Vergleich eines "glatten" und eines "rauhen" Stammes von Pseudomonas syringae pv. phaseolicola

Comparison of a “smooth” and a “rough” isolate of Pseudomonas syringae pv. phaseolicola The “smooth” (S) wild strain of Pseudomonas syringae pv. phaseolicola was compared with a “rough” (R) variant of low virulence. Both strains grew nearly equally well on a sucrose containing medium with yeast extract and casamino acids, and the strains did not differ markedly in the quantity of produced EPS (= extracellular polysaccharides). Principally the same results were obtained for high and medium concentrations of sucrose, or when sucrose was replaced by glucose or fructose. However, on glucose and fructose considerably lower quantities of EPS were produced. The biological activity of S-EPS was higher than that of R-EPS. This difference between the EPS preparations was not as marked as leaf inoculation with both bacterial isolates. After prolonged bacterial culture the EPS-production increased further, so that the differences between both strains decreased. A different EPS type was produced on the glycerol containing medium of KING B. Variations in the composition of this medium resulted in different morphology of the agar grown cultures, and the relative differences between S and R bacteria changed. When 62 different physiological tests for both bacterial strains were compared, the “rough” bacteria revealed a lowered range of positive reactions, with a few exceptions. However, it appeared unlikely that the reduced virulence of the “rough” bacteria was due to these differences. Obviously, defects in the extracellular products, but not in levan, were responsible for the reduction of virulence.     

Dynamics of bacterial growth and distribution within the liver during Salmonella infection

Salmonella enterica causes severe systemic diseases in humans and animals and grows intracellularly within discrete tissue foci that become pathological lesions. Because of its lifestyle Salmonella is a superb model for studying the in vivo dynamics of bacterial distribution. Using multicolour fluorescence microscopy in the mouse typhoid model we have studied the interaction between different bacterial populations in the same host as well as the dynamic evolution of foci of infection in relation to bacterial growth and localization. We showed that the growth of Salmonella in the liver results in the spread of the microorganisms to new foci of infection rather than simply in the expansion of the initial ones. These foci were associated with independently segregating bacterial populations and with low numbers of bacteria in each infected phagocyte. Using fast-growing and slow-growing bacteria we also showed that the increase in the number of infected phagocytes parallels the net rate of bacterial growth of the microorganisms in the tissues. These findings suggest a novel mechanism underlying growth of salmonellae in vivo with important consequences for understanding mechanisms of resistance and immunity.     

Efficiency of various selective media in determining Pythium population in soil

Of 15 selective media recommended for isolation and enumeration ofPythium spp. directly from soil, corn meal agar (CMA) supplemented with agar, sucrose, minor elements, thiamine, rose bengal, pimaricin, pentachloronitrobenzene and vancomycin (MPVM) was the most efficient. Streptomycin (30–50 ppm) and rose bengal (33–60 ppm) as used in certain tested media effectively suppressed development of bacteria and actinomycetes. However, these chemicals adversely affected germination of spores and mycelial growth and thereby the recovery ofPythium spp. from soil. Media containing pimaricin (5 to 100 ppm) were more effective than those with nystatin (40 ppm) in suppressing development of nonphycomycetous fungi on isolation plate. MPVM with pimaricin at 5 ppm was more efficient than that with 10 ppm of the antibiotic in recoveryingPythium from soil. However, there was no difference in recovery ofPythium by this medium containing rose bengal at 5 ppm or at 10 ppm, butPythium colonies were more dense and better delineated when the medium contained 10 ppm of rose bengal. CMA containing pimaricin (5–100 ppm) and vancomycin (200 ppm) permitted occasionally development of a large number ofMortierella and bacterial colonies from certain soils, that interfered with accurate determination of colonies of certainPythium spp. on the plates. Vancomycin at 300 ppm, as used in MPVM, substantially reduced development of bacterial colonies compared to 200 ppm of the antibiotic. Surface-soil dilution-plate was more effective than the soil-dilution-plate method in reducing bacteria andMortierella colonies on isolation plates without affecting recovery ofPythium. The importance of basal medium, complement of antimicrobial agents, and isolation methods for efficiency of selective medium in recovery ofPythium spp. directly from soil is discussed.     

