Secretion of an antibacterial factor during resuscitation of dormant cells inMicrococcus luteus cultures held in an extended stationary phase

A high proportion ofMicrococcus luteus cells in cultures starved for 3–6 months in spent medium following growth to stationary phase in batch culture lost the ability to grow and form colonies on agar plates, but could be resuscitated from dormancy by incubation in liquid medium containing supernatant taken from the late log phase of viable cultures of the same organism (Kaprelyants et al. 1994). In the present work, we found that during the first 50–70 h of such resuscitation the dormant cells actually divide for 10–17 generations in lactate minimal medium containing yeast extract whilst remaining nonculturable on agar plates. Further incubation results in a decrease in the total cell number in liquid medium. The addition of viable (culturable)Micrococcus luteus cells in concentrations of up to 10⁴ ml^–1 to test tubes containing either resuscitating cells or supernatant from these cultures revealed the excretion of a factor or factors which inhibited the proliferation of otherwise viable cells. The maximum production of this factor took place after some 96 h of incubation of starved cells in resuscitation medium. Supernatant from late logarithmic phase batch cultures ofM. luteus abolished the antibacterial effect of starved cultures incubated in resuscitation medium. It is concluded that the stimulating effect of viable cells, and of supernatant taken from batch cultures, on the resuscitation of dormant cells might be connected in part with overcoming the activity of an antibacterial factor causing self-poisoning of dormant cells during their resuscitation.     

Formation of nonculturable Mycobacterium tuberculosis and their regeneration

Nonculturable cells were found to occur in populations of Mycobacterium tuberculosis cells during the long post-stationary phase. These cells were small (0.6-0.8 micron) ovoid and coccoid forms with intact cell walls and negligible respiratory activity, which allows them to be regarded as dormant cells. Nonculturable cells were characterized by low viability after plating onto solid medium; a minor part of the population of these cells could be cultivated in liquid medium. Cell-free culture liquid of an exponential-phase Mycobacterium tuberculosis culture or the bacterial growth factor Rpf exerted a resuscitating effect, increasing substantially the growth capacity of the nonculturable cells in liquid medium. During resuscitation of nonculturable cells, a transition from ovoid to rodlike cell shape occurred. At early stages of resuscitation, ovoid cells formed small aggregates. The recovery of culturability was associated with the formation of rod-shaped cells in the culture. The data obtained demonstrate the in vitro formation of dormant cells of Mycobacterium tuberculosis, which do not grow on solid media but can be resuscitated in liquid medium under the effect of substance(s) secreted by actively growing cells.     

Formation of Nonculturable Cells of Mycobacterium tuberculosis and Their Resuscitation

Nonculturable cells were found to occur in populations of Mycobacterium tuberculosis cells during the long poststationary phase. These cells were small (0.6–0.8 m) ovoid and coccoid forms with intact cell walls and negligible respiratory activity, which allows them to be regarded as dormant cells. Nonculturable cells were characterized by low viability after plating onto solid medium; a minor part of the population of these cells could be cultivated in liquid medium. Cell-free culture liquid of an exponential-phase Mycobacterium tuberculosisculture or the bacterial growth factor Rpf exerted a resuscitating effect, increasing substantially the growth capacity of the nonculturable cells in liquid medium. During resuscitation of nonculturable cells, a transition from ovoid to rodlike cell shape occurred. At early stages of resuscitation, ovoid cells formed small aggregates. The recovery of culturability was associated with the formation of rod-shaped cells in the culture. The data obtained demonstrate the in vitro formation of dormant cells of Mycobacterium tuberculosis, which do not grow on solid media but can be resuscitated in liquid medium under the effect of substance(s) secreted by actively growing cells.     

