Selective Isolation of Leptospiras from Contaminated Material by Incorporation of Neomycin to Culture Media

Incorporation of neomycin to the culture medium was found to be effective in inhibiting Escherichia coli contaminants without interfering with the growth of serotype L. autumnalis. The growth of 12 other Leptospira serotypes was unaffected by the addition of 300 mug of neomycin per ml to Ellinghausen medium or 5 mug/ml to Fletcher medium. Neomycin-containing medium was found to be of value in the isolation of leptospiras from cultures of blood from infected laboratory animals. A higher percentage of isolates was obtained in swine kidneys from an abattoir in medium containing neomycin than resulted from the same medium without antibiotic or with 5-fluorouracil. Contaminated leptospiral cultures growing in media with 5-fluorouracil were purified by subculturing into neomycin-containing media.     

手工和仪器两种血培养结果差异性分析

目的对手工法双相血培养瓶和BACTEC9120全自动血培养仪的阳性率作回顾性分析。方法将血液标本同时接种双相血培养基和BACTEC9120全自动血培养仪配套血瓶中,将阳性结果移种血平板,如为阴性再移种巧克力平板。结果370例血培养,双相血培养瓶阳性25例,阳性率为6．76％（25／370）,树脂需氧（儿童）瓶BACTEC9120报警显示阳性59例,阳性率为15．9％（59／370）,阳性标本移种到血平板及巧克力平板阳性54例,阳性率为14．6％（54／370）,假阳性5例,假阳性率为1．4％（5／370）,共有29例树脂需氧（儿童）瓶阳性,而双相血培养瓶为阴性,P〈0．001。结论BACTEC9120全自动血培养仪提高阳性率,缩短阳性的报告时间优于传统的双相血培养基。     

Immunological analysis of extracellular products and cell surface components of motile Aeromonas isolated from fish

The present work describes the characterization of antigens present in the extracellular products (ECP) and cell wall of strains of motile Aeromonas isolated from rainbow trout culture systems. The relationships among virulence for fish, O-serogroup and profile of LPS were also examined. The slide agglutination test showed that most of the virulent strains of motile Aeromonas (72%) were included in the serotypes O3, O6, O11 and O19 (Guinée and Jansen System). However, there were also non-pathogenic strains within these groups. Electrophoretic analysis of lipopolysaccharides (LPS) and proteins from cell envelope and ECP showed heterogeneity not only among the different serogroups but also within the same serotype. Immunoblot assays of cell envelope components, and of LPS present in the ECP demonstrated a close relationship among Aeromonas strains from the same serotype, while strains from different serotypes were not immunologically related. Moreover, this assay showed that motile Aeromonas belonging to distinct serotypes produced extracellular proteins immunologically related. On the other hand, antigenic cross reactivity was observed between the LPS obtained from cell envelope and those obtained from the ECP. The present results point out the need to include strains representative of each of the serotypes which predominates in a particular area and their ECPs in the formation of vaccines against motile Aeromonas septicaemia.     

Cell aggregation: a mechanism of pathogenic Leptospira to survive in fresh water.

Transmission of leptospirosis is facilitated by the survival of pathogenic leptospires in moist environments outside their mammalian host. In the present study, the survival mechanisms of Leptospira interrogans serovar Canicola in aqueous conditions and lack of nutrients were investigated. In distilled water, leptospires were able to remain motile for 110 days (pH 7.2). However, when incubated in a semi-solid medium composed of distilled water and 0.5% purified agarose (pH 7.2), they survived 347 days. In this viscous environment, aggregates of live spirochetes were observed. Neither antibiotics (e.g. tetracycline and ampicillin) nor nutrients inhibited leptospiral aggregation. Immunoblot analysis suggested that cells incubated in water down-regulate the expression of LipL31, an inner-membrane protein, but retain expression of other membrane proteins. These studies provide insights into the mechanisms by which pathogenic Leptospira survives for prolonged periods of time in natural aqueous environments, a key stage in the leptospiral lifecycle.     

