Reduced redox-dependent mechanism and glucose-mediated reversal in gentamicin-resistant Vibrio alginolyticus

Strategy of managing antibiotic-resistant Vibrio alginolyticus, a bacterial pathogen that threatens human health and animal farming, is not available due to the lack of knowledge about the underlying mechanism of antibiotic resistance. Here, we showed that gentamicin-resistant V. alginolyticus (VA-R_GEN) has four mutations on metabolism and one mutation on a two-component system by whole-genome and PCR-based sequencing, indicating the metabolic shift in VA-R_GEN. Thus, metabolic profile was investigated by GC–MS based metabolomics. Glucose was identified as a crucial biomarker, whose abundance was decreased in VA-R_GEN. Further analysis with iPath, and gene expression and enzyme activity of the pyruvate cycle (the P cycle) demonstrated a global depressed metabolic pathway network in VA-R_GEN. Consistently, NADH, sodium-pumping NADH:ubiquinone oxidoreductase (Na(+)-NQR) system, membrane potential and intracellular gentamicin were decreased in VA-R_GEN. These findings indicate that the reduced redox state contributes to antibiotic resistance. Interestingly, exogenous glucose potentiated gentamicin to efficiently kill VA-R_GEN through the promotion of the P cycle, NADH, membrane potential and intracellular gentamicin. The potentiation was further confirmed in a zebrafish model. These results indicate that the gentamicin resistance reduces the P cycle and Na(+)-NQR system and thereby decreases redox state, membrane potential and gentamicin uptake, which can be reversed by exogenous glucose.     

溶藻弧菌的毒力相关基因及其对小鼠的致病力

【目的】通过多重PCR检测和小鼠动物实验,对溶藻弧菌环境分离株的毒力因子进行评估,以期获得较强致病菌株和弱致病菌株之间的差别,并初步探讨该菌毒力因子对小鼠的致病机理。【方法】采用多重PCR体系检测毒力相关基因,我妻氏血平板溶血实验和平板酶活实验检测溶藻弧菌株的溶血素和胞外酶;以昆明小白鼠为实验动物,攻毒方式为灌胃和腹腔注射,根据小鼠的致病症状和死亡情况来分析和对比溶藻弧菌的胞外分泌物以及菌体本身的毒性。【结果】10株溶藻弧菌产淀粉酶、卵磷脂酶的比例为100%,脂肪酶、明胶酶次之(为70%),脲酶均未被检出;神奈川现象阳性菌株率为60%。毒力基因检测的结果显示10株溶藻弧菌中toxR、Collagenase、tlh、FlaA、ompW、AspA、fur这些与毒力有关的基因均有分布,而toxS、trh、tdh、UreR并未检出。10株溶藻弧菌中的VA009对小鼠显示了较强的致病性,能造成腹腔积液,经腹腔注射感染此菌后7 d内死亡率高达80%。【结论】不同的溶藻弧菌对小鼠的致病性存在较大差异,溶藻弧菌菌体本身比胞外分泌物对其毒性的贡献要大,而副溶血弧菌的毒性则由其胞外分泌物起主要作用;比较我们筛选出的强致病菌株与弱致病菌株,其上述毒力基因的分布并没有差别,说明溶藻弧菌可能存在一套与副溶血弧菌不同的独立的毒力基因系统。     

Peptone Induction and Rifampin-Insensitive Collagenase Production by Vibrio alginolyticus

Vibrio alginolyticus produces an extracellular collagenase which requires specific induction by collagen or its high-molecular-weight fragments. Peptone also induces collagenase during the late exponential and early stationary growth phases. The peptone inducers have been shown to have a broad molecular weight range between 1,000 and 60,000. The peptone inducers supported slow growth of V. alginolyticus when supplied as the sole nitrogen source in minimal medium. Digestion of the peptone inducers with purified V. alginolyticus collagenase resulted in a decrease in their inducing ability, whereas digestion with trypsin or alpha-chymotrypsin did not. This indicated that induction by the inducers required the presence of collagenase-sensitive bonds. Prolonged digestion of the inducers with collagenase did not completely eliminate the inducing ability of the inducers. The peptone inducers acted as inhibitors of collagenase. A minimal medium induction system has been developed which involves resuspending cells at high density in a medium containing succinate, (NH(4))(2)SO(4), KH(2)PO(4), and the peptone inducer. Cells grown in minimal medium induce earlier than cells grown on peptone, Casamino Acids, or tryptone. Collagenase production was shown to occur for 30 to 60 min in the presence of rifampin at levels which completely inhibit the incorporation of [(3)H]uracil into trichloroacetic acid-precipitable material. Chloramphenicol completely and immediately abolished collagenase production, which together with labeling studies has confirmed that collagenase production involves de novo synthesis of the enzyme. Both glucose and Casamino Acids repressed collagenase production, although synthesis of the enzyme continued for 30 to 60 min after their addition. The repression of collagenase production by glucose and Casamino Acids was more severe than the inhibition of enzyme formation due to addition of rifampin.     

