Cell-free Culture Supernatant Fluids Research Articles

Screening for lactic acid bacteria capable of inhibiting Campylobacter jejuni in in vitro simulations of the broiler chicken caecal environment

Thermotolerant Campylobacter spp., specifically Campylobacter jejuni and Campylobacter coli, are the most common bacterial causes of human gastroenteritis in developed countries. Consumption of improperly prepared poultry products and cross contamination are among the main causes of human campylobacteriosis. The aim of this study was to identify lactic acid bacterial (LAB) strains capable of inhibiting C. jejuni growth in initial in vitro trials ('spot-on-lawn' method), as well as in batch fermentation studies mimicking the broiler caecal environment. These experiments served as an indication for using these strains to decrease the capability of Campylobacter to colonise and grow in the chicken caeca during primary production, with the aim of reducing the number of human campylobacteriosis cases. A total of 1,150 LAB strains were screened for anti-Campylobacter activity. Six strains were selected: members of the species Lactobacillus reuteri, Lactobacillus agilis, Lactobacillus helveticus, Lactobacillus salivarius, Enterococcus faecalis and Enterococcus faecium. After treatment with catalase, proteinase K and a-chymotrypsin, anti-Campylobacter activity of cell-free culture supernatant fluid (CSF) for all six strains was retained, which indicated that activity was probably not exerted by bacteriocin production. Based on the activity found in CSF, the compounds produced by the selected strains are secreted and do not require presence of live bacterial producer cells for activity. During initial in vitro fermentation experiments, the E. faecalis strain exhibited the highest inhibitory activity for C. jejuni and was selected for further fermentation experiments. In these experiments we tested for therapeutic or protective effects of the E. faecalis strain against C. jejuni MB 4185 infection under simulated broiler caecal growth conditions. The best inhibition results were obtained when E. faecalis was inoculated before the C. jejuni strain, lowering C. jejuni counts at least one log compared to a positive control. This effect was already observed 6 h after C. jejuni inoculation.

English

A total of 450 different colonies, isolated from 25 samples of dromedary milk collected from Laâyoune region of Morocco, were tested for antimicrobial compounds production. Out of these, 30 were determined to be lactic acid bacteria (LAB) and able to inhibit the growth of the indicator strain Listeria innocua CECT 4030. Seven isolates were selected by the large and clear zones of inhibition when tested by the agar well diffusion assay. They were classified by phenotypic and biochemical analysis as two Enterococcus durans (E204 and E214), two Lactococcus lactis (R75 and R76), one Enterococcus faecium R111, one Lactococcus cremoris R112 and one Enterococcus avium R122. Their antimicrobial compounds were detected in cell-free culture supernatant fluids under conditions that eliminate acid and hydrogen peroxide inhibition. The antimicrobial activity was altered after treatment with trypsin, -chymotrypsin, pepsin or papain which confirms the proteinaceous nature of the inhibition. It was heat stable even at autoclaving temperature (121°C for 15 min) and also active over a wide pH range (2 to 10). This fact suggests that bacteriocin-like produced by the seven LAB strains may find application as biopreservatives in food products. Key words: Dromedary milk, lactic acid bacteria, bacteriocin-like substances, antimicrobial activity.

Purification and Partial Characterization of Novel Bacteriocin L23 Produced by Lactobacillus fermentum L23

Lactobacillus fermentum strain L23 produced a small bacteriocin, designated bacteriocin L23, with an estimated molecular mass of < 7000 Da. Isolation, purification, and partial characterization of bacteriocin L23 are described. It displayed a wide inhibitory spectrum including both Gram-negative and Gram-positive pathogenic strains and two species of Candida. The antibacterial activity of cell-free culture supernatant fluid was not affected by catalase or urease but was abolished by the proteolytic enzymes trypsin and protease VI. Bacteriocin L23 was heat stable (60 min at 100 degrees C) and showed inhibitory activity over a wide pH range (4.0 to 7.0). The proteinaceous compound was isolated from cell-free culture supernatant fluid and purified. Crude bacteriocin sample was prepared by a process of ammonium sulfate precipitation, gel filtration, thin-layer chromatography, bioautography, and reversed-phase HPLC.

