Broth Culture Filtrates Research Articles

Cytotoxin production by Helicobacter pylori from patients with upper gastrointestinal tract diseases.

A cytotoxin produced by some Helicobacter pylori strains has recently been identified. The cytotoxin induces intracellular vacuolization of cultured cells. The aim of the present study was to examine the frequency of occurrence of cytotoxin-producing strains of H. pylori from subjects with upper gastrointestinal disease including nonulcer dyspepsia, gastric and duodenal ulcer disease, gastroesophageal reflux disease, and gastric cancer. Broth culture filtrates of clinical isolates of H. pylori recovered from 175 patients were used to inoculate Vero and HeLa cell monolayers for the detection of vacuolating cytotoxin activity. The results obtained demonstrated that the highest percentage of strains producing cytotoxin were found in subjects with peptic ulcer disease (gastric ulcer, 65%; duodenal ulcer, 66%; P < 0.01 compared with nonulcer dyspepsia, 38%). Of the 11 patients with gastroesophageal reflux disease, 4 of 5 patients in this group who had esophageal ulcers, were found to be infected with strains that produced cytotoxin. Three of the four patients with carcinoma of the stomach were also found to be infected with cytotoxic strains of H. pylori. With increasing severity of mucosal damage in subjects with a normal upper gastrointestinal tract, macroscopic gastritis, duodenitis, and peptic ulceration, there were corresponding increase in the proportion of strains producing cytotoxin; these increases were 32, 46, 50, and 66%, respectively. H. pylori strains from subjects with ulcer disease commonly produced vacuolating cytotoxin, suggesting that it may be a virulence factor in the pathogenesis of peptic ulcer disease.

Characteristics of Pseudomonas aeruginosa strains isolated from urinary tract infections.

Thirty-three uropathogenic strains of Pseudomonas aeruginosa were investigated for hemolytic activity in both bacterial broth culture filtrates and isolate lyzates, resistance to bactericidal activity of fresh human serum, resistance to six antibiotics and plasmid DNA profile. Twenty-four of the 33 (73%) bacterial filtrates showed lysis of rabbit erythrocytes, as did the three after guinea-pig erythrocyte treatment. Twelve of 33 isolate lysates showed in parallel lysis of both types of erythrocytes used. Serum resistance was found in 17 (52%) isolates, intermediate resistance in 15 (45 %) isolates and only one isolate showed serum sensitivity. Resistance to antibiotics was detected as follows (in %): tetracycline 94, kanamycin 79, chloramphenicol 76, septrin 73, ampicillin 64, streptomycin 45, gentamicin 18. None of the isolates investigated showed resistance to colistine. With the exception of one isolate, plasmid DNA was detected in all P. aeruginosa strains.

Cytotoxicity of Helicobacter pylori on human gastric epithelial cells in vitro

Objective To evaluate the respective roles of vacuolating cytotoxin(s) and urease-mediated ammonia production in Helicobacter pylori-induced cytotoxicity on cultured human gastric epithelial cells. Methods Monolayers of MKN 28 cell line were incubated with broth culture filtrates of two urease-positive H. pylori strains: CCUG 17874, cytotoxin-producing and C21, non-cytotoxin-producing. After incubation, highly sensitive quantitative assays were used to measure the ammonia levels in the cell medium, cell vacuolation and cell death. In order to evaluate the cytotoxic role of ammonia more accurately, we performed an ‘ammonia simulation’ by measuring cell vacuolation and cytolethality induced by incubation with definite concentrations of ammonium chloride. Cell damage was also studied morphologically. Results Both H. pylori strains produced ammonia and induced cell vacuolation and cytolethality. With the G21 strain, both cell vacuolation and cytolethality appeared virtually identical to ammonia simulation. With the CCUG 17874 strain, which induced a higher degree of cell vacuolation and cell death compared with the G21 strain, cell vacuolation and cytolethality appeared largely ammonia-independent. The vacuoles induced by each H. pylori strain and by ammonia simulation were similar to each other. In less severely affected cells, vacuole formation and ammonia simulation were regularly limited by smooth membranes and morphologically and topographically resembled dilated endosomes or trans Golgi vesicles. In more severely affected cells, large autophagic-like vacuoles containing cell debris were also prominent. Conclusions The vacuolating cytotoxin(s) and ammonia are major H. pylori virulence factors. The cytotoxin(s) seem to play a more prominent role than ammonia in cell vacuolation. Nevertheless, ammonia seems to damage cells by other mechanisms in addition to vacuolation. A common pathway for cytotoxin- and ammonia-induced cell vacuolation seems likely.

