Carbapenem-resistant Acinetobacter baumannii (CRAB) is a serious threat, and resistance is emerging to cefiderocol, one of the few remaining treatment options. However, how host micronutrients drive bacterial adaptation under antibiotic pressure is poorly defined. Using the AB5075 and CDC AR Bank #0033 (M27835) CRAB isolates, we combined time-kill assays, whole-genome sequencing, gene expression analysis, and phenotyping to test how vitamin B12 promotes small-colony variants (SCVs) -like adaptive derivatives and alters cefiderocol response. Vitamin B12 reproducibly selected slow-growing SCV-like adaptive derivatives that showed incomplete cefiderocol killing and frequent regrowth across drug concentrations in both genetic backgrounds. These derivatives carried genetic alterations affecting cell-surface structures, nutrient transport and TonB-dependent iron uptake, and energy balance, with capsule/biofilm remodeling and altered aminoglycoside killing. Co-exposure to vitamin B12 and cefiderocol further enriched SCV-like adaptive derivatives with changes in siderophore receptors and the BfmRS sensor BfmS. These findings identify vitamin B12 as an environmental cue that promotes the emergency of SCV-like adaptive derivatives and modulates cefiderocol response in CRAB, highlight nutrient availability as an underrecognized factor influencing antibiotic efficacy.

Carbapenem-resistant Acinetobacter baumannii (CRAB) is a global health threat with few effective treatment options remaining. Cefiderocol, a last-resort siderophore-cephalosporin antibiotic, exploits bacterial iron transport systems via TonB-dependent receptors (TBDRs) to gain cellular entry. However, treatment failures and the emergence of resistance highlight concerns with in vivo efficacy. In this study, we report an unanticipated cefiderocol resistance mechanism where vitamin B12, a micronutrient supplement, modulates cefiderocol susceptibility. Our work revealed that vitamin B12 (methylcobalamin) affects and interacts with TBDRs and other metabolic and adaptation processes that contribute to increased cefiderocol MIC levels and the emergence of persistence phenotypes. We demonstrate that vitamin B12 supplementation elicits strain-specific transcriptomic responses in the AB5075 and AMA17 CRAB strains, characterized by the downregulation of genes encoding siderophore-mediated iron acquisition functions, stress responses, metabolic reprogramming, and biofilm biogenesis. Structural modeling and molecular docking reveal overlapping binding sites for vitamin B12 and cefiderocol within TBDRs such as CirA and PirA, suggesting competitive inhibition. Additionally, vitamin B12 exposure increases cefiderocol MICs across a panel of A. baumannii clinical and reference strains, enhances survival in time-kill assays, and promotes the emergence of small-colony variants displaying persistent phenotypes. Notably, this effect is stable, dose dependent, and further enhanced in the presence of host-derived fluids. These findings describe a previously unrecognized host-pathogen-drug interaction with potential clinical implications, suggesting that vitamin B12 exposure could contribute to cefiderocol treatment failure. Our results underscore the urgent need to consider the potential contribution of vitamin supplements to antimicrobial therapy and management strategies for CRAB infections.IMPORTANCECefiderocol, a last-line antibiotic for treating carbapenem-resistant Acinetobacter baumannii (CRAB) infections, uses iron-uptake receptors to enter bacterial cells. Our work demonstrates that vitamin B12, a common supplement in outpatients and hospitalized adults, can antagonize cefiderocol by affecting TonB-dependent receptor expression and competing at shared entry sites. As a result, cefiderocol MICs are raised, thus promoting persistent small-colony variants. This dose-dependent, strain-specific effect is amplified by host fluids, revealing a clinically plausible pathway leading to treatment failure that current susceptibility testing assays do not consider. Recognizing vitamin B12 exposure and incorporating physiological B12/iron conditions into antimicrobial susceptibility testing and models could improve decision-making for treatment regimens. More broadly, our findings highlight nutrient-antibiotic interactions as overlooked drivers of CRAB's resistance and persistence.

