pubs.acs.

org/journal/aidcbc Article

Small Molecule IITR08367 Potentiates Antibacterial Efficacy of

Fosfomycin against Acinetobacter baumannii by Efflux Pump
Inhibition
Mahak Saini, Amit Gaurav, Arsalan Hussain, and Ranjana Pathania*
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ABSTRACT: Fosfomycin is a broad-spectrum single-dose therapy

approved for treating lower urinary tract infections. Acinetobacter
baumannii, one of the five major UTI-causing pathogens, is
intrinsically resistant to fosfomycin. Reduced uptake and active
efflux are major reasons for this intrinsic resistance. AbaF, a major
facilitator superfamily class of transporter in A. baumannii, is
responsible for fosfomycin efflux and biofilm formation. This study
describes the identification and validation of a novel small-
molecule efflux pump inhibitor that potentiates fosfomycin efficacy
against A. baumannii. An AbaF inhibitor screening was performed
against Escherichia coli KAM32/pUC18_abaF, using the non-
inhibitory concentration of 24 putative efflux pump inhibitors. The
inhibitory activity of IITR08367 [bis(4-methylbenzyl) disufide]
against fosfomycin/H+ antiport was validated using ethidium bromide efflux, quinacrine-based proton-sensitive fluorescence, and
membrane depolarization assays. IITR08367 inhibits fosfomycin/H+ antiport activity by perturbing the transmembrane proton
gradient. IITR08367 is a nontoxic molecule that potentiates fosfomycin activity against clinical strains of A. baumannii and prevents
biofilm formation by inhibiting efflux pump (AbaF). The IITR08367-fosfomycin combination reduced bacterial burden by > 3 log10
in kidney and bladder tissue in the murine UTI model. Overall, fosfomycin, in combination with IITR08367, holds the potential to
treat urinary tract infections caused by A. baumannii.
KEYWORDS: screening, efflux pump inhibitor, urinary tract infection, C. elegans, biofilm inhibition, quinacrine assay

Urinary tract infections (UTIs) are the most prevalent type of fosfomycin is one of the most effective treatment options
infectious disease worldwide. In 2020, about 82.5% of the total prescribed worldwide.10 Fosfomycin treatment is very effective
bacterial infection cases were attributed to UTIs.1 Major against all UTIs-causing pathogens except A. baumannii.11,12
causative organisms of UTIs are Escherichia coli, Klebsiella spp., A. baumannii, a Gram-negative nosocomial bacterium, is one
Enterococcus spp., Pseudomonas spp., and Acinetobacter of the five major urinary infection-causing pathogens.13
baumannii.2 The emergence of antimicrobial resistance Treatment of multidrug-resistant A. baumannii infection is
phenotypes among UTI pathogens is rising rapidly.3 Addi- shrinking rapidly.14 Intrinsic resistance of A. baumannii against
tionally, the development of new antibacterials has decreased fosfomycin is primarily due to ineffective concentration
alarmingly in the last few decades.4 A dearth of new accumulation of this drug inside the bacterial cells.15 One of
antibacterial chemical entities and a rise in bacterial antibiotic the reasons for low cellular concentration is inefficient uptake,
resistance is leading to therapy failure even in uncomplicated as A. baumannii lacks fosfomycin active uptake transporters like
infections. To deal with this current alarming situation, there is GlpT and UhpT.16 Additionally, A. baumannii can also actively
an urgent need to discover new antibacterial chemical efflux out fosfomycin using A. baumannii-specific fosfomycin
entities.5,6 Besides discovering new antibacterials, revival of
efflux pump, AbaF.17 AbaF plays a critical role in the intrinsic
old antibiotics using adjuvant molecules may serve as an
attractive strategy.7−9
Due to the increasing incidence of drug resistance, clinicians Received: January 25, 2024
are exploring older antibiotics. Fosfomycin is one such Revised: March 16, 2024
example, as it has remained active against both Gram-positive Accepted: March 22, 2024
and Gram-negative multiple drug resistant (MDR) and Published: April 2, 2024
extensively drug resistant (XDR) bacteria. Fosfomycin is
used for the treatment of uncomplicated UTIs. Single-dose

© 2024 American Chemical Society https://doi.org/10.1021/acsinfecdis.4c00077

1711 ACS Infect. Dis. 2024, 10, 1711−1724
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Figure 1. Effect of pH on the susceptibility of fosfomycin against A. baumannii. (a) Lowering the system pH by one unit enhances fosfomycin
activity up to 16-fold in clinical strains of A. baumannii. The experiment was performed with three independent biological replicates. (b)
Fosfomycin (128 mg/L) treatment enhances biofilm formation in all tested clinical strains of A. baumannii. The dotted line represents the biofilm
formation of each strain without any treatment. Each data point represents an independent biological replicate. (c) Relative biofilm formation of A.
baumannii strains: WT, ΔabaF, and pabaF represent A. baumannii ATCC 17978, A. baumannii ATCC 17978:ΔabaF and abaF complimented in A.
baumannii ATCC 17978:ΔabaF respectively. Normalization was performed with respect to A. baumannii ATCC 17978. Each data point represents
an independent biological replicate. Error bars represent ±s.d. * indicates p-value < 0.05 (unpaired student’s t-test followed by Mann−Whitney
posthoc test).

resistance of fosfomycin in A. baumannii. Thus, it can serve as a transporter HlpT. It is known that A. baumannii does not use
target to revive the fosfomycin efficacy. It is also known that a glucose as a primary energy source due to inefficient uptake.20
slightly acidic pH of the system (human body) can facilitate Additionally, glucose uptake transporters GlpT or UhpT or
fosfomycin absorption via simple diffusion by keeping the their homologues are not characterized in A. baumannii.16 To
fosfomycin in a partially protonated state.18,19 ascertain the absence of these transporters in A. baumannii, we
In this study, we identified IITR08367, a potent efflux pump performed a nucleotide homology search of these genes in the
inhibitor against AbaF. We demonstrated inhibition of AbaF genome of Acinetobacter species. We did not find any sequence
activity by IITR08367 using ethidium bromide efflux assay. We with more than 5% homology. Thus, it was concluded that the
also validated efflux inhibition of fosfomycin/H+ antiport bacterial uptake of the fosfomycin depends solely on the simple
activity in everted membrane vesicles. Furthermore, diffusion of the partially charged fosfomycin molecules.
IITR08367 showed a reversal of minimum inhibitory Fosfomycin has two ionization pHs; the first ionization occurs
concentration (MIC) of fosfomycin against MDR clinical at pH 1.25, which makes the molecule partially ionized. The
isolates of A. baumannii below the clinical breakpoint. second ionization pH of the fosfomycin is 7.82; therefore,
Additionally, IITR08367 successfully prevented fosfomycin- more than 90% of fosfomycin molecules remain partially
induced biofilm formation of A. baumannii in clinically relevant charged at pH ≤ 6.5 (pKa data obtained from Chemicalize
conditions. The addition of IITR08367 to fosfomycin database (https://chemicalize.com/#/calculation) and can be
treatment in the murine UTI infection model showed a easily absorbed by simple diffusion.19 The relationship
significant reduction of the bacterial burden. Overall, this study between acidic pH and susceptibility to fosfomycin has been
identified a bona fide efflux pump inhibitor that potentiates previously explored.18,19,21 However, there is a lack of
fosfomycin efficacy against a clinically challenging pathogen, A. information on whether an acidic environment can enhance
baumannii. fosfomycin susceptibility against A. baumannii. Surprisingly,
our results show that reducing media pH by one unit (7.2 to

