沙门氏菌快速增菌培养的实验研究

用改良的沙门氏菌增菌培养基与02快速增菌培养基,比较研究了鼠伤寒沙门氏菌、伤寒沙门氏菌、甲型副伤寒沙门氏菌、乙型副伤寒沙门氏菌、猪霍乱沙门氏菌及纽因吞沙门氏菌的增殖能力。以OD值测定菌量,改良法较02法增长1.43—3.97倍。尤其是对鼠伤寒沙门氏菌和伤寒沙门氏菌的菌量增殖较多,生长速度较快,分别为02法的3.97和3.7倍。改良法可适合于这两种菌的增殖培养,其次是纽因吞沙门氏菌和乙型副伤寒沙门氏菌的培养,但对甲型副伤寒沙门氏菌和猪霍乱沙门氏菌的增长欠佳。改良的增菌培养基尚有抑制葡萄球菌和大肠杆菌生长的作     

沙门氏菌增菌培养基中细菌混合培养生长动力学的试验研究

本文就甘露醇亚硒酸盐和亚硒酸盐两种增菌培养基对沙门氏菌各血清变型的选择性增菌作用作了研究。研究述及37℃下沙门氏菌与埃希氏大肠杆菌、绿脓杆菌、普通变形杆菌三种竞争菌混合培养生长动力学过程,测出了评价增菌培养基优劣的客观指标EI值;按沙门氏菌传统分离方法,对加有终浓度为10~1、10~2、10~4、10~6、10~8个活菌/毫升的沙门氏菌株与正常人大便的两种增菌培养基,经37℃下孵育20至24小时后作SS琼脂平板划线分离和沙门氏菌血清学鉴定。通过两种培养基EI值及沙门氏菌分离结果的比较证实,甘露醇亚硒酸盐培养基对沙门氏菌株的选择性增菌作用明显优越于亚硒酸盐培养基(P<0.001)。     

酪酸菌对动物肠道致病菌体外拮抗作用的研究

探讨了酪酸菌对三种畜禽肠道致病菌的体外拮抗作用。将酪酸菌分别和猪大肠杆菌、鸡大肠杆菌、鸡白痢沙门氏菌三种畜禽肠道致病菌按不同的比例混合接种于CL液体培养基中进行厌氧培养,培养过程中三种致病菌的活菌数急剧下降,30h后的菌落数都逐渐降为零。说明酪酸菌在体外实验中对上述三种畜禽肠道致病菌都有较强的拮抗作用,其中酪酸菌对猪大肠杆菌、鸡大肠杆菌和鸡白痢沙门氏菌的最佳接种比例分别为：10：1～100：1、10：1～100：1和5：1～10：1。     

大肠杆菌增殖培养方法

本文介绍一种培养大肠杆菌的新方法：在普通肉汤和普通琼脂培养基中加入一定量的乳糖,可明显地促进大肠杆菌（E．coli）增殖。用平板计数法、麦氏比浊法及离心称重法,对6株禽源E．coli和2株猪源E．coli培养产物的含菌量、菌泥湿重测定,结果表明：在普通肉汤中加乳糖可使E．coli产量增加约100％;在普通琼脂中,加乳糖可使E．coli产量增加200％以上。本法可用于菌苗的生产和分子生物学中,具有经济、简单的特点。     

北京鸡传染性鼻炎病原菌分离及鉴定

北京A公司病鸡,根据其临床症状的观察和对病鸡眶下窦渗出物分离菌的形态、培养特性和生化反应,血清学特片和致病力及交叉免疫试验,表明病鸡为传染鼻炎,其病原菌是page A型副鸡嗜血杆菌(Hamophilious paragallinarum)。     

不同增菌和培养条件下沙门氏菌检出效果的比较

通过在鸡精样本中添加低含量的鼠伤寒沙门氏菌和干扰菌(弗氏柠檬酸杆菌和奇异变形杆菌),参考国标和美国药典中沙门氏菌的定性方法,研究比较了3种选择性增菌液、4个培养时间段以及4种选择性平板对于沙门氏菌的不同检出效果。结果表明,在RV肉汤及四硫磺酸钠煌绿增菌液(TTB)中培养18 h～20 h,使用沙门显色培养基及亚硫酸铋琼脂(BS平板)进行选择性培养沙门氏菌的检出效果最好。     