OBJECTIVE:Cefiderocol (FDC) is a siderophore cephalosporin active against carbapenem-resistant Klebsiella pneumoniae, but resistance has emerged, often involving iron-uptake pathways and β-lactamases. We investigated how an NDM-producing clinical isolate adapts to FDC under stepwise selective pressure. METHODS:A ST147 K. pneumoniae isolate (Kp-1) carrying blaNDM-5 and blaOXA-181, susceptible to FDC but resistant to other agents, was propagated in iron-depleted Mueller-Hinton broth with progressive FDC exposure. Population-based whole-genome sequencing tracked mutation dynamics, and reverse transcription-quantitative PCR profiled 17 genes related to FDC resistance, iron acquisition, and virulence, comparing the parental population (G0) to the first exposed generation (G1 + FDC). RESULTS:The mean number of mutations detected in generation G1 under FDC exposure was 24.3 ± 12.2, and in generation G2, it increased to 31.7 ± 6.4. Two nonsynonymous substitutions in baeS (V295G and T299P) were recurrently selected, with V295G reaching ≥ 70% frequency in independent lineages, indicating convergent evolution. reverse transcription-quantitative PCR revealed downregulation of iron-uptake and porin genes (iroN, cirA, fepA, kfu, ompK35) and upregulation of entB, ompK36, blaNDM-5, and capsule-related genes (wzm, wbbM); baeS/baeR transcript levels were unchanged. Early FDC exposure enhanced biofilm formation without significantly affecting capsule production. CONCLUSIONS:Short-term FDC exposure reproducibly selects baeS variants and triggers a transcriptional program reducing siderophore-receptor entry and remodelling the outer membrane. These adaptations-together with increased blaNDM-5 expression and biofilm formation-contribute to reduced FDC susceptibility. The recurrent baeS mutations (V295G/T299P) and decreased cirA/fepA expression may serve as early surveillance markers of FDC adaptation in K. pneumoniae.

Human serum albumin (HSA), the predominant plasma protein, influences bacterial physiology and antibiotic susceptibility. We previously showed that HSA exposure alters Acinetobacter baumannii transcriptional profiles, reducing biofilm formation and modulating the expression of β-lactam resistance genes, while decreasing cefiderocol (FDC) activity through downregulation of TonB-dependent receptors (TBDRs). Since FDC uptake relies on siderophore-mediated transport, we hypothesised that albumin's iron content could affect bacterial susceptibility to FDC. However, its effect on FDC activity across carbapenem-resistant Gram-negative pathogens remains underexplored. Here, we evaluated FDC bactericidal efficacy in the presence of physiological HSA concentrations and various human biological fluids against five different species of carbapenem-resistant strains. Time-kill assays revealed that HSA markedly attenuates FDC activity, permitting bacterial regrowth in species including A. baumannii, Klebsiella pneumoniae, Pseudomonas aeruginosa, Escherichia coli, and Achromobacter xylosoxidans. Human fluids with high protein content similarly reduced FDC efficacy. Transcriptional analyses indicated downregulation of iron uptake genes, suggesting that albumin-mediated iron availability modulates siderophore-dependent drug uptake. These findings highlight the significant role of host proteins and fluid environments in modulating FDC activity, underscoring the need to consider such factors when interpreting susceptibility information and optimising treatment strategies for infections caused by carbapenem-resistant pathogens.