Biological deinking of inkjet-printed paper using Vibrio alginolyticus and its enzymes

Recycling of office waste paper (photocopy, inkjet, and laser prints) is a major problem due to difficulty in removal of nonimpact ink. Biological deinking of office waste paper is reported using several microorganisms and their enzymes. We report here deinking and decolorization of the dislodged ink particles from inkjet printed paper pulp by a marine bacterium, Vibrio alginolyticus isolate no. NIO/DI/32, obtained from marine sediments. Decolorization of this pulp was achieved within 72 h by growing the bacterium in the pulp of 3–6% consistency suspended in seawater. Immobilized bacterial cells in sodium alginate beads were also able to decolorize this pulp within 72 h. The cell-free culture supernatant of the bacterium grown in nutrient broth was not effective in deinking. However, when the culture was grown in nutrient broth supplemented with starch or Tween 80, the cell-free culture supernatant could effectively deink and decolorize inkjet-printed paper pulp within 72 h at 30°C. The culture supernatant of V. alginolyticus grown in the presence of starch or Tween 80 showed 49 U ml⁻¹ and 33 U ml⁻¹ amylase and lipase activities, respectively. Dialysis of these culture supernatants through 10 kDa cut-off membrane resulted in a 35–40% reduction in their efficiency in decolorizing the pulp. It appears that amylase and lipase effectively help in dislodging the ink particles from the inkjet printed-paper pulp. We hypothesize that the bacterium might be inducing the formation of low molecular weight free radicals in the culture medium, which might be responsible for decolorization of the pulp.     

Lysis of halophilic Vibrio alginolyticus and Vibrio costicolus induced by chaotropic anions.

High concentration (1.0 M) of KSCN, but not of NaSCN, induced lysis of slightly halophilic Vibrio alginolyticus and moderately halophilic Vibrio costicolus, and the decrease in absorbance of the cell suspension was complete after 30 min at 25 degrees C. Replacement of K+ with Na+ effectively prevented the lysis by SCN-.K+ salts of NO3-, Br- and I-, however, induced no significant lysis. In electron micrographs, a prolonged exposure of the cells of V. alginolyticus to 1.0 M KSCN displaced the nucleoplasm to maintain close contact with the cell membranes. After 40 min of interaction, 50% of the cellular protein, 96% of RNA and 94% of DNA were recovered in the lysed cells. In contrast to lysis in hypotonic conditions, the lysis induced by KSCN is due mainly to a partial release of protein from the cells. V. costicolus was more susceptible to SCN- than V. alginolyticus, whereas nonhalophilic Escherichia coli was resistant to 1.0 M KSCN. Thus, lysis by SCN- is characteristic of halophilic bacteria and cell membranes of more halophilic bacteria are more susceptible to chaotropic anions. The protective effect of Na+ observed here was considered to be manifested by specific interactions of Na+ with components of cell membranes, thereby rendering their structures resistant to the action of chaotropic anions.     

Molecular characterisation and antimicrobial resistance of Vibrio vulnificus and Vibrio alginolyticus isolated from mussels (Mytilus galloprovincialis)

Twelve Vibrio vulnificus biotype 1 and 11 Vibrio alginolyticus isolated from mussels in Italy were analysed by antimicrobial resistance, plasmid profiles, random amplification of polymorphic DNA (RAPD), and single enzyme amplified fragment length polymorphism (sAFLP). Plasmid DNA was detected in three V. vulnificus and four V. alginolyticus cultures. All isolates were resistant to at least two antimicrobial agents: all isolates were resistant to ampicillin, carbenicillin and streptomycin, except one V. alginolyticus which was sensitive to carbenicillin and two V. alginolyticus which were sensitive to streptomycin. No association was detected between the presence of plasmid DNA and antimicrobial resistance. Seven of the twelve V. vulnificus and two of the eleven V. alginolyticus cultures were susceptible to the 10 microg of the vibriostatic compound O/129; all cultures were susceptible to the 150 microg of O/129. Both RAPD and sAFLP was found to be reproducible. Ten sAFLP and seven RAPD profiles were detected amongst the 12 V. vulnificus cultures: three cultures were identified as indistinguishable by both methods. RAPD and sAFLP analysis of V. alginolyticus generated nine and seven profiles respectively, and these two methods were independent. These results demonstrate extreme variability of V. vulnificus and V. alginolyticus isolated from mussels, and both RAPD and sAFLP provided information on intraspecific differences which will be useful for molecular epidemiological or ecological studies. A combination of methods gave optimal discrimination, although a single method could provide sufficient information to characterise V. vulnificus isolates.     