Antibacterial activity of lactic acid bacteria against spoilage and pathogenic bacteria isolated from the same meat small-scale facility: 1—Screening and characterization of the antibacterial compounds

A total of 87 lactic acid bacteria (LAB) (36 Lactobacillus sakei, 22 Enterococcus faecium, 16 Lactococcus garvieae, 11 Vagococcus carniphilus and 2 Enterococcus sp.) isolated from a small-scale facility producing traditional dry sausages were screened for antagonistic activity against other LAB and some spoilage and pathogenic microorganisms, also isolated from the same processing facility, except Listeria innocua (in lieu of Listeria monocytogenes) and Escherichia coli. The final goal was to investigate LAB antibacterial activity within the facility microbial ecosystem and to select interesting strains for the role of bio-preservatives. Twenty-one Ec. faecium, 6 Vc. carniphilus, 4 Lc. garvieae, 3 Lb. sakei and 2 Enterococcus sp. were shown to inhibit the growth of some indicator microorganisms in an agar well diffusion assay. Except 2 Lb. sakei and an Enterococcus sp., all these isolates exhibited antibacterial activity against L. innocua, but only 3 Ec. faecium, 5 Vc. carniphilus and 3 Lc. garvieae displayed also antagonistic activity against Staphylococcus aureus. The 5 Vc. carniphilus isolates were also found to be inhibitory for the Gram-negative bacterium Hafnia alvei. Isolates displaying antibacterial activity against L. innocua and/or Sc. aureus were investigated for the nature of antibacterial compounds synthesized against these indicator microorganisms. Bacteriocin-like production could be detected only on agar plated in overlay assays, and was unsuccessfully researched in cell-free culture supernatant fluids under conditions that eliminate acid and hydrogen peroxide inhibition. Results also showed that a Lb. sakei isolate displayed an additional inhibitory effect by H 2O 2 against L. innocua. These isolates will be investigated for their ability to repress the growth of undesirable bacteria in biofilms, i.e., the real mode of bacterial attachment. This is the first report on bacteriocin-like from Vc. carniphilus and on bacteriocin-like from Lc. garvieae active against both L. innocua and Sc. aureus species.

Population dynamics and antagonistic potential of enterococci colonizing the phyllosphere of grasses.

To investigate the spatial and temporal dynamics of enterococci colonizing forage grass and their ability to produce bacteriocins. Enterococci could be detected on above-ground plant parts throughout the growing season, with high continuity but low cell numbers (2.60 x 101-6.16 x 104 cfu g-1 fresh matter). A total of 750 strains were isolated and identified by their whole-cell protein patterns as Enterococcus faecalis (7.9%), Ent. mundtii (7.9%), Ent. casseliflavus (5.5%), Ent. faecium (5.2%) and Ent. sulfureus (0.1%). The vast majority of the strains (69.7%) formed a homogeneous 16S rDNA genotype that differed from those of known enterococci. A screening for antagonistic activity using an agar spot test revealed that 18.4% of all isolates were potential antagonists. Partially-purified proteins extracted from cell-free culture supernatant fluids of various species were characterized as pH- and heat-stable bacteriocins active against a wide range of lactic acid bacteria, clostridia and Listeria. The producing strains were antagonistically active even on 'phylloplane agar' at temperatures between 4 and 37 degrees C. Enterococci are a common part of the epiphytic microflora of grasses, displaying probably some antagonistic activity. The results provide new information on the distribution, species diversity and antagonistic potential of enterococci in the phyllosphere.

Purification and characterization of a bacteriocin produced by Lactobacillus acidophilus IBB 801.

Lactobacillus acidophilus IBB 801 produces a small bacteriocin, designated acidophilin 801, with an estimated molecular mass of less than 6.5 kDa. It displays a narrow inhibitory spectrum (only related lactobacilli but including the Gram-negative pathogenic bacteria Escherichia coli Row and Salmonella panama 1467) with a bactericidal activity. The antimicrobial activity of cell-free culture supernatant fluid was insensitive to catalase but sensitive to proteolytic enzymes such as trypsin, proteinase K and pronase, heat-stable (30 min at 121 degrees C), and maintained in a wide pH range. The proteinaceous compound was isolated from cell-free culture supernatant fluid and purified. Crude bacteriocin was isolated as a floating pellicle after ammonium sulphate precipitation (40% saturation) and partially purified by extraction/precipitation with chloroform/methanol (2/1, v/v). Further purification to homogeneity was performed by reversed phase Fast Performance Liquid Chromatography. The amino acid composition was determined. Amino acid sequencing revealed that the N-terminal end was blocked.