The ongoing antibiotic resistance crisis is among the most pressing public health chal-lenges. The rise and dissemination of multidrug-resistant bacterial pathogens is reaching the point when some become untreatable. Consequently, besides discovering novel antibiotics, alternative strategies must be explored to manage the problem. One of them is the development of inhibitors that can overcome resistance to antibiotics currently in use. Resistance to aminoglycosides such as amikacin is mainly due to the action of aminoglycoside modifying enzymes. Despite being refractory to most resistance en-zymes, the semisynthetic amikacin is inactivated by the action of aminoglycoside 6’-N-acetyltransferases type I [AAC(6')-I], of which AAC(6')-Ib is the most common in Gram-negative pathogens. The recent discovery that certain divalent and monovalent cations can interfere with enzymatic acetylation catalyzed by AAC(6')-Ib has opened up new possibilities for developing formulations that combine antibiotics with these cations to enhance their efficacy. Addition of CdCl2 to in vitro enzymatic assays inhibited the transfer of an acetyl group to the 6'-N position of amikacin, kanamycin, and tobramycin. This result demonstrated the potential of Cd2+ as an adjuvant to aminoglycosides that can become effective for treating infections resistant due to AAC(6')-Ib. It was initially dis-appointing that, as with other divalent cations, addition of CdCl2 to cultures of bacteria harboring AAC(6')-Ib did not reverse resistance. However, the inhibitory action of Cd2+ became evident when it was added to cultures in complex with the ionophore pyrithione. The complex efficiently inhibited resistance in Acinetobacter baumannii and Klebsiella pneumoniae harboring AAC(6')-Ib. Furthermore, the combination efficiently inhibited amikacin resistance in carbapenem-resistant K. pneumoniae clinical isolates. These results add another cation to the arsenal of potential adjuvants for aminoglycosides, which could be developed either alone or in coordination complexes with ionophores to treat multi-drug-resistant infections.

Background/Objectives: Carbapenem-resistant Klebsiella pneumoniae (CRKP) is an urgent public health threat due to its rapid dissemination and resistance to last-line antibiotics. Cefiderocol (FDC), a novel siderophore cephalosporin, targets resistant Gram-negative pathogens by exploiting bacterial iron uptake mechanisms. However, resistance to FDC is emerging among Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae strains. This study characterizes a spontaneous FDC-resistant subpopulation (IHC216) derived from a KPC-producing strain (KPNMA216) using comprehensive genomic, transcriptional, and phenotypic analyses. Methods: Given the whole-genome sequencing results, where mutations were identified in genes involved in transcriptional regulation and membrane permeability (ompC) among others, in the present work we further explore their potential implications and conduct a more detailed analysis of the IHC216 genome. A qRT-PCR analysis highlighted significant downregulation of classical siderophore-mediated iron acquisition systems (fepA, cirA, iroN) and upregulation of alternative iron uptake pathways (iucA, fiU), reflecting a switch in iron acquisition strategies. Results: A notable downregulation of blaKPC-163 correlated with restored susceptibility to carbapenems, indicating collateral susceptibility. Altered expressions of pbp2 and pbp3 implicated adaptive changes in cell wall synthesis, potentially affecting FDC resistance mechanisms. Furthermore, enhanced oxidative stress responses via upregulated sodC expression and increased capsule production were observed. Conclusions: These findings underscore the complex interplay of genetic and transcriptional adaptations underlying FDC resistance, highlighting potential therapeutic vulnerabilities.

The ongoing antibiotic resistance crisis is one of the most pressing public health challenges. Multidrug-resistant bacterial pathogens are reaching the point where some are becoming untreatable. Consequently, besides discovering novel antibiotics, alternative strategies must be explored to manage the problem. One approach is developing inhibitors that overcome resistance to antibiotics currently in use. Resistance to aminoglycosides such as amikacin is mainly due to aminoglycoside-modifying enzymes. Despite being refractory to most resistance enzymes, the semisynthetic amikacin is inactivated by aminoglycoside 6′-N-acetyltransferases type I [AAC(6′)-I], of which AAC(6′)-Ib is the most common in Gram-negative pathogens. The discovery that certain divalent and monovalent cations interfere with enzymatic acetylation catalyzed by AAC(6′)-Ib opens possibilities for developing formulations combining antibiotics with these cations to enhance efficacy. Addition of CdCl₂ to in vitro enzymatic assays inhibited transfer of an acetyl group to the 6′-N position of amikacin, kanamycin, and tobramycin. Hence, Cd2⁺ is a potential adjuvant to aminoglycosides for treating AAC(6′)-Ib-mediated resistant infections. It was initially disappointing that, as with other divalent cations, CdCl₂ addition to cultures of bacteria harboring AAC(6′)-Ib did not reverse resistance. However, the inhibitory action of Cd2⁺ became evident when combined with the ionophore pyrithione. The complex efficiently inhibited resistance in Acinetobacter baumannii and Klebsiella pneumoniae harboring AAC(6′)-Ib. Furthermore, the combination inhibited amikacin resistance in carbapenem-resistant K. pneumoniae clinical isolates. These results add another cation to the arsenal of potential aminoglycoside adjuvants, which could be developed alone or in coordination complexes with ionophores to treat multidrug-resistant infections.

