Luria-Bertani Research Articles

One-step synthesis of Ag nano-assemblies and study of their antimicrobial activities

Three kinds of Ag nano-assemblies have been fabricated separately in a one-step reaction. These are designated as 3D–3D, shell-3/1D and 3D–1D nano-architectures. These nano-architectures were synthesized by polyol reduction method in the presence of polyvinyl pyrrolidone (PVP) at 120, 140, and 160 °C, respectively. During the experiment, controlled temperature decrease has been carried out which successfully developed 3D–3D, and 3D–1D conjunction. The shell-3/1D nano-assembly was successfully developed at 140 °C by gradual decreasing and subsequently increasing of temperature. The size and morphology of each product was characterized by transmission electron microscope (TEM). Surface plasmon resonance (SPR) behavior was analyzed by UV–Vis spectroscopy. The antimicrobial activities were also analyzed for gram positive bacteria like Enterococcus faecalis and Staphylococcus aureus, and gram negative like E. coli 0157:H7 and DH5α by scanned optical density (OD) and Kirby-Bauer process. The OD was measured by taking scanned UV–Vis absorption from 700–300 nm wavelength in LB (Luria–Bertani) broth media. Similarly Kirby-Bauer process was applied over LB agar media. The material was found unique in terms of morphology as well as in displaying its antimicrobial activities both for gram positive and gram negative bacteria.

Cultural optimization and metal effects of Shewanella xiamenensis BC01 growth and swarming motility

Shewanella species belonging to dissimilatory metal bacteria were found to decolorize most textile dyes and had also attracted great interests in regard to bioremediation. However, studies have rarely been reported on Shewanella xiamenensis BC01, which was isolated as a biodecolorization and bioelectricity strain recently. In this study, the effect of cultivation conditions on S. xiamenensis BC01 was studied to explore how environmental conditions may influence S. xiamenensis growth and swarming motility. Shewanella xiamenensis BC01 grew over a wide range of pH (5.0–9.0) and mild temperatures (25–42 °C). The optimal conditions for cell growth were using Luria-Bertani (LB) as medium with shaking at 150 rpm, 37 °C, and pH 8.0 which had been confirmed by shift pH and temperature. S. xiamenensis BC01 was able to resist 1 mM concentrations of various metal ions, i.e., Ca2+, Mg2+, Cu2+, Zn2+, Mn2+, Fe3+, and Al3+, respectively. As shown in scanning electron microscopy (SEM) analyses, cell morphologies were slightly changed under metal stress. Swarming motility showed that the velocity ranking at 80 μM and 1 mM of metal was Al > Cr > LB > Zn > Fe > Cu and Mg > Mn > Ca, respectively. This study evaluates the impact of cultivation methods and metal ions on the activity of S. xiamenensis BC01 and provides an alternative to bioremediation of heavy metal-containing wastewaters by utilizing this strain.

Efflux pump-deficient mutants as a platform to search for microbes that produce antibiotics.

Pseudomonas putida DOT-T1E-18 is a strain deficient in the major antibiotic efflux pump (TtgABC) that exhibits an overall increased susceptibility to a wide range of drugs when compared with the wild-type strain. We used this strain as a platform to search for microbes able to produce antibiotics that inhibit growth. A collection of 2400 isolates from soil, sediments and water was generated and a drop assay developed to identify, via growth inhibition halos, strains that prevent the growth of DOT-T1E-18 on solid Luria–Bertani plates. In this study, 35 different isolates that produced known and unknown antibiotics were identified. The most potent inhibitor of DOT-T1E-18 growth was an isolate named 250J that, through multi-locus sequence analysis, was identified as a Pseudomonas sp. strain. Culture supernatants of 250J contain four different xantholysins that prevent growth of Gram-positive bacteria, Gram-negative and fungi. Two of the xantholysins were produced in higher concentrations and purified. Xantholysin A was effective against Bacillus, Lysinibacillus and Rhodococcus strains, and the effect against these microbes was enhanced when used in combination with other antibiotics such as ampicillin, gentamicin and kanamycin. Xantholysin C was also efficient against Gram-positive bacteria and showed an interesting antimicrobial effect against Pseudomonas strains, and a synergistic inhibitory effect with ampicillin, chloramphenicol and gentamicin.

