Microstructure Investigation and Cyclic Oxidation Resistance of Ce-Si-Modified Aluminide Coating Deposited by Pack Cementation on Inconel 738LC

The synergistic effect of Si and Ce addition on the oxidation resistance of a pack cementation aluminide coating applied on a Ni-based IN738LC superalloy substrate was investigated in this study. The structural and thermal influences of both Si and Ce, focusing on morphology, oxidation behavior, and scale spallation tendency, are accordingly discussed based on the experimental results using X-ray diffraction (XRD), scanning electron microscopy (SEM), and energy-dispersive X-ray analyses (EDX). For this purpose, the oxidation resistance of the modified coatings was evaluated by measuring the weight gain of the coated samples after 16 h for each cycle at 1100 °C for a total of 50 cycles of the oxidation process. The investigations indicated that Si addition to the modified aluminide coating improves the oxidation resistance through the formation of β-NiAl and δ-Ni2Al3 phases, and also δ-Ni2Si phases. Furthermore, the addition of 1% Ce to the modified aluminide coating enhances the formation of the fine-grained microstructure of the β-NiAl and δ-Ni2Al3 and reduces the outward/inward diffusion of elements (so-called blocking effect), which significantly modifies the cyclic oxidation resistance. The oxidation enhancement also may be attributed to synergistic effects of Ce and Si addition during the deposition process that reduce the inward oxygen diffusion and reduce the growth rate of α-Al2O3 during oxidation tests

Selected lactic acid-producing bacterial isolates with the capacity to reduce Salmonella translocation and virulence gene expression in chickens.

BACKGROUND:Probiotics have been used to control Salmonella colonization/infection in chickens. Yet the mechanisms of probiotic effects are not fully understood. This study has characterized our previously-selected lactic acid-producing bacterial (LAB) isolates for controlling Salmonella infection in chickens, particularly the mechanism underlying the control. METHODOLOGY/PRINCIPAL FINDINGS:In vitro studies were conducted to characterize 14 LAB isolates for their tolerance to low pH (2.0) and high bile salt (0.3-1.5%) and susceptibility to antibiotics. Three chicken infection trials were subsequently carried out to evaluate four of the isolates for reducing the burden of Salmonella enterica serovar Typhimurium in the broiler cecum. Chicks were gavaged with LAB cultures (10(6-7) CFU/chick) or phosphate-buffered saline (PBS) at 1 day of age followed by Salmonella challenge (10(4) CFU/chick) next day. Samples of cecal digesta, spleen, and liver were examined for Salmonella counts on days 1, 3, or 4 post-challenge. Salmonella in the cecum from Trial 3 was also assessed for the expression of ten virulence genes located in its pathogenicity island-1 (SPI-1). These genes play a role in Salmonella intestinal invasion. Tested LAB isolates (individuals or mixed cultures) were unable to lower Salmonella burden in the chicken cecum, but able to attenuate Salmonella infection in the spleen and liver. The LAB treatments also reduced almost all SPI-1 virulence gene expression (9 out of 10) in the chicken cecum, particularly at the low dose. In vitro treatment with the extracellular culture fluid from a LAB culture also down-regulated most SPI-1 virulence gene expression. CONCLUSIONS/SIGNIFICANCE:The possible correlation between attenuation of Salmonella infection in the chicken spleen and liver and reduction of Salmonella SPI-1 virulence gene expression in the chicken cecum by LAB isolates is a new observation. Suppression of Salmonella virulence gene expression in vivo can be one of the strategies for controlling Salmonella infection in chickens

Microencapsulation of Bacteriophage Felix O1 into Chitosan-Alginate Microspheres for Oral Delivery▿

