The Impact of Omega-3 Fatty Acids on the Evolution of Acinetobacter baumannii Drug Resistance

ABSTRACT The bacterial pathogen Acinetobacter baumannii has emerged as an urgent threat to health care systems. The prevalence of multidrug resistance in this critical human pathogen is closely associated with difficulties in its eradication from the hospital environment and its recalcitrance to treatment during infection. The development of resistance in A. baumannii is in part due to substantial plasticity of its genome, facilitating spontaneous genomic evolution. Many studies have investigated selective pressures imposed by antibiotics on genomic evolution, but the influence of high-abundance bioactive molecules at the host-pathogen interface on mutation and rates of evolution is poorly understood. Here, we studied the roles of host fatty acids in the gain in resistance to common antibiotics. We defined the impact of the polyunsaturated fatty acids arachidonic acid and docosahexaenoic acid on the development of resistance to erythromycin in A. baumannii strain AB5075_UW using a microevolutionary approach. We employed whole-genome sequencing and various phenotypic analyses to characterize microbe-lipid-antibiotic interactions. Cells exposed to erythromycin in the presence of the fatty acids displayed significantly lower rates of development of resistance to erythromycin and, importantly, tetracycline. Subsequent analyses defined diverse means by which host fatty acids influence the mutation rates. This work has highlighted the critical need to consider the roles of host fatty acids in A. baumannii physiology and antimicrobial resistance. Collectively, we have identified a novel means to curb the development of resistance in this critical human pathogen. IMPORTANCE The global distribution of multidrug resistance in A. baumannii has necessitated seeking not only alternative therapeutic approaches but also the means to limit the development of resistance in clinical settings. Highly abundant host bioactive compounds, such as polyunsaturated fatty acids, are readily acquired by A. baumannii during infection and have been illustrated to impact the bacterium’s membrane composition and antibiotic resistance. In this work, we show that in vitro supplementation with host polyunsaturated fatty acids reduces the rate at which A. baumannii gains resistance to erythromycin and tetracycline. Furthermore, we discover that the impact on resistance development is closely associated with the primary antimicrobial efflux systems of A. baumannii, which represent one of the major drivers of clinical resistance. Overall, this study emphasizes the potential of host macromolecules in novel approaches to circumvent the difficulties of multidrug resistance during A. baumannii treatment, with fatty acid supplements such as fish oil providing safe and cost-effective ways to enhance host tolerance to bacterial infections.

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The ASM Journals program strives for constant improvement in our submission and publication process. Please tell us how we can improve your experience by taking this quick Author Survey. In this manuscript, the authors examine the effects of an omega-3 and omega-6 fatty acid on the development of resistance to erythromycin and cross-resistance to tetracycline. Each fatty acid decreased the development of resistance after exposure to erythromycin. Resistance was obtained by mutations that overexpressed either the AdeABC or AdeIJK efflux systems. I have the following comments 1. It is stated that 8 ug/ml of erythromycin was subinhibitory, yet this condition selected for enhanced resistance via mutations. Therefore, there was selective pressure. Was a careful growth rate analysis done to confirm that 8 ug/ml was not inhibitory? If not, this should be done and added as a supplemental figure. 2. For AA and DHA, what was the rationale for using the concentrations chosen in this study? 3. The E0 clones + PUFAs have reduced levels of tetracycline resistance, what accounts for this if they had no growth on erythromycin and presumably had no mutations? 4. Figure 3C should include erythromycin MICs for each strain. 5. In Fig. S2, the E0T0 clone appears to have an Erm MIC above wild-type. What accounts for this? 6. Fig. 4A: Some discussion of why mutants other than AdeR I27S might not be isolated in the presence of PUFAs should be added to the discussion. Also the fact that the majority of adeRS mutants did not alter DHA sensitivity in Fig. 4A is not consistent with lines 311-312. 7. Lines 322-324. Supplementation may not be beneficial if it increases resistance by upregulating AdeIJK Reviewer #2 (Comments for the Author): This study evaluates an interplay between antibiotic resistance and exposure to polyunsaturated fatty acids in Acinetobacter baumannii. The authors report that exposure to fatty acids significantly reduces frequency of resistance to erythromycin and tetracycline and that resistance is associated with overexpression of AdeIJK or AdeABC efflux pumps. The study is welldesigned and thorough. The manuscript is well-written and clear. The conclusions are supported by presented data. The findings will be of interest to antibiotic resistance and bacterial physiology fileds. This reviewer noticed a few mistypes (for example on line 46 "association" should be "associated") but these could be fixed during editing.

