The Effects of Sub-inhibitory Antibiotic Concentrations on Pseudomonas aeruginosa: Reduced Susceptibility Due to Mutations

Pseudomonas aeruginosa chronically infects in the lungs of people with cystic fibrosis and other forms of lung disease. Infections are treated with antibiotics, but over time, the bacteria acquire mutations that reduce their antibiotic susceptibility. The effects of inhibitory amounts of antibiotics in selecting for antibiotic-resistant mutants have been well studied. However, the concentrations of antibiotics that reach infecting bacteria can be sub-inhibitory and but may nonetheless promote emergence of antibiotic-resistant bacteria. Therefore, the aim of this research was to investigate the effects of sub-inhibitory concentrations of antibiotics on the antibiotic susceptibility of P. aeruginosa. Two P. aeruginosa reference strains, PAO1 and PA14, and six isolates from individuals with cystic fibrosis were studied. The bacteria were passaged in the presence of antibiotics (ceftazidime, ciprofloxacin, meropenem or tobramycin) at sub-inhibitory amounts. Fifteen populations of bacteria (up to five per strain) were exposed to each of the four antibiotics. Antibiotic susceptibility was determined following 10 passages on agar supplemented with antibiotic and compared with susceptibility prior to antibiotic exposure. Antibiotic exposure resulted in susceptibility being significantly (>2-fold) reduced for 13 of the 60 populations. Seven samples had reduced susceptibility to ciprofloxacin, three to tobramycin, two to ceftazidime and one to meropenem. Whole-genome sequencing revealed the mutations arising following antibiotic exposure. Mutants with reduced antibiotic susceptibility had mutations in genes known to affect antibiotic resistance, including regulators of efflux pumps (mexR, mexS, mexZ and nalC) and the fusA1 gene that is associated with aminoglycoside resistance. Genes not previously associated with resistance, including gacS, sigX and crfX and two genes with no known function, were also mutated in some isolates with reduced antibiotic susceptibility. Our results show that exposure to sub-inhibitory amounts of antibiotics can select for mutations that reduce the susceptibility of P. aeruginosa to antibiotics and that the profile of mutations is different from that arising during selection with inhibitory antibiotic concentrations. It is likely that exposure to sub-inhibitory amounts of antibiotics during infection contributes to P. aeruginosa becoming antibiotic-resistant.

A Survey of Spontaneous Antibiotic-Resistant Mutants of the Halophilic, Thermophilic Bacterium Rhodothermus marinus

Rhodothermus marinus is a halophilic extreme thermophile, with potential as a model organism for studies of the structural basis of antibiotic resistance. In order to facilitate genetic studies of this organism, we have surveyed the antibiotic sensitivity spectrum of R. marinus and identified spontaneous antibiotic-resistant mutants. R. marinus is naturally insensitive to aminoglycosides, aminocylitols and tuberactinomycins that target the 30S ribosomal subunit, but is sensitive to all 50S ribosomal subunit-targeting antibiotics examined, including macrolides, lincosamides, streptogramin B, chloramphenicol, and thiostrepton. It is also sensitive to kirromycin and fusidic acid, which target protein synthesis factors. It is sensitive to rifampicin (RNA polymerase inhibitor) and to the fluoroquinolones ofloxacin and ciprofloxacin (DNA gyrase inhibitors), but insensitive to nalidixic acid. Drug-resistant mutants were identified using rifampicin, thiostrepton, erythromycin, spiramycin, tylosin, lincomycin, and chloramphenicol. The majority of these were found to have mutations that are similar or identical to those previously found in other species, while several novel mutations were identified. This study provides potential selectable markers for genetic manipulations and demonstrates the feasibility of using R. marinus as a model system for studies of ribosome and RNA polymerase structure, function, and evolution.

