Isolation of lytic bacteriophage against Vibrio harveyi

Aims: The isolation of lytic bacteriophage of Vibrio harveyi with potential for phage therapy of bacterial pathogens of phyllosoma larvae from the tropical rock lobster Panulirus ornatus. Methods and Results: Water samples from discharge channels and grow‐out ponds of a prawn farm in northeastern Australia were enriched for 24 h in a broth containing four V. harveyi strains. The bacteriophage‐enriched filtrates were spotted onto bacterial lawns demonstrating that the bacteriophage host range for the samples included strains of V. harveyi, Vibrio campbellii, Vibrio rotiferianus, Vibrio parahaemolyticus and Vibrio proteolyticus. Bacteriophage were isolated from eight enriched samples through triple plaque purification. The host range of purified phage included V. harveyi, V. campbellii, V. rotiferianus and V. parahaemolyticus. Transmission electron microscope examination revealed that six purified phage belonged to the family Siphoviridae, whilst two belonged to the family Myoviridae. The Myoviridae appeared to induce bacteriocin production in a limited number of host bacterial strains, suggesting that they were lysogenic rather than lytic. A purified Siphoviridae phage could delay the entry of a broth culture of V. harveyi strain 12 into exponential growth, but could not prevent the overall growth of the bacterial strain. Conclusions: Bacteriophage with lytic activity against V. harveyi were isolated from prawn farm samples. Purified phage of the family Siphoviridae had a clear lytic ability and no apparent transducing properties, indicating they are appropriate for phage therapy. Phage resistance is potentially a major constraint to the use of phage therapy in aquaculture as bacteria are not completely eliminated. Significance and Impact of the Study: Phage therapy is emerging as a potential antibacterial agent that can be used to control pathogenic bacteria in aquaculture systems. The development of phage therapy for aquaculture requires initial isolation and determination of the bacteriophage host range, with subsequent creation of suitable phage cocktails.     

Lead-Enhanced Siderophore Production and Alteration in Cell Morphology in a Pb-Resistant <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> Strain 4EA

A lead-resistant bacterial strain 4EA from soil contaminated with car battery waste from Goa, India was isolated and identified as Pseudomonas aeruginosa. This lead-resistant bacterial isolate interestingly revealed lead-enhanced siderophore (pyochelin and pyoverdine) production up to 0.5 mM lead nitrate whereas cells exhibit a significant decline in siderophore production above 0.5 mM lead nitrate. The bacterial cells also revealed significant alteration in cell morphology as size reduction when exposed to 0.8 mM lead nitrate. Enhanced production of siderophore was evidently detected by chrome azurol S agar diffusion (CASAD) assay as increase in diameter of orange halo, and reduction in bacterial size along with significant biosorption of lead was recorded by scanning electron microscopy coupled with energy dispersive X-ray spectrometry (SEM-EDX). Pseudomonas aeruginosa strain 4EA also exhibits cross tolerance to other toxic metals viz. cadmium, mercury, and zinc besides resistance to multiple antibiotics such as ampicillin, erythromycin, amikacin, cephalexin, co-trimoxazole, mecillinam, lincomycin, ciphaloridine, oleondamycin, and nalidixic acid.     

AI-2 does not function as a quorum sensing molecule inCampylobacter jejuniduring exponential growthin vitro

Background  

Campylobacter jejunicontains a homologue of theluxSgene shown to be responsible for the production of the signalling molecule autoinducer-2 (AI-2) inVibrio harveyiandVibrio cholerae. The aim of this study was to determine whether AI-2 acted as a diffusible quorum sensing signal controllingC. jejunigene expression when it is produced at high levels during mid exponential growth phase.     

Decolorization of Acid Black 210 by <Emphasis Type="Italic">Vibrio harveyi</Emphasis> TEMS1, a newly isolated bioluminescent bacterium from Izmir Bay,Turkey

The present study deals with the decolorization of Acid Black 210 by a bioluminescent bacterium, Vibrio harveyi TEMS1, isolated from coastal seawater of Izmir Bay, Turkey. Maximum rate of decolorization of Acid Black 210 was observed when Luria Bertani medium was used. Decolorization of Acid Black 210 was 38.9% and 93.9% at 24 h under shaking and static conditions, respectively. The optimum dye-decolorizing activity of the culture was obtained at 100 ppm initial dye concentration and incubation temperature of 20°C. Vibrio harveyi TEMS1 was also tested for its ability to decolorize four azo dyes (Acid Black 24, Acid Blue 7, Acid Green 20, Acid Yellow 36) in addition to Acid Black 210.     

