Mitochondrial activity as an indicator of fish freshness.

The current methods used to routinely assess freshness in the fishing industry reflect more a state of spoilage than a state of freshness. Mitochondria, the seat of cellular respiration, undergo profound changes in post mortem tissues. The objective of this study was to demonstrate that mitochondrial activity constitutes a putative early fish freshness marker. The structure of gilthead sea bream (Sparus aurata) muscle tissue was evaluated over time by transmission electron microscopy. Respiration was assessed in mitochondria isolated from sea bream fillets using oxygraphy. Membrane potential (ΔΨm) was determined by fluorescence (Rhodamine 123). Mitochondrial activity of fillets stored at +4 °C was studied for 6 days. Changes in mitochondrial cristae structure appeared from Day 3 highlighting the presence of dense granules. ΔΨm and mitochondrial activity were significantly disrupted in sea bream fillets after 96 h of storage at +4 °C. Mitochondrial activity constituted a reliable and early indicator of fish freshness.

Use of Ratiometric Probes with a Spectrofluorometer for Bacterial Viability Measurement.

Assessment of microorganism viability is useful in many industrial fields. A large number of methods associated with the use of fluorescent probes have been developed, including fluorimetry, fluorescence microscopy, and cytometry. In this study, a microvolume spectrofluorometer was used to measure the membrane potential variations of Escherichia coli. In order to estimate the sensitivity of the device, the membrane potential of E. coli was artificially disrupted using an ionophore agent: carbonyl cyanide 3-chlorophenylhydrazone. The membrane potential was evaluated using two ratiometric methods: a Rhodamine 123/4',6-diamidino-2-phenylindole combination and a JC-10 ratiometric probe. These methods were used to study the impact of freezing on E. coli, and were compared with the conventional enumeration method. The results showed that it was beneficial to use this compact, easy-to-use, and inexpensive spectrofluorometer to assess the viability of bacterial cells via their membrane potential.

Assessment of freshness and freeze-thawing of sea bream fillets (Sparus aurata) by a cytosolic enzyme: Lactate dehydrogenase.

The evaluation of freshness and freeze-thawing of fish fillets was carried out by assessment of autolysis of cells using a cytosolic enzyme lactate dehydrogenase. Autolysis plays an important role in spoilage of fish and postmortem changes in fish tissue are due to the breakdown of the cellular structures and release of cytoplasmic contents. The outflow of a cytosolic enzyme, lactate dehydrogenase, was studied in sea bream fillets and the Sparus aurata fibroblasts (SAF-1) cell-line during an 8day storage period at +4°C. A significant increase of lactate dehydrogenase release was observed, especially after 5days of storage. The ratio between the free and the total lactate dehydrogenase activity is a promising predictive marker to measure the quality of fresh fish fillets. The effect of freeze-thawing on cytosolic lactate dehydrogenase and lysosomal α-d-glucosidase activities was also tested. Despite the protecting effect of the tissue compared to the cell-line, a loss of lactate dehydrogenase activity, but not of α-d-glucosidase, was observed. In conclusion, lactate dehydrogenase may be used as a marker to both assess freshness of fish and distinguish between fresh and frozen-thawed fish fillets.

A comparative study of antibodies against proteins extracted from staphylococcal biofilm for the diagnosis of orthopedic prosthesis-related infections in an animal model and in humans.

Staphylococcus aureus and Staphylococcus epidermidis are the microorganisms most frequently seen in periprosthetic infections (PPI) with the capacity of forming biofilm. To find potential antigens for the diagnosis of PPI, the immunogenicity of protein components in biofilm from a model biofilm-positive strain (S. epidermidis RP62A) was investigated. A guinea pig animal model of PPI was developed and sera were obtained. Sera of patients with PPI and those of controls were also collected. Data generated with an enzyme-linked immunosorbent assay showed that there were significantly higher levels of anti-extracellular protein IgG in sera of infected animals than in controls. We also found significantly higher anti-extracellular protein IgG levels in infected patients, compared to the controls; however, receiver operating characteristic curves did not aid in diagnosing PPI.

