Heat-inactivated Culture Research Articles

The development and establishment of a heat inactivated preparation of Mycobacterium tuberculosis (H37Rv) as the first international standard for nucleic acid amplification techniques

A need to develop an inactivated Mycobacterium tuberculosis (Mtb) preparation, to be used as a DNA standard, as an important and urgent requirement for Tuberculosis (TB) diagnostics standardization was identified in 2018. A candidate material was generated using a heat inactivated culture of Mtb strain H37Rv. A lyophilised preparation was evaluated for its suitability as an International Standard for molecular detection of Mtb DNA in an international collaborative study. Together with the use of quantitative PCR assays and rapid diagnostic tests, this candidate standard was demonstrated to be fit-for-purpose. Based on the results from this collaborative study, it is proposed this lyophilised heat inactivated Mtb preparation (NIBSC code: 20/152) to be established by the World Health Organization Expert Committee on Biological Standardization, as the First WHO International Standard for Mycobacterium tuberculosis (H37Rv) for nucleic acid amplification techniques with an assigned unitage of 6.3 log10 or 2 × 106 International Units per vial.The intended uses of this IS are for calibration of secondary or in-house reference preparations used in the assays for the molecular detection of Mtb DNA. It may also be used for assay validation and monitoring the performance in terms of limit of detection of rapid diagnostic tests.

Immunomodulatory effect of non-viable components of probiotic culture stimulated with heat-inactivated Escherichia coli and Bacillus cereus on holoxenic mice.

BackgroundCompetition of probiotic bacteria with other species from the intestinal microbiota involves different mechanisms that occur regardless of probiotics’ viability. The objective of this paper was to assess the cytokine serum levels in holoxenic mice after oral administration of non-viable components (NVC) of Enterococcus faecium probiotic culture stimulated with heat-inactivated Escherichia coli and Bacillus cereus in comparison to NVC of unstimulated E. faecium probiotic culture.MethodsProbiotic E. faecium CMGb 16 culture, grown in the presence of heat-inactivated cultures of E. coli and B. cereus CMGB 102, was subsequently separated into supernatant (SN) and heat-inactivated cellular sediment (CS) fractions by centrifugation. Each NVC was orally administered to holoxenic mice (balb C mouse strain), in three doses, given at 24 hours. Blood samples were collected from the retinal artery, at 7, 14, and 21 days after the first administration of the NVC. The serum concentrations of IL-12 and tumor necrosis factor-alpha (TNF-α) interleukins were assessed by ELISA method.ResultsAfter the oral administration of SN component obtained from the probiotic culture stimulated with heat-inactivated cultures of B. cereus CMGB 102 and E. coli O28, the serum concentrations of IL-12 were maintained higher in the samples collected at 7 and 14 days post-administration. No specific TNF-α profile could be established, depending on stimulated or non-stimulated probiotic culture, NVC fraction, or harvesting time.ConclusionThe obtained results demonstrate that non-viable fractions of probiotic bacteria, stimulated by other bacterial species, could induce immunostimulatory effects mediated by cytokines and act, therefore, as immunological adjuvants.

Synthesis of gold nanoparticles from different cellular fractions of Fusarium oxysporum.

The addition of varying concentrations of precursor gold salt to different cellular fractions of Fusarium oxysporum, viz., the culture filtrate and the intracellular extract obtained in the growing and resting phase of the cells had a profound influence on the size, shape, and state of aggregation of the nanoparticles. Multiply-twinned nanoparticles were obtained when the culture filtrate was used for synthesizing nanoparticles while mostly irregular shapes were obtained with the intracellular extract. The time taken for the formation of gold nanoparticles in the culture filtrate of resting cells was very less (< 30 min) while it took more than 8 h when the intracellular extract was used for synthesis of nanoparticles. There was a reduction in size of the nanoparticles with decreasing concentration of the gold salt from 1 mM to 0.05 mM. With the intracellular extract, the initial rate of increase in surface plasmon absorption maximum was linearly proportional to the initial concentration of the gold salt used. Gold nanoparticles were also obtained with the heat-inactivated culture filtrate which suggests alternatively the role of peptides and amino acids besides proteins in reducing and/or stabilizing the nanoparticles.