Development of a haemolysin gene‐based multiplex PCR for simultaneous detection of Vibrio campbellii,Vibrio harveyi and Vibrio parahaemolyticus

Aim: To develop a haemolysin (hly) gene‐based species‐specific multiplex PCR for simple and rapid detection of Vibrio campbellii, V. harveyi and V. parahaemolyticus. Methods and Results: The complete hly genes of three V. campbellii strains isolated from diseased shrimps were sequenced and species‐specific PCR primers were designed based on these sequences and the registered hly gene sequences of Vibrio harveyi and Vibrio parahaemolyticus. Specificity and sensitivity of the multiplex PCR was validated with 27 V. campbellii, 16 V. harveyi, and 69 V. parahaemolyticus, 18 other Vibrio species, one Photobacterium damselae and nine other bacterial species. The detection limits of all the three target species were in between 10 and 100 cells per PCR tube. Conclusions: Specificity and sensitivity of the multiplex PCR is 100% each and sufficient to be considered as an effective tool in a prediction system to prevent potential disease outbreak by these Vibrio species. Significance and Impact of the Study: Because there is lack of simple, rapid and cost‐effective method to differentiate these closely related V. campbellii, V. harveyi and V. parahaemolyticus species, the multiplex PCR developed in this study will be very effective in epidemiological, ecological and economical points of view.     

Chemical Characterization,Crossfeeding and Uptake Studies on Hydroxamate Siderophore of <Emphasis Type="Italic">Alcaligenes faecalis</Emphasis>

We report the production of two types of siderophores namely catecholate and hydroxamate in modified succinic acid medium (SM) from Alcaligenes faecalis. Two fractions of siderophores were purified on amberlite XAD, major fraction was hydroxamate type having a λ_max at 224 nm and minor fraction appeared as catecholate with a λ_max of 264 nm. The recovery yield obtained from major and minor fractions was 297 and 50 mg ml⁻¹ respectively. The IEF pattern of XAD-4 purified siderophore suggested the pI value of 6.5. Cross feeding studies revealed that A. faecalis accepts heterologous as well as self (hydroxamate) siderophore in both free and iron complexed forms however; the rate of siderophore uptake was more in case of siderophores complexed to iron. Siderophore iron uptake studies indicated the differences between hydroxamate siderophore of A. faecalis and Alc E, a siderophore of Alcaligenes eutrophus.     

Cinnamaldehyde and cinnamaldehyde derivatives reduce virulence in <Emphasis Type="Italic">Vibrio</Emphasis> spp. by decreasing the DNA-binding activity of the quorum sensing response regulator LuxR

Background  

To date, only few compounds targeting the AI-2 based quorum sensing (QS) system are known. In the present study, we screened cinnamaldehyde and substituted cinnamaldehydes for their ability to interfere with AI-2 based QS. The mechanism of QS inhibition was elucidated by measuring the effect on bioluminescence in several Vibrio harveyi mutants. We also studied in vitro the ability of these compounds to interfere with biofilm formation, stress response and virulence of Vibrio spp. The compounds were also evaluated in an in vivo assay measuring the reduction of Vibrio harveyi virulence towards Artemia shrimp.     

Potential plant growth-promoting activity of <Emphasis Type="Italic">Serratia nematodiphila</Emphasis> NII-0928 on black pepper (<Emphasis Type="Italic">Piper nigrum</Emphasis> L.)

A potential bacterial strain designated as NII-0928 isolated from Western ghat forest soil with multiple plant growth promoting attributes, and it has been identified and characterized. Plant growth promoting traits were analyzed by determining the P-solubilization efficiency, Indole acetic acid production, HCN, siderophore production and growth in nitrogen free medium. It was able to solubilize phosphate (76.6 μg ml⁻¹), and produce indole acetic acid (58.9 μg ml⁻¹) at 28 ± 2°C. Qualitative detection of siderophore production and HCN were also observed. At 5°C it was found to express all the plant growth promotion attributes except HCN production. The ability to colonize roots is a sine qua non condition for a rhizobacteria to be considered a true plant growth-promoting rhizobacteria (PGPR). 16S rRNA gene sequencing reveals the identity of the isolate as Serratia nematodiphila with which it shares highest sequence similarity (99.4%). Seed bacterization with black pepper cuttings in greenhouse trials using Sand: Soil: FYM with three individual experimental sets with their respective control showed clearly the growth promoting activity. Hence, Serratia nematodiphila NII-0928 is a promising plant growth promoting isolate showing multiple PGPR attributes that can significantly influence black pepper cuttings. The result of this study provides a strong basis for further development of this strain as a bioinoculants to attain the desired plant growth promoting activity in black pepper growing fields.     

