甘草内生真菌分离及其抑菌活性初探

为了解药用植物甘草(Glycyrrhiza uralensis)内生真菌资源多样性及其抑菌活性特征,通过组织块法对内生真菌进行分离,并选择小麦全蚀病菌（Gaeumannomyces graminis var.tritici）、枸杞黑果病菌（Colletotrichum gloeosporioides）、番茄灰霉病菌（Botrytis cinerea）、黄瓜枯萎病菌（Fusarium oxysporium f.sp.cucumeris）、黄瓜立枯病菌（Rhizoctonia solani）5种植物病原真菌和枯草芽孢杆菌（Bacillus subtilis）、大肠杆菌（Escherichia coli）、金黄色葡萄球菌（Staphylococcus aureus）、铜绿假单胞菌（Pseudomonas aeruginosa）4种细菌作为供试指示菌,采用对峙法和改进的菌块法测定抑菌活性。试验结果显示,从甘草根、茎、叶中分离出20株内生真菌,其中根部最多,占分离菌株总数的65.0%,其次为茎部,叶部最少;经形态学初步分类鉴定归于2目2科5属,梭孢霉属为优势菌属,占分离菌株总数的70.0%;在分离的内生真菌中有15株菌对1种或1种以上供试植物病原真菌有不同程度的抑制作用,为分离内生真菌总数的75.0%,19株菌对1种或1种以上供试细菌有不同程度的抑制作用,为分离内生真菌总数的95.0%;有7株内生真菌对枯草芽孢杆菌拮抗活性差异显著（P＜0.05）,有 8株内生真菌对金黄色葡萄球菌拮抗活性差异显著,有3株内生真菌对大肠杆菌拮抗活性差异显著,有1株内生真菌对革兰氏阴性铜绿假单胞菌拮抗活性差异显著,其中菌株RLEFR015对番茄灰霉病菌、金黄色葡萄球菌拮抗活性差异显著,菌株RLEFR010对大肠杆菌、金黄色葡萄球菌拮抗活性差异显著,菌株RLEFR002对枯草芽孢杆菌、金黄色葡萄球菌供试细菌拮抗活性差异显著;菌株RLEFR015、RLEFR002、RLEFR010均为梭孢霉属,为高活性菌株。甘草内生真菌具有多样性和抑菌活性,多数菌株对供试病原真菌和病原细菌具有拮抗活性,对革兰氏阳性金黄色葡萄球菌和革兰氏阳性枯草芽孢杆菌拮抗活性较强,预示着甘草内生真菌具有重要的资源价值。     

枯草芽孢杆菌MA139类细菌素抑菌活性的研究

为测定枯草芽孢杆菌MA139产生的类细菌素的抑菌活性。本试验采用管碟法对枯草芽孢杆菌MA139产生的类细菌素对多株革兰氏阳性菌、革兰氏阴性菌和真菌的抑菌活性进行测定。结果表明:类细菌素不但对芽孢杆菌属的其他菌种有抗菌活性,对病原菌和霉菌也有拮抗作用;不同指示菌对类细菌素的敏感性不一样,其中金黄色葡萄球菌对其最敏感。该类细菌素具有广谱抗菌作用,有作为畜禽饲料添加剂的潜力。     

An agar diffusion method for the determination of antibodies against Staphylococcus aureus deoxyribonuclease.

On the addition of small concentrations of deoxyribonuclease, produced by Staphylococcus aureus, to Toluidine Blue DNA agar, a medium is produced on which antibodies against S. aureus deoxyribonuclease may be detected. When samples of milk, or blood serum, containing antibodies against S. aureus are applied into wells in the agar, the deoxyribonuclease activity is inhibited by the antibodies diffusing into the agar. As a result of this inhibition, blue zones are produced around the wells in the otherwise bluish-red agar. The diameters of the zones correspond to the concentrations of antibodies, and the method may consequently be used for qualitative and quantitative examinations of antibodies against S. aureus deoxyribonuclease in milk and serum. The procedure and certain limitations of the method are described.     

