Polyhydroxyalkanoates (PHAs), bioprocessing using waste oil

Pseudomonas oleovorans NCIMB 6576 and Ralstonia eutropha NCIMB 10442 were used for the production of Polyhydroxyalkanoates (PHA) from industrial waste cooking oils, the bacteria were cultured on tryptone soya broth (TSB) and Tryptone soya agar (TSA). P. oleovorans NCIMB6576 gave a better percentage PHB yield (8.2%) with PS oil as carbon source as compared to 6.45% with TS oil. However, a very low yield (0.64%) was recorded when P. oleovorans NCIMB6576 was grown on TSB without the oils as carbon source. Ralstonia eutropha NCIMB 10442 gave an appreciable yield of 13.63% and 14.80% with PS and TS oil samples respectively as carbon source with negligible variation in the yields. The results obtained across all experiments were compared with one another. The SEM images from the PHB samples generated from the experiments shows that there is a slight difference in the surface morphologies of the PHB with respect to the oil samples as well as the different bacteria used in the experiment.

Effect of Isothiocyanates on the Activity of Lactobacillus plantarum Exposed to Irradiation

Two isothiocyanates, i.e., sulforaphane (SFA) and sulforaphene (SFE), are suggested to be used as an alternative chemopreventive diet. This study was focused on the effect of SFA and SFE on Lactobacillus plantarum, which has been subjected to the irradiation (2-50 Gy). The cultures grown in De Man, Rogosa and Sharpe (MRS) and Tryptone Soya Broth (TSB) were compared in terms of bacteria physiological activity under tested conditions. Broth composition notably influenced the bacteria growth kinetic parameters, as well as culture response to the oxidative stress. Activity of L. plantarum cells after irradiation was evaluated by their dehydrogenase (DHA) and quinone-reductase (QR) activities. The enzyme activity was quantified in living cells. Bacterial cultures obtained in MRS and TSB broth, demonstrated contrasting characteristics in their enzyme activities. The MRS-grown culture did not show any QR activity, whereas the TSB-grown cells showed a non-linear response towards gamma-irradiation with a maximum inhibition being at 10 Gy. Addition of SFA or SFE in concentration of 1 µg/mL to the cultures before irradiation exposure recovered the QR activity from 23% in a non-amended variant up to 102% and 121%, respectively, taking the non-irradiated non-amended variant as 100%.

In Vitro Inhibition of Biofilm Formation by Staphylococcus Aureus Under the Action of Selected Plant Extracts

In our study we investigated the ability of selected plant extracts to inhibit the formation of biofilms produced by Staphylococcus aureus. In the first phase, we focused on the optimisation of conditions for the correct method of an approach. For optimisation, we standardized the culture media and the bacterial culture in order to obtain interpretable results. The TSB (Tryptone Soya Broth) medium was used for the preparation of an inoculum from the bacterial suspension. For the in vitro tests of antibiofilm activity against the species Staphylococcus aureus CCM 3953, we used propylene glycol (PG) plant extracts from sage and rosemary, prepared in three different concentrations of 0.01 %, 0.05 % and 0.1 %. The tests were implemented in microtitre plates using crystal violet dye at 0.1 % concentration for visualization of the intensity of a biofilm. The results were obtained, by spectrophotometric measurements at a wavelength of 550 nm. Both rosemary and sage plant extracts had a significant effect on the formation of a biofilm by S. aureus. The antibiofilm activity was concentration-dependent as the formation of biofilm was reduced more effectively with increasing concentration of the extracts. The best antibiofilm activity was observed with 0.1 % rosemary extract resulting in 94 % inhibition of the biofilm formation.

Thermal Inactivation of Listeria monocytogenes in Crab Meat

ABSTRACT Listeria monocytogenes is an important bacterial pathogen in seafood products, but limited information is currently available on the thermal resistance of relevant isolates in seafood. Thermal inactivation studies were undertaken (i) to provide much needed thermal inactivation data for L. monocytogenes in crab meat and (ii) to investigate whether tryptone soya broth (TSB) is representative of crab meat in thermal inactivation studies involving L. monocytogenes. D-values were obtained for a cocktail of two crab isolates (serotypes 1/2a and 4b) at 50, 55, and 60°C. In crab meat, D-values were 174.4, 28.2, and 1.6 min, respectively. Similar D-values of 176.4, 28.8, and 1.4 min were obtained in TSB. The corresponding z-values were 4.9°C (crab meat) and 4.8°C (TSB), respectively. The conclusions were that (i) current pasteurization conditions (e.g., 70°C for 2 min) would achieve complete destruction of any L. monocytogenes present in crab meat and (ii) TSB could be used as a model matrix for assessing the thermal inactivation of L. monocytogenes in crab meat.