Prevalence and Heterogeneity of Hemolysin Gene <Emphasis Type="Italic">vhh</Emphasis> Among Hatchery Isolates of <Emphasis Type="Italic">Vibrio harveyi</Emphasis> in India

Vibrio harveyi, pathogenic to fish, harbor a hemolysin gene vhh, the homologues of which are found in many species of the Genus Vibrio. In this study, we investigated the prevalence of vhh gene among V. harveyi isolated from Penaeus monodon hatcheries in India by polymerase chain reaction (PCR). The vhh was detected in 67 of the 70 V. harveyi isolates tested in this study using different combinations of PCR primers. A variant vhh gene detected in a minority of strains was cloned, sequenced, and the recombinant protein was expressed in Escherichia coli. The deduced amino acid sequence of the cloned gene was 86% similar to the previously reported amino acid sequences of VHH. The results of this study suggest that though V. harveyi strains invariably harbor vhh, the sequence variants of the hemolysin gene exist that may impede their detection by PCR.     

Bioluminescent assay for sphingolipid ceramide N-deacylase using <Emphasis Type="Italic">Vibrio harveyi</Emphasis> dark mutant M-17

A new bioluminescent assay method for the activity of sphingolipid ceramide N-deacylase (SCDase: EC 3.5.1.69) as well as ceramidase (CDase: EC 3.5.1.23) was developed using bioluminescent marine bacteria. Enzymatically synthesized ceramide (N-myristoyl sphigosine, C14:0-18:l) and commercial SCDase were used in this demonstration, and myristic (tetradecanoic, C14:0) acid produced by the SCDase hydrolysis was quantified using Vibrio harveyi M-17, a dark mutant of V. harveyi. The in vivo light intensity of M-17 was stimulated up to thousands fold in the presence of myristic acid, was used for this assay. SCDase activity with as little as 10 μU and 5 nM of myristic acid production were detected in less than one min. The assay worked well for the determination of Km and chromatographic fraction assay.     

Inhibitory activity of probiotics <Emphasis Type="Italic">Streptococcus phocae</Emphasis> PI80 and <Emphasis Type="Italic">Enterococcus faecium</Emphasis> MC13 against Vibriosis in shrimp <Emphasis Type="Italic">Penaeus monodon</Emphasis>

Occurrence of widespread epizootics among larval and cultured shrimp has put on viable preventive approaches such as application of probiotics on a high priority in aquaculture. In the present study, four probiotics bacteria were isolated from marine fish and shrimp intestine based on the antagonistic activity and nonpathogenic to the host. The isolates of probiotics strains Streptococcus phocae PI80, Enterococcus faecium MC13, Lactococcus garvieae LC149, B49 and one commercial probiotics (ECOFORCE) were fed to post larvae of Penaeus monodon obtained from two different hatcheries to analyze the growth and protection against Vibrio harveyi and V. parahaemolyticus. Growth of P. monodon post larvae fed with probiotic strain S. phocae PI80 was significantly (P < 0.001) higher when compared with control and other three strains in both experiments. The treatment of post larvae with B49 reduced the growth as well as Specific growth rate. Among the three probiotic strains S. phocae PI80 and E. faecium MC13 have effectively inhibited the pathogens. In experiment I high survival (92%) were observed in S. phocae PI80 treated post larvae when challenged with Vibrio harveyi followed by E. faecium MC13 (84%), B49 (76%) and ECOFORCE (68%) but PI80 did not protect the post larvae in the same experiment when they were exposed to V. parahaemolyticus. The probiotic isolate of MC13 has protected the post larvae against V. parahaemolyticus when compared to other probiotics and control. Similarly in the second experiment feeding of S. phocae enhanced the survival of larvae when challenged with V. harveyi. The laboratory studies proved that bacterial probionts S. phocae and E. faecium isolated from shrimp and brackishwater fish has potential applications for controlling pathogenic vibriosis in shrimp culture.     

