A comparison of thiosulphate-citrate-bile salts-sucrose (TCBS) agar and thiosulphate-chloride-iodide (TCI) agar for the isolation of Vibrio species from estuarine environments

AIMS: The purpose of this study was to compare a recently described medium, thiosulphate-chloride-iodide (TCI), for the isolation of estuarine vibrios with thiosulphate-citrate-bile salts-sucrose (TCBS). METHODS: A total of 492 colonies which developed on these media from estuarine water samples taken monthly over a 10-month period were examined. RESULTS: A much larger number of colonies developed on TCBS than TCI, and minimal taxonomic criteria indicated that a higher percentage (61%) of TCBS colonies could be identified as Vibrio spp. when compared with TCI (46%). SIGNIFICANCE: This study suggests that TCBS is a superior medium when compared with TCI for the isolation of Vibrio spp. from estuarine waters. Because of the public health risk presented by V. vulnificus, V. parahaemolyticus, V. cholerae and other vibrios, the selection of the most appropriate medium for their isolation is extremely important.     

Evaluation of selective media for isolation and enumeration of vibrios from estuarine waters

We have compared the effectiveness of four media developed in the last years together with the medium GSTC(glucose-salt-tellurite-crystal violet), devised in our laboratory, for the recovery of vibrios from estuarine waters. In addition, a number of reference Vibrio and non-Vibrio strains have been tested for growth on the five media. TCBS and GSTC were the most selective media for reference strains of Vibrio spp. However, when the media were tested with samples of water from three different sites of an estuary, only TCBS was effective enough to recover vibrios inhibiting the growth of non-Vibrio populations. We also report here the usefulness of TCBS for isolation of the fish pathogen V. anguillarum, since a total of 81 strains isolated from diseased fish and water in various parts of the world grew on this medium. In conclusion, we consider the TCBS as the best medium to isolate Vibrio species pathogenic for humans and fish, and for recovery of vibrios from estuarine waters.     

渤海水母体细菌的微生态分布及弧菌生物学特性

采用稀平板法对我国辽宁渤海海域水母体中细菌的微生态分布进行了考察,结果表明,在渤海水母体中各部位均有腐生性细菌及弧菌生长,其中腐生性细菌主要存在于水母体现,而弧菌除存在于水母体表外,有的则能在水母体内深层生长,水母体细菌微生态研究结果表明,弧菌的占细菌总数的90％以上,通过改进TCBAS培养基,从水母体中分离得到6株优势类群的细菌,对其菌落特征、菌体形态、生理生化特性进行了研究,它们都具备弧菌属的共同特点：革兰氏阴性,氧化酶阳性,兼性厌氧,TCBS培养基上能生长,对O／129敏感,初步鉴定为弧菌属,研究还发现,这几株菌都能产蛋白酶,其中JF2、JF4、JF5、JF6产蛋白酶的能力明显较强,它们极有可能是导致水母捕捞后快速解体腐败的主要原因。     

Potentiality of a new compound for in vitro differentiation between halophilic and non-halophilic vibrios

Sensitivity of 21 halophilic vibrios and 16 clinical isolates of non-halophilic vibrios was determined against a new possible antivibrio agent, a pyrimidine analogue, 4, 6-dimethylpyrimidine -2-thiol (4,6-DMPT). It appeared to be a vibriocidal agent, having a mean MIC and MBC of 32 microg/ml for halophilic strains and 64 microg/ml for non-halophilic strains and an LD50 of 300 mg/Kg body weight of mice. Thus, 4,6-DMPT may help an in vitro distinction between halophilic and non-halophilic vibrios. Sensitivity of these strains was also studied with respect to pteridine, crystal violet and Tween 80 hydrolysis as further markers distinguishing between these 2 groups which could also be differentiated by their growth on TCBS or/and CLED media.     

False-positive coliform readings using membrane filter techniques for seawater

Seawater samples that had been analysed by membrane filter techniques produced positive coliform readings that were found to be false. Apparently, the culture media and the incubation temperatures used in these methods permit the growth of vibrios which are abundant in the seawater. The vibrio colonies isolated by membrane filter were oxidase-positive Gram-negative short rods that grew on TCBS and required NaCl.     

