Persister cells and tolerance to antimicrobials

Bacterial populations produce persister cells that neither grow nor die in the presence of microbicidal antibiotics. Persisters are largely responsible for high levels of biofilm tolerance to antimicrobials, but virtually nothing was known about their biology. Tolerance of Escherichia coli to ampicillin and ofloxacin was tested at different growth stages to gain insight into the nature of persisters. The number of persisters did not change in lag or early exponential phase, and increased dramatically in mid-exponential phase. Similar dynamics were observed with Pseudomonas aeruginosa (ofloxacin) and Staphylococcus aureus (ciprofloxacin and penicillin). This shows that production of persisters depends on growth stage. Maintaining a culture of E. coli at early exponential phase by reinoculation eliminated persisters. This suggests that persisters are not at a particular stage in the cell cycle, neither are they defective cells nor cells created in response to antibiotics. Our data indicate that persisters are specialized survivor cells.     

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.     

Persister cells in a biofilm treated with a biocide

This study investigated the physiology and behaviour following treatment with ortho-phthalaldehyde (OPA), of Pseudomonas fluorescens in both the planktonic and sessile states. Steady-state biofilms and planktonic cells were collected from a bioreactor and their extracellular polymeric substances (EPS) were extracted using a method that did not destroy the cells. Cell structure and physiology after EPS extraction were compared in terms of respiratory activity, morphology, cell protein and polysaccharide content, and expression of the outer membrane proteins (OMP). Significant differences were found between the physiological parameters analysed. Planktonic cells were more metabolically active, and contained greater amounts of proteins and polysaccharides than biofilm cells. Moreover, biofilm formation promoted the expression of distinct OMP. Additional experiments were performed with cells after EPS extraction in order to compare the susceptibility of planktonic and biofilm cells to OPA. Cells were completely inactivated after exposure to the biocide (minimum bactericidal concentration, MBC = 0.55 ± 0.20 mM for planktonic cells; MBC = 1.7 ± 0.30 mM for biofilm cells). After treatment, the potential of inactivated cells to recover from antimicrobial exposure was evaluated over time. Planktonic cells remained inactive over 48 h while cells from biofilms recovered 24 h after exposure to OPA, and the number of viable and culturable cells increased over time. The MBC of the recovered biofilm cells after a second exposure to OPA was 0.58 ± 0.40 mM, a concentration similar to the MBC of planktonic cells. This study demonstrates that persister cells may survive in biocide-treated biofilms, even in the absence of EPS.     

Immunofluorescent detection of double-stranded RNA in cells infected with reovirus, infectious pancreatic necrosis virus, and infectious bursal disease virus

Urea treatment of ethanol-fixed virus-infected cells exposed nucleic acid antigens for immunofluorescence. Three double-stranded (ds) RNA-containing viruses showed bright fluorescence using antibodies against dsRNA. Three single-stranded RNA-containing viruses showed less intense fluorescence with anti-dsRNA. Four out of five cell lines persistently infected with various RNA-containing viruses showed no dsRNA detectable by immunofluorescence.     

Persister cells, the biofilm matrix and tolerance to metal cations in biofilm and planktonic Pseudomonas aeruginosa

In this study, we examined Pseudomonas aeruginosa ATCC 27853 biofilm and planktonic cell susceptibility to metal cations. The minimum inhibitory concentration (MIC), the minimum bactericidal concentration (MBC) required to eradicate 100% of the planktonic population (MBC 100), and the minimum biofilm eradication concentration (MBEC) were determined using the MBEC trade mark-high throughput assay. Six metals - Co(2+), Ni(2+), Cu(2+), Zn(2+), Al(3+) and Pb(2+)- were each tested at 2, 4, 6, 8, 10 and 27 h of exposure to biofilm and planktonic cultures grown in rich or minimal media. With 2 or 4 h of exposure, biofilms were approximately 2-25 times more tolerant to killing by metal cations than the corresponding planktonic cultures. However, by 27 h of exposure, biofilm and planktonic bacteria were eradicated at approximately the same concentration in every instance. Viable cell counts evaluated at 2 and 27 h of exposure revealed that at high concentrations, most of the metals assayed had killed greater than 99.9% of biofilm and planktonic cell populations. The surviving cells were propogated in vitro and gave rise to biofilm and planktonic cultures with normal sensitivity to metals. Further, retention of copper by the biofilm matrix was investigated using the chelator sodium diethlydithiocarbamate. Formation of visible brown metal-chelates in biofilms treated with Cu(2+) suggests that the biofilm matrix may coordinate and sequester metal cations from the aqueous surroundings. Overall, our data suggest that both metal sequestration in the biofilm matrix and the presence of a small population of 'persister' cells may be contributing factors in the time-dependent tolerance of both planktonic cells and biofilms to high concentrations of metal cations.     

