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The existence of airborne mycotoxins in mold-contaminated buildings has long been hypothesized to be a potential occupant health risk. However, little work has been done to demonstrate the presence of these compounds in such environments. The presence of airborne macrocyclic trichothecene mycotoxins in indoor environments with knownStachybotrys chartarumcontamination was therefore investigated. In seven buildings, air was collected using a high-volume liquid impaction bioaerosol sampler (SpinCon PAS 450-10) under static or disturbed conditions. An additional building was sampled using an Andersen GPS-1 PUF sampler modified to separate and collect particulates smaller than conidia. Four control buildings (i.e., no detectableS. chartarumgrowth or history of water damage) and outdoor air were also tested. Samples were analyzed using a macrocyclic trichothecene-specific enzyme-linked immunosorbent assay (ELISA). ELISA specificity was tested using phosphate-buffered saline extracts of the fungal generaAspergillus,Chaetomium,Cladosporium,Fusarium,Memnoniella,Penicillium,Rhizopus, andTrichoderma, fiveStachybotrysstrains, and the indoor air allergens Can f 1, Der p 1, and Fel d 1. For test buildings, the results showed that detectable toxin concentrations increased with the sampling time and short periods of air disturbance. Trichothecene values ranged from 1,300 pg/m3of sampled air. The control environments demonstrated statistically significantly (P< 0.001) lower levels of airborne trichothecenes. ELISA specificity experiments demonstrated a high specificity for the trichothecene-producing strain ofS. chartarum. Our data indicate that airborne macrocyclic trichothecenes can exist inStachybotrys-contaminated buildings, and this should be taken into consideration in future indoor air quality investigations.

The growth of indoor molds and their resulting products (e.g., spores and mycotoxins) can present health hazards for human beings. The efficacy of chlorine dioxide gas as a fumigation treatment for inactivating sick building syndrome-related fungi and their mycotoxins was evaluated. Filter papers (15 per organism) featuring growth of Stachybotrys chartarum , Chaetomium globosum , Penicillium chrysogenum , and Cladosporium cladosporioides were placed in gas chambers containing chlorine dioxide gas at either 500 or 1,000 ppm for 24 h. C. globosum was exposed to the gas both as colonies and as ascospores without asci and perithecia. After treatment, all organisms were tested for colony growth using an agar plating technique. Colonies of S. chartarum were also tested for toxicity using a yeast toxicity assay with a high specificity for trichothecene mycotoxins. Results showed that chlorine dioxide gas at both concentrations completely inactivated all organisms except for C. globosum colonies which were inactivated an average of 89%. More than 99% of ascospores of C. globosum were nonculturable. For all ascospore counts, mean test readings were lower than the controls ( P < 0.001), indicating that some ascospores may also have been destroyed. Colonies of S. chartarum were still toxic after treatment. These data show that chlorine dioxide gas can be effective to a degree as a fumigant for the inactivation of certain fungal colonies, that the perithecia of C. globosum can play a slightly protective role for the ascospores and that S. chartarum , while affected by the fumigation treatment, still remains toxic.

Highly respirable particles (diameter

Neuraminidases produced by 16 strains of Pasteurella multocida (serotypes 1 to 16) were characterized by molecular weight, substrate specificity, and antigenic identity. After growth in a chemically defined medium, stage I (lyophilized) culture supernatants were assayed for activity with N-acetylneuramin lactose, human alpha-1-acid glycoprotein, fetuin, colominic acid, and bovine submaxillary mucin. Neuraminidase produced by P. multocida A:3 was purified by a combination of salt fractionation, ion-exchange chromatography on DEAE-Sephacel, and gel filtration on Sephadex G-200. Purified P. multocida A:3 neuraminidase was employed to immunize rabbits, and the resulting antiserum reduced the activity of the P. multocida A:3 enzyme by 40.3%. This antiserum also reduced the activities of the neuraminidases produced by other serotypes by between 30.8 and 59.6%. Molecular weight estimates of the neuraminidases produced by the various serotypes were obtained by gel filtration chromatography on Sephadex G-200. Each of the 16 serotypes examined produced a neuraminidase with a molecular weight of approximately 500,000. In addition, all 16 high-molecular-weight neuraminidases showed similar substrate specificities. On the basis of these data, it appears that the high-molecular-weight neuraminidases produced by different P. multocida serotypes are quite similar.

