Bacteria in food packaging paper and board

The bacteria of food packaging paper and board were studied. Most of the aerobic strains were spore-formers; members of the genus Bacillus with B. cereus group (B. cereus, B. mycoides, B. thuringiensis), B. polymyxa group (B. polymyxa, B. circulans, B. macerans, B. pabuli), B. brevis and B. licheniformis predominated. The main source of spore-forming bacteria in paper and board was the broke (rejected paper or board, which is repulped and recycled into the process). Gram-negative bacteria were rare in paper and board in spite of their abundance in the stock. A strain of B. pumilus forming clumping, hairy spores may be of significance in aseptic packaging.     

Bacteria in food packaging paper and board

The bacteria of food packaging paper and board were studied. Most of the aerobic strains were spore-formers; members of the genus Bacillus with B. cereus group ( B. cereus, B. mycoides, B. thuringiensis ), B. polymyxa group ( B. polymyxa, B. circulans, B. macerans, B. pabuli ), B. brevis and B. licheniformis predominated. The main source of spore-forming bacteria in paper and board was the broke (rejected paper of board, which is repulped and recycled into the process). Gram-negative bacteria were rare in paper and board in spite of their abundance in the stock. A strain of B. pumilus forming clumping, hairy spores may be of significance in aseptic packaging.     

Fatty Acids in the Genus Bacillus I. Iso- and Anteiso-Fatty Acids as Characteristic Constituents of Lipids in 10 Species

Fatty acids produced by 22 strains of 10 species of the genus Bacillus were analyzed on a very efficient and selective gas-liquid chromatographic column. All of the 10 species, alvei, brevis, cereus, circulans, licheniformis, macerans, megaterium, polymyxa, pumilus, and subtilis, produced eight fatty acids, six branched (anteiso-C(15), anteiso-C(17), iso-C(14), iso-C(15), iso-C(16), and iso-C(17)) and two normal (n-C(14) and n-C(16)). In all cases, the six branched-chain fatty acids made up over 60% of the total fatty acids. In addition to the eight fatty acids, B. cereus produced four extra fatty acids, three branched (anteiso-C(13), iso-C(12), and iso-C(13)) and one monoenoic-n-C(16). Furthermore, there were distinct differences in the relative amounts of fatty acids produced between B. cereus and the remaining nine species. B. cereus produced iso-C(15) fatty acid in the largest amount on a glucose-yeast extract medium as well as on Pennassay Broth. On the other hand, for the remaining nine species, anteiso-C(15) fatty acid was the major fatty acid from the glucose-yeast extract medium, whereas the amount of iso-C(15) fatty acid from Penassay Broth became comparable to that of anteiso-C(15) fatty acid. Mechanisms and various factors affecting the fatty acid distribution pattern in the 10 Bacillus species are discussed.     

Inhibition of the growth of Paenibacillus larvae, the causal agent of American foulbrood of honeybees, by selected strains of aerobic spore-forming bacteria isolated from apiarian sources

The bacterium Paenibacillus larvae, the causative agent of American foulbrood disease of honeybee larvae, occurs throughout the world and is found in many beekeeping areas of Argentina. The potential as biocontrol agents of antagonic aerobic spore-forming bacteria isolated from honey samples and other apiarian sources were evaluated. Each isolate was screened against one strain of Paenibacillus larvae (ATCC 9545) by using a perpendicular streak technique. Ten randomly selected bacterial strains from the group that showed the best antagonistic effect to P. larvae ATCC 9545 were selected for further study. These were identified as Bacillus subtilis (m351), B. pumilus (m350), B. licheniformis (m347), B. cereus (mv33), B. cereus (m387), B. cereus (m6c), B. megaterium (m404), Brevibacillus laterosporus (BLAT169), B. laterosporus (BLAT170), and B. laterosporus (BLAT171). The antagonistic strains were tested against 17 P. larvae strains from different geographical origins by means of a spot test in wells. The analysis of variance and posterior comparison of means by Tukey method (P < 0.01) showed that the best antagonists were B. megaterium (m404), B. licheniformis (m347), B. cereus (m6c), B. cereus (mv33), and B. cereus (m387).     

