Effect of grape seed extract on quality and microbiota community of container-cultured snakehead (Channa argus) fillets during chilled storage.

Herein, the effects of grape seed extract (GSE) on the microflora and biochemical changes of container cultured snakehead (Channa argus) fillets during 11 days of chilled storage were investigated. The sensory analysis, the total number of viable colonies, the total amount of volatile basic nitrogen, and k-value analysis revealed that GSE retarded the deterioration of snakehead fillets. The degradation of inosine 5'-monophosphate and the accumulation of inosine and hypoxanthine in the GSE group were slower than these in the control group. Moreover, GSE treatment effectively decreased the accumulation of putrescine, cadaverine, and histamine. Illumina-MiSeq high throughput sequencing results showed that GSE inhibited the growth of Aeromonas on snakehead fillets. Based on the microbial enumeration, sensory analysis, and k-value, GSE prolonged the shelf life of fillets for 3 days, suggesting its potential for snakehead fillets preservation.

Application of Illumina-MiSeq high throughput sequencing and culture-dependent techniques for the identification of microbiota of silver carp (Hypophthalmichthys molitrix) treated by tea polyphenols.

This study evaluated the antimicrobial effects of tea polyphenols (TP) on changes in microbiota composition and quality attributes in silver carp fillets stored at 4 °C. During storage, TP treatment was found to be effective in enhancing sensory quality, inhibiting microbial growth, and attenuating chemical quality deterioration. Meanwhile, the composition of microbiota of silver carp fillets was investigated using culture-dependent and culture-independent methods. Initially, compared to the control, TP obviously decreased the relative abundance of Aeromonas, which allowed Acinetobacter and Methylobacterium to become the dominant microbiota in TP treated fillets on day 0. The controls, 0.5% TP-treated fillets, and 1% TP-treated fillets were rejected by sensory panelists on days 8, 12, and 12, respectively. At the time of sensory rejection, Aeromonas, followed by Acinetobacter and Pseudomonas, became the main spoilers in the control on day 8. However, TP treatment inhibited the growth of Aeromonas and Acinetobacter significantly. Consequently, Aeromonas followed by Pseudomonas and Shewanella became the predominant microbiota in all TP-treated fillets on day 12. Therefore, TP improved the quality of fillets during chilled storage, which was mainly due to their modulating effects on microbiota that resulted in the change in pattern and process of spoilage in fillets.

Novel application of Mahua (Madhuca sp.) flowers for augmented protease production from Aeromonas sp. S1.

The present study explored the utilization of Mahua (Madhuca sp.) flowers, a major non-timber forest product (NTFP) of India, as a low-cost, natural substrate for protease production under submerged fermentation. Bacterial strain Aeromonas sp. Si1, previously reported by us, was used as the protease producer. Using Mahua flower extract (MFE) as the medium additive, the protease production could successfully be enhanced by 5.6-fold (564.5 UmL-1) after 24 h of fermentation under optimized conditions compared with initial production of 99.9 UmL' in the absence of MFE. The cultural parameters for optimum production of protease were determined to be: incubation time-24 h; pH-7.0; MFE concentration-5% (v/v); inoculum size-0.3% (v/v) and agitation rate-200 rpm. The results obtained demonstrate the potential of cheaper and abundantly available Mahua flowers for induction of proteases, and thus offer a new approach for value addition to this biomass through industrial enzyme production.

Investigation into the physiologies of Aeromonas veronii in vitro and inside the digestive tract of the medicinal leech using RNA-seq.

Host-associated microbial communities are widespread in nature and vital to the health and fitness of the host. Deciphering the physiology of the microbiome in vivo is critical to understanding the molecular basis of the symbiosis. Recently, the development and application of high-throughput sequencing techniques, particularly RNA-seq, for studying microbial communities has enabled researchers to address not only which microbes are present in a given community but also how the community functions. For microbes that can also be cultivated in the laboratory, RNA-seq provides the opportunity to identify genes that are differentially expressed during symbiosis by comparing in vitro to in vivo transcriptomes. In the current study, we used RNA-seq to identify genes expressed by the digestive-tract microbiome of the medicinal leech, Hirudo verbana, and by one of the two dominant symbionts, Aeromonas veronii, in a rich medium. We used a comparative approach to identify genes differentially expressed during symbiosis and gain insight into the symbiont's physiology in vivo. Notable findings include evidence for the symbionts experiencing environmental stress, performing arginine catabolism, and expressing noncoding RNAs that are implicated in stationary phase survival, a state in which A. veronii persists for months within the host.