■ RESULTS
Acidic Environment Enhances Susceptibility of A.
6.2) can significantly (up to 16-fold) increase the susceptibility
of fosfomycin against clinical strains of A. baumannii (Figure
1a). We found increased fosfomycin susceptibility against all
baumannii against Fosfomycin. The bacterial uptake of tested clinical strains of A. baumannii (n = 22). Acidification of
fosfomycin in E. coli is facilitated via glucose uptake transporter the bacterial growth medium is an important aspect affecting
GlpT and a glucose-6-phosphate inducible hexose uptake the efficacy of fosfomycin against A. baumannii. However, we
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Figure 2. Screening and identification of IITR08367. (a) List of small molecules (n = 24) used as potential efflux pump inhibitors (used at 50 μM)
that can potentiate the activity of fosfomycin against AbaF expressing E. coli KAM32/pUC18_abaF. IITR08367 (50 μM) decreased the MIC of
fosfomycin to 2 mg/L (16-fold reduction). Inset shows the chemical structures of IITR08367 [bis(4-methylbenzyl) disufide] and fosfomycin. (b)
Checkerboard titration assay of fosfomycin with IITR08367 shows the potentiation of fosfomycin activity against AbaF expressing E. coli KAM32/
pUC18_abaF. (c) Time-kill kinetics assay of fosfomycin (32 mg/L) alone or in combination with 25 μM IITR08367. The limit of detection is 50
cfu/mL. Data points of three independent replicates are shown here. Error bars represent ±s.d. (d) The postantibiotic effect (PAE) of fosfomycin
(128 mg/L) alone or in combination with IITR08367 (50 μM). Data points of three independent replicates are shown here. The limit of detection
(LOD) represents 50 cfu/mL. Error bars represent ±s.d.

also observed an increase in biofilm formation capacity of these could also be efficacious against AbaF. We tested the efficacy of
clinical strains (n = 10) after treatment with fosfomycin these 24 potential inhibitors against AbaF-expressing strain E.
(Figure 1b). We observed a two- to sixfold increase in biofilm coli KAM32/pUC18_abaF, and surprisingly, nine molecules
in these tested strains. One of the key factors of A. baumannii out of 24 restored the efficacy of fosfomycin (Figure 2a).
involved in fosfomycin resistance as well as in biofilm IITR08367 [bis(4-methylbenzyl) disufide] (Figure 2a inset)
formation is an efflux pump called AbaF. AbaF plays an displayed the most potent efflux inhibition and potentiates
important role in fosfomycin resistance and extrusion of fosfomycin activity up to 16-fold against AbaF expressing
biofilm matrix out of the cells.17 We found that deletion of strain, E. coli KAM32/pUC18_abaF. IITR08367 itself did not
abaF abolished biofilm-forming capacity of A. baumannii kill any tested strains of E. coli and A. baumannii up to 400 μM
ATCC 17978, while plasmid-based gene complementation (the highest tested concentration). IITR08367 showed a dose-
can recover its biofilm formation potential (Figure 1c). This dependent potentiation of fosfomycin activity against AbaF
highlighted the fact that targeting AbaF could provide dual expressing strain, E. coli KAM32/pUC18_abaF (Figure 2b).
benefits in reviving efficacy of fosfomycin against intrinsically The potentiation of fosfomycin activity in the presence of
resistant A. baumannii. IITR08367 was further confirmed by a time kill-kinetics assay
IITR08367 Is a Potent Efflux Inhibitor of AbaF. (Figure 2c).
Recently, our group screened a library of 8000 small molecules Treatment of E. coli KAM32/pUC18_abaF cells with 32
and discovered 24 potential efflux pump inhibitors active mg/L fosfomycin showed a 2 log10 cfu decrease within 2 h,
against AbeM, a proton-driven multidrug and toxic compound which grew back similar to the untreated control within the
extrusion (MATE) family efflux pump of A. baumannii.22 Since next 16 h. However, treatment with 32 mg/L fosfomycin in
the MFS and MATE families use a similar proton-driven efflux combination with 25 μM IITR08367 demonstrated a
mechanism, we hypothesized if our 24 novel efflux inhibitors significant decrease of 4 log10 cfu within 2 h and slowed
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down the regrowth of cells up to 12 h. Overall, a > 3 log10 cfu

