Tryptic Soy Broth Research Articles

High Cell Density Cultivation of Escherichia coli DH5α in Shake Flasks with a New Formulated Medium

High cell density cultivation necessitates cell division and biomass formation, the mechanisms of which remain poorly understood, especially from the cellular energetics perspective. Specifically, the sensing of energy abundance and the channelling of nutritional energy into biomass formation and cell maintenance remains enigmatic at the sensory, effector and decision levels. Thus, optimization of cell growth remains an iterative trial and error process where the principal parameters are growth medium composition and incubation temperature. In this study, a new semidefined formulated medium was shown to be useful for high cell density cultivation of Escherichia coli DH5α (ATCC 53868). Comprising K2HPO4, 12.54; KH2PO4, 2.31; D-Glucose, 4.0; NH4Cl, 1.0; Yeast extract, 12.0; NaCl, 5.0; MgSO4, 0.24; the medium possessed a high capacity phosphate buffer able to moderate pH fluctuations during cell growth known to be detrimental to biomass formation. With glucose and NH4Cl providing the nutrients for initial growth, followed by a lag phase of 3 hours, a maximal optical density of 12.0 was obtained after 27 hours of cultivation at 37 oC and 230 rpm. Yeast extract provides a secondary source of carbon and nitrogen. Maximal optical density obtained in formulated medium was higher than the 10.1, 4.2, and 3.4 obtained in Tryptic Soy Broth, M9 with 1 g/L of yeast extract, and LB Lennox, respectively. Cultivation of E. coli DH5α in formulated medium with 6 g/L of glucose resulted in a longer lag phase of 8 hours and a longer time (68 hours) to attainment of maximal optical density, which marked the upper limit of glucose concentration beyond which biomass formation would be reduced. Specifically, glucose concentration above 6 g/L markedly reduced biomass formation possibly due to the environmental stress arising from low pH in the culture broth. Glucose concentration below 4 g/L, on the other hand, reduced biomass formation through a smaller pool of nutrients serving as biomass building blocks. Deviation from 1:1 molar ratio between glucose and NH4Cl was not detrimental to biomass formation and growth rates. Collectively, a semi-defined formulated medium could increase optical density of E. coli DH5α beyond that of LB Lennox and Tryptic Soy Broth, and may find use in cultivation of cells for applied microbiology research.

Whey protein influences the production and activity of extracellular protease from Pseudomonas fluorescens W3

The heat-resistant protease secreted by Pseudomonas in raw milk is an important reason for reducing the quality of dairy foods. Protease from Pseudomonas fluorescens W3 (P. fluorescens W3) performed higher activity in milk than that in tryptic soy broth (TSB). In this study, the effects of different milk components on the activity of protease from P. fluorescens W3 were explored. The results showed that whey protein was the main reason that influenced the protease activity secreted by P. fluorescens W3, which exert positive correlation with the concentration of whey protein content (from 0.3% to 1%). The zymogram and mass spectrometry proved that the protease secreted by P. fluorescens W3 was AprX, which had a better ability to hydrolyze casein than whey protein. The AprX hydrolysised casein by the suquence as follows: κ-casein > β-casein > α-casein. The results of RT-qPCR showed that whey protein could increase the expression of gene aprX by P. fluorescens W3. The results could help us better understand how milk promoted the protease activity of Pseudomonas fluorescens. Destroying whey protein may become the key method to inhibit the activity of protease from psychrotrophic bacteria, and reduce the waste of dairy foods.

