D. Padilla

Characterization of the probiotic strain Vagococcus fluvialis in the protection of European sea bass (Dicentrarchus labrax) against vibriosis by Vibrio anguillarum.

Aquaculture is one of the main sources of income in many countries worldwide. Intensive farms are often affected by different infectious diseases that can decrease their final production. To control this situation, several antibiotics are frequently used with known environmental consequences. The aim of this study was to analyze different bacterial strains isolated from of gilthead sea bream, sea bass, sole and meagre guts, for use as probiotics in aquaculture. The strains were evaluated in vitro through various mechanisms of selection, such as the production of antagonistic effects against pathogens, production of antibacterial substance, adhesion to the intestinal mucus, competition for nutrients or binding site, and growth in intestinal mucus. A total of 50 bacterial strains were analyzed and only one showed excellent in vitro results for consideration as a candidate to be analyzed in vivo. The strain, identified as Vagococcus fluvialis, showed good protection against Vibrio anguillarum 975-1 in vivo in the experimental challenge, showing a relative percent survival of 42.3% higher than positive control group. Therefore, in conclusion we consider this strain to be a good candidate for use as a future probiotic in aquaculture.

The in vitro effect of probiotic Vagococcus fluvialis on the innate immune parameters of Sparus aurata and Dicentrarchus labrax.

In this study we evaluated the effect of the probiotic Vagococcus fluvialis on the cellular immune unspecific system of two different fish species of great interest in aquaculture such as gilthead sea bream (Sparus aurata) and European sea bass (Dicentrarchus labrax). Leucocytes from head kidney of the two fish species were extracted and concentration adjusted to 10(7) cells ml(-1). Phagocytic and respiratory burst activity and the peroxidase content of leucocytes were observed 30 min after incubation with the probiotic Vagococcus fluvialis alive or inactivated with heat shock or UV-light at different concentrations of 10(7), 10(8), 10(9) cfu ml(-1) (final concentration 10(6), 10(7), 10(8) cfu ml(-1)). V. fluvialis produced dose-dependent increments in respiratory burst in sea bream leucocytes. The respiratory burst activity of sea bream head kidney leucocytes incubated with 10(6) cfu ml(-1) of live and inactivated bacteria was not stimulated. The highest values of peroxidase content were observed in sea bass cells with stimulation indexes higher than 1 in HK leucocytes incubated with 10(8) cfu ml(-1) of live and inactivated bacteria. Statistical analysis revealed that differences being only significant in sea bass leucocytes where 10(8) cfu ml(-1) bacteria denote statistically significant differences (P < 0.05) respect to other concentrations. Highest values of phagocytic activity were obtained in sea bass macrophages incubated with UV-light inactivated bacteria (27.33% ± 1.45), where significantly differences with sea bass HK leucocytes were detected. Our results suggest that the in vitro assays are a useful tool to optimize the effective dose of probiotic bacteria. Although in vivo studies are necessary to confirm the immunomodulatory effect of this strain.

Invasion and survival of Photobacterium damselae subsp. piscicida in non-phagocytic cells of gilthead sea bream, Sparus aurata L.

Fluorescence microscopy and gentamicin protection assays were used to investigate the ability of four Photobacterium damselae subsp. pisicida (Phdp) strains to adhere to and to invade the fish epithelial cell line, SAF-1, derived from Sparus aurata. All strains tested were detected intracellularly using both techniques, although internalization levels varied among strains. Treatment with cytochalasin D and experiments carried out at 4 degrees C demonstrated that a functional host cell cytoskeleton and active cell metabolism are necessary for bacterial internalization. Intracellular bacteria were detected for up to 7 days with a round morphology and were stained with DAPI, indicating that some bacterial cells may remain viable inside SAF-1 cells. Our in vitro findings indicate that Phdp are capable of adhering, entering and surviving within the non-phagocytic epithelial cell line SAF-1, which may be important for persistence and establishment of a carrier state in S. aurata.

Invasion and intracellular survival of Hafnia alvei strains in human epithelial cells.

