Characterization,Identification and Application of Lactic Acid Bacteria Isolated from Forage Paddy Rice Silage

There has been growing interest to develop forage rice as a new feed resource for livestock. This study was to characterize the natural population of lactic acid bacteria (LAB) and select potentially excellent strains for paddy rice silage preparation in China. One hundred and twenty-six strains were isolated and screened from paddy rice silage prepared using a small-scale fermentation system, and ninety-nine of these isolates were considered to be LAB based on their Gram-positive and catalase-negative morphology and the production of most of their metabolic products as lactic acid. These isolates were divided into eight groups (A-H) on the basis of their morphological and biochemical characteristics. The Group A to H strains were identified as Lactobacillus (L.) plantarum subsp. plantarum (species ratio: 8.1%), L. casei (5.1%), Leuconostoc (Ln.) pseudomesenteroides (11.1%), Pediococcus (P.) pentosaceus (24.2%), Enterococcus (E.) mundtii (12.1%), Lactococcus (Lc.) garvieae (15.2%), E. faecium (9.1%) and Lc. lactis subsp. lactis (15.2%) based on sequence analyses of their 16S rRNA and recA genes. P. pentosaceus was the most abundant member of the LAB population in the paddy rice silage. A selected strain, namely L. casei R 465, was found to be able to grow under low pH conditions and to improve the silage quality with low pH and a relatively high content of lactic acid. This study demonstrated that forage paddy rice silage contains abundant LAB species and its silage can be well preserved by inoculation with LAB, and that strain R 465 can be a potentially excellent inoculant for paddy rice silage.     

Influence of Lactobacillus spp. from an Inoculant and of Weissella and Leuconostoc spp. from Forage Crops on Silage Fermentation

Lactobacillus spp. from an inoculant and Weissella and Leuconostoc spp. from forage crops were characterized, and their influence on silage fermentation was studied. Forty-two lactic acid-producing cocci were obtained from forage crops and grasses. All isolates were gram-positive, catalase-negative cocci that produced gas from glucose, and produced more than 90% of their lactate in the d-isomer form. These isolates were divided into groups A and B by sugar fermentation patterns. Two representative strains from the two groups, FG 5 and FG 13, were assigned to the species Weissella paramesenteroides and Leuconostoc pseudomesenteroides, respectively, on the basis of DNA-DNA relatedness. Strains FG 5, FG 13, and SL 1 (Lactobacillus casei), isolated from a commercial inoculant, were used as additives to alfalfa and Italian ryegrass silage preparations. Lactic acid bacterium counts were higher in all additive-treated silages than in the control silage at an early stage of ensiling. During silage fermentation, inoculation with SL 1 more effectively inhibited the growth of aerobic bacteria and clostridia than inoculation with strain FG 5 or FG 13. SL 1-treated silages stored well. However, the control and FG 5- and FG 13-treated silages had a significantly (P < 0.05) higher pH and butyric acid and ammonia nitrogen contents and significantly (P < 0.05) lower lactate content than SL 1-treated silage. Compared with the control silage, SL 1 treatments reduced the proportion of d-(−)-lactic acid, gas production, and dry matter loss in two kinds of silage, but the FG 5 and FG 13 treatments gave similar values in alfalfa silages and higher values (P < 0.05) in Italian ryegrass silage. The results confirmed that heterofermentative strains of W. paramesenteroides FG 5 and L. pseudomesenteroides FG 13 did not improve silage quality and may cause some fermentation loss.Silage is now the most common preserved cattle feed in many countries, including Japan. It is well established that lactic acid bacteria (LAB) play an important role in silage fermentation. Epiphytic microflora, the microorganisms naturally present on forage crops, are responsible for silage fermentation and also influence silage quality (3, 11, 15). Lactobacilli and lactic acid-producing cocci, e.g., leuconostocs, lactococci, streptococci, pediococci, and Weissella species, are major components of the microbial flora in various types of forage crops (3). Stirling and Whittenbury (21) reported that leuconostocs were the most numerous and widely distributed on forages and that lactobacilli occurred mostly on grasses. Cai et al. (3) examined a large number of forage crops and grasses and also found that the predominant LAB were lactic acid-producing cocci and that lactobacilli were the least numerous and mostly homofermentative. Ruser (17) found that although all LAB groups were present in chopped-maize samples, homofermentative lactobacilli and heterofermentative leuconostocs were present in the highest numbers.In order to improve silage quality, many LAB-containing biological additives have been developed and are currently available (13, 20, 25). These inoculants may inhibit the growth of harmful bacteria and enhance lactic acid fermentation during ensiling periods. The epiphytic LAB influence the effectiveness of silage inoculants because the introduced bacteria must compete with these LAB (12). Therefore, the LAB species and their characteristics in the silage environment require further study. However, while an increasing number of studies have reported positive benefits from using some bacterial inoculants as silage additives, relatively few have reported the effect of epiphytic LAB, especially Leuconostoc and Weissella species, on silage fermentation. In the present study, the characterization of Leuconostoc and Weissella species isolated from forage crops and their influence on silage fermentation were examined.     