A comparative study for production of germ tube in Candida albicans of various pulmonary samples, by different methods in a tertiary care hospital of south west Rajasthan

Background: Systemic candidiasis is associated with a high crude mortality rate, even with first line antifungal therapy. C. albicans is the predominant cause of invasive fungal diseases which is a serious public health issue. The main objective was to assess the reliability of different media for germ tube production in Candida albicans isolated from various clinically diagnosed pulmonary samples.Methods: All Candida isolates were identified and speciated by conventional methods such as Gram’s staining, germ tube test, chlamydospore formation on corn meal agar, sugar fermentation test, sugar assimilation test, and growth on Hi-chrome candida agar.Results: Out of 108 clinical isolates of Candida albicans, 5 different methods were used for germ tube production. Pooled human sera showed 93/108 (86.1%) was the most sensitive method wherein YEPD (yeast extract peptone dextrose) broth 91/108 (84.7%) was the reliable and easy method for detection of germ tube, followed by trypticase soy broth 81/108 (81.4%); peptone water 80/108 (74.7%) and 2% sucrose 71/108 (65.7%).Conclusions: YPED broth is found to be a better serum free substrate and subsequently for the presumptive differentiation of C. albicans from non-albicans candida (NAC), without the extensive time required for the preparation and testing of pooled human serum. Furthermore, this medium is commercially available, more stable, effective, and is not bio hazardous.

Design of a Low-Power Radio Frequency Unit and Its Application for Bacterial Inactivation under Laboratory Conditions

A lab-scale low-power free-running radio frequency (RF) oscillator operating at a frequency of 27.12 ± 0.50 MHz was developed to be suitable for fundamental microbiological research topics. Calibration and validation were conducted for two common foodborne pathogens in relevant microbiological growth media, i.e., Salmonella Typhimurium and Listeria monocytogenes in Tryptic Soy Broth and Brain–Heart Infusion broth, respectively. The evolution of temperature, frequency, and power consumption was monitored during treatments, both with and without bacterial cells. The setup operated within the predefined frequency range, reaching temperatures of 71–76 °C after 15 min. The average power consumption ranged between 12 and 14 W. The presence of bacteria did not significantly influence the operational parameters. The inactivation potential of the RF setup was validated, demonstrating the absence of viable cells after 8 and 10 min of treatment, for S. Typhimurium and L. monocytogenes, respectively. In future studies, the setup can be used to conduct fundamental microbiological studies on RF inactivation. The setup can provide added value to the scientific field, since (i) no consensus has been reached on the inactivation mechanisms of RF inactivation of pathogens in foods and (ii) most commercial RF setups are unsuitable to adopt for fundamental studies.

Drops join to make a stream: high-throughput nanoscale cultivation to grasp the lettuce root microbiome.

Root endospheres house complex and diverse bacterial communities, of which many strains have not been cultivated yet by means of the currently available isolation techniques. The Prospector® (General Automation Lab Technologies, San Carlos, CA, USA), an automated and high-throughput bacterial cultivation system, was applied to analyse the root endomicrobiome of lettuce (Lactuca sativa L.). By using deep sequencing, we compared the results obtained with the Prospector and the traditional solid medium culturing and extinction methods. We found that the species richness did not differ and that the amount of previously uncultured bacteria did not increase, but that the bacterial diversity isolated by the three methods varied. In addition, the tryptic soy broth and King's B media provided a lower, but different, diversity of bacteria than that of Reasoner's 2A (R2A) medium when used within the Prospector system and the number of unique bacterial strains did not weigh up against those isolated with the R2A medium. Thus, to cultivate as broad a variety of bacteria as possible, divergent isolation techniques should be used in parallel. Thanks to its speed and limited manual requirements, the Prospector is a valuable system to enlarge root microbiome culture collections.

Quantitative flow chamber system for evaluating in vitro biofilms and the kinetics of S. aureus biofilm formation in human plasma media