Potential use of poly-N-acetyl-beta-(1,6)-glucosamine as an antigen for diagnosis of staphylococcal orthopedic-prosthesis-related infections.

Staphylococcus aureus and coagulase-negative staphylococci are microorganisms most frequently isolated from orthopedic-implant-associated infections. Their capacity to maintain these infections is thought to be related to their ability to form adherent biofilms. Poly-N-acetyl-beta-(1,6)-glucosamine (PNAG) is an important constituent of the extracellular biofilm matrix of staphylococci. In the present study, we explored the possibility of using PNAG as an antigen for detecting antibodies in the blood sera of patients with staphylococcal orthopedic-prosthesis-associated infections. First, we tested the presence of anti-PNAG antibodies in an animal model, in the blood sera of guinea pigs that developed an implant-associated infection caused by biofilm-forming, PNAG-producing strains of Staphylococcus epidermidis. Animals infected with S. epidermidis RP62A showed levels of anti-PNAG immunoglobulin G (IgG) significantly higher than those of the control group. The comparative study of healthy individuals and patients with staphylococcal prosthesis-related infections showed that (i) relatively high levels of anti-PNAG IgG were present in the blood sera of the healthy control group, (ii) the corresponding levels in the infected patients were slightly but not significantly higher, and (iii) only 1 of 10 patients had a level of anti-PNAG IgM significantly higher than that of the control group. In conclusion, the encouraging results obtained in the animal study could not be readily applied for the diagnosis of staphylococcal orthopedic-prosthesis-related infections in humans, and PNAG does not seem to be an appropriate antigen for this purpose. Further studies are necessary to determine whether the developed enzyme-linked immunosorbent assay method could serve as a complementary test in the individual follow-up treatment of such infections caused by PNAG-producing staphylococci.

Neither the presence of ica locus, nor in vitro-biofilm formation ability is a crucial parameter for some Staphylococcus epidermidis strains to maintain an infection in a guinea pig tissue cage model.

The pathogenesis of Staphylococcus epidermidis is thought to be based on its capacity to colonize medical devices by forming a biofilm. Biofilm formation is in part mediated by the polysaccharide intercellular adhesin (PIA), which is encoded by the icaADBC operon. We have previously investigated in vitro the correlation existing between biofilm formation (B+/-), presence of ica locus (I+/-) and PIA production (P+/-) in some clinical isolates of coagulase-negative staphylococci (CoNS). Here, we used a guinea pig model of subcutaneous implanted tissue cages to assess the implication of B, I and P parameters in the capacity of nine S. epidermidis and one S. carnosus strains to develop and maintain an infection in vivo. Using clinical isolates and a model strain of S. epidermidis, we showed that the "B+, I+, P+" type confers the ability to maintain an infection in vivo. Surprisingly, the opposite type "B-, I-, P-" tested with clinical and commensal isolates, presented infection rates ranging from 25% to 60%. Other clinical isolates having a "B+, I+, P-" type, were not able to cause an infection in the present model. These results showed that, depending on the strains the capacity to colonize the tissue cage might be independent of the ability to form biofilm.

Correlation between biofilm formation and production of polysaccharide intercellular adhesin in clinical isolates of coagulase-negative staphylococci.

The ability to form a biofilm seems to play an essential role in the virulence of coagulase-negative staphylococci (CoNS) by permitting them to cause persistent prosthetic device-related infections. The most clearly characterized component of staphylococcal biofilms is the polysaccharide intercellular adhesin (PIA) encoded by the icaADBC operon. In the present paper, we assess the link between the ability to form a biofilm (Bf+/-), to synthesize PIA (PIA+/-) and the presence of the ica locus (ica+/-). For this purpose, 66 CoNS strains were tested in vitro. Seventy three percent of all strains revealed presence of the ica locus (ica+), and therefore were potentially able to produce PIA and to form a biofilm. However, the characteristics observed indicated that 15% of all strains were biofilm forming without PIA production (Bf+, PIA-, ica+/-) while 8% were PIA producers without biofilm formation (Bf-, PIA+, ica+). On the basis of the obtained data we conclude that (i) PIA synthesis alone is not sufficient to produce a biofilm and (ii) staphylococci can also form a biofilm without producing PIA.