Microbiological Aspects of Ethylene Oxide Sterilization: II. Microbial Resistance to Ethylene Oxide

The death rate kinetics of several sporeforming and nonsporeforming microorganisms, including radiation-resistant cocci, were determined by exposing them to a mixture of ethylene oxide and dichlorodifluoromethane (500 mg of ethylene oxide per liter, 30 to 50% relative humidity, and 54.4 C). Spore survivor curves obtained from tests of inoculated and exposed hygroscopic and nonhygroscopic carriers showed that the spores of Bacillus subtilis var. niger are more resistant to ethylene oxide than are spores of Clostridium sporogenes, B. stearothermophilus, and B. pumilus. The decimal reduction times (expressed as D values at 54.4 C-500 mg of ethylene oxide per liter) obtained under the test conditions for B. subtilis var. niger spores on hygroscopic and nonhygroscopic carriers exceeded the values obtained for the other organisms considered, both sporeformers and nonsporeformers. The decimal reduction times for the vegetative cells of the radiation-resistant organisms (Micrococcus radiodurans and two strains of Streptococcus faecalis) and the ATCC strain of S. faecalis demonstrated comparable resistance to ethylene oxide with the spores of C. sporogenes, B. stearothermophilus, and B. pumilus, but not those of B. subtilis var. niger.     

应用多重PCR鉴定微生物肥料常用芽孢杆菌

[目的]枯草群芽孢杆菌中枯草芽孢杆菌(Bacillus subtilis)、解淀粉芽孢杆菌(B.amyloliq-wefaciens)、地衣芽孢杆菌(B.licheniformis)和短小芽孢杆菌(B.pumilus)是微生物肥料中常用菌种,用传统方法鉴定费时费力,有必要建立检测和鉴定这些芽孢杆菌的种特异性PCR方法.[方法]利用已登录的gyrA、rpoA和16s rRNA基因序列分别设计和筛选上述菌种的特异引物并建立多重PCR反应体系.[结果]以基因组DNA为模板,扩增芽孢杆菌、类芽孢杆菌和短芽孢杆菌3属15种的标准菌株(共33株),4个目标种分别产生了大小不同的唯一的产物,除个别种与短小芽孢杆菌引物有交叉反应外,其余参考菌株均为阴性.从23株枯草群菌株的基因组DNA扩增发现,PCR鉴定与常规鉴定结果一致.[结论]本文建立的多重PCR方法具有较好的特异性,可快速准确鉴定枯草群的4个种,在微生物肥料检测方面有良好的实用前景.     

Numerical taxonomy of Bacillus isolated from orally administered drugs

Numerical taxonomy procedures were used to study 118 strains of Bacillus isolated from non-sterile drugs prepared for oral administration. Similarities between pairs of strains were calculated by the simple matching coefficient of Sokal and Michener (SSM). Each strain was tested for 60 unit characters and three clusters were defined. The strains in each cluster presented a similarity level of at least 60%. Cluster A comprised the strains identified as Bacillus cereus (SSM = 93.13%), cluster B contained three subgroups corresponding to the species B. pumilus, B. subtilis and B. licheniformis (SSM = 84.35%) and cluster C also included three subgroups that belonged to the species B. firmus, B. lentus and B. badius (SSM = 80.14%). The most discriminating tests were selected to differentiate the clusters from the subgroups. The feature with the highest discriminating power between clusters A and B was the lack of acid production from arabinose and mannitol. The Voges-Proskauer, methyl red tests and sensitivity to polymyxin B clearly distinguished cluster A from C. The Voges-Proskauer test and acid production from arabinose were the best to differentiate between B and C. Bacillus pumilus and B. subtilis differed in starch hydrolysis and B. licheniformis in growing anaerobically. To discriminate B. firmus from B. lentus the most important tests were the acid production from glucose and sucrose; intermediate strains were found. Bacillus badius was differentiated from B. firmus by 10 tests, and from B. lentus by the production of urease.     