Stimulation of the multiplication of Micrococcus luteus by an autocrine growth factor

Viable cells of Micrococcus luteus secrete a proteineous growth factor (Rpf) which promotes the resuscitation of dormant, nongrowing cells to yield normal, colony-forming bacteria. When washed M. luteus cells were used as an inoculum, there was a pronounced influence of Rpf on the true lag phase and cell growth on lactate minimal medium. In the absence of Rpf, there was no increase in colony-forming units for up to 10 days. When the inoculum contained less than 10⁵ cells ml^–1, macroscopically observable M. luteus growth was not obtained in succinate minimal medium unless Rpf was added. Incubation of M. luteus in the stationary phase for 100 h resulted in a failure of the cells to grow in lactate minimal medium from inocula of small size although the viability of these cells was close to 100% as estimated using agar plates made from lactate minimal medium or rich medium. The underestimation of viable cells by the most-probable-number (MPN) method in comparsion with colony-forming units was equivalent to the requirement that at least 10⁵ cells grown on succinate medium, 10³ cells from old stationary phase, or approximately 10–500 washed cells are required per millilitre of inoculum for growth to lead to visible turbidity. The addition of Rpf in the MPN dilutions led to an increase of the viable cell numbers estimated to approximately the same levels as those determined by colony-forming units. Thus, a basic principle of microbiology –“one cell-one culture”– may not be applicable in some circumstances in which the metabolic activity of “starter” cells is not sufficient to produce enough autocrine growth factor to support cell multiplication. Received: 7 December 1998 / Accepted: 7 April 1999     

Influence of Viable Cells on the Resuscitation of Dormant Cells in Micrococcus luteus Cultures Held in an Extended Stationary Phase: the Population Effect

A high proportion of Micrococcus luteus cells in cultures which had been starved for 3 to 6 months lost the ability to grow and form colonies on agar plates but could be resuscitated from their dormancy by incubation in an appropriate liquid medium (A. S. Kaprelyants and D. B. Kell, Appl. Environ. Microbiol. 59:3187-3196, 1993). In the present work, such cultures were studied by both flow cytometry and conventional microbiological methods and were found to contain various numbers of viable cells. Pretreatment of such cultures with penicillin G, and subsequent dilution, was used to vary this number. When the initial number of colony-forming cells per 30-ml flask was approximately nine (±five) or more, resuscitation of 10 to 40% of the cells, and thus culture growth, was observed. The lag period before the appearance of a population of cells showing significant accumulation of the fluorescent dye rhodamine 123 (i.e., of cells with measurable membrane energization) decreased from 70 to 27 h when the number of viable cells was increased from 30 to 10⁵ per flask, while the lag period before an observable increase in the number of colony-forming cells occurred was almost constant (at some 20 h). Provided there were more than nine (±five) initially viable cells per flask, the number of initially viable cells did not affect the final percentage of resuscitable cells in the culture. The lag period could be ascribed in part to the time taken to restore the membrane permeability barrier of starved cells during resuscitation, as revealed by flow cytometric assessment of the uptake of the normally membrane-impermeant fluorescent DNA stain PO-PRO-3 {4-[3-methyl-2, 3-dihydro-(benzo-1, 3-oxazole)-2-methylidene]-1-(3′-trimethylammonium propyl)-pyridinium diiodide}. Although cell populations which contained fewer than nine ±five viable cells per flask failed to grow, 4 to 20% of the cells (of 1.2 X 10⁶) were able to accumulate rhodamine 123 after 80 to 100 h of incubation, showing the ability of a significant number of the cells in the population at least to display “metabolic resuscitation.” Resuscitation and cell growth under such conditions were favored by the use of a 1:1 mixture of fresh lactate medium and supernatant from late-logarithmic-phase M. luteus cultures as the resuscitation medium. We conclude that the presence of a small fraction of viable cells at the onset of resuscitation facilitates the recovery of the majority of the remaining (dormant) cells. The cell density dependence of the kinetics, or population effect, suggests that this recovery is due to the excretion of some factor(s) which promoted the transition of cells from a state in which they are incapable of growth and division to one in which they are capable of colony formation.     