培养基及培养工艺对双歧杆菌产量的影响

目的：对双歧杆菌生产培养基进行筛选,提高双歧杆菌的产量。方法：使用保蒲培养基,采用发酵罐培养工艺。结果：使用保蒲培养基较西红柿原汁培养基可使双歧杆菌的产量提高3.06-5.36倍。用保蒲培养基采用发酵罐培养工艺较立瓶静止培养工艺双歧杆菌的产量可提高4.91-54.8倍;发酵罐培养工艺较用西红柿原汁培养基、立瓶培养工艺产量提高17.33-154.29倍。结论：用保蒲培养基发酵罐培养可大大提高双歧杆菌的产量。     

细菌对肉鸡肠粘液的粘附作用

研究两歧双歧杆菌、嗜酸乳杆菌、禽大肠杆菌O78、大肠杆菌 ATCC 25922、鸡白痢沙门氏菌和鼠伤寒沙门氏菌与肉鸡不同部位肠粘液糖蛋白的粘附性能,探讨两歧双歧杆菌和嗜酸乳杆菌对所试病原菌的抗粘附作用。结果表明：在不同的肠道部位,两歧双歧杆菌、嗜酸乳杆菌、鸡白痢沙门氏菌和鼠伤寒沙门氏菌与肠粘液糖蛋白均有不同的粘附作用,而禽大肠杆菌O78、大肠杆菌 ATCC 25922在各肠段粘液上的粘附性能则相近;在相同的肠道部位,所试益生菌的粘附能力大于病原菌;两歧双歧杆菌和嗜酸乳杆菌对所试病原菌的粘附有不同的阻断作用,同时二者有时还存在互补抗粘附作用。     

青少年牙周炎细菌病因学研究

应用选择和非选择性培养基的方法,采集了43名青少年牙周炎患者,31名龈炎患者和13名牙周健康者的303个龈下菌斑标本进行了放线共生嗜血杆菌、嗜二氧化碳噬纤维菌和产黑色素类杆菌的分离培养。结果表明:放线共生嗜血杆菌、嗜二氧化碳噬纤维菌在青少年牙周炎组的检出率显著高于其它     

选择性富集沙门氏菌、副溶血弧菌和霍乱弧菌的共增菌培养基SVV

本研究通过单因素试验和响应面分析试验建立了能够选择性富集沙门氏菌、副溶血弧菌和霍乱弧菌的共增菌培养基SVV,采用平板计数法及三重荧光PCR技术验证了SVV的增菌效果。结果表明:SVV能同时富集以不同浓度比例混合的3种目标菌,37oC振荡培养18h后,菌体浓度达到105~108CFU/mL;SVV强烈抑制大部分的非目标菌;用荧光PCR方法检测经过37oC振荡培养18h的10份人工接种样品和608份实际样品,结果表明目标菌在SVV中增殖18h后菌量达到检测限以上,SVV联合荧光PCR检测方法的检出率为4.06%,比传统检测方法(3.78%)高,无假阴性和假阳性。SVV可望应用于水产品中沙门氏菌、副溶血弧菌和霍乱弧菌检测前的增菌处理,可简化检测过程,有效克服漏检,提高检出率。     

In vitro antibacterial activity of laboratory grown culture of Spirulina platensis

The hexane, ethyl acetate, dichloromethane, methanol extracts and spent media (extracellular substances) were tested in vitro for their antibacterial activity for which one Gram-positive bacterium (Staphylococcus aureus) and four Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa, Salmonella typhi, and Klebsiella pneumoniae) were used as test organisms. The methanol extract showed more potent activity than other organic extracts, spent medium of the culture exhibited little activity against E. coli only. No inhibitory effect was found against Klebsiella pneumoniae.The broth microdilution assay gave minimum inhibitory concentrations (MIC) values ranging from 1 to 512 μg/ml. The MIC of methanol extract against S. aureus and E. coli were 128 μg/ml and 256 μg/ml, respectively.     

