Selection of bacterial flora in the Chlortetracycline treated oil sardine (Sardinella longiceps), Indian mackerel (Rastrelliger kanagurta) and prawn (Metapenaeus dobsoni) during ice storage

The native flora of oil sardine and mackerel consisting of Pseudomonas spp; Moraxella spp., Acinetobacter spp. and Vibrio spp. underwent significant changes during ice storage. At the time of spoilage, Pseudomonas spp. were predominant. CTC treatment significantly reduced the Pseudomonas spp. in the initial stages of storage; but later Pseudomonas spp. reasserted and constituted the bulk of the spoilage flora. In prawn, the native flora was comprised of Pseudomonas spp., Acinetobacter spp., Moraxella spp. and Vibrio spp. At the time of spoilage a heterogeneous flora, consisting of Pseudomonas spp; Moraxella spp. and Acinetobacter spp. predominated. CTC treatment significantly changed the flora of prawns. During spoilage, Pseudomonas predominated in CTC treated prawns.

Bacterial flora of EDTA treated oil sardine (Sardinella longiceps), Indian mackerel (Rastrelliger kanagurta) and prawn (Metapenaeus dobsoni) in ice storage

The native flora of fresh oil sardine and mackerel consisted mainly of Pseudomonas spp., Moraxella spp., Acinetobacter spp. and Vibrio spp. During spoilage in ice, nearly 75% of their bacterial flora belonged to Pseudomonas spp. alone. But Na sub(2) EDTA treatment reduced the proportion of Pseudomonas spp. considerably and the major bacterial groups at the time of spoilage were Moraxella spp. and Acinetobacter spp. In the case of fresh prawn, the native flora was constituted by Pseudomonas spp., Moraxella spp., Acinetobacter spp. and Vibrio spp. At the time of spoilage of prawn in ice, Moraxella spp. and Acinetobacter spp. predominated, together constituting 74% of the total population. Na sub(2) EDTA treatment did not alter significantly the spoilage flora of prawns. Moraxella spp. and Acinetobacter spp. accounted for 86% of the spoilage flora in ice storage of Na sub(2) EDTA treated prawns.

The bacteriology of oil sardine (Sardinella longiceps) and mackerel (Rastrelliger kanagurta) from tropical waters off Cochin. 2. Qualitative aspects

80% of the flora of skin, gills and intestines of oil sardine and mackerel at isolation temperature 28 ± 2°C consisted of Gram negative asporogenous rods or cocci, belonging to the genera Vibrio, Pseudomonas, Moraxella, Acinetobacter and Flavobacteria/Cytophaga. Nearly 10% of the flora was constituted by Gram positives, Micrococcus and Arthrobacter. Incubation temperature of 36 ± 1°C recovered more Vibrio spp. and Gram positives, while at lower temperatures of 8 ± 1°C and 1 ± 1°C, more Pseudomonas, Acinetobacter and Moraxella spp. were recovered. Significant changes with respect to season were observed in the relative distribution of different genera.

Microbial Communities Associated with Healthy and White Syndrome-Affected Echinopora lamellosa in Aquaria and Experimental Treatment with the Antibiotic Ampicillin

Prokaryotic and ciliate communities of healthy and aquarium White Syndrome (WS)-affected coral fragments were screened using denaturing gradient gel electrophoresis (DGGE). A significant difference (R = 0.907, p < 0.001) in 16S rRNA prokaryotic diversity was found between healthy (H), sloughed tissue (ST), WS-affected (WSU) and antibiotic treated (WST) samples. Although 3 Vibrio spp were found inWS-affected samples, two of these species were eliminated following ampicillin treatment, yet lesions continued to advance, suggesting they play a minor or secondary role in the pathogenesis. The third Vibrio sp increased slightly in relative abundance in diseased samples and was abundant in non-diseased samples. Interestingly, a Tenacibaculum sp showed the greatest increase in relative abundance between healthy and WS-affected samples, demonstrating consistently high abundance across all WS-affected and treated samples, suggesting Tenacibaculum sp could be a more likely candidate for pathogenesis in this instance. In contrast to previous studies bacterial abundance did not vary significantly (ANOVA, F2, 6 = 1.000, p = 0.422) between H, ST, WSU or WST. Antimicrobial activity (assessed on Vibrio harveyi cultures) was limited in both H and WSU samples (8.1% ±8.2 and 8.0% ±2.5, respectively) and did not differ significantly (Kruskal-Wallis, χ2 (2) = 3.842, p = 0.146). A Philaster sp, a Cohnilembus sp and a Pseudokeronopsis sp. were present in all WS-affected samples, but not in healthy samples. The exact role of ciliates in WS is yet to be determined, but it is proposed that they are at least responsible for the neat lesion boundary observed in the disease.

