Replacement Of Antibiotics Research Articles

The Inhibitory Effects of Lactobacillus Agilis Against Pseudomonas Aeruginosa Biofilm Formation and Evaluation of PpyR (PA2663) and algD Gene Expression

Background and Aims: Hospital infections and their antibiotic resistance have become a global concern recently. One of the most prominent factors in hospital infections is Pseudomonas aeruginosa (P. aeruginosa), which can become resistant to many antibiotics due to its ability to form biofilms. Recently, scientists have tried to replace antibiotic therapy with alternative therapies such as probiotics which can reduce or eliminate the pathogenic bacteria's ability to form biofilms. Therefore, the present study revealed that some genes, such as algD and PpyR, were involved in biofilm formation in P. aeruginosa. Furthermore, the inhibitory effect of the supernatant of lactobacillus agilis on the biofilm formation of P. aeruginosa was evaluated in the current study.
 Materials and Methods: In this study, the effect of the supernatant of probiotic Lactobacillus agilis on the biofilm formation of P. aeruginosa and also the expression of two genes effective in biofilm formation (algD and PpyR) were investigated. Antibiograms were performed to detect the most resistant bacteria since there is a link between biofilm formation and antibiotic resistance. Further, the effects of probiotics on the expression of PpyR and algD genes were discussed.
 Results: Results showed that the biofilm formation of P. aeruginosa was significantly reduced in the presence of lactobacillus agilis.
 Conclusions: According to the current study, it could be concluded that because of antibiotics resistance and their associated mechanisms, probiotics could be used as a replacement for antibiotics in many treatments.

Effects of nonantibiotic growth promoter combinations on growth performance, nutrient utilization, digestive enzymes, intestinal morphology, and cecal microflora of broilers.

Given the ban on antibiotic growth promoters, the effects of nonantibiotic alternative growth promoter combinations (NAGPCs) on the growth performance, nutrient utilization, digestive enzyme activity, intestinal morphology, and cecal microflora of broilers were evaluated. All birds were fed pellets of two basal diets-starter (0-21 d) and grower (22-42 d)-with either enramycin (ENR) or NAGPC supplemented. 1) control + ENR; 2) control diet (CON, basal diet); 3) control + mannose oligosaccharide (MOS) + mannanase (MAN) + sodium butyrate (SB) (MMS); 4) control + MOS + MAN + Bacillus subtilis (BS) (MMB); 5) control + MOS + fruit oligosaccharide (FOS) + SB (MFS); 6) control + FOS + BS (MFB); 7) control + MOS + FOS + MAN (MFM); 8) control + MOS + BS + phytase (PT) (MBP). ENR, MOS, FOS, SB, MAN, PT, and BS were added at 100, 2,000, 9,000, 1,500, 300, 37, and 500 mg/kg, respectively. The experiment used a completely random block design with six replicates per group: 2400 Ross 308 broilers in the starter phase and 768 in the grower phase. All NAGPCs significantly improved body weight gain (P < 0.01), utilization of dry matter, organic matter, and crude protein (P < 0.05), villus height and villus height/crypt depth in the jejunum and ileum (P < 0.01), and decreased the feed conversion ratio (P < 0.01) at d 21 and 42. MMS, MMB, MFB, and MFM duodenum trypsin, lipase, and amylase activities increased significantly (P < 0.05) at d 21 and 42. On d 21 and 42, MMS, MMB, and MBP increased the abundance of Firmicutes and Bacteroides whereas MMB, MFB, and MBP decreased the abundance of Proteobacteria, compared to ENR and CON. Overall, the NAGPCs were found to have some beneficial effects and may be used as effective antibiotic replacements in broilers.

Manipulation of hydrophobic motifs and optimization of sequence patterns to design high stability peptides against piglet bacterial infections

The risk of resistance to traditional antibiotics has led to a shift in attention to novel antimicrobial functional biomaterials. Antimicrobial peptides (AMPs) have been recognized since the date of birth as a powerful candidate for antibiotic replacement materials. However, immature salt and protease stability, as well as systemic toxicity, hinder the translation of AMPs from the bench to the bedside. Herein, the original motif (KP)3(HYXP)n(KP)3-NH2 (X = Trp or Phe, Y=Ile or Leu, n = 2, 3, 4, 5, or 6) was designed based on the principles of manipulating hydrophobic motifs, optimizing sequence patterns, as well as circumventing cleavage sites of proteases. Original motif was derived a 20-member library for obtaining AMPs with excellent antimicrobial activity, high salt, and protease stability. Results indicated that the target peptides HLFP-5 and HLWP-4 obtained using a comprehensive and integrated screening process exhibited excellent antibacterial ability, salts stability, and protease stability. Membrane cleavage and cell cycle interference-dominated mechanisms of action make target peptides less susceptible to drug resistance than antibiotics. Additionally, advanced target peptides HLFP-5 and HLWP-4 were able to exert direct antimicrobial efficacy in vivo to treat piglets with a systemic bacterial infection. Collectively, these findings may provide novel insights for future design principles, and help drive the application of antibacterial biomaterials in animal husbandry for replacing antibiotics.

