Muhammad Naseem Khan

Application of the VPp1 bacteriophage combined with a coupled enzyme system in the rapid detection of Vibrio parahaemolyticus

For rapid and quantitative detection of Vibrio parahaemolyticus, a method combining the specific lysis of bacteriophages with a bacterial luciferase-flavin mononucleotide:nicotinamide adenine dinucleotide oxidoreductase bioluminescent system in vitro was developed. A V. parahaemolyticus detection system was established by optimizing three main influencing factors: bacteriophage titer, volume ratio of the bacteriophage to its host bacterium, and lysis time. A standard curve between the number of bacteria and the luminescence intensity of the coupled enzyme system was studied and revealed a good linear relationship. More than 10(7)colony-forming units (cfu)·ml(-1) bacteria in pure culture and >10(8) cfu·ml(-1) bacteria in oyster samples were readily detected without pre-enrichment. Furthermore, >10(0) cfu·ml(-1) bacteria in oyster samples were readily detected after 4h of enrichment culture. Because of its rapid detection, high specificity, and simplicity in operation, this method is an effective tool for detecting living bacteria in food and environmental samples.

Protein carbonylation during electron beam irradiation may be responsible for changes in IgE binding to turbot parvalbumin.

The present study aimed to investigate the relationship between protein carbonylation and changes of the IgE reactivity of turbot parvalbumin (PV) following electron beam (EB) irradiation. The concentration of protein carbonyls, specific IgE binding, and IgE binding inhibition between irradiated and oxidized PV were assessed. Irradiation resulted in a 3-fold enhancement in the protein carbonyl content. In purified PV irradiated with a 10-kGy dose, specific IgE binding was reduced by 91.2±6.2%. When raw PV was treated with reactive oxygen species (ROS), the protein carbonyl content increased 17.6-fold, with the specific IgE binding being reduced by 87.9±6.5% at an ROS concentration of 10 nmol/mL. The IgE binding inhibition between irradiated and oxidized PV was investigated using an inhibition ELISA. Results showed that oxidized PV can inhibit the binding between irradiated PV and specific IgE with an IC50 of 8.2-58 ng according to different doses of irradiation. These findings suggest that EB irradiation reduces specific IgE binding, probably by the induction of protein carbonylation.

Molecular species analysis of monosialogangliosides from sea urchin Strongylocentrotus nudus by RPLC-ESI-MS/MS.

Sea urchin gangliosides have been proved to contain neuritogenic activities, which related to their molecular compositions. This study reports a method utilizing reversed-phase chromatography coupled to mass spectrometry for structure investigation and molecular species determination of the monosialogangliosides from sea urchin Strongylocentrotus nudus. Two types of sulfated and nonsulfated monosialogangliosides were isolated from the sea urchin ovary. In MS(2) spectra of both nonsulfated monosialoganglioside and sulfated monosialoganglioside, 2-6 linked sialic acids were identified by the characteristic fragments of (0,4)A₂-CO₂ and (0,2)A₁. Fragment ions at m/z 139.1 and m/z 169.1 of nonsulfated monosialoganglioside might be characteristic for 8-sulfated sialic acid residue. Retention time of the molecules was effectively used in the characterization of unknown molecules, and molecules that differ in mass by only 0.04 Da were easily differentiated.

Effects of bacteriophage on the quality and shelf life of Paralichthys olivaceus during chilled storage.

BACKGROUND The microbiological spoilage of fishery foods is mainly due to specific spoilage organisms (SSOs), with Shewanella putrefaciens being the SSO of most chilled marine fish. Bacteriophages have shown excellent capability to control micro-organisms. The aim of this study was to determine a specific bacteriophage to prevent spoilage by reducing SSO (S. putrefaciens) levels in the marine fish Paralichthys olivaceus (olive flounder) under chilled storage. RESULTS Chilled flounder fillets were inoculated with S. putrefaciens and treated with different concentrations of bacteriophage Spp001 ranging from 10(4) to 10(8) plaque-forming units (pfu) mL(-1) . Bacterial growth (including total viable count and SSO) of the bacteriophage-treated groups was significantly inhibited compared with that of the negative control group (P < 0.05). Sensory evaluation and biochemical parameters revealed that the bacteriophage could extend the shelf life of chilled flounder fillets (from <4 to 14 days) with good esthetic quality, even at low temperature (4 °C). Furthermore, bacteriophage concentrations of 10(6) and 10(8) pfu mL(-1) were more effective than the chemical preservative potassium sorbate (5 g L(-1) ). CONCLUSION The bacteriophage Spp001 offered effective biocontrol of S. putrefaciens under chilled conditions, retaining the quality characteristics of spiked fish fillets, and thus could be a potential candidate for use in chilled fish fillet biopreservation.

Antibacterial fatty acids destabilize hydrophobic and multicellular aggregates of biofilm in S. aureus

Present study is based on 20 methicillin-resistant Staphylococcus aureus (MRSA) isolates recovered from different food items. These isolates were identified on the basis of colony morphology, Gram staining and growth on different selective and differential media. Studies on 16S RNA and positive reactions on DNase agar and Prolex Latex Agglutination system confirm it as Staphylococcus aureus. Oxacillin susceptibility testing and PCR with mecA gene-specific primer results showed that these isolates are MRSA-carrying mecA gene that belongs to SCCmecA type IV and also harbor agr type II. Phenotypic study revealed that these isolates adopt biofilm mode of growth after exposure to subinhibitory doses of oxacillin. The biofilm and cell surface hydrophobicity have a strong correlation. It was noticed that affinity to hexadecane (apolar-solvent) of planktonic cells was low, suggesting its hydrophilic character. However, as the cells are exposed to oxacillin, they adopt biofilm mode of life and the affinity to apolar solvent increases, indicating a hydrophobic character. In biofilm consortia, the cells with more hydrophobic surfaces show incomplete septation and produce multicellular aggregates. This is due to reduced expression of atl gene. This was confirmed by real-time PCR studies. Moreover, the planktonic or wild-type phenotypes of these isolates were more tolerant to antibacterial effect of the fatty acids used; that is, cis-2-decanoic acid and cis-9-octadectanoic acid. These fatty acids were more effective against biofilms. After exposure to these fatty acids, established biofilms were dispersed and surviving cells were unable to readopt biofilm mode of life. The planktonic or wild-type phenotypes produce fatty acid-modifying enzyme (FAME) to inactivate the bactericidal activity of fatty acids by esterification to cholesterol. The biofilm indwellers are metabolically inactive and unable to produce FAME; hence, they are vulnerable to antibiofilm effect of cis-2-decanoic acid and cis-9-octadectanoic acid.