Production of Citrinin by Penicillium viridicatum on Country-Cured Ham

Seven strains of Penicillium viridicatum isolated from country-cured ham produced citrinin in potato dextrose broth and on country-cured ham. None of the strains produced detectable amounts of citrinin at 10 C. The optimal temperature range for citrinin production was 25 to 30 C.     

Occurrence of Toxigenic Aspergillus versicolor Isolates and Sterigmatocystin in Carpet Dust from Damp Indoor Environments

Over the past decade, there has been growing concern regarding the role of toxigenic fungi in damp indoor environments; however, there is still a lack of field investigations on exposure to mycotoxins. The goal of our pilot study was to quantify the proportion of toxigenic Aspergillus versicolor isolates in native carpet dust from damp dwellings with mold problems and to determine whether sterigmatocystin can be detected in this matrix. Carpet dust samples (n = 11) contained from <2.5 × 10¹ to 3.6 × 10⁵ (median, 3.1 × 10⁴) A. versicolor CFU/g of dust, and the median proportion of A. versicolor from total culturable fungi was 18%. Based on thin-layer chromatography detection of sterigmatocystin, 49 of 50 A. versicolor isolates (98%) were found to be toxigenic in vitro. By using high-performance liquid chromatography-electrospray ionization tandem mass spectrometry, sterigmatocystin could be detected in low concentrations (2 to 4 ng/g of dust) in 2 of 11 native carpet dust samples. From this preliminary study, we conclude that most strains of A. versicolor isolated from carpet dust are able to produce sterigmatocystin in vitro and that sterigmatocystin may occasionally occur in carpet dust from damp indoor environments. Further research and systematic field investigation are needed to confirm our results and to provide an understanding of the health implications of mycotoxins in indoor environments.     

Processing-Dependent and Clonal Contamination Patterns of Listeria monocytogenes in the Cured Ham Food Chain Revealed by Genetic Analysis

The quantitative and qualitative patterns of environmental contamination by Listeria monocytogenes were investigated in the production chain of dry-cured Parma ham. Standard arrays of surfaces were sampled in processing facilities during a single visit per plant in the three compartments of the food chain, i.e., ham production (19 plants) and postproduction, which was divided into deboning (43 plants) and slicing (25 plants) steps. The numbers of sampled surfaces were 384 in ham production, with 25 positive for L. monocytogenes, and 1,084 in postproduction, with 83 positives. Statistical analysis of the prevalence of contaminated surfaces showed that in ham production, contamination was higher at the beginning of processing and declined significantly toward the end, while in postproduction, prevalence rose toward the end of processing. Prevalence was higher in the deboning facilities than in slicing facilities and was dependent on the type of surface (floor/drainage > clothing > equipment). The qualitative pattern of contamination was investigated through an analysis of the survey isolates and a set of isolates derived from routine monitoring, including longitudinal isolations. Pulsed-field gel electrophoresis (PFGE) and whole-genome single-nucleotide polymorphism (SNP) analysis revealed a remarkable clonality of L. monocytogenes within plants, with the detection of 16 plant-specific clones out of 17 establishments with multiple isolates. Repeated detections of clonal isolates >6 months apart were also observed. Six was the maximum number of between-isolate differences in core SNPs observed within these clones. Based on the same six-SNP threshold, three clusters of clonal isolates, shared by six establishments, were also identified. The spread of L. monocytogenes within and between plants, as indicated by its clonal behavior, is a matter of concern for the hygienic management of establishments.     

Production of sterigmatocystin by Aspergillus versicolor isolated from roughage

A total of 69 samples of hay and straw collected during the winter period of 1984/85 were surveyed for their contamination by Aspergillus versicolor. The percentage of A. versicolor-positive samples was 14.5%. Nineteen A. versicolor strains mainly isolated from roughage were tested for the production of sterigmatocystin. All of the isolates examined were capable of producing different levels of sterigmatocystin on a cracked corn substrate. The majority of these strains were highly toxigenic; 53% of the isolates produced more than 500 mg/kg of sterigmatocystin. These findings suggest that corn is a very suitable substrate for sterigmatocystin production and that particularly in the surface layers of feed stocks and corn silos such toxigenic strains of A. versicolor can produce considerable growth and possibly sterigmatocystin, too.     

