Preventing pathogenic bacteria from sensing nutrient starvation may present a new therapeutic approach to increasing antibiotic efficacy and preventing drug resistance, researchers claim. A team led by McGill University investigators has found that blocking an active mechanism used by bacteria to respond to starvation by slowing their growth significantly reduces the natural tolerance to antibiotics that infectious organisms develop when nutrient supplies become low.
Scientists have largely assumed that antibiotic resistance in nutrient-deprived bacteria occurs passively as the starved cells stop growing and the antibiotic target is inactivated. However, Pradeep K. Singh, Ph.D., Dao Nguyen, Ph.D., and colleagues now report that antibiotic tolerance resulting in nutrient-starved P. aeruginosa and biofilm cultures is in fact mediated by an active protective mechanism controlled by the starvation-signaling stringent response (SR). Their in vitro and in vivo studies showed that inactivating this protective mechanism sensitized biofilms to different classes of antibiotics, and significantly improved the effects of antibiotic treatment in mouse models of infection.
The investigators work is reported in Science in a paper titled “Active Starvation Responses Mediate Antibiotic Tolerance in Biofilms and Nutrient-Limited Bacteria.”
Bacteria are known to become tolerant to antibiotics when they are starved of nutrients, and during chronic infections this phenomenon occurs when bacteria grow into biofilms, the authors explain. Starvation in biofilms results because bacteria at the periphery of the biofilm clusters consume the available nutrients, effectively preventing the passage of nutrients to bacteria within the clusters. While these nutrient-deprived biofilm bacteria can be tolerant to almost all antibiotic classes, antibiotic sensitivity can be restored by supplying even limited substrates.
One of the main hypotheses put forward to explain starvation-induced antibiotic tolerance is that antibiotic targets become inactive in growth-arrested cells. Unfortunately reversing starvation as an approach to increasing antibiotic sensitivity isn’t an ideal approach in terms of therapeutic utility, comments Dr. Nguyen. “It presents a major dilemma. Sensitizing starved bacteria to antibiotics could require stimulating their growth, and this could be dangerous during human infections.”
Taking a slightly different view, the authors focused on the observation that growth arrest during starvation occurs alongside physiological changes induced by starvation responses, raising the possibility that tolerance depends on the adaptive responses (i.e., not just target inactivity due to growth arrest). This might be a more clinically relevant approach to increasing antibiotic activity in nutrient-starved biofilms.
In order to look more closely at the relative contributions of growth arrest and starvation physiology to antibiotic tolerance, the researchers devised a set of experiments in which nutrient-limited cells could be studied in the presence and absence of starvation responses. Their approach hinged on the ability of many bacteria to sense and respond to nutrient limitation through a regulatory mechanism known as the stringent response (SR), a phenomenon by which lack of nutrients induces relA and spoT gene products to synthesize the alarmone (p)ppGpp, as a signal to regulate genes and virulence. The team thus inactivated the SR in Pseudomonas aeruginosa bacteria by disrupting relA and spotT.
Their initial studies showed that administering the starvation-inducing serine analog serine hydroxamate (SHX) to wild-type P. aeruginosa reduced the cell-killing ability of the antibiotic ofloxacin by about 2,300-fold. In contrast, the cell-killing ability of ofloxacin in SHX-treated SR-inactivated ΔrelA spoT mutants only dropped about 34-fold. The numbers of ofloxacin-tolerant wild-type P. aeruginosa cells grown in stationary cultures and in biofilms was also significantly higher than the numbers of ofloxacin-tolerant ΔrelA spoT mutants. This observation didn’t appear to be due to any SR-mediated inhibition of growth and antibiotic target activity, because the growth curves of both stationary-phase cultures and biofilms indicated that both the wild-type and ΔrelA spoT mutant strains had ceased growing before antibiotics were added, the McGill team notes.
Interestingly, and despite being more sensitive to antibiotic administration, the biofilms formed by ΔrelA spoT mutants demonstrated similar rates of protein and RNA synthesis, and lower rates of DNA synthesis than the wild-type strain. “These data indicate that reduced drug target activity or growth arrest per se are not responsible for the tolerance of stationary-phase and biofilm bacteria, and that active SR-mediated responses are required.”
Focusing more closely on biofilms because they are clinically relevant to the persistence of chronic infections, the team subsequently found that SR inactivation sensitized biofilm bacteria to antibiotics with different mechanisms of action. Antibiotic sensitization was in addition still observed after extended antibiotic treatment time, and in biofilms grown for longer periods.