Luminescence, virulence and quorum sensing signal production by pathogenic Vibrio campbellii and Vibrio harveyi isolates

Aims:  To study the relationship between luminescence, autoinducer production and virulence of pathogenic vibrios. Methods and Results:  Luminescence, quorum sensing signal production and virulence towards brine shrimp nauplii of 13 Vibrio campbellii and Vibrio harveyi strains were studied. Although only two of the tested strains were brightly luminescent, all of them were shown to produce the three different types of quorum sensing signals known to be produced by Vibrio harveyi. Cell-free culture fluids of all strains significantly induced bioluminescence in the cholerae autoinducer 1, autoinducer 2 and harveyi autoinducer 1 reporter strains JAF375, JMH597 and JMH612, respectively. There was no relation between luminescence and signal production and virulence towards brine shrimp. Conclusions:  There is a large difference between different strains of Vibrio campbellii and Vibrio harveyi with respect to bioluminescence. However, this is not reflected in signal production and virulence towards gnotobiotic brine shrimp. Moreover, there seems to be no relation between quorum sensing signal production and virulence towards brine shrimp. Significance and Impact of the Study:  The results presented here indicate that strains that are most brightly luminescent are not necessarily the most virulent ones and that the lower virulence of some of the strains is not due to a lack of autoinducer production.     

Immunoproteomic analysis and identification of novel immunogenic proteins from Vibrio harveyi

Aims: The main aim of this study was to screen novel immunogenic proteins of Vibrio harveyi, which could be vaccine candidates. Methods and Results: Whole‐cell proteins of V. harveyi, strain Li01 and Huang01, were first separated by isoelectric focusing, followed by 2D‐PAGE, respectively. Immunogenic proteins were identified by Western blotting, using Epinephelus coioides antisera against V. harveyi strain Li01. Western blot analyses revealed 16 shared immunogenic protein spots in both strains. All of the immunogenic proteins were successfully identified and corresponded to 15 proteins. None of these proteins have been previously reported as immunogenic for V. harveyi. Of the 15 proteins, 11 are specific immunoreactive proteins and four are nonspecific immunoreactive proteins. Furthermore, outer membrane protein N (spot 2) and oligopeptide ATP‐binding cassette (ABC) transporter (spot 3) were used as immunogens to immunize E. coioides for investigation of their protective abilities and activities. The E. coioides immunized with OmpN has abilities to fight against infections caused by V. harveyi Li01 and Huang01. However, vaccination with oligopeptide ABC transporter induces low protective immune response in fish. Conclusions: Eleven novel specific antigens were found, and OmpN could potentially be used as vaccine candidate for the development of novel vaccine against V. harveyi. Significance and Impact of the Study: These data show that immunoproteomics methods can be successfully applied in identifying immunogenic proteins of V. harveyi, which helps to search for the protective antigens in future.     

Evaluation of mutagenic and antimutagenic properties of some bioactive xanthone derivatives using Vibrio harveyi test

Aims: Drug safety evaluation plays an important role in the early phase of drug development, especially in the preclinical identification of compounds’ biological activity. The Vibrio harveyi assay was used to assess mutagenic and antimutagenic activity of some aminoalkanolic derivatives of xanthone (1–5), which were synthesized and evaluated for their anticonvulsant and hemodynamic activities. Methods and Results: A novel V. harveyi assay was used to assess mutagenic and antimutagenic activity of derivatives of xanthone 1–5. Two V. harveyi strains were used: BB7 (natural isolate) and BB7M (BB7 derivative containing mucA and mucB genes on a plasmid pAB91273, products of these genes enhance error‐prone DNA repair). According to the results obtained, the most beneficial mutagenic and antimutagenic profiles were observed for compounds 2 and 3. A modification of the chemical structure of compound 2 by the replacement of the hydroxy group by a chloride improved considerably the antimutagenic activity of the compound. Thus, antimutagenic potency reached a maximum with the presence of tertiary amine and chloride atom in the side chain. Conclusions: Among the newly synthesized aminoalkanolic derivatives of xanthone with potential anticonvulsant properties, there are some compounds exhibiting in vitro antimutagenic activity. In addition, it appears that the V. harveyi assay can be applied for primary mutagenicity and antimutagenicity assessment of compounds. Significance and Impact of the Study: The obtained preliminary mutagenicity and antimutagenicity results encourage further search in the group of amino derivatives of xanthone as the potential antiepileptic drugs also presenting some antimutagenic potential. Furthermore, V. harveyi test may be a useful tool for compounds safety evaluation.     