Toxic Metabolite Produced by Aspergillus wentii

Mycelial extracts of an Aspergillus wentii strain grown on yeast-extract sucrose medium and initially isolated from country-cured ham were highly toxic when inoculated into chicken embryos or fed to mice. Moldy corn and rice were less toxic when fed to mice. Water extracts of moldy corn or rice or culture filtrates from yeast-extract sucrose medium were not toxic. Purification by thin-layer chromatography followed by crystallization yielded orange-red crystals that showed high toxicity and had a melting point of 285 to 286 C. Chloroform solutions of the crystals had absorption maxima at 270, 295, and 452 nm. The smallest amount of this component necessary to have zero hatchability of fertile eggs was 50 mug/egg.     

Assessment of aflatoxin and cyclopiazonic acid production by Aspergillus flavus isolates from Hungary

Thirty-two isolates of Aspergillus flavus were obtained from various sources in Hungary. All isolates were morphologically identified as A. flavus and three atypical variants were confirmed as A. flavus by comparing their DNA with an ex type culture of A. flavus. None of these isolates produced aflatoxins when tested on coconut agar or grown on rice medium and culture extracts examined by thin layer chromatography. Also, none of the isolates converted sterigmatocystin, O-methyl sterigmatocystin, norsolorinic acid, or sodium acetate to aflatoxin. However, 59% of the isolates produced cyclopiazonic acid based on thin layer chromatographic analysis of culture extracts. The isolates that lack the ability to produce both aflatoxin and cyclopiazonic acid are potential candidates for use in bicontrol studies.     

Chemically defined medium for high yields of sterigmatocystin

Isolate of Aspergillus versicolor strain produced 138 g/ml of sterigmatocystin in a complete synthetic medium containing sucrose, salts, 1-phenylalanine, and Ca-pantothenate. The SSP (sucrose salts phenylalanine) medium apparently provided all necessary ingredients for the production of high levels of sterigmatocystin. For optimal sterigmatocystin formation, the amounts of sucrose and 1-phenylalanine were found to be 200 g and 5 g per liter, respectively. When Ca-pantothenate (0.01 g per liter) added, much higher amounts of sterigmatocystin were recovered, whereas CaCl₂ addition (0.01%) drastically reduced the yield. The high levels of sterigmatocystin were recovered in the cultures which incubated stationarily at 26 to 29 °C for over 12 days. Seven strains or isolates tested yielded high levels of sterigmatocystin in the SSP medium, whereas in each other media such as YES medium and rice medium only one isolate yielded highest amount of sterigmatocystin was found.     

Sterigmatocystin production by nine newly described Aspergillus species in section Versicolores grown on two different media

Nine recently described Aspergillus species and four known species in section Versicolores were tested for their ability to produce sterigmatocystin on two liquid media, Czapek w/20 % Sucrose Broth and Yeast Extract Broth grown in the dark for 1 week at 25 °C. Detection and quantification of ST were performed by reversed-phase liquid chromatography coupled with electrospray ionization ion trap mass spectrometry. Limit of detection was 3 ng/mL and limit of quantification was 10 ng/mL. Nine newly described Aspergillus species from various substrates, A. amoenus, A. creber, A. cvjetkovicii, A. fructus, A. jensenii, A. puulaauensis, A. subversicolor, A. tennesseensis and A. venenatus in section Versicolores were found to produce sterigmatocystin. Production was confirmed in recently collected isolates of A. protuberus and A. versicolor. A. austroafricanus and A. tabacinus did not produce sterigmatocystin.     

A microbiological assay for sterigmatocystin,T-2 toxin and zearalenone

A survey was done to find microorganisms useful for assaying sterigmatocystin; T-2 toxin and zearalenone.Staphylococcus aureus was found to be sensitive to T-2 toxin and zearalenone;Bacillus cereus was found to be sensitive to T-2 toxin only; andEscherichia coli was sensitive to sterigmatocystin. The response of the organisms to sterigmatocystin; T-2 toxin and zearalenone was found to be linear between 4 and 100 μg with sterigmatocystin toE. coli; between 2 and 25 μg with T-2 toxin toStaph, aureus andB. cereus; and between 4 and 100 μg with zearalenone toStaph, aureus. The lower limits of sensitivity of the test were 2 μg T-2 toxin and zearalenone, and 4 μg sterigmatocystin. The assay is rapid (15–17 hrs); simple and inexpensive; and can be used to verify the toxicity of samples and to confirm thin layer chromatographic results.     