Control of the rootknot nematodemeloidogyne javanicabyBacillus cereus

Exposure of Meloidogyne javanica second‐stage juveniles to the bacterium Bacillus cereus in soil inhibited the penetration of the juvenile nematodes into tomato roots. Culture filtrate of the bacterium grown on nutrient broth and tryptic soy broth revealed nematocidal activity on M. javanica juveniles and eggs. Loss of the nematocidal activity of the media by lowering pH, boiling or dialysis raised the possibility that the active ingredient in the culture filtrate was ammonia, released during the breakdown process of peptides in the media by bacterial activity. Free ammonia (NH3) concentrations in the nutrient broth and tryptic soy broth culture filtrates measured after 48 h were 140 and 190 µg ml−1 respectively. Exposure of second‐stage juveniles to 9.3 µg ml−1 ammonia for 40 h in vitro was lethal to 95% of the nematode population. In a nitrate medium, nitrite accumulated up to 250 µg ml−1 during the growth of the bacterium, and its culture filtrate revealed nematocidal activity. The nematocidal activity...

Western immunoblot analysis and serologic characterization of Blastomyces dermatitidis yeast form extracellular antigens.

A major 98-kDa extracellular protein antigen of Blastomyces dermatitidis was shown in Western blot (immunoblot) analysis to be highly reactive with serum antibodies from patients with blastomycosis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of yeast form B. dermatitidis culture filtrates and cell extracts demonstrated over 50 proteins, only 24 of which were immunoreactive. Of these, a 98-kDa protein was found to be the most specific and was isolated. This protein was found in both broth culture filtrates and extracts of yeast forms. Western blot tests with this antigen detected serum antibodies in 91% of 32 patients with clinically proven blastomycosis, in contrast to an enzyme-linked immunosorbent assay (ELISA) with DEAE-purified antigen, which detected 88% of the cases. The Western blot test also exhibited lower reactivity with a panel of sera from patients with heterologous fungal infections that were cross-reactive in the ELISA with DEAE-purified B. dermatitidis antigen. The 98-kDa protein electroeluted from polyacrylamide gels was identical to the diagnostic A antigen used in the blastomycosis immunodiffusion test. Comparison of the 98-kDa antigen with a previously described 120-kDa yeast form cell wall protein antigen of B. dermatitidis showed that these two antigens are almost identical.

Cytopathic effects of Helicobacter pylori on cultured mammalian cells.

Cytopathic effects of broth-culture filtrates from eight clinical isolates and one reference strain of Helicobacter pylori on three cultured mammalian cell lines were investigated. All the strains, including NCTC 11637, produced cytotoxic factors that caused intracellular vacuolation on these cell lines. AGS and SflEp cells were more sensitive than HEp-2 cells. To examine the role of urease in the cytotoxic effect, a urease-negative mutant was produced. Filtrates from both wild-type and mutant strains produced similar vacuolation on SflEp cells in the absence of urea, suggesting that H. pylori produces a cytotoxic substance other than urease. In contrast, ammonia alone, or jack bean urease with urea, also induced rounding and detachment of SflEp cells, whereas ammonium salts induced the production of small vacuoles. The combination of the broth filtrate of the wild-type strain and urea induced vacuolation followed by rounding and detachment of SflEp cells. Evidence is presented that the latter changes are due to ammonia produced during incubation. Nevertheless, the amounts produced were less than that needed to induce cytopathic effects by itself. These results suggest that the cytotoxic substance induces intracellular vacuolation, and that the vacuolated cells are more susceptible to killing by ammonia. Thus both the cytotoxic substance and urease may contribute to the lethal cytotoxicity of H. pylori in vitro.