Experimental conditions that increase the production of HIV-1 by monocyte-derived macrophages: use of collagen matrix

Monocyte-derived macrophages (MDMs) from healthy blood donors were isolated by adherence to tissue culture-treated plasticware. They were cultured in vitro in medium supplemented with human serum and recombinant GM-CSF, then infected with the macrophage-tropic prototype strain HIV-1-PAR. Virus production was quantitated at various times after infection by measuring reverse transcriptase concentration in cell-free tissue culture supernatant fluids, using a sensitive nonradioactive assay. Virus production was significantly increased by culturing MDMs on plasticware previously coated with collagen 1. The increase in virus production was dependent upon collagen 1 concentration, with maximal value being encountered after coating with 1.5 μg/cm 2. These results indicate that the sensitivity of peripheral macrophages to HIV-1 infection might be influenced by contact-dependent interactions involving components of the extracellular matrix that take place during the process of monocyte extravasation and migration.

Endo-(1 → 3)β-D-glucanase activity secreted by Bacillus sp. no. 215

The production of an extracellular endo-(1 → 3)-β-D-glucanase by Bacillus sp. no. 215 was induced during growth with (1 → 3)-β-D-glucan (curdlan) from Cellulomonas flavigena strain KU as carbon and energy source. Maximum levels of activity (0.26 U ml-1 resp. 1.40 U mg-1) were detected in cell-free culture supernatant fluid after 25 h of aerobic growth at 55°C. The cells secreted an endo-(1 → 3)-β-D-glucanase with low electrophoretic mobility that used curdlan from C. flavigena strain KU and from Agrobacterium sp. (formerly Alcaligenes faecalis var. myxogenes) as substrates. The enzyme activity was highest at pH 7.0 and 55°C. It exhibited a remarkably low thermal stability with a half-life of 14 min at 55°C in the presence of substrate. Divalent metal cations were required for enzyme activity.

Structure of the autoinducer required for expression of Pseudomonas aeruginosa virulence genes.

In Pseudomonas aeruginosa the LasR protein is required for activation of lasB and several other virulence genes. A diffusible signal molecule, the P. aeruginosa autoinducer (PAI), produced by the bacterial cell and released into the growth medium, is required for activity of LasR. By cloning a lasB::lacZ fusion and a lasR gene under control of the lac promoter in Escherichia coli, we have developed a quantitative bioassay for PAI. We have used this assay to follow the purification of PAI from cell-free culture supernatant fluids in which P. aeruginosa or E. coli containing the P. aeruginosa gene required for autoinducer synthesis, lasI, had been grown. Chemical analyses indicated the purified material was 3-oxo-N-(tetrahydro-2-oxo-3-furanyl)dodecanamide. To confirm this assignment, the compound was synthesized and the synthetic compound was shown to have chemical and biological properties identical to those of PAI purified from culture supernatant fluids. The elucidation of the PAI structure suggests therapeutic approaches toward control of P. aeruginosa infections.

Pulmonary Lesions in Mice Inoculated with Actinobacillus pleuropneumoniae Hemolysin and Lipopolysaccharide

Non-Swiss albino (CF1) male mice were used as a model to study the effects of specific toxic components of Actinobacillus pleuropneumoniae on the respiratory system. Mice were divided into five groups of ten each and were inoculated by the intranasal route with whole-cell Actinobacillus pleuropneumoniae serotype 1, cell-free culture supernatant fluid (CFCSF) containing hemolysin protein, purified lipopolysaccharide (LPS), heat-treated CFCSF, or phosphate-buffered saline solution. Pulmonary lesions were evaluated at 6 and 12 hours after inoculation. Mice inoculated with whole cells, CFCSF, and LPS developed severe purulent bronchiolitis and alveolitis. Focal pulmonary necrosis was also observed in these three groups but was most consistent in the CFCSF-inoculated mice. Ultrastructurally, lungs from mice inoculated with whole cells, LPS, and CFCSF were characterized by severe degenerative changes in type I and type II pneumocytes. Alveolar spaces contained cellular debris and fibrin. Endothelial cells were swollen, and selected pulmonary capillaries were occluded with platelets and fibrin. Infiltrating neutrophils were often swollen, vacuolated, and degranulated. Although inoculation with relatively large numbers of A. pleuropneumoniae are required to kill mice, the mouse lung appears quite sensitive to the toxic components produced by these bacteria. The elaboration of these two toxic components by A. pleuropneumoniae may be responsible for the characteristic pulmonary inflammatory and necrotic lesions observed in infected swine.