Perkembangan Resistensi Escherichia coli terhadap Oksitetrasiklin

Antimicrobial resistance is amongst the primary concern in the field of veterinary medicine worldwide. The research was conducted to evaluate the resistance of Escherichia coli due to inappropriate antibiotic treatment. Escherichia coli in this research was cultured in vitro using 5-ml Tryptone Soya Broth (TSB), which was mixed with an antibiotic, then was incubated at 37 0C for four days (stage I). Subsequently, a part of the culture was transferred into another 5-ml TSB and incubated at 37 0C for four days (stage II). This procedure was undertaken continuously for stage III, IV, V,and VI (until day 24). Antibiotics in this research was oxytetracycline with three various doses, which are lower dose (1 mg/ml), normal dose (2 mg/ml), and higher dose (8 mg/ml). The development of the antrimocobial resistance was evaluated every four days, using disk diffusion method and the data were analysed descriptively. The results showed that the normal and higher doses of oxytetracycline has the same rate (day 16) in causing E. coli resistant to oxytetracycline. Therefore, the treatment of oxytetracycline with a normal and higher doses continuously could accelerate the emergence of antimicrobial resistance in Escherichia coli.

A study on the antimicrobial resistant patterns of Pseudomonas Aeruginosa isolated from raw milk samples in and around Tirupati, Andhra Pradesh.

This study was aimed to detect the prevalence of Pseudomonas aeruginosa in milk (n=125) samples which were collected from local vendors, private dairy farms in and around Tirupati. Pre-enrichment was done by taking 10ml of each sample and inoculated in 90 ml of Tryptone Soya broth and incubated at 370C for 24hrs. A loopful of culture was taken from broth and streaked on nutrient agar and Cetrimide agar plates and incubated at 370C for 24hrs which were further confirmed by biochemical tests. The nineteen positive samples for P.aeruginosa were further tested for antimicrobial susceptibility which has shown multi drug resistant ranging from four to twelve antimicrobials and Multiple Antibiotic Resistance (MAR) index ranges from 0.33 to 1.The isolates of P.aeruginosain the present study are highlyresistant to ampicillin, penicillin, and oxacillin (100%) and maximum sensitive to Vancomycin (5.3%) followed by Tetracycline (10.5%).

Effect of nutrient and stress factors on polysaccharides synthesis in Proteus mirabilis biofilm.

The extracellular matrix in biofilm consists of water, proteins, polysaccharides, nucleic acids and phospholipids. Synthesis of these components is influenced by many factors, e.g. environment conditions or carbon source. The aim of the study was to analyse polysaccharides levels in Proteus mirabilis biofilms after exposure to stress and nutritional conditions. Biofilms of 22 P. mirabilis strains were cultivated for 24, 48, 72 hours, 1 and 2 weeks in tryptone soya broth or in modified media containing an additional amount of nutrients (glucose, albumin) or stress factors (cefotaxime, pH 4, nutrient depletion). Proteins and total polysaccharides levels were studied by Lowry and the phenol-sulphuric acid methods, respectively. Glycoproteins levels were calculated by ELLA with the use of selected lectins (WGA and HPA). For CLSM analysis dual fluorescent staining was applied with SYTO 13 and WGA-TRITC. In optimal conditions the levels of polysaccharides were from 0 to 442 μg/mg of protein and differed depending on the strains and cultivation time. The agents used in this study had a significant impact on the polysaccharides synthesis in the P. mirabilis biofilm. Among all studied components (depending on tested methods), glucose and cefotaxime stimulated the greatest production of polysaccharides by P. mirabilis strains (more than a twofold increase). For most tested strains the highest amounts of sugars were detected after one week of incubation. CLSM analysis confirmed the overproduction of N-acetyloglucosamine in biofilms after cultivation in nutrient and stress conditions, with the level 111-1134%, which varied depending on the P. mirabilis strain and the test factor.