Oregano extract fed to pre-weaned dairy calves. Part 2: Effect on ruminal and intestinal morphology of pre-weaned calves

The aim of this study was to evaluate the effects of oregano extract (Origanum vulgare) supplementation on the histological and morphological variables of the gastrointestinal tract (GIT) of suckling dairy calves. Ten Holsteins calves from commercial dairy farms were fed 6 L/day of milk replacer, calf starter ad libitum from the first day of the trial, as well were fed hay ad libitum from the third week of the start of the study period. Calves had free access to drinking water during the trial. Calves were distributed in two treatments: (1) basal diet without additive (CON); (2) the same diet, but containing 60 mg/kg body weight/day of oregano extract (OR). Weaning occurred on the 53rd day of the trial (56th day of life). On the 54th day of the trial, all calves were euthanized and the full and empty tracts were weighed and tissue samples from the rumen, jejunum, cecum and colon were also taken for histological analysis. The variables observed were: full and empty weight of the gastrointestinal tract; papillae height, tunica of the submucosa, number of papillae, number of branches, branch height and number of papillae/branch in the rumen; number of glands, villi length, mucosal thickness, submucosal thickness, muscle thickness in the jejunum, cecum and colon. Oregano extract increased the empty weight of the abomasum without changing the weight of the other segments. In the rumen, OR-supplemented calves showed lower number of branches, branch height and number of papillae/branches. Calves in CON tended to have thicker muscle layer at the jejunum than calves in OR. Nutrient fractions intake were positively correlated with empty weights of rumen, reticulum, omasum, abomasum and large intestine, but they did not correlate with full and empty weights of small intestine. The intake of the solid diet was not related with GIT organs’ weight. Rumen papillae length, jejunum mucosal and submucosal layer thickness values were positively related with nutrients intake, while rumen submucosal layer thickness were negatively related with nutrients intake. Most histological variables evaluated were not affected by OR supplementation. Essential oils are promising replacements for antibiotics. However, the dose, administration routes and supply time deserve further studies, allowing for better performance and health of the animal to be achieved.

CIM monolithic chromatography as a useful tool for endotoxin reduction and purification of bacteriophage particles supported with PAT analytics

Bacteriophages represent immense potential as therapeutic agents. Many of the most compelling applications of bacteriophages involve human therapy, some pertinent to gene therapy, others involving antibiotic replacement. Phages themselves are considered safe for humans. However, phage lysates may contain many kinds of harmful by-products, especially endotoxins of gram-negative bacteria and protein toxins produced by many pathogenic bacterial species. In bacteriophage research and therapy, most applications ask for highly purified phage suspensions, as such it is crucial to reduce proteins, endotoxins, DNA and other contaminants.In this article we present an efficient two-step chromatographic purification method for P. aeruginosa bacteriophage PP-01, using Convective Interaction Media (CIM®) monoliths, that is cGMP compliant and easy to scale-up for most stringent production of the therapeutic phage. First chromatographic step on CIMmultus OH resulted in 100% bacteriophage recovery with a reduction of 98 % protein and more than 99 % DNA content. Polishing was conducted using three different column options, CIMmultus with QA, H-Bond and PrimaS ligands. For PP-01 bacteriophage all three different options worked, but multimodal ligands H-Bond and PrimaS outperformed traditional QA in endotoxin removal (7 log step reduction). Additionally, an HPLC analytical method was developed to estimate phage concentration and impurity profile in different in-process samples. The HPLC method shows good correlation with drop assay titration, provides useful insights and can be run very fast with just 20 min per sample analysis.

Portulaca oleracea L. organic acid extract inhibits persistent methicillin-resistant Staphylococcus aureus in vitro and in vivo.