The properties of an extracellular neuraminidase produced by a Pasteurella multocida A:3 strain that was isolated in a case of bovine pneumonia were examined during growth in a defined medium. This enzyme (isolated from concentrated culture supernatants of P. multocida A:3) was active against N-acetylneuramin lactose, human alpha-1-acid glycoprotein, fetuin, colominic acid, and bovine submaxillary mucin. Enzyme elaboration was correlated with the growth of the organism in a defined medium, with maximum quantities produced in the stationary phase. The enzyme was purified by a combination of ammonium sulfate fractionation, ion exchange on DEAE-Sephacel, and gel filtration on Sephadex G-200. The purified neuraminidase possessed a specific activity of 9.36 mumol of sialic acid released per min per mg of protein against fetuin. The enzyme possessed a pH optimum of 6.0 and a Km of 0.03 mg/ml. The P. multocida A:3 neuraminidase had a molecular weight of approximately 500,000 as estimated by gel filtration. The enzyme was stable at 4 and 37 degrees C for 3 h. Approximately 75% of the neuraminidase activity was lost within 30 min at 50 degrees C. Greater than 90% of the enzyme activity was destroyed within 10 min at temperatures of > or = 65 degrees C. The P. multocida neuraminidase does not appear to be serologically related to the Pasteurella haemolytica A1 neuraminidase since antiserum prepared against the purified P. haemolytica enzyme did not neutralize the P. multocida enzyme.

Neuraminidases produced by 16 strains of Pasteurella haemolytica (serotypes 1 to 16) were characterized by molecular weight, antigenic identity, and substrate specificity. After growth in a chemically defined medium, stage I (lyophilized) culture supernatants were assayed for activity with N-acetylneuramin lactose, human alpha-1-acid glycoprotein, fetuin, colominic acid, and bovine submaxillary mucin. Neuraminidase produced by P. haemolytica serotype A1 (Ph A1) was purified by a combination of salt fractionation, ion-exchange chromatography on DEAE-Sephacel, and gel filtration on Sephadex G-200. Purified Ph A1 neuraminidase was used to immunize rabbits, and the resultant antiserum reduced the activity of Ph A1 neuraminidase by 46%. This antiserum also reduced the activity of neuraminidase produced by the other serotypes by between 15 and 66%. Molecular weight estimates of the neuraminidases produced by the various serotypes were obtained by gel filtration chromatography on Sephadex G-200. Fifteen of the 16 serotypes examined produced a neuraminidase with a molecular weight of approximately 150,000 to 200,000. One serotype (serotype 11) produced no material with neuraminidase activity. In addition, all 15 high-molecular-weight neuraminidases showed similar substrate specificities. That is, they were all most active against N-acetylneuramin lactose and least active against bovine submaxillary mucin. On the basis of these results, it appears that the high-molecular-weight neuraminidases produced by the different P. haemolytica serotypes are quite similar.

The properties of an extracellular neuroaminidase produced by a Pasteurella haemolytica A1 strain (isolated from a case of bovine pneumonia) during growth in a defined medium were examined in this investigation. This enzyme, isolated from concentrated culture supernatants of P. haemolytica A1, was active against N-acetylneuramin lactose, human alpha 1-acid glycoprotein, fetuin, and bovine submaxillary mucin. Neuraminidase production paralleled bacterial growth in a defined medium and was maximal in the stationary phase of growth. The enzyme was purified to homogeneity by a combination of salt fractionation, ion-exchange chromatography on DEAE-Sephacel, and gel filtration on Sephadex G-200. These procedures yielded an enzyme preparation that possessed a specific activity of 100.62 mumol of sialic acid released per min per mg of protein against human alpha 1-acid glycoprotein. The Km value for this enzyme with human alpha 1-acid glycoprotein as the substrate was 1.1 mg/ml, and the enzyme possessed a pH optimum of 6.5. The P. haemolytica A1 neuraminidase had a molecular weight of approximately 150,000 as estimated by gel filtration and approximately 170,000 when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme was stable at 4 degrees C for 3 h. At 37 degrees C for 3 h, 25% of enzymatic activity was lost. Approximately 55% of the enzyme activity was lost within 30 min at 50 degrees C, with greater than 70% of the enzyme activity being destroyed within 10 min at temperatures of > or = 65 degrees C.