BackgroundIt has been well established that biofilm formation on orthopaedic implants is a critical event in the pathogenesis of orthopaedic infections, yet the natural history of this process with respect to bacterial adhesion, proliferation, and glycocalyx matrix production remains poorly understood. Moreover, there are no quantitative methods yet available to assess the differences in biofilm formation between different bacterial strains or implant materials. Consequently, this study aimed to investigate the natural history of S. aureus in in vitro biofilm formation in human plasma media using a flow chamber system. Bioluminescent S. aureus strains were used to better understand the bacterial growth and biofilm formation on orthopaedic materials. Also, the effects of human plasma media were assessed by loading the chamber with Tryptic Soy Broth with 10% human plasma (TSB + HP).ResultsScanning electron microscopy (SEM) was utilized to assess the morphological appearance of the biofilms, revealing that S. aureus inoculation was required for biofilm formation, and that the phenotypes of biofilm production after 24 h inoculation with three tested strains (SH1000, UAMS-1, and USA300) were markedly different depending on the culture medium. Time course study of the bioluminescence intensity (BLI) and biofilm production on the implants due to the UAMS-1 and USA300 strains revealed different characteristics, whereby UAMS-1 showed increasing BLI and biofilm growth until peaking at 9 h, while USA300 showed a rapid increase in BLI and biofilm formation at 6 h. The kinetics of biofilm formation for both UAMS-1 and USA300 were also supported and confirmed by qRT-PCR analysis of the 16S rRNA gene. Biofilms grown in our flow chamber in the plasma media were also demonstrated to involve an upregulation of the biofilm-forming-related genes icaA, fnbA, and alt. The BLI and SEM results from K-wire experiments revealed that the in vitro growth and biofilm formation by UAMS-1 and USA300 on stainless-steel and titanium surfaces were virtually identical.ConclusionWe demonstrated a novel in vitro model for S. aureus biofilm formation with quantitative BLI and SEM outcome measures, and then used this model to demonstrate the presence of strain-specific phenotypes and its potential use to evaluate anti-microbial surfaces.

Attachment of Shiga Toxin-Producing Escherichia coli (STEC) to Pre-Chill and Post-Chill Beef Brisket Tissue.

Shiga toxin-producing Escherichia coli (STEC) has caused numerous foodborne illness outbreaks where beef was implicated as the contaminated food source. Understanding how STEC attach to beef surfaces may inform effective intervention applications at the abattoir. This simulated meat processing conditions to measure STEC attachment to adipose and lean beef tissue. Beef brisket samples were warmed to a surface temperature of 30 °C (warm carcass), while the remaining samples were maintained at 4 °C (cold carcass), prior to surface inoculation with an STEC cocktail (O26, O45, O103, O111, O121, O145, and O157:H7). Cocktails were grown in either tryptic soy broth (TSB) or M9 minimal nutrient medium. Loosely and firmly attached cells were measured at 0, 3, 5, and 20 min and 1, 3, 8, 12, 24 and 48 h. TSB-grown STEC cells became more firmly attached throughout storage and a difference in loosely versus firmly attached populations on lean and adipose tissues was observed. M9-grown STEC demonstrated a 0.2 log10 CFU/cm2 difference in attachment to lean versus adipose tissue and variability in populations was recorded throughout sampling. Future research should investigate whether a decrease in intervention efficacy correlates to an increase in firmly attached STEC cells on chilled carcasses and/or subprimals, which has been reported.

Protective activity of specific transfer factor against Salmonella typhimurium infection

Specific transfer factor (TF) extracted from spleens of sensitized and non-sensitized guinea pigs to study the efficacy of transfer of cellular immunity specific for salmonella . Two groups each of five guinea pigs were used for in vivo TF preparation. The First group was inoculated with 1 ml of aromatic dependent Salmonella typhimurium SL 1479 vaccine at a dose of 10' cfu/ml intramuscularly twice at two weeks intervals. The second group was injected with trypticase soy broth similarly . These two groups used as a donor for TF, and TFn respectively. Twenty one recipient guinea pigs were divided into three groups, the first group was TF at a dose of 1 ml equivalent to 5X10 cell intramuscularly three times/2 days intervals, Similarly the second group was given TF, where as the third group was given PBS- Cell mediated immunity in recipient animal was evaluated by delayed type hypersensitivity - skin test, Macrophage migration inhibition test (MIF) and then challenged with virulent Salmonella typhimurium. The TFt recipient group induced skin test and showed migration indices less than 0.8 and overcome the challenge organism. Contrary to TFn & PBS recipient groups which did not show any response for skin test and given migration indices more than 0.8 and did not show resistance for virulent Salmonella typhimurium .

