hipA, a newly recognized gene of Escherichia coli K-12 that affects frequency of persistence after inhibition of murein synthesis.

Except for a small fraction of persisters, 10(-6) to 10(-5), Escherichia coli K-12 is killed by prolonged inhibition of murein synthesis. The progeny of persisters are neither more resistant to inhibition of murein synthesis nor more likely to persist than normal cells. Mutants have been isolated in which a larger fraction, 10(-2), persists. The persistent response of the mutants, Hip (high persistence), is to inhibition of murein synthesis at early or late steps by antibiotics (phosphomycin, cycloserine, and ampicillin) or by metabolic block (starvation for diaminopimelic acid). Killing of the parent strain by each of the four inhibitors has two phases: The first is rapid and lasts about 30 min; the second is slower, but still substantial, and lasts 3 to 4 h. The first phase also occurs in the Hip mutants, but then viability of the mutants remains constant after about 30 min. Neither tolerance, resistance, impaired growth, nor reversion of spheroplasts accounts for high-frequency persistence. Two of the mutations map at 33.8 min in a region containing few other recognized functions. This position and the phenotypes define hipA as a newly recognized gene. Transposons Tn5 and Tn10 have been inserted close to hipA making it possible to explore the molecular genetics of persistence, a long recognized but poorly understood phenomenon.     

Emergence of Pseudomonas aeruginosa strains producing high levels of persister cells in patients with cystic fibrosis

The majority of cystic fibrosis (CF) patients succumb to a chronic infection of the airway with Pseudomonas aeruginosa. Paradoxically, pathogenic strains are often susceptible to antibiotics, but the infection cannot be eradicated with antimicrobial therapy. We find that in a majority of patients with airway infections, late isolates of P. aeruginosa produce increased levels of drug-tolerant persister cells. The genomes of a clonal pair of early/late isolates from a single patient have been previously sequenced, and the late isolate (obtained at age 96 months) showed a 100-fold increase in persister levels. The 96-month isolate carries a large number of mutations, including a mutation in mutS that confers a hypermutator phenotype. There is also a mutation in the mexZ repressor controlling the expression of the MexXY-OprM multidrug pump, which results in a moderate increase in the ofloxacin, carbenicillin, and tobramycin MICs. Knocking out the mexXY locus restored the resistance to that of the parent strain but did not affect the high levels of persisters formed by the 96-month isolate. This suggests that the late isolate is a high-persister (hip) mutant. Increased persister formation was observed in exponential phase, stationary phase, and biofilm populations of the 96-month isolate. Analysis of late isolates from 14 additional patients indicated that 10 of them are hip mutants. Most of these hip mutants did not have higher drug resistance. Increased persister formation appears to be their sole mechanism for surviving chemotherapy. Taken together, these findings suggest a link between persisters and recalcitrance of CF infection and identify an overlooked culprit-high-persister mutants producing elevated levels of drug-tolerant cells. Persisters may play a similarly critical role in the recalcitrance of other chronic infections.     

Specialized persister cells and the mechanism of multidrug tolerance in Escherichia coli

Bacterial populations produce persisters, cells that neither grow nor die in the presence of bactericidal agents, and thus exhibit multidrug tolerance (MDT). The mechanisms of MDT and the nature of persisters have remained elusive. Our previous research has shown that persisters are largely responsible for the recalcitrance of biofilm infections. A general method for isolating persisters was developed, based on lysis of regular cells by ampicillin. A gene expression profile of persisters contained toxin-antitoxin (TA) modules and other genes that can block important cellular functions such as translation. Bactericidal antibiotics kill cells by corrupting the target function (for example, aminoglycosides interrupt translation, producing toxic peptides). We reasoned that inhibition of translation will lead to a shutdown of cellular functions, preventing antibiotics from corrupting their targets, giving rise to MDT persister cells. Overproduction of the RelE toxin, an inhibitor of translation, caused a sharp increase in persisters. Functional expression of a putative HipA toxin also increased persisters, while deletion of the hipBA module caused a sharp decrease in persisters in both stationary and biofilm populations. HipA is thus the first validated persister-MDT gene. We suggest that random fluctuation in the levels of MDT proteins leads to the formation of rare persister cells. The function of these specialized dormant cells is to ensure the survival of the population in the presence of lethal factors.     

