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

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

Future challenges to microbial food safety

Despite significant efforts by all parties involved, there is still a considerable burden of foodborne illness, in which micro-organisms play a prominent role. Microbes can enter the food chain at different steps, are highly versatile and can adapt to the environment allowing survival, growth and production of toxic compounds. This sets them apart from chemical agents and thus their study from food toxicology. We summarize the discussions of a conference organized by the Dutch Food and Consumer Products Safety Authority and the European Food Safety Authority. The goal of the conference was to discuss new challenges to food safety that are caused by micro-organisms as well as strategies and methodologies to counter these. Management of food safety is based on generally accepted principles of Hazard Analysis Critical Control Points and of Good Manufacturing Practices. However, a more pro-active, science-based approach is required, starting with the ability to predict where problems might arise by applying the risk analysis framework. Developments that may influence food safety in the future occur on different scales (from global to molecular) and in different time frames (from decades to less than a minute). This necessitates development of new risk assessment approaches, taking the impact of different drivers of change into account. We provide an overview of drivers that may affect food safety and their potential impact on foodborne pathogens and human disease risks. We conclude that many drivers may result in increased food safety risks, requiring active governmental policy setting and anticipation by food industries whereas other drivers may decrease food safety risks. Monitoring of contamination in the food chain, combined with surveillance of human illness and epidemiological investigations of outbreaks and sporadic cases continue to be important sources of information. New approaches in human illness surveillance include the use of molecular markers for improved outbreak detection and source attribution, sero-epidemiology and disease burden estimation. Current developments in molecular techniques make it possible to rapidly assemble information on the genome of various isolates of microbial species of concern. Such information can be used to develop new tracking and tracing methods, and to investigate the behavior of micro-organisms under environmentally relevant stress conditions. These novel tools and insight need to be applied to objectives for food safety strategies, as well as to models that predict microbial behavior. In addition, the increasing complexity of the global food systems necessitates improved communication between all parties involved: scientists, risk assessors and risk managers, as well as consumer

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.</p

Multiplication of <i>ampC</i> upon Exposure to a Beta-Lactam Antibiotic Results in a Transferable Transposon in <i>Escherichia coli</i>

Plasmids play a crucial role in spreading antimicrobial resistance genes. Plasmids have many ways to incorporate various genes. By inducing amoxicillin resistance in Escherichia coli, followed by horizontal gene transfer experiments and sequencing, we show that the chromosomal beta-lactamase gene ampC is multiplied and results in an 8–13 kb contig. This contig is comparable to a transposon, showing similarities to variable regions found in environmental plasmids, and can be transferred between E. coli cells. As in eight out of nine replicate strains an almost completely identical transposon was isolated, we conclude that this process is under strict control by the cell. The single transposon that differed was shortened at both ends, but otherwise identical. The outcome of this study indicates that as a result of exposure to beta-lactam antibiotics, E. coli can form a transposon containing ampC that can subsequently be integrated into plasmids or genomes. This observation offers an explanation for the large diversity of genes in plasmids found in nature and proposes mechanisms by which the dynamics of plasmids are maintained

Antibiotic Killing through Incomplete DNA Repair

Two recent studies show that incomplete repair of DNA damage due to oxidized nucleotides is crucial for reactive oxygen species (ROS)-related antimicrobial lethality. Using widely different experimental approaches they both reach the same conclusions on the role of downstream ROS production in cell killing upon exposure to bactericidal antimicrobials