桑叶不同极性溶剂提取物的总多酚含量与抑菌活性

用不同极性溶剂萃取桑叶粉中的活性物质,分别获得乙酸乙酯提取物、正丁醇提取物和水提取物。采用琼脂打孔扩散法评价桑叶各极性溶剂提取物的抑菌效果,结果表明桑叶不同极性溶剂提取物对供试革兰阳性菌和革兰阴性菌均有一定的抑制作用,其中乙酸乙酯提取物的抑菌活性显著高于其它溶剂提取物,对大肠杆菌(Escherichia coli)的最低抑菌浓度为6.3mg/mL,对金黄色葡萄球菌(Staphyloccocus aureus)、枯草芽孢杆菌(Bacillus subtilis)、沙门氏菌(Salmonella sp.)的最低抑菌浓度均为12.5 mg/mL。测定乙酸乙酯提取物中的总多酚质量比为(11.91±0.67)mg/g,显著高于其它溶剂提取物,表明桑叶不同极性溶剂提取物的抑菌活性与其总多酚的含量成显著剂量效应关系(P<0.05)。     

Occurrence of staphylococcal species in clinically healthy domestic animals

The incidence of staphylococcus species in healthy animals was investigated in young and adult individuals of cattle, in pigs and in domestic fowl, using the method of selective isolation of strains. A total of 6066 samples was examined; 4567 strains were revealed and they included all known species, except Staphylococcus caseolyticus, S. saccharolyticus, S. schleiferi and S. lugdunensis. 3.8% of strains failed to be identified with any species. The test samples were taken from slaughtered animals, only in calves intravital smears of tonsils were examined. The species most frequently isolated from the tonsil tissue in adult cattle were as follows: S. aureus (19.9%), S. saprophyticus (12.4%), S. hyicus (8.8%) and S. hominis (8.6%). The following species were isolated from the lung tissue: S. saprophyticus (28.8%), S. cohnii (10.6%) and S. epidermidis (10.2%); from the mammary gland parenchyma these were the species S. saprophyticus (17.4%), S. xylosus (13.1%) and S. epidermidis (10%). In the tonsil tissue of pigs the percent proportions of S. aureus and S. hyicus were 44.5% and 31.8%, respectively; the most frequent species detected from the lungs were S. aureus (20.6%), S. saprophyticus (12.3%), S. hyicus (12.1%) and S. epidermidis (11.5%). In domestic fowl the most frequently occurring species were S. epidermidis (23.7%), S. gallinarum (16.5%) and S. aureus (15.4%). In calves the incidence of S. xylosus (37%), S. saprophyticus (19.2%) and S. cohnii (15.2%) was found to be highest. The indicence of the other staphylococcus species in all test animals can be expressed by low percentages.     

Comparison of Different Agar Diffusion Methods for the Detection of Antimicrobial Residues in Slaughter Animals

The different agar diffusion methods were compared using antibiotic and sulphonamide-impregnated filter-paper discs and the kidneys of healthy and emergency-slaughtered pigs and cows after slaughter.No method used seemed to be sensitive to all antimicrobial drugs preimpregnated onto discs. Tetracycline yielded a greater zone of inhibition at pH 6 than at pH 8 and aminoglycosides, erythromycin, polymyxin B and lin cornycin at pH 8 than at pH 6. It therefore seems necessary to use different pHs (6 and 8). The addition of trimethoprim to the medium is necessary for the detection of sulphonamides. Bacillus subtilis BGA used as the test organism was more sensitive to sulphonamides on the “Test agar for the inhibitor test” containing trimethoprim than on the “Iso-Sensitest agar” also containing trimethoprim. The addition of trimethoprim to “Test agar for the inhibitor test” is recommended at pH 8 but not at pH 6 because false-positive cases (with inhibition zones > 2 mm) were observed at pH 6 with trimethoprim on the kidneys of healthy pigs.     