Rapid and robust evolution of collateral sensitivity in Pseudomonas aeruginosa antibiotic-resistant mutants

The analysis of trade-offs, as collateral sensitivity, associated with the acquisition of antibiotic resistance, is mainly based on the use of model strains. However, the possibility of exploiting these trade-offs for fighting already resistant isolates has not been addressed in depth, despite the fact that bacterial pathogens are frequently antibiotic-resistant, forming either homogeneous or heterogeneous populations. Using a set of Pseudomonas aeruginosa-resistant mutants, we found that ceftazidime selects pyomelanogenic tobramycin-hypersusceptible mutants presenting chromosomal deletions in the analyzed genetic backgrounds. Since pyomelanogenic resistant mutants frequently coexist with other morphotypes in patients with cystic fibrosis, we analyzed the exploitation of this trade-off to drive extinction of heterogeneous resistant populations by using tobramycin/ceftazidime alternation. Our work shows that this approach is feasible because phenotypic trade-offs associated with the use of ceftazidime are robust. The identification of conserved collateral sensitivity networks may guide the rational design of evolution-based antibiotic therapies in P. aeruginosa infections.

Slow growth determines nonheritable antibiotic resistance in Salmonella enterica

Bacteria can withstand killing by bactericidal antibiotics through phenotypic changes mediated by their preexisting genetic repertoire. These changes can be exhibited transiently by a large fraction of the bacterial population, giving rise to tolerance, or displayed by a small subpopulation, giving rise to persistence. Apart from undermining the use of antibiotics, tolerant and persistent bacteria foster the emergence of antibiotic-resistant mutants. Persister formation has been attributed to alterations in the abundance of particular proteins, metabolites, and signaling molecules, including toxin-antitoxin modules, adenosine triphosphate, and guanosine (penta) tetraphosphate, respectively. Here, we report that persistent bacteria form as a result of slow growth alone, despite opposite changes in the abundance of such proteins, metabolites, and signaling molecules. Our findings argue that transitory disturbances to core activities, which are often linked to cell growth, promote a persister state regardless of the underlying physiological process responsible for the change in growth.

Associations between Bacteriophage Resistance and Antibiotic Resistance Phenotypes in Laboratory and Clinical strains of Salmonella Typhimurium

Background Bacteriophages have received great attention as alternative over antibiotics due to the host specificity. Therefore, this study was designed to evaluate the associations between bacteriophage-insensitive (BI) and antibiotic-resistant mutants of Salmonella Typhimurium strains. Bacteriophage-sensitive Salmonella Typhimurium ATCC 19585 (BSSTWT), ciprofloxacin-induced S. Typhimurium ATCC 19585 (BSSTCIP), S. Typhimurium KCCM 40253 (BSSTLAB), and clinically isolated multidrug-resistant S. Typhimurium CCARM 8009 (BSSTMDR) were used to induce the bacteriophage-insensitive mutants (BISTWT, BISTCIP, BISTLAB, and BISTMDR) against bacteriophage P22. Results The numbers of BSSTWT, BSSTCIP, and BSSTLAB were reduced by P22 (>3 log), while the least lytic activity was observed for BSSTMDR. BSSTWT treated with P22 showed the large variation in the cell state (CV>40%) and highest mutant frequency (62%), followed by 25% for STCIP. The least similarities between BSSTWT and BISTWT were observed at P22 and PBST-13 (<12%). The antibiotic susceptibilities were not significantly changed or slightly increased against BISTWT, BISTCIP, BISTLAB, and BISTMDR. The relative expression levels of bacteriophage-binding receptor-related genes (btuB, fhuA, fliK, fljB, ompC, ompF, rfaL, and tolC) were decreased in BISTCIP and BSSTMDR. Conclusion The results could pave the way for the application of bacteriophages as an alternative to control antibiotic-resistant bacteria.

