Many bacteria can infect and persist inside their hosts for long periods of time. This can be due to immunosuppression of the host, immune evasion by the pathogen and/or ineffective killing by ...antibiotics. Bacteria can survive antibiotic treatment if they are resistant or tolerant to a drug. Persisters are a subpopulation of transiently antibiotic-tolerant bacterial cells that are often slow-growing or growth-arrested, and are able to resume growth after a lethal stress. The formation of persister cells establishes phenotypic heterogeneity within a bacterial population and has been hypothesized to be important for increasing the chances of successfully adapting to environmental change. The presence of persister cells can result in the recalcitrance and relapse of persistent bacterial infections, and it has been linked to an increase in the risk of the emergence of antibiotic resistance during treatment. If the mechanisms of the formation and regrowth of these antibiotic-tolerant cells were better understood, it could lead to the development of new approaches for the eradication of persistent bacterial infections. In this Review, we discuss recent developments in our understanding of bacterial persisters and their potential implications for the treatment of persistent infections.
Increasing concerns about the rising rates of antibiotic therapy failure and advances in single-cell analyses have inspired a surge of research into antibiotic persistence. Bacterial persister cells ...represent a subpopulation of cells that can survive intensive antibiotic treatment without being resistant. Several approaches have emerged to define and measure persistence, and it is now time to agree on the basic definition of persistence and its relation to the other mechanisms by which bacteria survive exposure to bactericidal antibiotic treatments, such as antibiotic resistance, heteroresistance or tolerance. In this Consensus Statement, we provide definitions of persistence phenomena, distinguish between triggered and spontaneous persistence and provide a guide to measuring persistence. Antibiotic persistence is not only an interesting example of non-genetic single-cell heterogeneity, it may also have a role in the failure of antibiotic treatments. Therefore, it is our hope that the guidelines outlined in this article will pave the way for better characterization of antibiotic persistence and for understanding its relevance to clinical outcomes.
Many bacterial infections are hard to treat and tend to relapse, possibly due to the presence of antibiotic-tolerant persisters. In vitro, persister cells appear to be dormant. After uptake of
...species by macrophages, nongrowing persisters also occur, but their physiological state is poorly understood. In this work, we show that
persisters arising during macrophage infection maintain a metabolically active state. Persisters reprogram macrophages by means of effectors secreted by the
pathogenicity island 2 type 3 secretion system. These effectors dampened proinflammatory innate immune responses and induced anti-inflammatory macrophage polarization. Such reprogramming allowed nongrowing
cells to survive for extended periods in their host. Persisters undermining host immune defenses might confer an advantage to the pathogen during relapse once antibiotic pressure is relieved.
Genetically susceptible bacteria can escape the action of bactericidal antibiotics through antibiotic tolerance or persistence. However, one major difference between the two phenomena is their ...distinct penetrance within an isogenic population. While with antibiotic persistence, susceptible and persister cells co-exist, antibiotic tolerance affects the entire bacterial population. Here, we show that antibiotic tolerance can be achieved in numerous non-specific ways
in vitro
and during infection. More importantly, we highlight that, due to their impact on the entire bacterial population, these tolerance-inducing conditions completely mask persistence and the action of its molecular determinants. Finally, we show that even though tolerant populations display a high survival rate under bactericidal drug treatment, this feature comes at the cost of having impaired proliferation during infection. In contrast, persistence is a risk-limiting strategy that allows bacteria to survive antibiotic treatment without reducing the ability of the population to colonize their host. Altogether, our data emphasise that the distinction between these phenomena is of utmost importance to improve the design of more efficient antibiotic therapies.
The host environment is of critical importance for antibiotic efficacy. By impacting bacterial machineries, stresses encountered by pathogens during infection promote the formation of phenotypic ...variants that are transiently insensitive to the action of antibiotics. It is assumed that these recalcitrant bacteria-termed persisters-contribute to antibiotic treatment failure and relapsing infections. Recently, we demonstrated that host reactive nitrogen species (RNS) transiently protect persisters against the action of β-lactam antibiotics by delaying their regrowth within host cells. Here, we discovered that RNS intoxication of persisters also collaterally sensitizing them to fluoroquinolones during infection, explaining the higher efficiency of fluoroquinolones against intramacrophage Salmonella. By reducing bacterial respiration and the proton-motive force, RNS inactivate the AcrAB efflux machinery of persisters, facilitating the accumulation of fluoroquinolones intracellularly. Our work shows that target inactivity is not the sole reason for Salmonella persisters to withstand antibiotics during infection, with active efflux being a major contributor to survival. Thus, understanding how the host environment impacts persister physiology is critical to optimize antibiotics efficacy during infection.