Induction of motility and alteration of surface membrane polypeptides in lymphocytes by contact with autologous and allogeneic fibroblasts

Contact with cultured fibroblasts induced and maintained motile behavior in autologous and allogeneic human lymphocytes. After 3 h of contact with fibroblasts, 50 +/- 19% of the autologous lymphocytes were motile and after 24 h the corresponding figure was 49 +/- 18%. On a plastic surface the number of motile lymphocytes in the same individuals generally persisted below 15%. SDS-PAGE of iodine-labeled lymphocytes indicated that contact with fibroblasts but not with plastic for a 3-h period caused the appearance of a 300-kda band and the disappearance of several bands of lower molecular weight. During the course of T-lymphocyte activation by concanavalin A or allogeneic cells on a plastic surface, the number of motile forms did not reach a maximum (30 to 50% in separate individuals) until after 2 to 4 days in culture. Thus, in terms of both rate of development and number of motile forms, the fibroblast-dependent motility mechanism was more effective than conventional lymphocyte activation to blast transformation. Conditioned medium from fibroblasts did not induce motile behavior in the lymphocytes and did not provoke alteration of surface membrane polypeptides as revealed by iodination. Fibroblasts also triggered lymphocyte locomotion in serum-free medium, but their triggering effect was enhanced markedly by serum. The development and maintenance of lymphocyte motility required protein synthesis. These data suggest that during contact with fibroblasts lymphocytes acquire locomotor capacity by a mechanism different from activation to blast transformation.     

A novel approach for medium formulation for growth of a microalga using motile intensity

Motile intensity of the cells, defined as the specific mean kinetic energy, was measured by image analysis and used to formulate a suitable medium for the cultivation of a motile microalga. Nitrogen source at 60 mg/L was used as the target component. The cells grown in a medium containing urea showed the highest motile intensities during cultivation when compared to the cells grown with NH(4)Cl or (NH(4))(2)SO(4) as the nitrogen source. The urea culture gave the highest biomass yield and lipid content of 1.06+/-0.12 g/L and 22.34+/-2.56%, respectively after 150 h of cultivation. These results indicated a strong dependence of motile intensity on the nitrogen source used in the growth medium. This concept, which requires only a small droplet of the sample, can find potential application in the design and optimization of culture media.     

The Influence of CO(2) Supply on Colonial Formation of Leptospires

The colonial formation of three serotypes of Leptospires on Cox's solid medium was promoted by microaerophilic incubation of one to three per cent of CO(2) supplied by carbon dioxide cylinder, sodium carbonate oxalic acid, and candle method. In anaerobic incubation Leptospira pomona grew the same as with CO(2) incubation. The pH of the medium was an important influence on the rate of colonial formation of Leptospires. Addition of hemoglobin and inactivation of rabbit serum was not an essential condition for rapid colonial formation. It was found that variation in the morphology of leptospiral colonies occurred with hemoglobin from different species and individuals.     

Growth of Saprophytic and Pathogenic Leptospira: Evaluation of Medium, Temperature, Inoculum, and Cost

Of four media tested, a tissue culture medium supplemented with a bovine serum albumin-oleic acid complex provided the best leptospiral growth and cost efficiency.     

A novel method for the isolation of motile bacteria using gradient culture systems.

Isolation of motile bacteria from stream water samples was achieved by using Lutrol F127 (poloxamer 407) as a gelling agent in culture media. This block copolymer has the property of repeatedly liquefying and solidifying at low and high temperatures, respectively. The ability of motile bacteria to move through liquid-state Lutrol F127 towards a higher nutrient concentration was exploited. After establishment of the nutrient gradient and inoculation, the system was cooled to liquefy the medium and kept liquid to allow motile bacteria to move. Raising the temperature allowed solidification and prevented further movement. Colonies could be easily removed. The proportion of motile isolates (determined by microscopic observation) increased from 42% in the indigenous population to 100% after isolation using the gradient system.     