Bacterial symbioses of the medicinal leech Hirudo verbana.

Gastrointestinal microbiomes play important roles in the health and nutrition of animals and humans. The medicinal leech, Hirudo verbana, serves as a powerful model for the study of microbial symbioses of the gut, due to its naturally limited microbiome compared with other popular models, the ability to cultivate the most abundant microbes, and genetically manipulate one of them, Aeromonas veronii. This review covers the relevance and application of leeches in modern medicine as well as recent discoveries detailing the nature of the gut microbiome. Additionally, the dual life-style of A. veronii allows one to do direct comparisons between colonization factors for beneficial and pathogenic associations, and relevant findings are detailed with respect to their role within the host and pathogenicity to other animals.

Lactobacillus plantarum (VR1) isolated from an ayurvedic medicine (Kutajarista) ameliorates in vitro cellular damage caused by Aeromonas veronii.

BACKGROUND: Lactobacillus plantarum is considered as a safe and effective probiotic microorganism. Among various sources of isolation, traditionally fermented foods are considered to be rich in Lactobacillus spp., which can be exploited for their probiotic attribute. Antibacterial property of L. plantarum has been demonstrated against various enteric pathogens in both in vitro and in vivo systems. This study was aimed at characterizing L. plantarum isolated from Kutajarista, an ayurvedic fermented biomedicine, and assessing its antagonistic property against a common enteropathogen Aeromonas veronii. RESULTS: We report the isolation of L. plantarum (VR1) from Kutajarista, and efficacy of its cell free supernatant (CFS) in amelioration of cytotoxicity caused by Aeromonas veronii. On the part of probiotic attributes, VR1 was tolerant to pH 2, 0.3% bile salts and simulated gastric juice. Additionally, VR1 also exhibited adhesive property to human intestinal HT-29 cell line. Furthermore, CFS of VR1 was antibacterial to enteric pathogens like Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli, Aeromonas veronii and clinical isolates of P. aeruginosa and E. coli. Detailed study regarding the effect of VR1 CFS on A. veronii cytotoxicity showed a significant decrease in vacuole formation and detrimental cellular changes in Vero cells. On the other hand, A. veronii CFS caused disruption of tight junction proteins ZO-1 and actin in MDCK cell line, which was prevented by pre-incubation with CFS of VR1. CONCLUSIONS: This is the first study to report isolation of L. plantarum (VR1) from Kutajarista and characterisation for its probiotic attributes. Our study demonstrates the antagonistic property of VR1 to A. veronii and effect of VR1 CFS in reduction of cellular damage caused by A. veronii in both Vero and MDCK cell lines.

Oxytetracycline treatment reduces bacterial diversity of intestinal microbiota of Atlantic salmon.

The effect of oxytetracycline (OTC) treatment on intestinal bacterial populations in juvenile Atlantic salmon Salmo salar was evaluated. Oxytetracycline was administered by way of medicated feed to fish held in experimental tanks. Restriction fragment length polymorphism and sequencing of 16S rDNA from isolates were used to analyze the intestinal microbiota before, during, and after OTC administration. The microbiota from untreated fish was more diverse, consisting mainly of Pseudomonas, Acinetobacter, Bacillus, Flavobacterium, Psycrobacter, and Brevundimonas spp. In contrast, the microbiota of the OTC-treated group was characterized by lower diversity and consisted only of Aeromonas, clustering with A. sobria and A. salmonicida. Antibiotic-resistant isolates were identified as Aeromonas spp.; sequencing the resistance determinant showed it to be the tetE gene. Overall, OTC treatment changed the composition of the intestinal microbiota of Atlantic salmon, as evidenced by a reduction in bacterial diversity. These results support the current concern that antibiotic treatment can facilitate the proliferation of opportunistic bacteria by eradicating competing microorganisms.

Identification of Aeromonas veronii genes required for colonization of the medicinal leech, Hirudo verbana.