Metabolic Compensation of Fitness Costs Is a General Outcome for Antibiotic-Resistant Pseudomonas aeruginosa Mutants Overexpressing Efflux Pumps

ABSTRACT It is generally assumed that the acquisition of antibiotic resistance is associated with a fitness cost. We have shown that overexpression of the MexEF-OprN efflux pump does not decrease the fitness of a resistant Pseudomonas aeruginosa strain compared to its wild-type counterpart. This lack of fitness cost was associated with a metabolic rewiring that includes increased expression of the anaerobic nitrate respiratory chain when cells are growing under fully aerobic conditions. It was not clear whether this metabolic compensation was exclusive to strains overexpressing MexEF-OprN or if it extended to other resistant strains that overexpress similar systems. To answer this question, we studied a set of P. aeruginosa mutants that independently overexpress the MexAB-OprM, MexCD-OprJ, or MexXY efflux pumps. We observed increased expression of the anaerobic nitrate respiratory chain in all cases, with a concomitant increase in NO3 consumption and NO production. These efflux pumps are proton/substrate antiporters, and their overexpression may lead to intracellular H+ accumulation, which may in turn offset the pH homeostasis. Indeed, all studied mutants showed a decrease in intracellular pH under anaerobic conditions. The fastest way to eliminate the excess of protons is by increasing oxygen consumption, a feature also displayed by all analyzed mutants. Taken together, our results support metabolic rewiring as a general mechanism to avoid the fitness costs derived from overexpression of P. aeruginosa multidrug efflux pumps. The development of drugs that block this metabolic “reaccommodation” might help in reducing the persistence and spread of antibiotic resistance elements among bacterial populations. IMPORTANCE It is widely accepted that the acquisition of resistance confers a fitness cost in such a way that in the absence of antibiotics, resistant populations will be outcompeted by susceptible ones. Based on this assumption, antibiotic cycling regimes have been proposed in the belief that they will reduce the persistence and spread of resistance among bacterial pathogens. Unfortunately, trials testing this possibility have frequently failed, indicating that resistant microorganisms are not always outcompeted by susceptible ones. Indeed, some mutations do not result in a fitness cost, and in case they do, the cost may be compensated for by a secondary mutation. Here we describe an alternative nonmutational mechanism for compensating for fitness costs, which consists of the metabolic rewiring of resistant mutants. Deciphering the mechanisms involved in the compensation of fitness costs of antibiotic-resistant mutants may help in the development of drugs that will reduce the persistence of resistance by increasing said costs. IMPORTANCE It is widely accepted that the acquisition of resistance confers a fitness cost in such a way that in the absence of antibiotics, resistant populations will be outcompeted by susceptible ones. Based on this assumption, antibiotic cycling regimes have been proposed in the belief that they will reduce the persistence and spread of resistance among bacterial pathogens. Unfortunately, trials testing this possibility have frequently failed, indicating that resistant microorganisms are not always outcompeted by susceptible ones. Indeed, some mutations do not result in a fitness cost, and in case they do, the cost may be compensated for by a secondary mutation. Here we describe an alternative nonmutational mechanism for compensating for fitness costs, which consists of the metabolic rewiring of resistant mutants. Deciphering the mechanisms involved in the compensation of fitness costs of antibiotic-resistant mutants may help in the development of drugs that will reduce the persistence of resistance by increasing said costs.

The spatial profiles and metabolic capabilities of microbial populations impact the growth of antibiotic-resistant mutants

Antibiotic resistance adversely affects clinical and public health on a global scale. Using the opportunistic human pathogen Pseudomonas aeruginosa , we show that increasing the number density of bacteria, on agar containing aminoglycoside antibiotics, can non-monotonically impact the survival of antibiotic-resistant mutants. Notably, at high cell densities, mutant survival is inhibited. A wide range of bacterial species can inhibit antibiotic-resistant mutants. Inhibition results from the metabolic breakdown of amino acids, which results in alkaline by-products. The consequent increase in pH acts in conjunction with aminoglycosides to mediate inhibition. Our work raises the possibility that the manipulation of microbial population structure and nutrient environment in conjunction with existing antibiotics could provide therapeutic approaches to combat antibiotic resistance.