Cellulolytic bacteria from pig large intestine.

An anaerobic, cellulose-degrading, gram-negative rod and a gram-positive coccus, identified as Bacteroides succinogenes and Ruminococcus flavefaciens, respectively, were isolated from pig fecal samples. These organisms were isolated from cellulolytic most-probable-number dilutions which represented 4 or 6% of the viable bacterial count when pigs were fed a low- or high-fiber diet, respectively.     

烧伤病房患者创面金葡菌分布及耐药性分析

目的分析烧伤病房患者不同创面金葡菌的分布及耐药性,为临床合理选用抗菌药提供依据。方法对2006年1月至2013年12月间中国人民解放军第八五医院烧伤病房患者创面分离出金葡菌,采用K—B纸片扩散法进行药物敏感试验。分析金葡菌的耐药性,并对难愈性创面、非难愈性创面的耐甲氧西林金葡菌（MRSA）与甲氧西林敏感金葡菌（MSSA）的耐药性进行对比分析。结果分离出金葡菌112株,其中难愈性创面有70株MRSA和17株MSSA来自难愈性创面,16株MRSA和9株MSSA来自非难愈性创面。金葡菌对青霉素、红霉素、克林霉素的耐药率较高（分别为94．64％、81．25％和74．11％）,对复方新诺明、呋喃妥因的耐药率较低（分别为16．07％和1．79％）,对万古霉素、利奈唑烷的耐药率为0。MRSA的耐药率高于MSSA。来源于难愈性创面与非难愈性创面的MRSA仅在对利福平的耐药率上有明显差异,而来源于两创面的MSSA的耐药率无明显差异。结论创面金葡菌中MRSA的构成比高,难愈性创面MRSA耐药严重,应积极防控创面MRSA感染和扩散。     

Isolation and initial culture of porcine inner cell masses derived from in vitro-produced blastocysts

The present study was conducted to isolate and culture inner cell mass (ICM) primarily derived from in vitro-produced blastocysts and to develop the culture conditions for the ICM cells. In Experiment 1, immunosurgically isolated ICMs of blastocysts derived from in vitro fertilization (IVF), somatic cell nuclear transfer (SCNT) or parthenogenetic activation (PA) were seeded onto STO cells. Primary colonies from each isolated ICM were formed with a ratio of 28.9, 30.0 and 4.9%, respectively. In Experiment 2, blastocysts collected from IVF were directly seeded onto a feeder layer with or without zona pellucida (ZP), or were subjected to ICM isolation by immunosurgery. Primary colonies were formed in 36.8% of isolated ICMs and 19.4% in intact blastocysts without ZP. In Experiment 3, ICMs from IVF blastocysts were seeded onto STO cells, mouse embryonic fibroblast (MEF) or porcine uterine epithelial cells (PUEC). On STO and MEF cells, 34.5 and 22.2% of primary colonies were formed, respectively. However, no primary colony was formed on the PUEC or in feeder-free condition. In Experiment 4, ICMs from IVF blastocysts were cultured in DMEM + Ham's F10 (D/H medium), DMEM + NCSU-23 (D/N medium) or DMEM alone. When D/H medium or D/N medium was used, 21.7 or 44.4% of primary colony were formed, respectively, while no primary colony was formed in DMEM alone. These cells showed alkaline phosphatase activity and could be maintained for up to five passages. In suspension culture, cells formed embryoid bodies. These results demonstrate that porcine ICM could be isolated and cultured primarily from in vitro-produced blastocysts with a suitable culture system.     

Isolation of two trypsin inhibitors from resting seeds of the white bush (Cucurbita pepo var. patissonina) and their properties

Two trypsin inhibitors, CPPTI-I and CPPTI-II of Mr 3 250 and 7 850, respectively, were isolated from resting white bush seeds. Both inhibitors are cysteine-rich proteins. In addition to trypsin, they inhibit a trypsin-like enzyme isolated from Streptomyces griseus proteinase but they do not act on chymotrypsin, kallikrein or subtilopeptidase A. The isolated inhibitors contain a lysine residue in position P1 of the reactive site.     

Resistance of Salmonellae Isolated in 1959 and 1960 to Tetracyclines and Chloramphenicol

The resistance of cultures of Salmonella typhimurium to tetracyclines and chloramphenicol has been examined periodically. Although none of 200 cultures isolated prior to 1948 was resistant to the antibiotics, 5% of 100 cultures from man and 9% of 100 cultures from fowls which were isolated in 1956 and 1957 were resistant to tetracyclines. Among 158 cultures isolated from man and 100 cultures isolated from fowls in 1959 and 1960, 13.9 and 29%, respectively, were resistant to tetracyclines. In the last series, cultures resistant to chloramphenicol were found for the first time. Among 150 cultures of other Salmonella serotypes from man and 137 similar cultures isolated from fowls in 1959 and 1960, 5.3 and 8%, respectively, were found resistant to tetracyclines. There is no obvious explanation for the higher percentage of resistant strains occurring in S. typhimurium than in other serotypes.     