Avaliação da contaminação microbiológica e da resposta ao stress ambiental em moluscos bivalves da Ria Formosa

Dissertação de mestrado, Tecnologia de Alimentos, Instituto Superior de Engenharia, Universidade do Algarve, 2015

Clams from Tagus estuary : microbiological, physiological and chemical responses to depuration, transpot and environmental stress

Tese de doutoramento, Biologia (Biologia Marinha e Aquacultuta), Universidade de Lisboa, Faculdade de Ciências, 2014

Studies on isolation and molecular characterization of plasmids from Vibrios

While the seriousness of the problem of antibiotic resistance is now recognized, the complex web of resistance linking humans, animals, and the environment is getting realized. More often, antibiotics are used as a preventive measure against diseases. Antibiotic use for agriculture leads to the increased resistance in the environment since antibiotics are inevitable element during agriculture/aquaculture and antibiotic residues are excreted as waste that is frequently spread onto farmland as organic fertilizer. Fecal bacteria survive long periods in the environment and spread through runoff into groundwater, rivers, and marine ecosystems.However, horizontal gene transfer occurs in the animals and guts of humans and in a variety of ecosystems, creating a pool of resistance in the rice fields and open waters. Even if people are not in direct contact with resistant disease through food animals, there are chances of contact with resistant fecal pathogens from the environment. Additionally, pathogens that are autochthonous to the environment can acquire resistance genes from the environment. Our study revealed that autochthonous , bacteria Vibrio spp gained antibiotic resistance in the environment. Further, it was evident that horizontal gene transfer occurs in Vibrio by means of plasmids, which further augments the gravity of the problem. Non-pathogenic bacteria may also acquire resistance genes and serve as a continuing source of resistance for other bacteria, both in the environment, and in the human gut. As the effectiveness of antibiotics for medical applications decline, the indiscriminate use of in aquaculture and in humans can have disastrous conditions in future due to horizontal gene transfer and the spread of resistant organisms: We must recognize and deal with the threat posed by overuse of antibiotics.

Biochemical and molecular characterization of pathogenic vibrios from hatcheries and aquaculture farms

