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Ciprooxacin‑
and levooxacin‑loaded
nanoparticles eciently
suppressed uoroquinolone
resistance and biolm formation
in Acinetobacter baumannii
Alaa M. Aboelenin
1, Mohammed El‑Mowafy
1, Noha M. Saleh
2, Mona I. Shaaban
1* &
Rasha Barwa
1*
The spread of uoroquinolone (FQ) resistance in Acinetobacter baumannii represents a critical
health threat. This study aims to overcome FQ resistance in A. baumannii via the formulation of
polymeric nanoFQs. Herein, 80 A. baumannii isolates were obtained from diverse clinical sources. All
A. baumannii isolates showed high resistance to most of the investigated antimicrobials, including
ciprooxacin (CIP) and levooxacin (LEV) (97.5%). FQ resistance‑determining regions of the gyrA
and parC genes were the most predominant resistant mechanism, harbored by 69 (86.3%) and 75
(93.8%) of the isolates, respectively. Additionally, plasmid‑mediated quinolone resistance genes
aac(6′)-Ib and qnrS were detected in 61 (76.3%) and 2 (2.5%) of the 80 isolates, respectively. The
CIP‑ and LEV‑loaded poly ε‑caprolactone (PCL) nanoparticles, FCIP and FLEV, respectively, showed
a 1.5–6‑ and 6–12‑fold decrease in the MIC, respectively, against the tested isolates. Interestingly,
the time kill assay demonstrated that MICs of FCIP and FLEV completely killed A. baumannii isolates
after 5–6 h of treatment. Furthermore, FCIP and FLEV were found to be ecient in overcoming the FQ
resistance mediated by the eux pumps in A. baumannii isolates as revealed by decreasing the MIC
four‑fold lower than that of free CIP and LEV, respectively. Moreover, FCIP and FLEV at 1/2 and 1/4 MIC
signicantly decreased biolm formation by 47–93% and 69–91%, respectively. These ndings suggest
that polymeric nanoparticles can restore the eectiveness of FQs and represent a paradigm shift in the
ght against A. baumannii isolates.
Acinetobacter baumannii is an aerobic, non-motile Gram-negative coccobacillus that is considered one of
the most hazardous opportunistic pathogens. is organism can get resistance determinants as a result of its
genome plasticity, making the infections it causes dicult to treat1. e World Health Organization (WHO)
has recognized A. baumannii as one of the top three priority pathogens requiring urgent development of
new antimicrobials. Such dangerous pathogens have the potential to cause several diseases, including post-
neurosurgical meningitis, osteomyelitis, lung infections, urinary tract infections, and infections of traumatic
or surgical wounds2.
A. baumannii has developed extraordinary antimicrobial resistance mechanisms, including activated
multidrug eux pumps, increased outer membrane permeability, enzymatic modication of drugs, and target
gene mutation. e combined actions of those mechanisms have led to the development of multiple drug-
resistant (MDR) and extensively drug-resistant (XDR) strains of A. baumannii3. XDR is identied as resistance
to at least one agent in all but bacterial isolates remain susceptible to one or two categories4.
OPEN
1Department of Microbiology and Immunology, Faculty of Pharmacy, Mansoura University, PO Box 35516,
Mansoura, Egypt. 2Department of Pharmaceutics, Faculty of Pharmacy, Mansoura University, PO Box 35516,
Mansoura, Egypt. *email: mona_ibrahim@mans.edu.eg; rasha@mans.edu.eg
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Fluoroquinolones (FQs), such as ciprooxacin (CIP) and levooxacin (LEV), have been widely utilized to
treat A. baumannii infections by inhibiting DNA gyrase and topoisomerase IV. FQ resistance is mostly caused
by chromosomal mutations in the uoroquinolone resistance-determining regions (FQRDRs) of the DNA
gyrase genes (gyrA and gyrB) and/or topoisomerase IV genes (parC and parE), which reduce DNA gyrase or
topoisomerase’s anity for FQs5. Additionally, mutations in the regulatory genes that manage the expression
of eux pumps and outer membrane proteins (OMPs) are considered an important cause of FQ resistance.
Plasmid-mediated quinolone resistance (PMQR) plays a crucial role in the acquisition of resistance to FQs.
e PMQR genes aid in the selection of mutants with higher levels of resistance rather than conferring FQ
resistance6. ere are dierent types of PMQR determinants; qnr shields FQ targets (bacterial DNA gyrase and
topoisomerase IV) from inhibition. qnr proteins have been identied, including qnrS, qnrA, qnrB, qnrD, qnrC,
qnrVC, and the recently discovered qnrE, with numerous genetic variants7. e second PMQR gene is aac (6′)-
Ib. It is an aminoglycoside-modifying enzyme that transfers acetyl groups to some FQs, causing a decrease in
binding to the target site and the elimination of antibacterial eects8. e enhanced eux pumps produced by
plasmid genes for pumps qepA and oqxAB are another important mechanism of PMQR9.
Biolms oer an impenetrable barrier to antibacterial agents, providing the necessary conditions for bacterial
growth and colonization as well as the emergence of serious and health-threatening microbial infections.
Additionally, pathogenic bacteria form biolms that are encased in the exopolysaccharide matrix, and play a
signicant role in pathogenesis, and limit the eectiveness of available treatments10. As a result, antibacterial
therapy requires the search for ecient and biolm-preventing bactericidal drugs11. A smart delivery system has
the potential to improve the bactericidal eectiveness of existing antimicrobials and provide an eective solution
to combat the spread of resistant bacteria12. Developing new generations or derivatives of antimicrobials is a
very expensive investment process that takes a long time to distinguish in pharmaceutical production pipelines.
Nanosize carriers could provide the necessary chemical protection and the ecient delivery of antimicrobial
compounds12. is has drawn attention to their potential for preventing and eradicating biolm development,
and microbial resistance. Along with their ability to increase bacterial uptake, antimicrobial-loaded nanoparticles
have greater penetration power, which would help to prevent the emergence of MDR and XDR. Additionally,
they have greater invivo stability against biodegradation and require low therapeutic doses and less frequent
administration13.
Polymeric nanoparticles (NPs) are solid colloidal nano-based systems ranging in size from 10 to 1000nm.
Biodegradable polymers like alginate, chitosan, and polycaprolactone are commonly utilized for the preparation
of NPs. Poly ε-caprolactone (PCL) is a semicrystalline aliphatic polyester that degrades at a slower rate than
other biodegradable polymers. Such a property can be exploited to deliver antibiotics in a controlled manner
over time14. Unlike other polyesters, PCL degradation products do not elevate the acidity of the surrounding
environment with a minimum impact on homeostasis. PCL was chosen for the preparation of biodegradable NPs
in the current study due to its advantageous biocompatibility, biodegradability, and non-toxicity15.
e aim of this study is the molecular characterization of dierent mechanisms of FQ resistance in A.
baumannii clinical isolates. Additionally, CIP- and LEV-loaded polymeric nanoparticles were formulated using
PCL and further assessed their eectiveness in overcoming FQ resistance and biolm formation in A. baumannii
isolates.
Results
Identication of bacterial isolates
Microscopical characterization and biochemical reactions
In this study, a total of 550 specimens were collected, and 120 isolates were identied as Acinetobacter spp. using
standard microbiological techniques, including Gram staining, colony morphology, and biochemical reactions.