Biocontrol of Pythium aphanidermatum by the cellulolytic actinomycetes Streptomyces rubrolavendulae S4

Streptomyces rubrolavendulae (Yen) S4, isolated from a termite mound in Kanchanaburi, Thailand, was used to control the seedling damping-off disease of the horticultural plant Joseph's coat caused by Pythium aphanidermatum (Edson) Fitz. In a previous study, S. rubrolavendulae S4 was shown to strongly inhibit the growth of P. aphanidermatum on potato dextrose agar. This study investigated the modes of the antagonistic action of S. rubrolavendulae S4 on P. aphanidermatum. On carboxymethyl cellulose medium, S. rubrolavendulae S4 produced the highest cellulase activity of 65 U/ml in 5 days. The S. rubrolavendulae culture supernatant, with and without heat treatment, was tested for fungal growth inhibition using an agar well-diffusion method. The inhibitory effect was retained with the heat inactivated culture supernatant, indicating that the inhibition was derived from an antifungal compound rather than an enzyme produced by S. rubrolavendulae S4. The parasitic activity of S. rubrolavendulae S4 upon P. aphanidermatum was demonstrated by light and scanning electron micrographs. Joseph's coat (Amaranthus tricolor) seedlings were grown in peat mosses inoculated with S. rubrolavendulae S4 and P. aphanidermatum. In pots containing S. rubrolavendulae S4, the proportion of healthy seedlings was significantly increased. This was equivalent to the effect of the fungicide metalaxyl, and similar to the results from sterilized peat moss. Hence S. rubrolavendulae S4 can be used as an effective biological control agent in Joseph's coat cultivation.

Modulation of virulence and antibiotic susceptibility of enteropathogenic Escherichia coli strains by Enterococcus faecium probiotic strain culture fractions

The increasing rate of antimicrobial resistance drastically reduced the efficiency of conventional antibiotics and led to the reconsideration of the interspecies interactions in influencing bacterial virulence and response to therapy. The aim of the study was the investigation of the influence of the soluble and cellular fractions of Enterococcus (E.) faecium CMGB16 probiotic culture on the virulence and antibiotic resistance markers expression in clinical enteropathogenic Escherichia (E.) coli strains. The 7 clinical enteropathogenic E. coli strains, one standard E. coli ATCC 25,922 and one Bacillus (B.) cereus strains were cultivated in nutrient broth, aerobically at 37 °C, for 24 h. The E. faecium CMGB16 probiotic strain was cultivated in anaerobic conditions, at 37 °C in MRS (Man Rogosa Sharpe) broth, and co-cultivated with two pathogenic strains ( B. cereus and E. coli O28) culture fractions (supernatant, washed sediment and heat-inactivated culture) for 6 h, at 37 °C. After co-cultivation, the soluble and cellular fractions of the probiotic strain cultivated in the presence of two pathogenic strains were separated by centrifugation (6000 rpm, 10 min), heat-inactivated (15 min, 100 °C) and co-cultivated with the clinical enteropathogenic E. coli strains in McConkey broth, for 24 h, at 37 °C, in order to investigate the influence of the probiotic fractions on the adherence capacity and antibiotic susceptibility. All tested probiotic combinations influenced the adherence pattern of E. coli tested strains. The enteropathogenic E. coli strains susceptibility to aminoglycosides, beta-lactams and quinolones was increased by all probiotic combinations and decreased for amoxicillin-clavulanic acid. This study demonstrates that the plurifactorial anti-infective action of probiotics is also due to the modulation of virulence factors and antibiotic susceptibility expression in E. coli pathogenic strains.

PCR detection and identification of Alternaria species-groups in processed foods based on the genetic marker Alt a 1

In this study, a PCR method based on oligonucleotide primers targeting the Alt a 1 gene has been developed for the rapid detection of DNA from Alternaria spp. and for identification of Alternaria alternata, Alternaria porri, Alternaria radicina, and Alternaria infectoria species-groups in raw materials and processed food products. The assay design consists of two steps. First, a duplex PCR using primers Dir5cAlta1–Inv4Alta1 (that amplifies a specific DNA fragment of approximately 195 bp in all Alternaria spp.) and 18Sfweu–18Srveu (that amplifies a conserved 99 bp fragment on all the eukaryotic species), allows detection of Alternaria spp. DNA in foodstuffs with a high sensitivity and specificity. As a second step, identification of Alternaria species-groups is obtained through a seminested PCR method, without the need for sequencing PCR products. The specificity of the primer pairs designed was verified by PCR analysis of DNA from various Alternaria cultures, and also from several non target species. The detection limit of the method was approximately 10 2 CFU/ml, either in viable culture, heat inactivated culture or inoculated tomato pulp. Nevertheless, a sensitivity of 10 3 CFU/ml was obtained for tomato pulp inoculated with A. alternata or A. porri cultures heat inactivated at 90 °C for 5 min. PCR analysis of commercial foodstuff samples demonstrated the presence of DNA from A. alternata species-group in 100% of spoiled tomato samples, and 8% of tomato products, whilst 36.4% of cereal based infant food samples analyzed contained DNA from A. infectoria species-group.