Non-typhoidal Salmonella strains are responsible for invasive infections associated with high mortality and recurrence in sub-Saharan Africa, and there is strong evidence for clonal relapse following ...antibiotic treatment. Persisters are non-growing bacteria that are thought to be responsible for the recalcitrance of many infections to antibiotics. Toxin-antitoxin systems are stress-responsive elements that are important for Salmonella persister formation, specifically during infection. Here, we report the analysis of persister formation of clinical invasive strains of Salmonella Typhimurium and Enteritidis in human primary macrophages. We show that all the invasive clinical isolates of both serovars that we tested produce high levels of persisters following internalization by human macrophages. Our genome comparison reveals that S. Enteritidis and S. Typhimurium strains contain three acetyltransferase toxins that we characterize structurally and functionally. We show that all induce the persister state by inhibiting translation through acetylation of aminoacyl-tRNAs. However, they differ in their potency and target partially different subsets of aminoacyl-tRNAs, potentially accounting for their non-redundant effect.
Several important pathogens cause disease by surviving and replicating within host cells. Bacterial proliferation is the product of both replication and killing undergone by the population. However, ...these processes are difficult to distinguish, and are usually assessed together by determination of net bacterial load. In addition, measurement of net load does not reveal heterogeneity within pathogen populations. This is particularly important in persistent infections in which slow or nongrowing bacteria are thought to have a major impact. Here we report the development of a reporter system based on fluorescence dilution that enables direct quantification of the replication dynamics of Salmonella enterica serovar Typhimurium (S. Typhimurium) in murine macrophages at both the population and single-cell level. We used this technique to demonstrate that a major S. Typhimurium virulence determinant, the Salmonella pathogenicity island 2 type III secretion system, is required for bacterial replication but does not have a major influence on resistance to killing. Furthermore, we found that, upon entry into macrophages, many bacteria do not replicate, but appear to enter a dormant-like state. These could represent an important reservoir of persistent bacteria. The approach could be extended to other pathogens to study the contribution of virulence and host resistance factors to replication and killing, and to identify and characterize nonreplicating bacteria associated with chronic or latent infections.
Highlights ► Net bacterial growth rate does not necessarily reflect replication rate. ► Bacterial replication can be quantified directly by dilution methods. ► Single cell analysis of intracellular ...bacterial replication reveals heterogeneity. ► Heterogeneity of intracellular replication is caused by both host and pathogen. ► Non-replicating bacterial subpopulations illustrate the benefits of heterogeneity.
Mycobacterium tuberculosis infections result in a spectrum of clinical outcomes, and frequently the infection persists in a latent, clinically asymptomatic state. The within-host bacterial population ...is likely to be heterogeneous, and it is thought that persistent mycobacteria arise from a small population of viable, but non-replicating (VBNR) cells. These are likely to be antibiotic tolerant and necessitate prolonged treatment. Little is known about these persistent mycobacteria, since they are very difficult to isolate. To address this, we have successfully developed a replication reporter system for use in M. tuberculosis. This approach, termed fluorescence dilution, exploits two fluorescent reporters; a constitutive reporter allows the tracking of bacteria, while an inducible reporter enables the measurement of bacterial replication. The application of fluorescence single-cell analysis to characterize intracellular M. tuberculosis identified a distinct subpopulation of non-growing mycobacteria in murine macrophages. The presence of VBNR and actively replicating mycobacteria was observed within the same macrophage after 48 h of infection. Furthermore, our results suggest that macrophage uptake resulted in enrichment of non- or slowly replicating bacteria (as revealed by d-cycloserine treatment); this population is likely to be highly enriched for persisters, based on its drug-tolerant phenotype. These results demonstrate the successful application of the novel dual fluorescence reporter system both in vitro and in macrophage infection models to provide a window into mycobacterial population heterogeneity.
Bacterial populations can survive exposure to antibiotics through transient phenotypic and gene expression changes. These changes can be attributed to a small subpopulation of bacteria, giving rise ...to antibiotic persistence. Although this phenomenon has been known for decades, much remains to be learned about the mechanisms that drive persister formation. The RNA-binding protein ProQ has recently emerged as a global regulator of gene expression. Here, we show that ProQ impacts persister formation in Salmonella.
, ProQ contributes to growth arrest in a subset of cells that are able to survive treatment at high concentrations of different antibiotics. The underlying mechanism for ProQ-dependent persister formation involves the activation of metabolically costly processes, including the flagellar pathway and the type III protein secretion system encoded on Salmonella pathogenicity island 2. Importantly, we show that the ProQ-dependent phenotype is relevant during macrophage infection and allows Salmonella to survive the combined action of host immune defenses and antibiotics. Together, our data highlight the importance of ProQ in Salmonella persistence and pathogenesis.
Bacteria can avoid eradication by antibiotics through a phenomenon known as persistence. Persister cells arise through phenotypic heterogeneity and constitute a small fraction of dormant cells within a population of actively growing bacteria, which is susceptible to antibiotic killing. In this study, we show that ProQ, an RNA-binding protein and global regulator of gene expression, promotes persisters in the human pathogen Salmonella enterica serovar Typhimurium. Bacteria lacking the
gene outcompete wild-type bacteria under laboratory conditions, are less prone to enter growth dormancy, and form fewer persister cells. The basis for these phenotypes lies in ProQ's ability to activate energy-consuming cellular processes, including flagellar motility and protein secretion. Importantly, we show that ProQ contributes to the persister phenotype during Salmonella infection of macrophages, indicating an important role of this global regulator in Salmonella pathogenesis.
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