Under the microscope, all 120 isolates of Acinetobacter spp. were seen as Gram-negative coccobacilli. On solid
media, colonies were smooth, occasionally mucoid, and non-lactose fermenters appeared as pale or beige colonies
on MacConkey agar. Metallic reddish colonies were detected using CHROMagar Acinetobacter media aer
overnight incubation at 37°C. Furthermore, isolates of Acinetobacter spp. were positive for catalase and citrate
utilization but were negative for indole, oxidase, methyl red, Voges-Proskauer, and lactose fermentation.
Identication of A. baumannii by PCR
All the microbiologically identied isolates were conrmed to belong to the Acinetobacter genus, as revealed
by the detection of a 425 base pairs (bp) amplicon corresponding to the recA gene (Supplementary Fig.S1).
Furthermore, 80 isolates were identied as A. baumannii species, given the codes Ab1-80, via detection of
the characteristic 208bp fragment of the 16S–23S rRNA gene intergenic spacer (ITS) region in such species
(Supplementary Fig.S1).
e 80 isolates were recovered from blood (n = 28, 35%), sputum (n = 26, 32.5%), wounds (n = 22, 27.5%),
and urine (n = 4, 5%), as shown in Supplementary TableS1.
Determination of antimicrobial susceptibility
We tested the 80 A. baumannii isolates and 2 A. baumannii standard strains, ATCC 19606 and ATCC 17987,
for susceptibility to β-lactams, FQs, aminoglycosides, a sulfa drug, and tetracyclines using the Kirby–Bauer
disc diffusion technique on Mueller–Hinton agar media (Supplementary TableS2). High resistance was
detected in all isolates to most of the investigated antimicrobials (Fig.1). e highest resistance was observed to
ceazidime (100%), cefepime (99%), cefotaxime (99%), tazobactam/piperacillin (99%), sulbactam/ampicillin
(97.5%), imipenem (97.5%), ciprooxacin (97.5%), levooxacin (97.5%), amikacin (95%), gentamicin (92.5%),
and trimethoprim–sulfamethoxazole (83.8%). e lowest level of resistance was observed to doxycycline
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(55%) and minocycline (48.8%) (Supplementary TableS2). e A. baumannii standard strain ATCC 19606
showed sensitivity to all investigated antimicrobials, excluding ceazidime, cefepime, cefotaxime, tazobactam/
piperacillin, sulbactam/ampicillin, and sulfamethoxazole/trimethoprim, while the standard strain ATCC 17987
showed sensitivity to all except sulfamethoxazole/trimethoprim (Supplementary TableS2).
e results demonstrated that 78 isolates (97.5%) were XDR and FQ-resistant isolates (Supplementary
TableS2). Eight representative XDR and FQ-resistant isolates (Ab29, Ab30, Ab36, Ab60, Ab65, Ab71, Ab72, and
Ab77) were selected to study the eect of the CIP- and LEV-loaded nanopreparations, FCIP, and FLEV, respectively,
on their susceptibility in comparison with free CIP and LEV antimicrobial agents.
Molecular characterization of FQ resistance mechanisms in A. baumannii isolates
FQRDRs and target site mutation
In order to characterize the FQ resistance of the 80 A. baumannii isolates and the 2 A. baumannii standard strains,
the FQRDRs in their genomes were further evaluated. e presence of mutations in gyrA and parC genes, major
FQRDRs, was detected by PCR followed by HinfI digestion, which resulted in successful digestion for the PCR
products with original sequences but not the ones with mutations (Supplementary TableS3). gyrA and parC genes
were detected in A. baumannii with amplicon sizes of 343bp and 327bp, respectively. A total of 69 (86.3%) and 75
(93.8%) of the 80 isolates harbored the gyrA and parC genes, respectively, as shown in Fig.2a. Non-digested PCR
Figure1. Resistance percentage of the 80 recovered A. baumannii isolates to every antimicrobial agent.
CAZ ceazidime, FEP cefepime, CTX cefotaxime, TZP tazobactam/piperacillin, SAM sulbactam/ampicillin,
IPM imipenem, CIP ciprooxacin, LEV levooxacin, AK amikacin, CN gentamicin, SXT sulfamethoxazole/
trimethoprim, DO doxycycline, MIN minocycline.
Figure2. Distribution of uoroquinolone (FQ) resistance genes among A. baumannii isolates; (a)
uoroquinolone resistance-determining regions (FQRDRs) and (b) plasmid-mediated quinolone resistance
genes (PMQRs).
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products were obtained aer HinfI digestion of gyrA and parC amplicons in all A. baumannii isolates, indicating
mutations in the FQRDR of both genes (Supplementary Fig.S2). In the two standard A. baumannii strains, ATCC
19606 and ATCC17987, obtaining two fragments aer HinfI digestion, at 291 and 52bp conrmed the absence of
mutation in gyrA, while at 206 and 121bp indicated the absence of mutation in parC (Supplementary TableS3).
DNA sequencing of the FQRDRs of gyrA and parC genes in A. baumannii (Ab30, Ab60, and Ab72) isolates
showed gyrA mutations at Ser83 to Leu and parC mutations at Ser80 to Leu. e obtained sequences were depos-
ited in the gene bank with accession numbers OR289925, OR289926, and OR289927.
PMQR genes and eux pump genes
e PMQR genes qnrA, qnrB, qnrS, qnrC, qnrD, and aac(6′)-Ib, as well as eux pump-encoding genes oqxAB
and qepA were evaluated via PCR (Supplementary TableS3) using the relevant primers shown in Supplementary
TableS4. e aac(6′)-Ib and qnrS genes were detected in A. baumannii with amplicon sizes of 480bp and 427bp,
respectively (Supplementary Fig.S3). Among 80 A. baumannii isolates, the aac(6′)-Ib gene was the most detected
PMQR in 61 (76.3%) isolates (Fig.2b). Two A. baumannii isolates (2.5%), coded Ab60 and Ab65, harbored the
qnrS and aac(6′)-Ib genes. Nevertheless, the other PMQR- and eux pump-encoding genes, qnrA, qnrB, qnrC,
qnrD, qepA, and oqxAB, were absent in all isolates, as shown in Fig.2b. Additionally, no PMQR genes or eux
pump-encoding genes were detected in the 2 standard A. baumannii strains.
Preparation and physicochemical evaluation of antimicrobial-loaded PCL nanoparticles
e particle size, polydispersity index (PDI), and zeta potential (ZP) are shown in Table1. e determination of
the above parameters was necessary to assess the capability of the preparation method for producing PCL NPs
successfully. e particle size of the prepared NPs was unimodal, ranging from 263.30 ± 2.76 to 271.10 ± 9.14nm
with a very narrow size distribution (PDI ≤ 0.05). e surface of the PCL nanoparticles (NPs) showed a negative
charge of approximately 9mV. e entrapment eciency (EE%), antibiotic loading (AL%), and yield (Y%) of
the NPs are listed in Table1. CIP was entrapped more eciently (54.11%) than LEV (28.14%). Consequently,
the values of AL% and Y% of FCIP (8.64% and 73.65%, respectively) were higher than their corresponding values
for FLEV (5.09% and 64.88%, respectively; Table1).
