Potential risks of treating bacterial infections with a combination of ß-lactam and aminoglycoside antibiotics: A systematic quantification of antibiotic interactions in E. coli blood stream infection isolates.

BACKGROUND: Treatment of Blood Stream Infections (BSIs) with a combination of a ß-lactam and an aminoglycoside antibiotic is widely used in intensive care units (ICUs) around the world. However, no studies have systematically examined how these drugs interact and potentially influence the antimicrobial efficacy of the overall treatment. METHODS: We collected 500 E. coli isolates from the Uppsala University hospital that were isolated from blood of patients with suspicion of infection. Of those we tested the efficacy of combinations of 2 common ß-lactam antibiotics (Ampicillin and Cefotaxime) combined with 2 common aminoglycosides (Gentamicin and Tobramycin) on 254 isolates. The efficacy of all 4 pairwise combinations in inhibiting bacterial growth was then examined on all susceptible strains. That was done by quantifying the Fractional Inhibitory index (FICi), a robust metric for antibiotic combinatorial behaviour, of all possible treatments on every strain. When non additive interactions were identified, results of the original screen were verified with time kill assays. Finally, combination behaviours were analysed for potential cross correlations. FINDINGS: Out of the 4 antibiotic combinations screened none exhibited synergistic effects on any of the 254 strains. On the contrary all 4 exhibited important antagonistic effects on several isolates. Specifically, the combinations of AMP-GEN and CTX-GEN were antagonistic in 1.97% and 1.18% of strains respectively. Similarly, the combinations of AMP-TOB were antagonistic on 0.78% of all strains. PCA analysis revealed that an important factor on the responses to the combination treatments was the choice of a specific aminoglycoside over another. Subsequent cross correlation analysis revealed that the interaction profiles of combinations including the same aminoglycoside are significantly correlated (Spearman's cross correlation test p<0.001). INTERPRETATION: The findings of this study elucidate potential risks of the common combination treatment for blood stream infections. They also demonstrate, previously unquantified metrics on how antibiotics in combination therapies are not interchangeable with others of the same class. Finally, they reiterate the need for case-by-case testing of antibiotic interactions in a clinical setting. FUNDING: This work was funded by grants to DIA from the Swedish Research Council, the Wallenberg foundation and the Swedish Strategic Research Foundation.

Dynamics of Extensive Drug Resistance Evolution of Mycobacterium tuberculosis in a Single Patient During 9 Years of Disease and Treatment.

Mycobacterium tuberculosis is one of the hardest to treat bacterial pathogens with a high capacity to develop antibiotic resistance by mutations. Here we have performed whole-genome sequencing of consecutive M. tuberculosis isolates obtained during 9 years from a patient with pulmonary tuberculosis. The infecting strain was isoniazid resistant and during treatment it stepwise accumulated resistance mutations to 8 additional antibiotics. Heteroresistance was common and subpopulations with up to 3 different resistance mutations to the same drug coexisted. Sweeps of different resistant clones dominated the population at different time points, always coupled to resistance mutations coinciding with changes in the treatment regimens. Resistance mutations were predominant and no hitch-hiking, compensatory, or virulence-increasing mutations were detected, showing that the dominant selection pressure was antibiotic treatment. The results highlight the dynamic nature of M. tuberculosis infection, population structure, and resistance evolution and the importance of rapid antibiotic susceptibility tests to battle this pathogen.

Cellular hysteresis as a principle to maximize the efficacy of antibiotic therapy.

Antibiotic resistance has become one of the most dramatic threats to global health. While novel treatment options are urgently required, most attempts focus on finding new antibiotic substances. However, their development is costly, and their efficacy is often compromised within short time periods due to the enormous potential of microorganisms for rapid adaptation. Here, we developed a strategy that uses the currently available antibiotics. Our strategy exploits cellular hysteresis, which is the long-lasting, transgenerational change in cellular physiology that is induced by one antibiotic and sensitizes bacteria to another subsequently administered antibiotic. Using evolution experiments, mathematical modeling, genomics, and functional genetic analysis, we demonstrate that sequential treatment protocols with high levels of cellular hysteresis constrain the evolving bacteria by (i) increasing extinction frequencies, (ii) reducing adaptation rates, and (iii) limiting emergence of multidrug resistance. Cellular hysteresis is most effective in fast sequential protocols, in which antibiotics are changed within 12 h or 24 h, in contrast to the less frequent changes in cycling protocols commonly implemented in hospitals. We found that cellular hysteresis imposes specific selective pressure on the bacteria that disfavors resistance mutations. Instead, if bacterial populations survive, hysteresis is countered in two distinct ways, either through a process related to antibiotic tolerance or a mechanism controlled by the previously uncharacterized two-component regulator CpxS. We conclude that cellular hysteresis can be harnessed to optimize antibiotic therapy, to achieve both enhanced bacterial elimination and reduced resistance evolution.

Pharmacokinetics and Pharmacodynamics of Fosfomycin and Its Activity against Extended-Spectrum-ß-Lactamase-, Plasmid-Mediated AmpC-, and Carbapenemase-Producing Escherichia coli in a Murine Urinary Tract Infection Model.