A new selective, differential agar medium for isolation of Vibrio cholerae O1: PMT (polymyxin-mannose-tellurite) agar

A new selective and differential agar medium, polymyxin-mannose-tellurite (PMT) agar was devised to differentiate easily colonies of Vibrio cholerae O1 from those of V. cholerae non-O1. The differentiation between colonies of the two vibrios is based on mannose-fermentation. Colonies of V. cholerae O1 on the agar are agglutinated with O1 antiserum of V. cholerae much more easily than those on thiosulfate-citrate-bile salts-sucrose (TCBS) agar.     

Analysis of seawaters for the recovery of culturable Vibrio parahaemolyticus and some other vibrios

We investigated the recovery of dormant and injured cells along with the normally culturable cells of Vibrio species with special emphasis on V. parahaemolyticus using both selective and non-selective media at moderate (20 C) and standard (37 C) culture temperatures from a bay water environment. Culture temperatures (20 or 37 C) did not affect the recovery of V. parahaemolyticus but did for other vibrios. We observed similar seasonality of V parahaemolyticus as in most other environmental studies. V. parahaemolyticus and other Vibrio species were recovered in higher numbers by a replica plating method compared to most probable number (MPN) and direct TCBS (thiosulfate citrate bile-salt sucrose) agar counts. Even with the replica plating method, however, vibrios number goes down to a minimum level and V. parahaemolyticus was undetectable during the cool temperature period of the year, although total bacterial cells and CFU on nutrient agar (with 2% NaCl) did not vary so much during the study period.     

Low incidence of Vibrio vulnificus among Vibrio isolates from sea water and shellfish of the western Mediterranean coast

A specific search for Vibrio vulnificus in natural marine samples from the Spanish Mediterranean Sea was carried out by nested PCR and cultural approaches using thiosulphate-citrate-bile salts-sucrose agar (TCBS) and cellobiose-polymixin B-colistin agar (CPC), incubated at 40 degrees C, as selective media. Presumptive colonies were identified by PCR using specific primers against 23S rRNA sequences. This species was isolated from sea water and edible bivalves, mainly after preenrichment in alkaline peptone water (APW) at 40 degrees C followed by CPC agar. None of the V. vulnificus isolates identified corresponded to serovar E. Dominant Vibrio species on directly inoculated TCBS plates incubated at 25 degrees C were V. splendidus below 20 degrees C and V. harveyi and V. mediterranei above that temperature. Low percentages of several pathogenic vibrios were recorded but V. vulnificus was never recovered at this incubation temperature. The incidence of this species in the samples studied was lower than that described for other geographical areas, probably due to the high salinity values of the Mediterranean Sea.     

Isolation of medically significant Vibrio species from riverine sources in south east Queensland

Vibrio and Vibrio-like bacteria were isolated from water, sediments, plants and faeces from eight riverine sites in South East Queensland, Australia, using filtrations, enrichments and selective growth on thiosulphate-citrate-bile salts-sucrose (TCBS) and Simidu agars. Isolates (402 in toto) were classified by numerical analysis of phenotypic characteristics and comparison with fifteen reference cultures. Of the isolates 41 (10.2%) were identified as Vibrio cholerae, 33 (8.2%) as V. fluvialis and 118 (29.4%) as motile Aeromonas species. Other isolates resembled V. parahaemolyticus, V. alginolyticus, V. vulnificus and V. hollisae. No isolates were positively identified as V. damsela or Plesiomonas shigelloides. Vibrio species tolerant of low salt levels and aeromonads were widely distributed in riverine locations but more halophilic species were restricted to more saline areas. Simidu agar failed to select for vibrios as effectively as TCBS agar. Enrichment for 18 h produced more isolates than for 6 h.     

Occurrence and distribution of Vibrio spp., Listonella spp., and Clostridium botulinum in the Seto Inland Sea of Japan.