The effect of culture media on bacteriocin production in various strains of bacteria]

Some staphylococcus and enterococcus strains were used to investigate the effect of culture medium on bacteriocin production. Staphylococcus cohnii SC7, Staphylococcus sp. ZTJ 151, S. saprophyticus SS 877, Enterococcus faecium EF1 and E. faecalis EFG2 were isolated from the rumen wall and contents of lambs, calves and fallow deer, Enterococcus gallinarum EG10 and E. avium EA12 were isolated from the caecum of Japanese quail. The tested bacteria belong to producers with a wide antimicrobial effectiveness spectrum, they have low to medium adherence and urease activity (Tab. III). These culture media were used to test the effect of culture medium on bacteriocin production: nutrient agar no. 2 and VL agar enriched with 2% of glucose and lactose (ZAG, ZAL, VLG, VLL), agar for isolation of faecal streptococci (SA) and the base for blood agar no. 4 and no. 2 (KA4, KA2). The strains Streptococcus bovis AO 24/85 and Staphylococcus aureus Oxford 209 P were used as indicator bacteria. Tables I and II show the results of these tests. The tested strains produced the widest inhibition zones (6 mm) with both indicators on SA medium, and this indicates massive bacteriocin production. On ZAG medium, the zones of enterococci with the AO 24/85 strain were larger size than those of staphylococci, but the zones were dim. All strains with the 209P indicator produced dim zones of the 2mm size. The larger inhibition zones (2-5mm) in comparison with staphylococci were observed in enterococci on the ZAL medium with the AO24/85 strain. The production of tested strains was balanced on VLG agar with respect to the use of both indicators.(ABSTRACT TRUNCATED AT 250 WORDS)     

Bioelectrophoresis: a rapid procedure for the identification of antimicrobial agents

Sensitivity discs were placed on 1% Noble agar for electrophoresis in a 20 mM phosphate-buffered, pH 8.5-8.8 system. The discs were removed after electrophoresis, and the agar was overlaid with Bacillus subtilis spores in Mueller-Hinton agar. After incubation at 37 C, each individual antimicrobial agent produced a distinctive identifying pattern of B. subtilis growth inhibition. The few antimicrobial agents with similar patterns could be easily differentiated by the use of a strain of Escherichia coli containing a multiple-resistance factor. Filter discs impregnated with commercial antibiotic preparations in buffer or serum yielded growth-inhibition patterns which usually resembled those obtained from corresponding sensitivity discs.     

Examination of market carps for residues of antimicrobial agents]

The residues of inhibitory substances in the muscle, hepatopancreas, bile, and pyloric caeca of 30 carps from ponds south of Brno were examined by the microbiological diffusion method. The following testing micro-organisms were used: B. cereus var. mycoides (ATCC 11778), B. subtilis (ATCC 6633), Sarcina lutea (ATCC 9341), and Micrococcus flavus (ATCC 10240). Residues of inhibitory substances were not found in any of the muscle samples tested. The rest of the tested tissues showed, in some cases, small inhibition zones which seem to be due to the presence of natural antimicrobially active substances in the bile and gastro-intestinal tract of the fish rather than to the persistence of the residues of drugs given to the fish during their life.     

Residues of inhibitory agents in the tissues of slaughter-house animals--comparison of microbiological methods of agar diffusion

Three microbiological methods of agar diffusion were compared which are used to detect the residues of inhibitory substances: method using the strain Bacillus subtilis ATCC 6633 (B.s. 6633), method using the strain Bacillus stearothermophilus v. calidolactis C 953 (B. s. v. c. 953), and four-plate method. Using the compared methods, minimum inhibitory concentrations were determined for standard solutions of antibiotics and sulphadimidine. Inhibitory substances were detected parallely in the samples of the tissues of medicated and emergency-slaughtered animals, applying the three compared methods. In indicated cases inhibitory substances were identified by electrophoresis and chemical methods. The results indicate the following: 1) The B.s. 6633 method is the least sensitive of all to the residues of inhibitory substances. 2) For the purposes of detection of most antibiotics under investigation, the B.s.v.c. 953 method and the four-plate method are replaceable. The B.s.v.c. 953 method is more suitable for the detection of penicillin residues, in the case of tetracycline antibiotics it is the four-plate method. 3) Sulphadimidine residues can be detected only by the four-plate method. 4) The four-plate method enables to make the preliminary group identification of antibiotics and sulphonamides. 5) The detection limit of microbiological methods is not sufficiently sensitive to determine chloramphenicol residues; that is why the physico-chemical methods must be used.     