辽宁省溶藻弧菌REP-PCR和 PFGE分子分型、耐药谱的研究

目的利用PFGE和REP-PCR两种分型方法对溶藻弧菌进行分子分型和亲缘关系的研究;并将两种分型方法进行对比,并结合对抗生素耐药试验结果对其关联性进行初步探讨。方法应用脉冲场凝胶电泳(PFGE)方法对辽宁省2017年食品中分离的39株溶藻弧菌进行分型,应用BN(7.6版本)软件分析同源性。以基因外重复回文序列(REP)为引物,对40株溶藻弧菌DNA进行扩增,得到DNA指纹图谱,并利用SPSS 13.0统计软件对DNA扩增图谱进行聚类分析,同时将两种聚类分析结果进行比较,并用肉汤稀释法测定对15种抗生素的耐药性。结果 PFGE和REP-PCR两种方法的聚类分析结果均可分出两种优势带型A型和B型,且包含的菌株一致。PFGE分型方法在一些菌株中出现的DNA条带更多更复杂一些,分辨力指数(DI)为0.974,REP-PCR分型方法的DI为0.936。溶藻弧菌对头孢唑啉耐药率最高达57.5%,其次是氨苄西林(20.0%)和红霉素(12.5%),三重耐药菌比例达12.5%。结论 PFGE和REP-PCR两种方法均可以对溶藻弧菌进行分型,且均具有较好的分型能力,分辨力和再现性都很好,但PFGE分型方法的分辨力更优于REP-PCR分子分型。耐药菌株间带型具有高度同源性,优势带型菌株易耐药。综上所述,PFGE和REP-PCR两种分子分型方法对于评估溶藻弧菌流行及分布规律均是有用的,并且分型结果是一致的,都可以对菌株间亲缘关系进行有效溯源,因此在溶藻弧菌所致疾病爆发流行时,在普遍缺乏昂贵的PFGE专业设备和昂贵的配套BN软件以及专业操作人员的实验室,可以应用REP-PCR方法和SPSS统计软件代替PFGE方法和BN软件对菌株进行快速有效溯源。     

Two generalized transducing phages in Vibrio parahaemolyticus and Vibrio alginolyticus.

Two bacteriophages named phi VP253 and phi VP143 isolated after ultraviolet induction from lysogenic strains of Vibrio parahaemolyticus have been shown to be generalized transducing phages. So far, seven different auxotrophic markers of a V. parahaemolyticus strain could be transduced at the frequencies ranging from 2.2 x 10(-7) to 7.5 x 10(-5) per infected cell at the m.o.i. of approximately 1.0. The phage phi VP143, but not phi VP253, lysed 20 of the 28 strains of V. alginolyticus and the occurrence of generalized transduction by this phage in this Vibrio species has been confirmed. Molecular size of the genomes of both phages were estimated to be approximately 48 kb as judged from electrophoretic mobilities of the DNAs digested with HindIII endonuclease. The results and similarity of the two phages in morphology and other properties suggest very close relatedness of the phages.     

The immunostimulatory effects of sodium alginate and iota-carrageenan on orange-spotted grouper Epinephelus coicoides and its resistance against Vibrio alginolyticus

