Supernatant from Cultured Intestinal Cells Inhibits the Growth of Gram-Negative Bacteria

Bacterial growth chambers of Transwell units bearing intestinal epithelial monolayers (C2BBe) was consistently observed to be stagnant during the course of transmigration studies with Salmonella typhi. This limitation could not be explained by varying the bacterial load in the inoculum. Conditioned media produced by cultured C2BBe cells would not support bacterial growth. Growth support of the media was restored by heating to 95 C for 10 min. C2BBe conditioned media had bacteriostatic activity for a large variety of gram-negative, enteropathogenic bacteria but had no effect on gram-positive bacteria.     

Production and Degradation of N-Acylhomoserine Lactone Quorum Sensing Signal Molecules in Bacteria Isolated from Activated Sludge

N-Acylhomoserine lactones (AHLs) function as quorum-sensing signaling molecules in many Gram-negative bacteria. We isolated a total of 672 bacterial strains from activated sludge obtained from seven sewage treatment plants in Tochigi Prefecture, Japan, and screened for AHL-producing and degrading strains. Isolates (n=107) stimulated AHL-mediated purple pigment production in AHL reporter strains Chromobacterium violaceum CV026 and VIR07. Based on their 16S rRNA gene sequences, most of these AHL-producing isolates were assigned to the genus Aeromonas, and they were divided into six groups. Isolates (n=46) degraded N-decanoyl-L-homoserine lactone (C10-HSL) within 24 h. Based on their 16S rRNA gene sequences, the most dominant AHL-degrading isolates were assigned to the genus Acinetobacter and divided into six groups. Strains Ooi24, Omo91, and Uzu81, which showed higher C10-HSL-degrading activity, showed putative AHL-acylase activity.     

The Growth of Gram-Negative Bacteria in the Hen's Egg

Summary: Bacteriological and chemical methods were used to follow the course of infection in eggs, incubated at 27°, the air cells of which had been inoculated with a suspension of washed bacteria. In the 3–4 days following inoculation, limited bacterial multiplication occurred in the inner membrane of the air cell but very few organisms entered the albumen. These populations then remained static or decreased slightly until renewed multiplication occurred 12–30 days after inoculation. This was induced by contact of the yolk and the shell membranes: it occurred on the 12–20th day in eggs in which the yolk moved towards the site of inoculation, but later when the yolk moved in the opposite direction. At this time there was a general infection of the egg contents and significant changes occurred in the pH and glucose concentration in the albumen. In eggs that had been inoculated with chromogenic and/or proteolytic bacteria, the first macroscopic changes of the contents were seen at this time. The rate and extent of the initial multiplication was influenced by the composition of the fluid used to suspend the washed bacteria and, in all instances, the fastest multiplication occurred when iron was added to the inoculum. Moreover, renewed multiplication occurred when iron was added to the albumen of eggs in which the bacteria were in the stationary phase.     

细菌群体感应信号分子N-酰基高丝氨酸内酯的检测

群体感应是细菌生长到一定密度时相互感应,并进行基因表达及调控产生的独特、多样的群体行为现象。N-酰基高丝氨酸内酯(AHL)类化合物是革兰阴性菌群体感应中最重要的一类信号分子,调控许多生理特性基因的表达。快速、简便、有效地检测细菌能否产生AHL或产生何种信号分子,成为深入研究和了解细菌群体感应的重要手段。我们就细菌群体感应信号分子AHL检测的基本原理和方法及国内外研究进展进行了总结。     

生物量分配影响三种不同海拔起源的松树生长

松属的思茅松（Pinus kesiya var. langbianensis）、云南松（P. yunnanensis）和高山松（P. densata）是组成中国西南不同海拔针叶森林的主要树种,然而这三个树种在发育速度尤其是高生长方面表现出明显的差异。为了弄清引起这些变异的生理和形态学原因,本文将三种松树种植于同一环境下,对其光合作用、生物量分配、生长速率和叶片性状进行了研究。研究发现,与来源于高海拔的树种相比,低海拔的树种有更高的株高、以及更大的干物质重量、相对生长速率、叶质比、茎质比和比叶面积,但叶片氮含量、碳含量和根质比较低。高海拔树种的光合速率并不明显低于低海拔树种。相对生长速率和树高均与叶质比呈显著正相关,与根质比负相关,但与最大光合速率没有显著关系。这些结果表明,生物量的分配式样和长期的形态特性能够更好地预测不同海拔松树的生长表现。     

生物量分配影响三种不同海拔起源的松树生长

松属的思茅松（Pinus kesiya var．1angbianensis）、云南松（P．yunnanensis）和高山松（P．densata）是组成中国西南不同海拔针叶森林的主要树种,然而这三个树种在发育速度尤其是高生长方面表现出明显的差异。为了弄清引起这些变异的生理和形态学原因．本文将三种松树种植于同一环境下,对其光合作用、生物量分配、生长速率和叶片性状进行了研究。研究发现,与来源于高海拔的树种相比,低海拔的树种有更高的株高、以及更大的干物质重量、相对生长速率、叶质比、茎质比和比叶面积,但叶片氮含量、碳含量和根质比较低。高海拔树种的光合速率并不明显低于低海拔树种。相对生长速率和树高均与叶质比呈显著正相关,与根质比负相关,但与最大光合速率没有显著关系。这些结果表明,生物量的分配式样和长期的形态特性能够更好地预测不同海拔松树的生长表现。     

