Joran Michiels

Obg and Membrane Depolarization Are Part of a Microbial Bet-Hedging Strategy that Leads to Antibiotic Tolerance

Within bacterial populations, a small fraction of persister cells is transiently capable of surviving exposure to lethal doses of antibiotics. As a bet-hedging strategy, persistence levels are determined both by stochastic induction and by environmental stimuli called responsive diversification. Little is known about the mechanisms that link the low frequency of persisters to environmental signals. Our results support a central role for the conserved GTPase Obg in determining persistence in Escherichia coli in response to nutrient starvation. Obg-mediated persistence requires the stringent response alarmone (p)ppGpp and proceeds through transcriptional control of the hokB-sokB type I toxin-antitoxin module. In individual cells, increased Obg levels induce HokB expression, which in turn results in a collapse of the membrane potential, leading to dormancy. Obg also controls persistence in Pseudomonas aeruginosa and thus constitutes a conserved regulator of antibiotic tolerance. Combined, our findings signify an important step toward unraveling shared genetic mechanisms underlying persistence.

Frequency of antibiotic application drives rapid evolutionary adaptation of Escherichia coli persistence

The evolution of antibiotic resistance is a major threat to society and has been predicted to lead to 10 million casualties annually by 20501. Further aggravating the problem, multidrug tolerance in bacteria not only relies on the build-up of resistance mutations, but also on some cells epigenetically switching to a non–growing antibiotic-tolerant ‘persister’ state2–6. Yet, despite its importance, we know little of how persistence evolves in the face of antibiotic treatment7. Our evolution experiments in Escherichia coli demonstrate that extremely high levels of multidrug tolerance (20–100%) are achieved by single point mutations in one of several genes and readily emerge under conditions approximating clinical, once-daily dosing schemes. In contrast, reversion to low persistence in the absence of antibiotic treatment is relatively slow and only partially effective. Moreover, and in support of previous mathematical models8–10, we show that bacterial persistence quickly adapts to drug treatment frequency and that the observed rates of switching to the persister state can be understood in the context of ‘bet-hedging’ theory. We conclude that persistence is a major component of the evolutionary response to antibiotics that urgently needs to be considered in both diagnostic testing and treatment design in the battle against multidrug tolerance.

Use of Dual Marker Transposons to Identify New Symbiosis Genes in Rhizobium.

Rhizobium etli elicits nitrogen-fixing nodules on the roots of Phaseolus vulgaris. Using a composite dual-marker mini-Tn5 transposon carrying combinations of a constitutively expressed gfp gene and a promoterless gusA gene, we identified novel genes required for an efficient symbiosis. The induction of the gusA gene was used to determine the expression level of the different target genes under conditions partly mimicking the symbiotic environment ex planta. The green fluorescence was used to localize the bacteria in infection threads or inside the plant cells. Among the identified R. etli mutants, several produced a Nod− phenotype, whereas others were Fix− or displayed a reduced acetylene reduction activity during symbiosis. Partial sequence analysis of the mutated genes allowed us to classify them as nodulation genes, nitrogen fixation genes, genes possessing various enzymatic functions previously not yet associated with symbiosis, and genes displaying no similarity to any other sequence in the database. This methodology can be used to screen large numbers of mutants in the search for novel genes important for Rhizobium-legume symbiosis, and may be adapted to study other plant-bacterium interactions.

In vitro emergence of high persistence upon periodic aminoglycoside challenge in the ESKAPE pathogens

ABSTRACT Health care-associated infections present a major threat to modern medical care. Six worrisome nosocomial pathogens—Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.—are collectively referred to as the “ESKAPE bugs.” They are notorious for extensive multidrug resistance, yet persistence, or the phenotypic tolerance displayed by a variant subpopulation, remains underappreciated in these pathogens. Importantly, persistence can prevent eradication of antibiotic-sensitive bacterial populations and is thought to act as a catalyst for the development of genetic resistance. Concentration- and time-dependent aminoglycoside killing experiments were used to investigate persistence in the ESKAPE pathogens. Additionally, a recently developed method for the experimental evolution of persistence was employed to investigate adaptation to high-dose, extended-interval aminoglycoside therapy in vitro. We show that ESKAPE pathogens exhibit biphasic killing kinetics, indicative of persister formation. In vitro cycling between aminoglycoside killing and persister cell regrowth, evocative of clinical high-dose extended-interval therapy, caused a 37- to 213-fold increase in persistence without the emergence of resistance. Increased persistence also manifested in biofilms and provided cross-tolerance to different clinically important antibiotics. Together, our results highlight a possible drawback of intermittent, high-dose antibiotic therapy and suggest that clinical diagnostics might benefit from taking into account persistence.