Fosfomycin has become an attractive treatment alternative for urinary tract infections (UTIs) due to increasing multidrug resistance (MDR) in Escherichia coli In this study, we evaluated the pharmacokinetic (PK) and pharmacodynamic (PD) indices of fosfomycin and its in vivo activity in an experimental murine model of ascending UTI. Subcutaneous administration of fosfomycin showed that the mean peak plasma concentrations of fosfomycin were 36, 280, and 750 mg/liter following administration of a single dose of 0.75, 7.5, and 30 mg/mouse, respectively, with an elimination half-life of 28 min, and urine peak concentrations of 1,100, 33,400, and 70,000 mg/liter expected to be sustained above 1 mg/liter (MIC of the test strain, NU14) for 5, 8, and 9.5 h, respectively. The optimal PK/PD indices for reducing urine colony counts (number of CFU per milliliter) were determined to be the area under the concentration-time curve/MIC from 0 to 72 h and the maximum concentration/MIC on the basis of the dose-dependent bloodstream PK and the results of an evaluation of six dosing regimens. With a dosing regimen of 15 mg/mouse twice (every 36 h), fosfomycin significantly reduced the number of CFU per milliliter of all susceptible strains in urine, including clinical MDR strains, except for one clinical strain (P = 0.062). Variable degrees of reduction were observed in the bladder and kidneys. No significant reductions in the number of CFU per milliliter were observed with the resistant strains. In conclusion, fosfomycin shows concentration-dependent in vivo activity, and the results suggest that fosfomycin is an effective alternative to carbapenems in treating MDR E. coli in uncomplicated UTIs. The data on the effectiveness of fosfomycin against the MDR isolates along with the results of PK/PD modeling should facilitate the further development of improved recommendations for its clinical use.

Prediction of antibiotic resistance: time for a new preclinical paradigm?

Predicting the future is difficult, especially for evolutionary processes that are influenced by numerous unknown factors. Still, this is what is required of drug developers when they assess the risk of resistance arising against a new antibiotic candidate during preclinical development. In this Opinion article, we argue that the traditional procedures that are used for the prediction of antibiotic resistance today could be markedly improved by including a broader analysis of bacterial fitness, infection dynamics, horizontal gene transfer and other factors. This will lead to more informed preclinical decisions for continuing or discontinuing the development of drug candidates.

Resistance to the Cyclotide Cycloviolacin O2 in Salmonella enterica Caused by Different Mutations That Often Confer Cross-Resistance or Collateral Sensitivity to Other Antimicrobial Peptides.

Antimicrobial peptides (AMPs) are essential components of innate immunity in all living organisms, and these potent broad-spectrum antimicrobials have inspired several antibacterial development programs in the past 2 decades. In this study, the development of resistance to the Gram-negative bacterium-specific peptide cycloviolacin O2 (cyO2), a member of the cyclotide family of plant miniproteins, was characterized in Salmonella enterica serovar Typhimurium LT2. Mutants isolated from serial passaging experiments in increasing concentrations of cyO2 were characterized by whole-genome sequencing. The identified mutations were genetically reconstituted in a wild-type background. The additive effect of mutations was studied in double mutants. Fitness costs, levels of resistance, and cross-resistance to another cyclotide, other peptide and nonpeptide antibiotics, and AMPs were determined. A variety of resistance mutations were identified. Some of these reduced fitness and others had no effect on fitness in vitro, in the absence of cyO2. In mouse competition experiments, four of the cyO2-resistant mutants showed a significant fitness advantage, whereas the effects of the mutations in the others appeared to be neutral. The level of resistance was increased by combining several individual resistance mutations. Several cases of cross-resistance and collateral sensitivity between cyclotides, other AMPs, and antibiotics were identified. These results show that resistance to cyclotides can evolve via several different types of mutations with only minor fitness costs and that these mutations often affect resistance to other AMPs.

Dynamics of antibiotic resistant Mycobacterium tuberculosis during long-term infection and antibiotic treatment.

For an infecting bacterium the human body provides several potential ecological niches with both internally (e.g. host immunity) and externally (e.g. antibiotic use) imposed growth restrictions that are expected to drive adaptive evolution in the bacterium, including the development of antibiotic resistance. To determine the extent and pattern of heterogeneity generated in a bacterial population during long-term antibiotic treatment, we examined in a monoclonal Mycobacterium tuberculosis infection antibiotic resistant mutants isolated from one patient during a 9-years period. There was a progressive accumulation of resistance mutations in the infecting clone. Furthermore, apparent clonal sweeps as well as co-existence of different resistant mutants were observed during this time, demonstrating that during treatment there is a high degree of dynamics in the bacterial population. These findings have important implications for diagnostics and treatment of drug resistant tuberculosis infections.

Antibiotic treatment in vitro of phenotypically tolerant bacterial populations.

OBJECTIVES: Most pharmacodynamic models used for design of treatment regimens are based on time-kill data obtained with normal cells in the susceptible state without taking into account the killing kinetics of the antibiotic-tolerant cells in the population. We compared the microbiological efficacy of six antibiotics against tolerant cells and by mathematical modelling explored the potential clinical implications of tolerance. METHODS: Tolerant cells were obtained by filtration of bacterial cultures of Escherichia coli MG1655 after antibiotic exposure. Killing kinetics of the tolerant cells was compared with that of exponentially growing naive cells. To examine the nutrient dependency of the reversion from the tolerant state to the susceptible state, tolerant cells were re-suspended in Luria-Bertani and PBS and re-exposed to antibiotics. A mathematical model was used to explore the clinical implications of antibiotic tolerance. RESULTS: Streptomycin was the most efficient drug against tolerant cells. Ciprofloxacin and ampicillin had intermediate activity against tolerant cells while rifampicin, tetracycline and erythromycin had poor activity against tolerant cells. No correlation could be established between the microbiological efficacies against susceptible and tolerant cells. Reversion from tolerance to susceptibility was dependent on the presence of nutrients and growth. Computer simulations demonstrated that the efficacy of antibiotics against tolerant cell populations has a large influence on treatment outcome. CONCLUSIONS: The in vitro killing kinetics of tolerant cells is antibiotic-dependent and different from that of cells in the susceptible state. This difference in efficacy could have an influence on treatment outcome and tolerance should therefore be studied further in vivo.