Enhancement of biodegradation of 2,7-dichlorodibenzo-p-dioxin by addition of fungal culture filtrate

The hydrophilicity of 2,7-dichlorodibenzo-p-dioxin (2,7-DCDD), a model dioxin compound, increased when incubated with the culture filtrates of several strains of fungi. The possibility that the addition of these filtrates could enhance the biodegradation of 2,7-DCDD by the white-rot basidiomycetous fungus Phanerochaete sordida YK-624 was examined. The decrease of 2,7-DCDD after 3 weeks incubation in a YK-624 culture containing these filtrates was greater (30%) than that in the culture of YK-624 alone (15%). This is the first report describing the enhancement of dioxin decrease by the addition of a fungal filtrate.     

Effects of fungal culture filtrates of Verticillium lecanii (Lecanicillium spp.) hybrid strains on Heterodera glycines eggs and juveniles

Many nematode-antagonistic fungi produce secondary metabolites and enzymes that demonstrate toxicity against plant-parasitic nematodes. The objective of this study was to evaluate the effects of fungal culture filtrates of Verticillium lecanii hybrid strains on mature eggs, embryonated eggs (eggs fertilized but without development of juveniles), and second-stage juveniles (J2) of Heterodera glycines and to compare these effects with those of their parental strains. The fungal culture filtrates of certain hybrid strains inhibited egg hatch of mature eggs. Furthermore, the fungal culture filtrates of two hybrid strains, AaF23 and AaF42, exhibited high toxicity against embryonated eggs of H. glycines. However, most of the fungal culture filtrates of V. lecanii did not inactivate J2. These results suggested that enzymes or other active compounds produced by the fungal culture filtrates of V. lecanii exhibit activity against specific stages in the H. glycines life cycle. In addition, based on a visual assessment of the morphological changes in eggs caused by filtrates of each strain, there were differences between the hybrid strains and their respective parental strains with regard to the active substances produced by V. lecanii against the embryonated eggs. As a result of promoting recombination of whole genomes via protoplast fusion, several hybrid strains may have enhanced production of active substances that are different from those produced by their parental strains. It was concluded that natural substances produced by V. lecanii are one of the important factors involved in the suppression of H. glycines damage.     

Studies on Alkaloids of Streptomyces

A method was given for determination of alkaloids in culture filtrates using alkaloid reagents, e.g., modified Dragendorff’s and Mayer’s reagents, in conjunction with several pharmacological tests such as Magnus method employing the isolated guinea pig intestine and the influence on blood pressure etc. In a screening of about 1250 strains of actinomycetes, 8 strains yielded basic materials that gave positive reaction with both of the above reagents. Among them, HCI extract from the culture filtrate of Streptomyces strain No. FFD-101 caused a transient drop of blood pressure of rabbit. Furthermore one fraction of HCI extracts from the culture filtrate of Streptomyces strain 227 × 1 caused a continous rise of blood pressure.     

Antiviral effects of bacteria isolated from manure

The objectives of this study were to determine the role of microbial activity in inactivation of hepatitis A virus (HAV) and to learn how the virus is inactivated. Of 31 bacterial strains isolated from animal manure, 10 efficiently inactivated HAV in fluid thioglycollate medium, with D₁₀ values (time, in days, required for a 90% reduction of virus titer) of 10 at 30°C. The D₁₀ value of the control suspension without bacteria was 35.1. Most of the 10 strains raised the pH of the medium during growth; comparisons suggested that alkalinity was not a principal antiviral property of these cultures. Cell-free filtrates of nine of these strains caused net 90% inactivation of HAV within 6 days at 37°C; the other did not. The inactivation capacity of four of the nine culture filtrates was significantly reduced by incubation with selected protease inhibitors before the virus was added. These protease inhibitors did not affect the activities of the other five culture filtrates. Fractions prepared by ultrafiltration (nominal molecular weights <1,000) from two of these cultures inactivated HAV suggesting that their mode of action was not enzymatic. Correspondence to: D.O. Cliver. mis|Department of Animal Health and Biomedical Sciences     