Production of diarrheal enterotoxins and other potential virulence factors by veterinary isolates of bacillus species associated with nongastrointestinal infections

With the exceptions of Bacillus cereus and Bacillus anthracis, Bacillus species are generally perceived to be inconsequential. However, the relevance of other Bacillus species as food poisoning organisms and etiological agents in nongastrointestinal infections is being increasingly recognized. Eleven Bacillus species isolated from veterinary samples associated with severe nongastrointestinal infections were assessed for the presence and expression of diarrheagenic enterotoxins and other potential virulence factors. PCR studies revealed the presence of DNA sequences encoding hemolysin BL (HBL) enterotoxin complex and B. cereus enterotoxin T (BceT) in five B. cereus strains and in Bacillus coagulans NB11. Enterotoxin HBL was also harbored by Bacillus polymyxa NB6. After 18 h of growth in brain heart infusion broth, all seven Bacillus isolates carrying genes encoding enterotoxin HBL produced this toxin. Cell-free supernatant fluids from all 11 Bacillus isolates demonstrated cytotoxicity toward human HEp-2 cells; only one Bacillus licheniformis strain adhered to this test cell line, and none of the Bacillus isolates were invasive. This study constitutes the first demonstration that Bacillus spp. associated with serious nongastrointestinal infections in animals may harbor and express diarrheagenic enterotoxins traditionally linked to toxigenic B. cereus.     

Putative virulence factor expression by clinical and food isolates of Bacillus spp. after growth in reconstituted infant milk formulae

Forty-seven strains representing 14 different Bacillus species isolated from clinical and food samples were grown in reconstituted infant milk formulae (IMF) and subsequently assessed for adherence to, invasion of, and cytotoxicity toward HEp-2 and Caco-2 cells. Cell-free supernatant fluids from 38 strains (81%) were shown to be cytotoxic, 43 strains (91%) adhered to the test cell lines, and 23 strains (49%) demonstrated various levels of invasion. Of the 21 Bacillus cereus strains examined, 5 (24%) were invasive. A larger percentage of clinically derived Bacillus species (20%) than of similar species tested from the food environment were invasive. Increased invasion occurred after growth of selected Bacillus species in reconstituted IMF containing glucose. While PCR primer studies revealed that many different Bacillus species contained DNA sequences encoding the hemolysin BL (HBL) enterotoxin complex and B. cereus enterotoxin T, not all of these isolates expressed these diarrheagenic genes after growth in reconstituted IMF. Of the 47 Bacillus isolates examined, 3 isolates of B. cereus and 1 isolate of B. subtilis produced the HBL enterotoxin after 18 h of growth in brain heart infusion broth. However, eight isolates belonging to the species B. cereus, B. licheniformis, B. circulans, and B. megaterium were found to produce this enterotoxin after growth in reconstituted IMF when assessed with the B. cereus enterotoxin (diarrheal type) reversed passive latex agglutination (RPLA) kit. It is concluded that several Bacillus species occurring occasionally in clinical specimens and food samples are of potential medical significance due to the expression of putative virulence factors.     

Hemicellulases of Bacillus species: preliminary comparative studies on production and properties of mannanases and galactanases

A range of Bacillus subtilis strains and other Bacillus species were screened for mannanase, β-mannosidase and galactanase activities. Maximum mannanase activity, 106.2 units/ml, was produced by B. subtilis NRRL 356. β-Mannosidase and galactanase activities from all strains were relatively low. The effect of carbon and nitrogen source on mannanase and galactanase production by B. brevis ATCC 8186, B. licheniformis ATCC 27811, B. polymyxa NRRL 842 and B. subtilis NRRL 356 was investigated. Highest mannanase production was observed in the four strains tested when the mannan substrate, locust bean gum, was used as carbon source. Induction was most dramatic in the case of B. subtilis NRRL 356 where only basal enzyme levels were produced in the presence of other carbon sources. β-Mannosidase was induced in the four Bacillus cultures by locust bean gum. Results indicated that galactose acted as an inducer for production of galactanase. Organic and inorganic nitrogen sources resulted in induction of high mannanase titres in B. subtilis. Highest galactanase activity was produced by each organism in media containing sodium nitrate as nitrogen source. Mannanases from B. brevis, B. licheniformis, B. polymyxa and B. subtilis retained 100% residual activity after a 3 h incubation at 65°C, 65°C, 60°C and 55°C respectively. Galactanases retained more than 95% activity at 55°C after 3 h. The pH optima of mannanases ranged from 6.5–6.8 whereas galactanases ranged from 5.1 in the case of B. brevis to 7.0 for B. polymyxa.     