Staphylococcus aureus continues to be one of the most important pathogens capable of causing a wide range of infections in different sites of the body in humans and livestock. With the emergence of methicillin-resistant strains and the introduction of strict laws on antibiotic usage in animals, antibiotic replacement therapy has become increasingly popular. Previous studies have shown that Portulaca oleracea L. extract exerts a certain degree of bacteriostatic effect, although the active ingredients are unknown. In the present study, the antibacterial activity of the organic acid of P. oleracea (OAPO) against S. aureus was examined using a series of experiments, including the minimum inhibitory concentration, growth curve, and bacteriostasis curve. In vitro antibacterial mechanisms were evaluated based on the integrity and permeability of the cell wall and membrane, scanning electron microscopy, and soluble protein content. A mouse skin wound recovery model was used to verify the antibacterial effects of OAPO on S. aureus in vivo. The results showed that OAPO not only improved skin wound recovery but also decreased the bacterial load in skin wounds. Moreover, the number of inflammatory cells and cytokines decreased in the OAPO-treated groups. In summary, this study reports a botanical extract that can inhibit S. aureus in vitro and in vivo, indicating the potential use of OAPO to prevent and control S. aureus infection in the near future.

Xiaochaihu Decoction Treatment of Chicken Colibacillosis by Improving Pulmonary Inflammation and Systemic Inflammation.

Chicken colibacillosis-the most common disease of poultry, is caused mainly by avian pathogenic Escherichia coli (APEC). It has a major impact on the poultry industry worldwide. The present study was conducted to investigate the therapeutic effects of Xiaochaihu Decoction (XCHD) supplementation on clinical manifestation, organ index, bacterial load in organ and inflammatory mediators in a chicken model challenged with APEC. The results showed that all doses of XCHD significantly elevated the survival rate of infected chickens. XCHD improved the clinical signs of infected chickens, reduced the organ index, reduced the bacterial load of organs, and inhibited the secretion of serum and pulmonary inflammatory factors IL-1β, IL-6 and TNF- α. Taken together, this study demonstrates that XCHD had protective effects on APEC-infected chickens. Its mechanism includes anti-inflammatory and antibacterial effects. These findings may contribute to the further study of the mechanism of the formula and the prevention or treatment of colibacillosis in poultry. The significance of this study is that it provides a certain theoretical basis for the replacement of antibiotics by XCHD.

A synthetic peptide based on large yellow croaker (Larimichthys crocea) IFNG1R protein sequence has potential antimicrobial activity against Pseudomonas plecoglossicida

The overuse of antibiotics leads to the emergence of bacterial resistance, which poses a serious threat to aquaculture. Antimicrobial peptides (AMPs) display excellent antimicrobial activity and are thought to be the most feasible replacements for antibiotics. The usage of AMPs as feed additives has great application prospects in aquaculture. In this study, large yellow croaker interferon-γ related gene (IFNG1R) was cloned, and a 17-amino acids (aa) short peptide named SKL17-2 was synthesized based on its protein sequence. The synthesized SKL17-2 peptide possessed a strong antimicrobial activity against Pseudomonas plecoglossicida, which could cause visceral white nodules disease (VWND) in cultured marine fish, with a minimum inhibitory concentration (MIC) of 2 μM. SKL17-2 peptide also showed weak antimicrobial activities against other tested bacteria, indicating its narrow-spectrum antimicrobial activity. This suggested that SKL17-2 peptide may not kill probiotics in intestinal flora when used as the feed additive. Furthermore, SKL17-2 had broad temperature and pH stability, low cytotoxicity, and negligible hemolysis, indicating its good biosafety and stability. Mechanistically, the synthesized SKL17-2 peptide can form α-helical structure in a membrane environment and destroy P. plecoglossicida through membrane disruption. Thus, our data showed that SKL17-2 peptide may represent a potential feed additive used for prevention and treatment of VWND.

Investigation of the synergistic effect of glycolipid biosurfactant produced by Shewanella algae with some antibiotics against planktonic and biofilm forms of MRSA and antibiotic resistant Acinetobacter baumannii.