Age of inoculum strongly influences persister frequency and can mask effects of mutations implicated in altered persistence

The majority of cells transferred from stationary-phase culture into fresh medium resume growth quickly, while a few remain in a nongrowing state for longer. These temporarily nonproliferating bacteria are tolerant of several bactericidal antibiotics and constitute a main source of persisters. Several genes have been shown to influence the frequency of persisters in Escherichia coli, although the exact mechanism underlying persister formation is unknown. This study demonstrates that the frequency of persisters is highly dependent on the age of the inoculum and the medium in which it has been grown. The hipA7 mutant had 1,000 times more persisters than the wild type when inocula were sampled from younger stationary-phase cultures. When started after a long stationary phase, the two displayed equal and elevated persister frequencies. The lower persister frequencies of glpD, dnaJ, and surA knockout strains were increased to the level of the wild type when inocula aged. The mqsR and phoU deletions showed decreased persister levels only when the inocula were from aged cultures, while sucB and ygfA deletions had decreased persister levels irrespective of the age of the inocula. A dependency on culture conditions underlines the notion that during screening for mutants with altered persister frequencies, the exact experimental details are of great importance. Unlike ampicillin and norfloxacin, which always leave a fraction of bacteria alive, amikacin killed all cells in the growth resumption experiment. It was concluded that the frequency of persisters depends on the conditions of inoculum cultivation, particularly its age, and the choice of antibiotic.     

GlpD and PlsB participate in persister cell formation in Escherichia coli

Bacterial populations produce dormant persister cells that are resistant to killing by all antibiotics currently in use, a phenomenon known as multidrug tolerance (MDT). Persisters are phenotypic variants of the wild type and are largely responsible for MDT of biofilms and stationary populations. We recently showed that a hipBA toxin/antitoxin locus is part of the MDT mechanism in Escherichia coli. In an effort to find additional MDT genes, an E. coli expression library was selected for increased survival to ampicillin. A clone with increased persister production was isolated and was found to overexpress the gene for the conserved aerobic sn-glycerol-3-phosphate dehydrogenase GlpD. The GlpD overexpression strain showed increased tolerance to ampicillin and ofloxacin, while a strain with glpD deleted had a decreased level of persisters in the stationary state. This suggests that GlpD is a component of the MDT mechanism. Further genetic studies of mutants affected in pathways involved in sn-glycerol-3-phosphate metabolism have led to the identification of two additional multidrug tolerance loci, glpABC, the anaerobic sn-glycerol-3-phosphate dehydrogenase, and plsB, an sn-glycerol-3-phosphate acyltransferase.     

Control of bacterial persister cells by Trp/Arg-containing antimicrobial peptides

Persister cells are dormant phenotypic variants inherent in a bacterial population. They play important roles in chronic infections and present great challenges to therapy due to extremely enhanced tolerance to antibiotics compared to that of normal cells of the same genotype. In this study, we report that cationic membrane-penetrating peptides containing various numbers of arginine and tryptophan repeats are effective in killing persister cells of Escherichia coli HM22, a hyper-persister producer. The activities of three linear peptides [(RW)(n)-NH(2), where n is 2, 3, or 4] and a dendrimeric peptide, (RW)(4D), in killing bacterial persisters were compared. Although the dendrimeric peptide (RW)(4D) requires a lower threshold to kill planktonic persisters, octameric peptide (RW)(4)-NH(2) is the most effective against planktonic persister cells at high concentrations. For example, treatment with 80 μM (RW)(4)-NH(2) for 60 min led to a 99.7% reduction in the number of viable persister cells. The viability of persister cells residing in surface-attached biofilms was also significantly reduced by (RW)(4)-NH(2) and (RW)(4D). These two peptides were also found to significantly enhance the susceptibility of biofilm cells to ofloxacin. The potency of (RW)(4)-NH(2) was further marked by its ability to disperse and kill preformed biofilms harboring high percentages of persister cells. Interestingly, approximately 70% of the dispersed cells were found to have lost their intrinsic tolerance and become susceptible to ampicillin if not killed directly by this peptide. These results are helpful for better understanding the activities of these peptides and may aid in future development of more effective therapies of chronic infections.     

