Salt tolerance of lactose-grown Vibrio parahaemolyticus carrying Escherichia coli lac genes.

Lac- strains of Vibrio parahaemolyticus were converted to Lac+ on receiving a hybrid plasmid containing the lactose utilization genes of Escherichia coli K-12. A V. parahaemolyticus strain containing this hybrid plasmid exhibited optimal growth rates on glucose and other carbon sources in the presence of 0.2 to 0.4 M NaCl. Growth of the same strain on lactose was inhibited at similar concentrations of NaCl. The altered growth rate responses in lactose medium appeared to be attributable to effects of NaCl on the activity of lactose permease, and possibly on that of beta-galactosidase, rather than on the levels of these enzymes in V. parahaemolyticus cells.     

Growth of heat-injured Vibrio parahaemolyticus in media supplemented with various cations.

Mid- to late logarithmic growth phase cells of Vibrio parahaemolyticus grown in tryptic soy broth (TSB) containing 0.5, 3.0, and 7.5% NaCl were heated for 8 min at 45 degrees C in 0.1 M phosphate buffer (pH 7.2) containing 3% NaCl. Colony formation on thiosulfate-citrate-bile salts-sucrose agar (TCBS) containing 2% NaCl was greatest for unheated cells that had been grown in 7.5% NaCl-TSB; cells grown in 0.5% NaCl-TSB formed a greater number of colonies on 1.0% NaCl-TCBS. Thermal injury was evident in heated cells, regardless of the NaCl concentration in TSB growth medium. The effects of Mg2+, K+, and Li+ added as chlorides to 0.5% NaCl-TSB on the growth of nonheated and heated V. parahaemolyticus were studied. Lower levels of Mg2+ and slightly higher levels of K+ were required to replace Na+ in TSB inoculated with thermally injured cells that had been originally grown in 3.0 and 7.5% NaCl-TSB. LiCl had an inhibitory effect on both nonheated and heated cells when present in the recovery medium (0.5% NaCl-TSB) at concentrations as low as 0.5%. Increased numbers of colonies were formed by heated cells plated in MgCl2-supplemented TCBS, regardless of the NaCl concentration in the original growth medium. Potassium had little, if any, effect on colony formation by nonheated V. parahaemolyticus recovered on TCBS and may have had a detrimental effect on heat-injured cells.     

Isolation of a new drug-resistance plasmid from a strain of Vibrio parahaemolyticus

A new R plasmid, pSA55, with a molecular weight of 112 megadaltons (Md), was isolated from a strain of Vibrio parahaemolyticus with multiple drug resistance. The pSA55 plasmid conferred on its host resistance to chloramphenicol, tetracycline, streptomycin, kanamycin, ampicillin, trimethoprim and 2,4-diamino-6,7-diisopropyl pteridine, and belongs to incompatibility group C. The plasmid was transferable to Escherichia coli, V. parahaemolyticus, V. alginolyticus and NAG bivrio at a frequency of 10(-3) approximately -7, and was stably inherited by the transconjugants of these species. The conjugal transfer of pSA55 plasmid was significantly affected by the growth culture phase. The resistance pattern and resistance levels of transconjugants were the same as those of the donor strain. We did not observe fluctuations in minimal inhibitory concentrations with transfer, unlike the case of V. cholerae. The relationship between the pSA55 plasmid and the Kanagawa phenomenon was not clarified in the present study.     

Combined Effects of Water Activity, Solute, and Temperature on the Growth of Vibrio parahaemolyticus

Vibrio parahaemolyticus was grown at 36 C in tryptic soy broth (pH 7.8) containing added levels of NaCl ranging from 0.5 to 7.9% (wt/wt). The fastest generation time was 16.4 min in tryptic soy broth containing 2.9% NaCl (TSBS) which corresponded to a water activity (a(w)) of 0.992 (+/-0.005). Tryptic soy broth containing lower or higher levels of NaCl resulted in higher or lower a(w), respectively, and slower generation times. Growth was measured turbidimetrically at 36 C in TSBS containing added amounts of NaCl, KCl, glucose, sucrose, glycerol, or propylene glycol. The solutes used to reduce a(w) to comparable levels resulted in extended lag times of varied magnitude, dissimilar growth rates, and different cell numbers. Reduction of a(w) with glycerol was less inhibitory to growth than similar a(w) reductions with NaCl and KCl. Sucrose, glucose, and propylene glycol generally had the greatest effect on extending the lag times of V. parahaemolyticus when the addition of these solutes was made to establish similar a(w) levels lower than 0.992. Minimal a(w) for growth at 15, 21, 29, and 36 +/- 0.2 C for each of four strains of V. parahaemolyticus was tested in TSBS containing added solutes. Reduced a(w) was generally most tolerable at 29 C, whereas higher minimal a(w) for growth was required at 15 C. Solutes added to TSBS to achieve reduction in a(w), minimal a(w) for growth after 20 days, and incubation temperatures were as follows: glycerol, 0.937, 29 C; KCl, 0.945, 29 C; NaCl, 0.948, 29 C; sucrose, 0.957, 29 and 36 C; glucose, 0.983, 21 C; and propylene glycol, 0.986, 29 C. Each of the four strains tested responded similarly to investigative conditions. It appears that minimal a(w) for growth of V. parahaemolyticus depends upon the solute used to control a(w).     