difference was observed even after 24 h. Postantibiotic effect
(PAE), an important clinically relevant pharmacodynamic
parameter, helps in designing dosing intervals. PAE indicates
the time required by the bacterial population to overcome
antibiotic stress after the removal of an antibiotic from the
system. The addition of IITR08367 (50 μM) increased the
PAE of fosfomycin by 30 min (Figure 2d) with an overall
fosfomycin PAE of 1.6 h after 1 h treatment at 4× MIC
concentration. Noteworthy, we did not observe the same level
of potentiation in time-kill kinetics as well as in PAE
experiment with E. coli KAM32 harboring only pUC18
plasmid (only plasmid control) (Figure S1a,b).
IITR08367 Is a Bona Fide Efflux Pump Inhibitor That
Acts by Disrupting Proton Gradient. Ethidium bromide
(EtBr) is a common substrate for many efflux pumps. The
fluorescence of EtBr changes proportionally to its concen-
tration inside the cells. EtBr loses its fluorescence when
effluxed out due to quenching by water molecules.23 We
observed an enhanced EtBr accumulation in AbaF expressing
strain, E. coli KAM32/pUC18_abaF, after IITR08367 treat-
ment, thus validating its efflux inhibitory potential (Figure 3a).
To further test the effect of IITR08367 on bacterial Figure 3. IITR08367 is a bona fide efflux pump inhibitor that acts by
membrane potential, an anionic potentiometric bis-oxonol affecting the ΔpH component of the cell. (a) Effect of addition of
probe DiBAC4(3) [(bis(1,3-dibutylbarbituric acid)trimethine IITR08367 (100 μM, indicated by arrow) on ethidium bromide
oxonol)] was used. DiBAC4(3) undergoes voltage-dependent (EtBr) fluorescence in E. coli KAM32/pUC18_AbaF. The dotted line
partitioning between the aqueous phase and bacterial represents fluorescence values without IITR08367 treatment. Data
points represent an average of three independent replicates. E. coli
membrane; membrane depolarization decreases its uptake in KAM32/pUC18 (only plasmid) was used as a control. Error bars
the membrane; thus, an increase in fluorescence is observed. represent ±s.d. (b) Effect of IITR08367 (100 μM) on cell membrane
Phenylalanine−arginine beta-naphthylamide (PAβN) is a potential of E. coli KAM32/pUC18_AbaF. PAβN (25 μM) was used
known efflux pump inhibitor that also disrupts membrane as a positive control. Each data point represents independent
potential, and we also observed increased fluorescence in biological replicates. Data was normalized with respective to untreated
PAβN-treated cells (Figure 3b). Hyperpolarized membrane control. Error bars represent ±s.d. * indicates p-value < 0.05
increases its uptake in the membrane, leading to fluorescence (unpaired student t-test followed by Mann−Whitney posthoc test).
quenching.24 Bacterial cells treated with IITR08367 showed (c) Effect of IITR08367 on fosfomycin/H+ antiport. The fluorescence
signal of quinacrine (420 nm excitation and 500 nm emission) with
decreased fluorescence due to hyperpolarization of the cell respect to time in everted membrane vesicles of E. coli KAM32/
membrane (Figure 3b). Bacterial cells tend to hyperpolarize pUC18_AbaF. Potassium-lactate, fosfomycin, and IITR08367 were
when the H+ gradient gets disturbed to maintain overall PMF used at 5 mM, 50 μM, and 25 μM, respectively. Data points represent
across the membrane.25 It is also known that efflux pump an average of three independent replicates. Error bars represent ±s.d.
inhibitors, which perturb membrane potential, tend to have (d) Fold change in MIC of fosfomycin against E. coli KAM32
membrane-damaging effects.26 Thus, we tested whether expressing different efflux pumps of A. baumannii (AbaF�MFS class,
IITR08367 has any membrane permeabilizing activity, and AbeS�SMR class, AbeM�MATE class). Data points of three
we found that it does not cause membrane damage (Figure independent replicates are shown here.
S2). Overall, IITR08367 disrupts the H+ gradient across the
membrane, ultimately inhibiting the H+-gradient-driven efflux
pumps. output of the system (Figure 3c). This energy decoupling led
AbaF belongs to an MFS class of efflux pumps that works as to the inhibition of the H+-gradient-driven efflux pumps.
a fosfomycin/H+ antiporter, that is, it effluxes out fosfomycin Since many bacterial efflux pumps utilize proton gradient as
in exchange for proton ions. To understand the mechanism of an energy source, we were curious to test whether IITR08367
the inhibitory effect of IITR08367 on fosfomycin/H+ antiport could also inhibit other efflux pumps that utilize proton
movement, a quinacrine-based proton ion concentration- gradient. A. baumannii harbors multiple types of efflux pumps
sensitive fluorescence assay was performed in everted that utilize proton gradient as an energy source. AbeM (MATE
membrane vesicles. The monoprotonated form of quinacrine class) and AbeS (SMR) are two validated efflux pumps
quickly equilibrates inside out of vesicles via diffusion and responsible for providing resistance against fluoroquinolones
emits fluorescence. The addition of potassium lactate energizes and other antibiotics in A. baumannii.22 Hence, we also tested
everted membrane vesicles, which leads to fluorescence the activity of IITR08367 against AbeS and AbeM and found
quenching due to acidification (increased H+ concentration) that IITR08367 inhibits AbeS as well as AbeM, which resulted
inside the vesicles. A slight increase in fluorescence after the in MIC reversal of ciprofloxacin by four- and eightfold,
addition of fosfomycin is attributed to the decrease in respectively (Figure 3d). Overall, multiple efflux pumps
intravesicular H+ ion concentration due to AbaF-mediated inhibition by IITR08367 by disrupting the H+ gradient across
fosfomycin exchange. The addition of IITR08367 leads to bacterial membrane may prove beneficial for reviving multiple
disruption of the H+ gradient, thus increasing the fluorescence antibiotics which are actively effluxed out by A. baumannii.
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Figure 4. IITR08367 is effective against clinical strains of A. baumannii. (a) Reversal of MIC of fosfomycin in the presence of 50 μM IITR08367
against clinical isolates of A. baumannii. A. baumannii ATCC 17978 and A. baumannii ATCC 17978 ΔAbaF were used as positive and negative
controls, respectively. Dashed line represents the epidemiological cutoff (breakpoint) of fosfomycin against P. aeruginosa according to The
European Committee on Antimicrobial Susceptibility Testing (EUCAST); fosfomycin cutoff is not available (December 2023) against A.
baumannii. (b) Checkerboard titration isobologram represents dose-dependent potentiation of fosfomycin activity in the presence of IITR08367.
(c) Growth profile of A. baumannii RPTC-15 in human urine (pH ranges 6.5−7) supplemented with 20% (v/v) Cation adjusted Muller Hinton
broth. Control cells represent untreated cells. Fosfomycin was used at 64 mg/L (when used alone) or in the presence of 100 μM IITR08367. Data
points of a single human urine sample are shown here. (d) Time-kill kinetics assay of fosfomycin (256 mg/L) in combination with IITR08367 (100
μM) in the MH broth acidified (pH 6.9) with the addition of Vitamin C (100 mg/L). The LOD represents 50 cfu/mL. Data points of three
independent biological replicates are shown here. Error bars represent ±s.d.

IITR08367 Potentiates Fosfomycin Activity against nine clinical isolates, including A. baumannii ATCC 17978. No
Clinical Strains of A. baumannii. After getting encouraging change in fosfomycin MIC was observed in AbaF knockout
results of IITR08367 on E. coli KAM32/pUC18_AbaF, we and RPTC-10. A. baumannii RPTC-15 was selected as a
tested the efficacy spectrum of IITR08367 against 22 representative strain for further studies. A. baumannii RPTC-15
fosfomycin-resistant A. baumannii clinical strains harboring is resistant to multiple clinically important antibiotics. A 2D-
AbaF.17 IITR08367 potentiated the efficacy of fosfomycin in checkerboard assay reveals the dose-dependent effect of
21 clinical strains as well as ATCC 17978 (Figure 4a). A IITR08367 on fosfomycin activity against A. baumannii
maximum eightfold reversal of fosfomycin efficacy was RPTC-15 (Figure 4b).
observed against A. baumannii RPTC-11 and RPTC-15. A As fosfomycin is often prescribed for treating urinary tract
fourfold reversal of fosfomycin efficacy was observed against infections (UTIs), we were intrigued to test the clinical
ten clinical isolates, and a twofold reversal was observed against significance of IITR08367 in a simulated ex vivo urine
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Figure 5. IITR08367−fosfomycin combination is effective against A. baumannii biofilm. (a−f) Effect of IITR08367 on biofilm formation of A.
baumannii RPTC-15 under different clinically relevant simulated conditions. Fosfomycin and IITR08367 were used at 64 mg/L and 100 μM,
respectively. Data points of three independent replicates are shown here. Error bars represent ±s.d. *, **, and *** indicate p-values of 0.05, 0.01,
and 0.001 respectively (one-way ANOVA, with Tukey’s multiple comparison test). (g−n) SEM images of A. baumannii RPTC-15 biofilm on
urinary catheter show increased biofilm after fosfomycin (64 mg/L) treatment. Fosfomycin and IITR08367 were used at 64 mg/L and 100 μM,
respectively. Scale bar represents 5 μm.