Permeability factor,cytotoxicity and serotyping ofPseudomonas aeruginosa strains

We analyzed in detail the permeability and cytotoxic activity as well as the serotypes of 127Pseudomonas aeruginosa strains. Sixty-seven strains were isolated from immunocompromised patients (51 from patients with tumors and 16 from patients after transplantation) and 60 strains were isolated from patients’ ears. Culture filtrates of strains isolated from patients after transplantation were responsible for the highest part of permeability reactions corresponding to an intermediate toxin production (68.8%) (categories 2 and 3) and culture filtrates of strains isolated from patients with tumors caused the highest percentage of permeability reactions corresponding to a strong toxin production. Culture filtrates of strains isolated from ears of patients were responsible for the highest percentage of negative permeability reactions (15%). With positive permeability reaction size (categories 2–6) increased also the percentage of cytotoxicity as well as the intensity of morphological changes on Vero cells after 1 and 2 d. We did not observe any relationship between a particular permeability reaction category and the most frequent serotypes (O4, O6) or nontypable strains of the tested groups.     

Differentiated biological activity of Vero cytotoxins (VT) released by human and porcine Escherichia coli strains

Abstract We have investigated the biological activity in the filtered culture supernatants from 9 VT-producing Escherichia coli strains. The filtrates from 4 strains (3 of human and one of bovine origin), were cytotoxic on Vero and HeLa cells, and caused death in intraperitoneally injected adult mice. The 5 strains of porcine origin showed cytotoxic activity on Vero and Y-1 cells but not on HeLa cells. Filtrates of these latter strains were not lethal for adult mice. VT-cytotoxins produced by all strains were inactive in the infant mouse test and the filtrates from 7 of 8 VT-producing strains assayed in rabbit ileal loops caused fluid accumulation in at least one of the 3 rabbits employed.     

Serological diagnosis of petriellidiosis (Allescheriosis)

Soluble antigens in culture filtrates of three strains of Petriellidium boydii and three strains of Monosporium apiospermum were examined. Antigens were separated from concentrated crude filtrates by anion-exchange chromatography. A single major peak (Antigen 1), constituting a significant proportion of the total recoverable carbohydrate, was the only product isolated from each of four chromatographed filtrates. Depending on the fungus strain, Antigen 1 consisted of 90–96% carbohydrate, 3–4% protein, and 2–4% nucleic acid. Antigen 1 was found to consist of a population of molecules with a heterogeneous molecular size when assayed by gel filtration chromatography; however, isolated fractions of Antigen 1 proved to be immunologically identical when examined by Ouchterlony immunodiffusion. In addition, Antigen 1 from each strain was immunologically identical to similar preparations of Antigen 1 from the other five fungus strains. Chromatography of culture filtrates from two strains of M. apiospermum revealed a second peak (Antigen 2), which was found to consist of 70% carbohydrate, 16% protein, and 4% nucleic acid. Although Antigen 2 contained four times as much protein as Antigen 1, the two preparations were immunologically identical by immunodiffusion tests. Ion-exchange chromatography proved to be a useful procedure for isolating antigens of P. boydii and M. apiospermum from culture filtrates.     

Enzymatic hydrolysis of cellulose: Evaluation of cellulase culture filtrates under use conditions

Culture filtrates from three mutant strains of Trichoderma reesei grown on lactose and on cellulose were compared under use conditions on four cellulose substrates. Cellulose culture filtrates contained five to six times as much cellulase as lactose culture filtrates. Unconcentrated cellulose culture filtrates produced up to 10% sugar solutions from 15% cellulose in 24 h. Specific activity in enzyme assays and efficiency in saccharification tests were low for enzymes from all the mutants. Over a wide range the percent saccharification of a substrate in a given times was directly proportional to the logarithm of the ratio of initial concentrations of enzyme and substrate. As a result of this, dilute enzyme is more efficient than concentrated enzyme, but if high sugar concentrations are desired, very large quantities of enzyme are required. Since the slopes of these plots varied, the relative activity of cellulase on different substrates may be affected by enzyme concentration.     