The immune response of Taiwan abalone Haliotis diversicolor supertexta and its susceptibility to Vibrio parahaemolyticus at different salinity levels

Addition of NaCl at 2.5% to 3.5% to tryptic soy broth (TSB) significantly increased the growth of Vibrio parahaemolyticus. Taiwan abalone Haliotis diversicolor supertexta held in 30 per thousand seawater were injected with V. parahaemolyticus grown in TSB containing NaCl at 0.5, 1.5, 2.5, 3.5 and 4.5% at a dose of 1.6 x 10(5)colony-forming units (cfu) abalone(-1). After 48 h, the cumulative mortality was significantly higher for the abalone challenged with V. parahaemolyticus grown in 2.5% than those grown in 0.5 and 1.5% NaCl. In other experiments, abalones held in 30 per thousand seawater were injected with TSB-grown V. parahaemolyticus (1.6 x 10(5)cfu abalone(-1)), and then transferred to 20, 25, 30 and 35 per thousand seawater. All abalones held in 20 per thousand were killed in 48 h. The mortality of V. parahaemolyticus-injected abalone held in 30 per thousand was significantly lower over 24-120 h. Abalone held in 30 per thousand seawater and then transferred to 20, 25, 30 and 35 per thousand were examined for THC (total haemocyte count), phenoloxidase activity, respiratory burst, phagocytic activity and clearance efficiency of V. parahemolyticus after 24 and 72 h. The THC increased directly related with salinity levels. Phenoloxidase activity, respiratory burst, phagocytic activity and clearance efficiency of V. parahaemolyticus decreased significantly for the abalone in 20, 25 and 35 per thousand. It is concluded that the abalone transferred from 30 per thousand to 20, 25 and 35 per thousand had reduced immune ability and decreased resistance against V. parahaemolyticus infection.     

Demonstration of a plasmid-borne gene encoding a thermostable direct hemolysin in Vibrio cholerae non-O1 strains

Of 15 Vibrio cholerae non-O1 strains, 2 produced a hemolysin termed NAG-rTDH, which is very similar to the thermostable direct hemolysin of V. parahaemolyticus. These two strains contained DNA sequences which are homologous to a DNA probe for the V. parahaemolyticus thermostable direct hemolysin gene. A probe-positive 9-kilobase HindIII fragment was cloned from a plasmid of a V. cholerae non-O1 strain into plasmid pBR322, and the resulting Escherichia coli clones produced intracellular NAG-rTDH.     

Demonstration of a plasmid-borne gene encoding a thermostable direct hemolysin in Vibrio cholerae non-O1 strains.

Of 15 Vibrio cholerae non-O1 strains, 2 produced a hemolysin termed NAG-rTDH, which is very similar to the thermostable direct hemolysin of V. parahaemolyticus. These two strains contained DNA sequences which are homologous to a DNA probe for the V. parahaemolyticus thermostable direct hemolysin gene. A probe-positive 9-kilobase HindIII fragment was cloned from a plasmid of a V. cholerae non-O1 strain into plasmid pBR322, and the resulting Escherichia coli clones produced intracellular NAG-rTDH.     

Nucleotide sequence of the thermostable direct hemolysin gene of Vibrio parahaemolyticus.

The gene encoding the thermostable direct hemolysin of Vibrio parahaemolyticus was characterized. This gene (designated tdh) was subcloned into pBR322 in Escherichia coli, and the functional tdh gene was localized to a 1.3-kilobase HindIII fragment. This fragment was sequenced, and the structural gene was found to encode a mature protein of 165 amino acid residues. The mature protein sequence was preceded by a putative signal peptide sequence of 24 amino acids. A putative tdh promoter, determined by its similarity to concensus sequences, was not functional in E. coli. However, a promoter that was functional in E. coli was shown to exist further upstream by use of a promoter probe plasmid. A 5.7-kilobase SalI fragment containing the structural gene and both potential promoters was cloned into a broad-host-range plasmid and mobilized into a Kanagawa phenomenon-negative V. parahaemolyticus strain. In contrast to E. coli, where the hemolysin was detected only in cell lysates, introduction of the cloned gene into V. parahaemolyticus resulted in the production of extracellular hemolysin.     