Scaling Up the Production of Recombinant Antimicrobial Plantaricin E from a Heterologous Host, Escherichia coli.

Enhanced production of heterologously expressed plantaricin (plnE) from Escherichia coli BL21 (DE3) was achieved from a small- to large-scale batch culture. Starting from a 15-ml shake-flask culture grown in Luria-Bertani (LB) broth, the protein expression could be scaled up using 50 ml, 100 ml, 1 l, and 2 l batch culture. Using similar condition, plantaricin E (PlnE) was successfully expressed in a 30-l stirred fermenter. The protein was expressed as TRX-(His)6-fusion protein and separated by Ni(2+) affinity chromatography. Growth in two complex media, LB and Terrific broth (TB), was optimized and compared for the production of PlnE, which was higher in LB in comparison with that of TB. In the fermenter, 140 and 180 mg of PlnE could be produced from 12 l of culture volume at 30 and 25 °C, respectively. The yield of heterologously purified PlnE was found to be 1.2-1.5%, which was much higher in comparison with the plantaricins produced from the native strain of Lactobacillus plantarum (0.3-0.7%). Overproduction of PlnE with the help of heterologous expression can overcome the constraint of the low yield from producer strain and provides an easy and low-cost strategy for large-scale production.

Mercury (II) removal by resistant bacterial isolates and mercuric (II) reductase activity in a new strain of Pseudomonas sp. B50A

This study aimed to isolate mercury resistant bacteria, determine the minimum inhibitory concentration for Hg, estimate mercury removal by selected isolates, explore the mer genes, and detect and characterize the activity of the enzyme mercuric (II) reductase produced by a new strain of Pseudomonas sp. B50A. The Hg removal capacity of the isolates was determined by incubating the isolates in Luria Bertani broth and the remaining mercury quantified by atomic absorption spectrophotometry. A PCR reaction was carried out to detect the merA gene and the mercury (II) reductase activity was determined in a spectrophotometer at 340 nm. Eight Gram-negative bacterial isolates were resistant to high mercury concentrations and capable of removing mercury, and of these, five were positive for the gene merA. The isolate Pseudomonas sp. B50A removed 86% of the mercury present in the culture medium and was chosen for further analysis of its enzyme activity. Mercuric (II) reductase activity was detected in the crude extract of this strain. This enzyme showed optimal activity at pH 8 and at temperatures between 37 °C and 45 °C. The ions NH4(+), Ba(2+), Sn(2+), Ni(2+) and Cd(2+) neither inhibited nor stimulated the enzyme activity but it decreased in the presence of the ions Ca(2+), Cu(+) and K(+). The isolate and the enzyme detected were effective in reducing Hg(II) to Hg(0), showing the potential to develop bioremediation technologies and processes to clean-up the environment and waste contaminated with mercury.

Plate acoustic wave sensor for detection of small amounts of bacterial cells in micro-litre liquid samples

Ultrasonic acoustic waves propagating in thin piezoelectric plates with free faces are used for bacteria detection in micro-litre liquid samples deposited on one of the plate surface. The limits of the detection at normal conditions are as low as 0.04% for highly diluted rich cultural Luria–Bertani broth (LB-media) in distillate water, 0.07% for bacterial cells in distillate water, and 0.6% for bacterial cells in LB-media. For all analytes the most probable detection mechanism is the change in liquid conductivity. Because of no using any sorbent film the long-term stability of the detection is expected as very high.

