CFU Of Bacteria Research Articles

Rhizostimulation of Hydrocarbon Degrading Microorganisms Using Chrysopogon zizanioides and Eucalyptus camaldulensis Species During Bioattenuation of Hydrocarbon Polluted Soils

Background: This study was carried out to compare the rhizostimulation potentials of C. zizanioides and E. camaldulensis on microorganisms cfu during the phytoremediation of hydrocarbon contaminated potted soils. The objectives were to: i). determine the best treatment for the rhizostimulation of the microbial population in soils during the bioattenuation process. ii). examine the most efficient plant species in the Phytostimulation of hydrocarbon degrading microorganisms during the bioattenuation of hydrocarbon contaminated soils. iii). identify the various bacterial and fungal colonies associated with the bioattenuation of hydrocarbon. Materials and Method: The design was the Split-Split Plot experiment (4 x 4 x 2). The main plot factors were the crude oil contamination (4 levels), the sub-Plot factors were the soil amendments (4 levels) while the subsub-Plot factors were the plant species (2 levels). Results: Results indicated that there were significant differences (p < 0.05) in rhizostimulation potentials of the plant species in different crude oil contamination levels and in the different soil amendments for both bacteria and fungi. Although E. camaldulensis species was involved in biostimulation of microbial cfu, C. zizanioides was found better efficient (16.13 x 106 soil bacteria cfu g g -1 at 9 WAT and 10.04 x 106 soil fungal cfu g g -1 at 12 WAT respectively. Microorganisms found involved in bioattenuation of hydrocarbon include Bascillus spp., Staphylococcus spp., and Streptococcus spp for bacteria and Aspergillus spp., Rhizopus spp., and Penicillium spp for fungi. The most frequently occurred genus of microorganisms were Bacillus and Rhizopus. Conclusion: In conclusion, C. zizanioides was recommended as efficient rhizostimulating plant species. It was concluded that the mixture of both the grass and woody plant species are required for optimum growth and stimulation of hydrocarbon degrading microorganisms during phytoremediation procedure in the field. Additionally, bacterial and fungal cfu was observed to have increased with increasing hydrocarbon concentration under the influence of the two plant species and the applied landfarming treatments of a mixture of NPK (g g-1) and Cowdung (3:1 v/v) fertilizer. The most frequently occurred genus of microorganisms were Bacillus and Rhizopus.

First report of Pectobacterium carotovorum and Pectobacterium brasiliense causing bacterial soft rot of bok choy in Taiwan.

Bok choy (Brassica rapa var. chinensis) is one of the most popular leafy green vegetables in Asia (Wang et al. 2019; Zhang et al. 2014). In May 2022, disease resembling bacterial soft rot was observed in a commercial greenhouse located in Xiluo, Yunlin County, Taiwan. Affected plants exhibited maceration, primarily close to the base of the plants (Fig. S1). Almost all bok choy plants (about 1,800 plants in total) on site were symptomatic. Macerated tissues were collected from six plants. The samples were homogenized in 10 mM MgCl2 and bacteria were isolated on nutrient agar (NA) by streak plating. After 1 day of culturing at 28°C, creamy-white, round colonies were consistently grown on all the plates, and six strains (Br1 to Br6) were obtained; each isolated from a different plant. The strains were able to ferment glucose and induced maceration on potato tuber slices (Schaad et al. 2001) but could not produce indigoidine on NGM medium (NA added with glycerol and MnCl2; Lee and Yu 2006). The DNA samples of these strains were tested with Pectobacterium-specific primers Y1 and Y2 (Darrasse et al. 1994) and all samples produced the expected amplicon. To identify the isolated pathogens, 1,592-bp sequences concatenated from fragments of the leuS (452 bp), dnaX (492 bp), and recA (648 bp) genes (GenBank accession nos. OP360013-OP360021) were obtained for each strain as previously described (Portier et al. 2019). Three genotypes were detected, the sequences of strains Br1, Br2, Br4, and Br5 were identical, while strains Br3 and Br6 each belong to a different genotype. The sequence identity between Br3 and Br6 was 98.2%. The concatenated sequences (dnaX-leuS-recA), along with those of type strains from known Pectobacterium species, were subjected to maximum likelihood analysis. The reconstructed trees showed that strains Br1, Br2, Br4, and Br5 grouped with P. carotovorum CFBP2046T (Fig. S2); the sequence identity between the isolated strains and the type strain was 98.7%. Strains Br3 and Br6 clustered with P. brasiliense CFBP6617T (Fig S2); the sequence identity between CFBP6617T and Br3 and Br6 were 97.5% and 98.4%, respectively. The six strains were inoculated onto 55-day-old bok choy plants using previously described prick inoculation methods (Wei et al. 2019). Autoclaved toothpicks, each carrying 9.3 x 106- 5.6 x 107 cfu of bacteria, were used to inoculate the base of plant leaves. All six strains were tested, and each strain had three replicates. Plants in the control group were stabbed with bacteria-free toothpicks. The plants were enclosed in clear plastic bags during the assay to maintain humidity and kept in a growth chamber (27/25°C day/night; 14-h photoperiod). After 1 d, all inoculated plants produced soft rot symptoms resembling those observed in the sampling site. No noticeable differences were observed among symptoms produced by different strains. The controls were symptomless. One strain was re-isolated from each treatment group and their identity were confirmed by sequencing the dnaX gene. All re-isolated strains shared the same sequences with those of the original strains tested. This is the first report of P. brasiliense and P. carotovorum causing bacterial soft rot of bok choy in Taiwan. Importantly, the findings showed that different Pectobacterium species and genotypes could induce symptoms on a crop in the same facility at the same time, highlighting the potential complexity of interactions among different soft rot bacteria in the environment.