Identification of Osmophilic Aspergillus Isolated from Rice and Their Radio-sensitivity

Osmophilic Aspergillus responsible for spoilage of rice, corn, milo and wheat have been isolated and identified. Fifteen strains were classified as members of the Aspergillus glaucus group, and were subdivided into A. ruber, A. repens, A. mangini, A. chevalieri and A. montevidensis. Nine strains were classified as members of the A. restrictus group, and were subdivided into A. gracilis, A. vitricolae and A. casiellus. The other 7 strains were classified as A. versicolor in the A. versicolor group, A. sulphureus in the A. ochraceus group, and A. niveus in the A. flavipes group.

All of dose-survival curves obtained with the conidia of 10 strains showed the sigmoidal type having the D₁₀ values between 18 and 30 krad. The survival curves obtained with the ascospores of A. glaucus group also showed the sigmoidal type having the D₁₀ values of 54 krad. Radio-sensitivity of the dry conidia was similar to that of the dry ascospores, having D₁₀ values between 50 to 58 krad.     

Aflatoxin Production of Species and Strains of the Aspergillus flavus Group Isolated from Field Crops

Peanuts, cottonseed, rice, and sorghum from Texas were sampled over a 3-year period. To insure adequate isolation of alfatoxin-producing species of fungi, low-quality lots were sampled at a rate greater than their respective proportional representation. Aflatoxins were found each year in peanut and cottonseed and were found in 2 of 3 years in rice and sorghum. Aflatoxins were detected in all four crops. The Aspergillus flavus group was much more prevalent in peanut and rice than in cottonseed and sorghum. Of the isolates of the A. flavus group, 96% from peanuts, 79% from cottonseed, 49% from sorghum, and 35% from rice produced aflatoxins. The average toxin production of isolates from rice was much less than that from peanuts, cottonseed, or sorghum. More than 90% of all isolates of the A. flavus group were identified as the species A. flavus. A. parasiticus was isolated from all four crops. Only A. parasiticus produced aflatoxin G.     

Identification of Pseudomonas Species Isolated from Hospital Environment and Human Sources

Methods and procedures are described for the identification of aerobic pseudomonads isolated from clinical specimens. Fluorescence is used as a means of primary differentiation. Final identification is based on the observation of characteristics such as flagellation, storage of intracellular fat, growth at 4 C and at 41 C, denitrification, gelatin hydrolysis, arginine dihydrolase activity, and oxidase activity. The value of this schema to the diagnostic laboratory is discussed.     

Characterization of Fusobacterium Species Isolated from Soft Tissue Infections in Cats

A total of 35 strains of Gram negative anaerobes, belonging to the genus Fusobacterium and isolated from subcutaneous abscesses and pyothorax in cats, have been examined morphologically and biochemically. Gas chromatography and biochemical analysis placed them into four species— Fusobacterium necrophorum (seven strains), F. russii (22 strains), F. naviforme (four strains) and F. nucleatum (two strains). It is considered that subcutaneous abscesses and pyothorax result from contamination from the oral cavity and upper respiratory tract. The disproportionate numbers of isolation of F. russii may reflect differences in the predominant flora in this region of cats compared to man. The ambiguity of the concept in current literature of the ability of organisms to convert threonine to propionate is discussed.     

Molecular Characterization of Leishmania Species Isolated from Cutaneous Leishmaniasis in Yemen

Background

Cutaneous leishmaniasis (CL) is a neglected tropical disease endemic in the tropics and subtropics with a global yearly incidence of 1.5 million. Although CL is the most common form of leishmaniasis, which is responsible for 60% of DALYs lost due to tropical-cluster diseases prevalent in Yemen, available information is very limited.