New Bacillus thuringiensis strain isolated from the gut of Malabari goat is effective against Tetranychus macfarlanei

AbstractThis study illustrates a novel strain (designated as BPU5) of Bacillus thuringiensis (Bt) isolated from the rumen of Malabari goat, capable of producing polymorphic δ‐endotoxin crystals concomitantly with sporulation in Luria–Bertani medium (LB), and the δ‐endotoxin was efficient to combat Tetranychus macfarlanei, a devastating mite. Polymorphic δ‐endotoxin crystals produced were assessed by scanning electron microscopy and monitored its production concomitantly with sporulation in LB with or without sugar supplements. Toxicity of the δ‐endotoxin was assessed on T. macfarlanei using leaf disc bioassay method. Mortality rate was determined by comparing the survival of mites on the diet (prepared in 10% sucrose and powdered rice husk) containing different concentrations (1–10 mg/ml) of 72‐h‐old crude pellet (dried mixture of δ‐endotoxin (17 mg/g pellet), endospores and a few vegetative cells) or control diet with autoclaved pellet. The maximum production (1.39 mg/ml) of δ‐endotoxin was observed at 72 h in LB. Among the sugars (glucose, sucrose, maltose or lactose) tested as additional carbon source, glucose (8 g/l) enhanced (1.82 mg/ml) the production of δ‐endotoxin by 30%. The lethal concentration (LC50) required to kill 50% mites was estimated as 8.024 mg/ml. The δ‐endotoxin produced by B. thuringiensis BPU5 is shown to efficiently combat T. macfarlanei, a devastating mite infesting agricultural fields.

Acid environments affect biofilm formation and gene expression in isolates of Salmonella enterica Typhimurium DT104

The aim of this study was to examine the survival and potential virulence of biofilm-forming Salmonella Typhimurium DT104 under mild acid conditions. Salmonella Typhimurium DT104 employs an acid tolerance response (ATR) allowing it to adapt to acidic environments. The threat that these acid adapted cells pose to food safety could be enhanced if they also produce biofilms in acidic conditions. The cells were acid-adapted by culturing them in 1% glucose and their ability to form biofilms on stainless steel and on the surface of Luria Bertani (LB) broth at pH7 and pH5 was examined. Plate counts were performed to examine cell survival. RNA was isolated from cells to examine changes in the expression of genes associated with virulence, invasion, biofilm formation and global gene regulation in response to acid stress. Of the 4 isolates that were examined only one (1481) that produced a rigid biofilm in LB broth at pH7 also formed this same structure at pH5. This indicated that the lactic acid severely impeded the biofilm producing capabilities of the other isolates examined under these conditions. Isolate 1481 also had higher expression of genes associated with virulence (hilA) and invasion (invA) with a 24.34-fold and 13.68-fold increase in relative gene expression respectively at pH5 compared to pH7. Although genes associated with biofilm formation had increased expression in response to acid stress for all the isolates this only resulted in the formation of a biofilm by isolate 1481. This suggests that in addition to the range of genes associated with biofilm production at neutral pH, there are genes whose protein products specifically aid in biofilm production in acidic environments. Furthermore, it highlights the potential for the use of lactic acid for the inhibition of Salmonella biofilms.

Magnesium uptake of Arabidopsis transporters, AtMRS2-10 and AtMRS2-11, expressed in Escherichia coli mutants: Complementation and growth inhibition by aluminum

Magnesium (Mg2+) plays a critical role in many physiological processes. Mg2+ transport systems in Salmonella have been well documented, but those in Escherichia coli have not been fully elucidated. We examined the effects of corA, mgtA, yhiD and corC gene deletion on Mg2+ transport in E. coli. We obtained every combination of double, triple and quadruple mutants. The corA and mgtA double mutant required addition of 10mMMg2+ to Luria-Bertani (LB) medium for growth, and the corA, mgtA and yhiD triple mutant TM2 required a higher Mg2+ concentration. The Mg2+ requirement of the quadruple mutant was similar to that of TM2. The results demonstrated that either CorA or MgtA is necessary for normal E. coli growth in LB medium and that YhiD plays a role in Mg2+ transport under high Mg2+ growth conditions in E. coli. The Arabidopsis Mg2+ transporters, AtMRS2-10 and AtMRS2-11, were heterologously expressed in TM2 cells. TM2 cells expressing AtMRS2-10 and AtMRS2-11 could grow in LB medium that had been supplemented with 1mMMg2+ and without Mg2+ supplementation, respectively, and cell growth was inhibited by 2mM AlCl3. The results indicated that the growth of TM2 expressing AtMRS2-10 and AtMRS2-11 reflected these AtMRS2 function for Mg2+ and aluminum. The E. coli TM2 cells are useful for functional analysis of Arabidopsis MRS2 proteins.