Quantitative Analysis of the Physiological Heterogeneity within Starved Cultures of Micrococcus luteus by Flow Cytometry and Cell Sorting

A high proportion of Micrococcus luteus cells in cultures which had been starved for 3 to 6 months lost the ability to grow and form colonies on agar plates but could be resuscitated from their dormancy by incubation in an appropriate liquid medium (A. S. Kaprelyants and D. B. Kell, Appl. Environ. Microbiol. 59:3187-3196, 1993). We used flow cytometry and cell sorting to study populations of bacteria that had been starved for 5 months. These cells could be stained by the fluorescent lipophilic cation rhodamine 123, but such staining was almost independent of metabolically generated energy in that it was not affected by uncouplers. Two populations could be distinguished, one with a lower degree of rhodamine fluorescence (a degree of fluorescence referred to as region A and containing approximately 80% of the cells) and one with a more elevated degree of fluorescence (region B, approximately 20% of the cells). Subsequent incubation of starved cells in fresh medium in the presence of the antibiotic chloramphenicol (to which M. luteus is sensitive) resulted in the transient appearance of cells actively accumulating rhodamine 123 (and fluorescing in region B) and of larger cells exhibiting a yet-greater degree of fluorescence (region C). These more fluorescent cells accounted for as much as 50% of the total population, under conditions in which the viable and total counts were constant. Thus, metabolic resuscitation of at least one-half of the cells takes place under conditions in which cryptic growth cannot play any role. Sorting experiments revealed that the great majority of the viable cells in the starved population are concentrated in regions B and C and that the extent of rhodamine staining under conditions of starvation therefore reflects the physiological state of the cells. Physical separation of these cells from cells in region A resulted in an increase (of approximately 25-fold) in the viability of cells in regions B and C and of the population as a whole. Resuscitation of dormant cells in a most-probable-number assay in the presence of supernatant taken from growing M. luteus revealed the resuscitation of cells from regions B and C but not from region A. It is suggested that initially dormant (resuscitable) cells are concentrated in regions B and C.     

A comparison of solid and liquid media for resuscitation of starvation- and low-temperature-induced nonculturable cells of Aeromonas hydrophila

Like many other gram-negative bacteria, starved cells of Aeromonas hydrophila can be induced into a viable but nonculturable (VBNC) state by incubation at low temperature, as shown here by using various bacterial enumeration methods. Starved A. hydrophila strain HR7 cells at 4 degrees C reached the nonculturable stage in about 45 days. The cells were resuscitated by either a solid medium resuscitation method, using solid agar amended with H2O2-degrading agents, catalase or sodium pyruvate, or a liquid medium resuscitation method, by incubating nonculturable cells in liquid media containing these compounds before spreading onto plates. The liquid medium resuscitation method using catalase resulted in nearly complete recovery of nonculturable cells.     

Viability of escherichia coli cells under long-term cultivation in a rich nutrient medium

We investigated the viability of Escherichia coli cells during long-term cultivation in Brain Heart Infusion (BHI) medium and observed that the number of viable cells increased, then decreased, and increased again, in this medium, and finally the cells died out within about 10 days. This cell death may result from an increase in the pH of the medium. After repeated cultivation in BHI, bacterial cells that did not die out even under conditions of further cultivation were obtainable from cultures showing a stabilized viable count. We propose that long-term cultivation in BHI medium is a good system for studying growth phase-specific events in E. coli cells, because the total life-cycle of a population of E. coli, including exponential growth, stationary phase, and extinction, can be seen during a period of only about 10 days. Also, this system clearly allows detection of a phenotype that may not be detectable in other commonly used media. Moreover, in this report, we show that mutants displaying the GASP (growth advantage in stationary phase) phenotype appear at high frequency under long-term cultivation conditions.     

On resuscitation from the dormant state of Micrococcus luteus

It has been found previously that a significant number of Micrococcus luteus cells starved in a prolonged stationary phase (up to 2 months) and then held on the bench at room temperature without agitation for periods of up to a further 2–7 months can be resuscitated in liquid media which contained (statistically) no initially-viable (colony-forming) cells but which were fortified with sterile supernatant from the late logarithmic phase of batch growth. Here it was found that such resuscitation can be done only within a defined time period after taking the first sample from such cultures, necessarily involving agitation of the cells. The duration of this period depends on the age of the starved culture: cells kept on the bench for 3 months possess a 2 month period of resuscitability while cells starved for 6 months can be resuscitated only within 10 days after the beginning of sampling. It is suggested that the input of oxygen to the starved cultures while they are agitated may exert a negative influence on the cells, since cultures stored in anaerobic conditions (under nitrogen) had a more prolonged survival' time. The cells which experienced between 10 and 60 days of starvation on the bench could be resuscitated, although the number of resuscitable cells depended strongly on the concentration of yeast extract in the resuscitation medium. This concentration for cells stored on the bench for more than 2 months was 0.05% while 1-month-old cells displayed a maximum resuscitability in the presence of 0.01% of yeast extract. Application of the fluorescent probe propidium iodide revealed the formation of cells with a damaged permeability barrier if resuscitation was performed by using concentrations of yeast extract of 0.1% and above. Thus the successful resuscitation of bacterial cultures under laboratory conditions may need rather strictly defined parameters if it is to be successfully performed for the majority of cells in a population.     