The lysozyme activity, alternative complement activity (ACH50), respiratory burst, SOD (superoxide dismutase) activity and phagocytic activity of orange-spotted grouper Epinephelus coicoides were examined when the fish were injected intraperitoneally with sodium alginate at 10, 20, 30mgkg-1 and iota-carrageenan at 10, 20, 30mgkg-1, respectively after 24, 72 and 120 h. Serum ACH50 increased directly with dose after 24 and 72 h for both sodium alginate and iota-carrageenan treatments. The fish that received sodium alginate at 20mgkg-1 after 24 and 72 h, and the fish that received iota-carrageenan after 72 and 120 h showed significantly increased respiratory burst, SOD activity and phagocytic activity, respectively. In another experiment, E. coicoides which had been injected individually with sodium alginate and iota-carrageenan at 10, 20, 30mgkg-1, were challenged with Vibrio alginolyticus at 1.8x10(9) colony-forming units (cfu)fish-1 and then placed in seawater of 33 per thousand. The survival of fish that received sodium alginate at 20mgkg-1, and the fish that received iota-carrageenan at 30mgkg-1 was significantly higher than that of fish which received saline and the control fish after 48 h as well as at the termination of the experiment (120 h after the challenge). It is therefore concluded that E. coicoides which received sodium alginate at 20mgkg-1 or iota-carrageenan at 30mgkg-1 increased the non-specific immune response and resistance from V. alginolyticus infection.     

Regulation of exoprotease production by temperature and oxygen in Vibrio alginolyticus

The production of an extracellular collagenase and an alkaline protease by Vibrio alginolyticus during stationary phase was inhibited by a temperature shift from 30 to 37°C and by a lack of oxygen. The stability of the exoproteases was unaffected by incubation at 37°C and aeration. The optimum growth temperature for the V. alginolyticus strain was 33.5°C Aeration enhanced the rate of growth of exponential phase cells. Temperature and oxygen did not affect the growth of stationary phase cells when the exoproteases were being produced. Macromolecular synthesis in stationary phase cells was not affected by temperature. There was no rapid release of the exoproteases after temperature shift down and chloramphenicol inhibited the production of the enzymes when added at time of temperature shift down from 37 to 30°C. The regulation of exoprotease production by temperature and oxygen was specific and has implications regarding the ecology of V. alginolyticus. Cerulenin, quinacrine and O-phenanthroline inhibited the production of the exoproteases.     

Characterization and distribution of Vibrio alginolyticus and Vibrio parahaemolyticus isolated in Indonesia

Previous studies have shown that Vibrio alginolyticus and Vibrio parahaemolyticus can be isolated from similar types of marine samples. In this report, the results of an examination of 567 V. alginolyticus and V. parahaemolyticus strains, isolated from seawater in Jakarta Bay and from more than 30 types of seafood from markets in Jakarta, Indonesia, are presented. Most isolates were from mackerel, shrimp, or squid. Numerical taxonomic analyses clustered 337 isolates and three V. alginolyticus reference strains at S greater than or equal to 80%. These strains produced acid from sucrose, but only approximately 80% produced acetoin or grew in the presence of 10% NaCl. The frequency of occurrence of V. alginolyticus in seawater samples ranged from 0% (in February and March 1972) to 100% (in September and December 1972) and was highest in seafood samples from August to December 1972. A second cluster of 230 isolates and seven V. parahaemolyticus reference strains was observed at S greater than or equal to 82%. These strains did not produce acetoin or acid from sucrose, and approximately 20% grew in the presence of 10% NaCl. V. parahaemolyticus was detected in seawater samples each month, with the highest frequency of occurrence (83.3%) in May 1972. Twenty-nine K antigen serotypes were demonstrated in V. parahaemolyticus isolates, and another 40% were untypable. The modal antibiotic resistance pattern for each species included five drugs. Only 12% of the V. parahaemolyticus strains were Kanagawa positive, and 10% elicited fluid accumulation in ligated rabbit ileal loops. All of the 7 V. alginolyticus strains and 94 (70%) of the V. parahaemolyticus strains tested killed mice when inoculated intraperitoneally.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fucoidan effectively provokes the innate immunity of white shrimp Litopenaeus vannamei and its resistance against experimental Vibrio alginolyticus infection