Bacterial growth on lactose: an experimental investigation

A wild-type strain of Klebsiella oxytoca growing aerobically in batch culture has exhibited intermittent or oscillatory growth while growing on lactose at concentrations on the order of 1 g/L or less. In two-substrate experiments, preferred growth on glucose followed by growth on lactose also produced oscillatory growth behavior during the lactose growth phase at lactose concentrations of 1 g/L or less. Only oscillations in cell density have currently been observed. Alkalinization of the medium during growth on lactose indicated the presence of lactose active transport. The observed intermittent growth was reduced or removed during growth on lactose after preferred growth on galactose or in a medium containing 50 mM NaCl. Results suggested that the presence of an intracellular energy source or a sufficient DeltapH buffer may alleviate growth inhibition when transport and growth processes compete for essential energy resources during growth on lactose.     

Comparison of the Heme Iron Utilization Systems of Pathogenic Vibrios

Vibrio alginolyticus, Vibrio fluvialis, and Vibrio parahaemolyticus utilized heme and hemoglobin as iron sources and contained chromosomal DNA similar to several Vibrio cholerae heme iron utilization genes. A V. parahaemolyticus gene that performed the function of V. cholerae hutA was isolated. A portion of the tonB1 locus of V. parahaemolyticus was sequenced and found to encode proteins similar in amino acid sequence to V. cholerae HutW, TonB1, and ExbB1. A recombinant plasmid containing the V. cholerae tonB1 and exbB1D1 genes complemented a V. alginolyticus heme utilization mutant. These data suggest that the heme iron utilization systems of the pathogenic vibrios tested, particularly V. parahaemolyticus and V. alginolyticus, are similar at the DNA level, the functional level, and, in the case of V. parahaemolyticus, the amino acid sequence or protein level to that of V. cholerae.     

Molecular analysis of Vibrio parahaemolyticus isolated from human patients and shellfish during US Pacific north-west outbreaks

AIMS: The objective of this study was to investigate the occurrence and distribution of haemolysin genes, plasmid profile, serogroup analysis and cellular urease activity for Vibrio parahaemolyticus isolates from infected human patients and oysters from the Pacific north-western United States between 1988 and 1997. METHODS AND RESULTS: All of the clinical and environmental isolates tested in this study exhibited the presence of the thermolabile haemolysin gene, tl, confirming that all of the isolates were V. parahaemolyticus. Furthermore, the V. parahaemolyticus isolates that contained either the thermostable direct haemolysin gene, tdh, or the thermostable direct haemolysin-related gene, trh, or both, were also positive for urease. Isolates from infected human patients belong to serogroups O1 and O4, whereas, the isolates from oysters belong to serogroups O1, O4 and O5. These results suggest that the presence of a V. parahaemolyticus serogroup O1 and O4 could indicate the presence of a virulent strain of this pathogen. In this study, the presence of the haemolysin genes, serogroup profiles and urease production in V. parahaemolyticus isolated from human patients correlated with the oysters collected during the outbreaks. However, no significant correlation of the plasmid profiles was detected, based on their distribution and molecular weights, between V. parahaemolyticus isolated from infected human patients and from oysters collected during this outbreak. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF STUDY: It is apparent from this study that the identification of the haemolysin genes by multiplex PCR amplification, in conjunction with serogroup analysis and urease production, can be used to monitor shellfish for the presence of potentially pathogenic strains of V. parahaemolyticus.     