Biotransformation of sterigmatocystin and absence of aflatoxin biotransformation by blocked mutants ofAspergillus parasiticus

Four blocked aflatoxin mutants were used to test biotransformation of sterigmatocystin and aflatoxins. Three blocked anthraquinone-accumulating mutants (avn-1, avr-1, ver-1) were able to convert sterigmatocystin into both B and G aflatoxins. No conversions of sterigmatocystin were observed with autoclaved controls or with the fourth blocked mutant (fan). Under equivalent resting cell conditions, no interconversion of aflatoxins B₁, B₂, G₁, or G₂ was observed byver-1, fan, or autoclaved controls.     

Occurrence of toxigenic Aspergillus versicolor isolates and sterigmatocystin in carpet dust from damp indoor environments

Over the past decade, there has been growing concern regarding the role of toxigenic fungi in damp indoor environments; however, there is still a lack of field investigations on exposure to mycotoxins. The goal of our pilot study was to quantify the proportion of toxigenic Aspergillus versicolor isolates in native carpet dust from damp dwellings with mold problems and to determine whether sterigmatocystin can be detected in this matrix. Carpet dust samples (n = 11) contained from <2.5 x 10(1) to 3.6 x 10(5) (median, 3.1 x 10(4)) A. versicolor CFU/g of dust, and the median proportion of A. versicolor from total culturable fungi was 18%. Based on thin-layer chromatography detection of sterigmatocystin, 49 of 50 A. versicolor isolates (98%) were found to be toxigenic in vitro. By using high-performance liquid chromatography-electrospray ionization tandem mass spectrometry, sterigmatocystin could be detected in low concentrations (2 to 4 ng/g of dust) in 2 of 11 native carpet dust samples. From this preliminary study, we conclude that most strains of A. versicolor isolated from carpet dust are able to produce sterigmatocystin in vitro and that sterigmatocystin may occasionally occur in carpet dust from damp indoor environments. Further research and systematic field investigation are needed to confirm our results and to provide an understanding of the health implications of mycotoxins in indoor environments.     

Further Studies on Staphylococci in Meats, : III. Occurrence and Characteristics of Coagulase-positive Strains from a Variety of Nonfrozen Market Cuts

From 34 retail grocery stores and meat markets, 209 samples of nonfrozen meats were obtained and analyzed for coagulase-positive Staphylococcus aureus, employing six selective media. Sixty-seven (38.7%) of 173 samples obtained from 27 stores yielded S. aureus. No coagulase-positive S. aureus was isolated from 36 samples obtained from 7 of the stores. The 67 meats yielded 272 isolates from 10 different kinds of meats. There were 162 physiological strains represented when classified by store and 36 strains classified without regard to store of origin. The larger stores yielded fewer meats with staphylococci than the smaller stores. The meats from which S. aureus was recovered in the order of frequency of percentage recovery are as follows: chicken, pork liver, fish, spiced ham, round beef steak, hamburger, beef liver, pork chops, veal steak, and lamb chops. The following seven meats did not yield staphylococci: bologna, shucked oysters, olive and pickle loaf, salami, wieners, and chopped ham. Eighty-eight per cent of the isolates produced pigment, 85% were gelatinase positive, only 1 strain failed to form a precipitate on egg yolk agar, 92% formed deoxyribonuclease, 87% produced bound coagulase, 91% produced the α-hemolysin, 70% the δ-, 22% the β-, and 6% were nil in this regard. The isolates are compared with hospital and other food strains, and their possible source in the meats is discussed.     