environment. We tested the growth profile of A. baumannii 15 (Figure S4). Our time-kill kinetic assay showed that the
RPTC-15 in the presence of either fosfomycin alone (64−128 addition of vitamin C (100 mg/L) could further enhance the
mg/L) or in combination with 100 μM IITR08367 in freshly activity of the fosfomycin (256 mg/L)�IITR08367 combina-
collected filter sterile human urine (n = 21, pH ranges 6.5−7). tion (Figure 4d).
Our results showed that a combination of fosfomycin (64 mg/ IITR08367 Potentiates Fosfomycin Activity against A.
L) and IITR08367 (100 μM) could prevent the growth of A. baumannii Biofilms. Biofilms are one of the important
baumannii RPTC-15 (an MDR clinical strain), while arsenals that help the survival of A. baumannii under different
fosfomycin alone was unable to control growth (Figures 4c pathophysiological conditions.13 Previously, it has been
and S3). established that AbaF helps A. baumannii to establish
Considering our results that acidic pH potentiates biofilms.17 We tested whether IITR08367 could hamper
fosfomycin activity, it would be beneficial to add a pH biofilm formation by A. baumannii via inhibition of AbaF. To
regulator to ensure the effective uptake of fosfomycin via replicate hospital-like settings, we performed biofilm inhibition
simple diffusion. Vitamin C is a classical pH regulator widely assay on different surfaces such as glass, soft silicone pediatric
used as an adjuvant in various clinical therapies.27 Considering catheter, and silicone geriatric catheter under two simulated
vitamin C itself as a molecule with antibiotic activity,28 we used conditions (CAMHB and urine). Our biofilm assay indicated
50 times less concentration (100 mg/L) than its minimum that in addition to the potentiation of fosfomycin’s bactericidal
effective antimicrobial concentration (5 mg/mL) for the pH effect, adding IITR08367 to fosfomycin could significantly
regulation. We tested the effect of 100 mg/L vitamin C on the reduce the biofilm formation compared to fosfomycin alone
efficacy of 256 mg/L fosfomycin against A. baumannii RPTC- treatment via inhibition of AbaF (Figure 5a−f). We also
15 in a time-kill kinetic assay. Our results confirmed that the examined the effect of IITR08367−fosfomycin combination
tested concentration of vitamin C (100 mg/L) alone did not under high-resolution scanning electron microscopy (SEM),
affect the potency of fosfomycin against A. baumannii RPTC- and our data show that IITR08367 could abolish biofilm
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Figure 6. IITR08367−fosfomycin combination reduces biofilm matrix of A. baumannii. Fluorescence microscopy imaging and quantitative analysis
of biofilm matrix of A. baumannii RPTC-15 biofilm on a glass coverslip in the presence of pH-adjusted urine supplemented with 20% MH media.
(a) Fluorescence microscopy images of biofilm matrix formed on glass coverslip. Fosfomycin and IITR08367 were used at 64 mg/L and 100 μM,
respectively. Thioflavin-T (Th-T), WGA Alexa Fluor 647 (WGA-AF 647), and Hoechst 33258 were used to estimate amyloid proteins,
extracellular polysaccharides and extracellular nucleic acid of biofilm, respectively. Scale bar represents 100 μm. Scatter dot plot showing
quantitative analysis of fluorescence signal intensity of (b) Th-T (c) Hoechst 33258 and (d) WGA-AF 647. Four independent microscopic images
were analyzed for each sample using ImageJ software. Error bar represents ±sem. ns represents nonsignificant; * indicates a p-value of <0.05
(unpaired student’s t-test followed by Mann−Whitney posthoc test).