Numerical taxonomy of Bacillus isolated from orally administered drugs

Numerical taxonomy procedures were used to study 118 strains of Bacillus isolated from non-sterile drugs prepared for oral administration. Similarities between pairs of strains were calculated by the simple matching coefficient of Sokal and Michener (S_SM). Each strain was tested for 60 unit characters and three clusters were defined. The strains in each cluster presented a similarity level of at least 60%. Cluster A comprised the strains identified as Bacillus cereus (S_SM= 93·13%), cluster B contained three subgroups corresponding to the species B. pumilus, B. subtilis and B. licheniformis (S_SM= 84·35%) and cluster C also included three subgroups that belonged to the species B. firmus, B. lentus and B. badius (S_SM= 80·14%). The most discriminating tests were selected to differentiate the clusters from the subgroups. The feature with the highest discriminating power between clusters A and B was the lack of acid production from arabinose and mannitol. The Voges-Proskauer, methyl red tests and sensitivity to polymyxin B clearly distinguished cluster A from C. The Voges-Proskauer test and acid production from arabinose were the best to differentiate between B and C. Bacillus pumilus and B. subtilis differed in starch hydrolysis and B. licheniformis in growing anaerobically. To discriminate B. firmus from B. lentus the most important tests were the acid production from glucose and sucrose; intermediate strains were found. Bacillus badius was differentiated from B. firmus by 10 tests, and from B. lentus by the production of urease.     

Curie-point pyrolysis mass spectrometry applied to characterization and identification of selected Bacillus species

The use of pyrolysis mass spectrometry in the characterization and identification of Bacillus species was studied. Fifty-three strains of four closely related groups, Bacillus subtilis, B. pumilus, B. licheniformis and 'B. amyloliquefaciens', were used in a study of both sporulated and nonsporulated cultures. Pyrolysis was carried out using a Pyromass 8-80, a novel pyrolysis mass spectrometer specifically designed for fingerprinting complex samples. The pyrolysis data obtained were analysed using multivariate statistical techniques. All four groups could be differentiated using data from non-sporulated cultures but the data from sporulated cultures did not separate B. subtilis from 'B. amyloliquefaciens' or B. pumilus. In contrast, B. licheniformis was more clearly differentiated from the other three species using these data. Culture maturity affected the mass spectra obtained from non-sporulated cultures.     

鸡肠源芽胞杆菌的分离、鉴定和抗菌活性

从人工饲养的健康三黄肉鸡的盲肠和大肠中分离出10株芽胞杆菌,对其进行菌落形态观察、革兰染色、芽胞染色和生理生化特性鉴定,确定为枯草芽胞杆菌、地衣芽胞杆菌、蜡状芽胞杆菌、巨大芽胞杆菌、球形芽胞杆菌、短小芽胞杆菌、凝结芽胞杆菌。同时测定了它们对动物病原性大肠埃希菌和链球菌的抑菌活性。其中有9株芽胞杆菌对大肠埃希菌有抑制作用,8株芽胞杆菌对链球菌有抑制作用。结果表明芽胞杆菌分离株Y2、Y3、Y4、Y5、Y6、Y7、Y8具有作为益生菌开发的潜力。     

Distribution and characteristics of Bacillus bacteria associated with hydrobionts and the waters of the Peter the Great Bay, Sea of Japan

Bacilli of the species Bacillus subtilis, B. pumilus, B. mycoides, B. marinus and B. licheniformis (a total of 53 strains) were isolated from 15 invertebrate species and the water of the Vostok Bay, Peter the Great Bay, Sea of Japan. Bacilli were most often isolated from bivalves (22.7%) and sea cucumbers (18.9%); they occurred less frequently in sea urchins and starfish (13.2 and 7.5%, respectively). Most of bacilli strains were isolated from invertebrates inhabiting silted sediments. No Bacillus spp. strains were isolated from invertebrates inhabiting stony and sandy environments. The species diversity of bacilli isolated from marine objects under study was low. Almost all bacterial isolates were resistant to lincomycin. Unlike B. pumilus, B. subtilis isolates were mostly resistant to benzylpenicillin and ampicillin. Antibiotic sensitivity of B. licheniformis strains was variable (two strains were resistant to benzylpenicillin and oxacillin, while one was sensitive). A significant fraction of isolated bacilli contained pigments. Pigmented strains were more often isolated from seawater samples, while colorless ones predominated within hydrobionts. B. subtilis colonies had the broadest range of colors. In the Bacillus strains obtained, DNase, RNase, phosphatase, elastolytic, chitinase, and agarolytic activity was detected. Bacilli strains with hydrolytic activity occurred in invertebrates more often than in seawater.     

Biosynthesis of linkage units for teichoic acids in gram-positive bacteria: distribution of related enzymes and their specificities for UDP-sugars and lipid-linked intermediates.

