent multidrug efflux pump, was cloned from K. pneumoniae MGH78578 and ATCC10031, to investigate reasons for the high drug resistance of K. pneumoniae MGH78578, and the results revealed that AcrAB from K. pneumoniae ATCC10031 conferred weaker drug resistance than AcrAB from K. pneumoniae MGH78578. DNA sequencing revealed that acrB from K. pneumoniae ATCC10031 carried the nonsense mutation, UGA, which was not found in acrB from K. pneumoniae MGH78578. However, acrB from K. pneumoniae ATCC10031 conferred slightly elevated resistant levels to several antimicrobials. The intact length of AcrB was detected in K. pneumoniae ATCC10031 by Western blot analysis, even though its quantity was small. Therefore, the stop codon UGA in acrB was thought to be overcome to some extent in this strain. We artificially introduced the nonsense mutation,UGA to the cat gene on pACYC184, and the plasmid also elevated the MIC of chloramphenicol in K. pneumoniae ATCC10031. These results suggest that a mechanism to overcome the nonsense mutation in acrB sustained resistance against a few ß-lactams, dyes, and cholic acid in K. pneumoniae ATCC10031.

Characterization of all RND-type multidrug efflux transporters in Vibrio parahaemolyticus.

Resistance nodulation cell division (RND)-type efflux transporters play the main role in intrinsic resistance to various antimicrobial agents in many gram-negative bacteria. Here, we estimated 12 RND-type efflux transporter genes in Vibrio parahaemolyticus. Because VmeAB has already been characterized, we cloned the other 11 RND-type efflux transporter genes and characterized them in Escherichia coli KAM33 cells, a drug hypersusceptible strain. KAM33 expressing either VmeCD, VmeEF, or VmeYZ showed increased minimum inhibitory concentrations (MICs) for several antimicrobial agents. Additional four RND-type transporters were functional as efflux pumps only when co-expressed with VpoC, an outer membrane component in V. parahaemolyticus. Furthermore, VmeCD, VmeEF, and VmeYZ co-expressed with VpoC exhibited a broader substrate specificity and conferred higher resistance than that with TolC of E. coli. Deletion mutants of these transporter genes were constructed in V. parahaemolyticus. TM32 (ΔvmeAB and ΔvmeCD) had significantly decreased MICs for many antimicrobial agents and the number of viable cells after exposure to deoxycholate were markedly reduced. Strains in which 12 operons were all disrupted had very low MICs and much lower fluid accumulation in rabbit ileal loops. These results indicate that resistance nodulation cell division-type efflux transporters contribute not only to intrinsic resistance but also to exerting the virulence of V. parahaemolyticus.

Functionally cloned pdrM from Streptococcus pneumoniae encodes a Na(+) coupled multidrug efflux pump.

Multidrug efflux pumps play an important role as a self-defense system in bacteria. Bacterial multidrug efflux pumps are classified into five families based on structure and coupling energy: resistance-nodulation-cell division (RND), small multidrug resistance (SMR), major facilitator (MF), ATP binding cassette (ABC), and multidrug and toxic compounds extrusion (MATE). We cloned a gene encoding a MATE-type multidrug efflux pump from Streptococcus pneumoniae R6, and designated it pdrM. PdrM showed sequence similarity with NorM from Vibrio parahaemolyticus, YdhE from Escherichia coli, and other bacterial MATE-type multidrug efflux pumps. Heterologous expression of PdrM let to elevated resistance to several antibacterial agents, norfloxacin, acriflavine, and 4',6-diamidino-2-phenylindole (DAPI) in E. coli KAM32 cells. PdrM effluxes acriflavine and DAPI in a Na(+)- or Li(+)-dependent manner. Moreover, Na(+) efflux via PdrM was observed when acriflavine was added to Na(+)-loaded cells expressing pdrM. Therefore, we conclude that PdrM is a Na(+)/drug antiporter in S. pneumoniae. In addition to pdrM, we found another two genes, spr1756 and spr1877,that met the criteria of MATE-type by searching the S. pneumoniae genome database. However, cloned spr1756 and spr1877 did not elevate the MIC of any of the investigated drugs. mRNA expression of spr1756, spr1877, and pdrM was detected in S. pneumoniae R6 under laboratory growth conditions. Therefore, spr1756 and spr1877 are supposed to play physiological roles in this growth condition, but they may be unrelated to drug resistance.

Functional study of the novel multidrug efflux pump KexD from Klebsiella pneumoniae.