Orally Administered Bifidobacterium adolescentis Diminishes Serum Glutamate Concentration in Mice.

Several studies have described the contribution of glutamate-transforming microbiota to the development of chronic ailments. For instance, the blood concentration of glutamate is higher in some patients with fibromyalgia, chronic fatigue, and pain. Taking advantage of a naturally occurring strain of Bifidobacterium that is able to transform glutamate in γ-aminobutyric caid (GABA), B. adolescentis IPLA60004, we designed a placebo-controlled intervention to test if the presence of this GABA-producing bifidobacteria in mice was able to impact the concentration of glutamate in the blood in comparison with the administration of other strain of the same species lacking the genes of the glutamate decarboxylase (gad) cluster. Animals were fed every day with 8 log CFU of bacteria in a sterilized milk vehicle for 14 days. Samples from feces and blood were collected during this period, and afterwards animals were sacrificed, tissues were taken from different organs, and the levels of different metabolites were analyzed by ultrahigh-performance liquid chromatography coupled to mass spectrometry. The results showed that both bacterial strains orally administered survived in the fecal content, and animals fed B. adolescentis IPLA60004 showed a significant reduction of their glutamate serum concentration, while a nonsignificant decrease was observed for animals fed a reference strain, B. adolescentis LGM10502. The variations observed in GABA were influenced by the gender of the animals, and no significant changes were observed in different tissues of the brain. These results suggest that orally administered GABA-producing probiotics could reduce the glutamate concentration in blood, opening a case for a clinical trial study in chronic disease patients. IMPORTANCE This work presents the results of a trial using mice as a model that were fed with a bacterial strain of the species B. adolescentis, which possesses different active genes capable of degrading glutamate and converting it into GABA. Indeed, the bacterium is able to survive the passage through the gastric tract and, more importantly, the animals reduce over time the concentration of glutamate in their blood. The importance of this result lies in the fact that several chronic ailments, such as fibromyalgia, are characterized by an increase in glutamate. Our results indicate that an oral diet with this probiotic-type bacteria could reduce the concentration of glutamate and, therefore, reduce the symptoms associated with the excess of this neurotransmitter.

Surface decoration with leucine tetrapeptide: An antibacterial strategy against Gram-negative bacteria

Surface-associated microbe contamination by Gram-negative bacteria poses a serious problem in medical care. Cationic peptides or polymers are the main materials used for antibacterial surface coating, but the positive charge may lead to blood coagulation. Therefore, exploiting surface coating which is free of positive charge and is effective for Gram-negative bacteria inactivation is in urgent need. In this study, inspired by the affinity between lipopolysaccharides of Gram-negative bacteria and Toll-like receptors of immune cells, we develop a leucine-based tetrapeptide coating strategy for combating Gram-negative bacteria. The obtained surface has excellent bactericidal activity against Gram-negative bacteria like Pseudomonas aeruginosa and Escherichia coli. A 1 mm2 coated glass surface could kill > 9.9 × 104 CFU bacteria in 1 h and has nearly no damage to mammal cells. Moreover, this surface coating strategy could be applied on various surfaces like glass slices, glass capillary cavity and thermoplastic polyurethane slices. And the coated surface could largely mitigate the microbe contamination in an in vivo subcutaneous implantation. This work paves a new way for antibacterial surface-coating which is behaving no positive charge and is of great importance for biomedical devices.