Immunochemical detection of ochratoxin A in black Aspergillus strains

One hundred and fifty-seven strains belonging to Aspergillus section Nigri were tested for ochratoxin A production using three different methods: a relatively new immunochemical method based on an enzyme-linked immunosorbent assay (ELISA), thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC). The monoclonal antibody-based ELISA technique was successfully used to screen for low levels of ochratoxin A in the black Aspergilli without concentrating the culture filtrates. The results were confirmed by TLC and HPLC analysis and chemical derivatization. These latter methods required concentrated filtrates. Ochratoxin A was detected in the culture filtrates of five of the 12 A. carbonarius strains, none of the 45 A.japonicus strains and three of the 100 isolates in the A. niger aggregate (A. foetidus, A. awamori and A. niger).Abbreviations ELISA enzyme-linked immunosorbent assay - HPLC high-performance liquid chromatography - OA ochratoxin A - TLC thin-layer chromatography     

The Rice Culture Filtrate of Bacillus cereus Isolated from Emetic-Type Food Poisoning Causes Mitochondrial Swelling in a HEp-2 Cell

Rice culture filtrates of Bacillus cereus SA-50, an emetic-type strain, produced a toxin which caused cytoplasmic vacuole formation in HEp-2 and HeLa cells. Electron microscopic observation revealed that the apparent vacuoles in HEp-2 cells seen under a light microscope were actually swollen mitochondria. The oxygen consumption of HEp-2 cells was accelerated by the addition of the rice culture filtrate as was measured with a polarographic oxymeter; a respiratory control ratio was 1.0 for control cells, while 1.4 for ones with the filtrates. The culture filtrates showed a similar effect on the isolated mouse liver mitochondria; respiratory control ratios for the mitochondria with and without the filtrates were 3.6 and 1.0, respectively. The affecting manner of the culture filtrates on the oxygen consumption of mitochondria was similar to that of 2,4-dinitrophenol, suggesting that the culture filtrate contains a toxin acting as an uncoupler of oxidative phosphorylation in mitochondria. It is likely that the culture filtrates containing the emetic toxin of B. cereus causes mitochondrial swelling with a close relationship to the uncoupling of the oxidative phosphorylation of mitochondria.     

Butyrate: a cytotoxin for Vero cells produced by Bacteroides gingivalis and Bacteroides asaccharolyticus

Culture filtrates of B. gingivalis and B. asaccharolyticus are cytotoxic for Vero cells. It is shown that the cytotoxic effect is due to the butyrate concentrations present in the culture filtrates of these strains. This cytotoxic effect proved to be reversible. Strains of the B. melaninogenicus subspecies intermedius and melaninogenicus did not produce butyrate and did not show cytotoxic activity towards Vero Cells.The significance of the production of toxic concentrations of butyrate for the etiology of especially periodontal diseases is discussed.     

Diarrheal and Environmental Isolates of Aeromonas spp. Produce a Toxin Similar to Shiga-Like Toxin 1

Diarrheal and environmental isolates of 39 strains of Aeromonas spp. were studied for detection of virulence factors. Although these 39 strains did not produce either heat-labile or heat-stable enterotoxins, culture filtrates of 31 strains produced cytopathic effects on Vero cells. Among these, culture filtrates of three strains of Aeromonas hydrophila and one strain of Aeromonas caviae could be neutralized by Escherichia coli O157:H7 Shiga-like toxin 1 antiserum. A single band of plasmid DNA of 2.14 kbp was isolated from these strains of Aeromonas spp. and E. coli O157:H7, which could be amplified by the polymerase chain reaction (PCR), employing oligonucleotide primers from the Shiga-like toxin 1 (SLT1) gene of E. coli O157:H7. E. coli HB 101 cells when transformed with the same plasmid showed cytopathic effects on Vero cells, which indicates that the SLT 1 homolog gene(s) of Aeromonas spp. is plasmid encoded. These results suggest that Aeromonas spp. may also produce Shiga-like toxin 1, or at least a cytotoxin with some homology with the Shiga-like toxin 1 of E. coli O157:H7.     

Studies on Milk Clotting Enzymes Produced by Basidiomycetes

In the course of screening tests of Basidiomycete proteolytic enzymes, it was observed that some strains produced milk clotting enzymes with fairly weak proteolytic activities.

When sucrose-polypeptone and sucrose-corn steep liquor media were used, only 6 strains out of 44 strains tested showed weak milk clotting activities. Cheddar cheese making with culture filtrates of these 6 strains revealed that the culture filtrates of 2 strains, Irpex lacteus Fr. and Fomitopsis pinicola (Fr.) Karst., were able to produce Cheddar cheese of good quality.

