Survival of Aeromonas salmonicida in river water

Abstract By definition, Aeromonas salmonicida is found in fish but never in surface water. However, this does not explain the reason for explosive out-breaks of furunculosis among populations of salmonid fish which have never been exposed to the disease. Evidence is presented, from laboratory-based experiments, which show that A. salmonicida survives in freshwater, beyond the period necessary for plate counts to reach zero. These cells may subsequently be re-activated by the addition of nutrient. It may be assumed, therefore, that A. salmonicida survives outside of fish, by entering a dormant phase.     

Survival and Activity of lux-Marked Aeromonas salmonicida in Seawater

The fish pathogen Aeromonas salmonicida was chromosomally marked with genes encoding bacterial luciferase, luxAB, isolated from Vibrio fischeri, resulting in constitutive luciferase production. During exponential growth in liquid batch culture, luminescence was directly proportional to biomass concentration, and luminometry provided a lower detection limit of approximately 10(sup3) cells ml(sup-1), 1 order of magnitude more sensitive than enzyme-linked immunosorbent assay detection. In sterile seawater at 4(deg)C, lux-marked A. salmonicida entered a dormant, nonculturable state and population activity decreased rapidly. The activity per viable cell, however, increased by day 4, indicating that a proportion of the population remained active and culturable. Putative dormant cells were not resuscitated after the addition of a range of substrates.     

Protease and peptidase activities in growing and sporulating cells and dormant spores of Bacillus megaterium.

Peptidase and protease activities on many different substrates have been determined in several stages of growth of Bacillus megaterium. Extracts of log-phase cells, sporulating cells, and dormant spores of B. megaterium each hydrolyzed 16 different di- and tripeptides. The specific peptidase activity was highest in dormant spores, and the activity in sporulating cells and log-phase cells was about 1.2-fold and 2- to 3-fold lower, respectively. This peptidase acticity was wholly intracellular since extracellular peptidase activity was not detected throughout growth and sporulation. In contrast, intracellular protease activity on a variety of common protein substrates was highest in sporulating cells, and much extracellular activity was also present at this time. The specific activity of intracellular protease in sporulating cells was about 50- and 30-fold higher than that in log-phase cells and dormant spores, respectively. However, the two unique dormant spores proteins known to be the major species degraded during spore germination were degraded most rapidly by extracts of dormant spores, and slightly slower by extracts from log-phase or sporulating cells. The specific activities for degradation of peptides and proteins are compared to values for intracellular protein turnover during various stages of growth.     

Characterization of an ADP-ribosyltransferase toxin (AexT) from Aeromonas salmonicida subsp. salmonicida

An ADP-ribosylating toxin named Aeromonas salmonicida exoenzyme T (AexT) in A. salmonicida subsp. salmonicida, the etiological agent of furunculosis in fish, was characterized. Gene aexT, encoding toxin AexT, was cloned and characterized by sequence analysis. AexT shows significant sequence similarity to the ExoS and ExoT exotoxins of Pseudomonas aeruginosa and to the YopE cytotoxin of different Yersinia species. The aexT gene was detected in all of the 12 A. salmonicida subsp. salmonicida strains tested but was absent from all other Aeromonas species. Recombinant AexT produced in Escherichia coli possesses enzymatic ADP-ribosyltransferase activity. Monospecific polyclonal antibodies directed against purified recombinant AexT detected the toxin produced by A. salmonicida subsp. salmonicida and cross-reacted with ExoS and ExoT of P. aeruginosa. AexT toxin could be detected in a wild type (wt) strain of A. salmonicida subsp. salmonicida freshly isolated from a fish with furunculosis; however, its expression required contact with RTG-2 rainbow trout gonad cells. Under these conditions, the AexT protein was found to be intracellular or tightly cell associated. No AexT was found when A. salmonicida subsp. salmonicida was incubated in cell culture medium in the absence of RTG-2 cells. Upon infection with wt A. salmonicida subsp. salmonicida, the fish gonad RTG-2 cells rapidly underwent significant morphological changes. These changes were demonstrated to constitute cell rounding, which accompanied induction of production of AexT and which led to cell lysis after extended incubation. An aexT mutant which was constructed from the wt strain with an insertionally inactivated aexT gene by allelic exchange had no toxic effect on RTG-2 cells and was devoid of AexT production. Hence AexT is directly involved in the toxicity of A. salmonicida subsp. salmonicida for RTG-2 fish cells.     