Glucomannan as a polysaccharide adjuvant improved immune responses against Staphylococcus aureus: Potency and efficacy studies

Staphylococcus aureus is a gram-positive bacterium, representing one of the most important nosocomial pathogens. The treatment of infections, caused by S. aureus, has become increasingly intricate due to the emergence of highly resistant strains. Therefore, it is obvious that an effective prevention strategy against this bacterium could significantly decrease such infections. In the present study, the protective efficacy and immunological properties of recombinant autolysin, formulated in Montanide ISA266 and Alum adjuvants with Glucomannan as a polysaccharide, were assessed in the systemic mouse model of infection. Mice were immunized with the purified recombinant protein in various formulations in different groups and, subsequently, mice were challenged with 5 × 108 CFU of bacteria for the evaluation of their survival and bacterial clearances in the internal organs. ELISA was performed to determine the type of induced immunity, cytokine secretion (IFN-γ, IL-4, IL-2, and IL-17), and isotyping (IgG1 and IgG2a). In addition, we measured the opsonophagocytic activities of the antibodies.Results showed that immunization with r-autolysin + Alum + Glucomannan and r-autolysin + MontanideISA266+Glucomannan formulations significantly increased total IgG and isotypes (IgG1 and IgG2a), as compared with other vaccinated and control groups. Furthermore, the formulation of r-autolysin in Alum and MontanideISA266 adjuvants with Glucomannan enhanced IFN-γ, IL-4, and IL-17 cytokine secretion as well as protectivity, following experimental challenge.We concluded that Glucomannan has the potential to induce immune responses and would be used as an adjuvant factor in vaccine formulation.

Efficacy of Lactobacillus animalis and Propionibacterium freudenreichii-Based Feed Additives in Reducing Salmonella-Associated Health and Performance Effects in Commercial Beef Calves.

Salmonella enterica, which causes typhoid fever, is one of the most prevalent food-borne pathogens. Salmonellosis in cattle can greatly impact a producer’s income due to treatment costs, decreased productivity of the herd, and mortality due to disease. Current methods of treatment and prevention for salmonellosis consist of antibiotics and vaccinations, but neither of these options are perfect. Probiotics, categorized as antibiotic alternatives, are living microorganisms that are added to animal feeds in appropriate quantities in order to benefit health and productivity in adult and newborn livestock. The objective of this study was to demonstrate that Lactobacillus animalis and Propionibacterium freudenreichii, when used as a direct-fed microbial, was effective in reducing the adverse effects of experimentally induced Salmonella infection in beef calves. We conducted a single site efficacy study with masking using a randomized design comprising two groups of ten beef calves allocated to two treatment groups (control and probiotic). Procedures such as determining general health scores and body weight and collecting fecal samples were carried out following the experimental challenge of calves with Salmonella Typhimurium. The presence of at least one CFU of bacteria in feces was significantly higher among animals in the control than in the probiotic group, which was higher on days 0 to 7 than on days 8 to 14 (p = 0.012). Animals in the control group had a significantly higher presence of abnormal diarrhea scores than animals in the probiotic group (p < 0.001). Most notably, other health benefits in probiotic-fed group calves were obviously better than those for control calves and further substantiates the potential economic and health benefits of feeding effective probiotics.

Biogat-fert technique for maximizing water and fertilizer units productivity for potato crop

Biogat-fert technique ensure to maximizing water and fertilizer unit productivity for potato crop, Two field experiments were carried out during growing seasons 2019-2020 and 2020-2021, at research farm of National Research Centre in Nubaria region, Egypt to study management of biogat-fert technique on potato crop under drip irrigation system in sandy soil conditions. Study factors were fertigation techniques (fertigation, biogat-fert and fertilizers). The following parameters were studied to evaluate the effect of study factors: (1) mineral and biological fertilizers effect of soil, (2) microbial number, (3) NPK ratio, (4) Yield of potato, (5) water use efficiency of potato, (6) Crop water production function. The results conducted that soil fertility was enhanced greatly in bio-fertilizer treatment. The average total N, total P and total K in 0–40 cm soil layers increased to 29.17, 26.67and 36.7% respectively. The average CFU of bacteria in soil layers increased by 15 Time. After drip irrigation, the CFU of bacteria had increased further, and reached more than 7.8× 107 CFU/g dry soil in the 0–40 cm soil layers. The results showed that the application of different bio-fertilizer along with fertilizers revealed significant positive impact on crop yield.

ВЛИЯНИЕ ПОРОДЫ КРУПНОГО РОГАТОГО СКОТА НА КАЧЕСТВО РЯЖЕНКИ

The aim of the work was to conduct a comparative assessment of the quality of fermented baked milk obtained from the milk of two different breeds of cows: Simmental and black-and-white. The research was conducted in the Lipetsk region. The milk of the Simmental breed was fatter, less dense, with less acidity, a large amount of protein and skimmed milk residue. The rennet sample of milk of both breeds was less than 15 minutes, which indicates good coagulability. According to the reductase test, the milk of both breeds is of satisfactory quality, belongs to Class 2 and con-tains from 500 thousand to 4 million. CFU of bacteria per 1 ml. The content of somatic cells in the milk of the Simmental breed of cattle exceeded the norm (no more than 500) by 83 thousand cells/ml. More dense, homogeneous and dense was the fermented baked milk of the Simmental breed. The fermented baked milk from black-and-white cows was more acidic, and after 4 days of storage at a temperature of +4 oC, this trend persisted, while it also became more liquid. The ap-pearance of rod-shaped pathogenic bacteria was not detected in the fermented milk from the milk of both breeds of cows. The shelf life of fermented baked milk from both breeds of cows at a tem-perature of + 4 oC was 14 days. It is established that the breed of cows has shown itself to be the most important factor in influencing the technological properties of milk and the quality of the fermented milk product produced from it - ryazhenka.