Listeria monocytogenes L Forms I. Induction, Maintenance, and Biological Characteristics

L forms were induced from 15 of 16 strains of Listeria monocytogenes on penicillin gradient plates incubated under aerobic conditions. The culture medium for maintenance of these L forms must contain an electrolyte in a concentration of 1% or sucrose in a concentration of 10%. The electrolytes NaCl, KCl, or MgSO(4) were used in both induction and maintenance media. Induction of L forms occurred more rapidly on media containing KCl. Listeria L forms had the same fermentation reactions as the parent bacterium. The L-form growth in liquid medium was slow, not extensive, and appeared as clumps on the bottom of culture tubes. The morphology of Listeria L forms was similar to that reported for other bacterial L forms. The L forms derived from strain 10403, serotype 1, were stable after two or more passages on penicillin media. They did not revert to the bacterial form after 40 subcultures on penicillin-free media. Some L-form colonies derived from strain 10403 did revert to the bacterial form when transferred directly from induction plates to penicillin-free media. Studies of the growth characteristics for L forms derived from strain 10403 gave the following results: an optimal temperature of 30 C, high electrolyte or sucrose concentration necessary for induction and maintenance, and no requirement for serum.     

Kinetics of infection induced byYersinia

Yersinia enterocolitica of different serotypes andY. intermedia, Y. frederiksenii, andY. kristensenii, in a total of nine strains, were inoculated intragastrically and intravenously into Swiss mice. The animals were observed daily to check for clinical alterations. Groups of five were killed intermittently at 6-h, and 3-, 6-, 10-, 15-,and 21-day periods or more after the inoculation; possible macroscopic alterations of the organs and tissues were checked. Development of infection at these periods was followed by performing viable bacterial counts on homogenates of selected tissues and the kinetics of infection was established. Clinical and pathologic alterations occurred only in the animals inoculated with the human strains ofY. enterocolitica 0:3 and 0:8, independent of the route of infection. After intragastric inoculation, theY. enterocolitica strains considered to be adapted to man were isolated from all organs and tissues, with the exception of the blood, from which only serotype 0:8 was isolated; otherYersinia strains were found only in the cecal content. After intravenous challenge, all the strains infected the organs and tissues at different times and in varied intensity, with exception of Peyer's patches and mesenteric lymph nodes, which were not infected by all theYersinia strains.     

Cloning and characterization of endosymbiotic algae isolated fromParamecium bursaria

Summary The green parameciumParamecium bursaria has many endosymbiotic algae in its cytoplasm. Here, we cloned and characterized endosymbiotic algae fromP. bursaria and examined in detail the interaction between the cloned algae and algae-free paramecia. Homogenates ofP. bursaria were cultured on agar plates containing various kinds of media to establish clones of the endosymbiotic algae. Many algal colonies were obtained from poorly nutritious medium (CA medium) after one month in culture. Algae were picked up from these colonies and inoculations were repeated 9 times on agar plates containing CA medium. On enriched media including bacto-peptone, glucose, proteose-peptone and/or yeast extract, however, bacteria and mold grew rapidly and no algal colonies were formed. When the cloned algae were cultured in liquid CA medium, they grew faster than on agar plates and the numbers stayed constant at 1 × 10⁷ algae/ml after 7 days in culture. They revealed high infectivity to algae-free paramecia, and an incubation period of 24 h and at least 1 × 10³ algae/paramecium were required to achieve successful infection (80–90%). The growth and infection rate did not change through 74 repeated inoculations of algae in liquid CA medium. Optical microscopic observations revealed marked morphological similarity between endosymbiotic algae and free-livingChlorella, but the latter showed no infectivity to algae-free paramecia. The cloned endosymbiotic algae presented here will provide an excellent opportunity to examine the mechanism of symbiont-host interaction.     