Production of a Cytotoxin by Helicobacter pylori

Helicobacter pylori produces a cytotoxin that was initially detected as the ability of broth culture filtrates of this bacterium to induce intracellular vacuolation of cultured cells. Fifty-three percent of more than 200 isolates of H. pylori tested produce the cytotoxin, which appears to be unique to H. pylori. Results of characterization studies suggest that the cytotoxin is a high-molecular-weight protein. Detection of serum antibody to the cytotoxin by neutralization or immunoblotting indicates that this toxin is produced in vivo. The cytotoxin is more frequently associated with cases of peptic ulceration than with cases of gastritis; this finding suggests that the toxin contributes to the severity of disease associated with H. pylori infection.

Hepatocidol toxicity ofListeriaspecies

A novel rat hepatocidal test, based on morphological changes in monolayer culture and the percentage of lactate dehydrogenase (LDH) released into the medium after exposure to culture filtrates of Listeria spp. was used to determine listerial toxicity and pathogenicity. Primary cultures of rat hepatocytes exposed to brain heart infusion (BHI) culture filtrates from ATCC strains of Listeria monocytogenes and L. ivanovii, released 91–92% and 95% of LDH after 3 h and 18.5 h, respectively. Cultured monolayers changed from normal hepatocytes into nonviable round forms. Brain heart infusion broth and BHI culture filtrates of other Listeria spp. were nontoxic to hepatocytes. The rat hepatocidal test is a quantitative and rapid system for studying listerial toxicity and pathogenicity.

Hepatocidol toxicity of Listeria species

A novel rat hepatocidal test, based on morphological changes in monolayer culture and the percentage of lactate dehydrogenase (LDH) released into the medium after exposure to culture filtrates of Listeria spp. was used to determine listerial toxicity and pathogenicity. Primary cultures of rat hepatocytes exposed to brain heart infusion (BHI) culture filtrates from ATCC strains of Listeria monocytogenes and L. ivanovii, released 91–92% and 95% of LDH after 3 h and 18.5 h, respectively. Cultured monolayers changed from normal hepatocytes into nonviable round forms. Brain heart infusion broth and BHI culture filtrates of other Listeria spp. were nontoxic to hepatocytes. The rat hepatocidal test is a quantitative and rapid system for studying listerial toxicity and pathogenicity.

Production of hemolysin and other extracellular enzymes by clinical isolates of Pseudomonas pseudomallei

One hundred clinical isolates of Pseudomonas pseudomallei from humans were tested for their ability to produce extracellular, biologically active substances which are thought to contribute to the virulence of Pseudomonas species. All isolates produced at least on extracellular enzyme; 91 strains were positive for lecithinase, lipase, and protease; but none was positive for elastase. Ninety-three strains produced a hemolysin which was detectable around the heavy growth on saline-washed sheep erythrocyte brain heart infusion agar but not demonstrable around individual colonies or in broth culture filtrate. In contrast, a hemolysin which was cytolytic around individual colonies of P. pseudomallei on the assay plate and in broth culture filtrate was exhibited by four strains. By using one of these four isolates as the test strain, the latter hemolysin was characterized further. It was heat labile, most active in an acid environment (pH 5.5), and cytolytic in broth culture filtrate for a variety of animal and human erythrocytes. Sterols, particularly cholesterol and 7-dehydrocholesterol, inhibited its hemolytic activity, but the activity was not enhanced by reducing agents or suppressed by reagents which modify sulfhydryl-activated hemolysins. A nonhemolytic mutant of the test strain of P. pseudomallei retained the extracellular enzymes of its parent, indicating that the hemolysin was not a lecithinase, lipase, or protease.

