Role of RelA-synthesized (p)ppGpp and ROS-induced mutagenesis in de novo acquisition of antibiotic resistance in E. coli.

The stringent response of bacteria to starvation and stress also fulfills a role in addressing the threat of antibiotics. Within this stringent response, (p)ppGpp, synthesized by RelA or SpoT, functions as a global alarmone. However, the effect of this (p)ppGpp on resistance development is poorly understood. Here, we show that knockout of relA or rpoS curtails resistance development against bactericidal antibiotics. The emergence of mutated genes associated with starvation and (p)ppGpp, among others, indicates the activation of stringent responses. The growth rate is decreased in ΔrelA-resistant strains due to the reduced ability to synthesize (p)ppGpp and the persistence of deacylated tRNA impeding protein synthesis. Sluggish cellular activity causes decreased production of reactive oxygen species (ROS), thereby reducing oxidative damage, leading to weakened DNA mismatch repair, potentially reducing the generation of mutations. These findings offer new targets for mitigating antibiotic resistance development, potentially achieved through inhibiting (p)ppGpp or ROS synthesis.

The Effect of the Stringent Response and Oxidative Stress Response on Fitness Costs of De Novo Acquisition of Antibiotic Resistance.

Resistance evolution during exposure to non-lethal levels of antibiotics is influenced by various stress responses of bacteria which are known to affect growth rate. Here, we aim to disentangle how the interplay between resistance development and associated fitness costs is affected by stress responses. We performed de novo resistance evolution of wild-type strains and single-gene knockout strains in stress response pathways using four different antibiotics. Throughout resistance development, the increase in minimum inhibitory concentration (MIC) is accompanied by a gradual decrease in growth rate, most pronounced in amoxicillin or kanamycin. By measuring biomass yield on glucose and whole-genome sequences at intermediate and final time points, we identified two patterns of how the stress responses affect the correlation between MIC and growth rate. First, single-gene knockout E. coli strains associated with reactive oxygen species (ROS) acquire resistance faster, and mutations related to antibiotic permeability and pumping out occur earlier. This increases the metabolic burden of resistant bacteria. Second, the ΔrelA knockout strain, which has reduced (p)ppGpp synthesis, is restricted in its stringent response, leading to diminished growth rates. The ROS-related mutagenesis and the stringent response increase metabolic burdens during resistance development, causing lower growth rates and higher fitness costs.

Reactive oxygen species accelerate de novo acquisition of antibiotic resistance in E. coli.

Reactive oxygen species (ROS) produced as a secondary effect of bactericidal antibiotics are hypothesized to play a role in killing bacteria. If correct, ROS may play a role in development of de novo resistance. Here we report that single-gene knockout strains with reduced ROS scavenging exhibited enhanced ROS accumulation and more rapid acquisition of resistance when exposed to sublethal levels of bactericidal antibiotics. Consistent with this observation, the ROS scavenger thiourea in the medium decelerated resistance development. Thiourea downregulated the transcriptional level of error-prone DNA polymerase and DNA glycosylase MutM, which counters the incorporation and accumulation of 8-hydroxy-2'-deoxyguanosine (8-HOdG) in the genome. The level of 8-HOdG significantly increased following incubation with bactericidal antibiotics but decreased after treatment with the ROS scavenger thiourea. These observations suggest that in E. coli sublethal levels of ROS stimulate de novo development of resistance, providing a mechanistic basis for hormetic responses induced by antibiotics.

The influence of oxygen and oxidative stress on de novo acquisition of antibiotic resistance in E. coli and Lactobacillus lactis.