Most digestive tracts contain a complex consortium of beneficial microorganisms, making it challenging to tease apart the molecular interactions between symbiont and host. The digestive tract of Hirudo verbana, the medicinal leech, is an ideal model system because it harbors a simple microbial community in the crop, comprising the genetically amenable Aeromonas veronii and a Rikenella-like bacterium. Signature-tagged mutagenesis (STM) was used to identify genes required for digestive tract colonization. Of 3,850 transposon (Tn) mutants screened, 46 were identified as colonization mutants. Previously we determined that the complement system of the ingested blood remained active inside the crop and prevented serum-sensitive mutants from colonizing. The identification of 26 serum-sensitive mutants indicated a successful screen. The remaining 20 serum-resistant mutants are described in this study and revealed new insights into symbiont-host interactions. An in vivo competition assay compared the colonization levels of the mutants to that of a wild-type competitor. Attenuated colonization mutants were grouped into five classes: surface modification, regulatory, nutritional, host interaction, and unknown function. One STM mutant, JG736, with a Tn insertion in lpp, encoding Braun's lipoprotein, was characterized in detail. This mutant had a >25,000-fold colonization defect relative to colonization by the wild-type strain at 72 h and, in vitro, an increased sensitivity to sodium dodecyl sulfate, suggesting the presence of an additional antimicrobial property in the crop. The classes of genes identified in this study are consistent with findings from previous STM studies involving pathogenic bacteria, suggesting parallel molecular requirements for beneficial and pathogenic host colonization.

Spatial and temporal population dynamics of a naturally occurring two-species microbial community inside the digestive tract of the medicinal leech.

The medicinal leech, Hirudo verbana, is one of the simplest naturally occurring models for digestive-tract symbioses, where only two bacterial species, Aeromonas veronii bv. sobria (gamma-Proteobacteria) and a Rikenella-like bacterium (Bacteroidetes), colonize the crop, the largest compartment of the leech digestive tract. In this study, we investigated spatial and temporal changes of the localization and microcolony structure of the native symbionts in the crop, after ingestion of a sterile blood meal, by fluorescence in situ hybridization. The population dynamics differed between the two symbiotic bacteria. A. veronii was detected mainly as individual cells inside the intraluminal fluid (ILF) during 14 days after feeding (daf) unless it was found in association with Rikenella microcolonies. The Rikenella-like bacteria were observed not only inside the ILF but also in association with the luminal surface of the crop epithelium. The sizes of Rikenella microcolonies changed dynamically through the 14-day period. From 3 daf onward, mixed microcolonies containing both species were frequently observed, with cells of both species tightly associating with each other. The sizes of the mixed microcolonies were consistently larger than the size of either single-species microcolony, suggesting a synergistic interaction of the symbionts. Lectin staining with succinylated wheat germ agglutinin revealed that the planktonic microcolonies present in the ILF were embedded in a polysaccharide matrix containing N-acetylglucosamine. The simplicity, symbiont-symbiont interaction, and mixed microcolonies of this naturally occurring, digestive-tract symbiosis lay the foundation for understanding the more complex communities residing in most animals.

Novel role for Aeromonas jandaei as a digestive tract symbiont of the North American medicinal leech.

The gut bacteria of the North American medicinal leech, Macrobdella decora, were characterized. Biochemical tests and DNA sequences indicated that Aeromonas jandaei is the dominant culturable symbiont in leeches from a broad geographic area. In this work we identified a new habitat for A. jandaei, and here we suggest that there is unexpected specificity between leeches and Aeromonas species.

Influence of different concentrations of nitrogen and phosphorous on Aeromonas spp. growth.

This paper evaluates the influence of nitrogen and phosphorous on the growth of Aeromonas previously isolated from irrigation water. Three strains respectively of three species of Aeromonas (A. hydrophila, A. caviae, A. sobria) (nine strains) were individually inoculated in a microcosm added with ammonium, nitrate and phosphate using different combinations (NH4-N + NO3-N and NH4-N + NO3-N + PO4-P) and concentrations (NH4-N: from 20 to 2500 microg l-1; NO3-N: from 200 to 22000 microg l-1; PO4-P: from 20 to 1200 microg l-1). A positive correlation was found between bacterial growth and NH4-N charge, but not a strict correlation with NO3-N. The addition of PO4-P at concentrations from 20 to 200 microg l-1 stimulated the bacterial growth; inversely, concentrations of 500 and 1200 microg l-1 were inhibitory. Ammonium, nitrate and phosphate influence in different way Aeromonas growth; so, their various concentrations can partially explain the different recovery of these bacteria from aquatic environments. There is discordance in literature about the influence of environmental parameters on Aeromonas growth in aquatic ecosystems. Therefore, this study contributes to a better understanding about interactions of some chemical parameters and Aeromonas growth, which are at the basis of the varied relationship described for environmental factors governing aeromonads densities.

