A survey of microorganisms for thermonuclease production

A total of 1204 cultures comprising 16 genera were surveyed for production of thermonuclease (TNase) in milk. Cultures other than Staphylococcus capable of TNase production were restricted to two genera, Streptococcus and Bacillus. Nineteen percent of 338 group D streptococci comprising four species (85% of which were Streptococcus faecalis) and 17% of 60 streptococci belonging to other groups produced TNase. Nine percent of 130 Bacillus cultures comprising six species produced the enzyme. On the other hand, 99% of coagulase-positive staphylococci produced TNase and only 18% of the coagulase-negative staphylococci produced the enzyme. The amount of TNase produced by streptococci and bacilli was significantly lower than that produced by coagulase-positive staphylococci. The pH profile of the streptococci and Bacillus TNases was similar to that of the staphylococcal TNase; each enzyme exhibited a minor peak at pH 7.0 and a broad major peak ranging from pH 8.5 to 10. The nuclease produced by coagulase-positive Staphylococcus was more heat stable than the nucleases produced by Streptococcus and Bacillus; there was little loss in activity of the staphylococcal enzyme after 60 min at 100 degrees C, whereas 50% of the activity of the streptococcal and Bacillus nucleases was destroyed in 40-60 min and 60-80 min, respectively.     

Occurrence of Coagulase-Positive Staphylococci in Cheddar Cheese

Samples (13) from several lots of cheddar cheese incriminated in staphylococcal food poisoning and 343 samples of cheddar cheese purchased over a 3-year period in retail markets were examined quantitatively for coagulase-positive staphylococci with the smear plate technique. Of the food-poisoning samples, 11 contained coagulase-positive staphylococci in numbers that ranged from 50 to several million per g. Of the 343 market cheese samples, 20% contained coagulase-positive staphylococci in concentrations ranging from less than 50 to more than 200,000 per g. The phage patterns of 64 of 89 cultures isolated from the food-poisoning samples placed them in the miscellaneous phage group (44A) or in phage group IV and the miscellaneous group (42D/44A); 14 had phage patterns that involved group III, the group with which food poisoning has usually been associated. In contrast, over 50% of 104 cultures from the market cheese, which were typed at 100 times the critical test dilution, had phage patterns that involved group III. Of nine selected cultures isolated from the food-poisoning cheese, three (all in phage group III) were positive for enterotoxin by intravenous injection test of cats.     

Effect of non-fat dry milk on recovery of staphylococcal thermonuclease from foods

Modification of the method of Tatini et al. (1976) by addition of non-fat dry milk (NFDM) to food samples and subsequent acid precipitation at pH 3.8 enhanced the recovery of staphylococcal thermonuclease (TNase) from most of 37 foods tested. The modified TNase assay method allowed detection of 10 ng (0.002 units) of the enzyme per gram of each of the following foods: ground beef, boiled egg products, whey powder, fruit-containing yogurt, dressings and spreads, potato and egg salads, and pastas, all of which gave false-negative results without NFDM.     

Design and application of a Staphylococcus-specific single strand conformation polymorphism-PCR analysis to monitor Staphylococcus populations diversity and dynamics during production of raw milk cheese

AIM: Development of a nested-PCR single strand conformation polymorphism (SSCP) assay targeting the 16S rRNA genes of the Staphylococcus genus, to monitor staphylococci in cheese. METHODS AND RESULTS: New primer sets to specifically amplify 16S rDNA of staphylococci were designed to be used in a nested-PCR SSCP assay. The method was efficient in discriminating the staphylococcal species most frequently found in cheese. It was validated by monitoring Staphylococcus populations in three productions of raw milk cheese. Analysis of milk samples revealed dominant SSCP peaks corresponding to Staphylococcus aureus, Staphylococcus equorum and Staphylococcus saprophyticus. After 12 h, the S. aureus peak became dominant. CONCLUSIONS: The combination of specific Staphylococcus nested-PCR and SSCP allows rapid and direct monitoring of staphylococci diversity and dynamics in milk and cheese. In the core of the cheeses studied, S. aureus may have ecological advantages against other Staphylococcus populations. SIGNIFICANCE AND IMPACT OF THE STUDY: This approach is a promising tool to study the ecology of staphylococci in cheeses and in other food samples.     