BACKGROUND: Bacteria can acquire resistance through DNA mutations in response to exposure to sub-lethal concentrations of antibiotics. According to the radical-based theory, reactive oxygen species (ROS), a byproduct of the respiratory pathway, and oxidative stress caused by reactive metabolic byproducts, play a role in cell death as secondary killing mechanism. In this study we address the question whether ROS also affects development of resistance, in the conditions that the cells is not killed by the antibiotic. RESULTS: To investigate whether oxygen and ROS affect de novo acquisition of antibiotic resistance, evolution of resistance due to exposure to non-lethal levels of antimicrobials was compared in E. coli wildtype and ΔoxyR strains under aerobic and anaerobic conditions. Since Lactococcus lactis (L. lactis) does not have an active electron transport chain (ETC) even in the presence of oxygen, and thus forms much less ROS, resistance development in L. lactis was used to distinguish between oxygen and ROS. The resistance acquisition in E. coli wildtype under aerobic and anaerobic conditions did not differ much. However, the aerobically grown ΔoxyR strain gained resistance faster than the wildtype or anaerobic ΔoxyR. Inducing an ETC by adding heme increased the rate at which L. lactis acquired resistance. Whole genome sequencing identified specific mutations involved in the acquisition of resistance. These mutations were specific for each antibiotic. The lexA mutation in ΔoxyR strain under aerobic conditions indicated that the SOS response was involved in resistance acquisition. CONCLUSIONS: The concept of hormesis can explain the beneficial effects of low levels of ROS and reactive metabolic byproducts, while high levels are lethal. DNA repair and mutagenesis may therefore expedite development of resistance. Taken together, the results suggest that oxygen as such barely affects resistance development. Nevertheless, non-lethal levels of ROS stimulate de novo acquisition of antibiotic resistance.

Porcine circovirus type 2 promotes Actinobacillus pleuropneumoniae survival during coinfection of porcine alveolar macrophages by inhibiting ROS production.

Actinobacillus pleuropneumoniae (APP) and porcine circovirus type 2 (PCV2) are both important pathogens of the porcine respiratory disease complex (PRDC), which results in significant worldwide economic losses. Recently, PCV2 and APP coinfection has been described in the worldwide pork industry, and represents an extremely complex situation in veterinary medicine. However, the mechanism of their coinfection has not been investigated. In this study, we found that PCV2 promoted APP adhesion to and invasion of porcine alveolar macrophages (PAMs) during coinfection. Additionally, PCV2 suppressed reactive oxygen species (ROS) production by inhibiting cytomembrane NADPH oxidase activity, which was beneficial for APP survival in PAMs in vitro. During coinfection, PCV2 weakened the inflammatory response and macrophage antigen presentation by decreasing TNF-α, IFN-γ and IL-4 expression, and reduced clearance of the invading bacteria. The host-cell experimental results were verified in a mouse model. The findings provide a deeper and novel understanding of porcine coinfection, and will be extremely helpful for the design of strategies for PRDC control.

The combination of NK and CD8+T cells with CCL20/IL15-armed oncolytic adenoviruses enhances the growth suppression of TERT-positive tumor cells.

Adoptive immunotherapy and targeted gene therapy have been extensively used to eliminate tumor cells. The combination treatment is capable of efficiently generating an effective antitumor immune response and disrupting tumor-induced tolerance. Moreover, effective antitumor immune responses are dependent on coordinate interaction among various effector cells. This study focused on whether the combination of cytotoxic effector cell-based adoptive immunotherapy and CCL20/IL15-armed oncolytic adenoviruses could induce enhanced antitumor activity. The CCL20/IL15-armed oncolytic adenovirus was constructed using homologous recombination with shuttle plasmids and full-length Ad backbones. We chose the telomerase reverse transcriptase promoter (TERTp) to replace the E1A promoter to drive the oncolytic adenoviral E1A gene. Thus, this CRAd-CCL20-IL15 could induce apoptosis in TERTp-positive tumor cells due to viral propagation, but these viruses could not replicate efficiently in normal cells. The combination of cytotoxic effector cells and CRAd-CCL20-IL15 showed greater antitumor potential than that of cytotoxic effector cells or CRAd-CCL20-IL15 alone. Moreover, the combined treatment could induce tumor-specific cytotoxicity of CTLs in vitro. Further analysis demonstrated that this combined treatment resulted in significant tumor regression in mouse models. This study has provided preclinical evidence that combined treatment with cytotoxic effector cells and CRAd-CCL20-IL15 may offer alternative treatment options for tumor therapy.