Aims:  The aim of this study was to investigate the invasion and intracellular survival of different Hafnia alvei strains in HeLa cells.

Biofilm Formation and Quorum-Sensing-Molecule Production by Clinical Isolates of Serratia liquefaciens.

ABSTRACT Serratia spp. are opportunistic human pathogens responsible for an increasing number of nosocomial infections. However, little is known about the virulence factors and regulatory circuits that may enhance the establishment and long-term survival of Serratia liquefaciens in the hospital environment. In this study, two reporter strains, Chromobacterium violaceum CV026 and VIR24, and high-resolution triple-quadrupole liquid chromatography–mass spectrometry (LC-MS) were used to detect and to quantify N-acyl-homoserine lactone (AHL) quorum-sensing signals in 20 S. liquefaciens strains isolated from clinical samples. Only four of the strains produced sufficient amounts of AHLs to activate the sensors. Investigation of two of the positive strains by high-performance liquid chromatography (HPLC)-MS confirmed the presence of significant amounts of short-acyl-chain AHLs (N-butyryl-l-homoserine lactone [C4-HSL] and N-hexanoyl-l-homoserine lactone [C6-HSL]) in both strains, which exhibited a complex and strain-specific signal profile that included minor amounts of other short-acyl-chain AHLs (N-octanoyl-l-homoserine lactone [C8-HSL] and N-3-oxohexanoyl-l-homoserine lactone [OC6-HSL]) and long-acyl-chain (C10, C12, and C14) AHLs. No correlation between biofilm formation and the production of large amounts of AHLs could be established. Fimbria-like structures were observed by transmission electron microscopy, and the presence of the type 1 fimbrial adhesin gene fimH in all strains was confirmed by PCR. The ability of S. liquefaciens to adhere to abiotic surfaces and to form biofilms likely contributes to its persistence in the hospital environment, increasing the probability of causing nosocomial infections. Therefore, a better understanding of the adherence properties of this species will provide greater insights into the diseases it causes.

Presence of C. albidus , C. laurentii and C. uniguttulatus in Crop and Droppings of Pigeon Lofts ( Columba livia )

Columba livia is an important reservoir and carrier of Cryptococcus neoformans, Cryptococcus uniguttulatus, Cryptococcus laurentii and Cryptococcus albidus. Upper digestive tract of this species is also known as a habitat for Cryptococcus neoformans. Given the increasing clinical interest of this microorganism, 331 swabs from crop and 174 dropping samples from pigeon lofts in Grand Canary Island have been studied. The obtained results show an extensive presence samples 81 positive (24.47%) of Cryptococcus spp. in analysed crops: 32 (9.66%) for C. neoformans, 24 (7.2%) for C. uniguttulatus, 23 (6.9%) for C. albidus and 2 (0.6%) for C. laurentii. In the same way, Cryptococcus spp was also isolated in 82 (47.13%), dropping samples: C. neoformans in 59 (33.9%), C. uniguttulatus, in 9 (5.17%), C. laurentii in 8 (4.59%) and C. albidus in 6 (3.44%) of the investigated samples, respectively. The cryptococcosis produced by species of cryptococci other than C. neoformans has become more important during the last decade, supporting the study on the role of pigeon in the epidemiology of this disease.

Mx expression in gilthead sea bream (Sparus aurata L.) in response to poly I:C, bacterial LPS and chromosomal DNA: preliminary study.