Characterization of Certain Gram-Negative Bacteria from Surface Waters

Cultures of gram-negative bacteria with oxidative glucose metabolism were isolated from surface waters by a highly selective technique, and were classified into 11 types. The predominant type, making up about 50% of the isolated cultures, was cytochrome oxidase-positive, produced fluorescent pigment, and failed to grow at 37 C. A similar type, which differed in being cytochrome oxidase-negative, constituted about 10% of the isolates. Both types of bacteria probably were composed of more than one species. A third type, composed of purple-pigmented pseudomonads, made up approximately 30% of the isolated cultures, and probably represented the predominant species. Other bacterial types isolated constituted less than 10% of the cultures examined.     

Multiplicity of Quorum Quenching Enzymes: A Potential Mechanism to Limit Quorum Sensing Bacterial Population

Bacteria express certain of their characteristics especially, pathogenicity factors at high cell densities. The process is termed as quorum sensing (QS). QS operates via signal molecules such as acylhomoserine lactones (AHLs). Other bacteria inhibit QS through the inactivation of AHL signals by producing enzymes like AHL-lactonases and -acylases. Comparative genomic analysis has revealed the multiplicity of genes for AHL lactonases (up to 12 copies per genome) among Bacillus spp. and that of AHL-acylases (up to 5 copies per genome) among Pseudomonas spp. This genetic evolution can be envisaged to enable host to withstand the attacks from bacterial population, which regulates its functioning through QS.     

A Reliable Method for Demonstrating Gram-Positive and Gram-Negative Bacteria

Combined Gram techniques have been reviewed in the interest of improving technical safety and reliability in the demonstration of bacteria, particularly the Gram-negative type. The many modifications of the technique present various difficulties (Brown and Brenn 193 1, Humberstone 1963, Taylor 1966, Luna 1968, Brown and Hopps 1973, Engbaek et al. 1979, Bancroft and Stevens 1982, Churukian and Schenk 1982).     

A New Growth Effecting Peptide of Bacillus Species from Soybean Cake

A new peptide, which has a marked effect on the growth of Bacillus species, was purified from soybean cake by a procedure employing ammonium sulfate fractionation, and active charcoal-, DEAE-Sephadex A–25- and Sephadex gel-column chromatographies. The purified peptide gave a single band on paper electrophoresis. The peptide possessed the following properties: specific extinction (E at 280nm), 11.9; N-terminal amino acid, glutamate; molecular weight, 8058; total number of amino acid, 70; chemical composition (%), C, 49.12; H, 6.26; N, 16.44; and S, 2.34; isoelectric point, pH4.3. The hydrolyzed peptide had no stimulative effect on the growth of Bacillus species. Especially, those microorganisms belonging to the genera of Bacillus, Arthrobacter and Xanthomonas were remarkably sensitive to the peptide.     

Microbial Communication,Cooperation and Cheating: Quorum Sensing Drives the Evolution of Cooperation in Bacteria

An increasing body of empirical evidence suggests that cooperation among clone-mates is common in bacteria. Bacterial cooperation may take the form of the excretion of “public goods”: exoproducts such as virulence factors, exoenzymes or components of the matrix in biofilms, to yield significant benefit for individuals joining in the common effort of producing them. Supposedly in order to spare unnecessary costs when the population is too sparse to supply the sufficient exoproduct level, many bacteria have evolved a simple chemical communication system called quorum sensing (QS), to “measure” the population density of clone-mates in their close neighborhood. Cooperation genes are expressed only above a threshold rate of QS signal molecule re-capture, i.e., above the local quorum of cooperators. The cooperative population is exposed to exploitation by cheaters, i.e., mutants who contribute less or nil to the effort but fully enjoy the benefits of cooperation. The communication system is also vulnerable to a different type of cheaters (“Liars”) who may produce the QS signal but not the exoproduct, thus ruining the reliability of the signal. Since there is no reason to assume that such cheaters cannot evolve and invade the populations of honestly signaling cooperators, the empirical fact of the existence of both bacterial cooperation and the associated QS communication system seems puzzling. Using a stochastic cellular automaton approach and allowing mutations in an initially non-cooperating, non-communicating strain we show that both cooperation and the associated communication system can evolve, spread and remain persistent. The QS genes help cooperative behavior to invade the population, and vice versa; cooperation and communication might have evolved synergistically in bacteria. Moreover, in good agreement with the empirical data recently available, this synergism opens up a remarkably rich repertoire of social interactions in which cheating and exploitation are commonplace.     

