Antimicrobial peptide exposure selects for Staphylococcus aureus resistance to human defence peptides

Abstract

Background: The clinical development of antimicrobial peptides (AMPs) is currently under evaluation to combat the rapid increase in MDR bacterial pathogens. However, many AMPs closely resemble components of the human innate immune system and the ramifications of prolonged bacterial exposure to AMPs are not fully understood.

Objectives: We show that in vitro serial passage of a clinical USA300 MRSA strain in a host-mimicking environment containing host-derived AMPs results in the selection of stable AMP resistance.

Methods: Serial passage experiments were conducted using steadily increasing concentrations of LL-37, PR-39 or wheat germ histones. WGS and proteomic analysis by MS were used to identify the molecular mechanism associated with increased tolerance of AMPs. AMP-resistant mutants were characterized by measuring in vitro fitness, AMP and antibiotic susceptibility, and virulence in a mouse model of sepsis.

Results: AMP-resistant Staphylococcus aureus mutants often displayed little to no fitness cost and caused invasive disease in mice. Further, this phenotype coincided with diminished susceptibility to both clinically prescribed antibiotics and human defence peptides.

Conclusions: These findings suggest that therapeutic use of AMPs could select for virulent mutants with cross-resistance to human innate immunity as well as antibiotic therapy. Thus, therapeutic use of AMPs and the implications of cross-resistance need to be carefully monitored and evaluated.

Figure 1.

AMP resistance in S. aureus can evolve without a fitness cost. (a) Extent of LL-37 resistance was determined by calculating percentage bacterial survival after LL-37 exposure. Fold changes over WT (DA28823) are indicated above each bar. (b) Differences in membrane potential are presented as the ratio of red/green fluorescence intensity following incubation with the fluorescent membrane potential indicator dye DiOC₂(3). Assay was verified using the protonophore CCCP and two strains derived from the Nebraska Transposon Mutant Library that display reduced membrane potential (ΔmenD, NE1345 and ΔhemB, NE1845). Fitness was determined in (c) TSB, (d) LB or (e) MIEM growth conditions. Relative fitness is presented as the maximum exponential generation time relative to WT with the broken line representing the relative WT growth rate. All values given are the mean ± SEM derived from at least three independent determinations. Relative fitness assays were also performed in technical triplicate. Statistical significance relative to WT (DA28823) was determined by one-way ANOVA with Fisher's least significant difference test used for post hoc analysis (***P < 0.001, **P < 0.01 or *P < 0.05). All relative fitness values were statistically significant with P < 0.001.

Figure 2.

Stable AMP resistance is conferred by reduced AMP uptake. Heatmaps of selected protein expression profiles grouped into categories based on their biological roles: (a) AMP resistance determinants; (b) electron transport chain; (c) metabolism; (d) transporters; and (e) virulence factors. S. aureus strains (AMP-resistant mutants or WT) are indicated above each column and protein expression is shown in rows. Proteomics analysis was performed using MS in bacterial cells derived from exponential growth in TSB medium in the absence of AMP selection as described in the Materials and methods section. Each column represents the average fold change calculated relative to WT (DA28823) for the indicated protein. All expression experiments were performed with two biological replicates. Changes in protein expression levels are represented in colour: yellow indicates increased protein levels and blue indicates decreased protein levels relative to WT. Data are available via ProteomeXchange with identifier PXD004036.

Figure 3.

AMP-resistant S. aureus are virulent. (a) Haemolytic activity was assessed following isolation on veal blood agar. (b) Carotenoid pigment production was assayed using methanol extraction. Values presented are the mean ± SEM derived from three independent determinations and statistical significance relative to WT was analysed using one-way ANOVA with Fisher's least significant difference test for post hoc analysis (***P < 0.001 or *P < 0.05). To assay in vivo virulence capacity, BALB/c mice were infected intraperitoneally with 0.5–2 × 10⁷ cfu of S. aureus and bacterial cells were enumerated from the (c) spleen, (d) liver and (e) kidney. Values presented are bacterial cfu counts normalized per gram of tissue (cfu/g) from three to four mice per bacterial strain. Boxes show the median and 25th and 75th percentiles, while error bars show the minimum and maximum values. Significant differences in bacterial colonization of AMP-resistant S. aureus strains were determined relative to WT using the Mann–Whitney U-test (*P < 0.05).

Figure 4.

AMP-resistant mutants are less susceptible to human defensins. (a) Susceptibility to the human α-defensins was determined by calculating percentage bacterial survival following 2 h of exposure to 10 μM peptide. Susceptibility to the human β-defensins (b) hBD1, (c) hBD2, (d) hBD3 and (e) hBD4 was determined as for α-defensins with 10 μM (hBD1, hBD2, hBD4) or 1 μM (hBD3) peptide concentrations. Limit of detection of the assay was 1% survival. Values given are the mean ± SEM derived from at least three independent determinations. Statistical significance relative to WT was determined by performing one-way ANOVA with Fisher's least significant difference test used for post hoc analysis on log-transformed data (***P < 0.001, **P < 0.01 or *P < 0.05).

Figure 5.

Possible consequences of clinical AMP usage. Widespread clinical use of host-derived AMPs to treat MDR S. aureus may cause a vicious cycle further limiting treatment options. Continuous exposure to host-derived AMPs results in a stable AMP-resistant phenotype with reduced susceptibility to human defence peptides allowing for enhanced colonization of humans. Persistently colonized individuals are more susceptible to invasive S. aureus infections, which require antibiotic treatment. However, AMP-resistant S. aureus is associated with decreased susceptibility to antibiotics thereby increasing the use of last resort drugs. Enhanced exposure to these antibiotics will increase pressure for the evolution of bacterial resistance yielding highly drug-resistant pathogens.