The distribution and substrate specificities of enzymes involved in the formation of linkage units which contain N-acetylglucosamine (GlcNAc) and N-acetylmannosamine (ManNAc) or glucose and join teichoic acid chains to peptidoglycan were studied among membrane systems obtained from the following two groups of gram-positive bacteria: group A, including Bacillus subtilis, Bacillus licheniformis, Bacillus pumilus, Staphylococcus aureus, and Lactobacillus plantarum; group B, Bacillus coagulans. All the membrane preparations tested catalyzed the synthesis of N-acetylglucosaminyl pyrophosphorylpolyprenol (GlcNAc-PP-polyprenol). The enzymes transferring glycosyl residues to GlcNAc-PP-polyprenol were specific to either UDP-ManNAc (group A strains) or UDP-glucose (group B strains). In the synthesis of the disaccharide-bound lipids, GlcNAc-PP-dolichol could substitute for GlcNAc-PP-undecaprenol. ManNAc-GlcNAc-PP-undecaprenol, ManNAc-GlcNAc-PP-dolichol, Glc-GlcNAc-PP-undecaprenol, Glc-GlcNAc-PP-dolichol, and GlcNAc-GlcNAc-PP-undecaprenol were more or less efficiently converted to glycerol phosphate-containing lipid intermediates and polymers in the membrane systems of B. subtilis W23 and B. coagulans AHU 1366. However, GlcNAc-GlcNAc-PP-dolichol could not serve as an intermediate in either of these membrane systems. Further studies on the exchangeability of ManNAc-GlcNAc-PP-undecaprenol and Glc-GlcNAc-PP-undecaprenol revealed that in the membrane systems of S. aureus strains and other B. coagulans strains both disaccharide-inked lipids served almost equally as intermediates in the synthesis of polymers. In the membrane systems of other B. subtilis strains as well as B. licheniformis and B. pumilus strains, however, the replacement of ManNAc-GlcNAc-PP-undecaprenol by Glc-GlcNAc-PP-undecaprenol led to a great accumulation of (glycerol phosphate)-Glc-GlcNAc-PP-undecaprenol accompanied by a decrease in the formation of polymers.     

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.     

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.     

Phylogeny of gamma-polyglutamic acid-producing Bacillus strains isolated from a fermented locust bean product manufactured in West Africa

Twenty-five Bacillus strains capable of producing gamma-polyglutamic acid (PGA) were isolated from fermented locust bean products manufactured in the savanna area of Ghana. To clarify the phylogeny of these PGA-producing strains, phylogenetic analyses based on sequences of 16S rDNA, rpoB (RNA polymerase beta-subunit) and fus (elongation factor G) genes were performed. A phylogenetic tree based on 16S rDNA indicated that ten isolates were clustered in the same group of Bacillus subtilis. Another ten isolates were located in the cluster of B. amyloliquefaciens, and the remaining isolates were identified as B. pumilus (three isolates) and B. licheniformis (two isolates), respectively. Phylogenetic trees based on the partial sequences of rpoB and fus genes were similar to the phylogeny based on 16S rDNA sequences. Thirty-four strains in 27 species belonging to the genus Bacillus and its neighbors were also investigated for PGA production. It was found that PGA was produced by B. amyloliquefaciens NBRC 14141 and NBRC 15535(T), B. atrophaeus NBRC 15539(T), B. licheniformis NBRC 12107, B. mojavensis NBRC 15718(T), B. pumilus NBRC 12094, B. subtilis NBRC 16449, and Lysinibacillus sphaericus NBRC 3525. Except for L. sphaericus, the above Bacillus species are very closely related in phylogeny, indicating that PGA-producing Bacillus strains constitute a cluster.     

Identification of Bacillus subtilis NRRL B-3275 as a Strain of Bacillus pumilus

The physiological and biochemical properties of a species of Bacillus previously identified as B. subtilis NRRL B-3275 (B-3275) were compared with those of seven strains of B. pumilus and five strains of B. subtilis. The biotin requirement of B-3275, its inability to hydrolyze starch, and its failure to reduce nitrate indicate that the organism is more closely related to the B. pumilus strains than to those of B. subtilis. Hybridization of deoxyribonucleic acid (DNA) from B-3275 with that of the strains of B. pumilus showed a binding efficiency (compared with the homologous reaction) of 58 to 99%, depending on the strain. Hybridization with the DNA from any of the strains of B. subtilis did not exceed 24%. DNA from B-3275 was unable to transform two amino acid auxotrophic markers to prototrophy in a highly competent strain of B. subtilis 168. We conclude that B-3275 is a strain of B. pumilus which we designate as B. pumilus NRRL B-3275.     