Changes in ovine erythrocyte morphology due to sphingomyelin degradation by Corynebacterium pseudotuberculosis phospholipase D

Corynebacterium pseudotuberculosis produces a sphingomyelin-specific phospholipase D exotoxin that is a major determinant in the pathogenesis of caseous lymphadenitis. We assessed the changes induced in the morphology and sphingomyelin concentration of ovine erythrocytes after incubation with broth culture filtrates or purified exotoxin. Sphingomyelin was hydrolysed nearly in parallel with shape change. Morphologically, spherostomatocytes were seen initially, and later these contained numerous vacuoles at the periphery. Vacuoles seen in thin sections with transmission electron microscopy appeared as pits with scanning electron microscopy. Pitting became progressively worse with time, leading to extensive scalloping of the membrane surface. Chemically, significant decreases ( P ⩽ 0.05) in erythrocyte membrane sphingomyelin content and significant increases ( P ⩽ 0.05) in the content of glycerophospholipids (i.e. phosphatidylglycerol) were observed in erythrocytes incubated with broth culture filtrates or purified exotoxin. These alterations may contribute to the pathophysiology of acute infections induced by C. pseudotuberculosis.

Comparative characterization of the leukocidic and hemolytic activity of Moraxella bovis

SUMMARY The cytotoxic effect of Moraxella bovis 118F on bovine neutrophils was evaluated and characterized by use of a 51Cr release assay. Neutrophils harvested from healthy adult cattle were labeled with 51Cr. The leukocidic activity produced by M bovis 118F, a hemolytic strain of M bovis, was heat-labile. A live culture of strain 118F, at a ratio of 100 bacteria/neutrophil, released 97.7% of the 51Cr from labeled neutrophils. Neither a heat-killed preparation of M bovis 118F nor a live or heat-killed preparation of M bovis IBH63 (a nonhemolytic and nonpathogenic strain) induced significant (P &gt; 0.05) release of 51Cr. Moraxella bovis 118F broth culture filtrates prepared for evaluation of leukocidic activity also were evaluated for hemolytic activity. These 2 toxic activities had several characteristics in common. Both were filterable, heat-labile, produced by a hemolytic strain, and were released during early logarithmic phase growth from broth cultures. Leukocidic and hemolytic activities were protected from degradation by phenylmethyl sulfonyl fluoride, a serine protease inhibitor. Leukocidic and hemolytic activities were dependent on calcium ions. Filtrate resulted in 54.1% 51Cr release from labeled neutrophils and contained 646.7 hemolytic U/ml, respectively, when saline (0.85% NaCl) + 10 mM CaCl2 solution was used as diluent. Neither saline solution nor saline + 10 mM MgCl2 solution supported leukocidic or hemolytic activity. Serum, obtained from several calves 10 to 38 days after M bovis inoculation, substantially neutralized leukocidic and hemolytic activities, compared with paired preinoculation serum samples. In addition, no significant difference (P &gt; 0.05) was detected when the ability of each calf's postinfection serum to neutralize leukocidic activity was compared with the ability of the serum to neutralize hemolytic activity.

Pneumolysin Produced by Streptococcus pneumoniae Damages Human Respiratory Epithelium in vitro

Streptococcus pneumoniae is an important human pathogen responsible for lower respiratory tract infections, septicemia, and meningitis. It elaborates a number of proteins, including a hemolytic thiol-activated toxin called pneumolysin. We studied the effect of S pneumoniae culture filtrates on biopsy specimens of human respiratory epithelium in vitro by a photometric technique which measures ciliary beat frequency (CBF), and by transmission electron microscopy. Broth culture filtrates from 11 of 15 clinical isolates caused significant (p<0.001) slowing (16 to 48% control) of CBF.

Cytotoxic activity in broth-culture filtrates of Campylobacter pylori.