Twelve strains of serotype III group B streptococci (8 isolated from cases of neonatal disease, 3 isolated from asymptomatically colonized infants, and 1 laboratory reference strain) were examined for the vitro production of three potential extracellular virulence products: type-specific antigen, neuraminidase, and protease. In addition, virulence in a mouse model, expressed as 50% lethal dose, was determined for the 12 strains to determine whether a relationship existed between the production of any of the three extracellular products and virulence. Only production of extracellular type-specific antigen showed a correlation with virulence in the mouse model. The high producers of extracellular type-specific antigen were an average of 166-fold more virulent for mice than low producers of the same component. There was no correlation between virulence and either neuraminidase or protease production, nor was there a correlation between either of these two extracellular products and the levels of extracellular type-specific antigen. When levels of group B streptococci of each type (a high and low producer of extracellular type-specific antigen) in organs of infected mice were examined, comparable levels of organisms were found in the brain, spleen, and lungs of mice near death regardless of the initial inoculum. However, the high producer of extracellular type-specific antigen caused death in mice with a 2 to 3 log lower inoculum than the low producer, suggesting that these strains may be more invasive.

Six strains of viridans streptococci isolated from confirmed cases of subacute bacterial endocarditis were studied for production of extracellular material. All six strains, when grown to the exponential phase, produced exoproducts that had similar elution profiles on a G-100 Sephadex column. Since essential nutrients, such as amino acids, may be periodically growth limiting to streptococci in the fibrin-covered lesions on heart valves, the potential to elaborate extracellular protein and other material by streptococci that were deprived of essential amino acids was studied. Examination of supernatant fluids from cultures of Streptococcus MG intermedius deprived of glutamate and cystine revealed the presence of a complex mixture of extracellular materials in amounts comparable to those produced by normallly growing cells, Although only a slight (21 to 24%) increase in total protein occurred during amino acid deprivation of 12 h, the extracellular material contained numerous protein components, several of which demonstrated proteolytic activity. On a cell dry weight basis, the amino acid-deprived cells produced four-to eightfold more protease(s) than did exponential cells grown in complete medium. These results demonstrate that viridans streptococci are capable of elaborating potentially damaging compounds even when their multiplication has been arrested by nutritional deprivation.

Transtracheal instillation of clinical isolate Klebsiella pneumoniae serotype 1 (KP1) into the lungs of rats resulted in the production of a characteristic, chronic lobar pneumonia. To further examine this phenomenon, two variants of this organism were employed in this experimental model. These variants differed markedly in capsule size, colony morphology, and in virulence, as determined by mouse lethality tests. The ability of these strains to establish a lobar pneumonia in rats correlated with the virulence of the respective organisms as monitored by intraperitoneal injection in mice. The 50% lethal doses in mice were 4.9 x 10(1) colony-forming units (CFU) for the more virulent KP1 strain (KP1-O) and 1.42 x 10(5) CFU for the less virulent variant (KP1-T). In the rat lung model, marked lung pathology was evident by day 6 with a KP1-O inoculum of 5 x 10(2) CFU, whereas KP1-T caused little or no lung pathology when delivered transtracheally at a concentration of 7 x 10(6) CFU. Two relatively nonvirulent variants of K. pneumoniae serotype 2 were also used in this rat lung model and were found not to produce a lobar pneumonia even when delivered in large doses. These results indicate that a chronic lobar pneumonia can be established in a rat model if the appropriate organism is employed and the virulence of K. pneumoniae injected intraperitoneally into mice is an excellent indicator of an organism's potential to cause lobar pneumonia in rats.

Ten clinical isolates of Pseudomonas cepacia from the sputum of cystic fibrosis patients were examined for the ability to produce lipase. Lipase substrates used included egg yolk agar, four different polyoxyethylene sorbitans (Tweens), and p-nitrophenylphosphorylcholine, a chromogenic substrate used to assay for phospholipase C. Lipase activity was detected in the filtrates of organisms grown to the exponential phase in either tryptose minimal medium or chemically defined medium. Lipase activity increased in the filtrates if the cultures were allowed to proceed into the stationary phase. None of the isolates produced phospholipase C. Lipase activity on Tween 20 ranged from 41.6 X 10(-3) to 640.0 X 10(-3) U/micrograms of protein. The activity was similar or slightly lower when Tween 40, 60, or 80 was used as the substrate. There was no correlation between lipase activity on Tween and that demonstrated on egg yolk agar. Lipase activity increased as pH increased from 7.0 to 9.0. Boiling for 5 min resulted in 66% loss of enzyme activity. The remaining activity continued to decrease with increasing boiling time. The enzyme was purified by gel filtration on Sephadex G-200, and the resultant preparation, when subjected to polyacrylamide gel electrophoresis, resulted in a single protein band (molecular weight, approximately 25,000) from which lipase activity could be eluted. The purified lipase was not cytotoxic to HeLa cells, nor was it toxic when injected intravenously into mice.