Solid-state characterization was performed using attenuated total reectance-Fourier transform infrared
spectroscopy (ATR-FTIR) and dierential scanning calorimetry (DSC). ATR-FTIR was conducted to evaluate
any possible chemical interaction between PCL and CIP or LEV. Moreover, the crystallinity or amorphousness
of the developed polymeric matrices of FCIP and FLEV was examined using DSC. e spectra and thermograms of
the investigated samples are shown in Fig.3a,b, respectively. Both CIP and LEV spectra presented characteristic
peaks at 1700 (C=O acid), 1620 (C=O carbonyl), 2850–2930 (aromatic H), and 1510–1530 cm−1 (piperazinyl
group). e PCL spectrum showed characteristic peaks at 1723 (C=O), 1239, 1165 (C–O–C), 2865, and 2944 cm−1
(C–H). e spectra of the binary physical mixtures, PCL/CIP and PCL/LEV illustrated the peaks of the polymer,
while the peaks of the antimicrobial appeared with diminished intensities or even vanished. e spectra of FCIP
and FLEV demonstrated the disappearance of the characteristic peaks of CIP and LEV, respectively.
ermograms of the investigated samples are shown in Fig.3b. e pure CIP was shown to be in crystalline
state, as evidenced by an endothermic peak at 150°C. Additionally, other endothermic peaks of the antimicrobi-
als appeared between 300–320°C. In the thermograms of PCL and binary mixtures (PCL/CIP and PCL/LEV),
an endothermic peak of PCL at approximately 57°C was observed. ermograms of the binary mixtures also
exhibited the melting peaks of the antimicrobial that appeared with reduced intensity. In contrast, the thermo-
grams of FCIP and FLEV demonstrated the amorphousness of their matrices, with no distinctive melting peaks.
e morphology of FCIP, FLE V, and plain NPs was studied using transmission electron microscopy (TEM) as
shown Fig.3c–e, respectively. e results conrmed the formation of perfectly spherical, discrete, and uniform
NPs with a smooth surface. Individual NPs of FCIP and FLEV had a dense core of entrapped antimicrobials embed-
ded in the polymeric matrix of PCL (core–shell architecture) which was not the case for plain NPs (monophasic
polymeric matrix) due to the absence of antimicrobials. Hence, the size of plain NPs was observed to be smaller
than that of the antimicrobial-loaded NPs. However, the actual size of the FCIP and FLEV NPs was consistent with
that measured by dynamic light scattering (DLS) (approximately 270nm).
Table 1. Physicochemical evaluation of CIP- and LEV-loaded nanoparticles. CIP ciprooxacin, LEV
levooxacin, FCIP CIP-loaded nanoparticles, FLEV LEV-loaded nanoparticles, plain NPs plain PCL nanoparticles,
PDI polydispersity index, ZP zeta potential, EE% entrapment eciency, AL% antibiotic loading, Y% yield.
Parameter
Formula
FCIP FLEV Plain NPs
Size (nm) 268.3 ± 4.15 271.1 ± 9.14 263.3 ± 2.76
PDI 0.03 ± 0.01 0.05 ± 0.02 0.03 ± 0.01
ZP (mV) − 8.88 ± 0.5 − 8.67 ± 0.41 − 8.67 ± 0.9
EE% 54.11 ± 2.66 28.14 ± 4.56 –
AL% 8.64 ± 0.42 5.09 ± 0.83 –
Y% 73.65 ± 12.68 64.88 ± 11.41 66.7 ± 12.94
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In vitro release study and kinetic modeling
e invitro release experiment was conducted using the dialysis bag diusion method. is study was performed
to compare the release proles of FCIP and FLEV to the corresponding ones of free CIP and LEV, respectively. e
release behaviors of CIP and LEV are expressed as cumulative percentages released from FCIP and FLEV, as shown
in Fig.3f,g, respectively. Rapid release rates of 94.4 ± 2.5% and 97.86 ± 2% were exhibited by free CIP and LEV
within 6h, respectively. In contrast, the controlled release patterns of CIP and LEV from FCIP and FLEV extended
for 10days to reach 100 ± 0.3% and 87.09 ± 3.3%, respectively, without burst release (Fig.3f,g). e release proles
showed that 20.2% and 20.6% of the loaded-CIP and LEV, respectively, released aer 24h incubation and that
were even much lower than free ones.
e release data were tted to kinetic models. It was found that FCIP and FLEV followed non-Fickian anomalous
diusion with diusion exponent (n) values of 0.72 and 0.52, respectively. On the other hand, the release of the
free antimicrobials followed Case II transport with n values of 1.08 and 0.97, respectively.
Figure3. Solid-state characterization of CIP, LEV, PCL, a physical mixture of PCL and CIP, a physical
mixture of PCL and LEV, FCIP, and FLEV; ATR-FTIR (a) and DSC (b). Morphologies of FCIP (c), FLEV and (d)
Plain NPs (e) by TEM and invitro release of FCIP (f) and FLEV (g). CIP ciprooxacin, LEV levooxacin, PCL
poly ε-caprolactone, FCIP CIP-loaded nanoparticles, FLEV LEV-loaded nanoparticles, plain NPs plain poly
ε-caprolactone nanoparticles, DSC dierential scanning calorimetry, ATR-FTIR attenuated total reectance-
Fourier transform infrared spectroscopy, TEM transmission electron microscopy.
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In vitro antimicrobial activity of the prepared nanoantimicrobials FCIP and FLEV
e minimum inhibitory concentration (MIC) values were measured using the broth microdilution technique
for FCIP and FLEV compared to CIP and LEV, respectively, and displayed in Table2. Eight representative XDR- and
FQ-resistant A. baumannii isolates and 2 A. baumannii standard strains were selected. e MIC values of CIP
ranged from 32 to 128μg/ml, and the MIC values of LEV ranged from 8 to 16μg/ml. e MIC values of FCIP and
FLEV ranged from 10.7 to 21.5 and 0.72 to 1.3, respectively. However, the MIC values of CIP, LEV, FCIP, and FLEV
for A. baumannii standard strains, ATCC 19606 and ATCC 17987 were 0.5 and 0.25μg/ml, respectively. FCIP and
FLEV were more eective than CIP and LEV, due to the increased eciency of CIP and LEV to be delivered into
the bacterial cells (Table2). e MIC of FCIP reduced by 1.5- to 6-fold compared to that of free CIP, while the
MIC of FLEV decreased by 6- to 12-fold compared to that of free LEV. At the same time, the plain NPs revealed no
activity on bacterial growth, indicating that the antimicrobial eects were only obtained from the encapsulated
drug itself (Supplementary TableS5).
Eux pump suppression by FCIP and FLEV
e eux pump inhibitor carbonyl-cyanide-m-chlorophenylhydrazone (CCCP) was used on the 8 selected
representative A. baumannii isolates and 2 standard A. baumannii strains. Only 3 out of 8 A. baumannii
isolates, Ab30, Ab30, and Ab72, of which the eux mechanism was demonstrated to be one of the FQ-resistant
mechanisms were used to study the eect of FCIP and FLEV on eux activity (Supplementary TableS5). at
was conrmed by a four-fold or more reduction in MIC (MIC decrease factor (MDF value of more ≥ 4)) in the
case of CIP and LEV, while no change in MIC was observed regarding FCIP and FLEV for the same isolates in the
presence of CCCP (Table3). e plain NPs had no antimicrobial (Supplementary TableS5) when used alone
or in combination with free CIP or LEV with or without CCCP (Supplementary TableS5). e standard A.
baumannii strains, had no eux activity as no change in their MIC of CIP nor LEV with co-treatment with CCCP
(Supplementary TableS5). When we used CCCP with strains (Ab29, Ab36, Ab65, Ab71, and Ab77) which do not
exhibit an eux pump related FQ resistance, the addition of the CCCP did not aect the MIC either with free
Table 2. e MICs for FCIP and FLEV compared to CIP and LEV against uoroquinolone (FQ)-resistant A.
baumannii isolates and standard strains A. baumannii ATCC 19606 and 17978. MIC minimum inhibitory
concentration, CIP ciprooxacin, LEV levooxacin, FCIP CIP-loaded nanoparticles, FLEV LEV-loaded
nanoparticles. *e mean MIC value was reported aer experiments were carried out in duplicate.
