Effect of biofilm formation on antimicrobial tolerance of Flavobacterium psychrophilum

Treatment of bacterial fish diseases can be complicated by resistant bacterial biofilms harbouring pathogenic bacteria and causing recurrent exposure of fish to infections. In this study, the effect of biofilm formation on antimicrobial tolerance was examined using three bacterial isolates of the fish pathogen Flavobacterium psychrophilum and two antimicrobial agents, oxytetracycline and flumequine, commonly used in aquaculture. Planktonic and biofilm cells were exposed to a minimum inhibitory concentration (MIC), to a 3 × MIC concentration and to an environmental concentration level of each antimicrobial in 96-well microtitre plates after which growth on agar plates was measured. The type strain NCIMB1947 of F. psychrophilum was further used to study the development of antimicrobial resistance in biofilm cells. The results suggest that at high bacterial densities (>10(7) CFU mL(-1)), biofilm cells of F. psychrophilum are less susceptible to antimicrobial agents. Furthermore, the results imply that biofilm cells of F. psychrophilum may rapidly develop resistance to both oxytetracycline and flumequine if exposed to subinhibitory concentrations of these antimicrobials.     

Comparison of 5′-inosine monophosphate and p-nitrophenyl phosphate degrading activities among red,pink, and white muscle fibers of cultured carp

ABSTRACT:   As part of a study to clarify the differences in the temporal change in K -value among fish species, the temporal change in K -value and the 5'-inosine monophosphate (5'-IMP) and p-nitrophenol phosphate (p-NPP) degrading activities in the red, pink, and white muscle fibers in the dorsal muscle of the carp were compared. The temporal change in K -value was fastest in red, followed by pink, and white muscle fibers, at both 0°C and 32°C. Moreover, the 5'-IMP and p-NPP degrading activities were highest in red, followed by pink, and white muscle fibers at near optimum pH concentrations. The 5'-IMP degrading activity at pH 7.0 had a positive correlation with the increasing rate of K -value at 32°C for all types of muscle fibers. These results suggest that differences in increasing rates of K -values between red, pink, and white muscle fibers corresponded to the 5'-IMP degrading activities.     

Purification of chinook salmon (Oncorhynchus tshawytscha) GH for receptor study

A method for the purification of chinook Salmon (Oncorhynchus tshawytscha) GH, which retains its biological activity, is described. The biological activity was investigated with an established radioreceptor assay using liver membranes from pregnant rabbits and bovine GH as standard and labelled hormone. The enrichment of the preparation was checked with electrophoresis (SDS-PAGE). Extraction and further steps were carried out using low molarity alkaline buffer (pH 8–10, M = 100 mM). Three chromatography steps were performed (Concanavalin-A sepharose, Bio-gel P60, DEAE). Ion exchange chromatography was performed under isocratic conditions (using a 50 cm column). Two isoforms (sGH1 and sGH2) were isolated. The purification yield is 0.7% compared to lyophilized pituitaries. The molecule is homogeneous in SDS-PAGE. Contamination by prolactin, gonadotrophin and corticotrophin is negligible (< 0.5%). It could be demonstrated that the biological activity of the preparation is maintained since this preparation stimulates the growth of juvenile trout (Salmo gairdneri) and binds specifically (35%) to trout liver membranes.     

Water quality affects SDH activity, protein content and RNA:DNA ratios in fish (Catla catla, Labeo rohita and Oreochromis mossambicus) raised in ponds of a sewage-fed fish farm