Membrane protein profiling of Acidovorax avenae subsp. avenae under various growth conditions.

Membrane proteins (MPs) of plant pathogenic bacteria have been reported to be able to regulate many essential cellular processes associated with plant disease. The aim of the current study was to examine and compare the expression of MPs of the rice bacterial pathogen Acidovorax avenae subsp. avenae strain RS-1 under Luria-Bertani (LB) medium, M9 medium, in vivo rice plant conditions and leaf extract (LE) medium mimicking in vivo plant condition. Proteomic analysis identified 95, 72, 75, and 87 MPs under LB, in vivo, M9 and LE conditions, respectively. Among them, six proteins were shared under all tested growth conditions designated as abundant class of proteins. Twenty-six and 21 proteins were expressed uniquely under in vivo versus LB medium and LE versus M9 medium, respectively, with 17 proteins common among these uniquely induced proteins. Moreover, most of the shared proteins are mainly related to energy metabolism, transport of small molecules, protein synthesis and secretion as well as virulence such as NADH, OmpA, secretion proteins. Therefore, the result of this study not only suggests that it may be an alternate method to analyze the in vivo expression of proteins by using LE medium to mimic plant conditions, but also reveals that the two sets of differentially expressed MPs, in particular the common MPs between them, might be important in energy metabolism, stress response and virulence of A. avenae subsp. avenae strain RS-1.

First Report of Pantoea anthophila Causing Soft Rot Disease in Clausena lansium (Wampee) in China

Clausena lansium, also known as wampee (Clausena wampi), is a plant species native to China, Vietnam, the Philippines, Malaysia, and Indonesia, where it is widely cultivated, and also grown in India, Sri Lanka, Queensland, Florida, and Hawaii, but less frequently (3). The fruit can be consumed fresh or made into juice, jam, or succade. In summer to fall 2014, a soft rot disease was found in a wampee planting region in Yunan County, Guangdong Province, China. On Sept. 18, we collected diseased samples from a wampee orchard with about 20% disease incidence. The infected fruit initially showed pinpoint spots on the peel, water-soaked lesions, and light to dark brown discoloration. Spots expanded in 2 days, and tissues collapsed after 5 days. Severely affected fruit showed cracking or nonodorous decay. Five diseased samples were collected, and causal agents were isolated from symptomatic tissues 1 cm under the peel after surface sterilization in 0.3% NaOCl for 10 min and rinsing in sterile water three times. Tissues were placed on a Luria Bertani (LB) plate for culture. Ten representative isolates were selected for further characterization. No colony was isolated from healthy tissues. Colonies were round, smooth, with irregular edges, and produced a yellow pigment in culture. Biolog identification (Version 4.20.05) showed that all strains were gram negative, negative for indole production, and utilized glucose, maltose, trehalose, sucrose, D-lactose, and pectin but not sorbitol or gelatin. The isolates were identified as Pantoea agglomerans (SIM 0.69). Multilocus sequence analysis (MLSA) was conducted for rapid classification of the strains. Sequences of atpD, gyrB, infB, and rpoB were amplified using corresponding primers (2). All sequences of the 10 isolates were identical in each gene. BLASTn was performed, and maximum likelihood trees based on the concatenated nucleotide sequences of the four genes were constructed using MEGA6. Bootstrap values after 1,000 replicates were expressed as percentages. Results showed that the tested strain named CL1 was most homologous to P. anthophila, with 98% identity for atpD (KM521543), 100% for gyrB (KM521544), infB (KM521545), and rpoB (KM521546). The 16S rRNA sequence (KM521542) amplified by primers 27f and 1492r shared 99% identity with that of P. anthophila M19_2C (JN644500). P. anthophila was previously reclassified from P. agglomerans (3); therefore, we suggest naming this wampee pathogen P. anthophila. Subsequently, 10 wampee fruits were injected with 20 μl of bacterial suspension (1 × 108 CFU/ml) of strains CL1 and CL2, respectively, and another 10 were injected with 20 μl of LB medium as controls, all kept at 28°C for 4 days. Symptoms similar to those of natural infections were observed on inoculated fruits but not on the negative controls. Bacteria were isolated from diseased tissues and further identified as P. anthophila by gyrB sequencing. P. anthophila was reported to naturally infect balsam and marigold (1,2). To our knowledge, this is the first report of P. anthophila naturally causing soft rot disease and cracking on C. lansium (wampee).