The emergence of antimicrobial resistance in Acinetobacter species poses a significant clinical challenge, particularly in non-baumannii species, which are often overlooked in healthcare settings. In this study, we characterized two Acinetobacter clinical isolates, AMA204 and AMA207-identified as A. junii and A. haemolyticus, respectively-which exhibit uncommon resistance mechanisms that enable survival in the presence of cefiderocol, regardless of their initial minimum inhibitory concentration values. Whole-genome sequencing and comparative genomic analyses were performed to investigate the genetic determinants associated with their resistance profiles. Antimicrobial susceptibility testing confirmed multidrug resistance, with both isolates harboring key β-lactamase genes, including blaOXA-58, and blaNDM-1 in AMA204, and blaOXA-58 and blaPER-2 in AMA207. Phylogenomic analyses revealed genetic relatedness to geographically diverse isolates, suggesting possible evolutionary trends and transmission dynamics. Additionally, iron uptake systems were analysed, highlighting potential mechanisms contributing to cefiderocol resistance together with the presence of listed β-lactamase. This study underscores the clinical relevance of non-baumannii Acinetobacter species in antimicrobial resistance and emphasizes the need for continued surveillance and novel therapeutic strategies to combat these emerging threats.

Abstract Background Carbapenem-resistant Acinetobacter baumannii (CRAB) poses a serious threat to public health, as treatment options are becoming limited. IDSA Guidance has advocated combination therapies to combat CRAB. Sulbactam (SUL), a β-lactamase inhibitor with inherent antibacterial activity against CRAB, is recognized as an important component of combination therapy. Pairing avibactam (AVI) inhibitor with ceftazidime (CAZ), has also been successful in restoring its antibiotic efficacy against many Gram-negative pathogens but has not proven effective against CRAB when used alone. In vitro assays combining SUL and AVI have shown promise against CRAB with different genetic backgrounds. Our aim is to assess the in vitro efficacy of combining ampicillin-SUL (AMS) and CAZ-AVI (CZA) against CRAB. Figure 1 Methods Three different methodological strategies were used. 89 strains underwent MIC evaluation (agar dilution) against SUL plus AVI, AMS (2:1) plus AVI, and AMS plus CZA, with a constant concentration of AVI (4 mg/L) and CAZ (8 mg/L). Additionally, 30 CRAB strains with different genetic backgrounds were used to assess AMS/CZA (14 μg and 50 μg) synergy by the disk stacking method and MTS synergy. Results AVI reduced SUL MIC values to < =4 mg/L in 96.8% of the tested isolates, excluding NDM producers. For MBLs producers, SUL-AVI MIC values less < =4 mg/L were tested in 14.3% strains (Fig. 1A). The inclusion of AMP or CAZ in the SUL-AVI combination did not result in any alterations in MIC values (98% of strains with identical values). Using stacking assay, 59% of isolates showed >3 mm increase in halo diameter for AMS with both CZA disks (Fig. 1B). Additionally, we calculated the FICI by using gradient strips (MTS) obtaining a synergistic or additive effect in 69% and 2% of strains, respectively. Antagonism was only seen in two tested NDM-1 positive isolates. Conclusion Our results demonstrated synergy between AMS and CZA in geographically diverse CRAB, including extensively drug-resistant isolates, such as those producing widespread oxacillinases. Some methods, mainly disk-based, may underestimate the true extent of AMS-CZA synergy. These findings could facilitate the implementation AMS/CZA in clinical settings and provide a potential alternative in regions where the latest approved drugs are not accessible. Disclosures David Paterson, Infectious Diseases Physician, AMR Action Fund: Board Member|Aurobac: Advisor/Consultant|bioMerieux: Grant/Research Support|bioMerieux: Honoraria|CARB-X: Advisor/Consultant|Entasis: Expert Testimony|Menarini: Honoraria|Merck: Grant/Research Support|Pfizer: Advisor/Consultant|Pfizer: Grant/Research Support|Shionogi: Grant/Research Support|Shionogi: Honoraria