Identification of enterotoxigenic staphylococci from sheep and sheep cheese

The total of 127 Staphylococcus aureus strains obtained from sheep and sheep cheese were examined for their biochemical activities, biotypes, phage patterns, and ability to produce enterotoxins. Of the 83 staphylococcal strains isolated from animals 77 (93%) were classified as the C biotype. Of this group of sheep-adapted strains, 61 (79%) were sensitive to phage 78, and 46 (60%) produced enterotoxin C exclusively. The three isolated belonging to the A biotype produced enterotoxin D, and two of the three unclassifiable strains produced enterotoxin A. Of the 44 staphylococcal strains isolated from sheep cheese, there were 37 (84%) identified as the C biotype. From this series, 31 (84%) strains were lysed with phage 78, 6 (16%) strains produced enterotoxin C, and 1 strain produced enterotoxin A. One of the six strains determined as the A biotype produced enterotoxin D. C biotype strains, especially of ovine origin, are an exception among animal staphylococci, because a large number of them are enterotoixgenic. The C antigenic type is the most usual of the known enterotoxins in staphylococci of animal provenance.     

Purification and Characterization of Cystathionine (beta)-Lyase from Lactococcus lactis subsp. cremoris B78 and Its Possible Role in Flavor Development in Cheese

An enzyme that degrades sulfur-containing amino acids was purified from Lactococcus lactis subsp. cremoris B78; this strain was isolated from a mixed-strain, mesophilic starter culture used for the production of Gouda cheese. The enzyme has features of a cystathionine (beta)-lyase (EC 4.4.1.8), a pyridoxal-5(prm1)-phosphate-dependent enzyme involved in the biosynthesis of methionine and catalyzing an (alpha),(beta)-elimination reaction. It is able to catalyze an (alpha),(gamma)-elimination reaction as well, which in the case of methionine, results in the production of methanethiol, a putative precursor of important flavor compounds in cheese. The native enzyme has a molecular mass of approximately 130 to 165 kDa and consists of four identical subunits of 35 to 40 kDa. The enzyme is relatively thermostable and has a pH optimum for activity around 8.0; it is still active under cheese-ripening conditions, viz., pH 5.2 to 5.4 and 4% (wt/vol) NaCl. A possible essential role of the enzyme in flavor development in cheese is suggested.     

Investigation of aminoglycoside modifying enzyme genes in methicillin-resistant staphylococci

Methicillin-resistant staphylococci may also be resistant to some other antibiotics as well as beta-lactams. In this study, co-existence of resistance to methicillin and aminoglycosides was genetically investigated in staphylococci. A total of 50 staphylococci from in-patients, 17 Staphylococcus aureus and 33 coagulase negative staphylococci (CNS) that contained mecA (gene encoding PBP 2a, an altered penicillin-binding protein) determined by polymerase chain reaction (PCR) were included in the study. Aminoglycoside modifying enzyme (AME) genes were investigated using multiplex-PCR. Aminocyclitol-6'-acetyltransferase-aminocyclitol-2'-phosphotransferase [aac(6')/aph(2')] gene (encoding bifunctional acetyltransferases/phosphotransferases) was determined in 66% of the isolates, aminocyclitol-4'-adenylytransferase (ant(4')-Ia) gene (encoding phosphotransferases) in 24%, and aminocyclitol-3'-phosphotransferase (aph(3')-IIIa) gene (encoding nucleotidyltransferases) in 8%. Two isolates contained all these three genes. Thirty-six (72%) isolates had at least one of these genes. Three CNS and one S. aureus isolates sensitive to oxacillin had the mecA gene. In conclusion, a high rate of aminoglycoside resistance was determined in methicillin-resistant staphylococci. The aac(6')/aph(2') was the most frequently detected.     