Cyclic Amp-Dependent Resuscitation of Dormant Mycobacteria by Exogenous Free Fatty Acids

One third of the world population carries a latent tuberculosis (TB) infection, which may reactivate leading to active disease. Although TB latency has been known for many years it remains poorly understood. In particular, substances of host origin, which may induce the resuscitation of dormant mycobacteria, have not yet been described. In vitro models of dormant (“non-culturable”) cells of Mycobacterium smegmatis (mc²155) and Mycobacterium tuberculosis H37Rv were used. We found that the resuscitation of dormant M. smegmatis and M. tuberculosis cells in liquid medium was stimulated by adding free unsaturated fatty acids (FA), including arachidonic acid, at concentrations of 1.6–10 µM. FA addition enhanced cAMP levels in reactivating M. smegmatis cells and exogenously added cAMP (3–10 mM) or dibutyryl-cAMP (0.5–1 mM) substituted for FA, causing resuscitation of M. smegmatis and M. tuberculosis dormant cells. A M. smegmatis null-mutant lacking MSMEG_4279, which encodes a FA-activated adenylyl cyclase (AC), could not be resuscitated by FA but it was resuscitated by cAMP. M. smegmatis and M. tuberculosis cells hyper-expressing AC were unable to form non-culturable cells and a specific inhibitor of AC (8-bromo-cAMP) prevented FA-dependent resuscitation. RT-PCR analysis revealed that rpfA (coding for resuscitation promoting factor A) is up-regulated in M. smegmatis in the beginning of exponential growth following the cAMP increase in lag phase caused by FA-induced cell activation. A specific Rpf inhibitor (4-benzoyl-2-nitrophenylthiocyanate) suppressed FA-induced resuscitation. We propose a novel pathway for the resuscitation of dormant mycobacteria involving the activation of adenylyl cyclase MSMEG_4279 by FAs resulted in activation of cellular metabolism followed later by increase of RpfA activity which stimulates cell multiplication in exponential phase. The study reveals a probable role for lipids of host origin in the resuscitation of dormant mycobacteria, which may function during the reactivation of latent TB.     

Incorporation of P and Growth of Pseudomonad UP-2 on n-Tetracosane

Cultures of the marine pseudomonad UP-2 growing on n-tetracosane contained both free cells and cells bound to the solid hydrocarbon. After separation by filtration through a Whatman no. 1 filter, the numbers of free and bound cells were estimated from the amount of P incorporated into each fraction and the determined value of P incorporation per viable cell in the filtrate (free cells). During the early exponential growth phase, over 80% of the cells were bound to large pieces of n-tetracosane; as the culture approached the stationary phase, the number of bound cells remained constant, whereas free cells continued to accumulate. Pulse-labeling experiments indicated that cells grew both on the surface of the solid and in the aqueous medium. During the growth cycle, a portion of the n-tetracosane which was initially nonfilterable was recovered in the filtrate in a form which was largely cell associated. This cell-associated n-tetracosane was preferentially utilized and could completely account for the observed growth of free cells.     

红螺菌(Rhodospirillum sp.)的生长及其饥饿存活的研究

红螺菌（Ｒｈｏｄｏｐｉｒｉｌｌｕｍ ｓｐ．）是在多种不同生境中广泛存在的光合细菌中的一类,它在水产养殖上也得到了广泛应用。报道了不同生长阶段红螺菌在饥饿环境中的存添能力。红螺菌在饥饿环境中的存活能力提高。分批培养过程中,同时测定红螺菌数和可培养活菌数的变化表明,静止期生长期后的红螺菌难以在固体培养基上形成菌落,进入非可培养状态,进入非可培养状态的红螺菌经复苏培养后仍可恢复在固体培养基上形成菌落的能     

Analysis of cell size and DNA content in exponentially growing and stationary-phase batch cultures of Escherichia coli.

