Miguel A. Moriñigo

Coliphages as an indicator of faecal pollution in water. Its relationship with indicator and pathogenic microorganisms

Abstract The study was designed to test the proposal that Escherichia coli specific bacteriophages might serve as universal faecal pollution indicators in water. A highly specific, sensitive and rapid technique for the detection and quantification of these virus particles was developed. The numerical relationship between E. coli and its parasitic phages was investigated in three different aqueous ecosystems such as sea water in the vicinity of sewage outfalls, river water contaminated by domestic and industrial sewage discharges, and estuarine waters, and found to be very close. In addition, the results obtained indicate that the coliphages are good indicators of the presence of the pathogenic microorganisms studied. In nearly all the water samples tested, the results suggest that coliphages are better indicators of faecal pollution than the classical indicator systems currently employed.

Changes in intestinal morphology and microbiota caused by dietary administration of inulin and Bacillus subtilis in gilthead sea bream (Sparus aurata L.) specimens

Changes produced in gilthead sea bream (Sparus aurata L.) intestinal morphology and microbiota caused by dietary administration of inulin and Bacillus subtilis have been studied. Gilthead sea bream specimens were fed diets containing 0 (control), inulin (10 g kg(-1)), B. subtilis (10(7) cfu g(-1)), or B. subtilis + inulin (10(7) cfu g(-1) + 10 g kg(-1)) for four weeks. Curiously, fish fed the experimental diets (inulin, B. subtilis, or B. subtilis + inulin) showed the same morphological alterations when studied by light and electron microscopy, while significant differences in the signs of intestinal damage were detected by the morphometric study. All of the observed alterations were present only in the gut mucosa, and intestinal morphometric study revealed no effect of inulin or B. subtilis on the intestinal absorptive area. Furthermore, experimental diets cause important alterations in the intestinal microbiota by significantly decreasing bacterial diversity, as demonstrated by the specific richness, Shannon, and range-weighted richness indices. The observed alterations demonstrate that fish fed experimental diets had different signs of gut oedema and inflammation that could compromise their body homeostasis, which is mainly maintained by the epithelial lining of the gastrointestinal tract. To our knowledge, this is the first in vivo study regarding the implications of the use of synbiotics (conjunction of probiotics and prebiotics) on fish gut morphology and microbiota.

Survey of bacterial pathologies affecting farmed gilt-head sea bream (Sparus aurata L.) in southwestern Spain from 1990 to 1996

Abstract A bacteriological survey in three fish farms with intensive culture of gilt-head sea bream (Sparus aurata L.) was conducted in southwestern Spain (provinces of Cadiz, Malaga, and Huelva) from 1990 to 1996. All the disease problems occurring during this period in the S. aurata were investigated, focusing on the isolation, characterization, antimicrobial resistance and pathogenicity of the bacterial isolates affecting the fish. The main pathogenic microorganisms isolated were Vibrio (67.8%), Pseudomonas (13.5%), Photobacterium damsela subsp. piscicida (6.7%), Cytophaga/Flexibacter-like bacteria (4.8%), Aeromonas (0.5%), and Gram positive bacteria (6.7%). Although the highest percentages of isolates corresponded to Vibrio and Pseudomonas spp., the strains of P. damsela subsp. piscicida and Cytophaga/Flexibacter-like bacteria caused epizootics with highest degree of mortalities. From the present study, on the basis of the pathogenicity test, we suggest the consideration of V. anguillarum, V. alginolyticus, V. harveyi and V. splendidus as primary pathogenic species for gilt-head sea bream.

Evaluation of different bacteriophage groups as faecal indicators in contaminated natural waters in southern England

Abstract River and seawater affected by faecal discharges were analysed to evaluate the reliability of coliphages and F-specific RNA bacteriophages as indicators of the microbiological quality of waters. F-specific RNA bacteriophages showed no direct relationship with the levels of faecal pollution, and this group was never detected in samples with a low level of enteroviruses (1–10 pfu/10 l.). In contrast, coliphages were constantly detected in the same samples. The concentration of coliphages detected in the samples with 1–10 and > 10 pfu/101. of enteroviruses were similar and higher than the Economical European Community guide and imperative levels of faecal coliforms (100 and 2000 FC/100 ml, respectively). Therefore, coliphages would be considered as an optimal indicator of the microbiological quality of the natural waters.