RANDOMIZED, DOUBLE-BLIND, PLACEBO (PBO)-CONTROLLED, SINGLE- AND MULTIPLE-DOSE PHASE 1 STUDY OF VE202, A DEFINED BACTERIAL CONSORTIUM FOR TREATMENT OF IBD: SAFETY AND COLONIZATION DYNAMICS OF A NOVEL LIVE BIOTHERAPEUTIC PRODUCT (LBP) IN HEALTHY ADULTS

Abstract IBD patients can have reduced gut commensal abundance, relative expansion of pro-inflammatory taxa, and reduced microbial diversity and short-chain fatty acid production. FMT leads to clinical remission in some UC patients, but is a complex, nonscalable process with inconsistent composition/quality that has led to death from transmission of pathogens undetected in screening. VE202 is a rationally defined LBP of 16 beneficial Clostridia from clusters IV, XIVa, and XVIII, with properties including association with healthy human microbiomes, promotion of colonic Treg cells, production of beneficial and immunoregulatory metabolites, efficacy in mouse colitis models, and lack of overt pathogenic or toxigenic features. These properties could help restore a healthy microbiome in UC patients. Another rationally defined LBP, VE303, showed activity in a Phase 2 study for prevention of recurrentC. difficile. The goal of the current Phase 1 study was to evaluate safety and colonization of VE202, to inform planned Phase 2 trials in UC patients. METHODS: After 5 days of vancomycin pretreatment at 125mg po QID to decrease gut microbial burden, 31 healthy adults received 1 dose at 2 dosage levels (6 VE202, 3 PBO per dosage level) or a 14-day course (9 VE202, 4 PBO) of the lower dose. Each VE202 delayed-release enteric capsule contained ∼1x109 CFU of bacteria. Safety was monitored through Week 12, with phone follow-up at Week 24. Stool was collected for LBP strain detection and exploratory microbiome, metabolomic, and immune markers. RESULTS: VE202 was well tolerated with no dose-dependent pattern of AEs. The only MedDRA organ classes with ≥20% reported AEs in VE202 recipients were: —Single-dose cohorts (N=12): GI (n=6; 50%), nervous system (n=4; 33.3%), investigations and infections/infestations (n=3 each; 25%) —14-day cohort (N=9): GI (n=5; 55.6%), general/administration site, musculoskeletal/connective tissue and nervous system (n=3 each; 33.3%), and skin/subcutaneous tissue (n=2; 22.2%) In VE202 recipients (N=21), the only AEs seen in ≥10% of subjects were abdominal discomfort, diarrhea, headache (n=4 each; 19.0%), and fatigue (n=3; 14.3%). No severe AEs or clinically significant trends in lab values were seen. Two SAEs, neither treatment-related, occurred several weeks after completion of VE202 dosing: abdominal pain caused by a suspected passed bile duct stone (Day 71) and hip pain leading to hip replacement (Day 120). Sustained VE202 strain detection (Figure 1) and relative abundance (Figure 2) in the vancomycin-perturbed gut was seen as soon as Day 2, sustained through 2 weeks after dosing completion, then declined slowly but remained substantially above baseline through Week 24. A dose- and duration-dependent trend in colonization was seen. CONCLUSION: VE202 is well tolerated in healthy adults and provides robust, durable, abundant colonization of LBP strains.

Understanding the transmission dynamics of Escherichia coli O157:H7 super-shedding infections in feedlot cattle.