Cross-Species Induction of Antimicrobial Compounds, Biosurfactants and Quorum-Sensing Inhibitors in Tropical Marine Epibiotic Bacteria by Pathogens and Biofouling Microorganisms

Enhancement or induction of antimicrobial, biosurfactant, and quorum-sensing inhibition property in marine bacteria due to cross-species and cross-genera interactions was investigated. Four marine epibiotic bacteria (Bacillus sp. S3, B. pumilus S8, B. licheniformis D1, and Serratia marcescens V1) displaying antimicrobial activity against pathogenic or biofouling fungi (Candida albicans CA and Yarrowia lipolytica YL), and bacteria (Pseudomonas aeruginosa PA and Bacillus pumilus BP) were chosen for this study. The marine epibiotic bacteria when co-cultivated with the aforementioned fungi or bacteria showed induction or enhancement in antimicrobial activity, biosurfactant production, and quorum-sensing inhibition. Antifungal activity against Y. lipolytica YL was induced by co-cultivation of the pathogens or biofouling strains with the marine Bacillus sp. S3, B. pumilus S8, or B. licheniformis D1. Antibacterial activity against Ps. aeruginosa PA or B. pumilus BP was enhanced in most of the marine isolates after co-cultivation. Biosurfactant activity was significantly increased when cells of B. pumilus BP were co-cultivated with S. marcescens V1, B. pumilus S8, or B. licheniformis D1. Pigment reduction in the quorum-sensing inhibition indicator strain Chromobacterium violaceum 12472 was evident when the marine strain of Bacillus sp. S3 was grown in the presence of the inducer strain Ps. aeruginosa PA, suggesting quorum-sensing inhibition. The study has important ecological and biotechnological implications in terms of microbial competition in natural environments and enhancement of secondary metabolite production.     

Characterization of Bacillus probiotics available for human use

Bacillus species (Bacillus cereus, Bacillus clausii, Bacillus pumilus) carried in five commercial probiotic products consisting of bacterial spores were characterized for potential attributes (colonization, immunostimulation, and antimicrobial activity) that could account for their claimed probiotic properties. Three B. cereus strains were shown to persist in the mouse gastrointestinal tract for up to 18 days postadministration, demonstrating that these organisms have some ability to colonize. Spores of one B. cereus strain were extremely sensitive to simulated gastric conditions and simulated intestinal fluids. Spores of all strains were immunogenic when they were given orally to mice, but the B. pumilus strain was found to generate particularly high anti-spore immunoglobulin G titers. Spores of B. pumilus and of a laboratory strain of B. subtilis were found to induce the proinflammatory cytokine interleukin-6 in a cultured macrophage cell line, and in vivo, spores of B. pumilus and B. subtilis induced the proinflammatory cytokine tumor necrosis factor alpha and the Th1 cytokine gamma interferon. The B. pumilus strain and one B. cereus strain (B. cereus var. vietnami) were found to produce a bacteriocin-like activity against other Bacillus species. The results that provided evidence of colonization, immunostimulation, and antimicrobial activity support the hypothesis that the organisms have a potential probiotic effect. However, the three B. cereus strains were also found to produce the Hbl and Nhe enterotoxins, which makes them unsafe for human use.     

Occurrence of cold-labile NAD-specific glutamate dehydrogenase in Bacillus species

A nicotinamide adenine dinucleotide-specific glutamate dehydrogenase (NAD-GluDH; EC 1.4.1.3) inactivated by incubation at low temperatures was detected in several species of the genus Bacillus, including strains of B. cereus, B. laterosporus, B. lentus, B. panthotenicus, B. pasteurii, B. sphaericus, B. stearothermophilus, B. subtilis and B. thuringiensis. Incubation of cell-free extracts of these strains at 0 degrees C resulted in an 80-100% inactivation of NAD-GluDH activity within 120 min. The addition of 20% glycerol protected the enzyme from this inactivation in the cold. Strains of B. fastidiosus, B. licheniformis, B. macerans, B. megaterium and B. pumilus were found to lack NAD-GluDH activity.     