Helicobacter pylori induce neutrophil transendothelial migration: Role of the bacterial HP-NAP

Continuous recruitment of neutrophils into the inflamed gastric mucosal tissue is a hallmark of Helicobacter pylori infection in humans. In this study, we examined the ability of H. pylori to induce transendothelial migration of neutrophils using a transwell system consisting of a cultured monolayer of human endothelial cells as barrier between two chambers. We showed for the first time that live H. pylori, but not formalin-killed bacteria, induced a significantly increased transendothelial migration of neutrophils. H. pylori conditioned culture medium also induced significantly increased transendothelial migration, whereas heat-inactivated culture filtrates had no effect, suggesting that the chemotactic factor was proteinaceous. Depletion of H. pylori-neutrophil activating protein (HP-NAP) from the culture filtrates resulted in significant reduction of the transmigration. Culture filtrates from isogenic HP-NAP deficient mutant bacteria also induced significantly less neutrophil migration than culture filtrates obtained from wild-type bacteria. HP-NAP did not induce endothelial cell activation, suggesting that HP-NAP acts directly on the neutrophils. In conclusion, our results demonstrate that secreted HP-NAP is one of the factors resulting in H. pylori induced neutrophil transendothelial migration. We propose that HP-NAP contributes to the continuous recruitment of neutrophils to the gastric mucosa of H. pylori infected individuals.

HVMNE, a novel lymphocryptovirus related to Epstein-Barr virus, induces lymphoma in New Zealand White rabbits

HV(MNE) is a novel Epstein-Barr (EBV)-like virus isolated from a Macaca nemestrina with CD8(+) T-cell mycosis fungoides-cutaneous T-cell lymphoma. Here it is demonstrated that intravenous inoculation of irradiated HV(MNE)-infected T cells or cell-free virus from the J94356(PBMC) cell line in New Zealand White rabbits results in seroconversion to the viral capsid antigen (VCA) of EBV; all animals that seroconverted to VCA developed malignant lymphoma within months of inoculation. In contrast, control rabbits, inoculated with heat-inactivated culture supernatants from the same cell line, failed to seroconvert to VCA and did not develop disease. Disseminated lymphoma cells of mixed origin were detected in most vital organs, including the spleen, liver, lungs, kidneys, and heart of the affected rabbits. Neoplastic infiltrates were also observed in lymph nodes, thymus, skin, and subcutaneous tissues. HV(MNE) DNA and EBV-like RNA expression was demonstrated in the lymphomatous organs and in 2 transformed T-cell lines, one established from the lymph node and the other from the blood of the 2 lymphomatous animals. Analysis of one of these T-cell lines demonstrated the persistence of HV(MNE) DNA, expression of an LMP1-like protein, and acquisition of interleukin-2 independence, and constitutive activation of the Jak/STAT pathway. Thus, HV(MNE) in rabbits provides a valuable animal model for human T-cell lymphoma whereby genetic determinants for T-cell transformation by this EBV-like animal virus can be studied.

Effects of Bordetella avium Toxin on Turkey Tracheal Organ Cultures as Measured with a Tetrazolium-Reduction Assay

Turkey tracheal organ cultures (TOCs) were exposed to one of the following Bordetella avium fractions or controls: live B. avium, formalin-killed B. avium, B. avium sonicate, heat-inactivated sonicate, culture supernatant, heat-inactivated culture supernatant, phosphate-buffered saline, or brain-heart infusion broth. After the TOCs were incubated for 2 hours with the bacterial fractions, the cellular metabolism of each TOC was evaluated using a tetrazolium chloride reduction assay, and cellular morphology was determined by light microscopy. Additionally, bacterial fractions and controls were injected into turkeys to test lethality. Although the bacterial sonicate was lethal for turkeys, neither the sonicate nor any other B. avium fraction significantly affected the metabolism or morphology of turkey TOCs.

Proteoglycan production by chick embryonic chondrocytes is inhibited by culture filtrate of Bacteroides gingivalis.

Culture filtrate of Bacteroides gingivalis was tested in vitro for its effect on the proteoglycan synthesis (35S‐sulfate incorporation) of chick embryonic chondrocytes. The chondrocytes were cultured in high density aggregates. The culture medium (M 199 with 10% fetal calf serum) was supplemented with 20% culture filtrate, 20% heat inactivated culture filtrate (100°C, 10 min), or 20% of a low molecular fraction (M &lt; 1000) of the culture filtrate. Control aggregates were cultured with 20% bacterial medium added to the culture medium. Exposure of chondrocytes to culture filtrate resulted in a rapid reduction of the synthesis of proteoglycans within 5 hours. After 24 hours of culture proteoglycan synthesis was decreased by 80%. The small amount of proteoglycans still synthesized was mainly deposited into the medium whereas in control aggregates most proteoglycans were deposited around the cells. Protein synthesis was not changed and DNA synthesis was slightly increased by the culture filtrate. When after 24 hours of culture the medium with culture filtrate was replaced by control medium, this resulted in a rapid and total repair of the proteoglycan synthesis. The effect of culture filtrate on the proteoglycan synthesis did not change after heat inactivation or using the low molecular fraction, indicating that small, non‐proteinous molecules are responsible for the described effects.