Broth-culture filtrates of Campylobacter pylori induced non-lethal cytopathic effects in vitro in 7 of 9 mammalian cell lines tested. Transmission electronmicroscopy revealed that the response consisted of intracellular vacuolisation. Intestine 407 cells were among the most responsive and were used for routine assay. About 55% of isolates of C. pylori tested, originating from four geographic regions worldwide, produced cytotoxic activity. The activity was neutralisable by specific antisera to broth-culture filtrates or to sonicated bacteria but not by antisera to other bacterial preparations. Cytotoxic activity was heat-labile (70 degrees C for 30 min), was protease-sensitive and ammonium-sulphate precipitable. It did not pass through an ultrafiltration membrane with a nominal mol.-wt limit of 100 X 10(3). It was concluded that C. pylori can produce a factor that alters cultured cells in vitro. The relevance of this factor to the pathogenesis of gastritis associated with C. pylori remains to be determined.

Investigation into the pathogenesis of atrophic rhinitis in pigs. I. Atrophic rhinitis caused by Bordetella bronchiseptica and Pasteurella multocida and the meaning of a thermolabile toxin of P. multocida.

In two groups of swine herds, herds with and without clinical AR the presence of Atrophic Rhinitis (AR) correlated with the presence of toxinogenic Pasteurella multocida (PM) and not with the Bordetella bronchiseptica (BB) infection. Six BB- and eighteen PM-strains have been investigated for AR pathogenicity. Broth cultures were injected intradermally in guinea-pigs (GPST) or intranasally in 3-week-old colostrum deprived specific pathogen free (SPF) piglets. The average atrophy of the ventral conchae (AVC) correlated with the GPST in 4 BB-and 7 PM-strains. One BB- and 2 PM-strains were qualified as doubtful, the others as non-AR pathogenic. With AR pathogenic BB-and PM-strains clinical AR could be induced in 3-and 6-week-old piglets. AVC lesions (gradation greater than 1) could be induced with BB in piglets of 6 and with pathogenic PM in 16-week-old piglets. Six of seven AR pathogenic PM-strains resembled Carter-type D and one resembled type A. No significance was found between AR pathogenicity and somatic serotypes. Intranasal instillations of cell-free broth culture filtrates of AR pathogenic PM-strains also caused AR in piglets. These filtrates also caused lethality in piglets and in mice lethalitytest (MLT) and induced a positive GPST. After heating the pathogenic effects of the filtrates disappeared. The name AR toxin has been introduced for this thermolabile, haemorrhagic dermonecrotic (HDNT) fraction of the AR inducing filtrates. The severity of the AR lesions depended on the amount of the AR toxin intranasally instilled in pigs. Cross protecting antibodies obtained in rabbits against the AR toxins of two PM strains could be demonstrated by a toxin neutralisation test in the MLT and the GPST. Broth cultures were injected intradermally in guinea-pigs (GPST) or intranasally in 3-week-old colostrum deprived specific pathogen free (SPF) piglets.

Enterotoxin production in relation to taxonomic grouping and source of isolation of Aeromonas species.

A total of 19 of 20 (95%) strains of Aeromonas hydrophila biovar hydrophila and 16 of 17 (94%) strains of Aeromonas sobria isolated from a variety of clinical and environmental sources were found to be enterotoxin positive. Only 2 of 18 (11%) A. hydrophila biovar anaerogenes and 2 of 13 (15%) unidentified Aeromonas strains from a similar variety of sources produced enterotoxin. No association was apparent between the source of isolation, in particular diarrheal stools, and enterotoxigenicity; 41% of the isolates from diarrheal stools were enterotoxin negative. A strong correlation was noted between ability to produce enterotoxin and positive results in six characters: lysine decarboxylase and Voges-Proskauer reactions, production of gas from glucose, gluconate oxidation, xanthine hydrolysis, and hemolysis of human erythrocytes. In the majority of cases (35 of 39 strains), enterotoxigenicity was detected using cell-free filtrates of brain heart infusion broth cultures grown at 36 degrees C for 15; however, the other four positive isolates were detected after growth in the same broth at 30 degrees C or in Casamino Acids-yeast extract broth at 30 or 37 degrees C. It is recommended that for enterotoxin tests, strains should be grown in both media at both temperatures. The infant mouse test was found to be a simple and reliable method for detection of the enterotoxin. The toxin proved to be heat labile and not neutralized by cholera antitoxin.