Biphasic culture strategy based on hyperosmotic pressure for improved humanized antibody production in Chinese hamster ovary cell culture

Hyperosmotic pressure increased specific antibody productivity (q(Ab)) of recombinant Chinese hamster ovary (rCHO) cells (SH2-0.32) and it depressed cell growth. Thus, the use of hyperosmolar medium did not increase the maximum antibody concentration substantially. To overcome this drawback, the feasibility of biphasic culture strategy was investigated. In the biphasic culture, cells were first cultivated in the standard medium with physiological osmolality (294 mOsm/kg) for cell growth. When cells reached the late exponential growth phase, the spent standard medium was replaced with the fresh hyperosmolar medium (522 mOsm/kg) for antibody production. The q(Ab) in growth phase with the standard medium was 2.1 microg per 10(6) cells/d, whereas the q(Ab) in antibody production phase with the hyperosmolar medium was 11.1 microg per 10(6) cells/d. Northern blot analysis showed a positive relationship between the relative contents of intracellular immunoglobulin messenger ribonucleic acid and q(Ab). Because of the enhanced q(Ab) and the increased cell concentration in biphasic culture, the maximum antibody concentration obtained in biphasic culture with 522 mOsm/kg medium exchange was 161% higher than that obtained in batch culture with the standard medium. Taken together, the simple biphasic culture strategy based on hyperosmotic culture is effective in improving antibody production of rCHO cells.     

Leptospiral antibodies in white-tailed deer of the southeastern United States

Serum samples from 1,544 white-tailed deer (Odocoileus virginianus) collected in nine southeastern states were examined for leptospiral antibodies. Significant titers of 1:100 or greater were found in 292 deer. The highest prevalence of leptospiral antibodies was in Virginia, where 108 of 351 deer had significant titers. The most frequently encountered serotypes of Leptospira interrogans were: grippotyphosa (210 positive), pomona (81), and canicola (26). Other serotypes disclosed were australis (15), icterohaemorrhagiae (10), pyrogenes (6), tarassovi (hyos) (5), georgia (4), ballum (4), sejroe (3), bataviae (2) and autumnalis (2).     

Agrobacterium tumefaciens twin-arginine-dependent translocation is important for virulence,flagellation, and chemotaxis but not type IV secretion

This study characterized the contribution of the twin-arginine translocation (TAT) pathway to growth, motility, and virulence of the phytopathogen Agrobacterium tumefaciens. In contrast to wild-type strain A348, a tatC null mutant failed to export the green fluorescent protein fused to the trimethylamine N-oxide reductase (TorA) signal sequence or to grow on nitrate as a sole electron acceptor during anaerobic growth. The tatC mutant displayed defects in growth rate and cell division but not in cell viability, and it also released abundant levels of several proteins into the culture supernatant when grown in rich medium or in vir induction minimal medium. Nearly all A348 cells were highly motile in both rich and minimal media. By contrast, approximately 0.1% of the tatC mutant cells were motile in rich medium, and <0.01% were motile in vir induction medium. Nonmotile tatC mutant cells lacked detectable flagella, whereas motile tatC mutant cells collected from the edge of a motility halo possessed flagella but not because of reversion to a functional TAT system. Motile tatC cells failed to exhibit chemotaxis toward sugars under aerobic conditions or towards nitrate under anaerobic conditions. The tatC mutant was highly attenuated for virulence, only occasionally (approximately 15% of inoculations) inciting formation of small tumors on plants after a prolonged incubation period of 6 to 8 weeks. However, an enriched subpopulation of motile tatC mutants exhibited enhanced virulence compared to the nonmotile variants. Finally, the tatC mutant transferred T-DNA and protein effectors to plant cells and a mobilizable IncQ plasmid to agrobacterial recipients at wild-type levels. Together, our findings establish that, in addition to its role in secretion of folded cofactor-bound enzymes functioning in alternative respiration, the TAT system of A. tumefaciens is an important virulence determinant. Furthermore, this secretion pathway contributes to flagellar biogenesis and chemotactic responses but not to sensory perception of plant signals or the assembly of a type IV secretion system.     