Effect of pooling bovine fecal samples on the sensitivity of detection of E. coli O157:H7

To assess the effect of pooling fecal samples on the sensitivity of detection of E. coli O157:H7, 12 calves, inoculated orally with 10(8)cfu per calf of nalidixic acid resistant E. coli O157:H7, were used to provide positive fecal samples. After inoculation, calves were sampled twice weekly. Negative fecal samples were from calves at a local dairy. Samples from inoculated calves were incubated without pooling or were mixed with known negative fecal samples in a 1:4 ratio or a 2:3 ratio (positive:negative) for detection of E. coli O157:H7. Samples were enriched 6h in Gram negative broth with vancomycin, cefixime, and cefsoludin, underwent immunomagnetic separation with Dynabeads, and were plated onto sorbitol MacConkey agar with cefixime, and tellurite (SMACct). Morphologically typical colonies were plated onto blood agar, incubated overnight at 37 degrees C and an indole test was performed on each colony. Indole positives colonies were plated on SMAC agar with 20 microg/ml nalidixic acid (SMACnal). Colonies that grew on SMACnal were confirmed by O157 agglutination. Sensitivity of detection in non-pooled samples was 77%. Samples pooled 1:4 and 2:3 with negative samples were 55 and 52% sensitive, respectively. Pooling decreased sensitivity of detection for E. coli O157:H7 in bovine fecal samples (P<0.01). A deterministic binomial probability model was developed to assess the probability of detecting pens of cattle shedding E. coli O157 using a pooling protocol or individual samples. Pooling decreased sensitivity of detection at low pen prevalence compared to individual samples but was similar at high prevalence.     

Clinical efficacy of local administration of ceftiofur in a Staphylococcus aureus infection in tissue cages in ponies

Ceftiofur concentrations in an infected and uninfected environment were compared and the efficacy of locally administered ceftiofur was evaluated in an experimental infection with Staphylococcus aureus in tissue cages. Eight ponies had tissue cages (TCs) implanted s.c. on each side of the neck. Into one of the cages 150 mg of ceftiofur was administered and fluid samples were taken to determine ceftiofur concentrations. After 1 week the other TC was infected with S. aureus and subsequently treated with 150 mg ceftiofur administered locally into the TC once daily for 21 days. Samples of fluid were taken to determine ceftiofur concentrations and for bacterial counts. Ceftiofur concentrations did not differ significantly in the infected and uninfected environments after single dose of 150 mg of ceftiofur. Concentrations were considerably in excess of the minimum inhibitory concentration (MIC) of the S. aureus strain used. A marked decrease of viable bacteria in tissue cage fluid (TCF) occurred. In five of seven ponies; however, the infection was not eliminated and abscess formation occurred. Therefore, local application of ceftiofur alone is not advisable for infections with S. aureus in secluded sites in horses, but should be used only with adjunctive therapy.     

东亚飞蝗抗菌活性物质的萃取及抗菌效应研究

试验研究东亚飞蝗成虫抗菌活性物质的萃取参数与抑菌效应。采用针刺蘸大肠杆菌菌悬液方法,以抗菌活性为指标,用杯碟法测定临床多重耐药菌的抑菌活性,确定最佳诱导时间,初步研究抗菌谱。结果表明,东亚飞蝗抗菌活性物质粗提液对革兰氏阳性菌(如金黄色葡萄球菌、枯草芽孢杆菌)或革兰氏阴性菌(如大肠杆菌、水稻白叶枯病原菌)均有一定的抑制作用。其抗菌谱为:金黄色葡萄球>水稻白叶枯病原菌>枯草芽孢杆菌>大肠杆菌,其中以对金黄色葡萄球菌的抑菌效果最明显。抗菌活性物质浓度为11.782mg/mL,蝗虫抗菌活性物质收率为8.623%。经纯化后的抗菌活性物质增强对革兰氏阳性菌或革兰氏阴性菌的抑制作用。采用针刺蘸大肠杆菌方式可成功诱导东亚飞蝗抗菌物质的表达,尤其以针刺蘸大肠杆菌诱导后饲养24h得到的抗菌活性物质抑菌活性最强。结果表明东亚飞蝗虫体内含有抗菌活性物质可被诱导表达,其抗菌作用机理有进一步研究。     