BackgroundThe presence of Escherichia coli O157:H7 (E. coli O157:H7) super-shedding cattle in feedlots has the potential to increase the overall number (bio-burden) of E. coli O157:H7 in the environment. It is important to identify factors to reduce the bio-burden of E. coli O157 in feedlots by clarifying practices associated with the occurrence of super-shedders in feedlot cattle.MethodsThe objective of this study is to (1) identify host, pathogen, and management risk factors associated with naturally infected feedlot cattle excreting high concentrations of E. coli O157:H7 in their feces and (2) to determine whether the ingested dose or the specific strain of E. coli O157:H7 influences a super-shedder infection within experimentally inoculated feedlot cattle. To address this, (1) pen floor fecal samples and herd parameters were collected from four feedlots over a 9-month period, then (2) 6 strains of E. coli O157:H7, 3 strains isolated from normal shedder steers and 3 strains isolated from super-shedder steers, were inoculated into 30 one-year-old feedlot steers. Five steers were assigned to each E. coli O157:H7 strain group and inoculated with targeted numbers of 102, 104, 106, 108, and 1010 CFU of bacteria respectively.ResultsIn the feedlots, prevalence of infection with E. coli O157:H7 for the 890 fecal samples collected was 22.4%, with individual pen prevalence ranging from 0% to 90% and individual feedlot prevalence ranging from 8.4% to 30.2%. Three samples had E. coli O157:H7 levels greater than 104 MPN/g feces, thereby meeting the definition of super-shedder. Lower body weight at entry to the feedlot and higher daily maximum ambient temperature were associated with increased odds of a sample testing positive for E. coli O157:H7. In the experimental inoculation trial, the duration and total environmental shedding load of E. coli O157:H7 suggests that the time post-inoculation and the dose of inoculated E. coli O157:H7 are important while the E. coli O157:H7 strain and shedding characteristic (normal or super-shedder) are not.DiscussionUnder the conditions of this experiment, super-shedding appears to be the result of cattle ingesting a high dose of any strain of E. coli O157:H7. Therefore strategies that minimize exposure to large numbers of E. coli O157:H7 should be beneficial against the super-shedding of E. coli O157:H7 in feedlots.

Біотестування полімерних відходів вилучених з ТПВ

Introduction. Annually, the volume of accumulated polymers in landfills in Ukraine is growing by more than 1 million tons, but the volume of recycled polymer packaging over the past ten years has not exceeded 3%. The goal is to establish the degree of toxicological impact on biosphere objects of polymers extracted from solid waste, to assess the safety of using household polymer waste as a secondary raw material for further processing. The task is to establish the degree of biological and toxicological safety of polymer wastes extracted from solid waste. Methods and techniques: toxicological - determination of water toxicity on acute Daphnia magna according to DSTU 4173: 2003 (ISO 6341: 1996, MOD) and chronic according to DSTU 4166: 2003 (ISO 10706: 2000, MOD), and Paramecium caudatum ciliates, soil microflora reactions (saprotrophic soil bacteria CFU / g) according to MR 2609-82, the assessment of phytotoxic effects on higher plants was carried out by vegetation methods according to ISO 17402-2008, ISO 17126-2005a, ISO 22030: 2005b, ISO 11269 -1:2012a. Results. The research results show that aqueous extracts from waste polymers extracted from solid waste and their mixture do not have a pronounced toxic effect on aquatic organisms. There is no significant effect on soil bacteria. The study of the effect of polymer waste on seed germination did not reveal phytotoxic effects for any crop. A slight phytotoxic effect was observed during the study of PVC and PS waste. Wheat and mustard were the most sensitive. The level of phytotoxic effect was within acceptable limits and did not exceed 5.67%. Evaluation of phytotoxic effects on stem length showed the presence of effects from all processed products except PVC. The impact, characterized as weak, ranged from - 2.06% (PP) to - 13.27% (PS). The effect on root length was found for samples with PS waste (-7.23%), which was characterized as weak and PVC (-43.52%) - medium. Watercress and mustard were the most sensitive plants to the effects of polymer waste. Conclusions. The studied samples of polymer waste do not show hygienically significant toxic effects on water and soil test objects, even in concentrations of 1: 1, so the impact on the above test organisms polymer waste removed from solid waste is classified as hazard class 4. According to the assessment of phytotoxic effect, polymer waste removed from solid waste is classified as hazard class 4, except for PVC - hazard class 3.

​The Effects of Mesorhizobium ciceri Inoculation and Different Doses of Vermicompost Treatments on the Yield Components and Yield of Chickpea (Cicer arietinum L.) under Semi-arid Mediterranean Highland Condition of Turkey

Background: This study was conducted in 2016-2017 to determine the effects of Mesorhizobium ciceri inoculation and different doses of vermicompost applications on the yield components and yield of chickpea under semi-arid Mediterranean highland condition of Turkey. Methods: Mesorhizobium ciceri inoculant were applied to seeds (at 108 cfu bacteria per seed dose) as microbial fertilizer in the experiments. Vermicompost doses were 0, 1000, 2000 and 3000 kg ha-1. The trials were set up with three replications according to the randomized complete blocks design. Conclusion: Plant height, first pod height, pod number per plant, number of seeds per plant, 100 grain weight and grain yield were determined as 56.1-61.9 cm, 29.4-34.9 cm, 31.4-46.3 pods plant-1, 32.9-44.0 seed plant-1, 30.4-37.4 g and 1463-2072 kg ha-1, respectively. Co-application of 1000 kg ha-1 vermicompost with Mesorhizobium ciceri inoculation produced the highest values for all examined parameters for both years. Further applications of vermicompost reduced yield and related components. Control parcels and excess vermicompost applications (2000 and 3000 kg ha-1) produced lowest values. Mesorhizobium ciceri inoculation produced medium values between Co-application of 1000 kg ha-1 vermicompost with Mesorhizobium ciceri and control and excess vermicompost applied conditions. In conclusion, use of Mesorhizobium ciceri + 1000 kg ha-1 vermicompost was recommended in chickpea cultivation for maximum yield in the ecological conditions of Siirt province of Turkey or in similar ecologies.