Green synthesis and characterization of Carica papaya leaf extract coated silver nanoparticles through X-ray diffraction, electron microscopy and evaluation of bactericidal properties

The evolution of nanotechnology and the production of nanomedicine from various sources had proven to be of intense value in the field of biomedicine. The smaller size of nanoparticles is gaining importance in research for the treatment of various diseases. Moreover the production of nanoparticles is eco-friendly and cost effective. In the present study silver nanoparticles were synthesized from Carica papaya leaf extract (CPL) and characterized for their size and shape using scanning electron microscopy and transmission electron microscopy, respectively. Fourier transform infrared spectroscopy (FTIR), Energy dispersive X-ray spectroscopy (EDS/EDX) and X-ray diffraction spectroscopy (XRD) were conducted to determine the concentration of metal ions, the shape of molecules. The bactericidal activity was evaluated using Luria Bertani broth cultures and the minimum inhibition concentration (MIC) and minimum bactericidal concentration (MBC) were estimated using turbidimetry. The data analysis showed size of 50–250nm spherical shaped nanoparticles. The turbidimetry analysis showed MIC and MBC was >25μg/mL against both Gram positive and Gram negative bacteria in Luria Bertani broth cultures. In summary the synthesized silver nanoparticles from CPL showed acceptable size and shape of nanoparticles and effective bactericidal activity.

High-yield recombinant expression of the chicken antimicrobial peptide fowlicidin-2 in Escherichia coli.

The antimicrobial peptide fowlicidin-2 identified in chicken is a member of the cathelicidins family. The mature fowlicidin-2 possesses high antibacterial efficacy and lipopolysaccharide (LPS) neutralizing activity, and also represents an excellent candidate as an antimicrobial agent. In the present study, the recombinant fowlicidin-2 was successfully produced by Escherichia coli (E. coli) recombinant expression system. The gene encoding fowlicidin-2 with the codon preference of E. coli was designed through codon optimization and synthesized in vitro. The gene was then ligated into the plasmid pET-32a(+), which features fusion protein thioredoxin at the N-terminal. The recombinant plasmid was transformed into E. coli BL21(DE3) and cultured in Luria-Bertani (LB) medium. After isopropyl-β-D-thiogalactopyranoside (IPTG) induction, the fowlicidin-2 fusion protein was successfully expressed as inclusion bodies. The inclusion bodies were dissolved and successfully released the peptide in 70% formic acid solution containing cyanogen bromide (CNBr) in a single step. After purification by reverse-phase high-performance liquid chromatography (RP-HPLC), ∼6.0 mg of fowlicidin-2 with purity more than 97% was obtained from 1 litre of bacteria culture. The recombinant peptide exhibited high antibacterial activity against the Gram-positive and Gram-negative bacteria, and even drug-resistant strains. This system could be used to rapidly and efficiently produce milligram quantities of a battery of recombinant antimicrobial peptides as well as for large-scale production.

A D, D-carboxypeptidase is required for Vibrio cholerae halotolerance.