Abstract Background The aminoglycoside 6′-N-acetyltransferase type Ib [AAC(6’)-Ib] confers resistance to amikacin and other relevant aminoglycosides in Gram-negatives. To identify small molecule inhibitors of AAC(6’)-Ib that would overcome resistance, we utilized a strategy incorporating mixture-based combinatorial libraries, scaffold ranking, and positional scanning, followed by structure-activity relationship analysis. After identification of pyrrolidine pentamine by scaffold ranking, we isolated a compound substituted by two S-phenyl groups, an S-hydroxymethyl group, and a 3-phenylbutyl group at the R positions (compound 2700.001) (Fig 1). Herein, we describe a structure-relationship analysis of this compound. Figure 1 Chemical structure of compound 2700.001 Methods Initial inhibition assessment was done by measuring OD600 of A. baumannii A155 cultures at a single dose of compound. Checkerboard assays were done in triplicate, with each dose combination tested twice. The data were processed to eliminate any antibacterial effect exerted by the inhibitor. Results Single substitution of functionalities at R positions along the core scaffold of compound 2700.001 showed that position R5 is the most tolerant to changes. The replacement of 3-phenylbutyl by 4-phenylbutyl resulted in compound 2700.003, equally active to 2700.001. Modifications at all other locations produced weaker inhibitors, while truncations of the scaffold resulted in inactive compounds. Testing compounds with double or triple substitutions confirmed the critical role of the S-phenyl group at position R1. No compounds with higher inhibitory activity were found. Using molecular docking, the relationship between ΔG (Kcal/mol) values for each compound and inhibitory activity was studied, with results indicating a significant correlation. Conclusion Using mixture-based combinatorial libraries, the scaffold ranking approach, and the positional scanning strategy, we identified an inhibitor of AAC(6’)-Ib that can overcome resistance to amikacin in Gram-negatives. Furthermore, structure-activity relationship studies led to the identification of a second inhibitor, while also giving direction for further improvements to the molecule toward developing stronger inhibitors. Disclosures All Authors: No reported disclosures

Antisense interference with gene expression is usually achieved using nuclease-resistant oligonucleotide analogs that act by mRNA degradation, recruiting endogenous RNase H or RNase P, or steric hindrance of translation. Bridge nucleic acids (BNAs) are promising nucleotide analogs, and their chemical structure allows the development of new variants. Building on previous research, we evaluated gapmers composed of a short oligodeoxynucleotide flanked by BNA residues in a BNA5-DNA8-BNA4 configuration, using available BNA variants: the original locked nucleic acid (LNA; 2'-O-4'-methylene locked nucleic acid), cET (2'-O,4'-ethyl bridge), cMOE (2'-O,4'-methoxyethyl bridge), and BNANC (2'-O,4'-aminomethylene bridge). These gapmers were tested in vitro for their ability to direct cleavage of the aac(6')-Ib mRNA, which would restore susceptibility to clinically important aminoglycosides. The assays were carried out using gapmers that target a region of the mRNA previously identified as suitable for interaction with antisense oligomers. While all gapmers showed variable RNase H-mediated activity, only the LNA-containing gapmer (LDAA) elicited RNase P-dependent degradation, demonstrating ability to mimic both RNA and DNA. Coupled in vitro transcription-translation reactions using a cell lysate or a reconstituted system confirmed inhibition of expression and ruled out steric hindrance as mechanism of action. Gapmers with the LDAA structure can act as external guide sequences (EGSs), molecules that elicit RNAse P cleavage, and as antisense compounds that work via RNase H degradation. In contrast, gapmers targeting the ribosome binding site failed to recruit endogenous RNases but strongly inhibited expression by steric hindrance. Taken together, the results show that LNA-containing gapmers with the tested configuration can act through multiple mechanisms. A single molecule can elicit both RNase H- and RNase P-mediated degradation, and, when directed to other regions such as the ribosome binding site, inhibit expression through steric hindrance, supporting the potential for synergistic inhibition of gene expression when used in combination.