Bacterial Persister Cell Formation and Dormancy

Bacterial cells may escape the effects of antibiotics without undergoing genetic change; these cells are known as persisters. Unlike resistant cells that grow in the presence of antibiotics, persister cells do not grow in the presence of antibiotics. These persister cells are a small fraction of exponentially growing cells (due to carryover from the inoculum) but become a significant fraction in the stationary phase and in biofilms (up to 1%). Critically, persister cells may be a major cause of chronic infections. The mechanism of persister cell formation is not well understood, and even the metabolic state of these cells is debated. Here, we review studies relevant to the formation of persister cells and their metabolic state and conclude that the best model for persister cells is still dormancy, with the latest mechanistic studies shedding light on how cells reach this dormant state.     

LPS, TLR4 and infectious disease diversity

Innate immune receptors recognize microorganism-specific motifs. One such receptor-ligand complex is formed between the mammalian Toll-like receptor 4 (TLR4)-MD2-CD14 complex and bacterial lipopolysaccharide (LPS). Recent research indicates that there is significant phylogenetic and individual diversity in TLR4-mediated responses. In addition, the diversity of LPS structures and the differential recognition of these structures by TLR4 have been associated with several bacterial diseases. This review will examine the hypothesis that the variability of bacterial ligands such as LPS and their innate immune receptors is an important factor in determining the outcome of infectious disease.     

Dynamical properties of autoimmune disease models: tolerance, flare-up, dormancy

The mechanisms of autoimmune disease have remained puzzling for a long time. Here we construct a simple mathematical model for autoimmune disease based on the personal immune response function and the target cell growth function. We show that these two functions are sufficient to capture the essence of autoimmune disease and can explain characteristic symptom phases such as tolerance, repeated flare-ups and dormancy. Our results strongly suggest that a more complete understanding of these two functions will underlie the development of an effective therapy for autoimmune disease.     

Cytolytic CD4 cells: Direct mediators in infectious disease and malignancy

CD4 T cells have traditionally been regarded as helpers and regulators of adaptive immune responses; however, a novel role for CD4 T cells as direct mediators of protection against viral infections has emerged. CD4 T cells with cytolytic potential have been described for almost 40 years, but their role in host protection against infectious disease is only beginning to be realized. In this review, we describe the current literature identifying these cells in patients with various infections, mouse models of viral infection and our own work investigating the development of cytolytic CD4 cells in vivo and in vitro. CD4 CTL are no longer considered an artefact of cell culture and may play a physiological role in viral infections such as EBV, CMV, HIV and influenza. Therefore, vaccine strategies aimed at targeting CD4 CTL should be developed in conjunction with vaccines incorporating B cell and CD8 CTL epitopes.     

Disseminated tumor cells and dormancy in prostate cancer metastasis

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The role of gamma delta T cells in infectious disease]

There have been several lines of evidence that at least significant fraction of gamma delta--T cells are specialized to recognize epitopes on mycobacterial antigens including 65 KD heat shock protein(HSP). Since HSP is known to be highly conserved in amino acid sequences from prokaryotes to eukaryotes, it is possible that the HSP--specific gamma delta--T cells may recognize the endogenous HSP expressed by autologous cells. The broad--reactive gamma delta--T cells may be responsible for protection during the period between an early stage covered mainly by phagocytes and a late stage covered by typical immunities in terms of the time sequence after microbial invasions.     