We cloned a gene, kexD, that provides a multidrug-resistant phenotype from multidrug-resistant Klebsiella pneumoniae MGH78578. The deduced amino acid sequence of KexD is similar to that of the inner membrane protein, RND-type multidrug efflux pump. Introduction of the kexD gene into Escherichia coli KAM32 resulted in a MIC that was higher for erythromycin, novobiocin, rhodamine 6G, tetraphenylphosphonium chloride, and ethidium bromide than that of the control. Intracellular ethidium bromide levels in E. coli cells carrying the kexD gene were lower than that in the control cells under energized conditions, suggesting that KexD is a component of an energy-dependent efflux pump. RND-type pumps typically consist of three components: an inner membrane protein, a periplasmic protein, and an outer membrane protein. We discovered that KexD functions with a periplasmic protein, AcrA, from E. coli and K. pneumoniae, but not with the periplasmic proteins KexA and KexG from K. pneumoniae. KexD was able to utilize either TolC of E. coli or KocC of K. pneumoniae as an outer membrane component. kexD mRNA was not detected in K. pneumoniae MGH78578 or ATCC10031. We isolated erythromycin-resistant mutants from K. pneumoniae ATCC10031, and some showed a multidrug-resistant phenotype similar to the drug resistance pattern of KexD. Two strains of multidrug-resistant mutants were investigated for kexD expression; kexD mRNA levels were increased in these strains. We conclude that changing kexD expression can contribute to the occurrence of multidrug-resistant K. pneumoniae.

Quercetin diacylglycoside analogues showing dual inhibition of DNA gyrase and topoisomerase IV as novel antibacterial agents.

A structure-guided molecular design approach was used to optimize quercetin diacylglycoside analogues that inhibit bacterial DNA gyrase and topoisomerase IV and show potent antibacterial activity against a wide spectrum of relevant pathogens responsible for hospital- and community-acquired infections. In this paper, such novel 3,7-diacylquercetin, quercetin 6''-acylgalactoside, and quercetin 2'',6''-diacylgalactoside analogues of lead compound 1 were prepared to assess their target specificities and preferences in bacteria. The significant enzymatic inhibition of both Escherichia coli DNA gyrase and Staphylococcus aureus topoIV suggest that these compounds are dual inhibitors. Most of the investigated compounds exhibited pronounced inhibition with MIC values ranging from 0.13 to 128 µg/mL toward the growth of multidrug-resistant Gram-positive methicillin-resistant S. aureus, methicillin sensitive S. aureus, vancomycin-resistant enterococci (VRE), vancomycin intermediate S. aureus, and Streptococcus pneumoniae bacterial strains. Structure-activity relationship studies revealed that the acyl moiety was absolutely essential for activity against Gram-positive organisms. The most active compound 5i was 512-fold more potent than vancomycin and 16-32-fold more potent than 1 against VRE strains. It also has realistic in situ intestinal absorption in rats and showed very low acute toxicity in mice. So far, this compound can be regarded as a leading antibacterial agent.

SugE, a new member of the SMR family of transporters, contributes to antimicrobial resistance in Enterobacter cloacae.

We cloned a gene, sugE, from the chromosome of Enterobacter cloacae ATCC 13047. Analysis of the susceptibilities of the sugE-containing strain (Escherichia coli KAM32/pSUGE28) and sugE-deficient E. cloacae (EcΔsugE) showed that SugE confers resistance to cetyltrimethylammonium bromide, cetylpyridinium chloride, tetraphenylphosphonium, benzalkonium chloride, ethidium bromide, and sodium dodecyl sulfate. We also investigated expression of sugE. We confirm here that SugE from E. cloacae is an SMR family transporter as determined by observing its energy-dependent drug efflux activity.

Patch clamp analysis of the respiratory chain in Bacillus subtilis.

Bacillus subtilis is a representative Gram-positive bacterium. In aerobic conditions, this bacterium can generate an electrochemical potential across the membrane with aerobic respiration. Here, we developed the patch clamp method to analyze the respiratory chain in B. subtilis. First, we prepared giant protoplasts (GPs) from B. subtilis cells. Electron micrographs and fluorescent micrographs revealed that GPs of B. subtilis had a vacuole-like structure and that the intravacuolar area was completely separated from the cytoplasmic area. Acidification of the interior of the isolated and purified vacuole-like structure, due to H(+) translocation after the addition of NADH, revealed that they consisted of everted cytoplasmic membranes. We called these giant provacuoles (GVs) and again applied the patch clamp technique. When NADH was added as an electron donor for the respiratory system, a significant NADH-induced current was observed. Inhibition of KCN and 2-heptyl-4-hydroxyquinoline-N-oxide (HQNO) demonstrated that this current is certainly due to aerobic respiration in B. subtilis. This is the first step for more detailed analyses of respiratory chain in B. subtilis, especially H(+) translocation mechanism.

Design, synthesis, and biological evaluation of a novel series of quercetin diacylglucosides as potent anti-MRSA and anti-VRE agents.

A series of novel quercetin diacylglucosides were designed and first synthesized by Steglich esterification on the basis of MRSA strains inhibiting natural compound A. The in vitro inhibition of different multi-drug resistant bacterial strains and Escherichia coli DNA gyrase B was investigated. In the series, compound 10h was up to 128-fold more potent against vancomycin-resistant enterococci and more effective than A, which represents a promising new candidate as a potent anti-MRSA and anti-VRE agent.

