Desiccation survival in Salmonella enterica, Escherichia coli and Enterococcus faecium related to initial cell concentration and cellular components

Salmonella enterica is well-known for its ability to survive and persist in low-moisture environments. Previous studies have indicated a link between the initial cell concentration and the population of Salmonella that survive upon desiccation and subsequent storage; however, how the initial cell concentration affects survival is unknown. This study examined the basis of this phenomena and whether it occurred in other microorganisms, specifically Shiga toxigenic Escherichia coli (STEC), and Enterococcus faecium . Salmonella, STEC, and E. faecium were grown as lawns on TSAYE and harvested using buffered peptone water (BPW). To determine recovery at different initial cell levels, cultures were diluted to 9, 7, and 5 log CFU/mL and applied to filters. Filters were dried for 24 h, then stored for 28 d at 25°C/33% RH. During storage, cells were recovered from filters using BPW and cultivated on TSAYE. Both Salmonella and E. coli , but not E. faecium , showed non-proportional recovery. Less viability remained with lower initial starting population after 24 h desiccation such that ≥10 log CFU/mL were recovered when 11 log CFU/mL was desiccated, but <3 log CFU/mL were recovered when 5 log CFU/mL was desiccated. Once dried, persistence did not appear affected by initial cell concentration. When dead cells (heat-treated) were added to the diluent, recovery of Salmonella was proportional with respect to the initial cell concentration. To further examine the response on desiccation, Salmonella was diluted in BPW containing one of 11 different test cell components related to quorum sensing or known to affect desiccation resistance to assess recovery and persistence. Of the 11 additions only cell debris fractions, cell-free extract, and peptidoglycan improved recovery of Salmonella . Desiccation survival appears related to cell wall components, however, the exact mechanism affecting survival remains unknown.

Enhancement in Human Insulin Precursor Secretion by Pichia pastoris through Modification of Expression Conditions

Pichia pastoris is an alternative yeast expression system to produce heterologous proteins. It has excellent characteristics for an industrial cell factory, such as its ability to reach high cell densities, high secretory capacity, and a low level of native proteins. In our previous study, we introduced a synthetic insulin precursor (IP)-encoding gene constructed in a pD902 expression vector into P. pastoris. However, the P. pastoris recombinant strains expressed a little amount of IP protein. Here, we modified the expression conditions, including inoculum density, methanol concentration, methanol induction time, pH, and temperature, to obtain a higher amount of secreted IP than our previous result. Protein analysis for studying the five parameters was conducted by SDS-PAGE, and the protein amount was estimated by ImageJ applying lysozyme as standard. We successfully enhanced the IP expression by modifying expression conditions. The highest increased of up to 100 folds was achieved when methanol concentration for induction was arranged at 3% (v/v), and the initial cell density for methanol induction was set at an optical density at 600 nm (OD600) of approximately 10 compared to the standard procedure, where the expression was set at 0.5% (v/v) methanol induction and initial cell density at OD600 = 1.

The effects of substrate morphology by regulating pseudopods formation on cell directional alignment and migration

When cells are cultured on the micro- or nano- structure substrate, filamentous pseudopods are formed at specific locations due to the effects of substrate morphology and local membrane curvature, which provides a powerful method to guide cell migration and neurite orientation. However, it is unclear the effects of substrate surface morphology and initial cell membrane on pseudopod formation and growth. Here, we present a quantitative thermodynamic model to investigate the difficulty of pseudopod formation. Based on the established model, we studied the effects of substrate morphology and the curvature of the initial cell membrane on filamentous pseudopods formation by analyzing the magnitude of an average driving force. We find that the pseudopod-substrate adhesion and the larger curvature radius of the initial cell membrane can facilitate filamentous pseudopods formation due to the smaller minimum resistance energy. Furthermore, our theoretical results seem to show a broad agreement with experimental observations, which implies that these studies would provide useful guidance to control the pseudopods formation on substrate for biomedical applications.