Methodology/Principal Findings

This study was conducted to determine the molecular characterization of Leishmania species isolated from human cutaneous lesions in Yemen. Dermal scrapes were collected and examined for Leishmania amastigotes using the Giemsa staining technique. Amplification of the ribosomal internal transcribed spacer 1(ITS-1) gene was carried out using nested PCR and subsequent sequencing. The sequences from Leishmania isolates were subjected to phylogenetic analysis using the neighbor-joining and maximum parsimony methods. The trees identified Leishmania tropica from 16 isolates which were represented by two sequence types.

Conclusions/Significance

The predominance of the anthroponotic species (i.e. L. tropica) indicates the probability of anthroponotic transmission of cutaneous leishmaniasis in Yemen. These findings will help public health authorities to build an effective control strategy taking into consideration person–to-person transmission as the main dynamic of transmission of CL.     

Preparation and Characterization of Membranes from a Coryneform Isolated from Ratoon Stunted Sugarcane

Membranes from the coryneform were prepared by an optimized chemical procedure using EDTA and lysozyme and isolated upon equilibrium sucrose density gradient ultracentrifugation. Four bands, I to IV, of peak densities 1.15, 1.19, 1.21 and 1.245 g. cm^?3 respectively, were reproducibly obtained. Immunodiffusion of the isolated membrane bands against bacterial antiserum showed that the immunological integrity of the isolated membranes was retained. Membrane bands I and II had similar precipitin arcs having one major component. A shift towards greater immunological complexity in band IV was observed. Similarly, a change in polypeptide composition established by polyacrylamide gel electrophoresis was found. The major group of polypeptides inband I ranging in size from 39 to 67 kdal were successively reduced in bands II, III and IV whereas the polypeptide of 15.7 kdal and of those in the region of 140 kdal showed respective increases. Transmission electron microscopy revealed similar membrane, structures in all membrane bands with only minor differences between them.     

Identification of a Novel Afipia Species Isolated from an Indian Flying Fox

An old world fruit bat Pteropus giganteus, held in captivity and suffering from necrosis of its wing digits, failed to respond to antibiotic therapy and succumbed to the infection. Samples submitted to the National Centre for Foreign Animal Disease were tested for viral infection. Vero E6 cells exhibited minor but unique cytopathic effects on second blind passage, and full CPE by passage four. Utilizing an unbiased random amplification technique from cell culture supernatant, we identified a bacterium belonging to the Bradyrhizobiaceae. Purification of cell culture supernatant on TY media revealed a slow growing bacterial isolate. In this study using electron microscopy, 16S rRNA gene analysis and whole genome sequencing, we identify a novel bacterial species associated with the site of infection belonging to the genus Afipia. This genus of bacteria is very diverse, with only a limited number of species characterized. Afipia felis, previously described as the etiological agent to cause cat scratch disease, and Afipia septicemium, most recently shown to cause disease in humans, highlight the potential for members of this genus to form a branch of opportunistic pathogens within the Bradyrhizobiaceae. Increased utilization of next generation sequencing and genomics will aid in classifying additional members of this intriguing bacterial genera.     

RFLP分子标记及其在牧草研究中的应用

RFLP分子标记是用限制性酶(RE)消化不同个体的同源DNA分子,经电泳表现的限制性片段长度的差异。它反映了DNA分子水平上的变异,可能是限制性内切酶识别位点的改变,也可能是部分片段的缺失、插入、易位、倒位等,显示出丰富的多态性。本文综述了RFLP分子标记技术在牧草个体识别、绘制遗传图谱、目的基因定位、检测群体内或群体间序列差异程度以及辅助育种研究中的应用等,并对该技术在牧草研究中的应用作了展望。     

Genetic Characterization and Transmission Cycles of Cryptosporidium Species Isolated from Humans in New Zealand