Changes in Terminal Respiratory Pathways of Bacillus subtilis During Germination, Outgrowth, and Vegetative Growth

The chemical and enzymatic properties of the cytochrome system in the particulate preparations obtained from dormant spores, germinated spores, young vegetative cells, and vegetative cells of Bacillus subtilis PCI219 were investigated. Difference spectra of particulate fractions from dormant spores of this strain suggested the presence of cytochromes a, a(3), b, c(+c(1)), and o. All of the cytochrome components were present in dormant spores and in germinated spores and vegetative cells at all stages which were investigated. Concentrations of cytochromes a, a(3), b, and c(+c(1)) increased during germination, outgrowth, and vegetative growth, but that of cytochrome o was highest in dormant spores. As the cytochrome components were reducible by reduced nicotinamide adenine dinucleotide (NADH), they were believed to be metabolically active. Difference spectra of whole-cell suspensions of dormant spores and vegetative cells were coincident with those of the particulate fractions. NADH oxidase and cytochrome c oxidase were present in dormant spores, germinated spores, and vegetative cells at all stages after germination, but succinate cytochrome c reductase was not present in dormant spores. Cytochrome c oxidase and succinate cytochrome c reductase activities increased with growth, but NADH oxidase activity was highest in germinated spores and lowest in vegetative cells. There was no striking difference between the effects of respiratory inhibitors on NADH oxidase in dormant spores and those on NADH oxidase in vegetative cells.     

Susceptibility of Friend virus antigen-modulated erythroleukemic cells to lysis by T lymphocytes from mice with dormant Friend virus infections.

Spleen cells from DBA/2 mice with dormant Friend leukemia virus (FLV) infections or from mice immunized with x-irradiated FLC-745 erythroleukemic cells are not cytolytic for FLC-745 cells when tested directly, but acquire cytolytic activity in vitro by cultivation with x-irradiated FLC-745 cells. Spleen cells cultured from normal mice do not acquire such cytolytic activity. Cytolytic activity resides in the T lymphocyte population. Alloantiserum, but not antisera against FLV-virion polypeptides, inhibited the lysis of FLC-745 cells by cytolytic T lymphocytes. To understand further the role of cellular and humoral immune anti-FLV responses in mice with dormant FLV-infections, in vitro experiments were conducted that mimicked the in vivo FLV-specific immune environment of these mice. We found that FLV-immune serum from mice with dormant FLV-infections modulated FLV-antigen expression on the surfaces of FLC-745 cells without affecting their susceptibility to lysis by cytolytic T lymphocytes. These results suggest that cytolytic T lymphocyte restrain the outgrowth of FLV-antigen-modulated erythroleukemic cells in mice with dormant FLV infections and that the targets for cell-mediated lysis may be H-2d-associated antigens.     

Ultrastructure of Yersinia pseudotuberculosis in the process of their reversible transition into the dormant (non-culturable) state in association with blue-green algae

The ultrastructural organization of Y. pseudotuberculosis in the process of the transition of vegetative cells into the dormant (noncultivable) state in interaction with blue-green algae of the species Anabaena variabilis was studied by the method of transmission electron microscopy. The use of type specific Y. pseudotuberculosis serum made it possible to identify Y. pseudotuberculosis cells in the bacterial association and to find out whether their antigenic properties remained intact in time. The dormant forms of Y. pseudotuberculosis, recultivated by passage through the axenic culture of unfusoria (Tetrahymena pyryformis), were also studied with the use of electron microscopy. The revertants were found to be at different stages of restoration of their typical morphological characteristics and antigenic properties were partially retained. The fine structure of Y. pseudotuberculosis cells in the initial culture was shown to be similar to that of the revertants of dormant forms, morphological criteria of the dormant cell ultrastructure were established. The cyclic processes of reversible transition from vegetative forms to dormant ones in bacterial populations under the influence of hydrobios is regarded as an adaptive mechanism of their existence in the environment.     

New transfer ribonucleic acid species during sporulation of Bacillus subtilis.