Effets de la zearalenone sur la cellule bactérienne

Zearalenone, a macrocyclic lactone, shows a limited antibacterial activity which can be demonstrated in Grampositive spore-forming bacteria. In a sensitive Bacillus thuringiensis (Berliner), the mycotoxin disturbs various bacterial metabolisms: physiological damages (decreased growth rate, inhibition of sporulation and enzyme synthesis) and cellular alterations (filamentous forms or snake-cells) are noted. On the other hand, although zearalenone has been found to give a positive response in the presumptive screening test for mutagens measured by DNA-attacking ability on Bacillus subtilis, this mycotoxin failed to demonstrate mutagenicity in routine test using liver homogenate and Salmonella typhimurium strains.

     

实验猴主要细菌性感染疾病的病理特点分析

目的通过对实验猴细菌性感染疾病脏器病理改变的观察和分析,完善实验猴病理检测资料,为实验动物病理检测标准的制定提供依据。方法选取86例实验猴按5种必检细菌性感染疾病（沙门菌病;志贺菌病;结核杆菌病;小肠结肠炎耶尔森菌病;空肠弯曲菌病）病原种类分组,对脏器标本进行病理剖检,HE染色观察记录病变,建立实验猴必检细菌性疾病病理检测资料。结果病理检测结果显示：沙门菌病表现为伤寒肉芽肿,结核杆菌病表现为结核肉芽肿,小肠结肠炎耶尔森菌病表现为纵行溃疡、急性炎及化脓性肉芽肿;志贺菌病、空肠弯曲菌病表现为急性炎和表浅溃疡。结论感染5种必检细菌的实验猴分别表现出不同的病理变化,病理检测对疾病的分析诊断有重要价值,检测结果补充了实验猴细菌性疾病病理检测资料,为制定实验动物病理检测指南提供了相关依据。     

Photonic detection of bacterial pathogens in living hosts

The study of pathogenic is often limited to ex vivo assays and cell-culture correlates. A greater understanding of infectious diseases would be facilitated by in vivo analyses. Therefore, we have developed a method for detecting bacterial pathogens in a living host and used this method to evaluate disease processes for strains of Salmonella typhimurium that differ in their virulence for mice. Three strains of Salmonella were marked with bioluminescence through transformation with a plasmid conferring constitutive expression of bacterial luciferase. Detection of photons transmitted through tissues of animals infected with bioluminescent Salmonella allowed localization of the bacteria to specific tissues. In this manner progressive infections were distinguished from those that were persistent or abortive. We observed patterns of bio-luminescence that suggested the caecum may play a pivotal role in Salmonella pathogenesis. In vivo efficacy of an antibiotic was monitored using this optical method. This study demonstrates that the real time non-invasive analyses of pathogenic events and pharmacological monitoring can be performed in vivo .     

Preparation of protoplast-like structures of Pseudomonas putida and their reversion to bacterial forms

A technique is proposed for the preparation of Pseudomonas putida protoplast-like structures by treating the cells with lysozyme in a hypertonic medium containing mono-and divalent cations. Pretreatment of the cells at the logarithmic growth phase with sucrose (0.5 M) for 90 min at the pH 7.9 to 8.0 is a prerequisite for the formation of 'protoplasts'. Under these conditions, the yield of 'protoplasts' reached 99.9% of the total cell number. The protoplast-like structures are capable of reversing into the original bacterial forms in a minimal hypertonic medium containing amino acids which are components of the cell wall. The level of reversion reaches 10% for certain strains.     