Rapid Detection of Pathogenic Bacteria by the Naked Eye

Escherichia coli O157:H7 and Staphylococcus aureus are common pathogens. Gram-negative bacteria, such as E. coli, contain high concentrations of endogenous peroxidases, whereas Gram-positive bacteria, such as S. aureus, possess abundant endogenous catalases. Colorless 3,5,3′,5′-tetramethyl benzidine (TMB) changes to blue oxidized TMB in the presence of E. coli and a low concentration of H2O2 (e.g., ~11 mM) at pH of 3. Moreover, visible air bubbles containing oxygen are generated after S. aureus reacts with H2O2 at a high concentration (e.g., 180 mM) at pH of 3. A novel method for rapidly detecting the presence of bacteria on the surfaces of samples, on the basis of these two endogenous enzymatic reactions, was explored. Briefly, a cotton swab was used for collecting bacteria from the surfaces of samples, such as tomatoes and door handles, then two-step endogenous enzymatic reactions were carried out. In the first step, a cotton swab containing bacteria was immersed in a reagent comprising H2O2 (11.2 mM) and TMB for 25 min. In the second step, the swab was dipped further in H2O2 (180 mM) at pH 3 for 5 min. Results showed that the presence of Gram-negative bacteria, such as E. coli with a cell number of ≥ ~105, and Gram-positive bacteria, such as S. aureus with a cell number of ≥ ~106, can be visually confirmed according to the appearance of the blue color in the swab and the formation of air bubbles in the reagent solution, respectively, within ~30 min. To improve visual sensitivity, we dipped the swab carrying the bacteria in a vial containing a growth broth, incubated it for ~4 h, and carried out the two-stage reaction steps. Results showed that bluish swabs resulting from the presence of E. coli O157: H7 with initial cell numbers of ≥ ~34 were obtained, whereas air bubbles were visible in the samples containing S. aureus with initial cell numbers of ≥ ~8.5 × 103.

Effect of Initial Cell Concentration on Bio-Oxidation of Pyrite before Gold Cyanidation

Bio-oxidation of refractory sulfidic gold minerals has been applied at the commercial scale as a pre-treatment to improve gold yields and reduce chemical consumption during gold cyanidation. In this study, the effect of initial cell concentration on the oxidation of pyritic gold ore was evaluated with four aerated bioreactors at 30 °C with 10% pulp density and pH maintained at 1.4 with NaOH. Results of NaOH consumption and changes in soluble Fe and S concentrations indicated that increasing the initial cell concentration from 2.3 × 107 to 2.3 × 1010 cells mL−1 enhanced pyrite oxidation during the first week. However, by day 18 the reactor with the lowest initial cell concentration showed profound performance enhancement based on soluble Fe and S concentrations, sulfide-S and pyrite contents in the residues, and subsequent gold leaching of the bio-oxidation residues by cyanidation. Overall, the results showed that the cell concentration was clearly beneficial during the initial stages of oxidation (first 7–8 days).

Influence of the Initial Cell Number on the Growth Fitness of Salmonella Enteritidis in Raw and Pasteurized Liquid Whole Egg, Egg White, and Egg Yolk

Salmonella growth in egg and egg products has been widely studied, but there are still some aspects that are not fully known. The objective of this work was to study the influence of the initial cell number on the growth fitness of Salmonella Enteritidis in raw and pasteurized egg products. Growth curves of five Salmonella Enteritidis strains in raw and pasteurized egg products, starting from different initial numbers, were obtained and fitted to the Baranyi and Roberts model. The results revealed that lower initial numbers led to longer lag phases (λ) and lower maximum specific growth rates (μmax) in raw liquid whole egg. Similar results were observed in raw egg white (except for one strain). Conversely, no influence (p > 0.05) of the initial concentration on Salmonella growth parameters in raw egg yolk was observed. On the other hand, no influence of the initial number of cells on Salmonella growth fitness in commercial pasteurized liquid whole egg was observed. The results obtained demonstrate that the disappearance of this initial-dose dependency phenomenon was dependent on the intensity of the thermal treatment applied. Finally, the influence of the initial number was, in general, lower in pasteurized than in raw egg white, but large differences among strains were observed.