The transfer ribonucleic acid (tRNA) populations from log-phase cells, sporulating cells (stage III), and dormant spores were compared by tRNA-deoxyribonucleic acid hybridization techniques. New tRNA species not found in log-phase cells were observed in stage III cells. Some of the tRNA made during sporulation were also present in dormant spores. Although the role and function of these new tRNA species cannot be ascribed directly to the sporulation process, their presence indicates that new tRNA genes can be transcribed during sporulation and suggests that translational control may be exerted during sporulation by tRNA.     

Employing a triple metabarcoding approach to differentiate active,dormant and dead microeukaryotes in sediments

Microbial communities are commonly characterised through the metabarcoding of environmental DNA. This DNA originates from both viable (including dormant and active) and dead organisms, leading to recent efforts to distinguish between these states. In this study, we further these approaches by distinguishing not only between viable and dead cells but also between dormant and actively growing cells. This is achieved by sequencing both rRNA and rDNA, in conjunction with propidium monoazide cross-linked rDNA, to partition the active, dormant and relic fractions in environmental samples. We apply this method to characterise the diversity and assemblage structure of these fractions of microeukaryotes in intertidal sediments during a wet-dry-rewet incubation cycle. Our findings indicate that a significant proportion of microeukaryotic phylotypes detected in the total rDNA pools originate from dormant and relic microeukaryotes in the sediments, both in terms of richness (dormant, 13 ± 2%; relic, 47 ± 5%) and read abundance (dormant, 20 ± 7%; relic, 14 ± 5%). The richness and sequence proportion of dormant microeukaryotes notably increase during the transition from wet to dry conditions. Statistical analyses suggest that the dynamics of diversity and assemblage structure across different activity fractions are influenced by various environmental drivers. Our strategy offers a versatile approach that can be adapted to characterise other microbes in a wide range of environments.     

Survival of Aeromonas salmonicida in lake water

The survival of Aeromonas salmonicida subsp. salmonicida in lake water was investigated by using a variety of techniques. They included acridine orange epifluorescence, respiration, cell culture, cell revival, flow cytometry, plasmid maintenance, and membrane fatty acid analysis. During a 21-day study, A. salmonicida became nonculturable in sterile lake water samples. Flow cytometry and direct microscopy indicated that cells were present. Although the nonculturable cells could not be revived, the recovery method did indicate that the presence of low numbers of culturable cells within samples could produce misleading results. Plasmid DNA, genomic DNA, and RNA were maintained in the nonculturable cells; in addition, changes in the fatty acid profiles were also detected. Although viability could not be proven, it was shown that the morphological integrity of nonculturable cells was maintained.     

Dormancy of Solitary Metastatic Cells

After arriving in a secondary site metastatic cells either begin proliferating, undergo apoptosis or remain as solitary dormant cells. The process of metastasis, although dangerous, is extremely inefficient with the majority of the cells undergoing apoptosis and thus becoming clinically irrelevant. Of the cells that begin proliferating, the few that make it past the micrometastasis stage may be of immediate clinical relevance. Dormant cells, while not of immediate clinical concern, are believed to be at least in part responsible for cancer recurrence that can occur decades after apparently successful initial treatment. Dormant solitary cells are different from “dormant” micrometastases, in which active proliferation is balanced by apoptosis. The mechanisms of cell cycle regulation and the function of the molecules regulating this process are well understood. However, there is relatively little known about the mechanisms controlling cell cycle regulation and dormancy of solitary metastatic cells. There are several inherent difficulties impeding the study of solitary cells. This review paper will examine the models used in the study of dormant solitary metastatic cells, methods of imaging and studying these cells, the molecular mechanisms believed to be responsible for solitary cell dormancy, and finally the unique treatment challenges posed by these cells.     