Evaluation of Cell Binding Activities of Leptospira ECM Adhesins

Pathogenic spirochetes of the genus Leptospira are the causative agents of leptospirosis, a zoonotic infection that occurs globally. The bacteria colonize the renal proximal tubules of many animals and are shed in the urine. Contact with the urine, or with water contaminated with the urine of infected animals can cause infection of new host animals, including humans. Mechanisms of colonization of the proximal tubule and other tissues are not known, but specific interactions between bacterial adhesins and host substrates are likely to be critical in this process. Several extracellular matrix (ECM) adhesins have been previously identified, but more recently, it has been shown that Leptospira bind more efficiently to cells than ECM. In this work, recombinant forms of five putative Leptospira ECM adhesins, namely LipL32, Loa22, OmpL1, p31/LipL45, and LenA were evaluated for binding to cells as well as an expanded variety of ECM components. Reproducible and significant adhesin activity was demonstrated only for OmpL1, which bound to both mammalian cell lines tested and to glycosaminoglycans (GAGs). While determination of biologically significant bacterial adhesion activity will require generation of site-directed mutant strains, our results suggest that OmpL1 is a strong candidate for future evaluation regarding the roles of the adhesin activity of the protein during L. interrogans infection.     

Profiling and identification of eubacteria in the stomach of Mongolian gerbils with and without Helicobacter pylori infection

Background. Mongolian gerbils are frequently used to study Helicobacter pylori‐induced gastritis and its consequences. The presence of an indigenous bacterial flora with suppressive effect on H. pylori may cause difficulties with establishing this experimental model. Aim. The aim of the present study was to determine bacterial profiles in the stomach of Mongolian gerbils with and without (controls) H. pylori infection. Methods. Gastric tissue from H. pylori ATCC 43504 and CCUG 17874 infected and control animals were subjected to microbial culturing and histology. In addition, gastric mucosal samples from H. pylori ATCC 43504 infected and control animals were analyzed for bacterial profiling by temporal temperature gradient gel electrophoresis (TTGE), cloning and pyrosequencing of 16S rDNA variable V3 region derived PCR amplicons. Results. Oral administration of H. pylori ATCC 43504, but not CCUG 17874, induced colonization and gastric inflammation in the stomach of Mongolian gerbils. Temporal temperature gradient gel electrophoresis (TTGE) and partial 16S rDNA pyrosequencing revealed the presence of DNA representing a mixed bacterial flora in the stomach of both H. pylori ATCC 43504 infected and control animals. In both cases, lactobacilli appeared to be dominant. Conclusion. These findings suggest that indigenous bacteria, particularly lactobacilli, may have an impact on the colonization and growth of H. pylori strains in the stomach of Mongolian gerbils.     

Spread and Transmission of Bacterial Pathogens in Experimental Populations of the Nematode Caenorhabditis elegans

Caenorhabditis elegans is frequently used as a model species for the study of bacterial virulence and innate immunity. In recent years, diverse mechanisms contributing to the nematode''s immune response to bacterial infection have been discovered. Yet despite growing interest in the biochemical and molecular basis of nematode-bacterium associations, many questions remain about their ecology. Although recent studies have demonstrated that free-living nematodes could act as vectors of opportunistic pathogens in soil, the extent to which worms may contribute to the persistence and spread of these bacteria has not been quantified. We conducted a series of experiments to test whether colonization of and transmission between C. elegans nematodes could enable two opportunistic pathogens (Salmonella enterica and Pseudomonas aeruginosa) to spread on agar plates occupied by Escherichia coli. We monitored the transmission of S. enterica and P. aeruginosa from single infected nematodes to their progeny and measured bacterial loads both within worms and on the plates. In particular, we analyzed three factors affecting the dynamics of bacteria: (i) initial source of the bacteria, (ii) bacterial species, and (iii) feeding behavior of the host. Results demonstrate that worms increased the spread of bacteria through shedding and transmission. Furthermore, we found that despite P. aeruginosa''s relatively high transmission rate among worms, its pathogenic effects reduced the overall number of worms colonized. This study opens new avenues to understand the role of nematodes in the epidemiology and evolution of pathogenic bacteria in the environment.     