Isolate code
MIC* (μg/ml)
Fold decrease in MIC
MIC* (μg/ml)
Fold decrease in MICCIP FCIP LEV FLEV
Ab29 32 10.7 3 8 0.72 11
Ab30 32 10.7 3 8 0.72 11
Ab36 32 21.5 1.5 8 0.72 11
Ab60 128 21.5 6 16 1.3 12
Ab65 64 21.5 3 16 1.3 12
Ab71 64 21.5 3 8 1.3 6
Ab72 64 21.5 3 8 1.3 6
Ab77 64 21.5 3 8 1.3 6
ATCC 19606 0.5 0.5 1 0.5 0.5 1
ATCC 17987 0.25 0.25 1 0.25 0.25 1
Table 3. Eect of CCCP on the MICs of FCIP, CIP, FLEV and LEV against three representative XDR isolates
of A. baumannii. MIC minimum inhibitory concentration, CIP ciprooxacin, LEV levooxacin, FCIP CIP-
loaded nanoparticles, FLEV LEV-loaded nanoparticles, MDF MIC decrease factor, CCCP carbonyl-cyanide-m-
chlorophenylhydrazone. *e mean MIC value was reported aer experiments were carried out in duplicate.
Isolate code
MIC* (μg/ml)
MDF
MIC (μg/ml)
MDFCIP CIP + CCCP FCIP FCIP + CCCP
Ab30 32 8 4 10.7 10.7 1
Ab60 128 16 8 21.5 21.5 1
Ab72 64 16 4 21.5 21.5 1
Isolate code
MIC* (μg/ml)
MDF
MIC (μg/ml)
MDFLEV LEV + CCCP FLEV FLEV + CCCP
Ab30 8 2 4 0.72 0.72 1
Ab60 16 4 4 0.17 0.17 1
Ab72 8 2 4 1.3 1.3 1
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or encapsulated FQ (Supplementary TableS5). erefore, the increased killing activity of the PCL nanoparticle-
encapsulated CIP and LEV is associated with pump inhibition.
Time killing assay
Incubation of A. baumannii isolates Ab30, Ab60, and Ab72 with the MICs of FCIP or FLEV caused a signicant
drop (P < 0.05) in bacterial growth within 2–3h of incubation and complete bacterial killing aer 5–6h, as shown
in Fig.4. Free LEV reduced the viable bacterial count with 10% inhibition aer 24h incubation at the same
concentrations while free CIP showed no eect upon treatment. Treating A. baumannii Ab30, Ab60 and Ab72
isolates with the MIC of FCIP caused a signicant decrease (P < 0.05) in bacterial growth within 2h of incubation
and complete bacterial killing aer 6h in the Ab30 isolate (Fig.4a) and aer 5h in the Ab60 and Ab72 isolates,
Figure4. Time kill assay of treated A. baumannii isolates with the MIC of FCIP and the MIC of FLEV compared
to CIP and LEV over a period of 24h; A. baumannii isolate Ab30 treated with (a) FCIP and CIP and (b) FLEV
and LEV, A. baumannii isolate Ab60 treated with (c) FCIP and CIP and (d) FLEV and LEV and A. baumannii
isolate Ab72 treated with (e) FCIP and CIP and (f) FLEV and LEV. Experiments represent three replicates and
are expressed as the mean ± SD. Statistical signicance was assessed by the one-way ANOVA test: P < 0.05
was considered signicant (*P < 0.05). MIC minimum inhibitory concentration, CIP ciprooxacin, LEV
levooxacin, FCIP CIP-loaded nanoparticles, FLEV LEV-loaded nanoparticles, plain NPs plain poly ε-caprolactone
nanoparticles.
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as shown in Fig.4c,e, respectively. Similarly, the count of such A. baumannii isolates signicantly reduced aer
2h of incubation with FLEV (P < 0.05), complete bacterial killing aer 6h in the Ab30 and Ab60 isolates (Fig.4b,d)
and aer 5h in the Ab72 isolate, as shown in Fig.4f. e plain NPs had no killing activity as there was no eect
on the viable count of such isolates aer 24h (Fig.4).
Biolm elimination by FCIP and FLEV
e 80 clinical isolates of A. baumannii were classied as strong producers (62.5%, n = 50), moderate producers
(21.3%, n = 17) and weak producers (13.8%, n = 11) based on their ability to form biolms. Only 2.5% (n = 2) of
these isolates failed to form biolms (Supplementary TableS6). e 2 standard A. baumannii strains, ATCC 19606
and ATCC17987, were strong biolm producers (Supplementary TableS6). e results of biolm adherence of 80
A. baumannii isolates are summarized in Supplementary TableS6. Among the 78 FQ-resistant isolates (97.5%), 50
(64.1%) were strong, 16 (20.5%) were moderate, 10 (12.8%) were weak and 2 (2.6%) were non-biolm forming,
as shown in Fig.5a.
Subinhibitory concentrations of FCIP and FLEV (1/2 and 1/4 MIC) led to a signicant dose-dependent decrease
in biolm formation in the selected strong biolm-forming isolates (Ab30, Ab60, and Ab72) and standard A. bau-
mannii strain ATCC19606 compared to cultures treated with CIP and LEV or treated with plain NPs (Fig.5b,c,
respectively). FCIP was associated with 60–93% and 47–80% inhibition in biolm formation using 1/2 and 1/4
MIC, respectively, in the investigated isolates. Additionally, 1/2 and 1/4 MIC of FLEV caused 83–91% and 69–89%
inhibition, respectively, of biolm formation in such isolates. e greatest reduction (93%) in biolm formation
was achieved using 1/2 MIC FCIP on the Ab72 isolate (Fig.5b). On the other hand, CIP and LEV were found to
be associated with 2–10% and 3–11% inhibition, respectively, when used at their subinhibitory concentrations
(1/2 and 1/4 MIC), which indicates that there is no eect of either 1/2 or 1/4 MIC of CIP or LEV on biolm
formation (Fig.5b,c).
Discussion
A. baumannii is one of the deadliest and most contagious Gram-negative bacteria that has an improved capac-
ity to escape human immune responses and resist several types of antimicrobials, resulting in potentially fatal
pneumonia and bacteremia1. FQs have been found to be eective against A. baumannii isolates over the past
40years, although resistance to these antimicrobial agents has emerged quickly2. ere have been several reports
of the spread of FQ resistance among A. baumannii isolates worldwide16,17.
In the present study, 80 isolates of A. baumannii were obtained from diverse clinical sources. Among the
eighty conrmed A. baumannii isolates, resistance to the investigated antimicrobial agents was highly prevalent
(Fig.1). Most of the isolates were XDR (97.5%), while 97.5% of the isolates were resistant to both LEV and CIP.
On the other hand, 37 out of 80 isolates were sensitive to minocycline, with the highest frequency (46.3%).