Infection of human THP-1 cells with dormant Mycobacterium tuberculosis

Dormant, non-replicating Mycobacterium tuberculosis H37Rv strain cultured in hypoxic conditions was used to infect THP-1 cells. CFUs counting, Kinyoun staining and electron microscopy showed that dormant bacilli infected THP-1 cells at a rate similar to replicating M. tuberculosis, but failed to grow during the first 6 days of infection. The absence of growth was specific to the intracellular compartment, as demonstrated by efficient growth in liquid medium. Quantification of β-actin mRNA recovered from infected cells showed that, in contrast with log-phase bacteria, infection with dormant bacilli determined a reduced THP-1 cell death. Gene expression of intracellular non-replicating bacteria showed a pattern typical of a dormant state. Intracellular dormant bacteria induced the activation of genes associated to a proinflammatory response in THP-1 cells. Though, higher levels of TNFα, IL-1β and IL-8 mRNAs compared to aerobic H37Rv infected cells were not paralleled by increased cytokine accumulation in the supernatants. Moreover, dormant bacilli induced a higher expression of inducible cox-2 gene, accompanied by increased PGE2 secretion. Overall, our data describe a new model of in vitro infection using dormant M. tuberculosis that could provide the basis for understanding how non-replicating bacilli survive intracellularly and influence the maintenance of the hypoxic granuloma.     

Survival of Aeromonas salmonicida in lake water.

The survival of Aeromonas salmonicida subsp. salmonicida in lake water was investigated by using a variety of techniques. They included acridine orange epifluorescence, respiration, cell culture, cell revival, flow cytometry, plasmid maintenance, and membrane fatty acid analysis. During a 21-day study, A. salmonicida became nonculturable in sterile lake water samples. Flow cytometry and direct microscopy indicated that cells were present. Although the nonculturable cells could not be revived, the recovery method did indicate that the presence of low numbers of culturable cells within samples could produce misleading results. Plasmid DNA, genomic DNA, and RNA were maintained in the nonculturable cells; in addition, changes in the fatty acid profiles were also detected. Although viability could not be proven, it was shown that the morphological integrity of nonculturable cells was maintained.     

Structural Changes in the Vascular Cambium of Pinus strobus L. during an Annual Cycle

The changes in the ultrastructure of cambial cells of easternwhite pine (Pinus strobus L.) during an annual cycle are observedand recorded as are relationships of cambial cells during dormancyand at resumption of cambial activity. Cambial activity wasresumed late in March or early in April, when a few cells dividedpericlinally. Cambial activity reached a maximum during thelatter part of May with 15 to 20 undifferentiated cells present.In July it declined markedly, and the number of undifferentiatedcells equalled that of the dormant period. The xylem and phloemtissue cells produced late in the annual cycle overwinteredat varying developmental stages. In October cambial cells structurallyresembled dormant cells. The number of dormant cells in easternwhite pine cambium varied from 6 to 10. Active cells were characterizedby a large central vacuole, by an abundance of all cell organelles,and by thin cell walls. Dormant cells were characterized bynumerous small vacuoles, by structurally and quantitativelymodified cell organelles, and by relatively thick cell walls.     

GAS6 Receptor Status Is Associated with Dormancy and Bone Metastatic Tumor Formation

Disseminated tumor cells (DTCs) are believed to lie dormant in the marrow before they can be activated to form metastases. How DTCs become dormant in the marrow and how dormant DTCs escape dormancy remains unclear. Recent work has shown that prostate cancer (PCa) cell lines express the growth-arrest specific 6 (GAS6) receptors Axl, Tyro3, and Mer, and become growth arrested in response to GAS6. We therefore hypothesized that GAS6 signaling regulates the proliferative activity of DTCs in the marrow. To explore this possibility, in vivo studies were performed where it was observed that when Tyro3 expression levels exceed Axl expression, the PCa cells exhibit rapid growth. When when Axl levels predominate, PCa cells remain largely quiescent. These findings suggest that a balance between the expression of Axl and Tyro3 is associated with a molecular switch between a dormant and a proliferative phenotype in PCa metastases.     

Model of Tumor Dormancy/Recurrence after Short-Term Chemotherapy

Although many tumors regress in response to neoadjuvant chemotherapy, residual tumor cells are detected in most cancer patients post-treatment. These residual tumor cells are thought to remain dormant for years before resuming growth, resulting in tumor recurrence. Considering that recurrent tumors are most often responsible for patient mortality, there exists an urgent need to study signaling pathways that drive tumor dormancy/recurrence. We have developed an in vitro model of tumor dormancy/recurrence. Short-term exposure of tumor cells (breast or prostate) to chemotherapy at clinically relevant doses enriches for a dormant tumor cell population. Several days after removing chemotherapy, dormant tumor cells regain proliferative ability and establish colonies, resembling tumor recurrence. Tumor cells from “recurrent” colonies exhibit increased chemotherapy resistance, similar to the therapy resistance of recurrent tumors in cancer patients. Previous studies using long-term chemotherapy selection models identified acquired mutations that drive tumor resistance. In contrast, our short term chemotherapy exposure model enriches for a slow-cycling, dormant, chemo-resistant tumor cell sub-population that can resume growth after drug removal. Studying unique signaling pathways in dormant tumor cells enriched by short-term chemotherapy treatment is expected to identify novel therapeutic targets for preventing tumor recurrence.     