To tackling antibiotic resistance and the appearance of multidrug-resistant (MDR) strains, one current approach is the combined use of biosurfactants with antibiotics to increase their efficacy. The antimicrobial ability of biosurfactant produced by Shewanella algae strain B12 was examined using the agar well diffusion method versus some resistant Gram-negative and Gram-positive bacteria. The Minimum Inhibitory Concentration (MIC) of Glycolipid-Biosurfactant of B12 (GBB12) was performed by the broth dilution technique. The inhibition of biofilm formation, disruption of biofilm, and reducing the population of viable cells in biofilm were evaluated by the microtiter plate method. Finally, Scanning Electron Microscopy (SEM) analysis was used to confirm the disruption of the cell membrane by GBB12. In all experiments, when GBB12 was added to antibiotics (except Amikacin), the antimicrobial activity was increased. The synergistic effects of GBB12 and antibiotics (Ciprofloxacin and Gentamycin) against Methicillin-Resistant Staphylococcus aureus (MRSA) and Acinetobacter baumannii were confirmed by the Fractional Inhibitory Concentration Index (FICI). GBB12-Gentamycin mixture almost completely inhibits the formation of A. baumannii biofilm, reaching 99.8% inhibition. Also, the rate of MRSA biofilm inhibition treated with GBB12-Ciprofloxacin mixture was found to be 99.4%. biosurfactant-antibiotic mixture could be adequate replacements for traditional antibiotics in the near future. This study shows the potential of GBB12 as antimicrobial and antibiofilm agent.

Chemical Composition, Antimicrobial Activity, and Withdrawal Period of Essential Oil-Based Pharmaceutical Formulation in Bovine Mastitis Treatment

Due to the emergence of antibiotic-resistant bacteria, the risk it represents to public health, and the possible consequences for animal health and welfare, there is an increasing focus on reducing antimicrobial usage (AMU) in animal husbandry. Therefore, a great interest in developing alternatives to AMU in livestock production is present worldwide. Recently, essential oils (EOs) have gained great attention as promising possibilities for the replacement of antibiotics. The current study aimed to test the potential of using a novel EO-based pharmaceutical formulation (Phyto-Bomat) in bovine mastitis treatment. The antibacterial activity was performed using the microdilution technique. Lactating dairy cows were treated with 15 mL of Phyto-Bomat in the inflamed quarter for 5 consecutive days in order to analyze blood and milk samples for thymol and carvacrol residues using gas chromatography and mass spectrometry (GC-MS). Antimicrobial activity expressed as the minimum inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) indicates that this formulation has the highest activity against Gram-positive strains. The dominant compounds in Phyto-Bomat were thymol and carvacrol, at 12.58 ± 1.23 mg/mL and 23.11 ± 2.31 mg/mL, respectively. The quantification of these two compounds in evaluated biological samples showed that 24 h after administration the concentration of thymol and carvacrol in milk samples was at the same level as before application. On the other hand, thymol and carvacrol were detectable in plasma samples even after 24 h post-treatment, with values ranging from 0.15-0.38 and 0.21-0.66 µg/mL, respectively. The tested formulation showed encouraging results of antibacterial activity against bovine mastitis pathogens, as well as the withdrawal period of dominant compounds, which implies that further testing regarding the bacteriological and clinical cure rates in clinical settings is needed.

Isolation, Characterization and Whole Genome Analysis of an Avian Pathogenic Escherichia coli Phage vB_EcoS_GN06.

Escherichia coli (O78) is an avian pathogenic Escherichia coli (APEC). It can cause perihepatitis, pericarditis, septicemia and even systemic infections in the poultry industry. With the incidence of antibiotic resistance reaching a crisis point, it is important to find alternative treatments for multidrug-resistant infections. The use of phages to control pathogens is a promising therapeutic option for antibiotic replacement. In this study, we isolated a lytic phage called vB_EcoS_GN06 from sewage. It lysed APEC GXEC-N22. Transmission electron microscopy showed that the phage belongs to family Siphoviridae. Phage GN06 has a 107,237 bp linear double-stranded DNA genome with 39.2% GC content and 155 coding sequences. It belongs to the genus Tequintavirus, subfamily Markadamsvirinae. The multiplicity of infection of 0.01 and the one-step growth showed that the latent time is 60 min and the burst size is 434 PFU/cell. Temperature and pH stability tests showed that phage GN06 was stable in the range of 4 °C-60 °C and pH 5-9. GN06 showed significant inhibition of APEC both within the liquid medium and in biofilm formation. These results suggest that phage GN06 has the potential to control bacterial pathogens. Thus, GN06 has the potential to be a new potential candidate for phage therapy.