Cells infected with a virus are stimulated to produce and secrete IFNs, which in turn induce a complex pattern of physiological changes. The IFNs provide vertebrates with a first line of defence against viral infection. Fish are known to produce molecules with IFN activity as measured by a cell protection test [1]. The induction of IFN-I includes dsRNA virus infection, LPS and some bacterial components [2]. Mx proteins interfere with virus replication by inhibiting viral polymerases in the nucleus and binding viral components in the cytoplasm [3]. Mx genes have been cloned and characterised from several other mammals, birds and fish species [4], such as rainbow trout, Atlantic salmon, Atlantic halibut, Japanese flounder, pufferfish, sea bream [5] and Senegalese sole [6]. Production of IFN-like activity and Mx gene/protein expression in fish has been demonstrated in cells, organs and serum from several fish species [7]. Viral dsRNA and synthetic dsRNA polyinosinic: polycytidylic acid (poly I:C) are very potent inducers of IFN. Most viruses produce dsRNA at some time in their replication, and it seems that animals have evolved the ability to recognise and respond to these molecules by this innate mechanism [7]. IFN-like activity can also be stimulated by other factors such as bacterial lipopolysaccharide (LPS), bacterial DNA and inactivated vaccines against bacteria [8].

The in vitro immunomodulatory effect of extracellular products (ECPs) of Vagococcus fluvialis L21 on European sea bass (Dicentrarchus labrax) leucocytes.

The immune associated genes, interleukin-1β (IL-1β), interleukin-6 (IL-6), interleukin 10 (IL-10), tumor necrosis factor-α (TNF-α), ciclo-oxigenase-2 (COX-2), and Mx gene were studied by real-time PCR in head-kidney leucocytes of sea bass after incubation with the extracellular products (ECPs) of the probiotic strain Vagococcus fluvialis L21 and polyinosinic:polycytidylic acid (POLY I:C), at different times (T1.5, T6, T12, T24, T48 and T72). In general, we can observe how pro-inflammatory cytokines IL-1β, TNF-α, IL-6 and COX-2 studied displayed a strong peak after stimulation with 1.5 h of ECPs of V. fluvialis L21, significant differences (P < 0.05) exist with other periods and with the POLY I: C at the same time. Similarly to the case of IL-10 also produced a statistically significant (P < 0.05) peak of expression on leukocytes that were stimulated with the ECPs of V. fluvialis L21. In the case of Mx gene expression, we note that in almost all sampling times there is an up-regulation of the Mx gene in leucocytes incubated with ECPs and POLY I:C compared to the control and Mx expression was higher in leucocytes that were stimulated with the ECPs of V. fluvialis for all times, except in T24. With these results we can consider that the ECPs of V. fluvialis L21 have a great power of stimulating the in vitro expression of immune-related genes and may even be useful as adjuvants for vaccine in aquaculture.

Rhodococcus equi human clinical isolates enter and survive within human alveolar epithelial cells.

Rhodococcus equi is an emerging opportunistic human pathogen associated with immunosuppressed people, especially those infected with the human immunodeficiency virus (HIV). This pathogen resides primarily within lung macrophages of infected patients, which may explain in part its ability to escape normal pulmonary defense mechanisms. Despite numerous studies as a pulmonary pathogen in foals, where a plasmid seems to play an important role in virulence, information on the pathogenesis of this pathogen in humans is still scarce. In this study, fluorescence microscopy and vancomycin protection assays were used to investigate the ability of R. equi human isolates to adhere to and to invade the human alveolar epithelial cell line A549. Our findings indicate that some R. equi clinical strains are capable of adhering, entering and surviving within the alveolar cell line, which may contribute to the pathogen persistence in lung tissues.

Temperature influences the expression of fimbriae and flagella in Hafnia alvei strains: an immunofluorescence study

Hafnia alvei, a Gram negative bacillus related to the Enterobacteriaceae family, is considered an opportunistic pathogen of several animal species and humans. In this communication, we describe fimbrial-like structures from different strains of H. alvei that cannot be easily ascribed to any of the previously reported fimbrial types in this species (type I or type III). Polymerase chain reaction (PCR) and immunofluorescence assays were carried out to study fimbriae and flagella in H. alvei strains isolated from different sources. No correlation between the results obtained by PCR and those obtained by phenotypic methods were found, and the antibodies used gave cross or different recognition patterns of the surface structures present in these strains. We report as well that strain and growth temperature influence fimbriation and expression of flagella in human and animal isolates of H. alvei. This study also indicates that the absence of fimbriae have a significant positive influence on the initial adhesion of H. alvei to human epithelial cells.