Long-term Storage of Bacterial Isolates by Using Tryptic Soy Broth with 15% Glycerol in The Deep Freezer (-70 to -80 °C)

For different bacterial preservation techniques, there is no single method applicable for all bacteria. This study aimed to assess the viability of seven species/species groups of clinical bacteria isolates on the long-term storage (more than 5 years) by using Tryptic Soy Broth with 15% glycerol in the deep freezer (-70 to -80°C). A total 10,654 clinical bacteria isolates used as samples in this study. The isolates consisted of seven species/species groups (i.e. Escherichia coli, Campylobacter spp, Shigella spp, Vibrio spp, Salmonella spp, Aeromonas hydrophila, and Neisseria gonorhoeae). The isolates were collected from some previous studies and preserved in the Tryptic Soy Broth (TSB) with 15% glycerol and stored in the deep freezer (-70 to -80°C) for more than five years. The samples were revived on the suitable medium to evaluate the viability of bacteria. Identification conducted by microscopic examination, biochemical test, and latex agglutination. The study showed that the viability of Salmonella spp, Shigella spp, Aeromonas hydrophila and E. coli was 100%, while Campylobacter spp, Vibrio spp, and N. gonorhoeae were 66.7%, 66.4%, and 52.5% respectively. We concluded that viability of Salmonella spp, Shigella spp, A. hydrophila, and E. coli was optimum thus better than Campylobacter spp, Vibrio spp, and N. gonorhoeae for more than 5 years storage by using TSB with 15% glycerol in the deep freezer (-70 to -80 °C).

Growth of Bacillus toyonensis in Tofu Wastewater

Bacillus toyonensis has been isolated in Dumai mangrove ecosystem of Riau Province. One of factors affecting the growth of the bacteria is growth substrate. Tofu wastewater is rich in nutrition which can be used as substrate for bacterial growth. This research aimed to observe the growth of B. toyonensis in different concentration of tofu wastewater. The bacteria was grown in tofu wastewater at concentrations 8%, 10% and 12% was supplemented with 0.1 g K2HPO4, 0.15 g KH2PO4, 0.15 g NaCl and 0.5 g vitamin B12 in 100 mL distilled water. The bacterial growth was observed by using spectrophotometer at λ 610 nm and by analysis the total plate counts on plate count agar (PCA) at 0, 24, 48, 72 and 96 hour cultivation. Spectrophotometric observation showed that the highest bacterial growth of all tofu wastewater treatments indicated by the addition of 12% tofu wastewater, although the absorbance value was lower than culture in tryptic soy broth (TSB) as control. Exponential growth occurred between 0-24 hour incubation, and the highest growth indicated in substrate contained 12% tofu wastewater. Similarly, total plate count (TPC) analysis indicated that the highest bacterial growth of all treatment occurred at 24 hours incubation, and the highest count was also indicated by treatment of 12% tofu wastewater (2.42±0.06×108 CFU/mL). In conclusion, tofu wastewater can be an alternative substrate for the bacterial growth.

Evaluation of Antibacterial Effects of Matrix-Induced Silver Ions against Antibiotic-Resistant ESKAPE Pathogens.

Recently, drug-resistant bacterial infections, especially ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.), have become a critical health issue worldwide, highlighting the emerging need for novel antibacterial agents. In this study, silver nanoparticles were extracted from silver-containing mesoporous bioactive glass (MBG-Ag) using four different matrixes, including water, phosphate buffer saline (PBS), tryptic soy broth (TSB), and taurine (Tau). The inductively coupled plasma-mass spectrometer (ICP-MS) results demonstrated that the silver concentration of Tau-Ag was the highest among the four matrixes. The Tau-Ag was also observed to have 87.35% silver ions in its X-ray photoelectron spectrometer (XPS) spectra. The micrograph of transmission electron microscope (TEM) displayed a uniform distribution of silver nanoparticles, which was confined in a smaller size compared to that in TSB-Ag. Moreover, the peak shifts observed in the Fourier-transform infrared spectrometer (FTIR) spectrum implied that the -SO32− and -NH groups in taurine may interact with silver. A low cytotoxicity was noted for Tau-Ag, with approximately 70% of cells surviving at 0.63 mg/mL. Compared to the other three matrix-induced silver agents, Tau-Ag represented a better antibacterial effect against methicillin-resistant Staphylococcus aureus, with a minimum inhibitory concentration (MIC) value of 0.63 mg/mL and a postponed growth of 0.31 mg/mL observed. Further antibacterial examinations illustrated the presence of remarkable antibacterial activities against vancomycin-resistant Enterococcus feacium, carbapenem-resistant Klebsiella pneumoniae, carbapenem-resistant Acinetobacter baumannii, and carbapenem-resistant Pseudomonas aeruginosa. Given our observations and multiple bioactive functions of taurine (prevent patients from inflammation and oxidative-stress injuries), we anticipate that taurine matrix-induced silver ions would be a biomedical material with a high potential for combatting drug-resistant ESKAPE pathogens.