On the other hand, when sucrose-distillers solubles media were used, a lot of strains showed high proteolytic activity in addition to high milk clotting activity. The ratio of milk clotting to proteolytic activities (MCA/PA) was assumed to be an important index for the selection of organism, and F. pinicola and Coriolus consors (Berk.) Imaz. were selected as the strain with high MCA/PA ratio.

As the investigation on culture conditions of 3 strains mentioned above showed that F. pinicola and I. lacteus, were richly productive of milk clotting enzymes, the 2 strains except C. consors were used for further studies on cheese making.

Cheddar cheese making with crude enzymes revealed that cheese products produced by the enzyme of F. pinicola had a slightly bitter taste after 5 months’ ripening but that those produced by the enzyme of I. lacteus had good quality.     

Screening of Nonfilamentous Bacteria for Production of Cutin-Degrading Enzymes

Two hundred thirty-two nonfilamentous bacterial strains, including saprophytes, plant pathogens, and opportunistic plant and human pathogens, were screened for the ability to produce cutinases (cutin-degrading esterases). Initially, esterase activity of culture filtrates of strains grown in nutrient broth-yeast extract medium supplemented with 0.4% apple or tomato cutin was determined by a spectrophotometric assay utilizing the model substrate p-nitrophenyl butyrate. The culture filtrates of the 10 Pseudomonas aeruginosa strains tested exhibited the highest esterase activity, with values of >500 nmol/min/ml. Of these 10 strains, 3 (K799, 1499A, and DAR41352) demonstrated significant induction (10-fold or above) of esterase activity by addition of cutin to nutrient broth-yeast extract medium. The ability of culture filtrates of the three strains to cause release of apple cutin monomers was confirmed by a novel high-performance liquid chromatography technique. Monomer identification was confirmed by gas chromatography-mass spectroscopy analyses. Addition of the nonionic detergent n-octylglucoside stimulated cutinase activity of culture filtrates from strains K799 and DAR41352, but not that of filtrates from strain 1499A. Time course studies in nutrient broth-yeast extract medium supplemented with apple cutin indicated maximal levels of cutinase in the culture fluids after cultures entered stationary phase. Incubation temperatures below the optimal temperature for growth (37°C) led to maximal production of cutinase.     

Efficacy of culture filtrates of <Emphasis Type="Italic">Metarhizium anisopliae</Emphasis> against larvae of <Emphasis Type="Italic">Anopheles stephensi</Emphasis> and <Emphasis Type="Italic">Culex quinquefasciatus</Emphasis>

Efficacy of culture filtrates of five strains of Metarhizium anisopliae isolated from insects were evaluated against Anopheles stephensi and Culex quinquefasciatus. The culture filtrates released from the strains of M. anisopliae in the YpSs and chitin broths were filtered and used for the bioassays after a growth of 7 days. Among the culture filtrates of five strains, M. anisopliae 892 was found to be more effective against both the mosquitoes. The LC(50) values of culture filtrates of M. anisopliae 892 in chitin broth was lower than the LC(50) of culture filtrates in YpSs broth against first and fourth instars of both the mosquitoes. The LC(50) values of culture filtrates were significantly different between first and fourth instars of A. stephensi (t test; P = 0.0001) and C. quinquefasciatus (t test; P = 0.02). The larvae of A. stephensi were more susceptible than C. quinquefasciatus except in two cases. This is the first report of efficacy of culture filtrates produced by M. anisopliae in chitin broth against mosquitoes and have potential as a biological control agent of mosquitoes.     

Degradation of insect cuticle by Paecilomyces farinosus proteases

The entomopathogenic fungus Paecilomyces farinosus showed proteolytic activity in both solid and semi-liquid culture with gelatin as sole N and C source. Semi-liquid cultures were used to characterise proteases. Zymography of crude culture filtrates showed several bands of gelatin degradation in electrophoresis gels. Gel filtration chromatography of these filtrates revealed two peaks of proteolytic activity. Ion-exchange absorption eliminated gelatin from culture filtrates while retaining activity and was used to semipurify P. farinosus proteases. Semipurified culture filtrates had basic pH (8.5 approx.) optimum for proteolytic activity. Treatment of these filtrates with effectors revealed that P. farinosus proteases are serine proteases containing sulphydryl groups. Isoelectrofocusing combined with zymography revealed the presence of several active basic isoforms. Larvae of the lepidopteran Galleria mellonella showed cuticle damage and protein release 1h after incubation with semipurified extracts of P. farinosus. These results indicate that proteolytic enzymes could be involved in insect host penetration by P. farinosus.     