The biological roles of low molecular weight penicillin-binding proteins (LMW PBP) have been difficult to discern in Gram-negative organisms. In Escherichia coli, mutants lacking these proteins often have no phenotype, and cells lacking all seven LMW PBPs remain viable. In contrast, we report here that Vibrio cholerae lacking DacA-1, a PBP5 homologue, displays slow growth, aberrant morphology and altered peptidoglycan (PG) homeostasis in Luria-Bertani (LB) medium, as well as a profound plating defect. DacA-1 alone among V. cholerae's LMW PBPs is critical for bacterial growth; mutants lacking the related protein DacA-2 and/or homologues of PBP4 or PBP7 displayed normal growth and morphology. Remarkably, the growth and morphology of the dacA-1 mutant were unimpaired in LB media containing reduced concentrations of NaCl (100 mM or less), and also within suckling mice, a model host for the study of cholera pathogenesis. Peptidoglycan from the dacA-1 mutant contained elevated pentapeptide levels in standard and low salt media, and comparative analyses suggest that DacA-1 is V. cholerae's principal DD-carboxypeptidase. The basis for the dacA-1 mutant's halosensitivity is unknown; nonetheless, the mutant's survival in biochemically uncharacterized environments (such as the suckling mouse intestine) can be used as a reporter of low Na(+) content.

A simple, fast and cost-effective method of synthesis of cupric oxide nanoparticle with promising antibacterial potency: Unraveling the biological and chemical modes of action

BackgroundGradual attainment of bacterial resistance to antibiotics led us to develop a robust method of synthesis of stable, colloidal cupric oxide nanoparticle of physiological pH with potential antibacterial action. MethodsCu(II) oxide NP was synthesized by reduction–oxidation of CuCl2, using polyvinyl alcohol as stabilizer. Characteristics and antibacterial activity of the particles were investigated by techniques like UV–Vis spectrophotometry, DLS, AFM, TEM, EDS, FTIR, AAS, agar plating, FACS, gel electrophoresis and XPS. ResultsThe NPs were about 50nm in size and cubic in shape with two surface plasmon peaks at 266 and 370nm and had semi-conducting behavior with a band gap of 3.40 and 3.96eV. About 80% of precursor CuCl2 was converted to NP. The minimum inhibitory and the minimum bactericidal concentrations of CuO-NP were respectively 120 and 160μg/mL for Escherichia coli and 180 and 195μg/mL for Staphylococcus aureus in Luria–Bertani medium. In growth media, the NPs got modified by media organics with displacement of the stabilizer PVA molecules. This modified NP (around 240nm) killed cells by generating ROS, which finally caused membrane lipid per-oxidation and chromosomal DNA degradation in NP-treated cells. ConclusionReports indicate that we are among the few who had prepared CuO-NP in colloidal form. The antibacterial potency of our particle in growth media was much promising than other reports. Our findings demonstrated that ‘particle-specific’ effect, not ‘ion-specific’ one, was responsible for the NP action. General significanceThe NP may be used as a sterilizing agent in various bioprocesses and as substituent of antibiotics, after thorough toxicological study.

Factors Derived From Escherichia Coli Nissle 1917, Grown in Different Growth Media, Enhance Cell Death in a Model of 5-Fluorouracil-Induced Caco-2 Intestinal Epithelial Cell Damage

We evaluated supernatants (SNs) from Escherichia coli Nissle 1917 (EcN) grown in commonly used growth media for their capacity to affect the viability of Caco-2 colon cancer cells in the presence and absence of 5-Fluorouracil (5-FU) chemotherapy. EcN was grown in Luria-Bertani (LB), tryptone soya (TSB), Man Rogosa Sharpe (MRS), and M17 broth supplemented with 10% (v/v) lactose solution (M17). Human Caco-2 colon cancer cells were treated with DMEM (control), growth media alone (LB, TSB, MRS, and M17) or EcN SNs derived from these 4 media, in the presence and absence of 5-FU. Cell viability, reactive oxygen species (ROS), and cell monolayer permeability were determined. EcN SN in LB medium reduced Caco-2 cell viability significantly, to 51% at 48 h. The combination of this EcN SN and 5-FU further reduced cell viability to 37% at 48 h, compared to 5-FU control. MRS broth and EcN SN in MRS, together with 5-FU, generated significantly lower levels of ROS compared to 5-FU control. However, all 5-FU treatments significantly disrupted the Caco-2 cell barrier compared to control; with no significant differences observed among any of the 5-FU treatments. EcN SNs (LB+) was most effective at decreasing the viability of Caco-2 cells. This could indicate a potential role for this EcN SN in chemoprevention for colon cancer.