Vibrio are important during hatchery rearing. aquaculture phase and post-harvest quality of shrimps. Vibrio spp are of concern to shrimp farmers and hatchery operators because certain species can cause Vibriosis. Vibrio species are of concern to humans because certain species cause serious diseases.With the progress in aquaculture, intensive systems used for shrimp aquaculture create an artificial environment that increases bacterial growth. To maintain the productivity of such an intensive aquaculture, high inputs of fish protein have to be employed for feeding together with high levels of water exchange and the massive use of antibiotics/ probiotics / chemicals. It seems that the combination of these conditions favours the proliferation of vibrios and enhances their virulence and disease prevalence. The risk of a microbial infection is high, mainly at larval stages. The effect and severity are related to Vibrio species and dose, water, feed, shrimp quality and aquaculture management.Consumption of seafood can occasionally result in food-bome illnesses due to the proliferation of indigenous pathogens like Vibrio.Of the l2 pathogenic Vibrio species, 8 species are known to be directly food associated. Strict quality guidelines have been laid by the importing nations, for the food products that enter their markets. The microbiological quality requirement for export of frozen shrimp products is that V.cholerae, V.parahaemolyticus and V. vulnificus should be absent in 25g of the processed shrimp (Export Inspection Council of India, 1995). The mere presence of these pathogenic Vibrios is sufficient for the rejection of the exported product.The export rejections cause serious economic loss to the shrimp industry and might harm the brand image of the shrimp products from the countiy.There is a need for an independent study on the incidence of different pathogenic vibrios in shrimp aquaculture and investigate their biochemical characteristics to have a better understanding about the growth and survival of these organisms in the shrimp aquaculture niche. PCR based methods (conventional PCR, duplex PCR, multiplex-PCR and Real Time PCR) for the detection of the pathogenic Vibrios is important for rapid post-harvest quality assessment. Studies on the genetic heterogeneity among the specific pathogenic vibrio species isolated from shrimp aquaculture system provide; valuable information on the extent of genetic diversity of the pathogenic vibrios, the shrimp aquaculture system.So the present study was undertaken to study the incidence of pathogenic Vibrio spp. in Penaeus monodon shrimp hatcheries and aquaculture farms, to carry out biochemical investigations of the pathogenic Vibrio spp isolated from P. monodon hatchery and. aquaculture environments, to assess the effect of salt (NaCl) on the growth and enzymatic activities of pathogenic Vibrio spp., to study the effect of preservatives, and chemicals on the growth of pathogenic Vibrio spp. and to employ polymerase chain reaction (PCR) methods for the detection of pathogenic V ibrio spp.Samples of water (n=7) and post-larvae (n=7) were obtained from seven Penaeus monodon hatcheries and samples of water (n=5), sediment (n=5) and shrimp (n=5) were obtained from five P. monodon aquaculture farms located on the East Coast of lndia. The microbiological examination of water, sediment, post-larvae and shrimp samples was carried out employing standard methods and by using standard media.The higher bacterial loads were obtained in pond sediments which can be attributed to the accumulation of organic matter at the pond bottom which stimulated bacterial growth.Shrimp head. (4.78 x 105 +/- 3.0 x 104 cfu/g) had relatively higher bacterial load when compared to shrimp muscle 2.7 x 105 +/- 1.95 x 104 cfu/g). ln shrimp hatchery samples, the post-larvae (2.2 x 106 +/- 1.9 x 106 cfu/g) had higher bacterial load than water (5.6 x 103 +/- 3890 cfu/ml).The mean E.coli counts were higher in aquaculture pond sediment (204+/-13 cfu/g) and pond water (124+/-88 cfu/ml). Relatively lower Escherichia coli counts were obtained from shrimp samples (12+/-11 to 16+/-16.7 cfu/g). The presence of E.coli in aquaculture environment might have been from the source water. E.coli was not detected in hatchery waters and post-larvae.

Pyocyanin (5-methyl-1-hydroxyphenazine) produced by Pseudomonas aeruginosa as antagonist to vibrios in aquaculture: overexpression, downstream process and toxicity

Pyocyanin is a versatile and multifunctional phenazine, widely used as a bio-control agent. Besides its toxicity in higher concentration, it has been applied as bio-control agents against many pathogens including the Vibrio spp. in aquaculture systems. The exact mechanism of the production of pyocyanin in Pseudomonas aeruginosa is well known, but the genetic modification of pyocyanin biosynthetic pathways in P. aeruginosa is not yet experimented to improve the yield of pyocyanin production. In this context, one of the aims of this work was to improve the yield of pyocyanin production in P. aeruginosa by way of increasing the copy number of pyocyanin pathway genes and their over expression. The specific aims of this work encompasses firstly, the identification of probiotic effect of P. aeruginosa isolated from various ecological niches, the overexpression of pyocyanin biosynthetic genes, development of an appropriate downstream process for large scale production of pyocyanin and its application in aquaculture industries. In addition, this work intends to examine the toxicity of pyocyanin on various developmental stages of tiger shrimp (Penaeus monodon), Artemia nauplii, microbial consortia of nitrifying bioreactors (Packed Bed Bioreactor, PBBR and Stringed Bed Suspended Bioreactor, SBSBR) and in vitro cell culture systems from invertebrates and vertebrates. The present study was undertaken with a vision to manage the pathogenic vibrios in aquaculture through eco-friendly and sustainable management strategies with the following objectives: Identification of Pseudomonas isolated from various ecological niches and its antagonism to pathogenic vibrios in aquaculture.,Saline dependent production of pyocyanin in Pseudomonas aeruginosa originated from different ecological niches and their selective application in aquaculture,Cloning and overexpression of Phz genes encoding phenazine biosynthetic pathway for the enhanced production of pyocyanin in Pseudomonas aeruginosa MCCB117,Development of an appropriate downstream process for large scale production of pyocyanin from PA-pUCP-Phz++; Structural elucidation and functional analysis of the purified compoundToxicity of pyocyanin on various biological systems.

