Alteration of adeS Contributes to Tigecycline Resistance and Collateral Sensitivity to Sulbactam in Acinetobacter baumannii

ABSTRACT The treatment of extensively drug-resistant (XDR) A. baumannii has emerged as a major problem. Tigecycline (TGC) and sulbactam (SUL) are both effective antibiotics against XDR A. baumannii. Here, we investigated the in-host evolution and mechanism of collateral sensitivity (CS) phenomenon in development of tigecycline resistance accompanied by a concomitant increase of sulbactam susceptibility. A total of four XDR A. baumannii strains were sequentially isolated from the same patient suffering from bacteremia. Core-genome multilocus sequence typing separated all the strains into two clusters. Comparative analysis of isolate pair 1 revealed that multiplication of blaOXA-23 within Tn2006 on the chromosome contributed to the change in the antimicrobial susceptibility phenotype of isolate pair 1. Additionally, we observed the emergence of CS to sulbactam in isolate pair 2, as demonstrated by an 8-fold increase in the TGC MIC with a simultaneous 4-fold decrease in the SUL MIC. Compared to the parental strain Ab-3557, YZM-0406 showed partial deletion in the two-component system sensor adeS. Reconstruction of the adeS mutant in Ab-3557 in situ suggested that TGC resistance and CS to SUL were mainly caused by the mutation of adeS. Overall, our study reported a novel CS combination of TGC and SUL in A. baumannii and further revealed a mechanism of CS attributed to the mutation of adeS. This study provides a valuable foundation for developing effective regimens and sequential combinations of tigecycline and sulbactam against XDR A. baumannii. IMPORTANCE Collateral sensitivity (CS) has become an increasingly common evolutionary trade-off during adaptive bacterial evolution. Here, we report a novel combination of tigecycline (TGC) resistance and CS to sulbactam (SUL) in A. baumannii. TGC and SUL are both effective antibiotics against XDR A. baumannii, and it is essential to reveal the mechanism of CS between TGC and SUL. In our study, the partial deletion of adeS, a two-component system sensor, was confirmed to be the key factor contributing to this CS phenomenon. This study provides a valuable foundation for developing effective regimens and sequential combinations of tigecycline and sulbactam against XDR A. baumannii.

The following are some comments/suggestions: -Please briefly describe your methods in addition to the references you provide (such as in lane 300). -How did you check the integrity of the RNA for qRT-PCR? -Did you use PrimeScript RT Reagent Kit with gDNA Eraser (Perfect Real Time)? If not, please provide information for DNase treatment.
-Please add the reference strains you used for qRT-PCR in the methods part.
-I think a statistical analysis could be added to the data in Figure 4. -In Figure 5a, please add some color or shading to the part of adeS that comes after region 95-157, as it gives the impression that it is a shorter version of the gene, rather than a completely different sequence (as in 5b).
-What is your theory about sulbactam susceptibility in the adeS mutant? What genes are known to be controlled by adeS in addition to adeABC? Are there any porins that might be up-regulated? -Please change "Enterococus" to "Enterococcus" (lane 70) Reviewer #2 (Comments for the Author): Please refer to attached world file Staff Comments:

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This study describes the properties of 4 multi-drug (XDR) resistant Acinetobacter baumannii isolates that were sequentially isolated from a patient who suffered from bacteremia and was subjected to sequential carbapenem (i.e. imipenem and meropenem) and tigecycline chemotherapy treatments. The strains were characterized through whole-genome sequencing and antimicrobial susceptibility testing. MLST categorized the 4 strains into two independent strain lineages. The strain of lineage 1, which was isolated later from the patient, gained a duplication of transposon Tn2006 carrying the β-lactamase resistance gene oxa-23, which may explain the observed changes in the antimicrobial susceptibility phenotypes reported in Table 1. The later isolate of strain lineage 2 gained a deletion within gene adeS encoding a sensor kinase that controls the adeABC multidrug efflux pump. This mutation may explain increased resistance to tigecycline in agreement with previous observations. Interestingly, the latter strain concomitantly acquired increased sensitivity to the β-lactamase inhibitor Sulbactam, which is known to also have antibacterial activity by itself towards A. baumannii. Thus, increased resistance to tigecycline through mutation of adeS appears to be associated with higher susceptibility to Sulbactam, which is a novel and interesting observation. The latter conclusion is also supported by an experiment in which the adeS mutation was genetically engineered in the parental strain, thereby conferring the identical tigecycline/Sulbactam resistance/susceptibility phenotype as observed in the clinically evolved strain.
However, there are some ambiguities in the manuscript that need to be addressed before the study can be published. Major: -Lines 116-120; Fig. 1: There is apparently a mistake in the timeline. If the total timeline was 14 days, i.e. 6 days IMP treatment followed by 17 days MEM treatment, we cannot have 30 days of TIG treatment overlapping with the former treatments.  Table 1, it is difficult for the reader to judge whether the criteria of an XDR strain are met. It would be helpful to include this information in Table 1.