Transformation of sterigmatocystin and O-methylsterigmatocystin by aflatoxigenic and nonaflatoxigenic field isolates of the Aspergillus flavus group

Transformation of sterigmatocystin and O-methylsterigmatocystin (two metabolic aflatoxin precursors) to aflatoxins by aflatoxigenic and nonaflatoxigenic field isolates of Aspergillus flavus was studied. The 24 nonaflatoxigenic isolates investigated failed to transform both precursors. Among the 8 aflatoxin-producing isolates used, 7 transformed both precursors whereas the remaining failed to transform both. According to these results, the usefulness of the measurement of enzymatic activities related to aflatoxin production in understanding the true status of conflictive field isolates is discussed.Abbreviations ST sterigmatocystin - OMST O-methylsterigmatocystin - AFB₁ aflatoxin B₁ - AFB₂ aflatoxin B₂ - AFG₁ aflatoxin G₁ - AFG₂ aflatoxin G₂ - GM growth medium of Adye and Mateles - RM replacement medium of Adye and Mateles     

Sterigmatocystin in dairy cattle feed contaminated with Aspergillus versicolor

Sterigmatocystin (7.75 micrograms/g of feed) and a high-propagule-density of Aspergillus versicolor were detected in feed associated with acute clinical symptoms of bloody diarrhea and death in dairy cattle. Nine isolates of A. versicolor from the feed produced 13 to 89 micrograms of sterigmatocystin per g on cracked corn and lower amounts in liquid culture. This is the first report of sterigmatocystin in dairy cattle feed in the United States.     

Sterigmatocystin in dairy cattle feed contaminated with Aspergillus versicolor.

Sterigmatocystin (7.75 micrograms/g of feed) and a high-propagule-density of Aspergillus versicolor were detected in feed associated with acute clinical symptoms of bloody diarrhea and death in dairy cattle. Nine isolates of A. versicolor from the feed produced 13 to 89 micrograms of sterigmatocystin per g on cracked corn and lower amounts in liquid culture. This is the first report of sterigmatocystin in dairy cattle feed in the United States.     

Aflatoxin biosynthetic pathway: Elucidation by using blocked mutants of Aspergillus parasiticus

Two mutant strains of Aspergillus parasiticus, both deficient in aflatoxin production, were used to elucidate the biosynthetic pathway of this mycotoxin. One of the mutants, A. parasiticus ATCC 24551, was capable of accumulating large amounts of averufin, and the other, A. parasiticus 1-11-105 wh-1, accumulated versicolorin A. The averufin producing mutant efficiently converted ¹⁴C-labeled versiconal acetate, versicolorin A, and sterigmatocystin into aflatoxin B₁ and G₁, indicating that averufin preceded these compounds in the aflatoxin biosynthetic pathway. In the presence of dichlorvos (dimethyl 2,2-dichlorovinyl phosphate), a known inhibitor of aflatoxin biosynthesis, the conversion of versicolorin A and sterigmatocystin was unaffected, but the conversion of versiconal acetate was markedly inhibited. The mutant accumulating versicolorin A incorporated ¹⁴C-labeled acetate, averufin, and versiconal acetate into versicolorin A. In the presence of dichlorvos, however, the major conversion product was versiconal acetate. This strongly suggested that dichlorvos inhibited the conversion step of versiconal acetate into versicolorin A. This mutant resumed production of aflatoxin B₁ if sterigmatocystin was added to the resting cell cultures, indicating that the mutant was blocked at the enzymatic step catalyzing the conversion of versicolorin A into sterigmatocystin, and as a result was incapable of aflatoxin production. The experimental evidence is thus provided for the involvement and interrelationship of three anthraquinones (averufin, versiconal acetate, and versicolorin A) and a xanthone (sterigmatocystin) in aflatoxin biosynthesis. A pathway for the biosynthesis of aflatoxin B₁ is proposed to be: acetate →→→ averufin → versiconal acetate → versicolorin A → sterigmatocystin → aflatoxin B₁.     