Purification and characterization of a novel bacteriocin produced by Acinetobacter movanagherensis AS isolated from goat rectum

AbstractThe present investigation focuses on the purification and characterization of a novel bacteriocin produced by Acinetobacter movanagherensis AS isolated from goat rectum, which demonstrated high antimicrobial activity against both gram‐positive and gram‐negative bacteria. It was identified as A. movanagherensis AS using biochemical and 16S rDNA sequencing. Tryptic soy broth supplemented with 1% yeast extract with pH 6.5 and incubation temperature of 37°C for 32 h were the optimal conditions for bacteriocin production. Bacteriocin was purified in three steps: 70% ammonium sulfate precipitation, ion‐exchange chromatography with diethylaminoethyl‐cellulose resin, and size exclusion chromatography with a Sephadex G75 column. Purified bacteriocin had a recovery of 7.93% with 23.43 purification fold, and a molecular weight of 12.11 kDa was determined by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis analysis. Its biochemical characterization showed good stability at a broad range of temperature (40–80°C) as well as pH (3–9). Inhibition of antimicrobial activity was observed after treatment with trypsin, pepsin, and protease, which confirmed the proteinaceous nature of the purified bacteriocin. Bioinformatic analysis suggests that the purified novel bacteriocin from A. movanagherensis AS may belong to the colicin family. Considering the antimicrobial potential of purified bacteriocin coated on low‐density polyethylene film, we assume that it could be potentially exploited for food preservation in food processing industries.

Aeromonas veroniidetection in Egyptian fish farms with summer tilapia mortality outbreaks and the role of formic acid in limiting its spread

From 45 private fish farms, 641 tilapia samples were collected, which suffered from tilapia summer mortality outbreaks at different localities. The findings of the conventional test and 16S rRNA gene sequence recognition indicated that the isolated bacterium from the examined farms is Aeromonas veronii. A 15-day feeding trial was conducted to determine the effects of formic acid (FA) on biochemical parameters, immunological status, cortisol levels and total gut bacterial population of A. veronii-challenged tilapia. Fish were divided into five groups. The first and second groups served as control-negative and control-positive groups, which were intraperitoneally injected with 0.2-ml sterilized tryptic soy broth and A. veronii (4.3 × 106), respectively, and both were fed with a basal diet. Groups 3–5 were intraperitoneally injected with 0.2-ml A. veronii (4.3 × 106), fed a basal diet until clinical signs appeared and fed on a medicated diet containing florfenicol 15 mg/kg fish body weight and 0.5% and 1% FA respectively. The results suggest that including 1% FA in the diet lowered mortality and relieved the pathogenic effects of A. veronii on the general health status of tilapia fish.