Gene cloning and characteristics of the RND-type multidrug efflux pump MuxABC-OpmB possessing two RND components in Pseudomonas aeruginosa.

muxA-muxB-muxC-opmB (formerly PA2528-PA2527-PA2526-opmB), encoding a putative resistance nodulation cell division (RND)-type multidrug efflux pump system, was cloned from Pseudomonas aeruginosa PAO1. Introduction of muxABC-opmB into P. aeruginosa YM64, a drug-hypersusceptible strain, led to elevated MICs of aztreonam, macrolides, novobiocin and tetracycline. Since muxB and muxC, both of which encode RND components, were essential for function, MuxABC-OpmB is thought to be a drug efflux pump with four components. One novobiocin-resistant mutant, PMX725, isolated from P. aeruginosa PMX7 showed elevated resistance not only to novobiocin but also to aztreonam, macrolides and tetracycline. Increased mRNA expression of muxABC-opmB was observed in the mutant PMX725 compared with the parental strain. Sequencing analysis revealed that a single-nucleotide insertion had occurred in the deduced promoter region for muxABC-opmB in PMX725. In this study, we have characterized the last RND-type multidrug efflux pump predicted from the genome sequence in P. aeruginosa.

Gene cloning and characterization of EfmA, a multidrug efflux pump, from Enterococcus faecium.

A DNA fragment responsible for resistance to antimicrobial agents was cloned from chromosomal DNA of Enterococcus faecium FN-1, a clinically isolated strain. Escherichia coli KAM32, a drug-hypersusceptible mutant, was used as a host for gene cloning. Cells of E. coli KAM32 harboring a recombinant plasmid (pTFM8) carrying the DNA fragment became resistant to fluoroquinolones, macrolides, ethidium bromide, 4',6-diamidino-2-phenylindole (DAPI) and tetraphenylphosphonium chloride (TPPCl). Three complete open reading frames (ORFs) were found in the DNA insert of pTFM8, and the deduced amino acid sequences of one of the ORFs showed high similarity to Mdt(A) from Lactococcus lactis. Mdt(A) is a multidrug efflux pump belonging to a major facilitator superfamily. We designated the ORF efmA. E. coli KAM32 cells harboring the efmA showed energy-dependent efflux of DAPI and TPP(+). We also observed norfloxacin/H(+) antiport due to EfmA. The mRNA expression of efmA was observed in E. faecium FN-1 grown without any exogenously added antimicrobial agents. Thus, we conclude that efmA is constitutively expressed under laboratory growth conditions and would contribute to intrinsic resistance against multiple antimicrobial agents in E. faecium FN-1.

Synergistic effect of kaempferol glycosides purified from Laurus nobilis and fluoroquinolones on methicillin-resistant Staphylococcus aureus.

In a previous study, we reported that two kaempferol glycosides isolated from Laurus nobilis L., kaempferol-3-O-alpha-L-(2'',4''-di-E-p-coumaroyl)-rhamnoside (C2) and kaempferol-3-O-alpha-L-(2''-E-p-coumaroyl-4''-Z-p-coumaroyl)-rhamnoside (C3), showed strong antibacterial activities against methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci. Thereafter we found that these compounds greatly reduced the minimum inhibitory concentrations (MICs) of some fluoroquinolones in MRSA. In other words, C2 and C3 greatly potentiated anti-MRSA activity of fluoroquinolones. The effect of C2 and C3 with fluoroquinolones was found to be synergistic. The potentiation activity was observed with hydrophilic fluoroquinolones, such as norfloxacin and ciprofloxacin, but not with hydrophobic quinolones. We also found that norfloxacin reduced MICs of C2 and C3. The effect was synergistic. Possible mechanism of the synergistic effect was discussed.

Modulation of Vibrio mimicus hemolysin through limited proteolysis by an endogenous metalloprotease.

Vibrio mimicus is a causative agent of human gastroenteritis and food poisoning, and this species produces an enterotoxic hemolysin (V. mimicus hemolysin) as a virulence determinant. Vibrio mimicus hemolysin is secreted as an 80 kDa precursor, which is later converted to the 66 kDa mature toxin through removal of an N-terminal propeptide via cleavage of the Arg151-Ser152 bond. In this article, we investigate the role of the endogenous metalloprotease (V. mimicus protease) in the maturation of V. mimicus hemolysin. In vitro experiments using purified proteins showed that, although it activated the precursor at the early stage via cleavage of the Asn157-Val158 bond, V. mimicus protease finally converted the activated and physiologically maturated toxin to a 51 kDa protein through removal of the C-terminal polypeptide. This 51 kDa derivative was unable to lyse erythrocytes because of its inability to bind to the erythrocyte membrane. Vibrio mimicus protease-negative strains were found to produce high levels of V. mimicus hemolysin at the logarithmic phase of bacterial growth and maintained high hemolytic activity even at the stationary phase. These findings indicate that, although it is not directly related to toxin maturation in vivo, V. mimicus protease can modulate the activity of V. mimicus hemolysin and/or its precursor through limited proteolysis.