A single point mutation in DNA gyrase is required for A. baumannii to be resistant to FQs; nevertheless,
simultaneous mutations in the FQRDR areas of the gyrA and parC genes are anticipated to dramatically increase
the level of FQ resistance. Many reports have shown that FQ resistance in A. baumannii is related to spontane-
ous mutations in the FQRDRs of the gyrA and parC genes18,19. e A. baumannii isolates were analyzed for their
FQRDRs using PCR, followed by HinfI digestion. Out of the 80 isolates in this study, 69 (86.3%) and 75 (93.8%)
carried the mutations in the gyrA (343bp) and parC (327bp) genes, respectively (Fig.2a). In A. baumannii
(Ab30, Ab60, and Ab72) isolates, DNA sequencing of the FQRDRs of the gyrA and parC genes revealed gyrA
mutations at Ser83 to Leu and parC mutations at Ser80 to Leu. All CIP- and LEV-resistant isolates were found
to harbor simultaneous mutations in the FQRDRs of both the gyrA and parC genes. It is suggested that these
two mutations are associated with CIP- and LEV-resistance, as previously mentioned in several reports20–22.
PMQR plays a signicant role in the development of resistance to FQ and may be a factor in the rise in spon-
taneous FQRDR mutations6. e three most well-known mechanisms of resistance to FQs related to PMQR
involve protecting the binding site in DNA-gyrase (qnr gene)7, altering the drug enzymatically (aac(6′)-Ib gene)8,
and expelling the agent by eux pumps (oqxAB and qepA genes)9. Numerous investigations have revealed that
FQ-resistant isolates of A. baumannii lack qnrA, qnrB, and qnrS genes23,24. Regarding PMQR genes in this study,
the aac(6′)-Ib gene (76.3%) was prevalent among the A. baumannii isolates, and two isolates (2.5%) harbored
the qnrS and aac(6′)-Ib genes. However, such isolates lacked other PMQR genes, qnrA, qnrB, qnrC, qnrD, qepA
and oqxAB, as shown in Fig.2b.
FQ-resistant A. baumannii causes serious public and nosocomial infections16. erefore, this study aims
to restore the antimicrobial properties of FQs to alter this problem, and polymeric nanoparticles would be the
methodology to achieve that. e double emulsication process is the method of choice for encapsulating hydro-
philic drugs inside NPs. Although the EE% of the hydrophilic water-soluble drugs is usually low, adjusting the
pH values of the internal and external aqueous phases could be applied to enhance the EE%. As FQ contains one
carboxylic group and three basic nitrogen sites, its solubility is pH-dependent. At pH 3 of the internal aqueous
phase (W1), the soluble cationic species of CIP and LEV predominated. On the other hand, neutral pH stimu-
lates the formation of the zwitterionic least soluble species in the external aqueous phase (W2, pH 7.5). Such
a preferential solubility pattern could secure ecient loading of the antimicrobials and prevent their diusion
out to the external aqueous phase during the formation of NPs. e higher EE% of FCIP could be attributed to
the lower solubility of CIP than LEV at pH 7.5 (W2). Hence, more LEV molecules might prefer their existence
in the external aqueous phase rather than entrapping inside the core of the FLEV25. e negative surface charge
of the NPs can be attributed to the ionization of the surface free carboxylic groups of PCL (matrix polymer) in
their aqueous dispersion26.
e characteristic peaks of CIP, LEV, and PCL in their individual ATR-FTIR spectra in Fig.3a agree with
those reported by other investigators27,28. Nevertheless, the diminished intensities of the antimicrobial peaks in
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the binary physical mixture spectra could be related to the dilution of CIP and LEV with PCL. In the ATR-FTIR
spectra of FCIP and FLEV, the absence of antimicrobial characteristic peaks indicated their entrapment within
the cores of the NPs. However, the bands that appeared in the range of 2800–3500 cm−1 were attributed to the
characteristic absorption peaks of the hydroxyl groups in polyvinyl alcohol (PVA) (the NP stabilizer).
In the DSC thermogram, the endothermic peaks of the antimicrobials that appeared between 300 and 320°C
in Fig.3b could be ascribed to the melting of CIP and LEV29. e reduction in the intensities of the melting peaks
of the antimicrobials exhibited in the thermograms of the binary mixtures could be attributed to the dilution
action of PCL. e absence of melting peaks in the thermograms of FCIP and FLEV indicated the amorphousness
of the polymeric matrices of the prepared NPs. e ndings of the solid-state characterization were consistent
Figure5. Biolm formation/inhibition assay; (a) Distribution of FQ-resistant A. baumannii isolates and
standard strains A. baumannii ATCC 19606 and 17978 according to biolm formation. (b,c) e impact
of subinhibitory concentrations (1/2 and 1/4 MIC) of FCIP and FLEV compared to CIP and LEV on biolm
formation of three representative isolates of A. baumannii that were both XDR and strong biolm producers
and standard A. baumannii strain ATCC19606: (b) e eect of FCIP and CIP. (c) e eect of FLEV and LEV.
Control: Culture treated with plain NPs. Experiments represent four replicates and are expressed as the
mean ± SD. Statistical signicance was assessed by the one-way ANOVA test: P < 0.01 was considered signicant
(**P < 0.01). MIC minimum inhibitory concentration, CIP ciprooxacin, LEV levooxacin, FCIP CIP-loaded
nanoparticles, FLEV LEV-loaded nanoparticles, plain NPs plain poly ε-caprolactone nanoparticles.
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with each other. e spherical core–shell morphology of FCIP and FLEV could secure the stealth of antimicrobials.
Moreover, the spherical geometry of the NPs could increase bacterial uptake by inuencing the contact area with
the cell membrane receptors more than rod-shaped particles30.
For invitro release, the sustained performance of FCIP and FLEV in Fig.3f,g, respectively, might be attributed
to the eective entrapment of antimicrobials within their cores. is conclusion agreed with the morphological
results, where the compact polymeric coat around the antimicrobial-loaded core likely prolonged antimicrobial
release. If the antimicrobial was not eciently entrapped, an unfavorable initial release (burst release) would
have occurred, which was the case with free CIP and LEV. Furthermore, the slow dissolution of the hydrophobic
PCL shell of FCIP and FLEV via the hydrolysis of ester bonds followed by pore creation would further prolong
the release of the entrapped antimicrobial, which extended for 10days in our study. is nding could oer the
method for the production of antimicrobial with long-lasting activities31.
e release data tted to the kinetic modeling revealed that the release of FCIP and FLEV was ruled mainly by
the diusion mechanism, which ensured a sustained release of the antimicrobials, as observed in their release
patterns. Alternatively, the release of free antimicrobials followed Case II transport (zero-order kinetics), which
means that the dissolution rather than diusion was the release limiting step32. e latter nding could be
attributed to formation of zwitterionic least soluble species of CIP and LEV at pH 7.4 of the release medium33.
ese ndings indicate that not only the release pattern but also the release kinetics could be modied by the
incorporation of CIP and LEV in PCL-based NPs; FCIP and FLEV, respectively.
Numerous studies have indicated improved antibacterial drug activity when entrapped in polymeric
nanoparticles31,34. ese results can be attributed to a variety of variables, such as improved drug delivery to the
site of action, increased drug stability when encapsulated into nanoparticles, and easier drug penetration into
bacterial cells35. In this study, the potency of CIP and LEV loaded into PCL nanoparticles against FQ-resistant
A. baumannii strains was increased compared to that of free CIP and LEV. Similarly, a CIP polymer-lipid hybrid
nanoformulation with greater antibacterial activity against a clinical E. coli isolate has been detected36.