Factors affecting biosurfactant production by oil degrading Aeromonas spp. isolated from a tropical environment.

An Aeromonas spp. was isolated from tropical estuarine water. The organism grew on crude oil and produced biosurfactant that could emulsify hydrocarbons. The peak growth and biosurfactant production was on the 8th day. The organism grew on a range of hydrocarbons that include crude oil and hexadecane while no growth was recorded on some hydrocarbons that include benzene. The biosurfactant produced by the organism emulsified a range of hydrocarbons with diesel (E24=65) as the best substrate and hexane (E24=22) as the poorest. After purification, the biosurfactant was found to contain about 38% carbohydrate and an unidentified lipid. No protein was present in the purified biosurfactant. Production of biosurfactant was highest in medium with glucose and lowest in the medium with diesel+acetate. Soybean was the best nitrogen source for biosurfactant production. The activity of the biosurfactant was enhanced optimally at NaCl concentration of 5%, pH of 8.0 and temperature of 40 degrees C. The biosurfactant retained 77% of its original activity after 120 min of exposure to heat at a temperature of 100 degrees C. Biosurfactant may be produced with this organism using non-hydrocarbon substrates such as glucose and soybean that are readily available and would not require extensive purification for use in food and pharmaceutical industries.

External decontamination of wild leeches with hypochloric acid.

BACKGROUND: Medicinal leech, Hirudo medicinalis, has been used in plastic and reconstructive surgery, to relieve venous congestion and to improve the microrevascularization of flaps. In many countries, wild leeches are still provided from local markets and utilised with antibiotic prophylaxies. In this research, results of identification of bacteria in the transport fluid is reported, oral and intestinal floras and the antibiograms of the identified microorganisms are investigated. Also, to avoid possible infections, the ability of hypochloric acid, a disinfectant, to suppress the relevant microorganisms without changing the life style and behavior of leeches in terms of sucking function, is investigated. METHODS: Bacterial identifications and antibiograms of oral and intestinal flora and transport medium were performed for 10 leeches. The optimum concentration of hypochloric acid which eliminated microorganisms without affecting the viability and sucking function of the leeches were determined by dilution of hypochloric acid to 100, 50, 25, 12.5, 6.25 ppm concentrations in different groups of 25 leeches. Finally, 20 leeches were applied atraumatically to the bleeding areas of rats, the duration of suction was determined and compared statistically between the leeches treated and not treated with hypochloric acid solution. RESULTS: Aeromonas hydrophilia was the most commonly identified microorganism and found to be resistant to first generation cephalosporins, frequently used in prophylaxis at surgical wards. In the next stages of the study, the leeches were subjected to a series of diluted hypochloric acid solutions. Although disinfection of the transport material and suppression of the oral flora of hirudo medicinalis were successful in 100, 50, 25, 12.5, 6.25 ppm concentrations; 12.5 ppm solution was the greatest concentration in which hirudo medicinalis could survive and sucking function was not affected significantly. CONCLUSIONS: External decontamination of wild leeches with 12.5 ppm hypochloric acid enables bacterial suppression without causing negative effects on leech sucking function and life.

Ingested blood contributes to the specificity of the symbiosis of Aeromonas veronii biovar sobria and Hirudo medicinalis, the medicinal leech.