The distribution of Vibrio species in samples of surface water, bottom water (water 2 m above the sediment), and sediment from the Seto Inland Sea was studied. A simple technique using a membrane filter and short preenrichment in alkaline peptone water was developed to resuscitate the injured cells, followed by plating them onto TCBS agar. In addition, a survey was conducted to determine the incidence of Clostridium botulinum in sediment samples. Large populations of heterotrophs were found in surface water, whereas large numbers of total vibrios were found in bottom water. In samples from various water sampling regions, high counts of all bacterial populations were found in the inner regions having little exchange of seawater when compared with those of the open region of the inland sea. In the identification of 463 isolates, 23 Vibrio spp. and 2 Listonella spp. were observed. V. harveyi was prevalent among the members of the Vibrio genus. Vibrio species were categorized into six groups; an estimated 20% of these species were in the so-called "pathogenic to humans" group. In addition, a significant proportion of this group was hemolytic and found in the Bisan Seto region. V. vulnificus, V. fluvialis, and V. cholerae non-O1 predominated in the constricted area of the inland sea, which is eutrophic as a result of riverine influence. It was concluded that salinity indirectly governs the distribution of total vibrios and analysis of variance revealed that all bacterial populations were distributed homogeneously and the variance values were found to be significant in some water sampling regions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Occurrence and distribution of Vibrio spp., Listonella spp., and Clostridium botulinum in the Seto Inland Sea of Japan

The distribution of Vibrio species in samples of surface water, bottom water (water 2 m above the sediment), and sediment from the Seto Inland Sea was studied. A simple technique using a membrane filter and short preenrichment in alkaline peptone water was developed to resuscitate the injured cells, followed by plating them onto TCBS agar. In addition, a survey was conducted to determine the incidence of Clostridium botulinum in sediment samples. Large populations of heterotrophs were found in surface water, whereas large numbers of total vibrios were found in bottom water. In samples from various water sampling regions, high counts of all bacterial populations were found in the inner regions having little exchange of seawater when compared with those of the open region of the inland sea. In the identification of 463 isolates, 23 Vibrio spp. and 2 Listonella spp. were observed. V. harveyi was prevalent among the members of the Vibrio genus. Vibrio species were categorized into six groups; an estimated 20% of these species were in the so-called "pathogenic to humans" group. In addition, a significant proportion of this group was hemolytic and found in the Bisan Seto region. V. vulnificus, V. fluvialis, and V. cholerae non-O1 predominated in the constricted area of the inland sea, which is eutrophic as a result of riverine influence. It was concluded that salinity indirectly governs the distribution of total vibrios and analysis of variance revealed that all bacterial populations were distributed homogeneously and the variance values were found to be significant in some water sampling regions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Quantitative study, identification and antibiotics sensitivity of someVibrionaceae associated to a marine fish hatchery

This study characterises the bacteria associated with a marine hatchery in Tunisian coastal marine waters. Presumptive vibrios (TCBS agar) and heterotrophic aerobic microflora (CFU) were studied at different stages within the hatchery: seawater, batches of algal cultures, rotifers andArtemia culture tanks. The bacterial strains were isolated on TCBS Agar plates and described using different bacteriological tests (standardised micromethods “API 20 E Strips”, exoenzymes production, growth at different temperatures, pH and salinity, vibriostatic agent O/129 and antibiotics susceptibility). Two dominant genera of bacteria were found (Vibrio andAeromonas) associated with some strains of thePseudomonadaceae family.Vibrio alginolyticus was the dominant bacteria (75% of total isolates) found in rotifers (Brachionus plicatilis) andArtemia cultures (Artemia salina). In larvae rearing tanks, an increase ofVibrionaceae was noted after larvae were fed withArtemia. Most of the studied bacteria used the skin mucus ofSparus aurata larvae as their sole source of carbon. All theV. alginolyticus strains were β-haemolytic, hydrolyse the DNA and were susceptible to several tested antibiotics.     