Changes in enterococcal population during storage of reconstituted infant food

A six-fold increase in the enterococcal population was observed in reconstituted infant food samples after storage for 2 h at 37 degrees C. The increase in enterococcal counts at 40 degrees C and 45 degrees C was approximately five-fold during the same period. However, the corresponding total viable counts increased by twelve fold at these temperatures after 2 h. After 12 h, the enterococcal and total viable counts increased to 39 x 10(4) and 36 X 10(7) colony forming units per ml, at 37 degrees C respectively. A similar pattern in enterococcal and total bacterial count was observed at 40 degrees and 45 degrees C. TNase was detected in reconstituted infant food samples held at 37 degrees, 40 degrees and 45 degrees C, after 12 h, while pH values declined to 5.0, 5.1 and 5.2, respectively at the above temperatures. From TNase positive samples, an isolate S. faecium IF-100 capable to produce TNase was recovered. Storage of reconstituted infant food samples in the refrigerator (5 degrees C) resulted in a gradual increase in enterococcal population which reached 39 X 10(3) c.f.u. per ml after 12 days. However, TNase was not detected in any of these samples.     

Eighteen-deoxyaldosterone and other less polar forms of 18-hydroxycorticosterone as aldosterone precursors in rat adrenals

Samples containing as precursors either 18-hydroxycorticosterone (18-OH-B) in its M form, or this converted to less polar forms at pH 2 (ACM), or M or ACM enclosed in liposomes from adrenal lipids were incubated at pH 7.4, 4.8 or 3.3 in the presence or absence of quartered rat adrenals for 1 and 2 h. Optimal (10%) yields of aldosterone were obtained when (a) ACM was incubated at pH 4.8 and (b) M enclosed in liposomes was suspended in buffer and shaken without enzyme at pH 3.3. When conditions (a) were supplemented with malate and NADP, 16% of ACM was converted to aldosterone. ACM contained 80% of a fraction which, according to 13C NMR spectroscopy, was identical to 18-deoxyaldosterone (18-DAL). Experiments in which radioactivity from corticosterone (B) or M was trapped by radioinert M or 18-DAL disclosed a pathway comprising sequentially B, 18-OH-B, 18-DAL and aldosterone, and the combined evidence of this work, an enzymatic hydroxylation of 18-DAL to aldosterone.     

Biodiversity of the bacterial flora on the surface of a smear cheese

The bacteria on the surface of a farmhouse smear-ripened cheese at four stages of ripening (4, 16, 23, and 37 days) from inoculated (i.e., deliberately inoculated with Brevibacterium linens BL2) and noninoculated (not deliberately inoculated with B. linens BL2) cheese were investigated. The results show that, contrary to accepted belief, B. linens is not a significant member of the surface flora of smear cheese and no microbial succession of species occurred during the ripening of the cheeses. Of 400 isolates made, 390 were lactate-utilizing coryneforms and 10 were coagulase-negative Staphylococcus spp. A detailed analysis of the coryneforms was undertaken using phenotypic analysis, molecular fingerprinting, chemotaxonomic techniques, and 16S rRNA gene sequencing. DNA banding profiles (ramdom amplified polymorphic DNA [RAPD]-PCR) of all the coryneform isolates showed large numbers of clusters. However, pulsed-field gel electrophoresis (PFGE) of the isolates from the cheeses showed that all isolates within a cluster and in many contiguous clusters were the same. The inoculated and noninoculated cheeses were dominated by single clones of novel species of Corynebacterium casei (50.2% of isolates), Corynebacterium mooreparkense (26% of isolates), and Microbacterium gubbeenense (12.8% of isolates). In addition, five of the isolates from the inoculated cheese were Corynebacterium flavescens. Thirty-seven strains were not identified but many had similar PFGE patterns, indicating that they were the same species. C. mooreparkense and C. casei grew at pH values below 4.9 in the presence of 8% NaCl, while M. gubbeenense did not grow below pH 5.8 in the presence of 5 to 10% NaCl. B. linens BL2 was not recovered from the inoculated cheese because it was inhibited by all the Staphylococcus isolates and many of the coryneforms. It was concluded that within a particular batch of cheese there was significant bacterial diversity in the microflora on the surface.     

Incidence of Clostridium botulinum in honey of various origins

By the dilution-centrifugation method, 270 honey samples, both domestic and imported, were examined and Clostridium botulinum was detected in 23 samples (8.5%); type A in 11 samples, type B in two, type C in 10, and type F in one. Of 58 domestic honey samples, six (10%) were positive; three gave type A and the other two type C. Among imported honey samples, Chinese honey gave 12% positives (types A, B, and C) and Argentina honey 20% positives (types A and F). The incidence was higher with samples taken from drums (18%) and from apiaries (23%) than marketing honey (5%). It was estimated that most positive samples contained spores in one per gram or lower concentrations. One sample contained 4 type A spores per gram and another 36-60 type F spores per gram. No distinct biochemical properties were found with the honey isolates.     