Effect ofActinobacillus pleuropneumoniae hemolysin and lipopolysaccharide on cultured porcine neutrophils

Cultured porcine neutrophils were incubated in the presence of eitherActinobacillus pleuropneumoniae hemolysin protein (HP) in cell-free culture supernatant fluid or highly purified lipopolysaccharide (LPS) for times ranging from 2 to 15 min, followed by fixation and embedding for electron microscopy. Compared with untreated controls, neutrophils incubated in the presence of HP showed a progressive degeneration as evidenced by swelling, loss of cytoplasmic extensions, cytoplasmic vacuolation, and clumping of granules. Nuclear changes included the appearance of perinuclear spaces, nuclear membrane discontinuities, and disappearance of heterochromatin. These degenerative changes were cumulative and complete by 8 min of incubation time. In contrast, neutrophils incubated in LPS were unaffected compared with controls. Changes observed in neutrophils exposed to HP were similar to observed progressive changes in alveolar neutrophils from pigs exposed to aerosols ofA. pleuropneumoniae.

Temperature-sensitive production of azoverdin, the pyoverdin-like sid rophore of Azomonas macrocytogenes ATCC 12334

SUMMARY: Azoverdin, a visible yellow, blue--white fluorescent compound produced by iron-limited Azomonas macrocytogenes ATCC 12334, was isolated from cell-free culture supernatant fluid and purified in the ferrated form to 98% purity by ion-exchange chromatography and reverse-phase high-performance liquid chromatography, thereby separating it from several related iron-binding fluorescent compounds. Purified ferrated azoverdin exhibited a pH-independent absorption spectrum which became pH-dependent following deferration, typical of a pyoverdin-like siderophore. Azoverdin enhanced 55Fe3+ assimilation by iron-limited A. macrocytogenes and therefore functioned as a siderophore. Iron-limited cells were unable to produce azoverdin when grown at 34 °C rather than 28 °C. However, these cells still expressed a 74 kDa and a 70 kDa iron-repressible outer membrane protein and were capable of azoverdin-mediated iron transport. The use of A. macrocytogenes cells grown at 34 °C eliminated endogenous azoverdin production during iron uptake assays, which made it possible to accurately determine azoverdin-mediated 55Fe3+ transport rates. Azoverdin-mediated 55Fe-uptake proceeded with an apparent K m of 0·2 μM and a V of 0·46ng Fe3+ (108 cells)−1 min−1.

Production of 3,4-dihydroxybenzoic acid by Azomonas macrocytogenes and Azotobacter paspali

Four strains of Azomonas macrocytogenes and a strain of Azotobacter paspali were found to produce a common extracellular, iron-binding, phenolic compound. This compound was purified from cell-free culture supernatant fluids and chemically identified as 3,4-dihydroxybenzoic acid (protocatechuic acid). 3,4-Dihydroxybenzoic acid was produced by cells growing in a simple defined medium containing acetate and glucose or sucrose as sole carbon sources. This compound promoted the solubilization of iron from the minerals olivine, glauconite, pyrite, and marcasite. Mineral-free medium prepared by preincubation with olivine or glauconite as the only iron source supported enhanced growth yields of Azomonas macrocytogenes (ATCC 12334) when 3,4-dihydroxybenzoic acid was present during the preincubation period. This suggests that 3,4-dihydroxybenzoic acid may play a role in solubilizing iron from some natural iron sources; however, 3,4-dihydroxybenzoic acid can not be considered a true siderophore since it was produced by Azomonas macrocytogenes and Azotobacter paspali growing in iron-limited and iron-sufficient medium.