Synergistic inactivation of Escherichia coli O157:H7 and Staphylococcus aureus by gallic acid and thymol and its potential application on fresh-cut tomatoes

Antibacterial activity against Escherichia coli O157:H7 and Staphylococcus aureus of five typical plant-derived compounds [gallic acid (G.A), citral (Cit), thymol (Thy), salicylic acid (S.A), lauric acid (L.A)] were investigated by determining the minimum inhibitory concentration (MIC) and the fractional inhibitory concentration index (FICI). The results showed that only a combination of Thy and G.A (TGA), with a concentration of 0.1 and 1.25 mg/mL, respectively, had a synergistic effect (FICI = 0.5) on both E. coli O157:H7 and S. aureus. The amount of Thy and G.A in mixture were four-fold lower than the MICs of the individuals shown to cause the equivalent antimicrobial activity in trypticase soy broth (TSB). The microbial reduction obtained in TSB with addition of TGA were significantly higher (P < 0.05) than the reduction shown for the broth supplemented with the separated phenolics. TGA caused the changes of morphology and membrane integrity of bacteria. Additionally, the application of TGA on fresh-cut tomatoes are investigated. Fresh-cut tomatoes inoculated with E. coli O157:H7and S. aureus were washed for 2min, 5min, 10min at 4 °C, 25 °C, 40 °C in 0.3% NaOCl, or water containing TGA at various concentrations. Overall, the reduction of TGA achieved against S. aureus is higher than E. coli O157:H7. Same concentrations of combined antimicrobials at a temperature of 40 °C further increased the degree of microbial inactivation, with an additional 0.89–1.51 log CFU/g reduction compared to that at 25 °C. Moreover, 1/2MICThy+1/2MICG.A at 25 °C for 10min or 40 °C for 5min were generally acceptable with sensorial scores higher than 7. Our results showed that TGA could work synergistically on the inactivation of the tested bacteria and may be used as an alternative disinfectant of fresh produce.

Multiplex PCR method for the detection of human norovirus, Salmonella spp., Shigella spp., and shiga toxin producing Escherichia coli in blackberry, coriander, lettuce and strawberry

A multiplex PCR method was developed for the simultaneous detection of murine norovirus (MNV-1) as a surrogate for human norovirus (HuNoV) GI and GII, Salmonella spp., Shigella spp., and Shiga toxin producing Escherichia coli (STEC) in fresh produce. The toxicity of the glycine buffer on bacterial pathogens viability was evaluated. The growth of each of the three pathogens (previously stressed) was evaluated at 35 and 41.5 °C in modified buffered peptone water (mBPW) and trypticase soy broth (TSB), supplemented with vancomycin, novobiocin and brilliant green at two concentration levels. The selected conditions for simultaneous enrichment were: 41.5 °C/mBPW/supplemented with 8 ppm vancomycin, 0.6 ppm novobiocin and 0.2 ppm brilliant green. The pathogens and aerobic plate count (APC) growth was evaluated in the enrichment of lettuce, coriander, strawberry and blackberry under the best enrichment conditions. Starting from 1 to 10 CFU/mL, Salmonella reached from 7.63 to 8.91, Shigella 6.81 to 7.76 and STEC 7.43 to 9.27 log CFU/mL. The population reached for the APC was 5.11–6.56 log CFU/mL. Simultaneous detection by PCR was done using designed primers targeting invA, ipaH, stx1 and stx2 genes, and MNV-1. The detection sensitivity was 10–100 PFU for the MNV-1 and 1–10 CFU for each pathogenic bacteria. This protocol takes 6 h for MNV-1 and 24 h for Salmonella spp., Shigella spp., and STEC detection from the same food portion. In total, 200 samples were analyzed from retail markets from Queretaro, Mexico. Two strawberry samples were positive for HuNoV GI and one lettuce sample was positive for STEC. In conclusion, the method developed in this study is capable of detecting HuNoV GI and GII, Salmonella spp., Shigella spp and STEC from the same fresh produce sample.

Evaluation of the rCIM for carbapenemase detection in Enterobacterales and Pseudomonas aeruginosa and description of the TSBrCIM, an optimized variant