Use of bioluminescent Escherichia coli O157:H7 to study intra-protozoan survival of bacteria within Tetrahymena pyriformis

A method was developed that enabled real-time monitoring of the uptake and survival of bioluminescent Escherichia coli O157 within the freshwater ciliate Tetrahymena pyriformis. Constitutively bioluminescent E. coli O157 pLITE27 was cocultured with T. pyriformis in nutrient-deficient (Chalkley's) and in nutrient-rich (proteose peptone, yeast extract) media. Non-internalised bacteria were inactivated by addition of colistin, indicated by a decline in bioluminescence. Protozoa were subsequently lysed with Triton X-100 which lead to a further drop in bioluminescence, consistent with release of live internal bacteria from T. pyriformis into the colistin-containing environment. Bioluminescence measurements for non-lysed cultures indicated that internalised E. coli O157 pLITE27 cells were only slowly digested by T. pyriformis, in both media, over the time period studied. The results suggest that bioluminescent bacteria are useful tools in the study of bacterial intra-protozoan survival.     

The probiotic Escherichia coli strain Nissle 1917 interferes with invasion of human intestinal epithelial cells by different enteroinvasive bacterial pathogens

The probiotic Escherichia coli strain Nissle 1917 (Mutaflor) of serotype O6:K5:H1 was reported to protect gnotobiotic piglets from infection with Salmonella enterica serovar Typhimurium. An important virulence property of Salmonella is invasion of host epithelial cells. Therefore, we tested for interference of E. coli strain Nissle 1917 with Salmonella invasion of INT407 cells. Simultaneous administration of E. coli strain Nissle 1917 and Salmonella resulted in up to 70% reduction of Salmonella invasion efficiency. Furthermore, invasion of Yersinia enterocolitica, Shigella flexneri, Legionella pneumophila and even of Listeria monocytogenes were inhibited by the probiotic E. coli strain Nissle 1917 without affecting the viability of the invasive bacteria. The observed inhibition of invasion was not due to the production of microcins by the Nissle 1917 strain because its isogenic microcin-negative mutant SK22D was as effective as the parent strain. Reduced invasion rates were also achieved if strain Nissle 1917 was separated from the invasive bacteria as well as from the INT407 monolayer by a membrane non-permeable for bacteria. We conclude E. coli Nissle 1917 to interfere with bacterial invasion of INT407 cells via a secreted component and not relying on direct physical contact with either the invasive bacteria or the epithelial cells.     

Evolution of the ferric enterobactin receptor in gram-negative bacteria.

Using sodium dodecyl sulfate-polyacrylamide gel electrophoresis of iron-deficient and replete cell envelopes, 59Fe-siderophore uptake studies, and Western immunoblots and cytofluorimetric analyses with monoclonal antibodies (MAbs), we surveyed a panel of gram-negative bacteria to identify outer membrane proteins that are structurally related to the Escherichia coli K-12 ferric enterobactin receptor, FepA. Antibodies within the panel identified FepA epitopes that are conserved among the majority of the bacteria tested, as well as epitopes present in only a few of the strains. In general, epitopes of FepA that are buried in the outer membrane bilayer were more conserved among gram-negative bacteria than epitopes that are exposed on the bacterial cell surface. The surface topology and tertiary structure of FepA are quite similar in E. coli and Shigella flexneri but differ in Salmonella typhimurium. Of the 18 different genera tested, 94% of the bacteria transported ferric enterobactin, including members of the previously unrecognized genera Citrobacter, Edwardsiella, Enterobacter, Haemophilus, Hafnia, Morganella, Neisseria, Proteus, Providencia, Serratia, and Yersinia. The ferric enterobactin receptor contains at least one buried epitope, recognized by MAb 2 (C. K. Murphy, V. I. Kalve, and P. E. Klebba, J. Bacteriol. 172:2736-2746, 1990), that is conserved within the structure of an iron-regulated cell envelope protein in all the bacteria that we have surveyed. With MAb 2, we identified and determined the Mr of cell envelope antigens that are immunologically related to E. coli FepA in all the gram-negative bacteria tested. Collectively, the library of anti-FepA MAbs showed unique patterns of reactivity with the different bacteria, allowing identification and discrimination of species within the following gram-negative genera: Aeromonas, Citrobacter, Edwardsiella, Enterobacter, Escherichia, Haemophilus, Hafnia, Klebsiella, Morganella, Neisseria, Proteus, Providencia, Pseudomonas, Salmonella, Serratia, Shigella, Vibrio, and Yersinia.     