Mycotoxins and toxigenic fungi in sago starch from Papua New Guinea

AIMS: To assay sago starch from Papua New Guinea (PNG) for important mycotoxins and to test fungal isolates from sago for mycotoxin production in culture. METHODS AND RESULTS: Sago starch collected from Western and East Sepik Provinces was assayed for aflatoxins, ochratoxin A, cyclopiazonic acid, sterigmatocystin, citrinin and zearalenone and all 51 samples were negative. Frequently isolated species of Penicillium (13), Aspergillus (five) and Fusarium (one) were cultured on wheat grain, and tested for the production of ochratoxin A, cyclopiazonic acid, sterigmatocystin, citrinin, patulin and penicillic acid. All 12 isolates of P. citrinin and one of two A. flavipes isolates produced citrinin. A single isolate of A. versicolor produced sterigmatocystin. No other mycotoxins were detected in these cultures. CONCLUSIONS: No evidence was found of systemic mycotoxin contamination of sago starch. However, the isolation of several mycotoxigenic fungi shows the potential for citrinin and other mycotoxins to be produced in sago stored under special conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: Sago starch is the staple carbohydrate in lowland PNG and the absence of mycotoxins in freshly prepared sago starch is a positive finding. However, the frequent isolation of citrinin-producing fungi indicates a potential health risk for sago consumers, and food safety is dependant on promoting good storage practices.     

Three new sterigmatocystin analogues from marine-derived fungus Aspergillus versicolor MF359

During the systematic screening of active compounds from marine-derived fungi, the extract of a strain of Aspergillus versicolor MF359 isolated from a marine sponge of Hymeniacidon perleve was identified for detailed chemical investigation. Three new secondary metabolites, named hemiacetal sterigmatocystin (1), acyl-hemiacetal sterigmatocystin (2), and 5-methoxydihydrosterigmatocystin (3), together with a known compound, aversin (4), were characterized. 1 represents a first structure of sterigmatocystin hemiacetal from nature. The antibacterial activities of these identified compounds were evaluated against Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, Bacillus subtilis, and Pseudomonas aeruginosa. Compound 3 showed activity against S. aureus and B. subtilis with MIC values of 12.5 and 3.125 μg/mL, respectively.     

Identification and Characterization of Pathogenic Yersinia enterocolitica Isolates by PCR and Pulsed-Field Gel Electrophoresis

Approximately 550 to 600 yersiniosis patients are reported annually in Sweden. Although pigs are thought to be the main reservoir of food-borne pathogenic Yersinia enterocolitica, the role of pork meat as a vehicle for transmission to humans is still unclear. Pork meat collected from refrigerators and local shops frequented by yersiniosis patients (n = 48) were examined for the presence of pathogenic Yersinia spp. A combined culture and PCR method was used for detection, and a multiplex PCR was developed and evaluated as a tool for efficient identification of pathogenic food and patient isolates. The results obtained with the multiplex PCR were compared to phenotypic test results and confirmed by pulsed-field gel electrophoresis (PFGE). In all, 118 pork products (91 raw and 27 ready-to-eat) were collected. Pathogenic Yersinia spp. were detected by PCR in 10% (9 of 91) of the raw pork samples (loin of pork, fillet of pork, pork chop, ham, and minced meat) but in none of the ready-to-eat products. Isolates of Y. enterocolitica bioserotype 4/O:3 were recovered from six of the PCR-positive raw pork samples; all harbored the virulence plasmid. All isolates were recovered from food collected in shops and, thus, none were from the patients' home. When subjected to PFGE, the six isolates displayed four different NotI profiles. The same four NotI profiles were also present among isolates recovered from the yersiniosis patients. The application of a multiplex PCR was shown to be an efficient tool for identification of pathogenic Y. enterocolitica isolates in naturally contaminated raw pork.     

Metabolic precursor regulation of aflatoxin formation in toxigenic and non-toxigenic strains ofAspergillus flavus

Non-aflatoxin-producing isolates ofAspergillus flavus from nature and isolates ofA. flavus that had lost their toxigenic trait following laboratory transfer were compared biochemically. After the addition of aflatoxin B₁ precursors sterigmatocystin or O-methylsterigmatocystin to whole cell cultures, the non-toxin producing isolates from nature remained non-toxigenic while toxigenicity was restored in the nontoxigenic laboratory strains. Results imply a lack of enzymes needed for biochemical conversions of precursors to aflatoxin B₁ in natural non-producers and suppression of these enzymes in the nonproducing laboratory strains.