Isolation of acid-resistant urinary trypsin inhibitors by high performance liquid chromatography and their characterization by N-terminal amino-acid sequence determination

Two crude fractions of acid-resistant trypsin inhibitors (apparent molecular masses 44 and 20 kDa, respectively) were prepared from human urine by gel permeation chromatography. From both preparations the pure inhibitors were isolated by high performance liquid chromatography (HPLC). Their N-terminal amino-acid sequences were determined and compared with those of HI-30 and HI-14 as isolated by reversible binding to either immobilized trypsin or immobilized chymotrypsin. The N-terminal amino-acid sequence of the high-molecular mass inhibitor UI-I isolated by HPLC was identical with those of HI-30 and UI-C-I isolated via immobilized trypsin or chymotrypsin, respectively. The low-molecular mass inhibitors UI-II and UI-C-II differ from HI-14 by the N-terminal extension Glu-Val-Thr-Lys-when obtained by HPLC or by the extension Thr-Lys-when obtained via immobilized chymotrypsin, respectively. The comparison of these N-termini with the amino-acid sequence of HI-30 (Ala1-...-Val16-Thr-Glu-Val-Thr-Lys-HI-14) defines the low molecular urinary trypsin inhibitors as proteolytic degradation products of the high-molecular urinary inhibitor. Proteolysis may occur at different bonds. The existing discrepancies in molecular architecture and in molecular masses of the urinary trypsin inhibitors are discussed.     

Nonenzymatic isolation of Trichinella pseudospiralis infective L1 larvae at pH 7.4

Over half of the number of Trichinella pseudospiralis infective L1 larvae recovered from host carcasses by pepsin-HCl digestion were isolated from homogenized carcasses incubated in HBSS. More worms isolated by the latter method were viable compared to those isolated by pepsin-HCl digestion. When host carcasses infected with T. pseudospiralis were diced into pieces and incubated in HBSS, 30% more worms were recovered than from homogenized carcasses incubated in HBSS as above, and the majority of worms acquired by the former method were viable. The infectivity of T. pseudospiralis infective L1 larvae isolated from homogenized muscle in HBSS was 3.9 times greater than that for larvae recovered from homogenized carcasses by pepsin-HCl digestion. Only 4% and 0.8% of the number of T. spiralis recovered from homogenized muscle by pepsin-HCl digestion were isolated from homogenized or diced muscle incubated in HBSS, respectively. Fewer T. spiralis isolated from homogenized tissue in HBSS were viable compared to those recovered from homogenized carcasses digested in pepsin-HCl or diced carcasses incubated in HBSS.     

Fecal coliforms of wastewater treatment plants: antibiotic resistance, survival on surfaces and inhibition by sodium chloride and ascorbic acid.

Three hundred and sixteen fecal coliform strains isolated from raw sewage and final effluents of two representative wastewater treatment plants were examined for antibiotic resistance. For plant A (which was disinfecting its effluents before discharge), 55% and 41% of the strains isolated from the influent and the effluent, respectively were resistant to one or more antibiotics. For plant B (which was not disinfecting its effluents before discharge), 33% and 58% of the strains isolated from the influent and the effluent, respectively were resistant to one or more antibiotics. A considerable proportion of these bacteria were resistant to three or more antibiotics. NaCl (3%) and ascorbic acid (0.1%) reduced the growth rate of both sensitive and resistant strains, and more inhibitory effects on the sensitive strains than on the resistant strains were observed. All resistant strains tested survived on various types of surfaces (e.g. glass, stainless steel, agar, cabbage, parsley and sand) significantly better than sensitive strains.     

Antibiotic Resistance of Enterococci in American Bison (Bison bison) from a Nature Preserve Compared to That of Enterococci in Pastured Cattle

Enterococci isolated from a bison population on a native tall-grass prairie preserve in Kansas were characterized and compared to enterococci isolated from pastured cattle. The species diversity was dominated by Enterococcus casseliflavus in bison (62.4%), while Enterococcus hirae was the most common isolate from cattle (39.7%). Enterococcus faecalis was the second most common species isolated from bison (16%). In cattle, E. faecalis and Enterococcus faecium were isolated at lower percentages (3.2% and 1.6%, respectively). No resistance to ampicillin, chloramphenicol, gentamicin, or high levels of vancomycin was detected from either source. Tetracycline and erythromycin resistance phenotypes, encoded by tetO and ermB, respectively, were common in cattle isolates (42.9% and 12.7%, respectively). A significant percentage of bison isolates (8% and 4%, respectively) were also resistant to these two antibiotics. The tetracycline resistance genes from both bison and cattle isolates resided on mobile genetic elements and showed a transfer frequency of 10⁻⁶ per donor, whereas erythromycin resistance was not transferable. Resistance to ciprofloxacin was found to be higher in enterococci from bison (14.4%) than in enterococci isolated from cattle (9.5%). The bison population can serve as a sentinel population for studying the spread and origin of antibiotic resistance.     