Antisense inhibition of gene expression is usually achieved using nuclease-resistant oligonucleotide analogs that promote mRNA degradation through RNase H or RNase P, or by steric hindrance of translation. Bridge nucleic acids (BNAs) are nucleotide analogs available in a few chemical variants. We evaluated gapmers composed of an oligodeoxynucleotide flanked by BNA residues in a BNA5-DNA8-BNA4 configuration, using the available variants: the original locked nucleic acid (LNA; 2′-O,4′-methylene bridge), cET (2′-O,4′-ethyl bridge), cMOE (2′-O,4′-methoxyethyl bridge), and BNANC (2′-O,4′-aminomethylene bridge). These gapmers were tested in vitro for their ability to induce cleavage of the model aac(6′)-Ib mRNA. All gapmers complementary to a previously identified region suitable for interaction with antisense oligomers induced RNase H-mediated degradation. Instead, only the LNA-containing gapmer also elicited RNase P-dependent cleavage, demonstrating dual RNA- and DNA-mimicking capability. In vitro coupled transcription–translation assays using cell lysates or reconstituted systems confirmed inhibition of expression and ruled out steric hindrance as the mechanism. In contrast, gapmers targeting the ribosome-binding site strongly inhibited expression by steric hindrance. These findings demonstrate that LNA-containing gapmers can exert their effects through multiple mechanisms, depending on the targeted mRNA region, thereby supporting their potential for synergistic inhibition of gene expression.

OBJECTIVE:Klebsiella pneumoniae carbapenemase (KPC) variants, predominantly KPC-2 and KPC-3, are significant global resistance mechanisms, conferring resistance to many β-lactams, including carbapenems, while remaining susceptible to ceftazidime-avibactam (CZA). Recently, new KPC variants have developed resistance to CZA through mutations, insertions, or deletions in regions such as the Ω-loop, 240-loop (237-243 aa), and 270-loop (266-275 aa). This study investigated collateral resistance to cefiderocol (FDC) and cefepime/zidebactam (FPZ) in isolates with these mutations. METHODS:Fifteen clinical KPC-producing Klebsiella spp. isolates representing 15 distinct variants were analysed. Antimicrobial susceptibility testing determined the MICs for CZA, carbapenems, FDC, FPZ, and other antibiotics. Synergy between CZA and FDC was assessed. Whole-genome sequencing (WGS) was used to identify resistance-related mutations. RESULTS:CZA resistance was confirmed in 12/15 variants. Collateral resistance to FDC occurred in eight isolates, with five exhibiting spontaneous resistant subpopulations. Six FDC-resistant strains had mutations in the 270-loop (266-275 aa). FPZ resistance was seen in three KPC variants, especially those with mutations in the 270-loop, though many Ω-loop and 240-loop (237-243 aa) mutants remained susceptible. WGS of FDC-resistant subpopulations revealed additional mutations in ompC, rpoC, dksA, and cirA. CONCLUSIONS:Emerging CZA-resistant KPC variants often exhibit collateral FDC resistance, with FPZ seen less frequently. Mutations in blaKPC, cirA, and other genes contribute to resistance. Understanding these emerging resistant patterns linked with new KPC variants is crucial to inform therapeutic decisions, as emerging resistance may limit last-line treatment options in clinical settings.