Escherichia coli strains were grown in batch cultures in different media, and cell size and DNA content were analyzed by flow cytometry. Steady-state growth required large dilutions and incubation for many generations at low cell concentrations. In rich media, both cell size and DNA content started to decrease at low cell concentrations, long before the cultures left the exponential growth phase. Stationary-phase cultures contained cells with several chromosomes, even after many days, and stationary-phase populations exclusively composed of cells with a single chromosome were never observed, regardless of growth medium. The cells usually contained only one nucleoid, as visualized by phase and fluorescence microscopy. The results have implications for the use of batch cultures to study steady-state and balanced growth and to determine mutation and recombination frequencies in stationary phase.     

Resuscitation of viable but nonculturable Legionella pneumophila Philadelphia JR32 by Acanthamoeba castellanii.

Legionella pneumophila is an aquatic bacterium and is responsible for Legionnaires' disease in humans. Free-living amoebae are parasitized by legionellae and provide the intracellular environment required for the replication of this bacterium. In low-nutrient environments, however, L. pneumophila is able to enter a non-replicative viable but nonculturable (VBNC) state. In this study, L. pneumophila Philadelphia I JR 32 was suspended in sterilized tap water at 10(4) cells/ml. The decreasing number of bacteria was monitored by CFU measurements, acridine orange direct count (AODC), and hybridization with 16S rRNA-targeted oligonucleotide probes. After 125 days of incubation in water, the cells were no longer culturable on routine plating media; however, they were still detectable by AODC and by in situ hybridization. The addition of Acanthamoeba castellanii to the dormant bacteria resulted in the resuscitation of L. pneumophila JR 32 to a culturable state. A comparison of plate-grown legionellae and reactivated cells showed that the capacity for intracellular survival in human monocytes and intraperitoneally infected guinea pigs, which is considered a parameter for virulence, was not reduced in the reactivated cells. However, reactivation of dormant legionellae was not observed in the animal model.     

Analysis of plating technique for viability and drug-sensitivity determinations using Rosa cells

Rosa Paul's Scarlet'cell suspension cultures were used as a test system for working out a method of viability and drug-sensitivity determination based on plating efficiency. High plating efficiencies (80–95%) were obtained on a simple synthetic medium when aggregates of a mean size of c . 100 cells/unit from exponential phase cultures were plated at a density of 1500 units/plate in the middle layer (5 ml) of three layers of the agar-solidified medium (total = 30 ml). This 3-layer plating technique produces homogeneous colony growth and simplifies the microscopical evaluation of plating efficiencies. The reduction of plating efficiencies seen when the smaller aggregates of stationary phase cultures were plated was mainly due to low cell density and could be overcome by enriching the medium with various supplements. Reconstitution experiments using mixtures of inactivated and non-inactivated aggregates demonstrated that plating efficiency can be taken as a goodmeasure of viability. The described plating technique was found to be more sensitive and reliable compared to two other methods for determining p -fluorophenylalanine-sensitivity of Rosa cells.     

The enumeration of Leptospira interrogans serovar pomona by a bioluminescence ATP assay

A rapid method for enumerating viable Leptospira interrogans serovar pomona cells was investigated using a bacterial adenosine triphosphate (ATP) assay. The ATP was assayed by the luciferin-luciferase bioluminescence reaction. Samples of serovar pomona grown in liquid polysorbate 80-bovine albumin (P80-BA) medium for 1-3 days were analysed for ATP content, culture density (nephelometry), direct cell count and most probable number of viable cells (MPNVC) as determined by the dilution tube technique. A linear relationship was found between ATP content and the number of viable cells over the range of 4 X 10(8) to 8 X 10(9) leptospires/ml. Over this range the correlation coefficient for ATP content versus viable cells (0.96) was similar to the coefficient for culture density versus the number of viable cells. The coefficient for direct counts versus the number of viable cells was smaller. The bioluminescence assay of bacterial ATP is a promising method for enumerating viable leptospires in pure culture.     