Stimulatory Effect of Pseudomonas aeruginosa on Mucin Secretion by the Respiratory Epithelium

Cell-free filtrates of broth cultures from 11 of 18 strains of Pseudomonas aeruginosa isolated from patients with cystic fibrosis (CF) (or from patients without CF) increased substantially the secretion of mucin by explants of trachea from guinea pigs and human tracheal tissue obtained at autopsy. The effect was not related to the mucoid nature of the isolates, was not destroyed by heating at 90 degrees C for 30 minutes, and was not dependent on serotype. As P aeruginosa commonly infects the respiratory tracts of persons with CF, elaboration of an extracellular product that stimulates mucin secretion could play a role in the pathogenesis of the disease in the airways.

Evaluation of a commercial exoantigen test system for the rapid identification of systemic fungal pathogens.

Seventy-nine mycelial-form stock cultures of Blastomyces dermatitidis, Coccidioides immitis, Histoplasma capsulatum, and morphologically similar fungi were extracted and tested by using commercial macroimmunodiffusion exoantigen test kits and the Centers for Disease Control (CDC) reference system for identifying fungal isolates. Results showed 100% correlation between the two systems. Specific exoantigens of C. immitis and H. capsulatum extracted from agar slant cultures (slant extraction method) readily were identified. In eight of 26 cultures of B. dermatitidis, broth culture filtrates (broth-shake-flask method) were required to demonstrate the specific bands of identity. No false-negative reactions or cross-reactivity among the pathogens and other fungi were observed. The commercial test kits provided a rapid and specific method for identifying or confirming suspected fungal pathogens.

Isolation and characterization of thirteen intestinal microorganisms capable of 7 alpha-dehydroxylating bile acids.

Thirteen anaerobic bacteria capable of performing the 7 alpha-dehydroxylation of both cholic acid and chenodeoxycholic acid were isolated from human feces and also from sewage. Ten organisms from heat-treated samples were species of Clostridium identical or closely related to the Clostridium bifermentans-C. sordellii group and consisted of four strains elaborating 7 alpha-dehydroxylase alone and six strains capable of catalyzing both 7 alpha-dehydrogenation and 7 alpha-dehydroxylation. The remaining three organisms, recovered from fresh human feces, were gram-positive, nonflagellated, nonsporeforming, anaerobic rods and comprised two distinct species. Strain HD-17, still unidentified, had both activities, but was unique in that it exclusively 7 alpha-dehydroxylated cholic acid while biotransforming chenodeoxycholic acid, preferably though 7 alpha-dehydrogenation. Two unclassified strains, b-8 and c-25, metabolized both acids though 7 alpha-dehydroxylation and 7 alpha-dehydrogenation. Except for strains b-8 and c-25, all of th 7 alpha-dehydroxylating bacteria split the conjugated bile acid series, and hydrolases were detected in cell-free filtrates of early stationary-phase broth cultures.

Pathogenic Activities of Aeromonas hydrophila biovar hydrophila (Chester) POPOFF and VERON, 1976 to Fishes

A total 291 strains of motile members of the genus Aeromonas were classified according to the taxonomic scheme proposed by POPOFF and VERON (1976). Forty three were indetified as A. hydrophila biovar hydrophila, 9 as A. hydrophila biovar anaerogenes, and 52 as A. sobria. The remaining 187 strains could not be identified. Thirty seven (86%) of 43 A. hydrophila biovar hydrophila, 3 (33 %) of 9 A. hydrophila biovar anaerogenes, 32 (62 %) of 52 A. sobria, and 65 (35%) of the 187 unidentified originated from fishes.All 6 A. hydrophila biovar hydrophila examined fell in the category of high virulence. The Japanese loach injected with them were killed earlier than those injected with the other motile Aeromonas, and the difference of mean time to death was statistically significant (P<0.01). The difference of average value of proteolytic activity of the broth culture filtrate between A. hydrophila biovar hydrophila and the other was significant (P<0.01), also. Exophthalumus and scale protrusion were evident only among fish injected with the sonicated cell extracts of A. hydrophila biovar hydrophila.