Intestinal Microbiota Diversity of the Flat Fish Solea senegalensis (Kaup, 1858) Following Probiotic Administration

Pleuronectiforms are an important group of fish, and one of their species, Solea senegalensis (Kaup 1858), has been extensively studied at different levels, although information about its intestinal microbiota and the effects of different factors on it is very scarce. Modern aquaculture industry demands strategies which help to maintain a microbiologically healthy environment and an environmentally friendly aquaculture. In this context, probiotics seem to offer an attractive alternative. The intake of probiotics could modify the composition of the intestinal microbiota, which is a key component in excluding potential invaders and maintaining health. The aim of this study was to evaluate by 16S rRNA gene analysis using polymerase chain reaction-denaturing gradient gel electrophoresis the effect of administering fresh or lyophilized cells of Pdp11 on the intestinal microbiota of farmed Senegalese sole, using sodium alginate to facilitate the incorporation of bacterial cells to the feed. The results obtained showed that the composition of fish intestinal microbiota was affected when fish received a diet supplemented with sodium alginate and fresh or lyophilized probiotic cells. In all cases, the dominant bacterial groups belonged to γ-Proteobacteria and mainly the Vibrio species. The use of sodium alginate reduced the incidence of populations with <97% 16S rRNA gene sequence identity to uncultured microorganisms in the intestinal microbiota until non-detected limits. On the other hand, the supplementation of the diet with probiotics produced an increase of the predominant species related to Vibrio genus.

Coliphages as an indicator of faecal pollution in water : their survival and productive infectivity in natural aquatic environments

The capability of coliphages as indicators of faecal pollution was tested, on the basis of their survival and infectivity in two natural aquatic environments (marine and river). The results obtained indicate that coliphages show a similar inactivation rate to Salmonella in all types of water studied. On the other hand, the phage productive infection of Escherichia coli cells in water and environmental conditions depends on the physiological characteristics of the host bacteria, which generally cannot grow optimally in these conditions. In conclusion, the coliphages may be considered good indicators of faecal pollution in natural waters.

Bacteria recovered from diseased cultured gilthead sea bream (Sparus aurata L.) in southwestern Spain

A bacteriological study of 25 outbreaks affecting cultured gilthead sea bream (Sparus aurata L.) in southwestern Spain from 1997 to 2000 has been carried out. Each year, the highest number of outbreaks occurred during winter and affected fish with sizes ranging between 2 and 25 g. The most frequently isolated microorganisms were identified as Vibrio (69.90%). Other microorganisms belonging to Pseudomonas spp., Photobacterium damselae ssp. piscicida, Cytophaga/Flexibacterlike and Aeromonas spp. were isolated with a frequency lower than 10%. The outbreaks with the highest mortalities of cultured gilthead sea bream were due to P. damselae ssp. piscicida. An increase in the number of resistances to different antimicrobials has been observed in this study with respect to previous studies carried out in the same area. This increase was very high in the case of some species of Vibrio, Pseudomonas spp. and P. damselae ssp. piscicida. D 2003 Elsevier Science B.V. All rights reserved.

Relationships between salmonella spp and indicator microorganisms in polluted natural waters

Abstract The relationships between Salmonella spp and indicator microorganisms (total coliforms, faecal coliforms, faecal streptococci, Clostridium perfringens and coliphages) were investigated in three different aquatic environments affected by wastewater discharges. The results indicated that the statistical correlations obtained depended, basically, on the source of faecal discharges. The microorganisms most closely correlated with Salmonella spp were faecal coliforms and C. perfringens , the latter also yielding the highest linear regression slope values. The detection percentages of Salmonella spp were high even at a low degree of pollution, which allowed detection of the pathogens in the absence of classical indicator microorganisms in the sample.

Influence of salinity and pH on the adhesion of pathogenic Vibrio strains to Sparus aurata skin mucus

Abstract It is generally known that adhesion of bacteria to mucosal surfaces is an initial step for many microbial infections. Nevertheless, the role played by the mucus layer of fish, especially that of Sparus aurata, during colonization of fish skin by saprophytic and pathogenic bacteria has not been established yet. In this work, the influence of salinity and pH on the adhesion abilities of Vibrio strains isolated from diseased fish to S. aurata skin mucus has been studied. Forty percent of the 30 strains tested attached to mucus-coated glass slides. Attachment was directly related to the ability of the strains to form pellicle in broth, but not to the degree of hydrophobicity of the strains. The highest adhesion rate was achieved with seawater (3.5% salinity) and glass slides coated with mucus solutions with a pH range of 8.2–8.5. The results obtained indicate that seawater and mucus at the normal pH of seawater (pH 8.1) were the most appropriate conditions for the attachment of pathogenic Vibrio strains to S. aurata skin mucus.

Microbial colonization of different support materials used to enhance the methanogenic process.

Macrobial colonization of the different support materials used to enhance methane production in anaerobic digestors is rapid and occurs in the first 24 h of sludge incubation. Scanning electron microscopy studies reveal a predominant presence of filamentous methanogenic forms, closely resemblingMethanosaeta (Methanothrix), which are located on the outer layer and in the bacterial framework of the biofilm. These findings are consistent with the results obtained from microbial counts using both the most probable number and epifluorescence microscopic techniques, which show an increase in the numbers of aceticlastic methanogens compared to other microbial groups involved, such as sulphate-reducing bacteria, the numbers of which are similar to those obtained under the initial conditions. Moreover, a sharp increase in the bacterial counts is observed by using the epifluorescence microscopic technique applied to homogenized samples, probably due to the count of bacteria released from the support materials.