Isolation of Staphylococcus aureus from raw fish in relation to culture methods

Five hundred and fifty fish samples from various stages in the course of distribution in Hyogo Prefecture (209 retailed in super markets, 173 obtained from fishery cooperatives at a harbor, 91 caught by trawling and 77 caught by rod fishing) were examined for contamination with Staphylococcus aureus (S. aureus). S. aureus was detected in 41 (19.6%) of the retail fish samples and 46 (26.6%) of the samples from the fishery cooperatives. No S. aureus was isolated from the live fish (91 trawled and 77 fished by rod). With regard to the retail fish, the contamination rate of processed fish (26.0%) was significantly higher than that of unprocessed fish (14.2%). For 88 samples, the efficacy of the selective medium was compared using Baird-Parker agar and mannitol salt agar supplemented with egg yolk (MSEY agar) by the direct plate and enrichment culture methods. Using the direct culture method, the S. aureus positive rate with the Baird-Parker agar (30.7%) was significantly higher (P<0.01) than that with the MSEY agar (6.8%). The enrichment culture method remarkably raised the S. aureus detection rate. Seventy-eight (85.7%) of 91 isolates belonged to the human ecovar. Sixty-two (68.1%) of the 91 isolates had some enterotoxin genes, including 44 (48.4%) with the sea gene. These data showed that the fish were contaminated with S. aureus after landing and that Baird-Parker agar had an advantage in detecting S. aureus with a direct plate culture.     

Prevalence, antibiotic resistance and molecular characterisation of Staphylococcus aureus in pigs at agricultural fairs in the USA

Fairs and petting zoos have been associated with outbreaks of zoonotic disease. Previously, the presence of meticillin-resistant Staphylococcus aureus (MRSA) was documented in commercial pigs; therefore, it was hypothesised that antibiotic-resistant S aureus may also occur in pigs exhibited at agricultural fairs. To test this hypothesis, 157 pigs were swabbed at two state fairs in 2008 to 2009. Both nares were sampled and cultures were grown in enrichment broth, then plated onto selective MRSA plates and blood plates. S aureus was confirmed using phenotypic and molecular methods, and was analysed using spa typing, gene-specific polymerase chain reaction and antibiotic susceptibility testing. The presence of S aureus was confirmed in samples collected from pigs exhibited at USA pig shows. Twenty-five of 157 (15.9 per cent) samples were positive for S aureus. Two isolates (8 per cent) were resistant to meticillin; 23/25 (92 per cent), 14/25 (56 per cent) and 15/25 (60 per cent) were resistant to tetracycline, erythromycin and clindamycin, respectively. spa typing revealed multiple isolates of spa type t034 (9/25, 36 per cent) and t337 (7/25, 28 per cent) and singletons of t002, t209, t526, t1236, t1334, t1683, t3075, t5784 and t5883. These results verify the presence of antibiotic-resistant S aureus in pigs exhibited at USA fairs, suggesting that pigs are a potential reservoir for S aureus within this environment.     

10种中药提取物对耐药金黄色葡萄球菌抑菌作用的研究

为了解研究中药提取物对耐药菌的抑菌作用,采用纸片法和倍比稀释法研究10种中药水提取液对耐氟喹诺酮类药物金黄色葡萄球菌的体外抑菌作用;用点种法和药敏纸片法研究耐药抑转作用。结果显示,10种中药水提物中黄芩水提取物对耐氟喹诺酮类药物金黄色葡萄球菌抑制效果最明显。耐药性抑制试验结果表明,只有黄芩水提取物对Sa1的耐药性有明显的抑制作用,抑制率达65%,但对其他菌株的抑制作用则表现不明显;药敏纸片法显示黄芩对细菌耐药性有最强抑制作用。表明中药水提取物对耐氟喹诺酮类药物金黄色葡萄球菌有抑制作用,且部分药物抑菌作用明显;黄芩水提取物对Sa1菌株具有耐药逆转作用。     

艾叶提取液体外抑菌及耐药抑制作用研究

目的：探讨艾叶提取液对耐药性金黄色葡萄球菌的体外抑菌作用及耐药抑制作用。方法：采用中药药敏纸片法、微量稀释法、琼脂打孔培养法和抗菌药药敏纸片法考察艾叶提取液对耐药性金黄色葡萄球菌的体外抑菌及耐药抑制作用。结果：艾叶提取液对耐药性金黄色葡萄球菌有良好的体外抑菌作用。耐药逆转试验结果表明,艾叶提取液对耐药性金黄色葡萄球菌有一定的耐药逆转作用。结论：艾叶提取液对耐药金黄色葡萄球菌有明显的抑菌和耐药逆转作用。     