Synechocystis MCCB 114 and 115 as putative probionts for Penaeus monodon post-larvae

Synechocystis MCCB 114 and 115 were segregated as putative probionts for shrimp larvae from a collection of 54 cyanobacterial cultures enriched from seawater. On feeding Penaeus monodon post-larvae with the cyanobacteria, the generic diversity of the intestinal bacterial flora could be enhanced with substantial reduction or total absence of Vibrio spp. A significant difference (p < 0.001) in the percent survival of batches of post-larvae fed on the cyanobacterial cultures was observed and, on repeated challenge with V. harveyi, the relative percent survival of those batches of larvae fed on Synechocystis MCCB 114 and 115 was significantly higher. The Synechocystis MCCB 114 and 115 cultures were found to contain high levels of protein (34 to 43%), in addition to carotenoids

OCCURRENCE OF PATHOGENIC MICROORGANISMS IN FISH SOLD IN SAO PAULO, BRAZIL

This study investigated the presence of potentially human pathogenic strains of Vibrio spp., Aeromonas spp., Escherichia coli, Salmonella spp. and Staphylococcus aureus in fish commercialized in street markets of Sao Paulo city, Brazil. Twenty fish of different species were analyzed for foodborne pathogens using conventional methods. High levels of fecal contamination were detected in 25% of samples. S. aureus was isolated from 10% of samples. All were negative for Salmonella. Vibrio species, including Vibrio cholerae non-O1/non-O139, were observed in 85% of samples although Vibrio parahaemolyticus was not found in this study. Aeromonas spp., including A. hydrophila, was isolated from 50% of fish samples. The occurrence of these pathogens suggests that the fish commercialized in Sao Paulo may represent a health risk to the consumers.

Dynamics of Aeromonas species isolated from wastewater treatment system

Aeromonas are widely distributed in the aquatic environment, and are considered to be emerging organisms that can produce a series of virulence factors. The present study was carried out in a sanitary sewage stabilization pond treatment system, located in Lins, State of Sao Paulo, Brazil. Most probable number was applied for estimation of the genus Aeromonas. Colony isolation was carried out on blood agar ampicillin and confirmed by biochemical characterization. Aeromonas species were isolated in 72.4% of influent samples, and in 55.2 and 48.3% of effluent from anaerobic and facultative lagoons, respectively. Thirteen Aeromonas species were isolated, representing most of the recognized species of these organisms. Even though it was possible to observe a tendency of decrease, total elimination of these organisms from the studied system was not achieved. Understanding of the pathogenic organism`s dynamics in wastewater treatment systems with a reuse potential is especially important because of the risk it represents.

Microbiological responses to depuration and transport of native and exotic clams at optimal and stressful temperatures