This study describes the properties of 4 multi-drug (XDR) resistant Acinetobacter baumannii
isolates that were sequentially isolated from a patient who suffered from bacteremia and was subjected to sequential carbapenem (i.e. imipenem and meropenem) and tigecycline chemotherapy treatments. The strains were characterized through whole-genome sequencing and antimicrobial susceptibility testing. MLST categorized the 4 strains into two independent strain lineages. The strain of lineage 1, which was isolated later from the patient, gained a duplication of transposon Tn2006 carrying the β-lactamase resistance gene oxa-23, which may explain the observed changes in the antimicrobial susceptibility phenotypes reported in Table 1. The later isolate of strain lineage 2 gained a deletion within gene adeS encoding a sensor kinase that controls the adeABC multidrug efflux pump. This mutation may explain increased resistance to tigecycline in agreement with previous observations. Interestingly, the latter strain concomitantly acquired increased sensitivity to the β-lactamase inhibitor Sulbactam, which is known to also have antibacterial activity by itself towards A. baumannii. Thus, increased resistance to tigecycline through mutation of adeS appears to be associated with higher susceptibility to Sulbactam, which is a novel and interesting observation. The latter conclusion is also supported by an experiment in which the adeS mutation was genetically engineered in the parental strain, thereby conferring the identical tigecycline/Sulbactam resistance/susceptibility phenotype as observed in the clinically evolved strain.
However, there are some ambiguities in the manuscript that need to be addressed before the study can be published.

Major Comment:
COMMENT: Lines 116-120; Fig. 1: There is apparently a mistake in the timeline. If the total timeline was 14 days, i.e. 6 days IMP treatment followed by 17 days MEM treatment, we cannot have 30 days of TIG treatment overlapping with the former treatments.

RESPONSE:
We agree with the criticism. The total timeline of strain isolation was 14 days, but the timeline of drug usage is not only 14 days. So we have deleted the numbers in the paratheses and revised the right bracket to avoid misunderstanding (Fig 1).

RESPONSE:
We agree with your suggestion, and we have deleted the Figure 2. COMMENT: Lines 138-140: As there is no sensitive reference strain included in Table 1, it is difficult for the reader to judge whether the criteria of an XDR strain are met. It would be helpful to include this information in Table 1.

RESPONSE:
We agree with your viewpoint, and we have added a sensitive reference strain ATCC 25922 to Table 1. AdeS in our study is located on the sensor domain, and will lead to the loss of its sensor domain and phosphorylation activity of kinase. The deletion within AdeS in our study like many mutation types of AdeS (GLY186, SER188, GLU121), will result in the prevention of auto-phosphorylation of AdeS, and inhibit/slow the transfer of the phosphate group to AdeR, which can not be phosphorylated in other ways. Thus, AdeR may be binding loosely to the intercistronic spacer region leading to an overexpression of AdeABC.
In addition, the mutant AdeS in our study leads to the decrease of susceptibility to tigecycline, minocycline and levofloxacin, and substrates for the AdeABC efflux pump confirmed their roles in increasing pump expression. The description of the process of AdeS mutation affect AdeABC efflux pump have been added in the discussion part in Line 236-240.

RESPONSE:
We thank the reviewer for pointing out this possible theory about how the overexpression of AdeABC efflux pump increase the susceptible of sulbactam. Actually, AdeBC efflux pump will pump the sulbactam from the inner of cell to the outside, and increase concentration of sulbactam in the exterior surrounding the cells [9]. However, sulbactam will enter the cell through channels on the outer membrane, like some porins [10,11]. PBPs, as the antibacterial target of sulbactam, located on the peptidoglycan inside the outer membrane.
Therefore, if just increase the external concentration of sulbactam, it probably won't affect the amount of sulbactam that binds to PBP (Fig 3 listed below).
In our study, the transcriptomic analysis of Ab-3557-adeS-mutation revealed the gene expression of Outer-membrane protein A(OmpA) was up-regulated, which may be also regulated by AdeRS. OmpA is β-barrel porin in A. baumannii and also play a role in antimicrobial resistance and virulence in A.bauamnnii. OmpA is mainly distributed on the outer membrane and the outer membrane vesicle (OMV) [11], so we speculated that the sulbactam susceptibility restoring might be mediated by the following mechanism: (a) Change the distribution of OmpA: increasing the distribution of OmpA on the outer membrane enlarges the channel of sulbactam into bacteria; (b) Decrease the secretion of OMV containing β-lactamase: because sulbactam is easily hydrolyzed by β-lactamses. The extracellular hydrolysis of sulbactam is reduced by decreasing the formation of OMV and the secretion of β-lactamases. The above hypothesis is currently being tested in a large number of experiments.