Indeed, the features of FQs with low molecular weight and zwitterion composition are primarily responsible
for their ability to pass through the membrane of Gram-negative bacteria37. Additionally, the bacteria have
developed resistance to CIP and LEV due to chromosomal mutations that alter the target enzymes. FQ resist-
ance may occur by increasing eux or decreasing uptake, leading to reduced drug accumulation. Moreover,
plasmid-acquired resistance genes can produce proteins protecting bacterial molecules, antimicrobial metaboliz-
ing enzymes, or drug eux pumps5,6,9. e MICs of FCIP and FLEV against the investigated A. baumannii isolates
decreased by 1.5–6- and 6–12-fold, respectively, by encapsulating the drugs into nanoparticles (Table2). Such
a decrease in the MIC levels in our study may be due to enhanced drug penetration by nanoparticles into the
bacterial cell, which prevented bacterial development38. is behavior can be explained by the fact that NPs
can act as carriers for antimicrobials, eectively concealing them, enhancing their penetration of bacterial cell
walls, and helping to overcome resistance mechanisms of XDR bacteria39. erefore, the increased membrane
permeability obtained by nanosize-encapsulated CIP and LEV may account for the observed improvement in
the antibacterial action of FCIP and FLEV. Additionally, the prepared particle size aects membrane permeability40;
therefore, FCIP (268.3 ± 4.15nm) and FLEV (271.1 ± 9.14nm) nanoparticles were able to penetrate bacterial cells.
e nanosize and charge of the CIP and LEV formulations, as well as the bacterial hydrophobic anity for the
PCL polymer, which aids in rapid permeation across the bacterial outer membrane, may be the cause of the
instantaneous microbial killing induced by FCIP and FLEV41.
FQ resistance in A. baumannii isolates may be inuenced by eux-based systems42. CCCP increases the sensi-
tivity of several MDR bacteria, including A. baumannii, to various antimicrobials by inhibiting eux pumps43,44.
A four-fold or greater decline in MIC when CCCP was added to CIP and LEV served as evidence of its signi-
cance in increasing CIP and LEV resistance in some isolates45. e MICs of FCIP and FLEV nanopreparations were
not changed for the same isolates by co-treated CCCP. We thought that the reason for such a result is that the
nanoprepared antimicrobials totally inhibited the eux pump activity, so that the CCCP had no longer eect
on the MIC value when added to the culture (Table3). e eux-resistant mechanism was defeated by FCIP and
FLEV, as evidenced by the fact that their MIC was four times lower than that of free CIP and LEV, respectively,
on A. baumannii isolates with eux pump activity. NPs can bypass eux pumps by acting as a Trojan horse,
delivering antimicrobials, or by interacting with eux pumps to create irreversible blockage31,46. is notion is
strongly supported by the result that MIC for XDR strains without drug eux pumps was not aected by co-
treatment of CCCP with LEV and CIP, either, free or encapsulated into NPs. Similarly, zinc oxide nanoparticles
were demonstrated to have a unique eux pump inhibitory action on S. aureus eux pumps47. Additionally,
azithromycin poly lactic-co-glycolic acid nanoparticles (AZI-PLGA NPs) found to eectively counter the eux-
resistant mechanism exhibited by AZI-resistant bacteria. is was evidenced by a fourfold decrease inthe MIC
of NPs compared to free AZI48.
e rate of bacterial killing aer antimicrobial treatment is critical for preventing the emergence of antimi-
crobial resistance34. e killing activity of CIP and LEV was tremendously enhanced by PCL coating, as revealed
by the complete killing of the bacterial cells aer 5–6h of treatment with FCIP and FLEV compared to CIP and
LEV, respectively (Fig.4). On the other hand, aer 24h of incubation at the same concentrations, LEV slightly
(10%) decreased the viable bacterial count.
Biolm is a dispersed microbial growth that is challenging to penetrate and becomes resistant to conventional
treatment10. A variety of tactics have been studied to improve antibiolm activity, particularly in relation to
biolms that develop on medical devices that have been implanted. In the present study, 50 of 78 FQ-resistant
A. baumannii clinical isolates were strong biolm producers (Fig.5a). Subinhibitory concentrations of FCIP
and FLEV nanopreparations (1/2 and 1/4 MIC) signicantly reduced biolm formation by 47–93% and 69–91%
in strong biolm-forming isolates, whereas CIP and LEV, at their subinhibitory concentrations, aect biolm
formation by 10–17% (Fig.5b,c). Antimicrobial agents’ penetration and eectiveness are improved by the
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formulation of nanotherapy, which enhances the solubility and minimises the agglomeration of antimicrobials.
Nanoformulations of CIP and LEV were found to reduce their particle size and increase their antimicrobial-loaded
penetration. Moreover, the hydrophobic properties of PCL chains, which speed up the antimicrobial’s penetration
and cause the bacterial cell wall to burst, inhibit the growth of biolms and stop microbial colonization49. At the
same instance, the eect of metallic nanoparticles on A. baumannii biolms was demonstrated, as they inhibited
the biolm of A. baumannii by 88%50. Curcumin NPs, aluminium oxide NPs, silver NPs and other nanoparticles
were also found to suppress the growth of A. baumannii biolms51–53.
In conclusion, this is the rst report to study the inuence of CIP- and LEV-loaded nanoparticles on FQ
resistance and biolm inhibition ofclinical A. baumannii isolates in Egypt. e CIP- and LEV-loaded nano-
particles were found to be highly eective in killing A. baumannii and inhibiting biolm formation. Moreover,
it was demonstrated that encapsulation of CIP or LEV within NPs was a promising strategy to overcoming
eux-resistant mechanism towards FQs and improve their antibacterial eect. In future work, FQ-loaded PCL
nanoparticles will be investigated for ecacy invivo using adequate animal models.
Methods
Materials
Poly ε-caprolactone (PCL, Mn 80,000kDa), polyvinyl alcohol (PVA, Mw 14kDa), and methylene chloride (Mw
84.93) were purchased from Aldrich-Sigma Chemical Company, USA. Pharmaceutical grades of ciprooxacin
HCl (CIP) and levooxacin HCl (LEV) were kindly presented from EPICO and AMOUN pharmaceutical com-
panies in Egypt, respectively. In the nanopreparations procedures, deionized water (Millipore®, 18.2Mcm) was
used as the source of water.
Bacterial isolates
A. baumannii isolates from clinical specimens were obtained from the Central Microbiology Laboratory of
Mansoura University Hospital (MUH), Egypt, between December 2018 and November 2019. ese isolates
were obtained from diverse clinical specimens from patients in the intensive care unit (ICU), including wounds,
sputum, urine, and blood, according to hospital records. is work complies with the ethical guidelines of the
Research Ethics Committee in the Faculty of Pharmacy, Mansoura University, Egypt (Permit Number: 2022-193).
A. baumannii isolates were puried from the obtained specimens according to standard microbiological
culture techniques. All specimens were streaked on the chromogenic culture media Acinetobacter (CHRO-
Magar Acinetobacter Media, Paris, France) and incubated at 37°C for 48h. Isolates were also identied as the
Acinetobacter genus according to standard microbiological techniques, including colony morphology, Gram
stain, and biochemical reactions54. e standard strains, A. baumannii ATCC 19606 and 17978, were used as
positive controls.