Evidence for the Involvement of Pathogenic Bacteria in Summer Mortalities of the Pacific Oyster <Emphasis Type="Italic">Crassostrea gigas</Emphasis>

A study was conducted to investigate the involvement of bacteria in oyster mortalities during summer. Moribund and apparently healthy oysters were sampled during mortality events along the French coast and in rearing facilities, usually when temperature reached 19°C or higher, and oysters were in the gonadal maturation phase. Hemolymph samples were aseptically withdrawn and submitted to bacteriological analysis. In healthy oysters, bacteria colonized hemolymph at low concentrations depending on the location. In most moribund oysters, bacteria were present in hemolymph and other tissues. These bacterial populations were more often diverse in oysters originating from the open sea than from facilities where animals were generally infected by a single type of bacterium. Only the dominant colonies were identified by phenotypic and genotypic characters (RFLP of GyrB gene and partial sequence of 16S rRNA gene). They belonged to a limited number of species including Vibrio aestuarianus, members of the V. splendidus group, V. natriegens, V. parahaemolyticus, and Pseudoalteromonas sp. The most frequently encountered species was V. aestuarianus (56% of isolates), which was composed of several strains closely related by their 16S rRNA gene but diverse by their phenotypic characters. They appeared intimately linked to oysters. The species within the V. splendidus group were less prevalent (25% of isolates) and more taxonomically dispersed. A majority of the dominant strains of V. aestuarianus and V. splendidus group injected to oysters induced mortality, whereas others belonging to the same species, particularly those found in mixture, appeared innocuous.     

Host tissues as microhabitats for Wolbachia and quantitative insights into the bacterial community in terrestrial isopods

Animal–bacterial symbioses are highly dynamic in terms of multipartite interactions, both between the host and its symbionts as well as between the different bacteria constituting the symbiotic community. These interactions will be reflected by the titres of the individual bacterial taxa, for example via host regulation of bacterial loads or competition for resources between symbionts. Moreover, different host tissues represent heterogeneous microhabitats for bacteria, meaning that host‐associated bacteria might establish tissue‐specific bacterial communities. Wolbachia are widespread endosymbiotic bacteria, infecting a large number of arthropods and filarial nematodes. However, relatively little is known regarding direct interactions between Wolbachia and other bacteria. This study represents the first quantitative investigation of tissue‐specific Wolbachia–microbiota interactions in the terrestrial isopod Armadillidium vulgare. To this end, we obtained a more complete picture of the Wolbachia distribution patterns across all major host tissues, integrating all three feminizing Wolbachia strains (wVulM, wVulC, wVulP) identified to date in this host. Interestingly, the different Wolbachia strains exhibited strain‐specific tissue distribution patterns, with wVulM reaching lower titres in most tissues. These patterns were consistent across different host genetic backgrounds and might reflect different co‐evolutionary histories between the Wolbachia strains and A. vulgare. Moreover, Wolbachia‐infected females carried higher total bacterial loads in several, but not all, tissues, irrespective of the Wolbachia strain. Taken together, this quantitative approach indicates that Wolbachia is part of a potentially more diverse bacterial community, as exemplified by the presence of highly abundant bacterial taxa in the midgut caeca of several A. vulgare populations.     

Induction and migration of cryptic/defective <Emphasis Type="Italic">Salmonella enterica</Emphasis> prophages as a consequence of infection with lytic phages is an additional factor in stability of a coevolutionary vector

The influence of infection of natural isolates of Salmonella enterica with lytic (nonlysogenic) phages on the expression of resident cryptic or defective prophages in host bacteria was studied. The induction of defective/cryptic phages after infection with nonlysogenic phages and packaging of bacterial chromosomal fragments in capsids of defective phages is demonstrated. This may lead to migration and wide distribution of both the genomes of defective phages per se and various fragments of the bacterial chromosome (including pathogenic islands) in new bacterial strains with concomitant change of their properties, the acquired new features of pathogenicity among them.     