Antimicrobial effect of extract from root of Arctium tomentosum Mill. (woolly burdock) against several reference strains

Microorganisms are one of the main reasons for infectious diseases, however conditionally pathogenic ones are members of the normal human microbiome. Certain circumstances make them become pathogenic to organism. Treatment of microbial diseases majorly is carried out by antibiotics, using of which may lead to antibiotic resistance. Moreover it causes deficiency of gut microflora, subsequent dysbiosis and weakening of immunity. Thus, replacement of antibiotics by naturally derived medicinal drugs will be an optimal way of fighting pathogenic microorganisms. Since plants are rich in various biologically active compounds, extracts from them will exert antimicrobial, anti-inflammatory, antioxidant properties and etc. In this investigation extract from Arctium tomentosum Mill. obtained by supercritical carbon dioxide extraction was studied for antimicrobial effect. Four reference microbial strains, including gram positive (Staphylococcus aureus), gram negative (Staphylococcus epidermidis, Escherichia coli) and fungi (Candida albicans) were treated. Ampicillin, Chloramphenicol and Nystatin were used for positive control. Procedure of antimicrobial assay was carried out by the method of two-fold serial dilutions in corresponding nutrient medium. As a result minimum bactericide and fungicide concentrations of the extract from A. tomentosum Mill. were determined. Against S. aureus it was 10.4 mg/ml and 41.7 mg/ml against S. epidermidis, while against E. coli it was 20.8 mg/ml. Minimum fungicidal concentration was 5.2 mg/ml. Also ranges of concentration of the studied extract that stop reproduction and development of selected microorganisms were determined. Thus concentration range of bacteriostatic activity for S. aureus was 2.6 – 5.2, while for S. epidermidis was 2.6 – 20.8 mg/ml. Bacteriostatic activity of the studied extract against E.coli was not observed, however concentration range of fungistatic activity was 1.3 – 2.6 mg/ml. Obtained results show that extract from Arctium tomentosum Mill. has antimicrobial activity, so it can serve as a base for formation of phytopreparate for the treatment of diseases with microbial origin.

Lactobacillus Substitution as a Chicken Antibiotic Growth Promotor: A Systematic Review

Antibiotic usage in chickens can help to prevent disease and increase their growth rate. However, many farmers ignore the doses and the withdrawal time of antibiotics usage for chickens so that it can cause antibiotic residue in chicken meat. This antibiotic residue can cause health problems, so it is necessary to take action to reduce or find other alternatives for antibiotic usage in chickens. Therefore, our research objective was to summarize the records of tetracycline residue in broiler chickens. In addition, this research also identifies the effect of Lactobacillus usage as an antibiotic replacement. This research was a systematic review. The systematic review intended to synthesize in detail the information about natural ingredients for antibiotic substitutes and their effect on chicken’s growth from Google scholar, Science Direct, and Pub Med with the keywords “Tetracycline effect,” “Probiotics antibiotic,” “Lactobacillus antibiotics,” “Chicken antibiotics,” “Tetracycline residue” and “Chicken growth.” The results obtained from this systematic literature review were that the residual levels found in chicken samples from Indonesia and Egypt ranged from 0.7 to 61.05 mg/kg. In addition, the addition of Lactobacillus had a positive effect on the weight of chickens, namely the addition of 23.3 to 1421.67 g. This study concluded that the residual levels of tetracycline in chickens exceed the safe consumption limits set by the Standard Nasional Indonesia (SNI). The usage of Lactobacillus was common as feed additives.

Porcine Interleukin-17 and 22 Co-Expressed by Yarrowia lipolytica Enhance Immunity and Increase Protection against Bacterial Challenge in Mice and Piglets