Structure and glycosylation of lipoteichoic acids in Bacillus strains.

The occurrence, structure, and glycosylation of lipoteichoic acids were studied in 15 Bacillus strains, including Bacillus cereus (4 strains), Bacillus subtilis (5 strains), Bacillus licheniformis (1 strain), Bacillus polymyxa (2 strains), and Bacillus circulans (3 strains). Whereas in the cells of B. polymyxa and B. circulans neither lipoteichoic acid nor related amphipathic polymer could be detected, the cells of other Bacillus strains were shown to contain lipoteichoic acids built up of poly(glycerol phosphate) backbone chains and hydrophobic anchors [gentiobiosyl(beta 1----1/3)diacylglycerol or monoacylglycerol]. The lipoteichoic acid chains of the B. licheniformis strain and three of the B. subtilis strains had N-acetylglucosamine side branches, but those of the B. cereus strains and the remaining two B. subtilis strains did not. The membranes of the B. licheniformis strain and the first three B. subtilis strains exhibited enzyme activities for the synthesis of beta-N-acetylglucosamine-P-polyprenol and for the transfer of N-acetylglucosamine from this glycolipid to endogenous acceptors presumed to be lipoteichoic acid precursors. In contrast, the membranes of the other strains lacked both or either of these two enzyme activities. The correlation between the occurrence of N-acetylglucosamine-linked lipoteichoic acids and the distribution of these enzymes is consistent with the previously proposed function of beta-N-acetylglucosamine-P-polyprenol as a glycosyl donor in the introduction of alpha-N-acetylglucosamine branches to lipoteichoic acid backbone chains.     

Classification of the spoilage flora of raw and pasteurized bovine milk, with special reference to Pseudomonas and Bacillus

Eighty-one bacterial strains isolated from refrigerated raw milk, 124 from pasteurized milk and cream stored at 5°C and 7°C, and 19 type and reference strains of Pseudomonas spp. and Bacillus spp. were characterized by numerical phenotypic analysis. Data were processed with simple matching ( S _SM) and Jaccard ( S _J) coefficients, and UPGMA clustering. Fourteen clusters of Gram-negative bacteria were formed at S _J= 79% ( S _SM= 90%). Raw milk was exclusively spoilt by Gram-negative bacteria, the majority of which were Pseudomonas fluorescens biovar I, Ps. fragi, Ps. lundensis and Ps. fluorescens biovar III. Minor groups in raw milk included Enterobacteriaceae spp. and Acinetobacter spp. Pasteurized milk was spoilt by essentially the same Gram-negative organisms in 65% (5°C) and 50% (7°C) of the cases. The phenotypic characteristics of Gram-negative bacteria are given. Bacillus polymyxa (both temperatures) and B. cereus (only at 7°C) were responsible for 77% of samples spoiled by the Gram-positive organisms. Minor milk spoilage groups included other Bacillus spp. and lactic acid bacteria. All Bacillus spp. grew fermentatively in milk, and most strains denitrified. It is suggested that: (i) industrial recontamination tests of pasteurized milk are directed against Pseudomonas; (ii) milk is stored at 5°C or lower to avoid growth of B. cereus ; and (iii) the significance of gas-producing and nitrate/nitrite-reducing Bacillus strains is recognized in cheese production.     

Differentiation of dairy strains of the Bacillus cereus group by phage typing, minimum growth temperature, and fatty acid analysis

A total of 130 Bacillus strains were isolated from dairy products, the dairy environment and from packaging boards and board-producing machines. Ninety-eight of these were members of the B. cereus group ( B. cereus, B. mycoides and B. thuringiensis ) as determined by whole cell fatty acid composition. Fatty acid composition did not differentiate between the three species. Of the 98 strains, which were indistinguishable by biochemical tests, 87 could be assigned into 21 different phage types (11 strains remained untypable) when tested with 12 B. cereus, B. mycoides and B. thuringiensis phages. The distribution of phage types between strains from different sources showed that the source of contamination of the dairy products was of milk origin and not from the packaging materials. Most strains isolated from the dairy products were able to grow below 10°C, whereas strains from the dairy environment and from board mills had higher minimum growth temperatures.     