Hirudo medicinalis, the medicinal leech, usually carries in its digestive tract a pure culture of Aeromonas veronii bv. sobria. Such specificity is unusual for digestive tracts that are normally colonized by a complex microbial consortium. Important questions for the symbiotic interaction and for the medical application after microvascular surgery are whether other bacteria can proliferate or at least persist in the digestive tract of H. medicinalis and what factors contribute to the reported specificity. Using a colonization assay, we were able to compare experimentally the ability of clinical isolates and of a symbiotic strain to colonize H. medicinalis. The symbiotic A. veronii bv. sobria strain proliferated well and persisted for at least 7 days inside the digestive tract. In contrast, the proliferation of Pseudomonas aeruginosa and Staphylococcus aureus was inhibited inside the animal compared to growth in the in vitro control, indicating that the ingested blood was modified within the digestive tract. However, both strains were able to persist in the digestive tract for at least 7 days. For an Escherichia coli strain, the viable counts decreased approximately 1, 000-fold within 42 h. The decrease of viable E. coli could be prevented by interfering with the activation of the membrane-attack complex of the complement system that is present in blood. This suggests that the membrane-attack complex remained active inside H. medicinalis and prevented the proliferation of sensitive bacteria. Thus, antimicrobial properties of the ingested vertebrate blood contribute to the specificity of the A. veronii-H. medicinalis symbiosis, in addition to modifications of the blood inside the digestive tract of H. medicinalis.

Inhibition of Aeromonas caviae and A. sobria by sodium choloride, citric acid, ascorbic acid, potassium sorbate and extracts of Thymus vulgaris.

The respective and combined effects of sodium chloride, ascorbic acid, citric acid, potassium sorbate, and Thymus vulgaris extract on the growth of Aeromonas caviae and Aeromonas sobria were investigated. Sodium chloride (3%) significantly reduced the growth and 4% NaCl inhibited growth of the tested strains. Ascorbic acid (0. 1%), potassium sorbate (0.05%), and citric acid (0.03%) slightly inhibited growth. T. vulgaris extract (0.3%) greatly reduced the growth. Various combinations of these compounds prevented growth of the tested strains. A combination of NaCl (3%) and ascorbic acid (0. 1%), citric acid (0.03%) and potassium sorbate (0.05%), or citric acid (0.03%) and ascorbic acid (0.1%) inhibited growth of A. caviae and A. sobria. In fish homogenates, the addition of ascorbic acid (0. 1%) and citric acid (0.03%) was the most effective combination tested.

Symbiosis of Aeromonas veronii biovar sobria and Hirudo medicinalis, the medicinal leech: a novel model for digestive tract associations.

Hirudo medicinalis, the medicinal leech, is applied postoperatively in modern medicine. Infections by Aeromonas occur in up to 20% of patients unless a preemptive antibiotic treatment is administered. The associated infections demonstrate the need for a better understanding of the digestive tract flora of H. medicinalis. Early studies reported the presence of a single bacterial species in the digestive tract and suggested that these bacteria were endosymbionts contributing to the digestion of blood. In this study, we cultivated bacteria from the digestive tract and characterized them biochemically. The biochemical test results identified the isolates as Aeromonas veronii biovar sobria. This species identification was supported by sequence comparison of a variable region of the genes coding for 16S rRNA. In a colonization assay, a rifampin-resistant derivative of a symbiotic isolate was fed in a blood meal to H. medicinalis. The strain colonized the digestive tract rapidly and reached a concentration similar to that of the native bacterial flora. For the first 12 h, the in vivo doubling time was 1.2 h at 23 degreesC. After 12 h, at a density of 5 x 10(7) CFU/ml, the increase in viable counts ceased, suggesting a dramatic reduction in the bacterial growth rate. Two human fecal isolates, identified as Aeromonas hydrophila and A. veronii biovar sobria, were also able to colonize the digestive tract. These data demonstrate that the main culturable bacterium in the crop of H. medicinalis is A. veronii biovar sobria and that the medicinal leech can be used as a model for digestive tract association of Aeromonas species.

[Studies on enrichment broth for verotoxin-producing Escherichia coli (VTEC) O157].