Effect of Lactic Acid Bacteria on Growth of Staphylococcus aureus

Cultures of lactic acid bacteria, mostly from foods, were tested for their effect on the growth of Staphylococcus aureus in Trypticase Soy Broth (BBL). Some of the effectors, e.g., Streptococcus faecalis, S. faecium, Lactobacillus lactis, L. brevis, and Leuconostoc mesenteroides, stimulated growth of S. aureus during early hours of growth, especially at higher temperatures of incubation, but most cultures were inhibitory, and some (S. faecium and L. mesenteroides) were even killing by the time of attainment of the maximal phase of growth of the Staphylococcus. Low-temperature meat lactobacilli and Leuconostoc dextranicum inhibited S. aureus at 10, 15, 20, and 25 C throughout its growth. Streptococcus faecalis var. liquefaciens inhibited at these temperatures and at 30 and 37 C, as well. When the ratio of effectors to staphylococci in the inoculum was 100:1, the three enterococci, the meat Lactobacillus, and L. dextranicum prevented the attainment of 5 x 10(6) staphylococci per milliliter at 15 C, and all but the meat Lactobacillus did so at 22 C. A ratio of 1:1 accomplished similar results at 15 C, except that S. aureus was only delayed for 12 hr by S. faecalis. A ratio of 1:100 usually was ineffective. In general, the more effector bacteria there were in the inoculum, the greater was the overall inhibition (or stimulation) of S. aureus. Inhibition was most effective at 10 or 15 C, less so at 20 or 25 C, and least at 30 or 37 C, whereas stimulation during early growth was greater at the higher temperatures. Results with different strains of the effectors and with two strains of S. aureus were similar, for the most part.     

Effect of Bile and Desoxycholate on Gram-Negative Anaerobic Bacteria

The bile tests for characterizing gram-negative anaerobic bacilli were reevaluated in prereduced anaerobically sterilized peptone-yeast-glucose broth, in thioglycollate broth, and on blood agar plates. Blood agar plates were unsatisfactory. The combination of 20% bile with 0.1% desoxycholate inhibited Fusobacterium, Bacteroides melaninogenicus, and B. oralis and sometimes Sphaerophorus necrophorus, but not B. fragilis or other Sphaerophorus species studied. Ten per cent bile with 0.05% desoxycholate was less satisfactory. There was no significant difference between fresh and commercial powdered bile. Desoxycholate (0.1% in thioglycollate broth) inhibited B. fragilis, Fusobacterium, B. melaninogenicus, B. oralis, and S. necrophorus, but not S. varius or S. mortiferus/S. ridiculosus. The bile and desoxycholate tests are simple to perform and helpful for characterization and classification of gram-negative anaerobic bacilli.     

Saccharomyces cerevisiae-Based Molecular Tool Kit for Manipulation of Genes from Gram-Negative Bacteria

A tool kit of vectors was designed to manipulate and express genes from a wide range of gram-negative species by using in vivo recombination. Saccharomyces cerevisiae can use its native recombination proteins to combine several amplicons in a single transformation step with high efficiency. We show that this technology is particularly useful for vector design. Shuttle, suicide, and expression vectors useful in a diverse group of bacteria are described and utilized. This report describes the use of these vectors to mutate clpX and clpP of the opportunistic pathogen Pseudomonas aeruginosa and to explore their roles in biofilm formation and surface motility. Complementation of the rhamnolipid biosynthetic gene rhlB is also described. Expression vectors are used for controlled expression of genes in two pseudomonad species. To demonstrate the facility of building complicated constructs with this technique, the recombination of four PCR-generated amplicons in a single step at >80% efficiency into one of these vectors is shown. These tools can be used for genetic studies of pseudomonads and many other gram-negative bacteria.     

具细菌群体感应抑制活性海洋细菌的筛选鉴定

目的:从海洋环境中筛选对细菌群体感应有抑制作用的活性菌株,为以致病菌群体感应系统为靶点的新型疗法提供新的药用资源。方法:从海水中分离纯化细菌菌株,采用根癌农杆菌平板筛选模型筛选细菌群体感应抑制活性细菌,对筛选出的海洋细菌进行生理生化和16S rDNA序列测定,根据《伯杰氏手册》进行菌种分类鉴定。结果:从217株海洋细菌中筛选出1株能显著抑制细菌群体感应效应的海洋细菌Y2,该海洋细菌具有蜡样芽孢杆菌(Bacillus cereus)的典型特征,其16S rDNA序列与GenBank中蜡样芽孢杆菌16S rDNA的部分序列有100%的同源性。结论:海洋环境中也存在具有抑制细菌群体感应活性的微生物。     

Ethyl Violet. A Selective Dye for the Isolation of Gram-Negative Bacteria

Minimal inocula of Gram-negative and positive bacteria were seeded into tryptose broth containing varying concentrations of dyes. Three dyes were used, namely crystal violet, brilliant green and ethyl violet. Growth rates were determined for 2, 4 and 6 hours incubation. All three dyes were equally effective in inhibiting Gram positive bacteria. Ethyl violet showed markedly less toxicity toward Gram negative bacteria than did either crystal violet or brilliant green.