The spatial and seasonal variability of the respiratory enzyme succinate dehydrogenase and the protein content were examined in different tissues of fish cultured in three ponds along the effluent gradient of a sewage-fed fish farm. Indian major carp Catla catla (150-230 g) and Labeo rohita (60-190 g) cultured in both the middle and last points of the sewage effluent (stocking pond 1) and (stocking pond 4) and Oreochromis mossambicus (50-160 g), a naturally growing fish of the inlet (facultative pond) and the out let of the sewage effluent (stocking pond 4) were procured every month during the period of January-December, 2005 and were subjected to determination of succinate dehydrogenase activity, total protein, DNA and RNA contents from gill, liver and muscle tissue respectively. The SDH activity of all three test fishes (Catla catla, Labeo rohita and Oreochromis mossambicus) was reduced significantly (ANOVA; P < 0.05) when cultured in SP-4 compared to SP-1 in the case of Catla catla and Labeo rohita and in facultative pond in the case of Oreochromis mossambicus.Conspicuous differences in the SDH activity of fish between the last and first stocking pond or the facultative pond were clearly due to the result of the differences in water quality. There was a direct relationship between SDH activity in gill tissue of any of the fish investigated and ammonia-N concentration of water or water pH. This shows that the respiratory activity of these fishes was strongly affected by the ammonia and pH of water. In other words, this suggests that as the distance from the point source increases, there was a substantial improvement of water quality in the ponds located along the sewage effluent gradient. Evidently, there is a progressive pattern of growth, survival and physiological health of fish and abundance of favorable diversity of food organisms with rich biodiversity.     

Effect of dietary vitamin C supplementation on the oxygen consumption, ammonia-N excretion and Na/K ATPase of Macrobrachium nipponense exposed to ambient ammonia

The 96-h LC₅₀ of ammonia-N and the effects of dietary vitamin C on oxygen consumption, ammonia-N excretion and Na⁺/K⁺ ATPase activity of Macrobrachium nipponense exposed to ambient ammonia were investigated. The results showed that the 96-h LC₅₀ of ammonia-N was 36.6 mg l⁻¹ for the freshwater prawn, M. nipponense, at pH 8.0. When prawns were exposed to high ambient ammonia-N concentrations, the oxygen consumption rate increased and ammonia excretion decreased. Dietary vitamin C supplementation led to higher oxygen consumption and lower ammonia excretion. Na⁺/K⁺ ATPase activity increased with increased ambient ammonia-N exposure in the range of 0–18.3 mg l⁻¹, and then was reduced at ambient ammonia-N 36.6 mg l⁻¹. Na⁺/K⁺ ATPase activities of prawns fed a vitamin C-supplemented diet were significantly lower than those of prawns fed a diet which was not supplemented with vitamin C.     

栉孔扇贝血淋巴中酯酶活性及其性质

采用紫外分光光度法测定栉孔扇贝（Chlamysfarreri）血淋巴中酯酶的活性，并研究底物、抑制剂、激活剂、温度、pH对酯酶活力的影响及酶的热稳定性。结果表明，栉孔扇贝血清与血细胞中均存在酯酶，且血细胞中的酶活力高于血清。血清与血细胞中酯酶的最适作用底物有所不同，血清中为α-醋酸萘酯，而血细胞中为α-丁酸萘酯。当以α-醋酸萘酯为底物时，血清中酯酶的最适温度为45℃，最适pH值为7．5；以α-丁酸萘酯为底物时，血清中酯酶的最适温度为40℃，最适pH值为8．0。以α-醋酸萘酯及α-丁酸萘酯为底物分别测定酶的热稳定时，酶活力变化相似，在20℃时活力最高，而后缓慢下降。栉孔扇贝血淋巴中酯酶属中温型中性酶类，低温下较稳定，底物专一性不强，血清中的酯酶可能来自血细胞，该酶在贝类的免疫防御中町发挥重要的作用。     

Intestinal phytase I: Detection and preliminary characterization of activity in the intestinal brush border membrane of hybrid striped bass Morone saxatilis × M. chrysops