Rapid extraction of genomic DNA, PCR conditions, and purication of PCR products
e genomic DNA of A. baumannii isolates was prepared by suspending fresh colonies in 100µl of distilled water,
followed by heating at 95°C for 10min. e bacterial suspension was centrifuged at 12,000rpm for 5min, and
the clear supernatant was transferred to a new tube and stored at − 20°C.
Unless otherwise specied, Dream Taq polymerase (Fermentas) was used for all routine PCRs. A reaction
mixture (25μl) containing 0.5μl of each primer (10μM), 12.5μl Dream Taq Green PCR Master Mix (2 ×), 1μl
of extracted DNA, and 9.5μl nuclease-free water was prepared. All PCRs were carried out under the following
conditions: primary denaturation at 95°C for 5min, followed by 35 cycles of denaturation at 95°C for 30s,
annealing for 30s at the temperature specic for each primer pair (Supplementary TableS4), and extension at
72°C for 1min, followed by one cycle of nal extension at 72°C for 10min. e PCR products were analyzed
by agarose gel electrophoresis (1.5% w/v agarose gel) and visualized by a UV transilluminator aer ethidium
bromide staining.
In the case of experiments that required purication of PCR products, digestion of PCR products, and
sequencing, a PCR purication kit (ermo, USA, Catalog number: K0701) was utilized according to the manu-
facturer’s directions.
Molecular identication
e isolates were conrmed as A. baumannii using a one-tube multiplex PCR method of the recA gene (charac-
teristic of the Acinetobacter genus) and ITS region (specic for A. baumannii spp.) by the primers listed in Sup-
plementary TableS455. e target amplicons of the recA gene and ITS region were 425 and 208bp, respectively.
e standard strains, A. baumannii ATCC 19606 and 17978, were used as positive controls.
Detection of antimicrobial susceptibility
e antimicrobial susceptibility prole was determined using the Kirby–Bauer disc diusion technique on Muel-
ler–Hinton agar media. e following antimicrobial discs (Oxoid, UK) were used to dene resistance proles
among A. baumannii clinical isolates: ceazidime (CAZ, 30µg), cefepime (FEP, 30µg), cefotaxime (CTX, 30µg),
piperacillin-tazobactam (TZP, 100μg/10µg), ampicillin-sulbactam (SAM, 10μg/10μg), imipenem (IPM, 10μg),
ciprooxacin (CIP, 5μg), levooxacin (LEV, 5μg), amikacin (AK, 30µg), gentamicin (CN, 10μg), trimetho-
prim–sulfamethoxazole (SXT, 1.25μg/23.75μg), doxycycline (DO, 30µg) and minocycline (MIN, 30µg). e
inhibition zone diameter was determined and interpreted consistent with the recommendations of the Clinical
and Laboratory Standards Institute guidelines56.
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Molecular characterization of FQ resistance mechanisms in A. baumannii isolates
e FQRDRs of gyrA and parC, besides the PMQR genes qnrA, qnrB, qnrS, qnrC, qnrD, and aac(6′)-Ib and the
eux pump-encoding genes oqxAB and qepA, were amplied via conventional PCR using the relevant primers
shown in Supplementary TableS4.
e PCR of the FQRDRs of gyrA and parC was performed using the specic primers listed in Supplementary
TableS4. e purication of amplicons was carried out using a PCR purication kit (ermo, USA, catalog
number: K0701). e puried amplicons were digested with the HinfI digestion enzyme (ermo Scientic,
USA) according to the manufacturer’s protocol. e digested PCR products were then separated by agarose gel
electrophoresis (1.5% w/v agarose gel). e separated fragments were analyzed for mutations in the FQRDRs20,21.
Regarding gyrA, a single undigested PCR fragment (343bp) demonstrates the presence of a mutation at Ser83,
whereas two fragments, indicating digestion, at 291 and 52bp conrm the absence of mutation. In the case of
parC, a single undigested PCR band (327bp) conrms the presence of a mutation at Ser80, while digestion
generating two fragments at 206 and 121bp indicates the absence of a mutation.
ree representative XDR isolates, Ab30, Ab60, and Ab72, were selected for sequencing of the FQRDRs in
both the gyrA and parC genes. e target sequences in the gyrA and parC genes were amplied using Phusion
High-Fidelity DNA Polymerase (ermo Scientic, USA) and the specied primers in Supplementary TableS4.
PCRs were done according to the manufacturer’s instructions. e puried PCR products were sent to Sigma
Scientic Service Technical Support Company in Cairo, Egypt for sequencing by an Applied Biosystems 3500 XL
Genetic Analyzer and PCR primers specic for each gene. FinchTV program was used to analyze and visualize
chromatograms.
Preparation of NPs
e double emulsion/solvent evaporation method was used to prepare NPs. e ingredients of the prepared
NPs are shown in Supplementary TableS7. CIP or LEV was dissolved in sterile deionized water to form W1 at
a concentration of 2% w/v. PCL was dissolved in methylene chloride at a concentration of 15mg/ml to serve
as the organic phase (O). e pH of W1 was adjusted to 3, and then 1ml of it was emulsied in 10ml of O. A
primary emulsion (W1/O) was formed via ultrasonication for 1min at 100% amplitude in pulse mode (2s on
and 1s o) (Sonics Vibra Cell, Sonic & Materials, INC, USA) in an ice bath. W1/O was added to 200ml of a pH-
adjusted aqueous solution of 0.5% w/v PVA (W2, pH 7.5). is mixture was rapidly sonicated for 3min under
the above conditions to make a double emulsion (W1/O/W2). Aer solvent evaporation, the NPs were isolated
by centrifugation (Benchtop Centrifuge, Sigma Laborzentrifugen, Germany) at 10,000rpm for 1h, washed,
and centrifuged at the same speed for 30min to remove a clear supernatant. e NP pellets were dispersed in
deionized water then freeze-dried at − 80°C using a Freeze Dryer (SIM FD8-8T, SIM International, USA). e
lyophilized NPs were collected and refrigerated at 4°C for further evaluation. Plain NPs were prepared by the
same procedure as that for drug-free W1 at pH 3.
Physicochemical evaluation of the prepared NPs
A particle size analyzer (Malvern Instruments Ltd., England) equipped with DLS was used to determine the size
and surface charge, or ZP, of the NPs. e EE% of NPs was determined using an indirect method. Briey, the
concentration of unentrapped antimicrobials in the supernatants recovered aer centrifugation was measured
and subtracted from that of the total antimicrobials. Spectrophotometric measurements of CIP and LEV were
conducted using the supernatant of the plain NP as a blank at 275 and 278nm, respectively (UV–VIS Spectro
double beam, Labomed Inc., USA). e weight of lyophilized NPs was determined to calculate the AL% and Y%
of NPs. EE%, AL%, and Y% were calculated using the following equations:
Spectral analysis of PCL, pure CIP, pure LEV, and binary physical mixtures of each antimicrobial with PCL,
FCIP, and FLEV was conducted using ATR-FTIR (ermo Fisher Scientic, Inc., Waltham, MA, USA). DSC was
used to assess the crystallinity of the abovementioned samples (DSC, Pyris 6 DSC, Perkin Elmer, USA). e
morphology of the NPs was determined by TEM (TEM, JEOL 1010; JEOL Ltd, Tokyo, Japan). Briey, a 200-mesh
copper grid coated with carbon was placed with an aqueous drop of the NP dispersion, and any surplus liquid
was absorbed using lter paper. Subsequently, the samples were dried at room temperature so that they could
be observed under a 200kV voltage.