5-Oxo-prolinase activity in tobacco suspension cultures: Regulation by sulfate nutrition

In heterotrophic and photoheterotrophic tobacco ( Nicotiana tabacum L., var. Samsun) suspensions cultured with growth-limiting amounts of sulfate, 5-oxo-prolinase activity declines at the same time as the growth rate of the cells decreases. However, 5-oxo-prolinase activity is reduced to a greater extent than growth. As a result, the specific activity of 5-oxo-prolinase also declines when sulfur is scarce. The decrease in both growth and 5-oxo-prolinase activity can be prevented by adding sulfate to the suspensions during exponential growth. Addition of sulfate after the exponential growth phase restored neither growth nor 5-oxo-prolinase activity. These observations show that 5-oxo-prolinase activity in tobacco cells is regulated by the sulfate supply in the medium. Such a regulation is an essential prerequisite, but not a proof, for a role of 5-oxo-prolinase as the rate-limiting factor in glutathione degradation.
During exponential growth the average specific activity of 5-oxo-prolinase in heterotrophic tobacco cells is twice as high as in photoheterotrophic cells. This difference is consistent with the idea that green cells are equipped for glutathione synthesis and export, and chloroplast-free cells for uptake and degradation of this peptide.     

Persister cells and the riddle of biofilm survival

This review addresses a long standing puzzle in the life and death of bacterial populations—the existence of a small fraction of essentially invulnerable cells. Bacterial populations produce persisters, cells that neither grow nor die in the presence of bactericidal agents, and thus exhibit multidrug tolerance (MDT). The mechanism of MDT and the nature of persisters, which were discovered in 1944, have remained elusive. Our research has shown that persisters are largely responsible for the recalcitrance of infections caused by bacterial biofilms. The majority of infections in the developed world are caused by biofilms, which sparked a renewed interest in persisters. We developed a method to isolate persister cells, and obtained a gene expression profile of Escherichia coli persisters. The profile indicated an elevated expression of toxin-antitoxin modules and other genes that can block important cellular functions such as translation. Bactericidal antibiotics kill cells by corrupting the target function, such as translation. For example, aminoglycosides interrupt translation, producing toxic peptides. Inhibition of translation leads to a shutdown of other cellular functions as well, preventing antibiotics from corrupting their targets, which will give rise to tolerant persister cells. Overproduction of chromosomally-encoded toxins such as RelE, an inhibitor of translation, or HipA, causes a sharp increase in persisters. Deletion of the hipBA module produces a sharp decrease in persisters in both stationary and biofilm cells. HipA is thus the first validated persister/MDT gene. We conclude that the function of toxins is the exact opposite of the term, namely, to protect the cell from lethal damage. It appears that stochastic fluctuations in the levels of MDT proteins lead to formation of rare persister cells. Persisters are essentially altruistic cells that forfeit propagation in order to ensure survival of kin cells in the presence of lethal factors.Translated from Biokhimiya, Vol. 70, No. 2, 2005, pp. 327–336.Original Russian Text Copyright © 2005 by Lewis.This revised version was published online in April 2005 with corrections to the post codes.     

(Z)-4-Bromo-5-(bromomethylene)-3-methylfuran-2(5H)-one sensitizes Escherichia coli persister cells to antibiotics