formation capacity of A. baumannii with or without fosfomycin activity of IITR08367 was due to its ability to disrupt the
(Figure 5g−n). Additionally, our SEM analysis revealed that A. proton gradient across the bacterial membrane. Proton
baumannii forms structurally distinct microstructures when disruptors can be toxic to eukaryotic cells. Hence, we tested
supplemented with urine (Figure 5g,k). the biocompatibility of IITR08367 toward freshly isolated
Bacterial biofilms are composed of aggregations of bacteria human erythrocytes and peripheral blood mononuclear cells
adhered to surfaces and/or to one another, encapsulated (PBMCs). Our results indicate that the IITR08367 does not
within a self-generated matrix. This matrix comprises various show any toxicity to human erythrocytes (Figure S5a).
substances, including proteins (such as fibrin), polysaccharides Additionally, IITR08367 did not show much toxicity to
(such as alginate), and extracellular DNA (eDNA). Various human PBMCs at the effective concentration ranges (Figure
efflux pumps are also known to be involved in extrusion of S5b).
different biofilm matrix components.17,29 Considering the IITR08367 Potentiates Fosfomycin and Reduces In
effective biofilm inhibition and efflux pump inhibitory activity Vivo A. baumannii Burden in C. elegans. Caenorhabditis
of IITR08367, we further investigated the effect of IITR08367 elegans is a lower eukaryotic model organism used to study the
in reducing these matrix components in detail. We employed toxicity and efficacy of novel small molecules.30 Hence, we
fluorescence microscopy to measure amyloid proteins, tested toxicity of IITR08367 and its combination with
extracellular polysaccharides, and eDNA using their specific fosfomycin. Our results indicate that IITR08367 displays
fluorescent probes. We found a significant reduction in only marginal toxicity to C. elegans at a very high concentration
amyloid proteins of biofilm matrix after treatment with (400 μM). Similarly, fosfomycin either alone (512 mg/L) or in
IITR08367 (Figure 6b). This indicates that AbaF may be combination with IITR08367 (fosfomycin at 512 mg/L &
responsible for the extrusion of proteinaceous components of IITR08367 at 400 μM) only shows marginal toxicity (Figure
biofilm matrix. The combination of fosfomycin and IITR08367 7a). A combination of two different pharmaceutically active
significantly reduced all three components of biofilm matrix compounds can lead to enhanced toxicity in test organisms;
when compared with fosfomycin alone treatment (Figure 6b− however, our results indicate that combination of fosfomycin
d). and IITR08367 does not show any enhanced adverse effect.
IITR08367 Is a Nonhemolytic and Noncytotoxic Efflux C. elegans is a promising model for studying A. baumannii
Pump Inhibitor. IITR08367 showed great potential in pathogenesis.31 Hence, we evaluated in vivo efficacy of
reviving fosfomycin efficacy against intrinsically resistant A. IITR08367 along with fosfomycin in C. elegans-A. baumannii
baumannii via inhibiting fosfomycin efflux pump, AbaF. infection model.31,32 Our in vivo efficacy results indicate two
Furthermore, we observed that the efflux pump inhibition important evidence. First, in vivo efficacy of fosfomycin is
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Figure 7. In vivo toxicity and efficacy of fosfomycin-IITR08367 combination (a) Bar graph showing percentage survival of C. elegans (n = 15−20)
under different treatment regimens. Survival was calculated at the end of 72 h. Error bars represent ±s.d. ns represents nonsignificant (one-way
ANOVA, with Tukey’s multiple comparison test) (b) Bar graph showing A. baumannii RPTC-15 (a pathogenic clinical strain) reduction in C.
elegans (n = 15−20) under different treatment regimens. Data points of three independent replicates are shown here. Error bars represent ±s.d. ns
represents nonsignificant; *, **, and *** indicate p-values of 0.05, 0.01, and 0.001, respectively (one-way ANOVA, with Tukey’s multiple
comparison test). (c) A. baumannii RPTC-15 load in the kidney of mice after UTI infection. The LOD corresponds to 30 cfu/mL. Data points of
five individual mice are shown here. Error bars represent ±s.d. ** indicates a p-value of <0.01 (nonparametric one-way ANOVA, with Kruskal−
Wallis multiple comparison test). (d) A. baumannii RPTC-15 load in bladder of mice after UTI infection. Data points of five individual mice are
shown here. Error bars represent ±s.d. * indicates a p-value of <0.05 (nonparametric one-way ANOVA, with Kruskal−Wallis multiple comparison
test). (e) Histopathology of mice kidney under different treatment regimens. GC, PCT, and DCT are indicated by black arrow. Scale bar represents
100 μm.

better than what is in an in vitro assay. Our test pathogen, A. IITR08367 Potentiates Fosfomycin and Reduces In
baumannii RPTC-15, shows in vitro susceptibility at 256 mg/L. Vivo A. baumannii Burden in Murine UTI Model.
Surprisingly, fosfomycin (at 256 mg/L) shows ≈1 log10 cfu Motivated by our in vivo efficacy results in C. elegans, we
reduction in in vivo conditions, even after it has to go under extended the efficacy study in the murine UTI model. We
ADME (absorption, distribution, metabolism, and excretion) selected A. baumannii RPTC-15 (MDR clinical isolate) for
pathway. Second, IITR08367 shows potentiation of fosfomycin murine UTI infection and found that it can successfully
activity in the C. elegans model, as the combination of colonize the mice bladder. Our efficacy data show that
fosfomycin and IITR08367 displayed ≈3 log10 cfu reduction (p fosfomycin-IITR08367 combination successfully decreased the
< 0.001). Addition of a noninhibitory concentration of vitamin A. baumannii RPTC-15 burden by ∼3 log10, both in the kidney
and in the bladder (Figure 7c,d). On the other hand,
C (100 mg/L) to the fosfomycin-IITR08367 combination
fosfomycin alone only decreased A. baumannii RPTC-15
further reduces the bacterial load in the C. elegans infection
burden in the bladder by ∼1 log10; moreover, it did not reduce
model (Figure 7b). Additionally, we also tested the effect of any bacterial load in the kidney. Next, we performed
IITR09367-fosfomycin combination on the pathogenicity of A. histopathological examinations of kidney. We found that A.
baumannii in C. elegans infection model. We found better baumannii RPTC-15 caused extensive damage to functional
survivability of C. elegans under fosfomycin-IITR08367 treat- units of kidney, that is, glomerular capsule (GC), proximal
ment regimen. Notably, 80% of the C. elegans survived the convoluted tubules (PCT), and distal convoluted tubules
virulent A. baumannii infection even when treated with (DCT) in untreated mice (Figure 7e). Additionally, there was
subinhibitory concentration of fosfomycin-IITR08367 combi- very high infiltration of neutrophils, leading to tissue
nation, while only 40% of the population survived under only inflammation. Mice treated with either only fosfomycin or
fosfomycin treatment regimen (Figure S6). IITR08367 showed marked damage in PCT, DCT linings, and
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minor damage was observed in GC. However, mice treated mg/L) can be maintained for 24 h with a single oral dose of 50
with a combination of fosfomycin and IITR08367 showed mg/kg.45 These pharmacokinetic properties of fosfomycin
significant improvement, and tissue ultrastructures were similar make it the first choice for the treatment of uncomplicated
to uninfected mice (Figure 7e). In conclusion, IITR08367, UTIs. There is an increasing appreciation of efficacy of
along with fosfomycin, can play a major role in the fight against fosfomycin for the treatment of critically ill patients.
A. baumannii infections. Several physiological factors, such as plasma-binding