Experimental Evolution of Escherichia coli Persister Levels Using Cyclic Antibiotic Treatments

Persister cells are difficult to study owing to their transient nature and their usually small number in bacterial populations. In the past, numerous attempts have been made to elucidate persistence mechanisms. However, because of the challenges involved in studying persisters and the clear redundancy in mechanisms underlying their generation, our knowledge of molecular pathways to persistence remains incomplete. Here, we describe how to use experimental evolution with cyclic antibiotic treatments to generate mutants with an increased persister level in stationary phase, ranging from the initial ancestral level up to 100 %. This method will help to unravel molecular pathways to persistence, and opens up a myriad of new possibilities in persister research, such as the convenient study of nearly pure persister cultures and the possibility to investigate the role of time and environmental aspects in the evolution of persistence.

Should we develop screens for multi-drug antibiotic tolerance?

The rampant spread of antibiotic resistance endangers the continued use of antibiotics in modern medicine for effective treatment and prophylaxis of bacterial infections. For some pathogens, we have already run out of antibiotic therapeutic options. Due to the seemingly unstoppable spread of antibiotic resistance, a post-antibiotic era is imminent, jeopardizing modern health care [1]. Antibiotic resistance claims over 23,000 deaths per year in the USA alone [2], and is predicted to result in 10 million annual casualties around the globe by 2050, associated with a cost of up to 100 trillion USD [3]. When it comes to explaining treatment failure, genetic antibiotic resistance has always been considered the main culprit. Many different resistance mechanisms exist, but they all have in common that they prevent the antibiotic from binding to its cellular target. As a result, pathogens can grow at elevated antibiotic concentrations. Since resistant organisms are expected to be genetically stable and less responsive to treatment in vivo, antimicrobial susceptibility testing by assessing the minimum inhibitory concentration (MIC) in vitro has become mainstream to guide antimicrobial therapy. MIC values, however, do not always accurately predict treatment outcome. For example, MIC testing ignores possible interactions with host immune factors. Azithromycin, while lacking activity in standard susceptibility assays, possesses potent efficacy against multi-drug-resistant pathogens due to synergy with a host antimicrobial peptide [4]. On the other hand, many pathogens are documented to be highly susceptible in laboratory testing yet unresponsive in clinical therapy. The latter likely results from antibiotic tolerance, an alternative strategy employed by bacteria to withstand antibiotics. Contrary to resistance, tolerance does not increase the MIC, but instead enhances survival of the pathogen facing lethal antibiotics without increasing growth capacity. We argue that the impact of antibiotic tolerance on treatment outcome is being underestimated in the current antibiotic crisis, as it may impede pathogen clearance in patients, allowing recurrence of infection upon cessation of therapy. This particularly holds true when the immune response is suppressed, e.g. in immunocompromised hosts, in infections of body sites with restricted immune access, or when pathogens are occluded in biofilms. Under these circumstances, a strong and effective bactericidal action of the antibiotic is necessary to clear the infection. Not long after the introduction of penicillin, it was demonstrated that susceptible populations of Staphylococcus aureus are not completely sterilized after treatment with high antibiotic concentrations in vitro [5]. Later, prolonged antibiotic therapy of Mycobacterium tuberculosis infections in mice was found to result in viable pathogens that were highly susceptible upon subsequent in vitro testing [6]. Numerous studies also directly compared treatment outcomes of tolerant and nontolerant strains using animal infection models. In a rabbit endocarditis model, tolerant strains were consistently more difficult to cure [7]. In Streptococcus pneumoniae, loss-offunction of a sensor histidine kinase (VncS) causes high tolerance to vancomycin in rabbit, while the MIC remains unchanged [8]. Strikingly, similar mutations were also found in clinical isolates. Indeed, tolerance is common among clinical isolates and has been reported for at least 20 different pathogens [9]. While drug-resistant M. tuberculosis were found in 21% of recrudescent tuberculosis, the remaining 79% of relapse was not caused by resistance [1]. Salmonella bloodstream infections are prevalent among HIV-infected individuals, and survivors frequently experience multiple rounds of relapse. Isolates from initial and recurrent episodes are genetically identical, consistent with relapse of the primary strain due to tolerant survivors rather than reinfection [10]. Similarly, 20–30% of women affected by urinary tract infections experiences relapse of infection within 3–4 months [11]. Tolerance was also associated with failure to eradicate susceptible group A streptococci in pharyngitis [12], and in a child with recrudescent pneumococcal meningitis the isolated strain was highly susceptible (easily growth-inhibited by low antibiotic concentrations, thus low MIC values), yet also highly tolerant (growth-inhibited cells were nevertheless not killed) [13]. In recent years, the phenomenon of persistence has gained considerable attention and rekindled the clinical interest in antibiotic tolerance. Bacterial persistence refers to the presence of a small fraction of cells (persisters) within a