Prevalence of Escherichia coli O157:H7 and Salmonella in beef steers consuming different forage diets

AIMS: To compare the prevalence of faecal shedding of Escherichia coli O157:H7 and Salmonella in growing beef cattle consuming various forages. METHODS AND RESULTS: In Experiment I, faecal samples were collected from steers grazing either endophyte-infected (E+) tall fescue or common bermudagrass (CB). Steers grazing E+ tall fescue were confined to a dry-lot pen and fed CB hay ad libitum for 10 days. In Exp. II, faecal samples were collected from steers grazing either E+ or novel endophyte-infected (NE) tall fescue and treated with one of two anthelmintics: ivermectin (I) or fenbendazole (F). In Exp. I, prevalence of E. coli O157:H7 was less in E+ tall fescue steers fed CB hay than steers grazing CB. More I-treated steers shed Salmonella than F-treated steers at 42-day postanthelmintic treatment but shedding of Salmonella was similar between anthelmintics at day 63 in Exp. II. CONCLUSIONS: Faecal shedding of pathogenic bacteria was not affected by grazing E+ tall fescue. Alterations of forage diets may influence the prevalence of E. coli O157:H7, and anthelmintic treatment could affect faecal shedding of Salmonella. SIGNIFICANCE AND IMPACT OF THE STUDY: Knowledge of factors that influence shedding of pathogenic bacteria in cattle is necessary to develop on-farm intervention strategies aimed at reducing pathogen shedding.     

Transposon Tn10-like tetracycline resistance determinants in Haemophilus parainfluenzae.

Tetracycline resistance (Tcr) determinants from three different strains of Haemophilus parainfluenzae expressed 10-fold higher levels of resistance when mated into Escherichia coli. No plasmid was found in any of the E. coli recipients, even in matings in which a plasmid was identified in the donor Haemophilus sp. The Tcr determinant from Haemophilus sp. caused instability of resident plasmids in the recipient E. coli: all plasmids were lost within 30 generations in antibiotic-free media. However, by serial subculture in antibiotics, stable resident plasmids were obtained which carried the Tcr determinant from Haemophilus sp. and were transferable by conjugation and transformation among E. coli strains. All Haemophilus determinants hybridized with a probe for the Tcr determinant on Tn10, which bears inducible Tcr. However, Haemophilus determinants were constitutively resistant to tetracycline in the Haemophilus donors and in the E. coli recipients. This constitutive expression was recessive to wild-type Tn10 in the same cell, indicating that the constitutive phenotype resulted from the absence of an active repressor. Restrictive enzyme analysis of various E. coli plasmid derivatives bearing a Tcr determinant from Haemophilus sp. demonstrated that the inserted DNA was of similar size (8.95 to 9.35 kilobases), close to that of Tn10. Heteroduplex analysis and DNA:DNA hybridization confirmed that the Tcr determinant from Haemophilus sp. had greater than 90% homology with the Tn10 determinant, including the DNA sequence for the repressor.     

Mode of Salmonella and Escherichia coli O157:H7 inactivation by a stabilized oxychloro-based sanitizer

AIM: To determine the mechanisms by which a stabilized oxychloro (SOC)-based sanitizer, applied to decontaminate seeds destined for sprout production, inactivates Escherichia coli O157:H7 ph1 and Salmonella serotype Meleagridis. MATERIALS AND RESULTS: The action of SOC on the metabolism, membrane and DNA integrity of Salmonella and E. coli O157:H7 was studied. In both pathogens, there was an oxidative burst and depletion of intracellular glutathione (GSH) upon initial exposure to 200 ppm SOC. Metabolic activity, measured via bioluminescence, decreased over a 4-h period in E. coli O157:H7 ph1 cells exposed to SOC. Membrane integrity, assessed through viability staining, decreased progressively over 23 h when exposed to SOC. The appearance of auxotrophic mutants suggested that DNA damage had also occurred. Enzymes rich in disulfide bonds (alkaline phosphatase and protease) were sensitive to the chlorite-based sanitizer. Through challenging other microbial types, it was found that Gram positive had higher tolerance to SOC than Gram negatives with the exception of Salmonella. MS2 bacteriophage was highly sensitive; however, Bacillus endospores were not inactivated by SOC. CONCLUSIONS: SOC inactivates E. coli O157:H7 and Salmonella through GSH oxidation and disruption of disulfide bonds. Ultimately, membrane damage resulting from prolonged exposure to SOC leads to the loss of cell viability. SIGNIFICANCE AND IMPACT OF THE STUDY: The results provide a basis for understanding why extended treatment times are required to inactivate bacteria using SOC.     