Proteolysis of canine apolipoprotein by acid proteases in canine liver lysosomes.

Canine liver lysosomes were purified by sucrose discontinuous density gradient centrifugation and then ruptured by sonication to obtain the soluble fraction. This soluble lysosomal fraction, which contained a 25-fold increase in acid phosphatase activity per mg of total protein when compared with the original homogenate, was incubated with a subfraction (1.110 less than d less than 1.210 g/cm3, HDL3) of canine high density lipoproteins (HDL) at pH 3.8. HDL3 proteolysis by lysosomal proteases, measured as the release of peptides and amino acids by the ninhydrin reaction, followed hyperbolic curves with straight lines (r = 0.99) obtained on Lineweaver-Burk plots. Km calculated from the Lineweaver-Burk plot was 635 mug of HDL3 protein per 0.5 ml of incubation mixture. Optimum HDL3 proteolysis was observed from pH 3.8 to 4.5. Incubation with the other subcellular organelle fractions did not result in HDL3 proteolysis. To evaluate the effects of enzyme inhibitors, iodoacetate, p-chloromercuribenzoate (both specific for the endopeptidase, cathepsin B (EC 3.4.22.1)) and pepstatin (specific for the endopeptidase, cathepsin D (EC 3.4.23.5) were tested. Iodoacetate and p-chloromercuribenzoate inhibited HDL3 proteolysis 100% and bovine serum albumin proteolysis 65%. Pepstatin inhibited HDL3 proteolysis 45% and bovine serum albumin proteolysis 70%. The in vitro data presented support the hypothesis that hepatic lysosomes play an important role in HDL3 catabolism in the dog. Furthermore, results obtained from enzyme inhibition studies suggest that a specific lysosomal endopeptidase, cathepsin B, may play the key role in HDL3 proteolysis.     

A study of the incidence of different fluorescent Pseudomonas species and biovars in the microflora of fresh and spoiled meat and fish, raw milk, cheese, soil and water.

Of 182 various foodstuffs and environmental samples examined, 86% had microflora containing fluorescent Pseudomonas in differing proportions. A computer-aided technique was used to identify most of the 445 fluorescent strains. Pseudomonas fluorescens biovar V-1 was most frequently isolated (24%); it either predominated or was present in all types of samples. Other strains, belonging to the other subgroups of biovar V (V-2, V-4, V-5, V-6 and V-7), together represented 14.3%. We also identified Ps. fluorescens biovars I-1 and I-2 (13.9%), II-1 and II-3 (3.6%), III-1 and III-2 (8.7%), IV-2 (0.7%); Ps. putida A and B (11%); Ps. lundensis (10.3%); group B3 (2%) and Ps. aeruginosa (0.7%). Unidentified strains accounted for 10.6% of the flora, many resembling Ps. fluorescens biovar V. Although the presence of Ps. fluorescens V-1 was often marked, other taxa predominated or were present in large quantities in some particular samples, such as Ps. fluorescens I-1 in raw milk and cheese, Ps. lundensis in spoiled meat and Ps. fluorescens III-1 in spoiled fish. Pseudomonas putida A and B were evident in environmental rather than in food samples.     

A study of the incidence of different fluorescent Pseudomonas species and biovars in the microflora of fresh and spoiled meat and fish, raw milk, cheese, soil and water

M. GENNARI AND F. DRAGOTTO. 1992. Of 182 various foodstuffs and environmental samples examined, 86% had microflora containing fluorescent Pseudomonas in differing proportions. A computer-aided technique was used to identify most of the 445 fluorescent strains. Pseudomonas fluorescens biovar V-1 was most frequently isolated (24%); it either predominated or was present in all types of samples. Other strains, belonging to the other subgroups of biovar V (V-2, V-4, V-5, V-6 and V-7), together represented 14.3%. We also identified Ps. fluorescens biovars I-1 and I-2 (13.9%), II-1 and II-3 (3.6%), III-1 and III-2 (8.7%), IV-2 (0.7%); Ps. putida A and B (11%); Ps. lundensis (10.3%); group B3 (2%) and Ps. aeruginosa (0.7%). Unidentified strains accounted for 10.6% of the flora, many resembling Ps. fluorescens biovar V. Although the presence of Ps. fluorescens V-1 was often marked, other taxa predominated or were present in large quantities in some particular samples, such as Ps. fluorescens I-1 in raw milk and cheese, Ps. lundensis in spoiled meat and Ps. fluorescens III-1 in spoiled fish. Pseudomonas putida A and B were evident in environmental rather than in food samples.     