Persisters are a small subpopulation of bacterial cells that are dormant and extremely tolerant to antibiotics. The intrinsic antibiotic tolerance of persisters also facilitates the development of multidrug resistance through acquired mechanisms based on drug resistance genes. In this study, we demonstrate that (Z)-4-bromo-5-(bromomethylene)-3-methylfuran-2(5H)-one (BF8) can reduce persistence during Escherichia coli growth and revert the antibiotic tolerance of its persister cells. The effects of BF8 were more profound when the pH was increased from 6 to 8.5. Although BF8 is a quorum sensing (QS) inhibitor, similar effects were observed for the wild-type E. coli RP437 and its ΔluxS mutant, suggesting that these effects did not occur solely through inhibition of AI-2-mediated QS. In addition to its effects on planktonic persisters, BF8 was also found to disperse RP437 biofilms and to render associated cells more sensitive to ofloxacin. At the doses that are effective against E. coli persister cells, BF8 appeared to be safe to the tested normal mammalian cells in vitro and exhibited no long-term cytotoxicity to normal mouse tissues in vivo. These findings broadened the activities of brominated furanones and shed new light on persister control.     

Persisters: a distinct physiological state of E. coli

Background  

Bacterial populations contain persisters, phenotypic variants that constitute approximately 1% of cells in stationary phase and biofilm cultures. Multidrug tolerance of persisters is largely responsible for the inability of antibiotics to completely eradicate infections. Recent progress in understanding persisters is encouraging, but the main obstacle in understanding their nature was our inability to isolate these elusive cells from a wild-type population since their discovery in 1944.     

Clonal variation in maximum specific growth rate and susceptibility towards antimicrobials

AIMS: To examine associations between growth rate within bacterial populations and survival patterns following treatment with antimicrobial agents. METHODS AND RESULTS: Time survival data were generated for the inactivation of Escherichia coli populations, grown as batch and continuous cultures, exposed to ciprofloxacin, benzalkonium chloride and tetracycline. Time-survivor plots were biphasic. Surviving cells were collected and immediately re-exposed to agent or were regrown and then re-exposed. Survivors were resistant to immediate challenge with any of the treatment agents. This resistance was lost on regrowth suggesting that survival reflects an expressed phenotype within a subset of the culture (persisters) rather than individual resistant clones or nonspecific quenching of the test agent. The fraction of persisters increased with decreasing growth rate when cultures were prepared in continuous culture. CONCLUSIONS: Clonal growth rates within populations were determined by culture of individual cells within microtitre plate wells. The fraction of clones, in batch cultures, growing maximally at rates below the apparent threshold for susceptibility to the test agents was sufficient to explain the results of continuous culture experiments. SIGNIFICANCE AND IMPACT OF THE STUDY: The presence of persisters in populations of bacteria relate to small subset of cells that are growing only slowly or are metabolically quiescent.     

Effect of growth phase on survival of bromegrass suspension cells following cryopreservation and abiotic stresses

BACKGROUND AND AIMS: Cryopreservation is a practical method of preserving plant cell cultures and their genetic integrity. It has long been believed that cryopreservation of plant cell cultures is best performed with cells at the late lag or early exponential growth phase. At these stages the cells are small and non-vacuolated. This belief was based on studies using conventional slow prefreezing protocols and survival determined with fluorescein diacetate staining or 2,3,5-triphenyltetrazolium chloride assays. This classical issue was revisited here to determine the optimum growth phase for cryopreserving a bromegrass (Bromus inermis) suspension culture using more recently developed protocols and regrowth assays for determination of survival. METHODS: Cells at different growth phases were cryopreserved using three protocols: slow prefreezing, rapid prefreezing and vitrification. Stage-dependent trends in cell osmolarity, water content and tolerance to freezing, heat and salt stresses were also determined. In all cases survival was assayed by regrowth of cells following the treatments. KEY RESULTS: Slow prefreezing and rapid prefreezing protocols resulted in higher cell survival compared with the vitrification method. For all the protocols used, the best regrowth was obtained using cells in the late exponential or early stationary phase, whereas lowest survival was obtained for cells in the late lag or early exponential phase. Cells at the late exponential phase were characterized by high water content and high osmolarity and were most tolerant to freezing, heat and salt stresses, whereas cells at the early exponential phase, characterized by low water content and low osmolarity, were least tolerant. CONCLUSIONS: The results are contrary to the classical concept which utilizes cells in the late lag or early exponential growth phase for cryopreservation. The optimal growth phase for cryopreservation may depend upon the species or cell culture being cryopreserved and requires re-investigation for each cell culture. Stage-dependent survival following cryopreservation was proportionally correlated with the levels of abiotic stress tolerance in bromegrass cells.     