Effect of Lysogeny on Serum Sensitivity

When Escherichia coli K-12 was infected with lambda phage and mutants of lambda characterized by the production of temperature-sensitive repressors, the lysogenic bacteria were significantly more resistant to normal serum than the uninfected organisms. Infection of E. coli K-12 with a lambdoid phage, phi80, whose prophage attachment site is different from that of lambda, did not result in a detectable change in serum resistance. Similarly, infection with certain Pseudomonas and Shigella phages caused no detectable differences in serum resistance. Finally, the well-known conversion of the Salmonella anatum serotype to S. newington by E(15) phage indicated that, despite the relatively greater roughness of S. anatum, S. newington was more sensitive to normal serum than S. anatum. Thus, the effects of lysogeny on the sensitivity of bacteria to the bactericidal action of serum mediated by the complement system may be quite variable.     

Mu-like prophage strong gyrase site sequences: analysis of properties required for promoting efficient mu DNA replication

The bacteriophage Mu genome contains a centrally located strong gyrase site (SGS) that is required for efficient prophage replication. To aid in studying the unusual properties of the SGS, we sought other gyrase sites that might be able to substitute for the SGS in Mu replication. Five candidate sites were obtained by PCR from Mu-like prophage sequences present in Escherichia coli O157:H7 Sakai, Haemophilus influenzae Rd, Salmonella enterica serovar Typhi CT18, and two strains of Neisseria meningitidis. Each of the sites was used to replace the natural Mu SGS to form recombinant prophages, and the effects on Mu replication and host lysis were determined. The site from the E. coli prophage supported markedly enhanced replication and host lysis over that observed with a Mu derivative lacking the SGS, those from the N. meningitidis prophages allowed a small enhancement, and the sites from the Haemophilus and Salmonella prophages gave none. Each of the candidate sites was cleaved specifically by E. coli DNA gyrase both in vitro and in vivo. Supercoiling assays performed in vitro, with the five sites or the Mu SGS individually cloned into a pUC19 reporter plasmid, showed that the Mu SGS and the E. coli or N. meningitidis sequences allowed an enhancement of processive, gyrase-dependent supercoiling, whereas the H. influenzae or Salmonella serovar Typhi sequences did not. While consistent with a requirement for enhanced processivity of supercoiling for a site to function in Mu replication, these data suggest that other factors are also important. The relevance of these observations to an understanding of the function of the SGS is discussed.     

Thermosensitive H1 plasmids determining citrate utilization.

Twelve thermosensitive H1 plasmids from strains of Salmonella typhi that had caused outbreaks of chloramphenicol-resistant typhoid fever in Vietnam, Thailand and India mediated citrate utilization (Cit+) in a prototrophic Escherichia coli K12 strain but not in the S. typhi strains from which they were derived. Four H1 plasmids from a similar outbreak in Mexico differed from the Far Eastern plasmids in not mediating citrate utlization but in mediating mercury resistance. H1 plasmids resembling the Far Eastern and the Mexican plasmids in regard to citrate utilization and mercury resistance were found in sewage in Britain. Citrate utilization was transferred to eight pathogenic strains of E. coli and to one strain each of Shigella flexneri and Shigella sonnei. Cultures of Cit+ bacteria grew more rapidly in citrate media at 28 degrees C than at 37 degrees C. Plasmid mutants that were more efficient at utilizing citrate were present in all such cultures--they grew equally well or better at 37 degrees C than at 28 degrees C. None of 222 strains of E. coli or Shigella that contained a variety of different plasmids were able to utilize citrate. This property was not transferred to the prototrophic E. coli K12 strain from Citrobacter (3 strains), Salmonella (39 strains), Proteus (44 strains), Klebsiella pneumoniae (33 strains) or Pseudomonas aeruginosa (44 strains).