Little is known about the genetic characteristics, distribution, and transmission cycles of Cryptosporidium species that cause human disease in New Zealand. To address these questions, 423 fecal specimens containing Cryptosporidium oocysts and obtained from different regions were examined by the PCR-restriction fragment length polymorphism technique. Indeterminant results were resolved by DNA sequence analysis. Two regions supplied the majority of isolates: one rural and one urban. Overall, Cryptosporidium hominis accounted for 47% of the isolates, with the remaining 53% being the C. parvum bovine genotype. A difference, however, was observed between the Cryptosporidium species from rural and urban isolates, with C. hominis dominant in the urban region, whereas the C. parvum bovine genotype was prevalent in rural New Zealand. A shift in transmission cycles was detected between seasons, with an anthroponotic cycle in autumn and a zoonotic cycle in spring. A novel Cryptosporidium sp., which on DNA sequence analysis showed a close relationship with C. canis, was detected in two unrelated children from different regions, illustrating the genetic diversity within this genus.     

Molecular Characterization of Various Trichomonad Species Isolated from Humans and Related Mammals in Indonesia

Trichomonad species inhabit a variety of vertebrate hosts; however, their potential zoonotic transmission has not been clearly addressed, especially with regard to human infection. Twenty-one strains of trichomonads isolated from humans (5 isolates), pigs (6 isolates), rodents (6 isolates), a water buffalo (1 isolate), a cow (1 isolate), a goat (1 isolate), and a dog (1 isolate) were collected in Indonesia and molecularly characterized. The DNA sequences of the partial 18S small subunit ribosomal RNA (rRNA) gene or 5.8S rRNA gene locus with its flanking regions (internal transcribed spacer region, ITS1 and ITS2) were identified in various trichomonads; Simplicimonas sp., Hexamastix mitis, and Hypotrichomonas sp. from rodents, and Tetratrichomonas sp. and Trichomonas sp. from pigs. All of these species were not detected in humans, whereas Pentatrichomonas hominis was identified in humans, pigs, the dog, the water buffalo, the cow, and the goat. Even when using the high-resolution gene locus of the ITS regions, all P. hominis strains were genetically identical; thus zoonotic transmission between humans and these closely related mammals may be occurring in the area investigated. The detection of Simplicimonas sp. in rodents (Rattus exulans) and P. hominis in water buffalo in this study revealed newly recognized host adaptations and suggested the existence of remaining unrevealed ranges of hosts in the trichomonad species.     

Characterization of bacteriocin produced by Pediococcus pentosaceus from wine

A.M. STRASSER DE SAAD AND M.C. MANCA DE NADRA. 1993. Twenty strains of Pediococcus pentosaceus isolated from wine were examined for production of bacteriocins. Only two of them showed inhibitory activity, Ped. pentosaceus N4p against the indicator strains of the same species and N5p against 19 strains of the three genera of lactic acid bacteria from wine. The antimicrobial substance from N5p strains was removed by membrane (0.2 μm) filtration, destroyed by organic solvents and proteolytic enzymes. It was stable for 60 min at 100C. The bacteriocin was produced early in the growth cycle and its production was maximum after 48 h of culture in tomato juice medium at an initial pH of 6.5. The bactericidal effect was observed.     

Biocontrol and Plant Growth Promotion Characterization of Bacillus Species Isolated from Calendula officinalis Rhizosphere

The phenotypic and genotypic diversity of the plant growth promoting Bacillus genus have been widely investigated in the rhizosphere of various agricultural crops. However, to our knowledge this is the first report on the Bacillus species isolated from the rhizosphere of Calendula officinalis. 15 % of the isolated bacteria were screened for their important antifungal activity against Fusarium oxysporum, Botrytis cinerea, Aspergillus niger, Cladosporium cucumerinium and Alternaria alternata. The bacteria identification based on 16S r-RNA and gyrase-A genes analysis, revealed strains closely related to Bacillus amyloliquefaciens, B. velezensis, B. subtilis sub sp spizezenii and Paenibacillus polymyxa species. The electro-spray mass spectrometry coupled to liquid chromatography (ESI-LC MS) analysis showed that most of the Bacillus isolates produced the three lipopeptides families. However, the P. polymyxa (18SRTS) didn’t produce any type of lipopeptides. All the tested Bacillus isolates produced cellulase but the protease activity was observed only in the B. amyloliquefaciens species (9SRTS). The Salkowsky colorimetric test showed that the screened bacteria synthesized 6–52 μg/ml of indole 3 acetic acid. These bacteria produced siderophores with more than 10 mm wide orange zones on chromazurol S. The greenhouse experiment using a naturally infested soil with Sclerotonia sclerotiorum showed that the B. amyloliquefaciens (9SRTS) had no significant (P > 0.05) effect on the pre-germination of the chickpea seeds. However, it increased the size of the chickpea plants and reduced the stem rot disease (P < 0.05).These results suggested that the Bacillus strains isolated in this work may be further used as bioinoculants to improve the production of C. officinalis and other crop systems.     