Protection activity of a novel probiotic strain of Bacillus subtilis against Salmonella Enteritidis infection

The activity of 240 bacterial isolates screened from the gastrointestinal tracts of native chickens were evaluated for use as a potential probiotic in food animal production in order to protect against animal diseases and reduce pathogenic contamination of human food products. In observing the antagonistic activity of 117 bacilli isolates, 10 of these isolates exhibited higher growth inhibition of seven foodborne pathogens, including Salmonella Enteritidis, Salmonella Typhimurium, Escherichia coli, Bacillus cereus, Staphylococcus aureus, Listeria monocytogenes, and Vibrio cholerae. Beneficial probiotic criteria from these isolates - which included non-pathogenicity, acid and bile salt tolerance, hydrophobicity, and adhesion to intestinal epithelial cells - exhibited that one isolate of NC11 had the most potential as a probiotic. 16S rRNA gene sequencing showed that this NC11 isolate was Bacillus subtilis. This B. subtilis NC11 was sensitive to all antibiotics and was not cytotoxic to intestinal epithelial cells. Reduction of S. Enteritidis attachment to the surfaces of intestinal epithelial cells via action of a cultured medium from B. subtilis NC11 was observed by scanning electron microscopy. B. subtilis NC11 cells, as well as the bacterial cultured medium or the cultured medium adjusted to pH 7, significantly inhibited S. Enteritidis invasion (P<0.01) of intestinal epithelial cells. This study indicates that B. subtilis NC11 has characteristics of a potential probiotic, and exhibits strong inhibition activity against S. Enteritidis infection to intestinal epithelial cells.     

Deoxyribonuclease inhibitory activity in pig intestinal contents

The inhibitory effect of pig intestinal contents on some microbial DNases and bovine pancreas DNase was examined employing an agar diffusion test. The molecular weight of 1 inhibitor as well as the electrophoretic patterns of the inhibitors were determined.DNases from Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pyogenes, Clostridium perfringens and bovine pancreas were inhibited by intestinal contents. DNases from Clostridium septicum, Serratia marcescens, Proteus mirabilis, Aeromonas hydropnila and Pseudomonas aeruginosa were not inhibited. The concentrations of the inhibitory substances were considerably higher in contents from the small intestine than from the large intestine.Using electrophoretic procedures on intestinal contents, 3 different fractions showing DNase inhibition were demonstrated. The molecular weight of one of the inhibitors was estimated to be about 30000.     

A comparison of selected public health criteria in milk from milk-shops and from a national distributor

Selected public health criteria of pasteurised milk available to the consumer from milk-shops in a pre-defined area of Pretoria compared with a national distributor's milk was evaluated. Of the 135 milk samples purchased from milk-shops, 87% were not fit for human consumption on the basis of the minimum standards prescribed in the Foodstuffs, Cosmetics and Disinfectants Act, 1972 (Act 54 of1972). The national distributor's milk (n = 79) did not contain any pathogens, toxins nor inhibitory substances and passed all the criteria laid down in the Act. Even though milk-shop milk was sold as having been pasteurised, 38.5% of samples were alkaline phosphatase positive, indicating probable inadequate pasteurisation. Milk-shop milk quality varied between milk-shops and between sampling days and differed significantly (P < 0.05) from the national distributor's milk. Total aerobic plate and coliform counts were generally high for all milk-shop milk samples. Somatic cell counts of milk-shop milk differed significantly (P < 0.05) from the national distributor's milk. Escherichia coli was detected in 1 ml of 17% of milk-shop milk, 95% of which originated from milk which was alkaline phosphatase positive. Salmonella spp. could not be detected in 1 ml in any of the E. coli-positive milk tested. Staphylococcus aureus was isolated from 40% of milk-shop milk samples, and S. aureus enterotoxins from 7.8% of 51 cultures. Inhibitory substances were detected in 54.1% of milk-shop milk. The presence of inhibitory substances and the isolation of E. coli and S. aureus (some of which were able to produce enterotoxins) indicated potentially unsafe milk and poses a serious public health risk to consumers.