Procedure for Isolation and Enumeration of Vibrio parahaemolyticus,

An evaluation of criteria used in the identification of Vibrio parahaemolyticus showed that cultural responses varied with respect to growth in broth with 10% NaCl, type of hemolysis, reactions in triple sugar-iron-agar, and serological reactions. With few or no exceptions, cultures were positive for cytochrome oxidase, utilized glucose fermentatively, were sensitive to pteridine (0/129) and novobiocin, and failed to grow in Trypticase soy broth (TSB) without NaCl. A procedure employing a direct plating technique, with or without prior enrichment, was designed for the isolation and enumeration of V. parahaemolyticus. The plating medium consisted of 2.0% peptone, 0.2% yeast extract, 1.0% corn starch, 7% NaCl, and 1.5% agar, with the pH adjusted to 8.0. The enrichment broth was TSB with 7% NaCl. Dilutions of food homogenates were either spread directly on the plates or inoculated into enrichment broth. TSB enrichments were incubated at 42 C for 18 hr. A loopful of the TSB tubes then was streaked onto the direct plating medium. Incubation of plates was at 42 C for 24 to 48 hr. Smooth, white to creamy, circular, amylase-positive colonies were then picked as suspect V. parahaemolyticus. Confirmation of gram-negative, fermentative, oxidase-positive, pleomorphic rods sensitive to pteridine 0/129 was made by a fluorescent-antibody technique. With this procedure, a satisfactory quantitative recovery of known V. parahaemolyticus from inoculated seafoods was made possible. V. parahaemolyticus was nto isolated from other salted foods.     

Suitability of some enrichment broths and diluents for enumerating cold- and heat-stressed Vibrio parahaemolyticus.

Vibrio parahaemolyticus cells were injured by chilling and heating, and their recovery was tested in glucose-salt-Teepol broth (GSTB), tryptic soy broth containing 7% NaCl (TSBS), Horie - arabinose - ethyl violet broth (HAEB), and water blue - alizarin yellow broth (WBAY). Exponential phase cells were more sensitive to cold shock than were stationary phase cells. Exposure of chill-injured V. parahaemolyticus to GSTB and TSBS resulted in 70 to 80% death; about 70% lethality was noted for heat-injured cells inoculated into TSBS. Neither HAEB nor WBAY enrichment media were lethal to stressed cells, although rates of growth were retarded. The 3% NaCl in 0.1 M potassium phosphate (pH 7.0) diluent proved to be most suitable for protecting against inactivation of cold- and heat-injured cells.     

Use of randomly amplified polymorphic DNA analysis to differentiate isolates of Vibrio parahaemolyticus from cockles (Anadara granosa)

A total of 35 Kanagawa-negative strains of Vibrio parahaemolyticus isolated from cockles (Anadara granosa) were investigated by randomly amplified polymorphic DNA fingerprinting with three primers and their plasmid profiles. Eighteen strains carried small plasmid(s) of 2.4 to 7.3kb that enabled the V. parahaemolyticus to be grouped into eight plasmid patterns. The three primers generated polymorphisms in all 35 strains of V. parahaemolyticus tested, producing bands ranging from 0.25 to 3.9kb. The RAPD profiles revealed a high level of DNA sequence diversity within the Vibrio parahaemolyticus strains tested, and that cockles in the study area are populated by genetically polymorphic strains of V. parahaemolyticus.     

Membrane filter procedure for enumeration of Vibrio parahaemolyticus.

A membrane filtration procedure has been developed for the enumeration of Vibrio parahaemolyticus in marine waters. Background microbial growth on the primary medium was decreased through the use of sodium cholate and copper sulfate, high pH, 3% NaCl, and an elevated incubation temperature. A series of in situ tests was employed to obviate the picking of colonies for identification; thereby, the enumeration of V. parahaemolyticus was accomplished within 30 h. Confirmation of typical colonies approached 95%. Relative to immediate plating on brain heart infusion agar spread plates, the recovery of V. parahaemolyticus cells suspended in phosphate-buffered saline or in seawater held for 24 h at 4 to 6 degrees C was about 90%. Assay variability did not exceed that expected by chance. Recoveries of V. parahaemolyticus from coastal and estuarine surface waters exceeded those obtainable by other methods examined.     

Method for the detection of injured Vibrio parahaemolyticus in seafoods.

The sensitivity of Vibrio parahaemolyticus cells to refrigeration and frozen storage and the development of a method for detecting injured and uninjured V. parahaemolyticus cells were studied. Cell suspensions in different kinds of seafood homogenates were either regrigerated (4 degrees C) or frozen (-20 degrees C), stored, and examined for cell survival during storage. V. parahaemolyticus cells were sensitive to both storage temperatures. Many cells died, and many survivors were sublethally injured. In general, refrigeration storage appeared to be more injurious than frozen storage. The initial recovery of the sublethally injured cells was highest in a nutritionally rich, nonselective liquid medium such as Trypticase soy broth, whereas maximum cell multiplication was observed in Trypticase soy broth containing 3% NaCl. The sublethally injured V. parahaemolyticus cells demonstrated sensitivity to the selective enrichment medium, glucose salt teepol broth. From these findings, a new method (designated as the "repair-detection" method) was developed for the isolation and enumeration of V. parahaemolyticus. Comparative studies between the recommended and the repair-detection methods showed that injured V. parahaemolyticus cells were present in commercial seafoods and that the repair-detection method was definitely more effective for the detection of total numbers of V. parahaemolyticus cells.     