Drug resistance in economic animals to pathogens is a matter of widespread concern due to abuse of antibiotics. In order to develop a safe and economical immunopotentiator to raise the immunity and antibacterial response as a replacement for antibiotics, a recombinant yeast co-expressing pig interleukin-17 (IL-17) and IL-22 was constructed and designated as Po1h-pINA1297-IL-17/22. To evaluate the immunoregulator activities of Po1h-pINA1297-IL-17/22, two experiment groups (oral inoculation with Po1h-pINA1297 or Po1h-pINA1297-IL-17/22) and a negative control group (PBS) were set up using 4-week-old female BALB/c mice (10/group). The level of cytokines, including IL-2, IL-4, IL-10, and IFN-γ, were detected by ELISA, and the circulating CD4+ and CD8+ lymphocytes were quantified by flow cytometry. The IgG and secretory IgA (SIgA) levels in both small intestine and fecal matter were also measured by ELISA. The results indicated that the IgG antibody titer and SIgA concentration increased significantly in the Po1h-pINA1297-IL17/22 group in comparison with the controls (p < 0.05) and so did the cytokine levels in the serum (IL-2, IL-4, IL-10, and IFN-γ). In addition, CD4+ and CD8+ T cells were also obviously elevated in the Po1h-pINA1297-IL17/22 group on 35th day (p < 0.05). After challenge with pathogenic Salmonella typhimurium, the Po1h-pINA1297-IL17/22 group showed a relatively higher survival rate without obvious infectious symptoms. On the contrary, the mortality of control group reached 80% due to bacterial infection. As for the piglet experiment, 30 healthy 7-day piglets were similarly attributed into three groups. The oral inoculation of piglets with Po1h-pINA1297-IL17/22 also markedly improved the growth performance and systemic immunity (up-regulations of IL-4, IL-6, IL-15, IL-17, IL-22, and IL-23). Overall, the results indicated that Po1h-pINA1297-IL17/22 effectively promoted the humoral and cellular immunity against bacterial infection. These proved the promising potential of Po1h-pINA1297-IL-17/22 to be a potent immunopotentiator for the prevention of microbial pathogen infections.

The Role of CISH in the Prevention of Blood-Brain Barrier Disruption Induced By Meningitic Escherichia coli

Backgrounds: Despite the availability of antibiotics over the last several decades, an unmet requirement still exists for new practical therapeutic approaches to treat pediatric infections, especially in developing countries. Most of these deaths were caused by bacterial sepsis and meningitis (BSM), which is characterized by high mortality and adverse prognosis. Recently, CHINET (China bacterial resistance monitoring network) reported that "as the most common gram-negative bacterial pathogens causing BSM, Escherichia coli (E. coli) exhibits an uncontrollable, increasing multi-antibiotics resistance" (http://www.chinets.com). Antibiotics are effective in treating pediatric BSM, but antibiotic overuse increases the risk of drug resistance. In addition, excessive antibiotic treatments in BSM patients were related to several adverse outcomes, including necrotizing enterocolitis (NEC), late-onset sepsis (LOS), and even death. Hence, the crosstalk between host-pathogen interactions has become a new strategy and research point for developing antibiotic replacement therapy. Published studies suggest that the suppressor of cytokine signaling proteins (SOCS) are the main regulators in the innate immune reaction induced by a microbial pathogen, avoiding excessive inflammation. Recent studies have found that SOCS proteins can function as executors and contribute to the downregulation of proinflammatory factors associated with infection. As an important member of the SOCS family, the CISH protein defense mechanism of Pediatric E. Coli-induced BSM was unclear. This study explored the underlying defense mechanism of CISH during pediatric E. coli-induced BSM. Methods and materials: Sixteen mice were randomly divided into two groups (control group, E. coli E44 group). Mice were given sterile normal saline and E. coli E44 (1×107CFU/mouse) by intraperitoneal administration, respectively. After infection for 8 h, mice in different groups were sacrificed to collect brain tissues and fixed in 10% formalin buffer solution for 24h. The sample were pretreated (dehydration, embedding, sectioning, hyalinization, H&E staining and sealing) and morphological observations. Human brain microvascular endothelial cells (HBMECs) in 6-well plates (1×105 cells/well) were cotransfected with GV492-CISH plasmid and siRNA using Lipofectamine 3000 reagent. The vector GV492-CISH (MW: 12.7 kb) was identified by restriction enzyme digestion and nucleotide sequencing. Then, the cells were incubated for 2-3 days to constructed the CISH overexpression HBMECs. HBMECs were inoculated in 96-well plates (excluding the edge effect) and divided into a control group, E. coli E44 infection group and E. coli E44+CISH siRNA pretreatment group. After E. coli. E44 infection for 2 h, the expression of Occludin was detected by Western Boltting. SPSS 20.0 software was used for statistical analysis. P <0.05 were considered statistically significant. Results: H&E staining showed that E. coli E44 could induce impairments in endothelial cells and cause brain microvascular damage. Histological damage was characterized by microvascular collapse, destruction of the bases, and RBC cells that were irregularly positioned into the tissue space. Few well-structured blood capillaries could be observed under a typical view. Expression of Occludin declined significantly in the E. coli E44 infection group compared with the uninfected group(P<0.01). In addition, compared with the untreated group, designed siRNA showed an acceptable inhibition effect of CISH with statistical significance (P<0.05). The expression of Oc-cludin in HBMECs treated with siRNA decreased significantly compared with that in HBMECs not treated with siRNA after infection (P<0.01). These results suggested that CISH defense mechanism during pediatric E. coli-induced BSM, CISH knockdown aggravated E. coli-induced blood brain barrier injury in BSM. Conclusions: In conclusion, the completion of this study reveals the protective mechanism of CISH in meningitic Escherichia coli-induced HBMECs injuries and BBB disruptions. CISH deficiency reduces the expression of Occludin, which aggravates the damage of BBB induced by Escherichia coli infected.(Acknowledgements: X.G. and J.Z. contributed equally to this work; Corresponding author: Hong Cao, gzhcao@smu.edu.cn; National Natural Science Foundation of China, No. 81871198 to H.C.)Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Applications of butyric acid in poultry production: the dynamics of gut health, performance, nutrient utilization, egg quality, and osteoporosis.