Engineered hypoxia-responding Escherichia coli carrying cardiac peptide genes, suppresses tumor growth, angiogenesis and metastasis in vivo

Development of engineered non-pathogenic bacteria, capable of expressing anti-cancer proteins under tumor-specific conditions, is an ideal approach for selectively eradicating proliferating cancer cells. Herein, using an engineered hypoxia responding nirB promoter, we developed an engineered Escherichia coli BW25133 strain capable of expressing cardiac peptides and GFP signaling protein under hypoxic condition for spatiotemporal targeting of mice mammary tumors. Following determination of the in vitro cytotoxicity profile of the engineered bacteria, selective accumulation of bacteria in tumor microenvironment was studied 48 h after tail vein injection of 108 cfu bacteria in animals. For in vivo evaluation of antitumoral activities, mice with establishment mammary tumors received 3 consecutive intravenous injections of transformed bacteria with 4-day intervals and alterations in expression of tumor growth, invasion and angiogenesis specific biomarkers (Ki-67, VEGFR, CD31and MMP9 respectively), as well as fold changes in concentration of proinflammatory cytokines were examined at the end of the 24-day study period. Intravenously injected bacteria could selectively accumulate in tumor site and temporally express GFP and cardiac peptides in response to hypoxia, enhancing survival rate of tumor bearing mice, suppressing tumor growth rate and expression of MMP-9, VEGFR2, CD31 and Ki67 biomarkers. Applied engineered bacteria could also significantly reduce concentrations of IL-1β, IL-6, GC-SF, IL-12 and TNF-α proinflammatory cytokines while increasing those of IL-10, IL-17A and INF-γ. Overall, administration of hypoxia-responding E. coli bacteria, carrying cardiac peptide expression construct could effectively suppress tumor growth, angiogenesis, invasion and metastasis and enhance overall survival of mice bearing mammary tumors.

Molecular cloning and immunogenicity evaluation of IsdE protein of methicillin resistant Staphylococcus aureus as vaccine candidates

Methicillin resistant Staphylococcus aureus is one of the most common causes of nosocomial infections. Current therapeutic approaches are not always effective in treatment of nosocomial infections, thus, there is a global demand for the development of novel therapeutic strategies. Staphylococcus aureus possesses various systems to uptake iron. One of the most important of them is iron regulated surface determinant (Isd) which can be an excellent candidate for immunization. Here, following the preparation of recombinant IsdE protein, 20 μg of r-IsdE prepared in various formulations were subcutaneously injected in different groups of mice. Two booster vaccinations were administered in two-week intervals, then, blood samples were collected two weeks after each injection. ELISA was used for the evaluation of total IgG and its isotypes (IgG1 and IgG2a) as well as quantity of IFN-γ, IL-4, IL-17, IL-2 and TNF-α cytokines on the serum samples. Meanwhile, the immunized mice were intraperitoneally inoculated with 5 × 108 CFU of bacteria then, their mortality rate and bacterial load were assessed. Our results showed that immunization with the r-IsdE in various formulations raised total IgG and isotypes (IgG1 and IgG2a) compared with the control groups. Moreover, r-IsdE formulation with MF59 and Freund adjuvants raised production of IFN-γ, IL-4, IL-17, IL-2 and TNF-α cytokines and provided an acceptable protection against Staphylococcus aureus infections. Results of present study suggest that r-IsdE which can easily be expressed by Escherichia coli BL21 system shows a great potential to develop a protective immunity against infections caused by Methicillin resistant Staphylococcus aureus.

Antibacterial effect of nanocurcumin inside the implant fixture: An in vitro study.