Studies on the Bacillus flora of milk and milk products

Bacillus licheniformis and B. cereus were the most commonly isolated species of Bacillus found in milk at all stages of processing. Bacillus licheniformis was ubiquitous in the farm environment and counts in raw milks heat-treated in the laboratory were higher during the winter months, whilst B. cereus was associated with cattle feed throughout the year, and tended to be more common in raw milks during the summer months. Although B. licheniformis was usually isolated in larger numbers than B. cereus, this pattern changed after raw and pasteurized milks and reconstituted milk powders were pre-incubated at ambient temperatures, and B. cereus came to dominate the Bacillus population, reaching levels associated with enterotoxin production. Investigation of the growth kinetics of strains of both species showed that B. cereus grew faster than B. licheniformis at ambient temperatures. It is suggested that post-pasteurization contamination, which is commonly blamed for spoilage of milk and milk products by B. cereus, is not necessarily the most important source of this organism.     

长江流域水稻根际芽孢杆菌属固氮菌株的分离与鉴定

生物固氮资源的研究、开发和利用有利于发展持续农业,水稻田的氮素肥力比一般旱地高得多,活跃的生物固氮被认为是主要因素之一.苏宝林、崔宗均等系统地研究了我国北方稻区的固氮菌资源,分离鉴定出8属18种共35株固氮菌.在此基础上,我们对长江流域水稻根际异养固氮菌资源进行了系统研究.本文报道芽孢杆菌属(Bacillus)固氮菌株分离与鉴定的结果.     

Diarrhoeal enterotoxin production by psychrotrophic Bacillus cereus present in reconstituted milk-based infant formulae (MIF)

One hundred reconstituted milk-based infant formulae (MIF) representative of 10 leading brands available in many European Economic Community countries were examined for psychrotrophic Bacillus cereus and for the presence of diarrhoeal enterotoxin. Of the 38 B. cereus isolates recovered from MIF, one, four and 16 strains grew at 4, 6 and 8 °C after 15 d. One (2·6%), two (5·3%) and six (15·8%) of the isolates were identified as potential psychrotrophic food poisoning strains as they were both enterotoxigenic and exhibited good growth at 4, 6 and 8 °C, respectively. Enterotoxin was not detected in MIF in which less than 5·36 log₁₀ cfu of B. cereus ml⁻¹ had grown. While psychrotrophic enterotoxigenic B. cereus strains occur occasionally in MIF, brief storage of reconstituted MIF at the recommended refrigeration temperature of 4 °C will allow this product to remain safe for consumption.     

Occurrence of a barbiturate-inducible catalytically self-sufficient 119,000 dalton cytochrome P-450 monooxygenase in bacilli

In a recent publication (Narhi, L.O. and Fulco, A.J.[1986] J. Biol. Chem. 261, 7160-7169) we described the characterization of a catalytically self-sufficient 119,000 Dalton cytochrome P-450 fatty acid monooxygenase (P-450BM-3) induced by barbiturates in Bacillus megaterium ATCC 14581. We have now examined cell-free preparations from 12 distinct strains of B. megaterium and from one or two strains each of B. alvei, B. brevis, B. cereus, B. licheniformis, B. macerans, B. pumilis and B. subtilis for the presence of this inducible enzyme. Using Western blot analyses in combination with assays for fatty acid hydroxylase activity and cytochrome P-450, we were able to show that 11 of the 12 B. megaterium strains contained not only a strongly pentobarbital-inducible fatty acid monooxygenase identical to or polymorphic with P-450BM-3 but also significant levels of two smaller P-450 cytochromes that were the same as or similar to cytochromes P-450BM-1 and P-450BM-2 originally found in ATCC 14581. Unlike the 119,000 Dalton P-450, however, the two smaller P-450s were generally easily detectable in cultures grown to stationary phase in the absence of barbiturates and, with some exceptions, were not strongly induced by pentobarbital. None of the non-megaterium species of Bacillus tested exhibited significant levels of either fatty acid monooxygenase activity or cytochrome P-450. The one strain of B. megaterium that lacked inducible P-450BM-3 was also negative for BM-1 and BM-2. However, this strain (ATCC 13368) did contain a small but significant level of another P-450 cytochrome that others have identified as the oxygenase component of a steroid 15-beta-hydroxylase system. Our evidence suggests that the BM series of P-450 cytochromes is encoded by chromosomal (rather than by plasmid) DNA.     