Bacteria associated with the rapid tissue necrosis of stony corals

The rapid tissue necrosis (RTN) is a common disease of both wild and captive stony corals, which causes a fast tissue degradation (peeling) and death of the colony. Here we report the results of an investigation carried out on the stony coral Pocillopora damicornis, affected by an RTN-like disease. Total abundance of prokaryotes in tissue samples, determined by epifluorescence microscopy, was significantly higher in diseased than in healthy corals, as well as bacterial counts on MB2216 agar plates. Further experiments performed by fluorescent in situ hybridization using a 16S rDNA Vibrio-specific probe showed that vibrios were significantly more abundant in diseased than in healthy corals. Accordingly, bacterial counts on TCBS agar plates were higher in diseased than in healthy tissues. 16S rDNA sequencing identified as Vibrio colonies from diseased tissues only. Cultivated vibrios were dominated by a single ribotype, which displayed 99% of similarity with Vibrio harveyi strain LB4. Bacterial ribotype richness, assessed by terminal-restriction fragment length polymorphism analysis of the 16S rDNA, was significantly higher in diseased than in healthy corals. Using an in silico software, we estimated that a single terminal restriction fragment, putatively assigned to a Vibrio sp., accounted for > 15% and < 5% of the total bacterial assemblage, in diseased and healthy corals respectively. These results let us hypothesize that the RTN in stony corals can be an infectious disease associated to the presence of Vibrio harveyi. However, further studies are needed to validate the microbial origin of this pathology.     

副溶血性弧菌显色培养基检测效果初步评价

副溶血性弧菌(Vibrio parahaemolyticus)是一种重要的食源性致病菌, 广泛存在于各种海产品中。由于传统培养基和检测方法费时费力, 本研究设计开发了一种新型显色培养基HKC vibrio, 通过应用于人工污染样品和实际样品检验, 以法国科玛嘉弧菌显色平板CHROMagar vibrio和柠檬酸钠-硫代硫酸钠-氯化钠-蔗糖琼脂平板(TCBS)为对照, 对显色培养基HKC vibrio的灵敏性、特异性和检测效果进行了初步评价。结果表明, HKC vibrio的灵敏性与CHROMagar vibrio和TCBS相当, 并具有较好的特异性, HKC vibrio是非常有价值的分离平板, 可大大提高副溶血性弧菌的检测效率。     

副溶血性弧菌显色培养基检测效果初步评价

副溶血性弧菌(Vibrio parahaemolyticus)是一种重要的食源性致病菌,广泛存在于各种海产品中.由于传统培养基和检测方法费时费力,本研究设计开发了一种新型显色培养基HKC vibrio,通过应用于人工污染样品和实际样品检验,以法国科玛嘉弧菌显色平板CHROMagar vibrio和柠檬酸钠-硫代硫酸钠-氯化钠-蔗糖琼脂平板(TCBS)为对照,对显色培养基HKC vibrio的灵敏性、特异性和检测效果进行了初步评价.结果表明,HKC vibrio的灵敏性与CHROMagar vibrio和TCBS相当,并具有较好的特异性,HKC vibrio是非常有价值的分离平板,可大大提高副溶血性弧菌的检测效率.     

Effect of probiotics on bacterial population and health status of shrimp in culture pond ecosystem

The artificially manufactured probiotics having beneficial bacteria, Bacillus spp. was applied regularly in a modified extensive shrimp (Penaeus monodon) culture pond, located on the bank of Vellar estuary, Parangipettai. The populations of total heterotrophic bacteria (THB), beneficial bacteria (Bacillus spp.) and pathogenic bacteria (vibrios) were monitored in water and sediment of the pond. The results were compared with a control pond, situated in the same location having same water spread area, stocking density, species managed with same technologies and optimum environmental parameters in which no probiotic was applied. The populations of THB and Bacillus spp. in the experimental pond increased and the vibrios decreased after each application of probiotics. But the result of the control pond showed an increasing trend of the populations of THB, Bacillus spp. and vibrios towards days of culture. The control pond had lower levels of THB and Bacillus spp. and higher levels of vibrios than the probiotic applied (experimental) pond. Also the probiotics maintained optimum transparency and low organic load in the experimental pond as compared to control. In general, water and sediment had almost equal number of Bacillus spp. and vibrios, but sediment had higher THB load than water. The applications of probiotics lesser pathogenic vibrios and enhance beneficial bacilli in the culture leading to improved water quality, promoted growth and survival rates and increased the health status of the shrimp without stress and disease outbreaks. Thus the application of probiotics could lead to disease-free and profitable shrimp culture operations which will be helpful for shrimp farmers as most of them are now-a-days severely affected by microbial diseases.     