Due to the increasing demand for antibiotic-free livestock products from the consumer side and the ban on the use of antibiotic growth promoters, the poultry feed industry is increasingly interested in developing more alternatives to cope with this problem. Organic acids (butyric acid) have many beneficial effects on poultry health, performance, and egg quality when used in their diet, thus they can be considered for the replacement of antibiotics in livestock production systems. Butyric acid is most efficacious against pathogenic bacteria such as Salmonella spp. and Escherichia coli, and stimulates the population of beneficial gut bacteria. It is a primary energy source for colonocytes and augments the differentiation and maturation of the intestinal cells. Collectively, butyric acid should be considered as an alternative to antibiotic growth promoters, because it reduces pathogenic bacteria and their toxins, enhancing gut health thereby increasing nutrient digestibility, thus leading to improved growth performance and immunity among birds. The possible pathways and mechanisms through which butyric acid enhances gut health and production performance are discussed in this review. Detailed information about the use of butyric acid in poultry and its possible benefits under different conditions are also provided, and the impacts of butyric acid on egg quality and osteoporosis are noted.

Direct Detection of Antibacterial-Producing Soil Isolates Utilizing a Novel High-Throughput Screening Assay.

The ever-increasing global threat of common infections developing resistance to current therapeutics is rapidly accelerating the onset of a primitive post-antibiotic era in medicine. The prevention of further antimicrobial resistance development is unlikely due to the continued misuse of antibiotics, augmented by the lack of discovery of novel antibiotics. Screening large libraries of synthetic compounds have yet to offer effective replacements for current antibiotics. Due to historical successes, discovery from large and diverse natural sources and, more specifically, environmental bacteria, may still yield novel alternative antibiotics. However, the process of antibiotic discovery from natural sources is laborious and time-consuming as a result of outdated methodologies. Therefore, we have developed a simple and rapid preliminary screening assay to identify antibacterial-producing bacteria from natural sources. In brief, the assay utilizes the presence or absence of luminescence in bioluminescent reporter bacteria and test bacterium co-cultures in a 96-well plate format to determine the absence or presence of antibacterial compound production. Our assay, called the bioluminescent simultaneous antagonism (BSLA) assay, can accurately distinguish between known antibacterial-producing and non-producing test bacteria. The BSLA assay was validated by screening 264 unknown soil isolates which resulted in the identification of 10 antibacterial-producing isolates, effectively decreasing the pool of isolates for downstream analysis by 96%. By design, the assay is simple and requires only general laboratory equipment; however, we have shown that the assay can be scaled to automated high-throughput screening systems. Taken together, the BSLA assay allows for the rapid pre-screening of unknown bacterial isolates which, when coupled with innovative downstream dereplication and identification technologies, can effectively fast-track antimicrobial discovery.

Effects of synbiotic supplementation as an antibiotic growth promoter replacement on cecal Campylobacter jejuni load in broilers challenged with C. jejuni