In this study, we examined the effect of fucoidan on the immune response of white shrimp Litopenaeus vannamei and its resistance against Vibrio alginolyticus infection. Fucoidan induced degranulation, caused changes in the cell morphology, and increased activation of prophenoloxidase (proPO) and the production of superoxide anions in vitro. Shrimp that received fucoidan via immersion at 100, 200, and 400 mg l^?1 after 3 h showed haemocyte proliferation and a higher mitotic index of haematopoietic tissue. In another experiment, the haemocyte count, phenoloxidase (PO) activity, and respiratory bursts (RBs) were examined after the shrimp had been fed diets containing fucoidan at 0 (control), 0.5, 1.0, and 2.0 g kg^?1 for 7–21 days. Results indicated that these parameters directly increased with time. The immune parameters of shrimp fed the 1.0 g kg^?1 diet were significantly higher than those of shrimp fed the 2.0 g kg^?1 diet after 14 and 21 days. Phagocytic activity and the clearance efficiency against V. alginolyticus were significantly higher in shrimp fed the 1.0 g kg^?1 diet compared to those of shrimp fed the 0, 0.5 and 2.0 g kg^?1 diets. In a separate experiment, shrimp that had been fed diets containing fucoidan for 21 days were challenged with V. alginolyticus at 10⁶ colony-forming units shrimp^?1. Survival rates of shrimp fed the 1.0 and 2.0 g kg^?1 diets were significantly higher than those of shrimp fed the 0 and 0.5 g kg^?1 diets for 96–120 h. We concluded that fucoidan provokes innate immunity of shrimp as evidenced by haemocyte degranulation, proPO activation, and the mitotic index of haematopoietic tissue, and that dietary administration of fucoidan at 1.0 g kg^?1 enhanced the immune response of shrimp and their resistance against V. alginolyticus infection.     

The immune stimulatory effect of sodium alginate on the white shrimp Litopenaeus vannamei and its resistance against Vibrio alginolyticus

The total haemocyte count (THC), differential haemocyte count (DHC), phenoloxidase activity, respiratory burst (release of superoxide anion), superoxide dismutase activity, phagocytic activity and clearance efficiency to the pathogen Vibrio alginolyticus were measured when the white shrimp Litopenaeus vannamei (9.4-11.3 g) were injected individually with sodium alginate at 10, 20 or 50 microg g(-1). No significant differences in THC, DHC and superoxide dismutase activity were observed among the shrimp injected with saline and those injected with sodium alginate at 10, 20 or 50 microg g(-1). However, L. vannamei injected with sodium alginate at 20 microg g(-1)increased its phenoloxidase activity and respiratory burst after 2 days and one day, respectively. L. vannamei injected with sodium alginate at 50 microg g(-1)maintained a higher phagocytic activity and clearance efficiency to V. alginolyticus after 4 days. In another experiment, L. vannamei which had been injected with sodium alginate, were challenged with V. alginolyticus at 2x10(5)colony-forming units (CFU) shrimp(-1)and then placed in seawater of 34 per thousand. The survival of shrimp that received sodium alginate at either dose was significantly higher than that of control shrimp at the termination of the experiment (6 days after the challenge). It is therefore concluded that L. vannamei received sodium alginate at 10 microg g(-1)or more and increased its immune ability and resistance from V. alginolyticus infection.     

Medium for Isolation and Differentiation of Vibrio parahaemolyticus and Vibrio alginolyticus

Trypticase soy agar supplemented with sucrose, sodium chloride, bile salts, and triphenyltetrazolium chloride is an improved plating medium for the isolation of Vibrio parahaemolyticus from samples of seawater, permitting better differentiation of this organism from Vibrio alginolyticus and other bacteria.     

Medium for isolation and differentiation of Vibrio parahaemolyticus and Vibrio alginolyticus

Trypticase soy agar supplemented with sucrose, sodium chloride, bile salts, and triphenyltetrazolium chloride is an improved plating medium for the isolation of Vibrio parahaemolyticus from samples of seawater, permitting better differentiation of this organism from Vibrio alginolyticus and other bacteria.     

Dietary administration of sodium alginate enhances the immune ability of white shrimp Litopenaeus vannamei and its resistance against Vibrio alginolyticus

Haemocyte count, phenoloxidase activity, respiratory burst (release of superoxide anion), superoxide dismutase (SOD) activity, glutathione peroxidase (GPX) activity, phagocytic activity and clearance efficiency to the pathogen Vibrio alginolyticus were measured in white shrimp Litopenaeus vannamei juveniles (12.3 +/- 1.2 g) which had been fed diets containing sodium alginate at 0.5, 1.0, 2.0 g kg(-1) after five months. L. vannamei fed a diet containing 2.0 g kg(-1) sodium alginate had increased phenoloxidase activity, respiratory burst and SOD activity, but decreased GPX activity significantly. L. vannamei fed a diet containing 2.0 g kg(-1) sodium alginate had increased phagocytic activity and the shrimp fed a diet containing sodium alginate at 0.5, 1.0 or 2.0 g kg(-1) had increased clearance efficiency to V. alginolyticus. In another experiment, L. vannamei, which had been fed control diet, or sodium alginate-containing diets after 5 months, were challenged with V. alginolyticus at 2 x 10(6) colony-forming units (CFU) shrimp(-1) and then placed in seawater of 15 per thousand. The survival of shrimp fed a diet containing 2.0 g kg(-1) after one day, and the survival of shrimp fed diets containing sodium alginate at 0.5 and 1.0 g kg(-1) after 2-4 days increased significantly, as compared to that of shrimp fed control diet. It is therefore concluded that administration of sodium alginate in the diet at 2.0 g kg(-1) or less could enhance the immune ability of L. vannamei and increase its resistance to V. alginolyticus infection.     