长江流域水稻根际芽杆菌属固氮菌株的分离与鉴定

Rice rhizosphere soil samples were colected from 10 sites of 7 provinces in the Yangzi River Valley, and from the soil samples 16 endospore-forming strains with ARA (Acetylene Reduction Activity) were isolated, the nitrogen fixing ability was tested by the method of 15N tracer and the atom 15N% excess are ranged from 0.0297% to 0.4714%. The strains were identified as Bacillus licheniformis, B. subtilis, B. azotoformans, B. cereus, B. pumilus, B. brevis, B. megaterium, B. firmu.     

Identification of bacteria in pasteurized zucchini purées stored at different temperatures and comparison with those found in other pasteurized vegetable purées

One hundred nineteen isolates from a commercial zucchini purée stored at 4, 10, and 20 to 25 degrees C were fingerprinted using repetitive sequence-based PCR (REP-PCR) and classified into 35 REP types. One representative isolate of each REP type was subsequently identified by API50CHB/20E profile and partial rrs gene sequence analysis. Nine REP types were misidentified by the API system. Strains were misidentified as being in the Bacillus circulans (group 2) API taxon or in taxa with a low number of positive API characters such as Brevibacillus brevis. A phylogenetic analysis pointed to one new species of Bacillus and three new species of Paenibacillus among the misidentified REP types. Bacterial components in zucchini purée were compared phenotypically with those obtained in previous work on broccoli, carrot, leek, potato, and split pea purées, based on simple matching coefficient and unweighted pair group method with averages cluster analysis. Out of 254 strains, 69 strains previously identified as B. circulans (group 2) or B. circulans/B. macerans/B. polymyxa were assigned to a new Paenibacillus taxon phylogenetically related to P. azotofixans. Storage conditions at 4 degrees C favored the development of "B. macroides/B. maroccanus" and Paenibacillus spp. in zucchini purées and Paenibacillus spp. in other purées. Storage conditions at 20 to 25 degrees C favored the development of B. subtilis group (B. licheniformis and B. subtilis) and B. cereus group strains. At 10 degrees C, Paenibacillus spp. were always present at high frequencies, whereas the occurrence of B. macroides/B. maroccanus (in zucchini purées), B. cereus, and B. pumilus varied with the experiment.     

Comparison of various properties of low-molecular-weight proteins from dormant spores of several Bacillus species.

Several properties of the major proteins degraded during germination of spores of Bacillus cereus, Bacillus megaterium, and Bacillus subtilis have been compared. All of the proteins had low molecular weights (6,000 to 13,000) and lacked cysteine, cystine, and tryptophan. The proteins could be subdivided into two groups: group I (B. megaterium A and C proteins, B. cereus A protein, and B. subtilis alpha and beta proteins) and group II (B. cereus and B. megaterium B proteins and B. subtilis gamma protein). Species in group II had lower levels of (or lacked) the amino acids isoleucine, leucine, methionine, and proline. Similarly, proteins in each group were more closely related immunologically. However, antisera against a B. megaterium group I protein cross-reacted more strongly with the B. megaterium group II protein than with group I proteins from other spore species, whereas antisera against the B. megaterium group II protein cross-reacted most strongly with B. megaterium group I proteins. Analysis of the primary sequences at the amino termini and in the regions of the B. cereus and B. subtilis proteins cleaved by the B. megaterium spore protease revealed that the B. cereus A protein was most similar to the B. megaterium A and C proteins, and the B. cereus B protein and the B. subtilis gamma protein were most similar to the B. megaterium B protein. However, amino terminal sequences within one group of proteins varied considerably, whereas the spore protease cleavage sites were more highly conserved.     