The increased cell-killing ability of antibiotics on SR-inactivated biofilms was similarly evident in both laboratory stains and clinical isolates, and in biofilms grown in microtiter wells and on filters on agar plates. However, antibiotic sensitization resulting from SR inactivation wasn’t evident in biofilms grown in a reactor system that flowed continuously.
Previous work has suggested that regardless of their primary targets, antibacterial drugs induce hydroxyl radical production and kill cells by oxidative damage. Further studies with biofilms supported this, by demonstrating that SR hydroxyl radical levels and also increased biofilm killing by the oxidants paraquat and phenazine methosulfate.
Observations that spontaneous cell death occurred in the centers of ΔrelA spoT colonies and biofilm clusters provided clues about the mechanisms involved in causing endogenous oxidative stress, because prior work had linked this type of autolysis in P. aeruginosa with bacterial overproduction of 4-hydroxy-2-alkylquinoline molecules (HAQ). Overexpression of HAQs in wild-type P. aeruginosa has been found to moderately increase antibiotic susceptibility.
LC-MS analysis confirmed that the ΔrelA spoT colonies did in fact produce higher levels of HAQs than wild-type strains, and inactivating the pqsA gene in the mutants to eliminate HAQ biosynthesis also abolished autolysis of colonies and restored wild-type hydroxyl radical levels in the mutant biofilms to those of wild-type bacteria. The effects of HAQ production on antibiotic susceptibility also appeared dose dependent, as modest increases in HAQ levels resulted in substantially enhanced antibiotic sensitivity (and hydroxyle radical levels) in ΔrelA spoT biofilms.
Interestingly, engineering wild-type P. aeruginosa to produce progressively increasing levels of HAQ had only minimal effects on antibiotic sensitivity, leading the researchers to put forward the notion that the ΔrelA spoT mutants might have impaired antioxidant defenses. Supporting this, studies showed that catalase and superoxide dismutase (SOD) activity was significantly depressed in SR-inactivated biofilms and also in the ΔrelA spoT pqsA triple mutant, which indicated that impaired oxidant defense occurred independently to HAQ overproduction.
Indeed, both the ΔpqsA and the ΔrelA spoT pqsA mutant biofilms displayed equivalent antibiotic sensitivity. This was particularly interesting because while neither strain expresses HAQ, the but ΔpqsA biofilms produce SOD and catalase at near wild-type levels, while the ΔrelA spoT pqsA biofilms produce only low levels of SOD and catalase. “Taken together, the data are consistent with a model in which the SR mediates the antibiotic tolerance of P. aeruginosa biofilms by both curtailing HAQ production and inducing antioxidant defenses,” the authors note.
Encouragingly, inactivating relA spoT in bacteria such as E. coli that don’t produce HAQs also reduced the numbers of antibiotic-tolerant bacteria 65-fold, and these mutant biofilms also demonstrated reduced catalase and higher hydroxyl radical levels.
For the findings thus far to have potential clinical utility, the researchers needed to see whether targeting SR would increase antibiotic activity in lethal infections. They infected mice with either stationary-phase wild-type P. aeruginosa, or ΔrelA spoT mutant P. aeruginosa, and then administered antibiotic therapy. This confirmed that whereas ofloxacin therapy had no effect on the survival of mice infected with wild-type bacteria, it was highly effective against the ΔrelA spoT strain. SR inactivation also increased antibiotic activity in a murine biofilm model, and eliminated the emergence of ofloxacin-resistant clones in conditions that would promote adaptive resistance.
The bacterial ability to sense starvation won’t affect the fact that the organisms will eventually stop growth and inactivate their antibiotic targets, Dr. Singh et al. conclude. But what this sensing process does allow is for bacteria to arrest their growth in a controlled manner that will maximize their chances of long-term survival. “Our data show that interfering with this orderly process sensitizes experimentally starved, stationary-phase, and biofilm bacteria to antibiotics, without stimulating their growth.” And importantly, the approach doesn’t involve directly tampering with antibiotic targets, but is independent to antibiotic activity.
“Antibiotic-tolerant states may depend on physiological adaptations without direct connections to antibiotic target activity or to drug uptake, efflux, or inactivation. Identifying these adaptations, and targeting them to enhance the activity of existing drugs, is a promising approach to mitigate the public health crisis caused by the scarcity of new antibiotics.”