副溶血弧菌LacZ报告基因融合实验方法的建立与应用

目的:建立副溶血弧菌(Vibrio parahaemolyticus,VP)的LacZ报告基因融合实验方法。方法:PCR扩增靶基因的整个启动子区序列,并将其直接克隆入pHRP309质粒中,构建重组质粒;将重组质粒VPA1513转入VP野生株(WT)和opaR突变株(ΔopaR)中,而后通过比较两者β半乳糖苷酶活性的差异,确定OpaR对VPA1513的调控关系,以检验实验的稳定性。结果:构建出9个VP生物膜或毒力相关基因的LacZ重组质粒;OpaR对VPA1513的转录具有抑制作用。结论:建立了VP的LacZ报告基因融合实验方法,为后续转录调控机制的研究奠定了基础。     

A Halophilic Vibrio Isolated from a Case of Chronic Cholecystitis

We found a halophilic vibrio in B bile from a 75-year-old female patient with chronic cholecystitis, and examined its biochemical characteristics. The organisms are gram-negative short rods or comma shaped, with some ring forms. They have a single polar flagellum, but no capsule. The strains can grow in peptone water with 1.0 to 4.0% NaCl, but not with no NaCl or 6.0% NaCl. The characteristics of the organisms are positive dextrose fermentation, catalase, oxidase, and ornithine decarboxylase, and negative lysine decarboxylase, arginine dihydrolase, and absence of gas from glucose. They are sensitive to 2,4-diamine-6,7-diisopropyl pteridine (0/129). These characteristics indicate that the isolated strain should be a halophilic vibrio. However, no growth on Salmonella-Shigella (SS), SS with added sucrose and bromcresol purple (SS-SB), MacConkey's or thiosulfate citrate bile salts (TCBS) agar plates was demonstrated. Nitrate reduction, Simmons' citrate agar, indole, o-nitrophenol-β-d-galactopyranoside (ONPG), motility and esculin hydrolysis were positive. Urease, gelatinase, Voges-Proskauer, phenylalanine deaminase and malonate reactions were negative. Acid was produced from amygdalin, arabinose, cellobiose, fructose, galactose, glucose, lactose, maltose, mannitol, salicin, starch, sucrose, trehalose, and xylose, but not from adonitol, dulcitol, inositol, mannose, melibiose, raffinose, rhamnose and sorbitol. From these characteristics the isolate is considered to be not identical with V. parahaemolyticus, V. cholerae, V. vulnificus or other vibrios. It can be presumed that this isolate represents another species of halophilic vibrio.     

In Vivo Cloning of lac Genes in Streptococcus lactis ML3

The isolation and characterization of a Streptococcus lactis ML3 strain which possessed a recombinant lactose plasmid is described. The recombination events generating this plasmid occurred in vivo in a recombination-deficient strain and appeared to be mediated by transposition events. Restriction mapping revealed that the recombinant plasmid, pDA0307, contained a region of the lactose plasmid, pSK08, linked to another resident plasmid, pSK07. Copy number determinations indicated that the lac genes were present at approximately 20 copies per cell in pDA0307, whereas the lac genes are normally present at approximately 10 copies per cell in pSK08. The strain containing pDA0307 displayed a 21 to 54% increase in the expression of the Lac enzyme phospho-beta-d-galactosidase. However, the strain containing pDA0307 both grew and produced lactic acid in milk at rates identical to that of a strain containing pSK08. This result suggests that lac gene dosage of plasmid-linked lac genes was not limiting the rate at which these derivatives of S. lactis ML3 fermented milk.     

副溶血性弧菌基因敲除方法的建立及应用

目的摸索出一套副溶血性弧菌基因敲除的可靠方案,副溶血性弧菌致病相关基因的敲除对深入研究其致病机制有重要意义。方法通过融合PCR技术将目的基因上下游同源臂融合并克隆到自杀载体pDS132上,将重组质粒转化大肠杆菌S17λpir中,再接合转移到副溶血性弧菌菌株内,经pDS132质粒上sacB基因的反向筛选得到突变株。结果成功构建了副溶血性弧菌RIMD2210633菌株ΔopaR,ΔtoxR和ΔaphA三个基因突变株。结论通过自杀载体同源重组成功获得精确敲除的无痕突变株更有利于基因功能的研究,使后续副溶血性弧菌突变株与野生株的对比研究成为可能。