Persistent bacterial infections, antibiotic tolerance, and the oxidative stress response

Abstract

Certain bacterial pathogens are able to evade the host immune system and persist within the human host. The consequences of persistent bacterial infections potentially include increased morbidity and mortality from the infection itself as well as an increased risk of dissemination of disease. Eradication of persistent infections is difficult, often requiring prolonged or repeated courses of antibiotics. During persistent infections, a population or subpopulation of bacteria exists that is refractory to traditional antibiotics, possibly in a non-replicating or metabolically altered state. This review highlights the clinical significance of persistent infections and discusses different in vitro models used to investigate the altered physiology of bacteria during persistent infections. We specifically focus on recent work establishing increased protection against oxidative stress as a key element of the altered physiologic state across different in vitro models and pathogens.

Keywords: antibiotic tolerance; biofilms; oxidative stress; persistent bacterial infections; persisters; small colony variants.

Figure 1. Persistence vs. environmentally induced antibiotic indifference. (A) Type I persistence. Slowly replicating, antibiotic tolerant cells result from passage through stationary phase, and are characterized by an extended lag time after dilution in fresh media (type I persisters). The number of type I persisters observed is directly proportional to the size of the stationary phase inoculum. When the diluted population is exposed to antibiotics the majority of cells die, but the persisters survive. Persister cells are represented by red ovals, and rapidly growing, antibiotic-sensitive cells are represented by white ovals. (B) Type II persistence. Slowly replicating, antibiotic tolerant cells also may be continuously generated during exponential growth (type II persisters). When the population is exposed to antibiotics, the majority of cells die, but the persisters survive. When the surviving cells are regrown in fresh media, the bacteria regain antibiotic sensitivity as this is a phenotypic change, but new persister cells may be generated. Persister cells are represented by black ovals and rapidly growing, antibiotic-sensitive cells are represented by white ovals. (C) Environmentally induced antibiotic indifference. Slowly replicating or non-replicating, antibiotic-tolerant cells may also result from environmental stresses like hypoxia or carbon starvation, which induce population-wide antibiotic indifference. When the population is exposed to antibiotics, all the cells survive. When the environmental stress is removed, the surviving cells resume growth and regain antibiotic sensitivity as this is a phenotypic, not genetic, change. Environmental-induced antibiotic-tolerant cells are represented by green ovals and rapidly growing, antibiotic-sensitive cells are represented by white ovals. This figure depicts theoretical extremes for persistence and environmental-induced antibiotic indifference. In reality, all three types of antibiotic-tolerant cells can co-exist depending on the conditions of growth and the culture.

Figure 2. The role of oxidative stress in different in vitro models for persistent infections. (A) Biofilms. Bacteria in biofilms are exposed to increased endogenous oxidative stress, resulting in upregulation of soxS, a regulator of the superoxide response. In addition, for bacteria in biofilms or exposed to starvation conditions, the RelA mediated stringent response results in increased expression of superoxide dismutases (SOD) and decreased expression of pro-oxidants (HAQ). As a result, bacteria exhibit higher tolerance to ROS. (B) Persisters. Persisters have been shown to exhibit differential sensitivity to ROS compared with rapidly growing, antibiotic susceptible bacteria. Proposed mechanisms include increased expression of efflux pumps, a component of the oxidative stress response, the secretion of indole, which may induce oxidative protective mechanisms in neighboring cells, and upregulation of the SOS response. (C) Intracellular infection. M. tuberculosis, an intracellular pathogen, expresses specific factors to counteract the ROS and RNS encountered in the phagosome, including expression of efflux pumps, superoxide dismutases and low molecular weight thiols. Antibiotic tolerance within the macrophages is mediated by the expression of efflux pumps in M. marinum. (D) Small colony variants. The small colony variant phenotype, which may represent an adaptation to facilitate intracellular survival and growth, is characterized by deficiencies in the electron transport chain. Repression of the electron transport chain results in the generation of fewer ROS within the cell as well as reduced ATP production and transmembrane potential, all of which may affect antibiotic efficacy. In all panels, antibiotic tolerant cells are represented by black ovals, and antibiotic sensitive bacteria are represented by white ovals.