ObjectivesInfections after implant placement are the main reasons for the failure of implant treatments. The present study aimed to evaluate the antibacterial effects of nanocurcumin inside the implant fixture against Escherichia coli, Staphylococcus aureus, and Enterococcus faecalis.Materials and MethodsTwenty seven implants were classified in three groups for testing the antibacterial effect of nanocurcumin, chlorhexidine (as negative control), and distilled water (as negative control). Each group was then divided into three subgroups to study the effect of the applied torque on the antimicrobial effect of nanocurcumin. All implant abutment assemblies were submerged in bacteria suspension and were incubated at 37°C for 24 hours. The contents of each implant were removed to count the colony of bacteria on the surface of plates containing nutrient agar.ResultsResults indicated that the inhibitory rate of bacteria by nanocurcumin was above 99% in all bacteria. Besides, by increasing the amount of applied torque from 10 to 35 N.cm, the CFU of bacteria in exposure to nanocurcumin significantly were decreased (p‐value < 0.01).ConclusionThe results of this study revealed that nanocurcumin can be used inside the implant fixture in order to use antimicrobial effects and further stabilization and success of the implant.

Synergistic Antibacterial Activity of Berberine in Combination with Amylmetacresol Against Enterococcus faecalis in vitro

To study the antibacterial effect of berberine combined with amylmetacresol on Enterococcus faecalis. Both dilution method and live bacteria CFU were used to determine the minimum inhibitory concentration (MIC) of berberine and amylmetacresol on E. faecalis. The killing effect of berberine and amylmetacresol on planktonic E. faecalis was detected by suspension quantitative germicidal test and live/dead bacteria staining. The effects of berberine and amylmetacresol on the structure of mature biofilm of E. faecalis was observed by scanning electron microscopy (SEM). The toxicity of berberine and amylmetacresol on human oral keratinocytes (HOK) was determined by CCK-8 cell proliferation and cytotoxicity assay and cytotoxicity LDH assay. The MIC of berberine was 512 μg/mL, and the MIC of amylmetacresol was 0.023 3%. 512 μg/mL berberine and 0.002 33% amylmetacresol had a weak killing effect on planktonic E. faecalis alone, while they showed a synergistic antibacterial effect in combination. Cell survival in the biofilm was only slightly changed by berberine and amylmetacresol. The structure of biofilm was obviously changed by berberine and amylmetacresol. 512 μg/mL berberine and 0.002 33% amylmetacresol alone or in combination showed the survival rate was much higher than the injury rate, suggesting berberine and amylmetacresol had a low cytotoxicity. Berberine and amylmetacresol had synergism against E. faecalis, and the biological safety of the combination use was better.

Timing of entry of Streptococcus uberis into the mammary gland of the dairy cow.

Streptococcus uberis do not colonise the teat canal and appear to invade the mammary gland of the dairy cow by direct entry though the canal. When they enter the mammary gland, and the early resulting processes, are unclear. Experimental infusions of the lactating mammary gland have been made to determine outcomes of infection, mastitis and disease. Infusion of 500 cfu bacteria was made immediately after milking (8 and 16 h intermilking interval) and 1, 4 or 12 h prior to milking. A mastitis resulted from all infusions, probably in response to the skim milk carrier. Infusions post milking resulted in clinical mastitis in more than half of the quarters, whereas infusion 1 h premilking created no clinical mastitis. Infusion 4 or 12 h pre milking resulted in the most severe reactions, with all quarters developing moderate to severe clinical mastitis. This was more rapid with the 4 h pre milking group. The results demonstrate that the initial inflammatory response caused by an invasion of the mammary gland is not necessarily protective against establishment of a pathogen, and that especially the response to invasion in the intermilking interval is often insufficient to prevent infection and/or disease.

P21 Novel ex-vivo model of septic arthritis identifies role of neutrophils in joint destruction and identifies a potential biomarker for diagnosis