Frequent isolation of Edwardsiella tarda and Pleisiomonas shigelloides from healthy Zairese freshwater fish: a possible source of sporadic diarrhea in the tropics.

The intestinal contents of 59 Zairese freshwater fish were examined for the presence of potential human enteric pathogens. Edwardsiella tarda and Plesiomonas shigelloides were isolated from 57 and 59% of them, respectively. For both microorganisms there was a significant difference between the isolation rates from lake and river fish: whereas E. tarda was much more frequently isolated from lake fish than was P. shigelloides, the reverse was observed for river fish. The authors hypothesize that sporadic cases of tropical diarrhea with E. tarda or P. shigelloides can be traced to contact with or consumption of freshwater fish.     

Isolation and identification of two protein-protein crosslinks within the two subunits of Bacillus stearothermophilus ribosomes

Bacillus stearothermophilus 30S and 50S ribosomal subunits were isolated and crosslinked with diepoxybutane. The crosslinked proteins were extracted with LiCl or with 67% acetic acid and purified by a combination of different high performance liquid chromatography techniques. The protein fractions were analysed by two-dimensional and diagonal polyacrylamide gel electrophoresis and by immunological methods. Two crosslinked protein pairs, one from the large and one from the small subunit, consisting of proteins L23-L29 and S13-S19 respectively, were isolated in milligram amounts for determination of the crosslinked amino acids.     

Isolation of varicella-zoster virus from pharyngeal and nasal swabs in varicella patients

Thirteen children aged 5 months to 4 years were observed during a varicella epidemic in an Infants' Hospital; except for two normal individuals, the children had various forms of congenital defects. Eleven of the children developed varicella. During the first 3 days of exanthem, a total of 17 VZ virus strains were isolated: 12 from vesicular fluid, 3 from 23 nasal and 2 from 22 pharyngeal swabs. No strain was isolated during the incubation period despite 57 and 56 swabs having been collected from the throat and nose, respectively; nor was VZ virus isolated from 6 pharyngeal and 7 nasal swabs taken on the first day of exanthem. Isolation attempts performed from vesicular fluid to control quality of the isolation conditions gave a positivity rate of 100%. Under these optimal isolation conditions VZ virus was found in the nose or throat alongside skin vesicles in four of the 11 ill children. Besides VZ virus, the pharyngeal and nasal swabs yielded, respectively, four and four cytomegalovirus strains. The cytomegalovirus infections were inapparent.     

浅部真菌病1948份临床标本的真菌学分析

目的 通过对浅部真菌病患者临床送检标本的病原真菌菌种进行系统分析,了解感染及病原真菌的分布情况。方法 采用直接镜检、培养及真菌鉴定等方法对临床送验标本进行检验和鉴定,大部分标本鉴定到种。结果 1948份临床送验标本中,直接涂片镜检阳性率53．41％,培养阳性率40．28％,而镜检＋培养的阳性率为66．98％。对上述3种方法的真菌检出率进行比较,均存在显著差异（χ^2检验P均〈0．005）。在培养的1944份标本中,共分离出18个属,36种真菌,其中,红色毛癣菌24．52％、须癣毛癣菌16．48％、白念珠菌12．64％。结论 ①镜检结合培养的阳性率显著高于单一的镜检或培养的阳性率。②在患者即时的真菌镜检阴性时,应选择培养方法进一步检测,不轻易排除浅部真菌病感染可能。③皮肤癣菌居患者浅部真菌病致病菌首位,而白念珠菌及酵母类菌也是重要病原菌。     

Comparative Studies of Bacillus Cereus Strains Isolated From Various Foods and Food Poisoning Outbreaks

Certain properties of 22 Bacillus cereus strains isolated from different foods and food poisoning episodes were investigated in order to evaluate possible différences between strains isolated from diarrhoeal and vomiting type food poisoning outbreaks. None of the strains isolated from vomiting type episodes produced acid from salicin and mannose, whereas 80 and 40 % of the strains from diarrhoeal type outbreaks were positive, respectively. No association between the antibiotic sensitivity pattern or the fatty acid composition and the source of a strain could be found, although some strains differed from the general pattern of B. cereus in some instances. No significant differences in the production of the skin factor between strains isolated from the two types of outbreaks were found either. The findings of this study support the observation that the food environment itself essentially affects the enterotoxin formation of B. cereus.     