■ DISCUSSION
Bacteria possess multiple arsenal to tackle antibiotics, such as
capacity, renal clearance, pH of the system, and so on, control
efficacy of a given antibiotic. Fluoroquinolones, aminoglyco-
sides, and trimethoprim are among the antibiotics that exhibit
target site protection and modification and enzymatic greater effectiveness in alkaline environments, contrary to
inactivation of antibiotics.33 However, efflux pumps are one antibiotics whose activity is favored in acidic media are
of the most important arsenals for a resistant bacterial fosfomycin, tetracycline, nitrofurantoin.47 Strategically, the
pathogen. 34 Efflux pumps play a critical role in the system’s pH can be controlled by providing systemic alkalizers
pathophysiology of many human pathogens.34−36 What or mild acidifiers. Vitamin C is one such acidifier used in
makes bacterial efflux pumps so special is broad substrate clinics.27 Surprisingly, potency of fosfomycin is enhanced
specificity, that is, a single type of efflux pump can expel under acidic pH, and we observed up to 16-fold susceptibility
multiple antibiotics. Active expression of a single efflux pump enhancement against clinical strains of A. baumannii in our
can cause treatment failure against multiple antibiotics.35,37 study. However, only pH does not solve the inherent problem
Furthermore, efflux pumps also play an important role in of reduced susceptibility of fosfomycin against A. baumannii,
biofilm formation by transporting various biofilm matrix because this bacterium tries to evade fosfomycin by making
components.29 robust biofilm which protects them against fosfomycin.29
Fosfomycin shows broad-spectrum antibacterial activity Moreover, A. baumannii has fosfomycin specific efflux pump,
against both Gram-positive and Gram-negative pathogens.38 AbaF.17 Taking into account these factors, we developed a
Its usage has been endorsed by the Infectious Diseases Society double-edged sword strategy to overcome this problem by
of America (IDSA) in USA.39 Despite the very high level of identifying IITR08367, a potent efflux inhibitor that targets
antibiotic resistance among E. coli, K. pneumoniae, Pseudomonas AbaF and thus decreases biofilm matrix as well as fosfomycin
aeruginosa, and E. faecalis, fosfomycin has retained its efflux. We also uncover that IITR08367 is a safe and
susceptibility in different countries against these human nonspecific inhibitor of multiple efflux pumps of A. baumannii,
pathogens.40 Additionally, it shows activity against some which may be advantageous for clinical settings.
unusual and difficult-to-treat pathogens like Klebsiella oxytoca, In order to further broaden the efficacy of fosfomycin against
Serratia marcescens, and Proteus mirabilis. The most noteworthy clinically challenging pathogens like A. baumannii, we screened,
fact regarding the efficacy of fosfomycin is its retained activity identified, and validated a novel efflux pump inhibitor of AbaF
against Enterobacteriaceae producing extended-spectrum β- in A. baumannii. IITR08367 potentiates the antibacterial
lactamase (ESBL), New Delhi metallo-β-lactamase-1 (NDM- efficacy of fosfomycin against A. baumannii. IITR08367
1), and KPC-2 enzymes.40 In contrast, fosfomycin appears to inhibits efflux pumps by disrupting the proton gradient across
be ineffective against notorious A. baumannii.13,15 Antibiotic the bacterial membrane. Relatively high activity/toxicity index
resistance in A. baumannii is increasing rapidly.3,41 A. of IITR08367 offers it as a potent adjuvant for fosfomycin
baumannii is intrinsically resistant to fosfomycin due to several treatment against A. baumannii.
factors. First is the lack or modification in the transport
systems for fosfomycin uptake, such as glycerol-3-phosphate
transporter system (GlpT) and the hexose phosphate trans-
porter (UhpT). Second is the presence of fosfomycin-
■ MATERIAL AND METHODS
Ethical Statement. All animal experiments were approved
inactivating enzymes such as FosX. Third is a mutation in by the Institutional Animals Ethics Committee of the Indian
UDP-N-acetylglucosamine enolpyruvyl transferase gene Institute of Technology Roorkee (Protocol no. BT/IAEC/
(murA). Fourth is an active efflux of fosfomycin by the AbaF 2014/08/REV). All the animal experiments were conducted as
(Acinetobacter baumannii Fosfomycin efflux) system.15,17 per Institutional Animals Ethics Committee (IAEC) guidelines
AbaF is a type of MFS transporter found in clinical strains (CPCSEA registration number 563/GO/Re/S/02/CPCSEA).
of A. baumannii and is primarily responsible for high-level The collection of human urine and blood samples from healthy
fosfomycin resistance in clinical settings.15,17 Hence, targeting volunteers was approved by the Institutional Human Ethics
AbaF would be an excellent choice for rejuvenating fosfomycin Committee of the Indian Institute of Technology Roorkee
against A. baumannii. (Protocol no. BT/IHEC-IITR/2019/7300).
Fosfomycin is primarily available as oral preparation for the Chemicals. All small molecules used as efflux pump
treatment of uncomplicated UTIs and other infections.42 inhibitors in this work were purchased from Maybridge
However, there is an increase in the usage of intravenous (Now Thermo Fisher Scientific, USA). DiBAC4(3), PAβN,
fosfomycin along with other antibiotics for severe infections as Thioflavin-T (Th-T), Hoescht 33258, and wheat germ
a combinatorial therapy.38,43,44 Fosfomycin has excellent oral agglutinin- Alexa Fluor 647 (WGA-AF 647) were purchased
bioavailability and tissue dissemination at different body sites from Thermo Fisher Scientific (USA). All other chemical
such as lungs, kidneys, liver, and cerebrospinal fluids.45,46 What probes and antibiotics were purchased from Sigma-Aldrich
makes fosfomycin unique for the treatment of UTIs is its (USA).
bioavailability at the bladder wall and in urine. Active Bacterial Strains and Media. All the bacterial strains used
concentration of fosfomycin in urine can reach 2500 mg/L in this study are listed in Table S1. All strains were maintained
within 2 h after a single oral dose of 50 mg/kg.45,46 on Cation-adjusted Muller Hilton (CAMH) Agar (Himedia,
Additionally, a very high urinary concentration (up to 700 India). During all experiments, cells were grown in Cation-
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adjusted Muller Hilton Broth (CAMHB) (Himedia, India) In Vitro Postantibiotic Effect. The PAE of IITR08367
until specified. was studied according to a previously published protocol with
Determination of MIC. MICs of various antibacterial and minor modifications.52,53 Briefly, the culture of E. coli KAM32/
small molecules were determined in 96-well clear bottom pUC18_abaF (approximately 107 cfu/mL) was treated with
plates with an initial inoculum of 105 cfu/mL as per Clinical fosfomycin alone (128 mg/L) or in combination with
and Laboratory Standards Institute (CLSI) guidelines in IITR08367 (50 μM) for 1 h at 37 °C with aeration (180
CAMH broth.48 Details are described in the Supporting rpm). Cells were washed thrice with fresh MH broth to
Information methods (Section S1). remove test compounds. Cells were further incubated in fresh
Biofilm Inhibition Assay. A biofilm inhibition experiment MH broth at 37 °C with aeration. Viable bacterial counts were
was performed with various clinical strains of A. baumannii, determined at regular time intervals (0, 2, 4, 6, and 8 h). The
ATCC 17978 (WT), A. baumannii ΔabaF (ΔabaF), and abaF PAE was estimated by the formula PAE = Tn − Tc where Tn is
complimented in A. baumannii ΔabaF (pabaF) according to the time required for a unit log10 bacterial growth (cfu/mL)
previously described method with minor modification.49 increase in a drug-treated sample, while Tc is the time required
Details are described in the Supporting Information methods for a unit log10 growth in an untreated sample.54
(Section S2). Ethidium Bromide Efflux Assay. Freshly harvested mid-
Screening of AbaF Inhibitors Using a Chemical log phase cells of E. coli KAM32/pUC18_abaF and E. coli
Genetic Approach. Screening of AbaF inhibitor was KAM32/pUC18 were washed and resuspended in 1x PBS to
performed against E. coli KAM32 harboring cloned copy of an OD600 ∼ 0.3. EtBr (10 μg/mL) was added to each cell
abaF in pUC18 plasmid (overexpressor strain of AbaF).17,50 E. suspension, followed by 30 min incubation at 37 °C with
coli KAM32 is deficient of important efflux pumps (YdhE, intermittent mixing. Cell suspension (200 μL) of each sample
AcrAB), making it an ideal host to test putative efflux pump or was added to a UV opaque flat bottom black 96-well microtiter
efflux pump inhibitors.50 E. coli KAM32 containing only plate (BRAND, Germany). 0.4% (w/v) glucose was added to
pUC18 was used as the control strain for this assay. A fold each well to initiate the efflux of EtBr, and fluorescence was
change reversal in MIC of fosfomycin was tested with 24 measured at 480 nm excitation and 610 nm emission at the 1
putative efflux pump inhibitors. All putative efflux pump min interval using the spectrofluorometer (SynergyH1,
inhibitors were used at a concentration of 50 μM, which is BioTek, USA).55 After 10 min, 100 μM IITR08367 was
noninhibitory for test strain. Molecules showing more than a added, and fluorescence measurement was continued for the
fourfold reduction in MIC of fosfomycin were considered next 10 min. Cells without IITR08367 treatment were used as
potential inhibitors of AbaF. a control for this experiment.
Evaluation of Synergistic Drug Interactions of Assessment of IITR08367 Effect on Membrane
Fosfomycin with Potential Inhibitors of AbaF Using Potential. Logarithmic-phase E. coli KAM32/pUC18_abaF