ObjectivesThe rapid Carbapenem Inactivation Method (rCIM) was evaluated with a strain collection of 164 and 69 carbapenem-resistant Enterobacterales and Pseudomonas aeruginosa, respectively, that produced various carbapenemases. For an improved carbapenemase detection in Enterobacterales, an optimized variant of the rCIM named TSBrCIM was developed. MethodsBacterial isolates were incubated with two meropenem disks in distilled water (rCIM) or tryptic soy broth (TSBrCIM). After centrifugation, the supernatant was incubated with a susceptible E. coli indicator strain in tryptic soy broth. Growth of the indicator strain implied carbapenemase activity in the test strain. ResultsThe rCIM detected 100/113 carbapenemase-producing Enterobacterales, resulting in a sensitivity of 88.5% and a specificity of 94.1%. For P. aeruginosa, sensitivity and specificity were 96.0% and 100%, respectively. The TSBrCIM was able to detect 105/113 carbapenemase-producing Enterobacterales, resulting in a sensitivity of 92.9% and a specificity of 96.1%. ConclusionThis study shows that the TSBrCIM can be valuable tool for detection of carbapenemases in Enterobacterales in the clinical laboratory, while the rCIM showed the best results for carbapenemase detection in P. aeruginosa.

Pyomelanin-Producing Brevundimonas vitisensis sp. nov., Isolated From Grape (Vitis vinifera L.).

A novel endophytic bacterial strain, designated GR-TSA-9T, was isolated from surface-sterilized grape (Vitis vinifera L.). 16S rRNA gene sequence analyses showed that the isolate was grouped within the genus Brevundimonas, displaying the highest similarity with Brevundimonas lenta DS-18T (97.9%) and Brevundimonas kwangchunensis KSL-102T (97.8%) and less than 97.5% similarity with other members of Brevundimonas. The strain GR-TSA-9T was a gram negative, rod shaped, facultatively anaerobic, catalase and oxidase positive, and motile bacterium. Its growth occurred at 10–37°C (optimally 25–30°C), at pH 7.0–8.0, and in NaCl 0–1% (optimally 0%). It contained ubiquinone-10 as a respiratory quinone, and the major cellular fatty acids (>10% of the total) were C16:0 (14.2%) and summed feature 8 (C18:1ω7c and/or C18:1ω6c, 65.6%). The polar lipids present in the strain were phosphoglycolipids, phosphatidylglycerol, 1,2-di-O-acyl-3-O-[d-glucopyranosyl-(1→4)-α-d-glucopyranuronosyl]glycerol, and unidentified lipids (L1, L2, and L4). The strain had one 2,976,716bp circular chromosome with a G+C content of 66.4%. The digital DNA–DNA hybridization value between strain GR-TSA-9T and B. lenta DS-18T was 20.9%, while the average nucleotide identity value was 76.7%. In addition, the dDDH and ANI values to other members in this genus, whose genome sequences are available, are less than 21.1 and 77.6%. Genome annotation predicted the presence of some gene clusters related to tyrosine degradation and pyomelanin formation. Strain GR-TSA-9T produced a brown melanin-like pigment in the presence of L-tyrosine-containing media. The highest pigment production (0.19g/L) was observed in tryptic soy broth with 1.0mg/ml L-tyrosine at 25°C for 6days of culture. Biophysical characterization by ultraviolet (UV)–visible spectroscopy, Fourier-transform infrared spectroscopy, and electrospray ionization mass spectrometry confirmed that the pigment was pyomelanin. Additionally, melanized GR-TSA-9T cells could protect the cells against UVC exposure. The phylogenetic, genomic, phenotypic, and chemotaxonomic features indicated that strain GR-TSA-9T represents a novel melanin-producing species of Brevundimonas. The type strain was GR-TSA-9T (KCTC 82386T=CGMCC 1.18820T).

Biological Transformation of Zearalenone by Some Bacterial Isolates Associated with Ruminant and Food Samples.