Evaluation of test-kits for the detection of Escherichia coli O157 in raw meats and cattle faeces.

Escherichia coli O157 detection limits in artificially contaminated beef and cattle faeces samples were determined using Dynabeads anti E. coli O157 immunomagnetic beads, VIDAS-UP, VIDAS-ICE, and real-time PCR (GeneDisc and LightCycler) systems. Dynabeads anti-E. coli O157 immunomagnetic separation (IMS) and the GeneDisc cycler were the most sensitive methods, and could detect an initial 1 CFU in 25g beef samples after 6h of incubation in modified tryptone soya broth with novobiocin (mTSB+n) or buffered peptone water (BPW). The VIDAS-UP method could detect an initial 10 CFU, while VIDAS-ICE and the LightCycler methods could only detect an initial 100 CFU. Higher detection rates were achieved with 18 hour incubations, where an initial 1 CFU in a 25g sample could be detected with all five methods. For cattle faeces enrichments, Dynabeads anti-E. coli O157 IMS could detect an initial 1 CFU after a 6 h incubation in mTSB+n, while the VIDAS-UP and VIDAS-ICE methods could detect an initial 10 CFU and both PCR methods could only detect an initial 100 CFU. Detection rates were lower in BPW, compared to mTSB+n, with thresholds of 100 CFU for VIDAS-ICE, VIDAS-UP and GeneDisc methods, and >100 CFU for the LightCycler method.

Evaluation of test-kits for the detection of Escherichia coli O157 in raw meats and cattle faeces.

Escherichia coli O157 detection limits in artificially contaminated beef and cattle faeces samples were determined using Dynabeads anti E. coli O157 immunomagnetic beads, VIDAS-UP, VIDAS-ICE, and real-time PCR (GeneDisc and LightCycler) systems. Dynabeads anti-E. coli O157 immunomagnetic separation (IMS) and the GeneDisc cycler were the most sensitive methods, and could detect an initial 1 CFU in 25g beef samples after 6h of incubation in modified tryptone soya broth with novobiocin (mTSB+n) or buffered peptone water (BPW). The VIDAS-UP method could detect an initial 10 CFU, while VIDAS-ICE and the LightCycler methods could only detect an initial 100 CFU. Higher detection rates were achieved with 18 hour incubations, where an initial 1 CFU in a 25g sample could be detected with all five methods. For cattle faeces enrichments, Dynabeads anti-E. coli O157 IMS could detect an initial 1 CFU after a 6 h incubation in mTSB+n, while the VIDAS-UP and VIDAS-ICE methods could detect an initial 10 CFU and both PCR methods could only detect an initial 100 CFU. Detection rates were lower in BPW, compared to mTSB+n, with thresholds of 100 CFU for VIDAS-ICE, VIDAS-UP and GeneDisc methods, and >100 CFU for the LightCycler method.

Bacterial biofilms resist oxidising agents due to the presence of organic matter

This study evaluated the susceptibility of planktonic and biofilm cells of Staphylococcus spp. (n = 87), Klebsiella spp. (n = 30), and Escherichia coli (n = 74) isolates originating from food contact surfaces of milk and meat processing plants to benzalkonium chloride (BAC), sodium hypochlorite (NaClO), chloramine B (CAB), and peracetic acid (PAA). Bacterial growth and reduction of viable cells in the presence of disinfectants were determined in tryptone soya broth (TSB) and water, respectively. Biofilm positive isolates (n = 73) were tested for the presence of selected qac genes. Unlike BAC, chlorine‑based disinfectants and PAA were poorly efficient in TSB, especially in the case of biofilms. However, when tested in water, the efficacy of NaClO, CAB and PAA substantially increased, which was particularly evident in biofilms. In water, staphylococcal biofilms were even more susceptible to CAB than planktonic cells. A part (23.3%) of the biofilm positive staphylococci carried the qac genes but did not express an increased resistance to BAC. This study showed that bacterial biofilms protected with organic matter could be one of the main reasons for disinfection failure.