Checkerboard Assays. A 2D checkerboard broth micro- cells were harvested, washed, and resuspended in 1× PBS,
dilution assay was performed using twofold serially diluted containing 0.4% glucose (w/v) to an OD600 ∼ 0.2. The cell
concentrations of fosfomycin with IITR08367 against E. coli suspension was treated with 100 μM IITR08367 for 2 h at 37
KAM32/pUC18_abaF as previously described.32 Cell growth °C. After 1 h, 1 μM DiBAC4(3) (Invitrogen, USA) was added
was monitored by measuring optical density at 600 nm and incubated for 15 min. The fluorescence of DiBAC4(3) was
(OD600nm) after 18 h of incubation at 37 °C. The fractional measured at an excitation of 490 nm and emission at 520 nm
inhibitory concentration (FIC) index was calculated by the using the spectrofluorometer (SynergyH1, BioTek, USA).
following formula Fluorescence was normalized by OD600 to calculate relative
fluorescence values (RFU).
l
o MIC of drug A in combination |o Preparation of Everted Membrane Vesicles for In
FIC index = o
m
o
o
}
o Vitro Fosfomycin/H+ Antiport Assay. Everted membrane
o
n MIC of drug A alone o
~ vesicles of E. coli KAM32/pUC18_abaF cells were prepared
l
o MIC of drug B in combination |
o according to the previously described method with slight
+om
o
o
}
o modifications.56,57 For fosfomycin/H+ antiport assay, quina-
o
n MIC of drug B alone o
~ (1) crine fluorescence quenching was estimated in a mixture of
membrane vesicles (0.2 mg/mL of protein) and quinacrine (1
Synergy was defined as an FIC index value of ≤0.5, and μM). Details are described in the Supporting Information
additivity or indifference was defined as an FICI value of ≥0.5 methods (Sections S3 and S4).
to <4, whereas antagonism was defined as an FICI value of Drug Susceptibility Assay/MIC Determination. The
≥4.51 MICs of different antibiotics (alone as well as in the presence
Time-Kill Kinetics Assay. The time-kill experiments were of 50 μM IITR08367) were determined against E. coli
performed in CAMHB (Himedia, India) according to the KAM32/pUC18_abaF,17 E. coli KAM32/pUC18_abeS,22 E.
previously described method with a slight modification.32 coli KAM32/pUC18_abeM,22 and clinical strains of A.
Briefly, E. coli KAM32/pUC18_abaF cells (approximately baumannii (Table S1). The MIC was determined according
∼105 cfu/mL) were treated either with fosfomycin alone (32 to Clinical and Laboratory Standards Institute (CLSI)
mg/L) or in combination with IITR08367 (25 μM) at 37 °C guidelines.48
with aeration for 24 h. For A. baumannii RPTC-15, fosfomycin Assessment of IITR08367 Effect on Bacterial Cell
was used at 256 mg/L when used alone or with 100 μM of Membrane Integrity. Membrane damage assay was
IITR08367. 100 μL of culture was removed at different time performed as previously described.58 Briefly, logarithmic
intervals (2, 4, 8, 12, and 24 h) and plated on CAMH agar after phase bacterial cells were harvested, washed, and resuspended
10-fold serial dilution. Viable colonies were counted after 24 h in 1× PBS, containing 0.4% w/v glucose up to an OD600 of 0.2.
of incubation and presented at a log10 scale over time. The cell suspension was treated with 100 μM IITR08367 and
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4 mg/L polymyxin B (positive control). After 2 h of incubation −4 and day −1 by injecting cyclophosphamide (150 mg/kg
at 37 °C, one mg/L propidium iodide (Sigma-Aldrich, USA) body wt.; I.P.) (TCI Chemical, Japan). On day 0, mice were
was added and incubated for 15 min. Optical density at 600 anesthetized with a 1:1 dose of ketamine (75 mg/kg body wt.;
nm and fluorescence at 535 nm (excitation) and 617 nm I.P.) (Themis Medicare, Ltd.) and xylazine (16 mg/kg body
(emission) wavelengths were measured by using the wt.; I.P.) (Indian Immunologicals, Ltd., India).64 Mice were
spectrofluorometer (SynergyH1, BioTek, USA). Fluorescence infected with A. baumannii RPTC-15 (105 cfu) by slowly
was normalized by OD600 to calculate RFU. injecting 10 μL culture into the urinary bladder using a
Ex Vivo Growth Kinetics Study of A. baumannii in neonatal I.V. cannula (26G, IVWP-26; Prime Healthcare
Human Urine. A growth kinetic was put forward in freshly Products Pvt. Ltd., India).65 Treatment was started after 6 h of
collected, pH-adjusted (6.5−7), and filtered sterile urine infection. Group 1 was given normal saline and served as
samples supplemented with 20% (v/v) CAMH broth. Each vehicle control (untreated); groups 2, 3, and 4 were treated
sample was inoculated with 105 cells of A. baumannii clinical with fosfomycin (10 mg/kg body wt.), IITR08367 (30 mg/kg
isolate RPTC-15. Each replicate was treated with fosfomycin body wt.), and an equivalent amount of fosfomycin and
(64 and 128 mg/L) alone as well as in the presence of IITR08367, respectively. Four doses of treatment were applied
IITR08367 (100 μM). Culture was incubated for 12 h at 37 °C every 12 h starting from T0 (6 h postinfection). All mice were
with shaking, and optical density was measured at 600 nm after sacrificed after 54 h by giving an overdose of anesthesia; the
30 min interval using the spectrofluorometer (SynergyH1, kidney and urinary bladder were excised, and the weight of
BioTek, USA). This experiment was performed with urine tissue was measured. One kidney from each mouse was fixed in
obtained from 21 healthy volunteers. 4% formalin solution and used for tissue histology (hematox-
Scanning Electron Microscopy of Biofilm on Urinary ylin and eosin staining). One kidney and bladder from each
Catheters. Biofilm of A. baumannii RPTC-15 was grown on mouse were homogenized with a tissue homogenizer (Genetix,
urinary catheters were prepared as previously described with India). 100 μL of each sample was 10-fold serially diluted and
some minor modifications.59 Briefly, a small section fixed plated on MH agar plates. CFU counts were enumerated after
biofilm-formed catheters were dehydrated with an ethanol incubating the plates for 24 h. The results were analyzed with
gradient and visualized under the scanning electron micro- nonparametric one-way ANOVA, with Kruskal−Wallis multi-
scope (Zeiss, Germany). Details are described in the ple comparison test.
Supporting Information (Section S5).
Fluorescence Microscopy and Quantitative Analysis
of Biofilm Matrix. Samples were prepared as previously
■
*
ASSOCIATED CONTENT
sı Supporting Information
described with some modification.60 Briefly, biofilm of A. The Supporting Information is available free of charge at
baumannii RPTC-15 on glass coverslips were treated with Th- https://pubs.acs.org/doi/10.1021/acsinfecdis.4c00077.
T, WGA-AF 647, and Hoechst 33258. Fluorescence imaging
was performed and the fluorescence intensity of each image (PDF)
was measured using ImageJ software.61 Details are described in
the Supporting Information (Section S6).
Erythrocytes (RBCs) Hemolysis and Cytotoxicity
Assay. Human RBC hemolysis and toxicity against freshly
■ AUTHOR INFORMATION
Corresponding Author
isolated PBMCs was performed as previously described with Ranjana Pathania − Department of Biosciences and
minor modifications.62 Briefly, erythrocytes and PBMCs were Bioengineering, Indian Institute of Technology, Roorkee,
treated with different concentrations of IITR08367. Details are Uttarakhand 247 667, India; orcid.org/0000-0002-
described in Supporting Information methods (Section S7). 7965-8176; Phone: +91 1332 285324;
Toxicity and Efficacy Studies of IITR08367 in Email: [email protected]
Caenorhabditis elegans Model. Toxicity and efficacy
Authors
studies of IITR08367 in C. elegans were performed according
to the previously described protocol with minor modifica- Mahak Saini − Department of Biosciences and Bioengineering,
tions.63 Infected or uninfected L2-stage worms were used. The Indian Institute of Technology, Roorkee, Uttarakhand 247
number of live and dead worms after 72 h for toxicity, and 667, India
bacterial load per worm was calculated for efficacy. Details are Amit Gaurav − Department of Biosciences and Bioengineering,
described in supplementary methods (Section S8). Indian Institute of Technology, Roorkee, Uttarakhand 247
C. elegans Survival Assay. Worms of C. elegans strain 667, India
AU37 were exposed to A. baumannii RPTC-15 in the presence Arsalan Hussain − Department of Biosciences and
of a subinhibitory concentration of fosfomycin alone as well as Bioengineering, Indian Institute of Technology, Roorkee,
in combination with IITR08367. The number of live and dead Uttarakhand 247 667, India
worms per well was recorded every 24 h for up to 7 days. Complete contact information is available at:
Details are described in supplementary methods (Section S9). https://pubs.acs.org/10.1021/acsinfecdis.4c00077
In Vivo Murine UTI Model. In vivo efficacy of fosfomycin-
IITR08367 combination in murine UTI model was performed Author Contributions
in 6-week-old BALB/c female mice (n = 5). Mice were R.P. and M.S. contributed to the conceptualization; M.S., A.G.
procured at the age of 5 weeks and housed for 7 days with free and A.H. contributed to the methodology; R.P. contributed to
access to food and water. RandoMice v1.1.7 software was used the resources:; M.S. and A.G. contributed to formal analysis;
to randomly distribute mice among four groups on the basis of R.P. contributed to funding acquisition; R.P. contributed to
urinary pH and body weight. Mice were provided with a 12 h supervision; R.P., M.S., A.G., and, A.H. contributed to
day−night light cycle. Mice were immunocompromised on day Writing�review and editing.
1721 https://doi.org/10.1021/acsinfecdis.4c00077
ACS Infect. Dis. 2024, 10, 1711−1724
ACS Infectious Diseases pubs.acs.org/journal/aidcbc Article