Abstract Background Septic arthritis (SA) caused by bacterial species, such as Staphylococcus aureus, has high morbidity and mortality. Currently diagnosis is often prolonged and unreliable, with no suitable near-patient biomarkers available. To generate more reliable biomarkers and to understand pathogenesis we sought to develop a novel ex vivo system to explore the effect of pathogenic Staph. aureus strains in promoting cartilage degradation. Methods Human cartilage explants were obtained from femoral heads being surgically removed following trauma. Explants were infected for 48h with 106 cfu bacteria from two Staph. aureus SA-derived patient isolates (28g &amp; 36v strains). In the final 24h of bacterial infection, neutrophils purified from healthy donor blood were added to explant cultures at 3 x 106 cells/well. Chondrocyte viability was assessed using CellTracker green CMFDA and propidium iodide. Images were captured using confocal microscopy (LSM880) and cells counted using Imaris software. Structural damage was measured by glycosaminoglycan (GAG) and a neo-epitope of MMP-mediated degradation of type II collagen (C2M) release. Statistical analysis was performed using GraphPad Prism software. Results When cartilage explants were co-cultured with bacteria +/- neutrophils, cell death was significantly increased compared to the negative control or addition of neutrophils alone, (Friedman multiple comparisons test, N = 3, negative control vs. bacteria - neutrophils p &amp;lt;0.05, negative control vs. bacteria + neutrophils p &amp;lt; 0.01). Cartilage breakdown, estimated via GAG release, was induced by Staph. aureus alone, whereas it was significantly enhanced upon neutrophil addition in the final 24h of co-culture (Friedman multiple comparisons test, N = 8, neutrophil vs. neutrophil + bacterial strain 36v p &amp;lt;0.0001, neutrophil vs. neutrophil + bacterial strain 28g p &amp;lt;0.0005). Therefore, it is the combined effect with neutrophils that results in significant cartilage destruction. Although cartilage is damaged with the bacterial infection, shown by GAG release, C2M was only released when the bacteria is co-cultured with neutrophils (Friedman multiple comparisons test, N = 8, 36v vs. 36v + neutrophil p &amp;lt;0.0001, 28g vs. 28g + neutrophil p &amp;lt;0.005). This indicates that C2M is a biomarker that could potentially be used to diagnose septic arthritis as neutrophils infiltrate the joint in response to a bacterial infection. Conclusion A co-culture model of septic arthritis has been developed which allows precise examination of the contribution of the host neutrophil response to cartilage damage. We used this to identify a collagen breakdown product as a biomarker of host response to infected cartilage. This novel model will be a valuable tool in understanding the pathology of joint infection and can be used for the identification of future diagnostic biomarkers. Disclosures K.E. McCall None. C. Atherton None. C. Thudium None. C. Goodyear Grants/research support; C.G. has received funding for research from Celgene, AstraZeneca, MedAnnex, UCB &amp; Jannsen. T. Evans None. N. Millar Grants/research support; Novartis. I. McInnes Consultancies; I.M. has received consultancies fees from BMS, Abbvie, Lilly, GSK &amp; Pfizer. Grants/research support; I.M received research funding from Calgene, Janssen, Novartis, Boehringer Ingelheim &amp; BMS.

Near-infrared-driven Au-decorated polymer-metal protein microfibers with bacterial filtration ability for use in photothermal sterilization

The use of protein nanostructures can be widely applied in medical devices and biomaterials. However, the synthesis of photothermic bovine serum albumin (BSA) microfibers remains a challenge. In this study, novel protein microfibers (MS@BDA@Au) with bacterial filtration abilities were developed for use in inactivating bacteria under near-infrared (NIR) irradiation. Three types of MS@BDA microfibers were fabricated through the assembly of proteins with M2+ (Fe2+, Cu2+, Mn2+)/Na2S and the assistance of dopamine (DA). These were further decorated with gold nanoparticles to produce MS@BDA@Au microfibers, which exhibited a filtering capacity of 1 × 108 CFU bacteria per milligram, and obvious antibacterial effects at concentrations of 0.5 mg/mL. This paper presents a method that can be used in the functional micro-crystallization of proteins and the development of new types of protein fibers that can be employed in bacteria filtration and photothermal killing.

Recombinant PBP2a of methicillin-resistant S. aureus formulation in Alum and Montanide ISA266 adjuvants induced cellular and humoral immune responses with protection in Balb/C mice

Staphylococcus aureus is an important cause of both hospital and community acquired infections worldwide. S.aureus can develop multidrug resistance; thus, immunotherapy can be a rational alternative. High level β-lactam resistance of S. aureus has been attributed to the penicillin binding protein 2a (PBP2a). In this study, we assessed the immunogenicity and protectivity of PBP2a formulated in Montanide ISA266 and Alum adjuvants.Recombinant PBP2a with a molecular weight of approximately 13 kDa was expressed and purified by nickel-nitrilotriacetic acid (NI-NTA) affinity chromatography and characterized by SDS-PAGE and Western blot.To investigate the immunogenicity and protective effects of recombinant protein, 20 μg of r-PBP2a in various formulations were subcutaneously injected in different groups. Two booster vaccinations were carried out in two-week intervals and blood samples were collected two weeks after each injection. To determine the type of induced immune response, sera and splenocytes were analyzed by ELISA for total IgG and isotypes (IgG1 and IgG2a) and cytokine secretion (IFN-γ, IL-4, IL-17 and TNF-α), respectively. Three weeks following the last immunization, experimental mice were challenged with 5 × 108 CFU of bacteria intraperitoneally and mortality rate and bacterial load were assessed.Interestingly, analysis of humoral immune responses revealed that administration of r-PBP2a with Montanide ISA266 significantly increased specific IgG responses and also IgG1 isotype compared to alum-adjuvanted vaccine group. Also, r-PBP2a formulation with alum and MontanideISA266 adjuvants raised IFN-γ, IL-4, IL-17 cytokines secretion, and protectivity following experimental challenge.The results of the present study provide evidences for immunogenicity and protectivity of PBP2a protein as a vaccine candidate.