Trypanosoma cruzi: isolation of cloned strains and characterization of their infectivity

Cloning of Trypanosoma cruzi strains Y, CL and Colombiana was achieved by plating on solid medium. Clones were obtained either from culture epimastigotes or from bloodstream trypomastigotes. In both cases the efficiency of plating was almost 100%. Clones from culture epimastigotes did not infect the albino mouse, while clones from bloodstream trypomastigotes remained infective even after several passages in a blood-agar/BHI biphasic medium, in which the amastigote-like forms prevail.     

Intermediate Energy Metabolism of Leptospira

Metabolic studies were performed on three representative serotypes of Leptospira: a water isolate designated B(16) and two pathogenic serotypes, pomona and schueffneri. Examination of whole cells of B(16) for their ability to oxidize various substrates revealed that oleate significantly stimulated oxygen uptake. The respiratory quotient of 0.7 implied that oleate was degraded to carbon dioxide and water. Other substrates, such as carbohydrates, alcohols, intermediates of the citric acid cycle, and short-chain acids, including selected amino acids, did not stimulate endogenous respiration of whole cells. No oxygen uptake could be measured when cell-free extracts were tested with the substrates used with whole cells. Enzymatic analyses of cell-free extracts of the three strains demonstrated enzymes of the citric acid cycle, enzymes of the glycolytic and pentose pathways, and the general acyl coenzyme A dehydrogenase required for beta-oxidation of fatty acids. Strain B(16) and the two pathogenic serotypes appeared to possess similar metabolic capabilities. Enzymatic data might also explain the apparent inability of B(16) to oxidize other substrates; kinases necessary for activation of common nonphosphorylated compounds were not detected in leptospiral extracts. These findings emphasized the dependence of leptospiral growth upon long-chain fatty acids.     

Serological Investigations of Leptospiral Deoxyribonucleases

Enzymoserological comparison of a selection of leptospira strains tested with sera from rabbits immunized with unpurified DNase of Leptospira interrogans, serotype canicola, indicates the production of DNase of serologically very similar properties by the serotypes canicola, autumnalis, icterohemorrhagiae and pomona. The DNase produced by serotype hyos was serologically different from the others, while the serotypes grippotyphosa and bataviae did not produce DNases at all. The method used made it possible to differentiate between leptospiral DNase and normally occurring DNases in the serum samples. Neither leptospira DNase nor specific leptospira-DNase-antibodies could be detected in dog sera with high agglutinationlysis titres after natural infection.     

A new antileishmanial compound, phaseolinone

Inclusion of phaseolinone, a newly described mycotoxin, at 20 micrograms per ml in a solid culture medium (blood agar overlay) and at 50 micrograms per ml in a liquid culture (medium 199) inhibited the growth of L. donovani promastigotes. About 90% of the motile promastigotes lost motility after exposure to 50 micrograms per ml of phaseolinone for 6-7 h and here 3-day-old culture was more sensitive than 7-day-old culture. In an in vitro assay, DNA dependent RNA polymerase activity of 3-day-old promastigotes was considerably inhibited in the presence of this toxin. Therefore, this key enzyme was suggested to be one of the sites of action of phaseolinone.     

Fed-batch culture of<Emphasis Type="Italic"> Bacillus thuringiensis</Emphasis> based on motile intensity

The operation of a fed-batch culture is more complicated than that of batch or continuous culture. Thus, an appropriate feeding strategy for fed-batch cultures should be carefully designed. In this study, a simple feeding strategy for fed-batch culture of Bacillus thuringiensis based on motile intensity is described. The feeding strategy consisted of two steps: (1) initiating feeding at the peak of motile intensity; (2) terminating feeding at low motile intensity (or non-motility) of the cells. In addition, the motile intensity of B. thuringiensis was used to determine the optimum environmental conditions (pH, temperature, and dissolved oxygen) and optimum medium composition. Using this fed-batch strategy, the production of thuringiensin increased 34% compared with batch culture using the same environmental conditions and medium composition. The proposed strategy for fed-batch culture helps to avoid overfeeding of substrate and facilitates on-line control. A comparison of several alternative strategies for fed-batch culture demonstrated that strategies such as glucose-stat and DO-stat result in a lower productivity than that obtained using the motility intensity method.     