Salmonella typhimurium strains carrying independent mutations display similar virulence phenotypes yet are controlled by distinct host defense mechanisms

The outcome of Salmonella infection in the mammalian host favors whoever succeeds best in disturbing the equilibrium between coordinate expression of bacterial (virulence) genes and host defense mechanisms. Intracellular persistence in host cells is critical for pathogenesis and disease, because Salmonella typhimurium strains defective in this property are avirulent. We examined whether similar host defense mechanisms are required for growth control of two S. typhimurium mutant strains. Salmonella pathogenicity island 2 (SPI2) and virulence plasmid-cured Salmonella mutants display similar virulence phenotypes in immunocompetent mice, yet their gene loci participate in independent virulence strategies. We determined the role of TNF-alpha and IFN-gamma as well as different T cell populations in infection with these Salmonella strains. After systemic infection, IFN-gamma was essential for growth restriction of plasmid-cured S. typhimurium, while SPI2 mutant infections were controlled in the absence of IFN-gamma. TNFRp55-deficiency restored systemic virulence to both Salmonella mutants. After oral inoculation, control of plasmid-cured bacteria substantially relied on both IFN-gamma and TNF-alpha signaling while control of SPI2 mutants did not. However, for both mutants, ultimate clearance of bacteria from infected mice depended on alphabeta T cells.     

Cytotoxicity and Calcium-Dependent Antigen of Yersinia

The relationship between invasiveness and calcium dependency was examined in various strains of Yersinia enterocolitica and Y. pseudotuberculosis by using established cell lines. Infection with calcium-dependent bacteria resulted in the formation of microvilli and the adherence of bacteria on the cell surface, and the adherent bacteria were ingested 1.5 hr after infection. Morphological changes in the cells became visible 2 to 3 hr after infection, and intracellular multiplication of the ingested bacteria was noted. When the cells were incubated with bacteria at 37 C for 1.5 hr and then at 25 C, however, the morphological changes in the infected cells were not observed. No isogenic strains that had lost calcium dependency for growth at 37 C were able to elicit the morphological changes in the cells, though they possessed the ability to adhere to and penetrate the cells. The antigen(s) supposedly related to cytotoxicity of the calcium-dependent Yersinia was sought by using antibodies prepared against calcium-dependent bacteria and then absorbed with calcium-independent bacteria and with calcium-independent bacterial cytosol. Double diffusion tests between the antisera and bacterial cytosol extracts revealed the presence of an antigen which was a cytoplasmic substance common to all calcium-dependent but not calcium-independent strains of Y. enterocolitica and Y. pseudotuberculosis.     

Influence of lipopolysaccharide on external hemolytic activity ofSalmonella typhimurium andKlebsiella pneumoniae

Excretion ofEscherichia coli -hemolysin was tested in rough and smooth strains ofKlebsiella pneumoniae andSalmonella typhimurium. Smooth strains harboring the hemolytic recombinant plasmid pANN202-312 showed a five- to tenfold increase in the hemolytic activity evaluated in the external medium compared with isogenic rough strains harboring the same plasmid.     

Transformation of forage legumes using Agrobacterium tumefaciens

Summary Galls were induced in six species of forage legumes following inoculation with wild-type strains of A. tumefaciens. The plant species was more influential than the bacterial strain in determining the type of tumour produced. Inoculation of Medicago sativa resulted in small, disorganised tumours. The three Trifolium species, T. repens, T. hybridum and T. pratense, formed galls which tended to produce roots and both Onobrychis viciifolia and Lotus corniculatus produced teratomatous galls. The shoots elongated in the latter species only. In L. corniculatus, tissues that were infected by five bacterial strains were capable of shoot regeneration when cultured on a hormone-free medium. The transformed nature of these shoots was confirmed by their failure to root, the production of callus from leaves cultured on hormone-free medium and the presence of opines.