The application of bioflocs technology to protect brine shrimp (Artemia franciscana) from pathogenic Vibrio harveyi

Aims: To study the potential biocontrol activity of bioflocs technology. Methods and Results: Glycerol‐grown bioflocs were investigated for their antimicrobial and antipathogenic properties against the opportunistic pathogen Vibrio harveyi. The bioflocs did not produce growth‐inhibitory substances. However, bioflocs and biofloc supernatants decreased quorum sensing‐regulated bioluminescence of V. harveyi. This suggested that the bioflocs had biocontrol activity against this pathogen because quorum sensing regulates virulence of vibrios towards different hosts. Interestingly, the addition of live bioflocs significantly increased the survival of gnotobiotic brine shrimp (Artemia franciscana) larvae challenged to V. harveyi. Conclusions: Bioflocs grown on glycerol as carbon source inhibit quorum sensing‐regulated bioluminescence in V. harveyi and protect brine shrimp larvae from vibriosis. Significance and Impact of the Study: The results presented in this study indicate that in addition to water quality control and in situ feed production, bioflocs technology could help in controlling bacterial infections within the aquaculture pond.     

Curdlan-like exopolysaccharide production by <Emphasis Type="Italic">Cellulomonas flavigena</Emphasis> UNP3 during growth on hydrocarbon substrates

Cellulomonas flavigena UNP3, a natural isolate from vegetable oil contaminated soil sample has been studied for growth associated exopolysaccharide (EPS) production during growth on glucose, groundnut oil and naphthalene. The EPS showed matrix formation surrounding the cells during scanning electron microscopy. Cell surface hydrophobicity and emulsifying activity studies confirmed the role of EPS as bioemulsifier. Emulsifying activity was found to increase with time (0.2 U/mg for 10 min to 0.27 U/mg for 30 min). Emulsification index, E₂₄ value increased with the increase in EPS concentration. Degradation of polyaromatic hydrocarbons was confirmed using gas chromatography analysis. FTIR analysis showed presence of characteristic absorbance at 895.10 cm⁻¹ for β-configuration of glucan. NMR studies also revealed EPS produced by C. flavigena UNP3 as a linear β-1, 3-d-glucan, and a curdlan like polysaccharide.     

Siderophore production by actinomycetes isolates from two soil sites in Western Australia

The actinomycetes are metabolically flexible soil micro-organisms capable of producing a range of compounds of interest, including siderophores. Siderophore production by actinomycetes sampled from two distinct and separate geographical sites in Western Australia were investigated and found to be generally similar in the total percentage of siderophore producers found. The only notable difference was the proportion of isolates producing catechol siderophores with only 3% found in site 1 (from the north-west of Western Australia and reportedly containing 40% magnetite) and 17% in site 2 (a commercial stone fruit orchard in the hills east of Perth with a soil base ranging from sandy loam to laterite). Further detailed characterization of isolates of interest identified a Streptomyces that produced extracellularly excreted enterobactin, the characteristic Enterobacteriaceae siderophore, and also revealed some of the conditions required for enterobactin production. Carriage of the entF gene, which codes for the synthetase responsible for the final assembly of the tri-cyclic structure of enterobactin, was confirmed by PCR in this isolate. Another separate Streptomyces produced a compound that matched the UV/VIS spectra of heterobactin, a siderophore previously only described in Rhodococcus and Nocardia.     

Unstable Lysogeny and Pseudolysogeny in Vibrio harveyi Siphovirus-Like Phage 1

Exposure of Vibrio harveyi (strain VH1114) to V. harveyi siphovirus-like phage 1 (VHS1) resulted in the production of a low percentage of lysogenized clones of variable stability. These were retrieved most easily as small colonies within dot plaques. Analysis revealed that VHS1 prophage was most likely carried by VH1114 as an episome rather than integrated into the host chromosome. In the late exponential growth phase, lysogenized VH1114 continuously produced VHS1 but also gave rise to a large number of cured progeny. The absence of phage DNA in the cured progeny was confirmed by the absence of VHS1 DNA in Southern blot and PCR assays. Curiously, these very stable, cured subclones did not show the parental phenotype of clear plaques with VHS1 but instead showed turbid plaques, both in overlaid lawns and in dot plaque assays. This phenotypic difference from the original parental isolate suggested that transient lysogeny by VHS1 had resulted in a stable genetic change in the cured clones. Such clones may be called pseudolysogens (i.e., false lysogens), since they have undergone transient lysogeny and have retained some resistance to full lytic phage development, despite the loss of viable or detectable prophage.