Cefiderocol, a siderophore-cephalosporine conjugate antibiotic, shows promise as a therapeutic option for carbapenem-resistant (CR) Acinetobacter infections. While resistance has already been reported in A. baumannii, combination therapies with avibactam or sulbactam reduce MICs of cefiderocol, extending its efficacy. However, careful consideration is necessary when using these combinations. In our experiments, exposure of A. baumannii and A. lwoffii to cefiderocol and sulbactam or avibactam led to the selection of cefiderocol-resistant strains. Three of those were subjected to whole genome sequencing and transcriptomic analysis. The strains all possessed synonymous and non-synonymous substitutions and short deletions. The most significant mutations affected efflux pumps, transcriptional regulators, and iron homeostasis genes. Transcriptomics showed significant alterations in expression levels of outer membrane proteins, iron homeostasis, and β-lactamases, suggesting adaptive responses to selective pressure. This study underscores the importance of carefully assessing drug synergies, as they may inadvertently foster the selection of resistant variants and complicate the management of CR Acinetobacter infections.IMPORTANCEThe emergence of carbapenem-resistant Acinetobacter strains as a serious global health threat underscores the urgent need for effective treatment options. Although few drugs show promise against CR Acinetobacter infections, resistance to both drugs has been reported. In this study, the molecular characterization of spontaneous cefiderocol-resistant variants, a CR A. baumannii strain with antagonism to sulbactam, and an A. lwoffii strain with antagonism to avibactam, provides valuable insights into the mechanisms of resistance to cefiderocol. Some mechanisms observed are associated with mutations affecting efflux pumps, regulators, and iron homeostasis genes. These findings highlight the importance of understanding resistance mechanisms to optimize treatment options. They also emphasize the importance of early evaluation of drug synergies to address the challenges of antimicrobial resistance in Acinetobacter infections.

Aminoglycosides are essential components in the available armamentarium to treat bacterial infections. The surge and rapid dissemination of resistance genes strongly reduce their efficiency, compromising public health. Among the multitude of modifying enzymes that confer resistance to aminoglycosides, the aminoglycoside 6 ' -N-acetyltransferase type Ib [AAC(6 ' )-Ib] is the most prevalent and relevant in the clinical setting as it can inactivate numerous aminoglycosides, such as amikacin. Although the mechanism of action, structure, and biochemical properties of the AAC(6 ' )-Ib protein have been extensively studied, the contribution of the intracellular milieu to its activity remains unclear. In this work, we used a fluorescent-based system to quantify the number of AAC(6 ' )-Ib per cell in Escherichia coli, and we modulated this copy number with the CRISPR interference method. These tools were then used to correlate enzyme concentrations with amikacin resistance levels. Our results show that resistance to amikacin increases linearly with a higher concentration of AAC(6 ' )-Ib until it reaches a plateau at a specific protein concentration. In vivo imaging of this protein shows that it diffuses freely within the cytoplasm of the cell, but it tends to form inclusion bodies at higher concentrations in rich culture media. Addition of a chelating agent completely dissolves these aggregates and partially prevents the plateau in the resistance level, suggesting that AAC(6 ' )-Ib aggregation lowers resistance to amikacin. These results provide the first step in understanding the cellular impact of each AAC(6 ' )-Ib molecule on aminoglycoside resistance. They also highlight the importance of studying its dynamic behavior within the cell. IMPORTANCE Antibiotic resistance is a growing threat to human health. Understanding antibiotic resistance mechanisms can serve as foundation for developing innovative treatment strategies to counter this threat. While numerous studies clarified the genetics and dissemination of resistance genes and explored biochemical and structural features of resistance enzymes, their molecular dynamics and individual contribution to resistance within the cellular context remain unknown. Here, we examined this relationship modulating expression levels of aminoglycoside 6 ' -N-acetyltransferase type Ib, an enzyme of clinical relevance. We show a linear correlation between copy number of the enzyme per cell and amikacin resistance levels up to a threshold where resistance plateaus. We propose that at concentrations below the threshold, the enzyme diffuses freely in the cytoplasm but aggregates at the cell poles at concentrations over the threshold. This research opens promising avenues for studying enzyme solubility's impact on resistance, creating opportunities for future approaches to counter resistance.