Synbiotic supplementation alters gut microbiota via competitive exclusion, improving production performance, decreasing cecal Campylobacter jejuni load and colonization, and improving immunological responses while offering an alternative to antibiotic growth promoters in poultry production. This study aimed to determine effects of synbiotic supplementation as an antibiotic replacement in C. jejuni challenged broilers. 336 one-day-old broiler chicks were distributed into four treatment groups: Control, Synbiotic, Antibiotic + Challenge, and Synbiotic + Challenge, with six replicate pens per treatment. Antibiotic (Virginiamycin) and synbiotic (PoultryStar® Me) were supplemented in feed 20 mg/kg and 20 g/1000 birds/day, respectively. At 21 days of age, birds were orally inoculated with PBS or 1 × 10 8 CFU/bird C. jejuni . Feed intake and body weight were measured on a weekly basis. On 3, 7, and 14 dpi, intestinal permeability, anti- C. jejuni IgA bile concentration, C. jejuni content (ceca, ileal tissue, and liver), splenocyte MNC nitric oxide production, cecal tonsil CD4 + :CD8 + and CD4 + :CD25 + ratios, and cecal cytokine immune gene and tight junction protein expression were quantified and statistically analyzed. We found that synbiotic supplementation limited cecal C. jejuni colonization more effectively than antibiotic supplementation, upregulated tight junction protein expression, and positively altered cytokine and chemokine gene expression in response to challenge. We conclude that synbiotic supplementation decreases C. jejuni loads in broilers and beneficially modulates the immune response to C. jejuni challenge, compared to antibiotic supplementation, and that synbiotics can be applied to replace antibiotic growth promoter usage during poultry C. jejuni infection.

Inhibition of TNBS-induced intestinal inflammation in crucian carp (Carassius carassius) by oral administration of bioactive Bioactive food derived peptides

Intestinal enteritis is a main issue in crucian carp production which results in massive economic loss. Traditional antibiotics used for disease prevention of crucian carp (Carassius carassius) have been banned, thus an alternative approach needs to be identified. In this study, the bioactive peptide was evaluated as a diet supplement for preventing intestinal inflammation in crucian carp. Intestinal inflammation was induced by intrarectal administration of a 2,4,6-trinitrobenzene sulfonic acid (TNBS) solution. The fish samples were fed with different diets for 14 days. The disease activity index (DAI), which included, fish swimming, food intake, anal inflammation, body surface, and ascites was determined daily. Intestine segments were stained with haematoxylin and eosin (H.E.) for histopathological analysis. The expression of cytokines, including interleukin-1β (IL-1β), interleukin-8 (IL-8), tumor necrosis factor α (TNF-α), and myeloperoxidase (MPO) in crucian carp were determined. In TNBS-induced groups, the DAI scores were dramatically increased compared to the control group. The histopathological analysis showed that the damage of the fish intestine after the injection of TNBS. The relative expression levels of pro-inflammation cytokines (TNF-α, IL-1β, IL-8, MPO) were significantly increased compared to the control group on day 1. In the TNBS-induced group feed with a diet supplemented with bioactive peptide, the symptoms of intestinal inflammation were relieved on day 3 and the mRNA expression levels of pro-inflammation cytokines (TNF-α, IL-1β, IL-8, MPO) were reduced compared to day 1. On day 7, the fish samples enrofloxacin group and bioactive peptide group were recovered from TNBS-induced intestinal inflammation. This study showed that the fish diet supplemented with bioactive peptide could help to prevent and recover from intestinal inflammation. Thus, the bioactive peptide can be used as a replacement for antibiotics to prevent disease in aquaculture production.

Investigation of Biological and Prooxidant Activity of Zinc Oxide Nanoclusters and Nanoparticles.

Zinc oxide (ZnO) nanomaterials offer some promising antibacterial effects. In this study, a new form of ZnO is synthesized, named ZnO nanocluster bars (NCs). Herein, ZnO NCs, ZnO nanoparticles (NPs), ZnO coated with silica (ZnO-SiOA, ZnO-SiOB), and SiO2 NPs were prepared, characterized, and their antimicrobial and prooxidant activity were tested. The prooxidant activity of all nanomaterials was studied according to free-radical oxidation reactions (pH 7.4 and pH 8.5) in chemiluminescent model systems. Each form of new synthesized ZnO nanomaterials exhibited a unique behavior that varied from mild to strong prooxidant properties in the Fenton`s system. ZnO NPs and ZnO NCs showed strong antibacterial effects, ZnO-SiOA NPs did not show any antibacterial activity representing biocompatibility. All tested NMs also underwent oxidation by H2O2. ZnO NCs and ZnO NPs exhibited strong oxidation at pH 8.5 in the O2-. generating system. While, SiO2, ZnO-SiOA andZnO-SiOB possessed pronounced 60-80% antioxidant effects, SiO2 NPs acted as a definitive prooxidant which was not observed in other tests. ZnO NCs are strong oxidized, assuming that ZnO NCs provide a slower release of ZnO, which leads to having a stronger effect on bacterial strains. Thus, ZnO NCs are an important antibacterial agent that could be an emergent replacement of traditional antibiotics.