The microbiological responses of two bivalves species from Tagus estuary, Venerupis pullastra (native clam) and Ruditapes philippinarum (exotic clam) were investigated during 48h of depuration and subsequent simulated transport in semi-dry conditions at two temperatures (4 and 22°C) until reaching 50% lethal time (LT50). Regardless of temperature and species, the maintenance of clams in water for 48h (depuration period) did not affect LT50 during transport. R.philippinarum showed higher survival rates than V.pullastra, always reaching LT50 later, especially at 4°C. Significant differences between clams' species were found in almost all microbiological parameters. This can be related with clams' biological activity and habitat environmental conditions since both clams do not coexist in Tagus estuary. Depuration was efficient to reduce the bacterial load, particularly Escherichia coli, but not efficient to remove Vibrio spp. In both species, the growth of Vibrio spp. was inhibited at 4°C, whereas exponential growth occurred at 22°C. Total viable counts significantly increased in most treatments, while E.coli counts significantly decreased to undetected levels, except for non-depurated R.philippinarum simulated transported at 4°C. Thus, this study highlights the importance of clams depuration for at least 24h in polluted estuarine areas, followed by transport at low temperatures (4°C). © 2013 Elsevier Ltd.

Physicochemical parameters of water sample the four coastal lagoons at the Peninsula of Yucatan

Study sites. Samples of surface water were taken from 4 coastal lagoons on the Yucatan Peninsula in Mexico: Celestun (20° 45' N - 90° 22' W), Chelem (21° 15' N - 89° 45' W), Rosada Lagoon (21º 19' N - 89º 19' W), and Sabancuy Estuary (18° 58' N - 91° 12' W). The sampling was performed from august to October of 2011 (Chelem 08/24; Laguna Rosada 09/06; Celestún 09/28; Sabancuy 10/25). The sampling was random without replacement and 10 samples of surface water were collected along a transect parallel to the coastal axis. Samples were deposited in sterile plastic bottles and conserved in refrigeration at 4°C. All samples were processed within 24 hours after sampling. According to the Mexican laws and regulations no permissions are required to obtain water and sediment samples from open public areas. Analysis of environmental and physicochemical parameters. Determinations of the environmental parameters were performed with a Hach 5465000 model 156 multi-parameter measuring instrument. The Lorenzen method was used to determine chlorophyll-a (21) with 90% acetone and the concentration was calculated according to the formula: Chla= 27.63 (OD665o - OD665a)(VA)/VM x L Where, OD665o: absorbance at 665 nm before acidification; OD665a: absorbance at 665 nm after acidification; VA: volume (ml) of acetone for extraction; VM: volume (ml) of filtered water; L: length (cm) of the photometric cell. Determinations of the physicochemical parameters (silicates, phosphates, nitrates, nitrites and ammonia) were performed using the spectrophotometric techniques described and modified by Strickland and Parsons (1972).

Growth, survival, gene expression, microbiota and tryptic activity during feeding of Scophthalmus maximus first feeding larvae with beta-glucan (MacroGard)

Reflecting the natural biology of mass spawning fish aquaculture production of fish larvae is often hampered by high and unpredictable mortality rates. The present study aimed to enhance larval performance and immunity via the oral administration of an immunomodulator, beta-glucan (MacroGard®) in turbot (Scophthalmus maximus). Rotifers (Brachionus plicatilis) were incubated with or without yeast beta-1,3/1,6-glucan in form of MacroGard® at a concentration of 0.5 g/L. Rotifers were fed to first feeding turbot larvae once a day. From day 13 dph onwards all tanks were additionally fed untreated Artemia sp. nauplii (1 nauplius ml/L). Daily mortality was monitored and larvae were sampled at 11 and 24 dph for expression of 30 genes, trypsin activity and size measurements. Along with the feeding of beta-glucan daily mortality was significantly reduced by ca. 15% and an alteration of the larval microbiota was observed. At 11 dph gene expression of trypsin and chymotrypsin was elevated in the MacroGard® fed fish, which resulted in heightened tryptic enzyme activity. No effect on genes encoding antioxidative proteins was observed, whilst the immune response was clearly modulated by beta-glucan. At 11 dph complement component c3 was elevated whilst cytokines, antimicrobial peptides, toll like receptor 3 and heat shock protein 70 were not affected. At the later time point (24 dph) an anti-inflammatory effect in form of a down-regulation of hsp 70, tnf-alpha and il-1beta was observed. We conclude that the administration of beta-glucan induced an immunomodulatory response and could be used as an effective measure to increase survival in rearing of turbot.