Environmental disease: environmental alteration and infectious disease

Humans have changed their environment to survive and to achieve a safer and more comfortable life. For example, drinking water and wastewater infrastructures are indispensable for civilized societies to flourish and to prevent water-borne infectious diseases. However, excessive loading on environments might disturb microbial ecosystems, resulting in outbreaks of pathogenic microbes and the expansion of infectious diseases. Clarifying the relationship between environmental alterations and changes in microbial ecosystems is thus important to prevent further outbreaks of infectious diseases. The present study aims to understand the links between the following factors: environmental alterations; ecosystem disturbance and the occurrence of infectious disease; and impact on society. We focus on legionellosis and nontuberculous mycobacterial diseases from the viewpoint of their environmental linkage. While Legionella spp. are ubiquitous in aquatic environments, Legionella pneumophila often increases in anthropogenic environments, such as cooling towers or spas, and can cause outbreaks of legionellosis. Recently, travel-associated Legionnaires’ disease has caused concern in many countries. The numbers of patients infected with nontuberculous Mycobacteria (NTM) have increased worldwide since the 1990s. Disturbances to microbial ecosystems caused by changes in water usage might be one cause of NTM diseases. Clarifying the dynamics of Legionella pneumophila and NTM in aquatic environments should help prevent outbreaks of diseases associated with these bacteria.     

Bringing systems biology to cancer,immunology and infectious disease

A report on the seventh annual ‘International Conference on Systems Biology of Human Disease’ held in Boston, Massachusetts, USA, 17–19 June, 2014.     

Seasonal infectious disease epidemiology

Seasonal change in the incidence of infectious diseases is a common phenomenon in both temperate and tropical climates. However, the mechanisms responsible for seasonal disease incidence, and the epidemiological consequences of seasonality, are poorly understood with rare exception. Standard epidemiological theory and concepts such as the basic reproductive number R0 no longer apply, and the implications for interventions that themselves may be periodic, such as pulse vaccination, have not been formally examined. This paper examines the causes and consequences of seasonality, and in so doing derives several new results concerning vaccination strategy and the interpretation of disease outbreak data. It begins with a brief review of published scientific studies in support of different causes of seasonality in infectious diseases of humans, identifying four principal mechanisms and their association with different routes of transmission. It then describes the consequences of seasonality for R0, disease outbreaks, endemic dynamics and persistence. Finally, a mathematical analysis of routine and pulse vaccination programmes for seasonal infections is presented. The synthesis of seasonal infectious disease epidemiology attempted by this paper highlights the need for further empirical and theoretical work.     

Production in FL cells of infectious and potentially infectious reovirus

Spendlove, Rex S. (California State Department of Public Health, Berkeley), Edwin H. Lennette, Charles O. Knight, and Jean N. Chin. Production in FL cells of infectious and potentially infectious reovirus. J. Bacteriol. 92:1036-1040. 1966.-A comparative study was made of the development in, and release from, FL cells of infectious and potentially infectious (chymotrypsin-activatable) reovirus (Lang strain). The latent period was shorter, the rate of synthesis was more rapid, and the total yield was more than 10-fold greater in potentially infectious virus as compared with infectious virus. Almost all of the potentially infectious virus, but only approximately one-third of the infectious virus, was released from the infected cells.     

Bacterial DNA topology and infectious disease

The Gram-negative bacterium Escherichia coli and its close relative Salmonella enterica have made important contributions historically to our understanding of how bacteria control DNA supercoiling and of how supercoiling influences gene expression and vice versa. Now they are contributing again by providing examples where changes in DNA supercoiling affect the expression of virulence traits that are important for infectious disease. Available examples encompass both the earliest stages of pathogen–host interactions and the more intimate relationships in which the bacteria invade and proliferate within host cells. A key insight concerns the link between the physiological state of the bacterium and the activity of DNA gyrase, with downstream effects on the expression of genes with promoters that sense changes in DNA supercoiling. Thus the expression of virulence traits by a pathogen can be interpreted partly as a response to its own changing physiology. Knowledge of the molecular connections between physiology, DNA topology and gene expression offers new opportunities to fight infection.