Culturable autochthonous gut bacteria in Atlantic salmon (Salmo salar L.) fed diets with or without chitin. Characterization by 16S rRNA gene sequencing, ability to produce enzymes and in vitro growth inhibition of four fish pathogens

The present investigation evaluated the effect of chitin (5% supplementation) on the adherent aerobic intestinal microbiota of Atlantic salmon (Salmo salar L.). One hundred and seventy three isolates were isolated but 34 isolates died prior to positive identification. Sixty four out of 139 autochthonous gut bacteria were further identified by 16S rRNA gene sequencing and further tested for protease, amylase, cellulase, phytase, lipase and chitinase activities. Moreover, the most promising enzyme-producing bacteria and intestinal lactic acid bacteria (LAB) were tested for in vitro growth inhibition of four important fish pathogens: Aeromonas salmonicida subsp. salmonicida, Vibrio (Listonella) anguillarum, Moritella viscosa and Carnobacterium maltaromaticum. Dietary chitin modulates the gut microbiota but not the portion of enzyme-producing gut bacteria. LAB were only isolated from fish fed the chitin supplemented diet and they were able to inhibit in vitro growth of 3 of the 4 pathogens. However, the most promising gut bacteria isolated in the present study with respect to enzyme production and in vitro growth inhibition showed high similarity to Bacillus thuringiensis by 16SrRNA gene sequencing

The effect of different feeding regimes on enzyme activities of gut microbiota in Atlantic cod (Gadus morhua L.)

The aims of this study were: (1) evaluate enzyme-producing bacteria isolated from the GI tract of Atlantic cod, (2) identify the most promising enzyme-producing bacteria by 16S rRNA gene sequencing and (3) to assess whether these bacteria have the ability to inhibit in vitro growth of four well known pathogenic bacteria; Aeromonas salmonicida subsp. salmonicida, V. (L.) anguillarum, Moritella viscosa and Carnobacterium maltaromaticum. In this study, 79 gut bacteria previously isolated from the GI tract of Atlantic cod fed fish meal (FM), soybean meal (SBM) and bioprocessed soybean meal (BPSBM) from the study of Ringø et al. (2006a), were randomly selected for further investigation. These bacteria had not previously been tested for enzyme-production, identified by 16S rRNA gene sequencing or tested for antagonistic activity. Determination of qualitative enzyme activities; protease, amylase, cellulase, phytase, lipase and chitinase were carried out as described by Ray, Roy, Mondal and Ringø (2010) and Askarian, Zhou, Olsen, Sperstad and Ringø (2011). These endogenous bacterial enzymes were selected as they might contribute to fish nutrition (Ray et al. 2011). Forty eight of the most promising enzyme-producing bacteria, 15, 16 and 17 isolated from the GI tract of Atlantic cod fed FM, SBM and BPSBM, respectively were further identified by 16S rRNA gene sequencing as described by Ringø, Sperstad, Myklebust, Mayhew and Olsen (2006b). All sequences were analysed and edited in BIOEDIT and blasted against the sequences available in GenBank. Gut bacteria showing low similarities (<94%) with known sequences in GenBank were treated as unknown

Effects of dietary soybean meal, inulin and oxytetracycline on gastrointestinal histological characteristics, distal intestine cell proliferation and intestinal microbiota in Atlantic salmon (Salmo salar L.)