Growth of verotoxin-producing Escherichia coli (VTEC) O157 in conventionally recommended pre-enrichment broth media at different temperatures was evaluated. In addition, sensitivity of VTEC O157 isolates to several antibacterial drugs, which were added to the selective enrichment broth, was tested. All five isolates of VTEC O157 tested grew well in trypticase soy broth (TSB) at 36 degrees C and 42 degrees C, while the growth of one isolate was markedly suppressed in TSB supplemented with cefixime (CFIX), potassium tellurite (PT), and vancomycin (TSB-CTV) even at 36 degrees C. A significant growth suppression was also observed in three of the isolates cultured in novobiocin (NB)-supplemented modified EC broth (mEC-NB) at 42 degrees C. In mEC-NB after 24-hr incubation at 36 degrees C, the five VTEC O157 isolates grew well, although one isolate was slightly suppressed during the first 8 hours. Minimum growth inhibitory concentrations of CFIX, NB and PT for a total of 90 clinical and environmental isolates of VTEC O157 were all above the concentrations usually prescribed for mEC-NB and TSB-CTV. These findings suggest that mEC-NB and TSB-CTV should be used at 36 degrees C for growth of VTEC O157 and that use of a nonselective pre-enrichment broth medium (i.e. TSB) together with a selective one (i.e. TSB-CTV or mEC-NB) is necessary for successful isolation of VTEC O157 from various specimens.

Fever and survival: the role of serum iron.

1. The effects of bacterial infection and temperature on serum iron levels were investigated in the lizard Dipsosaurus dorsalis. 2. Changes in body temperature from normal (38 degrees C) to febrile (41 degrees C) did not alter serum iron levels. Injection with Aeromonas hydrophila led to a significant reduction in serum iron levels, comparable to that found in mammals. This reduction in serum iron level was independent of the lizard's body temperature. 3. When grown in vitro, A. hydrophila grew equally well at afebrile (38 degrees C) and febrile (41 degrees C) temperatures. When the iron levels of the growth medium were reduced, the bacterial growth was diminished at the febrile temperature but was not significantly affected at the afebrile temperature. 4. The addition of iron supplements to bacterially infected lizards led to an increase in the percent mortality. 5. These results indicate that one of the mechanisms behind the beneficial, or adaptive value of fever in D. dorsalis is the decrease in iron available to the pathogenic micro-organisms.

Effect of four dispersants on biodegradation and growth of bacteria on crude oil.

Four chemical dispersants, Corexit 8666, Gamlen Sea Clean, G. H. Woods Degreaser-Formula 11470, and Sugee 2 were examined singly and in individual combinations with Arabian Crude Oil (1:1 ratio) at 10 and 25 C for their effects on the growth of bacteria indigenous to local marine waters, the bacterial population composition, and biodegradation of crude oil; in addition, their emulsifying capacities, at approximately 24 C, were determined. None of the dispersants used alone were toxic even at relatively high concentrations (1.25%), although Gamlen Sea Clean and G. H. Woods Degreaser-Formula 11470 did cause an increase in the lag phase which was more pronounced at 10 than at 25 C; addition of the crude oil reduced the lag phase increase. All of the dispersants used alone supported good growth of microorganisms, but qualitative population shifts were caused by the dispersant-oil combinations. The degrees of degradation of the n-alkane fraction of the crude oil varied depending upon the dispersant used. Under these test conditions, only Sugee 2, which had the poorest emulsifying capacity, promoted n-alkane degradation compared with the values obtained by using the crude oil alone.

Production and catabolite repression of the constitutive polygalacturonic acid trans-eliminase of Aeromonas liquefaciens.

Production of polygalacturonic acid (PGA) trans-eliminase was greatly stimulated under conditions of restricted growth of Aeromonas liquefaciens. This was accomplished either by substrate restriction in a continuous-feeding culture or by restricting divalent cations in a batch culture, with the use of PGA as the sole source of carbon in a chemically defined medium containing inorganic nitrogen. Slow feeding of glucose, glycerol, or PGA to carbon-limited cultures allowed PGA trans-eliminase to be formed at a maximum differential rate 500 times greater than in batch cultures with excess substrate present. The differential rate of enzyme formation obtained by slow feeding of these three substrances or of a mixture of PGA plus glucose was observed to be the same. Therefore, PGA trans-eliminase produced by A. liquefaciens, contrary to the current view, appears to be constitutive. These observations also indicate that production of PGA trans-eliminase is subject to catabolite repression and that limiting the substrate reverses this repression. It was also found that, under conditions of unrestricted growth, any compound which the bacteria can use as a source of carbon and energy repressed constitutive PGA trans-eliminase production. The heritable reversal of catabolite repression of PGA trans-eliminase synthesis was demonstrated by isolation of mutant strain Gc-6 which can readily synthesize the constitutive catabolic enzyme PGA trans-eliminase while growing in the presence of excess substrate.