Survival of nonculturable Aeromonas salmonicida in lake water.

The survival of Aeromonas salmonicida subsp. salmonicida was investigated in sterile and untreated lake water. In sterile lake water (filtered and autoclaved), it was found that cells of A. salmonicida entered a nonculturable but viable condition. Viability was determined by flow cytometry with the dye rhodamine 123, which is taken up and maintained within cells with a membrane potential. For survival studies in untreated lake water, A. salmonicida was marked with the xylE gene by using the plasmid pLV1013. Marked cells were detected by growth on tryptone soy agar and tryptone soy agar supplemented with kanamycin. Cells were also detected by polymerase chain reaction DNA amplification of the xylE gene and a chromosomal DNA fragment specific for A. salmonicida (pLV1013). The results indicated that A. salmonicida entered a nonculturable condition in untreated lake water over a 21-day study. The viability of nonculturable cells could not be determined in mixed samples; however, the presence of nonculturable cells containing both chromosomal and plasmid DNA was confirmed.     

Dormant Wnt-initiated mammary cancer can participate in reconstituting functional mammary glands

The minimal residual disease foci that beget breast cancer relapse after a period of disease dormancy remain uncharacterized despite their enormous clinical importance. To model dormant breast cancer in vivo, we employed a transgenic mouse model in which Wnt1-initiated mammary cancer is doxycycline dependent. After regression of Wnt-dependent cancers, subclinical disease lesions were propagated in vivo using classical tissue recombination techniques. Surprisingly, outgrowths derived from dormant malignant tissue reconstituted morphologically normal ductal trees in wild-type mammary fat pads. Whereas hyperplasia-derived outgrowths remained benign, outgrowths derived from dormant malignancy underwent a morphological transition suggesting single-step transformation following reactivation of Wnt signaling and rapidly yielded invasive mammary tumors. Remarkably, outgrowths derived from dormant malignancy could be serially propagated in vivo and retained the potential to undergo lobuloalveolar differentiation in response to hormones of pregnancy. Matching somatic H-Ras mutations shared by antecedent tumors and descendant mammary ductal outgrowths confirmed their clonal relatedness. Thus, propagation of epithelium that possesses a latent malignant growth program reveals impressive regenerative and developmental potential, supporting the notion that dormant mammary cancers harbor transformed mammary progenitor cells. Our results define an experimental paradigm for elucidating biological properties of dormant malignancy.     

Harnessing redox signaling to overcome therapeutic-resistant cancer dormancy

Dormancy occurs when cells preserve viability but stop proliferating, which is considered an important cause of tumor relapse, which may occur many years after clinical remission. Since the life cycle of dormant cancer cells is affected by both intracellular and extracellular factors, gene mutation or epigenetic regulation of tumor cells may not fully explain the mechanisms involved. Recent studies have indicated that redox signaling regulates the formation, maintenance, and reactivation of dormant cancer cells by modulating intracellular signaling pathways and the extracellular environment, which provides a molecular explanation for the life cycle of dormant tumor cells. Indeed, redox signaling regulates the onset of dormancy by balancing the intrinsic pathways, the extrinsic environment, and the response to therapy. In addition, redox signaling sustains dormancy by managing stress homeostasis, maintaining stemness and immunogenic equilibrium. However, studies on dormancy reactivation are still limited, partly explained by redox-mediated activation of lipid metabolism and the transition from the tumor microenvironment to inflammation. Encouragingly, several drug combination strategies based on redox biology are currently under clinical evaluation. Continuing to gain an in-depth understanding of redox regulation and develop specific methods targeting redox modification holds the promise to accelerate the development of strategies to treat dormant tumors and benefit cancer patients.