Superoxide dismutase and catalase levels in halophilic vibrios.

Superoxide dismutase (SOD) and catalase (CAT) levels were determined for several aerobically grown halophilic vibrios and compared with those found in aerobically grown Escherichia coli K-12. The SOD levels ranged from 25 to 103.6 U/mg of protein for the vibrios compared with 44.6 U/mg of protein for E. coli. The CAT levels ranged from 2.1 to 32.1 U/mg of protein. Electrophoretic analysis of cell extracts revealed that the halophilic vibrios tested possessed only one detectable SOD enzyme, except one strain which possessed two distinct enzymes, as compared with the three SOD enzymes in aerobically grown E. coli K-12. A comparison of anaerobically and aerobically grown vibrios revealed a three- to fourfold increase in SOD activity in the aerobic cells, suggesting that oxygen acts as an inducer for SOD in the vibrios as has been reported for E. coli. In one strain, Vibrio parahaemolyticus 27519, both SOD enzymes were observed in low levels in anaerobic and at higher levels in aerobically grown cells as compared with only one SOD enzyme in anaerobically grown E. coli. This suggests that differences in SOD regulation occur between the two genera. Our results indicate that halophilic vibrios possess SOD, which could enhance viruulence by allowing the organisms to survive in oxygenated environments.     

Potentially pathogenic vibrios in brackish waters and mussels

Water and mussel samples were collected from two brackish lakes, used as mussel farms, at different times of the year, for the quantitative analysis of Vibrio spp. and for the isolation of potentially pathogenic species. The isolates underwent cultural and biochemical tests selected for rapid identification. Glucose oxidizing-fermenting and O/129 sensitive strains were distinguished on the basis of the following tests: sucrose and cellobiose utilization, sulphatase activity and polymyxin B resistance performed, respectively, on TCBS, CPC and SPS media. Responses to the presence of beta-galactosidase, salt requirement and growth on triple sugar iron medium were also added. A total of 125 from 152 isolates were referred to the species Vibrio fluvialis (55 strains), V. alginolyticus (40), V. parahaemolyticus (11), V. vulnificus (10) and V. mimicus (9). The remaining 27 isolates were not identified. The isolation of potentially pathogenic vibrios from cultivated mussels is a risk for health of people consuming raw seafood. Therefore, a long-term monitoring programme should also include the search for these bacterial species.     

The use of the gene hybridization method for the identification of epidemiologically dangerous strains of cholera vibrios

Using the labeled DNA fragments containing the genes for cholera toxin the strains of cholera vibrios were studied for the presence of cholera toxin genes. Vibrio cholerae strains isolated from natural water reservoirs under the favourable epidemic situation do not contain the cholera toxin genes. The DNA hybridization method was compared with other methods used in research and practical work for estimation of epidemic importance of cholera vibrios.     

一种副溶血性弧菌显色培养基的应用

副溶血性弧菌(Vibri oparahemolyticus)是一种在海产品中广泛存在的嗜盐性细菌,也是重要的食源性致病菌。传统检验方法检测周期长、特异性低,为提高检测效率、缩短检测周期,开发出一种副溶血性弧菌显色培养基(HKMVPM),与国外同类产品KHVPM和TCBS在灵敏度、特异性及检测效果方面进行了比较和初步评价。结果显示,HKMVPM显色培养基和KHVPM显色培养基均具有较好的选择性和特异性,这3种培养基对天然污染样品的分离率高低顺序为HKMVPMKHVPMTCBS。显色培养基具有高检出率及高特异性的特点,有良好的应用前景。