Cyclic growth of lactobacilli in beer

J.L. FERNANDEZ AND W.J. SIMPSON 1994. Beer-spoilage lactic acid bacteria displayed unusual growth characteristics in beer. Cycles of growth and death occurred, as assessed by the ability of the organisms to form colonies on a range of solid growth media, or grow in liquid media, including beer and modified de Man, Rogosa and Sharpe medium. During the death phase, the viable cell count fell by more than 99.9%. The cells were dead rather than nonculturable. Staining with acridine orange showed that they remained physically intact during the death phase. It seems that cycles of 'cryptic growth' occur in beer. Cells isolated from different stages of the growth cycle produced similar growth curves when re-inoculated into beer, showing that selection of adapted cells does not occur.     

A new chemically-defined medium for Bacillus subtilis (168) NCIMB 12900

J. LEITCH AND P.J. COLLIER. 1996. Most chemically-defined media for the growth of Bacillus subtilis 168 in batch culture are unsuitable for use in nutrient limitation experiments due to either their low cell density yield or their high numbers of added amino-acids. The authors have developed a medium which gives relatively high cell densities and is highly chemically-defined, containing only one added amino-acid. Growth of B. subtilis in this new medium was observed over time using optical density measurements at 470 nm. The growth curve exhibited entry into exponential phase after only 1–2 h and stationary phase after 8 h. Cell density yields and comparison of growth rates with B. subtilis grown in Fang and Demain Medium (1989) suggest that this new medium is highly suitable for the growth of this micro-organism under both normal and nutrient limited conditions.     

Survival of Campylobacter jejuni during stationary phase: evidence for the absence of a phenotypic stationary-phase response

When Campylobacter jejuni NCTC 11351 was grown microaerobically in rich medium at 39 degrees C, entry into stationary phase was followed by a rapid decline in viable numbers to leave a residual population of 1% of the maximum number or less. Loss of viability was preceded by sublethal injury, which was seen as a loss of the ability to grow on media containing 0.1% sodium deoxycholate or 1% sodium chloride. Resistance of cells to mild heat stress (50 degrees C) or aeration was greatest in exponential phase and declined during early stationary phase. These results show that C. jejuni does not mount the normal phenotypic stationary-phase response which results in enhanced stress resistance. This conclusion is consistent with the absence of rpoS homologues in the recently reported genome sequence of this species and their probable absence from strain NCTC 11351. During prolonged incubation of C. jejuni NCTC 11351 in stationary phase, an unusual pattern of decreasing and increasing heat resistance was observed that coincided with fluctuations in the viable count. During stationary phase of Campylobacter coli UA585, nonmotile variants and those with impaired ability to form coccoid cells were isolated at high frequency. Taken together, these observations suggest that stationary-phase cultures of campylobacters are dynamic populations and that this may be a strategy to promote survival in at least some strains. Investigation of two spontaneously arising variants (NM3 and SC4) of C. coli UA585 showed that a reduced ability to form coccoid cells did not affect survival under nongrowth conditions.     

Effect of yeast growth conditions on yeast-mycelial transition in Candida albicans

When grown and induced to form germ tubes in liquid defined media, yeast cells of Candida albicans must reach stationary phase before acquiring ability to carry out the yeast-mycelial transition. This study examined the effect of the carbon source utilized for yeast growth on the inducibility of stationary phase yeast. When grown to the same stationary phase cell density as glucose cultures, cultures grown on citrate were fully inducible while cultures grown on galactose and mannose showed a small reduction. Cultures grown on ethanol were reduced 80% in morphological conversion. When glucose grown cells were induced in the presence of these carbon sources, hexoses supported full induction while ethanol reduced induction 80%. Induction in the presence of carboxylic acids was similar to induction in the absence of added carbon source. When induced on the same source used in yeast growth, germ tube formation was reduced for all carbon sources except hexoses. When induced in the absence of added carbon source, yeasts grown on citrate and ethanol were inhibited 80-100%. Cultures starved for glucose were more inhibited than cultures starved for NH4Cl when induced without added carbon source. These observations suggest that the metabolic state of the stationary phase cell is an important factor in the ability to respond to conditions inducing germ tube formation.