Characterization and distribution of Vibrio alginolyticus and Vibrio parahaemolyticus isolated in Indonesia.

Previous studies have shown that Vibrio alginolyticus and Vibrio parahaemolyticus can be isolated from similar types of marine samples. In this report, the results of an examination of 567 V. alginolyticus and V. parahaemolyticus strains, isolated from seawater in Jakarta Bay and from more than 30 types of seafood from markets in Jakarta, Indonesia, are presented. Most isolates were from mackerel, shrimp, or squid. Numerical taxonomic analyses clustered 337 isolates and three V. alginolyticus reference strains at S greater than or equal to 80%. These strains produced acid from sucrose, but only approximately 80% produced acetoin or grew in the presence of 10% NaCl. The frequency of occurrence of V. alginolyticus in seawater samples ranged from 0% (in February and March 1972) to 100% (in September and December 1972) and was highest in seafood samples from August to December 1972. A second cluster of 230 isolates and seven V. parahaemolyticus reference strains was observed at S greater than or equal to 82%. These strains did not produce acetoin or acid from sucrose, and approximately 20% grew in the presence of 10% NaCl. V. parahaemolyticus was detected in seawater samples each month, with the highest frequency of occurrence (83.3%) in May 1972. Twenty-nine K antigen serotypes were demonstrated in V. parahaemolyticus isolates, and another 40% were untypable. The modal antibiotic resistance pattern for each species included five drugs. Only 12% of the V. parahaemolyticus strains were Kanagawa positive, and 10% elicited fluid accumulation in ligated rabbit ileal loops. All of the 7 V. alginolyticus strains and 94 (70%) of the V. parahaemolyticus strains tested killed mice when inoculated intraperitoneally.(ABSTRACT TRUNCATED AT 250 WORDS)     

Microcalorimetric Measurements of Glucose Metabolism by Marine Bacterium Vibrio alginolyticus

Microcalorimetric measurements of heat production from glucose by Vibrio alginolyticus were made to assess the viability of calorimetry as a technique for studying the metabolism of marine bacteria at organic nutrient concentrations found in marine waters. The results show that the metabolism of glucose by this bacterium can be measured by calorimetry at submicromolar concentrations. A linear correlation between glucose concentration and total heat production was observed over a concentration range of 8 mM to 0.35 μM. It is suggested that these data indicate a constant efficiency of metabolism for this bacterium over the wide range of glucose concentrations studied.     

Downregulation of Na(+)-NQR complex is essential for Vibrio alginolyticus in resistance to balofloxacin

Increasingly isolated frequency of antibiotic-resistant V. alginolyticus strains in clinic and aquaculture has been reported, but the mechanisms of V. alginolyticus antibiotic resistance are largely absent. In the present study, native/SDS-PAGE based proteomics, which may provide information on protein-protein interaction, was utilized to investigate differential proteins of V. alginolyticus in resistance to balofloxacin. Ten proteins were altered, in which V12G01_04671, V12G01_00457, V12G01_15927, V12G01_15240, NqrA (spot 26), and NqrF (spot 30) were downregulated, while V12G01_22043, TolC, V12G01_15130, V12G01_19297 were upregulated. Importantly, the two components of Na(+)-NQR complex, NqrA and NqrF, were vertically lined and was further investigated. Western blotting assay indicated that downregulation of the two proteins contrasted sharply with upregulation of a control protein TolC, which was consistent with the result obtained from 2-DE gel analysis. Furthermore, overexpression of NqrA, NqrF and TolC resulted in decrease and elevation of bacterial survival ability in medium with balofloxacin, respectively. These results indicate that downregulation of Na(+)-NQR complex is essential for V. alginolyticus resistance to balofloxacin. This is the first report on the role of Na(+)-NQR complex in antibiotic resistance. This finding highlights the way to an understanding of antibiotic-resistant mechanisms in content of metabolic regulation.