While numerous studies have evaluated intestinal phytase activity in terrestrial monogastric species, little work has been conducted investigating the enzyme's activity in finfish. This experiment was conducted to determine if hybrid striped bass Morone saxatilis × M. chrysops possess intestinal phytase activity, and to perform a preliminary characterization of this activity in terms of pH, metal ion dependence and substrate kinetics. Hybrid bass do exhibit low levels of intestinal phytase activity. The activity has an acidic pH optimum between 3.5 and 4.5. Low concentrations of Mn²⁺ slightly enhance this activity. The divalent cations Mg²⁺ and Zn²⁺ were shown to either have no effect on the enzyme activity or to be inhibitory, particularly at high concentrations. The present results indicate that intestinal phytase activity in hybrid bass does follow Michaelis-Menten kinetics, with estimated K_m and V_max values of 2.5 mM phytate and 4.8 units mg⁻¹ protein, respectively. The physiologic significance of the enzyme's presence in hybrid striped bass is questionable due to the low activity as compared to terrestrial monogastrics, the acidic pH optimum and the low affinity the enzyme appears to have for phytate. These observations also raise the question of whether the intestinal phytase activity detected in hybrid bass is merely a manifestation of non-specific acid phosphatase activity. Further investigation is needed to determine the practical significance of phytase activity in hybrid bass in terms of phytate phosphorus utilization. Additional experiments should also be conducted which evaluate intestinal phytase activity in other important aquacultured finfish species. This revised version was published online in July 2006 with corrections to the Cover Date.     

Effect of shrimp head protein hydrolysates on the state of water and denaturation of fish myofibrils during dehydration

ABSTRACT:   To utilize fisheries waste products as food materials with functional properties, shrimp head protein hydrolysates (SHPH) from three species of shrimp, that is, Northern pink shrimp ( Pandalus eous ), Endeavour shrimp ( Metapenaeus endeavouri ) and Black tiger shrimp ( Penaeus monodon ), were produced by enzymatic hydrolysis using endopeptidase derived from Bacillus subtilis and exopeptidase derived from Aspergillus oryzae at a level of 0.1% (w/w). SHPH were rich in protein (90–91%) and amino acids (71–84%) but little fat (0.01–0.02%). The average molecular weight of SHPH was 300–1400. The effect of 5% SHPH (dry basis) addition on the state of water and denaturation of lizard fish myofibrils (Mf) during the dehydration process was evaluated by the desorption isotherm and the Ca-ATPase activity, and compared with the effect of sodium glutamate (Na-Glu). SHPH decreased the water activity and the Ca-ATPase inactivation, and increased monolayer sorbed water and multilayer sorbed water of Mf, although these effects of SHPH were smaller than those of Na-Glu. These findings suggest that the SHPH suppressed dehydration-induced denaturation of myofibrillar protein by stabilizing the hydrated water surrounding myofibrils.     

GST-MtDef4融合蛋白的原核表达及体外抑菌活性的检测

为研究苜蓿(Medicago truncatula)防卫素基因MtDef4的抑菌功能,以含有该基因片段的质粒pAND-MtDef4为模板,扩增出相应的目的基因,测序正确后将MtDef4基因片段连接到带有GST标签的原核表达载体pGEX6P-1中,转化大肠杆菌TransB(DE3),经IPTG诱导,Glutathione Sepharose 4B亲和纯化,通过SDS-PAGE电泳及Western blot确定融合蛋白的表达。体外抑菌试验表明,MtDef4能够抑制病原真菌Corynespora cassiicola而非Collectotrichum gloeosporioides的生长。本研究结果可为进一步培育转基因橡胶抗病品种奠定基础。     

Marine Pseudovibrio sp. as a Novel Source of Antimicrobials

Antibiotic resistance among pathogenic microorganisms is becoming ever more common. Unfortunately, the development of new antibiotics which may combat resistance has decreased. Recently, however the oceans and the marine animals that reside there have received increased attention as a potential source for natural product discovery. Many marine eukaryotes interact and form close associations with microorganisms that inhabit their surfaces, many of which can inhibit the attachment, growth or survival of competitor species. It is the bioactive compounds responsible for the inhibition that is of interest to researchers on the hunt for novel bioactives. The genus Pseudovibrio has been repeatedly identified from the bacterial communities isolated from marine surfaces. In addition, antimicrobial activity assays have demonstrated significant antimicrobial producing capabilities throughout the genus. This review will describe the potency, spectrum and possible novelty of the compounds produced by these bacteria, while highlighting the capacity for this genus to produce natural antimicrobial compounds which could be employed to control undesirable bacteria in the healthcare and food production sectors.