Microbial ecology of Bacillus thuringiensis: fecal populations recovered from wildlife in Korea

A total of 34 fecal samples, collected from 14 species of wild mammals in Korea, were examined for the occurrence of Bacillus thuringiensis. The organism was detected in 18 (53%) samples. Among the three food-habit groups, herbivorous animals yielded the highest frequency (69%) of samples positive for B. thuringiensis, followed by omnivorous animals (50%). Of the six fecal samples from carnivorous animals, only one sample contained B. thurin giensis. Among 527 isolates belonging to the Bacillus cereus - B. thuringiensis group, 43 (8%) were assigned to B. thurin giensis on the basis of the formation of parasporal inclusions. Of the 43 isolates, 13 were serologically allocated to the nine H-antigenic serotypes: H3ad (serovar sumiyoshiensis), H15 (dakota), H17/27 (tohokuensis/ mexicanensis), H19 (tochigiensis), H21 (colmeri), H29 (amagiensis), H31/49 (toguchini/muju), H42 (jinghongiensis), and H44 (higo). Other isolates were untestable or untypable by the 55 reference H antisera available. Insecticidal activity was associated with 23% of the fecal populations: three isolates killed larvae of the silkworm, Bombyx mori (Lepidoptera), and seven exhibited larvicidal activity against the mosquito, Aedes aegypti (Diptera). There was no larvicidal activity against the three lepidopterous insects: Plutella xylostella, Spodoptera exigua, and Spodoptera litura. The overall results suggest that wild animals in Korea are in contact with naturally occurring B. thuringiensis at high frequencies through the daily food intake of plants.     

Effect of pH on the Proportions of Polar Lipids, in Chemostat Cultures of Bacillus subtilis

Significant changes in the relative proportions of the individual polar lipids of two strains of Bacillus subtilis were observed when the pH of their chemostat cultures was varied. In phosphate- and magnesium-limited cultures of B. subtilis var. niger NCIB 8058. lysylphosphatidylglycerol was present in higher proportions at low pH (5.1) than at neutral pH. With magnesium-limited cultures of this strain harvested at pH 8.0, lysylphosphatidylglycerol and phosphatidylethanolamine were not detected. Phosphate-limited cultures of B. subtilis NCIB 3610 contained no phosphatidylethanolamine or lysylphosphatidylglycerol at neutral pH, but at low pH (5.1) both these lipids were present in substantial proportions. The proportions of phosphatidylglycerol in actively dividing cells of chemostat cultures of bacilli were always greater than those of lysylphosphatidylglycerol. The reverse is commonly found in batch cultures of bacilli and staphylococci harvested at low pH. Changes in the proportions of the other polar lipids present in these bacilli (diphosphatidylglycerol and diglucosyl diacylglycerol) with pH were also noted. Certain cultures of both strains of B. subtilis contained small proportions of a peptidolipid.     

Distribution of entomopathogenic nematodes in Southern Cameroon

A first survey of entomopathogenic nematodes (EPN) was conducted in three agro-ecological zones of Southern Cameroon in 2007 and 2008. Entomopathogenic nematodes were recovered from 26 of 251 soil samples (10.4%). Three species, Heterorhabditis baujardi, Steinernema sp. A and Steinernema sp. B were found. The two steinernematids were considered unidentified species. Among the positive samples, 23 samples contained only H. baujardi (88.5%), two contained Steinernema sp. A co-occurring with H. baujardi (7.7%), and one sample contained Steinernema sp. B (3.9%). H. baujardi was frequent in forest and fruit crop (cocoa and oil palm plantations). Steinernema sp. A was found in a tree plantation of teak, Steinernema sp. B in a forest habitat. Nematodes were mostly present in acidic soils with pH ranging from 3.7 to 7.0. The highest EPN presence was recorded in sandy loam, sandy clay loam, sandy clay and clay soils. EPNs were not recovered in sand, loamy sand and clay loam soils. Using principal component analysis for elucidating the major variation patterns among sampling sites, four factors explaining for 73.64% of the overall variance were extracted. Factors were a combination of geographical (latitude, longitude, altitude), soil (pH, contents of sand, silt and clay, organic carbon, texture), and moisture (wilting point, field capacity) parameters as well as climatic parameters (mean annual rainfall, mean air temperature). Logistic regression and redundancy analyses (RDA) revealed that soil pH, longitude, available water and altitude were associated with presence and absence of EPN. Both logistic regression and RDA indicated that, increasing soil pH and longitude, associated with decreasing altitude, led to higher percentages of samples containing entomopathogenic nematodes.     