Zearalenone (ZEA) is a secondary metabolite produced by Fusarium spp., the filamentous fungi. Food and feed contamination with zearalenone has adverse effects on health and economy. ZEA degradation through microorganisms is providing a promising preventive measure. The current study includes isolation of 47 bacterial strains from 100 different food and rumen samples. Seventeen isolates showed maximum activity of ZEA reduction. A bacterial isolate, RS-5, reduced ZEA concentration up to 78.3% through ELISA analysis and 74.3% as determined through HPLC. Ten of the most efficient strains were further selected for comparison of their biodegradation activity in different conditions such as incubation period, and different growth media. The samples were analyzed after 24 h, 48 h, and 72 h of incubation. De Man Rogosa Sharp (MRS) broth, Tryptic soy broth, and nutrient broth were used as different carbon sources for comparison of activity through ELISA. The mean degradation % ± SD through ELISA and HPLC were 70.77% ± 3.935 and 69.11% ± 2.768, respectively. Optimum reducing activity was detected at 72 h of incubation, and MRS broth is a suitable medium. Phylogenetic analysis based on 16S rRNA gene nucleotide sequences confirmed that one of the bacterial isolate RS-5 bacterial isolates with higher mycotoxin degradation is identified as Bacillus subtilis isolated from rumen sample. B05 (FSL-8) bacterial isolate of yogurt belongs to the genus Lactobacillus with 99.66% similarity with Lactobacillus delbrukii. Similarly, three other bacterial isolates, D05, H05 and F04 (FS-17, FSL-2 and FS-20), were found to be the sub-species/strains Pseudomonas gessardii of genus Pseudomonas based on their similarity level of (99.2%, 96% and 96.88%) and positioning in the phylogenetic tree. Promising detoxification results were revealed through GC-MS analysis of RS-5 and FSL-8 activity.

Development of a species‐specific monoclonal antibody for rapid detection and identification of foodborne pathogen Vibrio vulnificus

AbstractVibrio vulnificus is one of the major cause of foodborne illness related to seafood consumption. This study aimed to develop monoclonal antibodies (MAbs) to enable rapid detection and monitoring food contamination of this pathogen. Five groups of MAbs against V. vulnificus were generated from a mouse immunized with a clinical isolate of V. vulnificus, heat and formalin‐inactivated whole cells. The first two groups of MAbs were species‐specific to V. vulnificus, recognized all 40 isolates of the bacterium without cross‐reactivity to other Vibiro and non‐Vibrio species, but bound to different protein antigens. The MAbs in the third group recognized four of nine clinical isolates and two isolates from seafood samples and bound to lipopolysaccharide antigens. The MAbs in the fourth group bound only to all isolates of V. vulnificus and Vibrio alginolyticus, and the MAb in the fifth group was genus‐specific bound to all isolates of Vibrio species tested. Based on Dot‐enzyme‐linked immunosorbent assay, the range of detection sensitivity limits of these MAbs were 105 to 107 CFU/ml, which is 10–1,000 times less than that of direct PCR detection (104 CFU/ml). However, after pre‐enrichment of an oyster sample in tryptic soy broth for 6 hr, the detection limit was improved to 1 CFU/ml. This detection sensitivity limit is comparable to that of PCR protocols, which mostly require a pre‐enrichment step in sample preparation. The sets of MAbs could provide valuable tools for simple, fast, and low‐cost detection of V. vulnificus and Vibrio species in seafood, clinical, and environmental samples.

Effect of Glucose, Agar Supplementation and Bacterial Cell Density on the in vitro Liquid Culture of Steinernema jeffreyense

Entomopathogenic nematodes (EPNs) of the family Steinernematidae are effective biological control agents against important pest insects. The in vitro liquid culture method of mass production is used to produce EPNs of high quantity, quality and with reduced cost by upscaling. The first step in liquid mass production is the use of shake flasks to obtain monoxenic infective juvenile (IJ) inoculum for optimisation purposes, followed by upscaling to a desktop fermenter. This study was undertaken to assess the impact of the addition of agar and glucose to the culture medium, as well as assessing the impact of bacterial cell density inoculum on IJ recovery and yield. Steinernema jeffreyense was cultured in 250 ml Erlenmeyer flasks, with the mutualistic bacterium Xenorhabdus khoisanae. In this study the impact of four different agar and glucose concentrations showed negligible impact on nematode recovery and yield. Different initial bacterial inoculum densities inoculated to the culture medium showed a low inoculum density of 2 % (6 × 106 cells) of the bacteria culture to be the optimal inoculum concentration. A bacterial growth curve for X. khoisanae in tryptic soy broth, showed 40–44 h to be the optimum time for introduction of the initial nematode inoculum into the complex medium.