The properties of an extracellular neuraminidase produced by a Streptococcus sanguis strain (isolated from a confirmed case of subacute bacterial endocarditis) during growth in a defined medium was examined in this investigation. This enzyme, isolated from concentrated culture supernatants of S. sanguis biotype II, was active against human alpha-1 acid glycoprotein, N-acetylneuramin lactose, bovine submaxillary mucin, and fetuin. Neuraminidase production paralleled bacterial growth in defined medium and was maximal in the early stationary phase of growth but decreased dramatically, probably owing to protease production, during the late stationary phase. The enzyme was purified to near homogeneity by a combination of salt fractionation, ion-exchanged chromatography on DEAE-Sephacel, and gel filtration on Sephadex G-200. These procedures yielded an enzyme preparation that possessed a specific activity of 174.4 mumol of sialic acid released per min per mg of protein against human alpha-1 acid glycoprotein. The Km value for this enzyme with human alpha-1 acid glycoprotein as substrate was 2.5 X 10(-3) M, and the enzyme possessed a pH optimum of 6.5. The S. sanguis neuraminidase had a molecular weight of approximately 85,000 as estimated by gel filtration and approximately 90,000 when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme was stable at temperatures of 4 and 37 degrees C for 3 h, but approximately 50% of the enzymatic activity was lost within 30 min at 50 degrees C, with 100% of the enzymatic activity being destroyed within 10 min at temperatures of greater than or equal to 65 degrees C.

Neuraminidase (sialidase) activity in concentrated culture filtrates of group B streptococci was measured with bovine submaxillary mucin as substrate. Group B streptococcal neuraminidase was not active on human alpha-1 acid glycoprotein and did not show increased activity on bovine submaxillary mucin that had been O-deacetylated by alkaline treatment. The enzyme was produced in a variety of media, including a chemically defined medium (FMC; Terleckyj et al., Infect. Immun. 11:649-655, 1975) supplemented with bovine serum albumin or human serum albumin. Maximal levels of activity were present in filtrates from cells grown in a dialyzable fraction of Todd-Hewitt broth harvested during the late exponential phase of growth. Dramatic decreases were seen when filtrates from the late stationary phase were assayed. The decrease in specific activity during the stationary phase was shown to be due to proteolytic digestion of neuraminidase and not to the elaboration of an extracellular neuraminic acid aldolase.

Extracellular neuraminidase from a type III fresh clinical isolate of a group B streptococcus was purified by a combination of salt fractionation, affinity chromatography of Affi-Gel blue, ion-exchange chromatography on diethylaminoethylcellulose, and gel filtration on Sephacryl S-200. These procedures yielded enzyme which was purified approximately 1,000-fold compared with the enzyme found in the original supernatant fluid. This type III streptococcal neuraminidase had a molecular weight of approximately 125,000 as estimated by filtration on Sephacryl S-200 and approximately 106,000 when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In contrast to the majority of other bacterial neuraminidases, the type III group B streptococcal enzyme had no effect on colominic acid or N-acetylneuramin-lactose; however, it was quite active on bovine submaxillary mucin.

A simple and rapid procedure for the isolation in high yield (about a 30% recovery based on the total 30 to 60% ammonium sulfate recovery) of homogeneous purified group A streptococcal M-protein is described. M-proteins extracted from whole cells of group A streptococci by treatment with hot HCl were neutralized, fractionated with ammonium sulfate, dialyzed, lyophilized, and then subjected to treatment with hot 60% trichloroacetic acid. This was shown to produce an M-protein preparation, free of group A carbohydrate activity and extraneous antigens, in yields up to 10-fold higher than previous methods in about one-fifth the time. These M-protein preparations were shown to: (i) have similar amino acid compositions to their respective type-specific proteins purified by diethylaminoethyl and O -(carboxymethyl) cellulose chromatography, (ii) react with their respective type-specific antisera in Ouchterlony diffusion, (iii) produce antisera in rabbits capable of promoting streptococcal long-chain formation in vitro, and (iv) give only one major band on polyacrylamide gel disk electrophoresis. The data allow for an explanation of the hitherto described multiple banding M-proteins seen on acrylamide electrophoresis.