Purification and characterization of aspartic proteinase from sunflower seeds

Aspartic proteinases were purified from sunflower seed extracts by affinity chromatography on a pepstatin A-EAH Sepharose column and by Mono Q column chromatography. The final preparation contained three purified fractions. SDS-PAGE showed that one of the fractions consisted of disulfide-bonded subunits (29 and 9 kDa), and the other two fractions contained noncovalently bound subunits (29 and 9 kDa). These purified enzymes showed optimum pH for hemoglobinolytic activity at pH 3.0 and were completely inhibited by pepstatin A like other typical aspartic proteinases. Sunflower enzymes showed more restricted specificity on oxidized insulin B chain and glucagon than other aspartic proteinases. The cDNA coding for an aspartic proteinase was cloned and sequenced. The deduced amino acid sequence showed that the mature enzyme consisted of 440 amino acid residues with a molecular mass of 47,559 Da. The difference between the molecular size of purified enzymes and of the mature enzyme was due to the fact that the purified enzymes were heterodimers formed by the proteolytic processing of the mature enzyme. The derived amino acid sequence of the enzyme showed 30-78% sequence identity with that of other aspartic proteinases.     

Properties of the exo-1,4-beta-xylosidase of Aspergillus niger 15

Properties of exo-1,4-beta-xylosidase from the fungus Aspergillus niger 15 were investigated. The enzyme was homogeneous during gel filtration, electrophoresis in polyacrylamide gel in the presence and absence of Na dodecyl sulfate, ultracentrifugation and isoelectric focusing. The enzyme had a temperature optimum at 70 degrees, pH optimum 3.8-4.0 for p-nitrophenyl-beta-D-xylopyranoside (p-NPX), was stable at pH 3-8, retained its 100% activity for 1 hour at 50 degrees and 42% activity at 60 degrees. Km was 0.23 mM for p-NPX and 0.67 mM for xylobiose. Xylose was a competitive inhibitor of exo-1,4-beta-xylodidase with Ki = 2.9 mM. The enzyme showed a transglycosilase activity. The aminoacid analysis of exo-1,4-beta-xylosidase showed that the enzyme molecule contained predominantly dicarboxylic and hydrophobic amino acids as well as serine. The enzyme contained no carbohydrates. Its activity was inhibited by p-chloromercury benzoate.     

Mycoflora and Occurrence of Aflatoxin B 1 in Dried Yam Chips from Markets in Ogun and Oyo States,Nigeria

Seventy-six samples of dried yam chips locally called elubo isu were purchased in 2000 from markets in Ogun and Oyo States of southwestern Nigeria. The samples were assessed for pH, moisture content, associated fungi and aflatoxin B1 contamination. The pH of samples ranged from 5.6 to 6.1,while the moisture contents varied from 6.8 to 14.5% in Ogun samples, and 7.1 to 13.6% in samples from Oyo. Aspergillus and Penicillium were the two prevalent genera of fungi, and the number of colony forming units per gram of these two genera in the yam chips studied exceeded the tolerance limit in foodstuffs. The other fungal genera isolated included Botryodiplodia, Cladosporium, Fusarium, Rhizopus, Mucor, Aureobasidium and Paecilomyces. The two most frequent fungal species were A. niger and A. flavus. Thin layer chromatographic analysis showed that 17 samples or 22% contained aflatoxin B1 beyond the detection limit (5 ppb), but only three samples or 4% had toxin level above 30 ppb, the tolerance level in food for human consumption. The mean concentration of aflatoxin B1 in positive samples was 27.1 ppb.