环保微生物菌剂常用5种芽胞杆菌的PCR鉴定

枯草芽胞杆菌（Bacillus subtilis）、地表芽胞杆菌（Bacillus licheniformis）、巨大芽胞杆菌（Bacillus megate．rium）、短小芽胞杆菌（Bacillus pumilus）、环状芽胞杆菌（Bacillu scirculans）是5种环保微生物菌剂中常用的芽胞杆菌,我国每年有大量此类菌剂进口。针对这些菌的传统理化检测方法费时费力,检测部门急需建立一种快速、准确的检测方法。利用GenBank上已有gyrB、rpoB、gdh基因序列分别设计和筛选上述菌种的特异性引物,经优化建立PCR检测方法。结果显示,使用所设计的特异引物,每种茼均扩增得到特异单1条带,能准确鉴定上述菌种并且与其他对照芽胞杆菌及非芽胞杆菌无交叉反应,送检样品的PCR检测结果与常规方法检测结果一致。建立的特异PCR方法具有良好的重复性和准确性,可快速鉴定上述菌种,具有很好的推广应用前景。     

Sigma 29-like protein is a common sporulation-specific element in bacteria of the genus Bacillus.

A monoclonal antibody specific for an antigenic determinant on the Bacillus subtilis sporulation-induced sigma factor sigma 29 reacted with proteins similar in size to sigma 29 in extracts of sporulating Bacillus licheniformis, Bacillus amyloliquifaciens, Bacillus cereus, Bacillus natto, and Bacillus pumilus but not in extracts prepared from vegetatively growing cultures of these bacteria. These results indicate that RNA polymerase modifications, initially described for B. subtilis, are likely to be common among sporulating Bacillus spp. and that at least some of the specific modifications that are observed in sporulating B. subtilis are conserved among members of this genus.     

Bacillus subtilis (natto) plasmid pLS20 mediates interspecies plasmid transfer.

The 55-kilobase plasmid, pLS20, of Bacillus subtilis (natto) 3335 promotes transfer of the tetracycline resistance plasmid pBC16 from B. subtilis (natto) to the Bacillus species B. anthracis, B. cereus, B. licheniformis, B. megaterium, B. pumilus, B. subtilis, and B. thuringiensis. Frequency of pBC16 transfer ranged from 2.3 x 10(-6) to 2.8 x 10(-3). Evidence for a plasmid-encoded conjugationlike mechanism of genetic exchange includes (i) pLS20+ strains, but not pLS20- strains, functioned as donors of pBC16; (ii) plasmid transfer was insensitive to the presence of DNase; and (iii) cell-free filtrates of donor cultures did not convert recipient cells to Tcr. Cotransfer of pLS20 and pBC16 in intraspecies matings and in matings with a restriction-deficient B. subtilis strain indicated that pLS20 was self-transmissible. In addition to mobilizing pBC16, pLS20 mediated transfer of the B. subtilis (natto) plasmid pLS19 and the Staphylococcus aureus plasmid pUB110. The fertility plasmid did not carry a selectable marker. To facilitate direct selection for pLS20 transfer, plasmid derivatives which carried the erythromycin resistance transposon Tn917 were generated. Development of this method of genetic exchange will facilitate the introduction of plasmid DNA into nontransformable species by use of transformable fertile B. subtilis or B. subtilis (natto) strains as intermediates.     

Characterization of Bacillus strains of marine origin.

A total of twenty aerobic endospore-forming bacilli, isolated from marine invertebrates and sea water of different areas of the Pacific Ocean, were taxonomically characterized. Most of the bacilli (11 strains) of marine origin belonged to the species Bacillus subtilis, according to their phenotypic characteristics, antibiotic susceptibility profiles, and fatty acids patterns. A group of four alkaliphilic strains formed a separate cluster that was tentatively classified as B. horti. One isolate, KMM 1717, associated with a sponge from the Coral Sea was identified as B. pumilus. Two strains, Bacillus KMM 1916 and KMM 1918, showed antibiotic sensitivity profiles similar to B. licheniformis, but they had a distinct fatty acid composition and peculiar phenotypic traits. The taxonomic affiliation of KMM 1810 and KMM 1763 remained unclear since their fatty acid composition and antibiotic sensitivity patterns were not resembled with none of these obtained for Bacillus strains.     

THE GERMINATION OF SPORES OF BACILLUS SUBTILIS AND BACILLUS LICHENIFORMIS AT TEMPERATURES BELOW THE MINIMUM FOR VEGETATIVE GROWTH

SUMMARY: The germination in nutrient media of the spores of several strains of Bacillus subtilis and B. licheniformis at temperatures too low to permit vegetative growth has been investigated. Germination occurred with only 11 of 54 strains of B. subtilis and with none of 35 strains of B. licheniformis .