This paper reports the development of microencapsulated bacteriophage Felix O1 for oral delivery using a chitosan-alginate-CaCl2 system. In vitro studies were used to determine the effects of simulated gastric fluid (SGF) and bile salts on the viability of free and encapsulated phage. Free phage Felix O1 was found to be extremely sensitive to acidic environments and was not detectable after a 5-min exposure to pHs below 3.7. In contrast, the number of microencapsulated phage decreased by 0.67 log units only, even at pH 2.4, for the same period of incubation. The viable count of microencapsulated phage decreased only 2.58 log units during a 1-h exposure to SGF with pepsin at pH 2.4. After 3 h of incubation in 1 and 2% bile solutions, the free phage count decreased by 1.29 and 1.67 log units, respectively, while the viability of encapsulated phage was fully maintained. Encapsulated phage was completely released from the microspheres upon exposure to simulated intestinal fluid (pH 6.8) within 6 h. The encapsulated phage in wet microspheres retained full viability when stored at 4°C for the duration of the testing period (6 weeks). With the use of trehalose as a stabilizing agent, the microencapsulated phage in dried form had a 12.6% survival rate after storage for 6 weeks. The current encapsulation technique enables a large proportion of bacteriophage Felix O1 to remain bioactive in a simulated gastrointestinal tract environment, which indicates that these microspheres may facilitate delivery of therapeutic phage to the gut

<i>Lactobacillus zeae</i> Protects <i>Caenorhabditis elegans</i> from Enterotoxigenic <i>Escherichia coli</i>-Caused Death by Inhibiting Enterotoxin Gene Expression of the Pathogen

<div><p>Background</p><p>The nematode <i>Caenorhabditis elegans</i> has become increasingly used for screening antimicrobials and probiotics for pathogen control. It also provides a useful tool for studying microbe-host interactions. This study has established a <i>C. elegans</i> life-span assay to preselect probiotic bacteria for controlling K88⁺ enterotoxigenic <i>Escherichia coli</i> (ETEC), a pathogen causing pig diarrhea, and has determined a potential mechanism underlying the protection provided by <i>Lactobacillus</i>.</p><p>Methodology/Principal Findings</p><p>Life-span of <i>C. elegans</i> was used to measure the response of worms to ETEC infection and protection provided by lactic acid-producing bacteria (LAB). Among 13 LAB isolates that varied in their ability to protect <i>C. elegans</i> from death induced by ETEC strain JG280, <i>Lactobacillus zeae</i> LB1 offered the highest level of protection (86%). The treatment with <i>Lactobacillus</i> did not reduce ETEC JG280 colonization in the nematode intestine. Feeding <i>E. coli</i> strain JFF4 (K88⁺ but lacking enterotoxin genes of <i>estA</i>, <i>estB</i>, and <i>elt</i>) did not cause death of worms. There was a significant increase in gene expression of <i>estA</i>, <i>estB</i>, and <i>elt</i> during ETEC JG280 infection, which was remarkably inhibited by isolate LB1. The clone with either <i>estA</i> or <i>estB</i> expressed in <i>E. coli</i> DH5α was as effective as ETEC JG280 in killing the nematode. However, the <i>elt</i> clone killed only approximately 40% of worms. The killing by the clones could also be prevented by isolate LB1. The same isolate only partially inhibited the gene expression of enterotoxins in both ETEC JG280 and <i>E. coli</i> DH5α <i>in-vitro</i>.</p><p>Conclusions/Significance</p><p>The established life-span assay can be used for studies of probiotics to control ETEC (for effective selection and mechanistic studies). Heat-stable enterotoxins appeared to be the main factors responsible for the death of <i>C. elegans</i>. Inhibition of ETEC enterotoxin production, rather than interference of its intestinal colonization, appears to be the mechanism of protection offered by <i>Lactobacillus</i>.</p></div

Effect of feeding isolates LB1 (<i>Lactobacillus zeae</i>) and CL11 (<i>Lactobacillus casei</i>) on the survival of <i>C. elegans</i> infected with ETEC JG280 and on bacterial colonization of the nematode intestine.

<p>(A) Survival (%) of <i>C. elegans</i> in the presence or absence of <i>Lactobacillus</i>. (B) Colonization of ETEC JG280 in the intestine of worms. (C) Colonization of <i>Lactobacillus</i> in the intestine of worms. Control worms were fed <i>E. coli</i> OP50 only, either isolate LB1 or CL11 at 10⁸CFU/ml for 8 days. In other treatments, worms were first fed either <i>E. coli</i> OP50 or <i>Lactobacillus</i> (isolate LB1 or CL11) at 10⁸ CFU/ml for 18 h and then ETEC JG280 for the remaining days. Treatments:○, <i>E. coli</i> OP50 only; ▪, <i>E. coli</i> OP50 and then ETEC JG280; □, isolate LB1 only; •, isolate CL11 only; △, isolate LB1 and then JG280; × isolate CL11 and then JG280. The curves of two treatments (□ and ○) were almost overlapped in Panel A.</p