Isolation of variants of lymphocytopathic retroviruses from the peripheral blood and cerebrospinal fluid of patients with ARC or AIDS

LAV/HTLV-III/AAV viruses were isolated from 20 German patients with ARC/AIDS in order to investigate strain variation. Virus was isolated from the peripheral blood and/or cerebrospinal fluid (CSF) in umbilical cord peripheral blood lymphocyte (PBL) cultures. Isolates were identified by their cytopathic effect (CPE), by reverse transcriptase assays on cell-free infected culture supernatant fluid (SNF), and one or more of the following: immunofluorescence assays on infected cells for viral antigen using HTLV-III reference sera, Western blot analysis of cell-free infected culture SNF, electron microscopy of infected cells, and Southern blot restriction analysis and specific HTLV-III probing of DNA extracted from infected cultured PBL. The isolates could be classified into three groups according to differences in growth rate and cytopathic effect: Most showed what was regarded as the typical CPE, while some either grew rapidly and induced a striking CPE and others grew slowly with minimal CPE. In one patient, virus producing typical CPE was isolated from the peripheral blood while the isolate from his filtered cell-free CSF produced atypical slow CPE, suggesting that antigenic variation may occur with persistent infection or that superinfection may occur. Southern blot DNA restriction analysis of the DNA of three selected isolates showed that two of the isolates were similar but that the restriction pattern of all three differed from patterns previously published. Our results supplement the accumulating evidence of genetic variation among LAV/HTLV-III strains. The extent of this variation needs to be evaluated for any effect on the sensitivity of diagnostic tests, on the strategy of vaccine development, on tissue tropism by altering the viral surface receptor-binding sites, and possibly on the development of specific chemotherapy.

Streptokinase: cloning, expression, and excretion by Escherichia coli.

Genomic DNA from Streptococcus equisimilis strain H46A was cloned in Escherichia coli by using the bacteriophage lambda replacement vector L47 and an in vitro packaging system. A casein/plasminogen overlay technique was used to screen the phage bank for recombinants carrying the streptokinase gene ( skc ). The gene was present with a frequency of 1 in 836 recombinants, and 10 independent clones containing skc were isolated and physically characterized. One recombinant clone was used to subclone skc in E. coli plasmid vectors. Plasmid pMF2 [10.4 kilobases (kb)] consisting of pACYC184 with a 6.4-kb H46A DNA fragment in the EcoRI site and pMF5 (6.9 kb) carrying a 2.5-kb fragment in the Pst I site of pBR322 were among the recombinant plasmids determining streptokinase production in three different E. coli host strains. Expression of skc was independent of its orientation in either vector, indicating that its own promoter was present and functional in E. coli. However, expression in pBR322 was more efficient in one orientation than in the other, suggesting that one or both of the bla gene promoters contributed to skc expression. Several lines of evidence, including proof obtained by the immunodiffusion technique, established the identity of E. coli streptokinase. Testing cell-free culture supernatant fluids, osmotic shock fluids, and sonicates of osmotically shocked cells for streptokinase activity revealed the substance to be present in all three principal locations, indicating that E. coli cells were capable of releasing substantial amounts of streptokinase into the culture medium.

Detection of toxins produced by vibrio fluvialis.

The results of studies with cell-free extracts and culture supernatant fluids of Vibrio fluvialis (a recently recognized, potential enteric pathogen for humans) grown in the absence and presence of lincomycin indicated that the bacterium could produce (i) a factor which causes CHO cell elongation (CEF) similar to that elicited by V. cholerae enterotoxin and by the heat-labile enterotoxin of Escherichia coli, (ii) cytolysin(s) active against erythrocytes, (iii) nonhemolytic, CHO cell-killing factor(s), and (iv) protease(s) active against azocasein. The CEF was heat labile and ammonium sulfate precipitable, and it had an isoelectric point (estimated by sucrose density gradient electrofocusing) and molecular weight (estimated by gel filtration) of about 5.1 and 135,000, respectively.

Purification and characterization of staphylococcal pyrogenic exotoxin type B.