Identification and Characterization of Non-Tuberculous Mycobacteria Isolated from Tuberculosis Suspects in Southern-Central China

The incidence of non-tuberculous mycobacteria (NTM)-related death has increased globally recently. To obtain information of the species and characterization of pathogens involved in NTM pulmonary infection in Southern-central China, we identified 160 non-tuberculous infection cases from 3995 acid-fast bacilli (AFB)-positive tuberculous suspects. We then randomly selected 101 non-tuberculous patients, isolated bacteria from their sputa and genotyped the pathogens using the 16S rRNA gene and 16S-23S rRNA internal transcribed spacer sequences. M. intracellulare (32.67%, 33/101), M. abscessus (32.67%, 33/101) and M. fortuitum (7.92%, 8/101) are identified in these isolates. Surprisingly, non-mycobacteria including Gordonia (8.91%, 9/101), Nocardia (5.94%, 6/101) and Tsukamurella (0.99%, 1/101) are also discovered, and the case of Tsukamurella pulmonis infection is first discovered in Southern-central China. Moreover, species of M. mucogenicum group, M. chubuense, M. kansasii, M. gastri, M. avium, M. porcinum and M. smegmatis are identified. In addition, nine immune compromised cases (8.91%, 9/101), including type two diabetes mellitus and HIV/AIDS are found to be infected with non-tuberculous bacteria. This study revealed the distribution and characteristics of non-tuberculous AFB pathogen infection occurred in Southern-central China, and suggested that physicians should be alert of the emerging of NTM and non-mycobacteria infection in AFB positive cases and take caution when choosing chemotherapy for tuberculosis-like pulmonary infections. Generally, this study may help with the development of new strategy for the diagnosis and treatment of mycobacterial infection.     

Identification and Characterization of Leuconostoc fallax Strains Isolated from an Industrial Sauerkraut Fermentation

Lactic acid bacterial strains were isolated from brines sampled after 7 days of an industrial sauerkraut fermentation, and six strains were selected on the basis of susceptibility to bacteriophages. Bacterial growth in cabbage juice was monitored, and the fermentation end products were identified, quantified, and compared to those of Leuconostoc mesenteroides. Identification by biochemical fingerprinting, endonuclease digestion of the 16S-23S intergenic transcribed spacer region, and sequencing of variable regions V1 and V2 of the 16S rRNA gene indicated that the six selected sauerkraut isolates were Leuconostoc fallax strains. Random amplification of polymorphic DNA fingerprints indicated that the strains were distinct from one another. The growth and fermentation patterns of the L. fallax isolates were highly similar to those of L. mesenteroides. The final pH of cabbage juice fermentation was 3.6, and the main fermentation end products were lactic acid, acetic acid, and mannitol for both species. However, none of the L. fallax strains exhibited the malolactic reaction, which is characteristic of most L. mesenteroides strains. These results indicated that in addition to L. mesenteroides, a variety of L. fallax strains may be present in the heterofermentative stage of sauerkraut fermentation. The microbial ecology of sauerkraut fermentation appears to be more complex than previously indicated, and the prevalence and roles of L. fallax require further investigation.     

Characterization of Mycoplasma pulmonis Variants Isolated from Rabbits I. Identification and Properties of Isolates

Mycoplasma showing at least two colony types were isolated from the nares and oropharynx of New Zealand white rabbits. Two strains were purified by single-colony passages and characterized. Morphology by phase-contrast and electron microscopy was typical of Mycoplasmataceae. Both grew anaerobically as well as aerobically, caused hemolysis of guinea pig, sheep, and horse red blood cells, and fermented glucose. These characteristics are shared by members of the species M. pulmonis, commonly isolated from the respiratory tracts of laboratory rats and mice. By use of the growth-inhibition test and agar-gel double-diffusion tests, the two strains were found to be serologically related to each other and to M. pulmonis ATCC 14267 but not to other representative Mycoplasma species from man and animals.