The antibiotic effect of culture filtrates of some soil fungi on rhizobial growth in cultures

Summary Four strains ofRhizobium sp. from peanut (Arachis hypogaea L.) root nodules were tested for their sensitivity to metabolites (culture filtrates) of more than ten common soil inhabiting fungi, in yeast extract mannitol agar and broth cultures. Among the rhizobial strains tested strain CB-530, BU-1 and BU-2 were not sensitive to metabolites ofMyrothecium roridum andFusarium moniliforme whereas CB-1024 was sensitive. Culture filtrates ofGliocladium roseum, Thielavia basicola andDrechslera pedicellata inhibited the strains CB-530 and BU-2 but not CB-1024. Most of the soil inhabiting fungi tested were inhibitory to rhizobial growthin vitro and very few were stimulatory, their effect in either case being strain specific.     

Behavior and Pathogenicity of Tritrichomonas foetus in Chick Liver Cell Cultures*

SYNOPSIS. Clones of Tritrichomonas foetus, referred to as strains KV-1, DK-2, and UT-1, which were judged by subcutaneous mouse assay to differ in pathogenicity, had different effects on trypsin-dispersed liver cell cultures prepared from 3 lines (Light Brown Leghorn, Massachusetts Brown, Massachusetts Low Growth) of chicks. The general pattern of parasite-cell interaction was similar in all cell cultures, but the intensity of certain responses of the cultures to trichomonads, reflected best in the levels of macrophage activity, depended on the line of chicks. The mild UT-1 strain was readily engulfed and digested by the macrophages, caused very little damage to the epithelial cells and fibroblasts, and had a minimal inhibitory effect on the division rate of the latter. Many abnormal changes were seen, however, in the cytoplasm and nucleil of fibroblasts and epithelial cells in cultures exposed to strains of high (KV-1) and intermediate (DK-2) pathogenicity or to cell-free filtrates of rich active cultures (henceforth referred to as filtrates) of these strains. The changes included retraction of cytoplasm which caused cell-free spaces to appear in the sheets of fibroblasts and around the epithelial islands; the islands became detached from the surrounding fibroblast sheets and tended to form multilayered cell mounds. The flagellates and filtrates of KV-1 strain greatly inhibited fibroblast division, and similar, but less pronounced, inhibitory effects were exerted by DK-2 strain and its filtrates. Trichomonads of all 3 strains did not attach themselves to fibroblasts and epithelial cells. Thus the abnormal changes in cultures infected with KV-1 and DK-2 parasites apparently were caused by some toxic substances produced by the trichomonads, which had to be responsible also for the identical changes in cultures exposed to filtrates of these strains. KV-1 and DK-2 strains inhibited the phagocytic activity of the macrophages in the early stages of infection, the former causing stronger and longer lasting inhibition. Trichomonads of KV-1 strain multiplied actively within the phagocytes which developed degenerative changes and ultimately burst, releasing healthy parasites into the medium. DK-2 flagellates, altho incapable of dividing inside the macrophages, usually were not digested and caused degeneration of the cells which contained them.     

On the toxinogenicity of Pseudomonas aeruginosa strains

Investigation of 367 P. aeruginosa strains primarily isolated from clinical and other biological material as well as from the environment yielded results suggesting a substantial toxinogenic potential. 92.6% of the assayed culture filtrates derived from the strains under investigation proved positive in the early skin tests on rabbits. 49.7% of the assayed material induced cytotoxic alterations on Vero cells, the rates for Y1 and CHO cells being 50.3% and 43.5% respectively. 54.3% culture filtrates caused haemolysis of rabbit RBC and 52.7% lysed horse RBC. Gelatinase activity was found in 96.3% of tested material, protease in 89.8%, lecithinase in 62.4% and elastase in 29.6%. 12.6% of tested material induced fluid accumulation in a ligated intestinal loop. None of the culture filtrates elicited a positive reaction in the suckling mice test suggesting the absence of the thermostable enterotoxin.