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Reviewer #1:
In this manuscript, the authors examine the effects of an omega-3 and omega-6 fatty acid on the development of resistance to erythromycin and cross-resistance to tetracycline. Each fatty acid decreased the development of resistance after exposure to erythromycin. Resistance was obtained by mutations that overexpressed either the AdeABC or AdeIJK efflux systems. I have the following comments Q1. It is stated that 8 ug/ml of erythromycin was subinhibitory, yet this condition selected for enhanced resistance via mutations. Therefore, there was selective pressure. Was a careful growth rate analysis done to confirm that 8 ug/ml was not inhibitory? If not, this should be done and added as a supplemental figure.
A. We have performed growth analyses of AB5075_UW to examine the impact of various concentrations of erythromycin. These new data have been presented in Figure S2 (Lines 179 -180; track-changed version) and illustrate that AB5075_UW cells were able to propagate with minimal perturbation when treated with 8 μg.ml -1 erythromycin. Further, the terminology "subinhibitory concentration" has been omitted in the revised version of the manuscript.

Q2. For AA and DHA, what was the rationale for using the concentrations chosen in this study?
A. We agree with the reviewer and have taken the opportunity to include this important information in lines 85 -87 (track-changed version). The concentration of DHA was determined based on typical human plasma DHA levels in individuals on standard Western diet, versus those with higher fish consumption, thereby rendering the concentrations in our study physiologically relevant. The concentration of AA was adjusted for experimental consistency.

Q3. The E0 clones + PUFAs have reduced levels of tetracycline resistance, what accounts for this if they had no growth on erythromycin and presumably had no mutations?
A. This is an interesting observation made by the reviewer. We believe that mutations that lead to multidrug efflux pump overexpression have a relatively greater potential on tetracycline resistance as compared to erythromycin, since AB5075_UW is already resistant to erythromycin but remains relatively susceptible to tetracycline. This is reflected in Figure S3, where the gain of tetracycline resistance for the E2T2 clones were approximately 3-fold versus <2-fold for erythromycin. We anticipate that the E0T1 and E0T2 clones harbor mutations possibly associated with increased expression of the adeB or adeJ efflux systems, sufficient to increase tetracycline resistance, but not erythromycin. Hence, the impact of PUFAs on these groups may be of similar nature and magnitude compared to the E2T2 clones. Figure 3C should include erythromycin MICs for each strain. A. We thank the reviewer for this suggestion and have incorporated the data as Figure S3C, where the average MIC values of a larger number of E2T2 mutants has been presented, thereby allowing for appropriate comparison. Fig. S2, the E0T0 clone appears to have an Erm MIC above wild-type. What accounts for this? A. Rather than presenting the modus, we have amended Table 1 to reflect the range of the MIC for erythromycin in strain AB5075_UW. This range is similar across the 22 E0T0 clones ( Figure S3). Fig. 4A: Some discussion of why mutants other than AdeR I27S might not be isolated in the presence of PUFAs should be added to the discussion. Also the fact that the majority of adeRS mutants did not alter DHA sensitivity in Fig. 4A is not consistent with lines 311-312.

Q6.
A. We agree with the reviewer's comments and have expanded our discussion in lines 318 -331 (track-changed version). The lack of susceptibility towards DHA in other adeRS mutants, but their potential reduced incidence following PUFA co-treatment may be due to the direct impact of DHA upon the transporter, as reported in our previous studies (Zang et al. 2021 mBio).

Q7. Lines 322-324. Supplementation may not be beneficial if it increases resistance by upregulating AdeIJK
A. This is an excellent comment. We have included the following statement "Further, this highlights the potential risk of introducing resistance with PUFA treatment via adeIJK upregulation, and would require adequate selection of antimicrobial compounds that may bypass this particular efflux system." in lines 304 -306 (track-changed version).

Reviewer #2
This study evaluates an interplay between antibiotic resistance and exposure to polyunsaturated fatty acids in Acinetobacter baumannii. The authors report that exposure to fatty acids significantly reduces frequency of resistance to erythromycin and tetracycline and that resistance is associated with overexpression of AdeIJK or AdeABC efflux pumps. The study is well-designed and thorough. The manuscript is well-written and clear. The conclusions are supported by presented data. The findings will be of interest to antibiotic resistance and bacterial physiology fileds. This reviewer noticed a few mistypes (for example on line 46 "association" should be "associated") but these could be fixed during editing.
We thank the reviewer for the positive comments and have corrected the typographic errors in the revised manuscript.

Dr. Bart A Eijkelkamp Flinders University Sturt Rd Bedford Park, SA 5042 Australia
Re: Spectrum01455-21R1 (The impact of omega-3 fatty acids on the evolution of Acinetobacter baumannii drug resistance) Dear Bart: Your manuscript has been accepted, and I am forwarding it to the ASM Journals Department for publication. You will be notified when your proofs are ready to be viewed.
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