Comparative phenotypic and genotypic virulence of Salmonella strains isolated from Australian layer farms.

There are over 2500 Salmonella enterica serovars that circulate globally. Of these, serovars those classified into subspecies I are the most common cause of human salmonellosis. Many subspecies I Salmonella serovars are routinely isolated from egg farm environments but are not frequently associated with causing disease in humans. In this study, virulence profiles were generated for 10 strains of Salmonella enterica isolated directly from egg farm environments to investigate their potential public health risk. Three virulence parameters were assessed including in vitro invasion, in vivo pathogenicity and characterization of genomic variation within five specific pathogenicity islands. These 10 Salmonella strains exhibited significant differences in invasion into the human intestinal epithelial cell line, Caco2. Low, moderate, and high invasion patterns were observed and the degree of invasion was dependent on bacterial growth in a nutritive environment. Interestingly, two Salmonella strains, S. Adelaide and S. Bredeney had consistently low invasion. The S. Typhimurium definitive types and S. Virchow exhibited the greatest cell invasion following growth in Luria Bertani broth. Only the S. Typhimurium strains caused disease in BALB/c mice, yet the majority of serovars were consistently detected in feces over the 21 day experiment. Genomic comparison of the five specific pathogenicity islands has shown that variation in virulence is likely multifactorial. Sequence variability was observed primarily in strains with low virulence. In particular, genes involved in forming the structures of the SPI-1 and SPI-2 type 3 secretion systems as well as multiple effector proteins were among the most variable. This variability suggest that serovars with low virulence are likely to have both invasion and within host replication defects that ultimately limit their pathogenicity.

Heat resistance of Cronobacter sakazakii DPC 6529 and its behavior in reconstituted powdered infant formula

Cronobacter sakazakii is an opportunistic pathogen in neonates which can cause meningitis, septicaemia and enterocolitis related to the consumption of contaminated Powdered Infant Formula (PIF). C. sakazakii has an unusual ability to survive under dry conditions and it could be among the most thermotolerant members of the Enterobacteriaceae. Little is known about how Cronobacter species respond to heat stress and the mechanisms involved in the process. In the current study we determined the heat resistance of a particularly stress tolerant C. sakazakii strain, C. sakazakii DPC 6529, and monitored the behavior of a lux-tagged derivative under different reconstitution and handling scenarios in a commercial brand of PIF. Some of the molecular mechanisms involved in the heat stress response were investigated using a transposon mutagenesis approach. Survival curves of C. sakazakii DPC 6529 in Luria-Bertani (LB) broth and PIF at various temperatures (58, 60, 62 and 64°C) displayed an upward concavity and were fitted to the non-linear Weibull model. While at the highest treatment temperatures heat resistance was lower in PIF than in LB broth, at lower temperatures no significant differences in heat resistance were observed. Experiments in real time with artificially inoculated PIF reconstituted at different water temperatures (50, 55, 60, 65, 70°C) and cooled at different rates confirmed that C. sakazakii can survive for long time periods in powdered formula, and is capable of proliferating after reconstitution. The use of water at temperatures between 50 and 65°C for reconstitution did not provide a significant inactivation of C. sakazakii cells. Reconstitution at 70°C reduced the bacterium to levels below the detection limit, although survivors were able to proliferate and reached dangerous levels when the reconstituted product was stored for a long time at room temperature. The cooling rate had an important impact on survival and subsequent growth of C. sakazakii, which makes it advisable to avoid rapid cooling of baby formula. Transposon mutagenesis allowed the identification of some of the molecular mechanisms involved in the response of C. sakazakii DPC6529 to heat stress. Genes identified included the Ribosome Maturation Protein RimP and Outer Membrane Porin L (OmpL). Results suggest that de novo protein synthesis, and the uptake of cysteine for the formation of disulfide bonds for protein stabilization, are key processes.