Investigation of spore surface antigens in the genus Bacillus by the use of polyclonal antibodies in immunofluorescence tests

Fluorescein-conjugated rabbit antibodies to formalized spores of Bacillus anthracis were tested against strains of B. anthracis and other Bacillus species in a subjective immunofluorescence test. The lack of reaction of B. anthracis Vollum spores with conjugated antibody raised against B. anthracis Sterne spores indicated that spores of the Vollum strain lacked a major surface antigen present in most of the other anthrax strains tested, including the non-encapsulated strains Sterne and the Soviet ST1, variants cured of the pX01 plasmid that codes for the toxin, and several virulent strains. Four other antibody preparations, raised against B, anthracis Vollum, New Hampshire, Ames and Strain 15, reacted to an approximately similar degree with spores of all four strains and of Sterne, indicating that Vollum has at least one spore antigen in common with these other strains. The anti-Sterne and anti-Vollum conjugates both displayed cross-reactions with spores of strains of B. cereus, B. coagulans, B. subtilis, B. megaterium, B. polymyxa, B. pumilus and B. thuringiensis. Absorption of the anti-anthrax conjugates with B. cereus NCTC 8035 and NCTC 10320 removed all these cross-reactions, demonstrating the existence of spore antigens specific for anthrax.     

Investigation of spore surface antigens in the genus Bacillus by the use of polyclonal antibodies in immunofluorescence tests

Fluorescein-conjugated rabbit antibodies to formalized spores of Bacillus anthracis were tested against strains of B. anthracis and other Bacillus species in a subjective immunofluorescence test. The lack of reaction of B. anthracis Vollum spores with conjugated antibody raised against B. anthracis Sterne spores indicated that spores of the Vollum strain lacked a major surface antigen present in most of the other anthrax strains tested, including the non-encapsulated strains Sterne and the Soviet ST1, variants cured of the pX01 plasmid that codes for the toxin, and several virulent strains. Four other antibody preparations, raised against B. anthracis Vollum, New Hampshire, Ames and Strain 15, reacted to an approximately similar degree with spores of all four strains and of Sterne, indicating that Vollum has at least one spore antigen in common with these other strains. The anti-Sterne and anti-Vollum conjugates both displayed cross-reactions with spores of strains of B. cereus, B. coagulans, B. subtilis, B. megaterium, B. polymyxa, B. pumilus and B. thuringiensis. Absorption of the anti-anthrax conjugates with B. cereus NCTC 8035 and NCTC 10320 removed all these cross-reactions, demonstrating the existence of spore antigens specific for anthrax.     

Isolation and identification of nitrogen-fixing bacilli from plant rhizospheres in Beijing region

AIMS: To isolate and identify nitrogen-fixing bacilli from the plant rhizospheres in Beijing region of China. METHODS AND RESULTS: A total of 29 isolates were selectively obtained from the rhizospheres of wheat, maize, ryegrass and willow based on their growth on nitrogen-free medium and their resistance to 100 degrees C for 10 min. Of the 29 isolates, seven had nifH gene determined by PCR amplification. The seven isolates were found to belong to the genera Bacillus and Paenibacillus based on phenotypic characterization, 16S rDNA sequence, G+C content and DNA-DNA hybridization. Isolates T1 and W5 were identified as Bacillus cereus and Bacillus marisflavi respectively. Isolates G1, C4 and C5 were identified as Bacillus megaterium. Isolate G2 was identified as Paenibacillus polymyxa and isolate T7 as Paenibacillus massiliensis. CONCLUSIONS: This study suggests that nifH gene could be detected in the both genera Bacillus and Paenibacillus. These degenerate primers for nifH gene fragment used in this study were shown to be useful for identifying nitrogen-fixing bacilli. SIGNIFICANCE AND IMPACT OF THE STUDY: It is the first demonstration that nitrogen fixation exists in B. marisflavi and P. massiliensis and the first report of the sequences of the nifH gene from B. megaterium and B. cereus. The nitrogen-fixing bacilli obtained in this study will be used in our future research for investigating the mechanisms of nitrogen fixation in bacilli.