Abstract
Antimicrobial peptides (AMPs) offer a promising solution to the antibiotic resistance crisis. However, an unresolved serious concern is that the evolution of resistance to therapeutic AMPs may generate cross-resistance to host AMPs, compromising a cornerstone of the innate immune response. We systematically tested this hypothesis using globally disseminated mobile colistin resistance (MCR) that has been selected by the use of colistin in agriculture and medicine. Here, we show that MCR provides a selective advantage to Escherichia coli in the presence of key AMPs from humans and agricultural animals by increasing AMP resistance. Moreover, MCR promotes bacterial growth in human serum and increases virulence in a Galleria mellonella infection model. Our study shows how the anthropogenic use of AMPs can drive the accidental evolution of resistance to the innate immune system of humans and animals. These findings have major implications for the design and use of therapeutic AMPs and suggest that MCR may be difficult to eradicate, even if colistin use is withdrawn.
Generated Summary
This research article investigates the evolution of colistin resistance and its impact on bacterial resistance to host antimicrobial peptides (AMPs) and virulence. The study focuses on mobile colistin resistance (MCR) genes, which have become widespread due to the use of colistin in agriculture and medicine. The researchers tested the hypothesis that the evolution of resistance to therapeutic AMPs, such as colistin, might lead to cross-resistance to host AMPs, thereby compromising the innate immune response. The study employed a systematic approach, using experimental evolution and in vitro and in vivo assays, to assess the effects of MCR genes on bacterial resistance, growth, and virulence. The research examines the impact of MCR-1, a key MCR gene, on resistance to host AMPs, serum resistance, and virulence in a Galleria mellonella infection model. The researchers utilized various techniques, including measuring minimum inhibitory concentrations (MICs) of AMPs, in vitro competition assays, and in vivo virulence assays, to evaluate the effects of MCR plasmids on bacterial susceptibility, fitness, and virulence. The findings highlight the implications for the use of therapeutic AMPs and suggest the potential difficulty in eradicating MCR, even if colistin use is discontinued. The study’s scope includes testing the pleiotropic impacts of MCR genes that have become widely distributed in Escherichia coli due to anthropogenic use of colistin, analyzing the effect of MCR on resistance to host AMPs, assessing serum resistance, and evaluating virulence in a G. mellonella infection model.
Key Findings & Statistics
- The study found that MCR-1 imposes a significant fitness burden in the absence of an antimicrobial peptide (AMP) (p=1.174e-15).
- MCR-1 provided a significant competitive fitness advantage at concentrations of host AMPs between 1/4 and 1/2 of minimum inhibitory concentration (MIC).
- On average, MCR plasmids provided increased resistance to host AMPs by 62% (mean fold change in MIC = 1.62; SEM = 0.11; t = 5.615; p<0.0001).
- MCR plasmids increased resistance to most AMPs, but generated collateral sensitivities to both PROPH and PR39 (Figure 2b; F8,40 = 7.85; p<0.0001).
- The concentration of LL-37 required to select for MCR-1 (~3.4 µM) falls well within the reported physiological concentration range (up to 10 µM).
- The average change in resistance conferred by MCR plasmids varied significantly between AMPs.
- MCR generated large increases in resistance to colistin compared to host AMPs.
- The study demonstrated that MCR-1 is effective at providing protection against even complex mixtures of antimicrobials.
- MCR-1 conferred high levels of resistance to human serum (Figure 4a).
- MCR-1 carrying strain showed increased virulence compared to the control strain (Figure 4e, log-rank test, p=0.024).
- MCR-1 continued to increase virulence in LPS-treated larvae, suggesting that reduced host immunostimulation was not responsible for the increased virulence associated with MCR-1 expression (Figure 4f, log-rank test p=0.0074).
Other Important Findings
- The study revealed that sub-MIC doses of antimicrobial peptides (AMPs) generate selection for mobile colistin resistance (MCR).
- The research showed that MCR-1 expression vector (pSEVA:MCR-1) or an empty vector control (pSEVA:EV) were competed against a tester strain carrying a chromosomally integrated GFP across a range of AMP concentrations.
- MCR-1 provides a selective advantage to Escherichia coli in the presence of key AMPs from humans and agricultural animals by increasing AMP resistance.
- MCR promotes bacterial growth in human serum and increases virulence in a Galleria mellonella infection model.
- Host complement systems play a major role in bacterial killing by serum, suggesting that MCR-1 may provide resistance against the complement system.
- The presence of functional MCR-1 was not associated with increased resistance to C6-defficient serum.
- MCR-1-mediated modification of LPS can result in reduced stimulation of macrophages and limited release of inflammatory molecules.
Limitations Noted in the Document
- The study acknowledges the challenge of understanding the selective benefits of increased resistance to host immunity.
- The researchers were not able to quantitatively assess the fitness benefit provided by MCR-1 in serum, which made it difficult to estimate the selective advantage of increased serum resistance.
- The selective advantage of MCR-1 in Galleria or determine the extent to which increased virulence was driven by decreased susceptibility to insect AMPs in this system was not determined.
- The study used a single wild-type host strain for testing, which may not be representative of all E. coli strains due to the extensive genetic diversity.
- The study notes that the effects of MCR-1 in intracellular environments and the selective benefits in such conditions remain unclear.
- The study acknowledges that the population structure of the MCR-1-negative isolates in the study remains unknown, which limits the ability to draw conclusions about the association between MCR-1 and B2 in infection.
Conclusion
The research underscores the potential threat posed by the evolution of resistance to host AMPs due to the use of therapeutic AMPs, highlighting the need for caution in their application. The study found that the anthropogenic use of AMPs, such as colistin, can drive the evolution of resistance to key components of innate immunity. This raises concerns because many AMPs are currently in clinical trials, and their effectiveness could be compromised if resistance evolves. The research emphasizes that MCR-1 increases bacterial fitness and resistance in the presence of AMPs from humans and agricultural animals that are important sources of MCR-carrying E. coli. This highlights the risk of infections caused by MCR-E. coli. The findings imply that MCR-1 provides effective resistance against AMP cocktails found in host tissues. While MCR-mediated protection against other antimicrobials, like lysozyme and complement systems, may contribute to this protective phenotype. The study showed that MCR-1-mediated evasion of immunity may be important in clinical settings. The proportion of human infection isolates with MCR-1 remained constant following the ban on colistin use, in contrast to the decline in healthy human carriage isolates. This suggests that AMP resistance may help to maintain MCR-1 in humans and animals, even if colistin usage remains low. The study stresses the value of understanding the population structure of clinical pathogens when studying antimicrobial resistance, and it stresses the importance of further understanding the role of MCR in human infection. The findings of this study underscore the importance of assessing the impact of evolved resistance to therapeutic AMPs on resistance to host innate immunity and bacterial virulence during preclinical development using sensitive and quantitative assays. It is conceivable that mechanisms that have evolved to provide pathogenic bacteria with protection against host AMPs may also help to accelerate the evolution of resistance to therapeutic AMPs.