Staphylococcal pyrogenic exotoxin (PE) type B was purified and characterized biochemically and biologically. The exotoxin was purified from cell-free culture supernatant fluids by using differential precipitation with ethanol and resolubilization in pyrogen-free distilled water followed by preparative thin-layer isoelectric focusing. A final purification of 153-fold was achieved on the basis of the capacity of the exotoxin to produce fever. The toxin migrated as a homogeneous protein with a molecular weight of approximately 18 000 when tested with sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Hyperimmune antisera raised against the purified exotoxin reacted with partially purified toxin in an immuno-diffusion assay to form a single precipitin line. The isoelectric point of the PE was estimated to be 8.5. Alanine was identified as the N-terminal amino acid. The exotoxin contained significant amounts of lysine but few aromatic amino acids. The PE was pyrogenic and enhanced host susceptibility to lethal shock and myocardial damage by endotoxin. In addition, the exotoxin was a potent nonspecific lymphocyte mitogen and suppressed immunoglobulin M synthesis against sheep erythrocytes.

Rat alveolar macrophages are susceptible to activation by free and liposome-encapsulated lymphokines.

Alveolar macrophages (AM) obtained from F344 rats were rendered tumoricidal by incubation in vitro with cellfree culture supernatant fluids rich in macrophage-activating factor (MAF) activity harvested from mitogen-stimulated F344 rat lymphocytes. AM activated by this procedure destroyed syngeneic, allogeneic, and xenogeneic tumor cells but were not cytotoxic for nonneoplastic cells. MAF was encapsulated in multilamellar lipid vesicles (liposomes) and its ability to render AM tumoricidal was compared with that of free (unencapsulated) MAF. Liposome-encapsulated MAF rendered AM cytotoxic at concentrations up to 16,000 times lower than free MAF. These data demonstrate that AM can respond in vitro to lymphokines and that MAF encapsulated within liposomes is far more efficient in rendering AM tumoridical than free MAF.

Purification and physicochemical and biological characterization of a staphylococcal pyrogenic exotoxin

A staphylococcal pyrogenic exotoxin was purified and characterized biochemically and biologically. The organism producing the toxin was a group I Staphylococcus aureus strain which was isolated from a vaginal infection of a patient with mucocutaneous lymph node syndrome (Kawasaki's disease). The possible association of the toxin with the disease syndrome is discussed. The toxin was purified from cell-free culture supernatant fluids by means of differential precipitation with ethanol and resolubilization in pyrogen-free distilled water followed by preparative thin-layer isoelectric focusing. The pyrogenic exotoxin produced fevers in both rabbits and mice and enhanced host susceptibility to lethal shock and myocardial and liver damage by endotoxin. Also, the toxin was a potent nonspecific lymphocyte mitogen, stimulating rabbit spleen cells and human cord blood lymphocytes to proliferate. The toxin migrated as a homogeneous protein when tested with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (molecular weight, 12,000) and reisoelectric focusing (pI 5.3). Hyperimmune antisera raised against the purified toxin reacted with ethanol-precipitated toxin, using immunodiffusion to form a single precipitin arc. The toxin was distinguished from other staphylococcal toxins by a variety of methods. The amino acid composition was determined.

Cell-mediated immune response to products of Actinomyces viscosus cultures

Hartley strain guinea pigs were sensitized with 0.5 ml of concentrated cell-free Actinomyces viscosus culture supernatant fluids mixed with Freund complete adjuvant. Fourteen to 16 days later the animals were challenged by intradermal injection with 0.1 ml of the culture supernatant, and the reactions were observed at 4, 8, 16, 24, and 48 h. Peritoneal exudate cells from sensitized animals were used for determination of migration inhibition factor, and guinea pig peripheral blood served as a source of cells for determining the induction of mitogenesis by antigenic material. Skin responses were consistently positive to challenge with the test material, whereas reactions to noninoculated culture medium were negative. Sensitized cells, challenged with antigen, resulted in 60% or greater inhibition of migration of indicator cells in migration inhibition factor experiments. Tests for mitogenesis showed a greater than fourfold increase in isotope uptake when sensitized cells were challenged with test material. The data are consistent with the suggestion that A. viscosus culture supernatants contain substances that induce cell-mediated immune responses in guinea pigs.