Evaluation of a novel biphasic culture medium for recovery of mycobacteria: a multi-center study

Background

Mycobacterial culture and identification provide a definitive diagnosis of TB. Culture on Löwenstein-Jensen (L-J) medium is invariably delayed because of the slow growth of M. tuberculosis on L-J slants. Automated liquid culture systems are expensive. A low-cost culturing medium capable of rapidly indicating the presence of mycobacteria is needed. The aim of this study was to develop and evaluate a novel biphasic culture medium for the recovery of mycobacteria from clinical sputum specimens from suspected pulmonary tuberculosis patients.

Methods and Findings

The biphasic medium consisted of 7 ml units of L-J slant medium, 3 ml units of liquid culture medium, growth indicator and a mixture of antimicrobial agents. The decontamination sediments of sputum specimens were incubated in the biphasic culture medium at 37°C. Mycobacterial growth was determined based on the appearance of red granule sediments and the examination using acid-fast bacilli (AFB). The clinical sputum specimens were cultured in the biphasic medium, on L-J slants and in the Bactec MGIT 960 culture system. Among smear-positive specimens, the mycobacteria recovery rate of the biphasic medium was higher than that of the L-J slants (P<0.001) and similar to that of MGIT 960 (P>0.05). Among smear-negative specimens, the mycobacterial recovery rate of the biphasic medium was higher than that of L-J slants (P<0.001) and lower than that of MGIT 960 (P<0.05). The median times to detection of mycobacteria were 14 days, 20 days and 30 days for cultures grown in MGIT, in biphasic medium, on L-J slants for smear negative specimens, respectively (P<0.001).

Conclusions

The biphasic culture medium developed in this study is low-cost and suitable for mycobacterial recovery. It does not require any expensive detection instrumentation, decreases the time required for detection of M. tuberculosis complex, and increases the detection rate of M. tuberculosis complex.     

Influence of Carbohydrates on Growth and Sporulation of Clostridium perfringens Type A

Growth and sporulation of Clostridium perfringens type A in Duncan and Strong (DS) sporulation medium was investigated. A biphasic growth response was found to be dependent on starch concentration. Maximal levels of heat-resistant spores were formed at a starch concentration of 0.40%. Addition of glucose, maltose, or maltotriose to a sporulating culture resulted in an immediate turbidity increase, indicating that biphasic growth in DS medium may be due to such starch degradation products. Amylose and, to a lesser extent, amylopectin resulted in biphasic growth when each replaced starch in the sporulation medium. A levels of heat-resistant spores approximately equal to the control was produced with amylopectin but not amylose as the added carbohydrate. Addition of glucose or maltose to a DS medium without starch at stage II or III of sporulation did not alter the level of heat-resistant spores as compared with the level obtained in DS medium with starch. Omission of starch or glucose or maltose resulted in an approximately 100-fold decrease in the number of heat-resistant spores, although the percentage of sporulation (90%) was unaffected. The role of starch and amylopectin in the formation of heat-resistant spores probably involves the amyloytic production of utilizable short-chain glucose polymers that provide an energy source for the completion of sporulation.     

Enzyme patterns in the study of leptospira

An analysis of intracellular and extracellular leptospiral enzymes was made by use of starch-gel electrophoresis with natural and synthetic substrates. Of 37 serotypes examined for extracellular exterase, all had activity of varying mobility and degree. All extracellular preparations were negative for catalase, phosphatase, and naphthylamidase. Intracellularly, five serotypes were examined, including Leptospira biflexa Patoc I, L. biflexa Waz, L. canicola Moulton, L. icterohaemorrhagiae RGA, and L. pomona S91. Among the enzymes detected by this electrophoretic technique were transaminase and catalase, confirming the results of previous investigators. Further, other enzymes heretofore unreported have been detected. These include esterases, phosphatases, lactic, malic, glutamic, succinic, alpha-glycerophosphate, and 6-phosphogluconic dehydrogenases, and a naphthylamidase. The presence of these enzymes suggests the existence of tricarboxylic acid, glycolytic, and pentose-related pathways in Leptospira. In addition, enzyme patterns show promise in leptospiral classification.