Evaluation of the influence of deep geomagnetic impacting on growth rate and sensitivity to antibiotics in Escherichia coli

The short-term (1-7 days) and prolonged (30-90 days) adaptation of bacteria to hypomagnetic conditions was studied by observing changes in growth rate and resistance to antibiotics in E. coli used as an experimental model. The decrease of the growth rate of bacterial cells in the exponential phase was found to occur. The effect disappeared when the screened culture was placed under normal geomagnetic conditions for 1 hour daily. After prolonged screening an increase in the resistance of these bacteria to kanamycin and carbenicillin was registered. These changes were not hereditary: after the cessation of screening the sensitivity of the bacteria to antibiotics was restored.     

Tackling <Emphasis Type="Italic">Salmonella</Emphasis> Persister Cells by Antibiotic–Nisin Combination via Mannitol

Bacterial persisters (defined as dormant, non-dividing cells with globally reduced metabolism) are the major cause of recurrent infections. As they neither grow nor die in presence of antibiotics, it is difficult to eradicate these cells using antibiotics, even at higher concentrations. Reports of metabolites (which help in waking up of these inactive cells) enabled eradication of bacterial persistence by aminoglycosides, suggest the new potential strategy to improve antibiotic therapy. Here we propose, mannitol enabled elimination of Salmonella persister cells by the nisin–antibiotic combination. For this, persister cells were developed and characterized for their typical properties such as non-replicative state and metabolic dormancy. Different carbon sources viz. glucose, glycerol, and mannitol were used, each as an adjunct to ampicillin for the eradication of persister cells. The maximum (but not complete) killing was observed with mannitol–ampicillin, out of all the combinations used. However, significant elimination (about 78%) could be observed, when nisin (an antimicrobial peptide) was used with ampicillin in presence of mannitol, which might have mediated the transfer of antibiotic–nisin combination at the same time when the cells tried to grab the carbon molecule. Further, the effectiveness of the trio was confirmed by flow cytometry. Overall, our findings highlight the potential of this trio-combination for developing it as an option for tackling Salmonella persister cells.     

Biofilms and Planktonic Cells of Pseudomonas aeruginosa Have Similar Resistance to Killing by Antimicrobials

Biofilms are considered to be highly resistant to antimicrobial agents. Strictly speaking, this is not the case-biofilms do not grow in the presence of antimicrobials any better than do planktonic cells. Biofilms are indeed highly resistant to killing by bactericidal antimicrobials, compared to logarithmic-phase planktonic cells, and therefore exhibit tolerance. It is assumed that biofilms are also significantly more tolerant than stationary-phase planktonic cells. A detailed comparative examination of tolerance of biofilms versus stationary- and logarithmic-phase planktonic cells with four different antimicrobial agents was performed in this study. Carbenicillin appeared to be completely ineffective against both stationary-phase cells and biofilms. Killing by this beta-lactam antibiotic depends on rapid growth, and this result confirms the notion of slow-growing biofilms resembling the stationary state. Ofloxacin is a fluoroquinolone antibiotic that kills nongrowing cells, and biofilms and stationary-phase cells were comparably tolerant to this antibiotic. The majority of cells in both populations were eradicated at low levels of ofloxacin, leaving a fraction of essentially invulnerable persisters. The bulk of the population in both biofilm and stationary-phase cultures was tolerant to tobramycin. At very high tobramycin concentrations, a fraction of persister cells became apparent in stationary-phase culture. Stationary-phase cells were more tolerant to the biocide peracetic acid than were biofilms. In general, stationary-phase cells were somewhat more tolerant than biofilms in all of the cases examined. We concluded that, at least for Pseudomonas aeruginosa, one of the model organisms for biofilm studies, the notion that biofilms have greater resistance than do planktonic cells is unwarranted. We further suggest that tolerance to antibiotics in stationary-phase or biofilm cultures is largely dependent on the presence of persister cells.