Increasing plasmid-based DNA vaccine construct (16 kb pSVK-HBVA) production in Escherichia coli XL10-Gold through optimization of media component

At present, there are production processes to produce protein by Escherichia coli (E. coli) fermentation. Research on the design and optimization of the plasmid fermentation medium, however, is less advanced. The fermentation medium that is optimized for plasmid DNA production is different from the medium that is optimized for protein production. So, establishing a scientific and rational method to optimize the fermentation medium used for plasmid production is very important. Previously, our laboratory developed a novel therapeutic DNA vaccine (named pSVK-HBVA) for hepatitis B based on the alphavirus replicon, and found that E. coli XL10-Gold was the optimal host strain for the production of plasmid pSVK-HBVA. The aim of this study was to establish a scientific and rational method to optimize the fermentation medium used for plasmid production, and investigate the effect of growth medium composition on the production of plasmid pSVK-HBVA harboured in E. coli XL10-Gold, as well as to optimize the medium composition.The one-factor-at-a-time experiments demonstrated that Luria-Bertani (LB) was the optimal basic medium. The optimal carbon source and nitrogen source were glycerol and home-made proteose peptone, respectively. Based on the Plackett–Burman (PB) design, proteose peptone, glycerol and NH4Cl were identified as the significant variables, which were further optimized by the steepest ascent (descent) method and central composite design. Growth medium optimization in 500-mL shake flasks by response surface methodology resulted in a maximum volumetric yield of 13.61 mg/L, which was approximately 2.5 times higher than that obtained from the basic medium (LB).

Variation in Heat and Pressure Resistance of Verotoxigenic and Nontoxigenic Escherichia coli

This study evaluated the heat and pressure resistance of 112 strains of Escherichia coli, including 102 strains of verotoxigenic E. coli (VTEC) representing 23 serotypes and four phylogenetic groups. In an initial screening, the heat and pressure resistance of 100 strains, including 94 VTEC strains, were tested in phosphate-buffered saline (PBS). Treatment at 60°C for 5 min reduced cell counts by 2.0 to 5.5 log CFU/ml; treatment at 600 MPa for 3 min at 25°C reduced the cell counts by 1.1 to 5.5 log CFU/ml. Heat or pressure resistance did not correlate to the phylogenetic group or the serotype. A smaller group of E. coli strains was evaluated for heat and pressure resistance in Luria-Bertani (LB) broth. Generally, the levels of heat resistance of E. coli strains in LB and PBS were similar; however, the levels of pressure resistance observed for treatments in LB broth or PBS were variable. The cell counts of pressure-resistant strains of VTEC were reduced by less than 1.5 log CFU/ml after treatment at 600 MPa for 3 min. E. coli strains were also treated with 600 MPa for 3 min in ground beef or inoculated into beef patties and grilled to 63 or 71°C. The cell counts of the VTEC E. coli O26:H11 strain 05-6544 were reduced by 2 log CFU/g by pressure treatment in ground beef. The cell counts of the heat-resistant E. coli strain AW1.7 were reduced by 1.4 and 3.4 log CFU/g in beef patties grilled to internal temperatures of 63 and 71 °C, respectively. The cell counts of E. coli 05-6544 were reduced by less than 3 and 6 log CFU/g in beef patties grilled to internal temperatures of 63 and 71 °C, respectively. To study whether the composition of the beef patties influenced heat resistance, E. coli strains AW1.7, AW1.7ApHR1, MG1655, and LMM1030 were mixed into beef patties containing 15 or 35% fat and 0 or 2% NaCl, and the patties were grilled to an internal temperature of 63°C. The highest heat resistance of E. coli was observed in patties containing 15% fat and 2% NaCl.