Statistical analysis of the protection effect of lactic acid-producing bacterial isolates on <i>C. elegans</i> infected with ETEC JG280^a.

a<p>Summary of two or more separate experiments. Survival of worms on the last day (day 10) of the assays with 95% confidence interval (CI) was estimated with the Kaplan-Meier survival analysis.</p>b<p>E+JG280: treatment with <i>E. coli</i> OP50 and then with ETEC K88 strain JG280. In the assays with LAB isolates, the nematode was firstly treated with a LAB isolate and then with ETEC JG280.</p>c<p>C, isolates from chickens; P, isolates from pigs.</p>d<p>Putative species identity was determined by BLAST analysis of sequences of 16S rRNA genes. Sequence similarities between the isolates and the 16S rDNA database sequences were 98 to 100%. Among the thirteen isolates, CL10, CL11, CL12, S64 and LB1 have been reported previously (7).</p>e<p>DT50, the time at which half of the worms were dead.</p>f<p>Comparisons of survival curves. L+J, <i>C. elegans</i> was treated with <i>Lactobacillus</i> and then JG280. L+J vs <i>E. coli</i> OP50, the statistical difference between the group of <i>C. elegans</i> treated with <i>Lactobacillus</i> followed by JG280 and the group of <i>C. elegans</i> treated with <i>E. coli</i> OP50 only (control group). L+J vs E+J, the statistical difference between the group of <i>C. elegans</i> treated with <i>Lactobacillus</i> followed by JG280 and the group of <i>C. elegans</i> treated with <i>E. coli</i> OP50 followed by JG280.</p

Establishment of a life-span assay of <i>C. elegans</i> infected with K88⁺ ETEC strain JG280.

<p>The life-span is expressed as survival of <i>C. elegans</i> during the assay after infection with JG280 at different cell concentrations. In the assay, worms were fed one of the following for 10 days: ○, <i>E. coli</i> OP50 (food for <i>C. elegans</i>) at 10⁸ CFU/ml; +, JG280 at 10⁷ CFU/ml; ▪, JFF4 at 5×10⁸ CFU/ml; ▴, JG280 at 2×10⁸CFU/ml; ×, JG280 at 5×10⁸CFU/ml.</p

TEM images showing the intestine of <i>C. elegans</i> and the colonization of ETEC and <i>L. zeae</i> LB1 in the nematode intestine.

<p>(A) Cross section of the whole intestine of <i>C. elegans</i>. L: the lumen of intestine; W: the wall of intestine. (B) Colonization of ETEC JG280 in the intestine with a bacterial cell attached to the intestinal surface. E: ETEC JG280 cell; S: inner surface of the intestine; L: the lumen of intestine. (C) Co-existence of ETEC JG280 and <i>L. zeae</i> LB1 in the intestinal lumen of worms. E: ETEC JG280 cells; LAC: <i>L. zeae</i> LB1 cells. (D) Image of ETEC JG280 cells showing the inner and outer members of G-negative bacterium. IM: inner member; OM: outer member. The size of images is indicated by the scale bars.</p

Gene expression of enterotoxins in ETEC strain JG280 during the life-span assay in the presence or absence of <i>L. zeae</i> LB1.

<p>The baseline (Gridline bar) is the level of gene expression of three enterotoxins in ETEC JG280 just before mixing with <i>C. elegans</i> (day 0). (A), (B), and (C) represent the expression level of <i>estA</i> (STa), <i>estB</i> (STb), and <i>elt</i> (LT), respectively, produced by ETEC JG280, in the absence (black bars) or presence of <i>L. zeae</i> LB1 (grey bars) on day 1, 2, and 3. Relative expression was determined using the 2^−ΔΔCt method as the ratio of gene transcript level of each time point to zero time point ETEC JG280 (day 0 before inoculation of <i>C. elegans</i>) and expressed as fold changes. Data are presented as mean ± S.D. Means marked with different letters (a, b, c,) are significantly different at <i>P</i> values of <0.05 within the ETEC JG280 group. Means marked with different letters (A, B, C) are significantly different at <i>P</i> values of <0.05 within the <i>L. zeae</i> LB1 group. *Represents significant difference from the ETEC JG280 group within the same day.</p