Interaction of Mycoplasma bovis and Mycoplasma bovigenitalium with preimplantation bovine embryos

In vitro exposures of bovine embryos to Mycoplasma bovis and Mycoplasma bovigenitalium were conducted to determine if these organisms adhered to the zona pellucida-intact (ZP-I) bovine embryo, and standard procedures for washing and treating embryos were evaluated to determine their effectiveness for removing or killing mycoplasmas. Mycoplasma bovis and M. bovigenitalium were isolated from 19 of 19 and 24 of 24 ZP-I embryos, respectively, after in vitro exposure and subsequent washing, thus demonstrating adherence of the two species of Mycoplasma to the ZP. Additionally, M. bovis was isolated from 20 of 20 and 23 of 23 embryos, while M. bovigenitalium was isolated from 25 of 25 and 22 of 22 embryos after antibiotic and trypsin treatment, respectively. It was concluded that neither of the standard procedures currently used for cleansing embryos should be relied upon for insuring freedom from mycoplasmas.     

Isolation of Actinomycetales from the soil of Kazakhstan on selective media with antibiotics

About 3000 actinomycetes were isolated from various soil samples collected in 11 regions of Kazakhstan. 62.7 per cent of them proved to be antagonists. For isolation of the strains, selective media supplemented with antibiotics were used. Kanamycin promoted growth of Actinomadura and Nocardia. Rubomycin promoted growth of Actinomadura. Tavromycetin and roseofungin were used as selective agents for the first time. Tavromycetin favoured isolation of Actinomadura and Nocardia. Roseofungin favoured isolation of Actinomadura. Light chestnut and serozemic soils were the most rich in antagonists (67.1 and 61.3 per cent, respectively) while saline and chestnut soils were the poorest in antagonists (32.2 and 30.6 per cent, respectively). Actinomadura were more frequent in light-chestnut light-loamy and serozemic soils. Half of the antibiotics isolated in the form of concentrates were identified with the known antibiotics or classified as belonging to various groups. A culture producing a novel antibiotic was isolated.     

Circular DNA in human and boar spermatozoa

Circular DNA molecules were isolated from human and boar whole spermatozoa or spermatozoal nuclei and measured for size by electron microscopy. The DNA molecules derived from both mammals were heterogeneous in size ranging from 0.07 to 17 μm; nearly 75% of the molecules were ?0.5 μm in length. The mean lengths were 1.0 μm and 1.5 μm for circular DNAs isolated from human and boar spermatozoa, respectively. The origin and function of these molecules remains unknown.     

Measuring mitochondrial respiration in intact single muscle fibers

Measurement of mitochondrial function in skeletal muscle is a vital tool for understanding regulation of cellular bioenergetics. Currently, a number of different experimental approaches are employed to quantify mitochondrial function, with each involving either mechanically or chemically induced disruption of cellular membranes. Here, we describe a novel approach that allows for the quantification of substrate-induced mitochondria-driven oxygen consumption in intact single skeletal muscle fibers isolated from adult mice. Specifically, we isolated intact muscle fibers from the flexor digitorum brevis muscle and placed the fibers in culture conditions overnight. We then quantified oxygen consumption rates using a highly sensitive microplate format. Peak oxygen consumption rates were significantly increased by 3.4-fold and 2.9-fold by simultaneous stimulation with the uncoupling agent, carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP), and/or pyruvate or palmitate exposure, respectively. However, when calculating the total oxygen consumed over the entire treatment, palmitate exposure resulted in significantly more oxygen consumption compared with pyruvate. Further, as proof of principle for the procedure, we isolated fibers from the mdx mouse model, which has known mitochondrial deficits. We found significant reductions in initial and peak oxygen consumption of 51% and 61% compared with fibers isolated from the wild-type (WT) animals, respectively. In addition, we determined that fibers isolated from mdx mice exhibited less total oxygen consumption in response to the FCCP + pyruvate stimulation compared with the WT mice. This novel approach allows the user to make mitochondria-specific measures in a nondisrupted muscle fiber that has been isolated from a whole muscle.     

何首乌总RNA提取方法的比较及改进

目的：探讨不同提取方法对提取何首乌总RNA的质量影响,寻找适于何首乌成熟叶组织RNA的提取方法。方法：以何首乌成熟叶组织为材料,采用SDS／酸酚法、常规CTAB法及改良的TRIzol试剂法分别进行实验,并对所提取RNA的质量进行验证。结果：采用3种方法都能提取出RNA,但质量差异较大。其中改良的TRIzol试剂法能有效抑制次生物质的影响,提取的RNA产量可达70-110μg／g,纯度高于其他2种方法,D260nm／D280nm值为1．85～1．97。结论：改良的TRIzol试剂法操作简便,提取的RNA完整性和纯度较高,可以满足下一步实验的要求。