Soyabean meal (SBM)-induced enteritis in the distal intestine of the teleost Atlantic salmon (Salmo salar L.) and other salmonids may be considered a model for diet-related mucosal disorders in other animals and man. The role of the intestinal microbiota in its pathogenesis was explored. Compared to diets containing fishmeal (FM) as the sole protein source, responses to extracted SBM or the prebiotic inulin, with or without oxytetracycline (OTC) inclusion, were studied following a 3-week feeding trial. Intestinal microbiota, organosomatic indices and histology, as well as immunohistochemical detection of proliferating cell nuclear antigen (PCNA), heat shock protein 70 (HSP70) and caspase-3-positive cells in the distal intestine, were studied. Distal intestine somatic indices (DISI) were higher in inulin and lower in SBM compared to FM-fed fish. The low DISI caused by SBM corresponded with histological changes, neither of which was affected by OTC, despite a significant decrease in adherent bacteria count. Image analysis of PCNA-stained sections showed a significant increase in the proliferative compartment length in SBM-fed fish, accompanied by apparent increases in reactivity to HSP70 and caspase-3 along the mucosal folds, indicating induction of cellular repair and apoptosis, respectively. Fish fed the SBM diet had higher total number as well as a more diverse population composition of adherent bacteria in the distal intestine. Thus SBM-induced enteritis is accompanied by induction of distal intestinal epithelial cell protective responses and changes in microbiota. Putative involvement of bacteria in the inflammatory response merits further investigation

Bacterial diversity of aMasi, a South African fermented milk product, determined by clone library and denaturing gradient gel electrophoresis analysis

In the present study, we investigated the bacterial diversity of aMasi, a traditional South African fermented milk product, by 16S rRNA clone library and Denaturing Gradient Gel Electrophoresis (DGGE) analysis. Two hundred and eighty two clones from clone library were isolated and identified from aMasi, prepared from the milk of four cows from one herd in the EkuPindiseni Community, North West of Hluhluwe-iMfolozi Park in KwaZulu-Natal Province. The majority of the identified sequences corresponded to lactic acid bacteria (LAB), with the genus Lactococcus as major representative. The species Lactococcus lactis accounted for 179 of the identified clones. In addition, several species of Lactobacillus, Leuconostoc and Enterococcus were detected. Furthermore, several clones belonging to Acinetobacter, Aeromonas and genera within the Enterobacteriaceae were detected. It is important to note that human pathogens such as Klebsiella pneumoniae were identified in aMasi in the present study. Conversely, zoonotic bacteria such as Brucella abortus and Mycobacterium bovis were not detected in aMasi, although, they are present in the cattle population in the study area. Thirty (30) clones were identified as uncultured bacterial clones. Nine DGGE bands were successfully sequenced, of which four were classified as L. lactis with other bands belonging to lactobacilli, Clostridium acidurici, Enterobacter sp., Acinetobacter baumannii and an un-culturable bacterium. Even though there was some discrepancy between the two culture independent methods used to study the bacteriological community in aMasi, a general conclusion can be drawn, L. lactis may be considered as the dominant bacterium within a diverse bacterial community in this locally-produced dairy product

Paralithocins, Antimicrobial Peptides with Unusual Disulfide Connectivity from the Red King Crab, <i>Paralithodes camtschaticus</i>

As part of an ongoing exploration of marine invertebrates as a source of new antimicrobial peptides, hemocyte extracts from the red king crab, <i>Paralithodes camtschaticus</i>, were studied. Three cationic cysteine (Cys)-rich peptides, named paralithocins 1–3, were isolated by bioassay-guided purification, and their amino acid sequences determined by Edman degradation and expressed sequences tag analysis. Disulfide bond mapping was performed by high-resolution tandem mass spectrometry. The peptides (38–51 amino acids in length) share a unique Cys motif composed of eight Cys, forming four disulfide bridges with a bond connectivity of (Cys relative position) Cys1–Cys8, Cys2–Cys6, Cys3–Cys5, and Cys4–Cys7, a disulfide arrangement that has not been previously reported among antimicrobial peptides. Thus, paralithocins 1–3 may be assigned to a previously unknown family of antimicrobial peptides within the group of Cys-rich antimicrobial peptides. Although none of the isolated peptides displayed antimicrobial activity against the target strains <i>Escherichia coli</i>, <i>Pseudomonas aeruginosa</i>, or <i>Staphylococcus aureus</i>, they inhibited the growth of several marine bacterial strains with minimal inhibitory concentrations in the 12.5–100 μM range. These findings corroborate the hypothesis that marine organisms are a valuable source for discovering bioactive peptides with new structural motifs