Cefiderocol (CFDC) is a siderophore-cephalosporin antibiotic designed to combat highly resistant Gram-negative bacterial infections. Its mechanism involves a strong affinity for iron and active transport into bacterial cells, providing an alternative against strains resistant to common antibiotics. However, the emergence of CFDC resistance in Klebsiella is a growing concern. Recent reports highlight increasing CFDC resistance in K. pneumoniae, particularly associated with mutations in the cirA gene, responsible for encoding a siderophore receptor. Co-localization of blaNDM-like gene and cirA mutations correlates with higher CFDC resistance. The study focuses on a carbapenem-resistant K. pneumoniae strain (Kp-1) with carbapenemases blaNDM-5 and blaOXA-181, recovered from a post-surgery patient. The strain exhibited resistance to all tested antibiotics but susceptibility to CFDC. Heteroresistant populations with the halo on inhibition of CFDC were observed. Genomic analysis identified a novel mutation (W123*) in the cirA gene associated with CFDC resistance. Additionally, increased blaNDM-5 expression in Kp-1 IHC (intra-halo colony) compared to Kp-1 was noted. The coexistence of blaNDM-like and cirA variants, along with high blaNDM-5 expression, explains the observed 21-fold increase in Minimum Inhibition Concentration (MIC) in Kp-1 IHC. The study contributes to understanding the molecular mechanisms driving the emergence of cefiderocol resistance, emphasizing the significance of coexisting mutations in cirA and blaNDM-like genes.

Recently, considerable uncertainty has arisen concerning the appropriate susceptibility testing for cefiderocol in gram-negative bacilli, particularly in the context of its application to Acinetobacter spp. The optimal method for assessing the susceptibility levels of Acinetobacter spp. to cefiderocol remains a subject of debate due to substantial disparities observed in the values obtained through various testing procedures. This study employed four minimum inhibitory concentration (MIC) methodologies and the disk diffusion to assess the susceptibility of twenty-seven carbapenem resistant (CR)-Acinetobacter strains to cefiderocol. The results from our study reveal significant variations in the minimum inhibitory concentration (MIC) values obtained with the different methods and in the level of agreement in interpretation categories between the different MIC methods and the disk diffusion test. Among the MIC methods, there was relatively more consistency in reporting the interpretation categories. For European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints, the categorical agreement (CA) for MIC methods ranged between 66.7 and 81.5%. On the other hand, the essential agreement (EA) values were as low as 18.5-29.6%. The CA between MIC methods and disk diffusion was 81.5%. These results emphasize the need for a reliable, accurate, and clinically validated methodology to effectively assess the susceptibility of Acinetobacter spp. to cefiderocol. The wide variability observed in our study highlights the importance of standardizing the susceptibility testing process for cefiderocol to ensure consistent and reliable results for clinical decision-making.

The emergence of Gram-negative bacteria resistant to multiple antibiotics, particularly carbapenem-resistant (CR) Acinetobacter strains, poses a significant threat globally. Despite efforts to develop new antimicrobial therapies, limited progress has been made, with only two drugs—cefiderocol and sulbactam-durlobactam—showing promise for CR- Acinetobacter infections. Cefiderocol, a siderophore cephalosporin, demonstrates promising efficacy in the treatment of Gram-negative infections. However, resistance to cefiderocol has been reported in A. baumannii . Combination therapies, such as cefiderocol with avibactam or sulbactam, show reduced MICs against cefiderocol-non-susceptible strains with in vivo efficacy, although the outcomes can be complex and species-specific. In the present work, the molecular characterization of spontaneous cefiderocol-resistant variants, a CRAB strain displaying antagonism with sulbactam and an A. lwoffii strain showing antagonism with avibactam, were studied. The results reveal intriguing insights into the underlying mechanisms, including mutations affecting efflux pumps, transcriptional regulators, and iron homeostasis genes. Moreover, gene expression analysis reveals significant alterations in outer membrane proteins, iron homeostasis, and β-lactamases, suggesting adaptive responses to selective pressure. Understanding these mechanisms is crucial for optimizing treatment strategies and preventing adverse clinical outcomes. This study highlights the importance of preemptively assessing drug synergies to navigate the challenges posed by antimicrobial resistance in CR- Acinetobacter infections.