Notes (15) Leite, G. C.; Perdigão-Neto, L. V.; Ruedas Martins, R. C.;

The authors declare no competing financial interest. Rizek, C.; Levin, A. S.; Costa, S. F. Genetic factors involved in
fosfomycin resistance of multidrug-resistant Acinetobacter baumannii.

■ ACKNOWLEDGMENTS
We would like to thank Prof. T. Tsuchiya (Okayama
Infect., Genet. Evol. 2021, 93, 104943.
(16) Singkham-In, U.; Chatsuwan, T. Synergism of imipenem with
fosfomycin associated with the active cell wall recycling and
University) for providing us E. coli KAM32. This research heteroresistance in Acinetobacter calcoaceticus-baumannii complex.
received no specific grant from any funding agency in the Sci. Rep. 2022, 12 (1), 230.
public, commercial or not-for-profit sectors. The PhD (17) Sharma, A.; Sharma, R.; Bhattacharyya, T.; Bhando, T.;
fellowship of M.S. and A.H. was supported by the Ministry Pathania, R. Fosfomycin resistance in Acinetobacter baumannii is
of Human Resource Development (MHRD), Government of mediated by efflux through a major facilitator superfamily (MFS)
India. We would like to extend our gratitude to Prof. Debasis transporter-AbaF. J. Antimicrob. Chemother. 2017, 72 (1), 68−74.
(18) Pourbaix, A.; Guérin, F.; Burdet, C.; Massias, L.; Chau, F.;
Banerjee, Department of Chemistry, IIT Roorkee for helping
Cattoir, V.; Fantin, B. Unexpected Activity of Oral Fosfomycin against
with structure confirmation of IITR08367. Resistant Strains of Escherichia coli in Murine Pyelonephritis.

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