In vitro release study and kinetic modeling
e invitro release of CIP and LEV from the NPs was evaluated by the dialysis bag diusion method. e FCIP
or FLEV (equivalent to 2.3mg) was suspended in 1ml of deionized water and placed in pre-equilibrated dialysis
bags (Dialysis Sacks, Avg. Flat Width 35mm, MWCO 12kDa, Sigma-Aldrich). Each dialysis bag was immersed
in a beaker containing 100ml of phosphate buer (pH 7.4) to represent the release medium and maintained at
EE
%=
Total Antibiotic
−
Free Antibiotic
Total Antibiotic
×
100
AL
%=
Total Antibiotic
−
Free Antibiotic
Wt of NPs
×
100
Y
%=
Wt of NPs
Wt of Antibiotic
+
PCL
×
100
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37 ± 0.5°C in a shaking incubator at 100rpm (GFL Gesellscha für Labortechnik, Burgwedel, Germany). Every
predetermined time interval, 2ml aliquots were sampled and replaced with an equal fresh volume. e samples
were then ltered (0.45μm), appropriately diluted, and analyzed using spectrophotometry at 275 and 278nm
for CIP and LEV, respectively. Each experiment was done in triplicate, and the cumulative released percentage
of the antimicrobial was calculated at every time interval using preconstructed calibration curves. To explain the
release mechanisms of CIP and LEV, the release data were kinetically analyzed using the zero-order, rst-order,
Higuchi diusion mechanism, and Korsmeyer-Peppas model. e model with the highest correlation coecient
(r2) was the one that described the release mechanism.
Antimicrobial evaluation of the prepared NPs
e activity of FCIP and FLEV against eight representative XDR-resistant isolates was compared to CIP and
LEV, respectively. e microtiter plate assay method was used to determine MICs of CIP, LEV and their
nanopreparations56. Muller-Hinton broth medium (100μl) was pipetted into sterile microtiter plate wells. CIP,
FCIP, LEV, and FLEV were prepared as two-fold serial dilutions in Muller-Hinton broth medium ranging from 1024
to 0.5μg/ml. Inoculation of all dilutions was made with overnight cultures of the isolates at a nal inoculum of
5 × 105CFU/ml. Positive (culture only) and negative (medium only) controls were performed in all experiments.
Under the same conditions, the MICs of plain NPs were also determined. e plates were incubated at 37°C for
24h. Each experiment was performed in duplicate.
Eect of FCIP and FLEV on eux activity
To study the eect of nanoformulated FQs on FQ resistance via an eux pump mechanism, the MICs of CIP,
FCIP, LEV and FLEV were determined. e eux pump inhibitor CCCP was administered at a subinhibitory
concentration (20μg/ml) to each well. e wells were inoculated with diluted culture (5 × 105CFU/ml) of XDR
A. baumannii isolates (Ab30, Ab60, and Ab72) where the eux mechanism was demonstrated to be one of the
FQ-resistant mechanisms. Furthermore, the eect of CCCP on the MICs of plain NPs, CIP, and LEV against
representative FQ-resistant and XDR A. baumannii isolates and 2 standard strains A. baumannii was investigated.
A positive control for each isolate was included to investigate the viability of dierent isolates in the presence
of CCCP alone. e MDF was determined for each isolate in duplicate. Inhibition of the eux pump by CCCP
was deemed to have a considerable eect when the MDF value was 4 or above57.
Antimicrobial killing assay
e killing rate of FQ-resistant bacteria by FCIP and FLEV was determined and compared to CIP- and LEV-free
antimicrobials, respectively. e investigated XDR A. baumannii isolates (Ab30, Ab60, and Ab72) were propa-
gated until the bacterial count reached 5 × 106CFU/ml. e investigated isolates were treated with FCIP and FLEV
at the MIC and incubated at 37°C. Samples were collected at 0, 1, 2, 3, 4, 6, 10, and 24h, and each sample was
ten-fold serially diluted to determine the viable bacterial count. In the same instance, bacterial counts of cultures
treated with CIP and LEV were also performed under the same conditions. As a control, bacterial growth with-
out free antimicrobials (CIP or LEV) or nanoformulated antimicrobials (FCIP or FLEV) and plain NPs were also
investigated. e surface drop method was used in triplicate to calculate the number of bacteria that recovered
over time following treatment58. Following antimicrobial treatment, the number of recovered cells was plotted
against the CFU/ml over time. Each experiment was carried out in triplicate.
Eect of FCIP and FLEV on biolm formation
e capacity of biolm formation among 80 A. baumannii isolatesand2 standard A. baumannii strains, ATCC
19606 and ATCC17987, was assessed invitro using 96-well microtiter plates as previously mentioned59. e
formed biolm was stained with 1% w/v crystal violet followed by solubilization using glacial acetic acid (33%
v/v). e solubilized biolm was measured using an ELx808TM Absorbance Microplate Reader (BioTek Instru-
ments Inc., Winooski, VT) at OD490nm. A negative control of the medium was included in each experiment. e
mean OD490nm of each bacterial isolate from four independent experiments was calculated to assess the ability
of A. baumannii isolates to produce biolms. Biolm formation by the A. baumannii isolate was repeated in
quadruplicate.
To determine the biolm formation capacity of A. baumannii isolates, the cut-o optical density (ODc)
was established as three standard deviations above the mean OD of the inoculum-free negative control. Strains
were classied as follows: non-biolm producer (N) if OD ≤ ODc, weak biolm producer (W) if ODc < ODW ≤ 2
ODc, moderate biolm producer (M) if 2 ODc < ODM ≤ 4 ODc, and strong biolm producer (S) if ODS > 4 ODc.
ree representative strong biolm-forming, FQ-resistant, and XDR isolates (Ab30, Ab60, and Ab72) were
selected to assess the eect of FCIP and FLEV on biolm formation. Subinhibitory concentrations for CIP, FCIP,
LEV, and FLEV (1/2 and 1/4 MIC) were incorporated during bacterial incubation in 96-well plates60. Controls of
medium only or cultures without drugs were included in each experiment. e biolm-forming reference strain
of A. baumannii ATCC 19606 was used as a positive control.
Statistical data analysis
Statistical analysis involved calculating the mean and standard deviation of the values. All microbiological assays
were performed in duplicate except the antimicrobial killing assay, and the capacity for biolm formation was
repeated in triplicate and quadruplicate, respectively. A one-way ANOVA test was calculated, with the signi-
cance value set at P < 0.05 or P < 0.01 using the GraphPad Prism soware package (version 8.3.0).
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Scientic Reports | (2024) 14:3125 | https://doi.org/10.1038/s41598-024-53441-1
www.nature.com/scientificreports/
Ethics approval
e Research Ethics Committee at the Faculty of Pharmacy at Mansoura University in Egypt (Permit Number:
2022-193) has approved this work as ethically compliant.
Data availability
All data generated or analyzed during this study are included in this published article [and its supplementary
information le].
Received: 23 July 2023; Accepted: 31 January 2024
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Acknowledgements
Sincere gratitude is extended to Mansoura University’s clinical laboratory for donating clinical isolates.
Author contributions
A.A.: Methodology, data analysis and writing the manuscript. M.E.: Participation in data analysis and writing
of the manuscript. N.S.: Participation in the preparation, evaluation of nanoparticles, data analysis and writing
of manuscript. M.S.: Suggestion the idea of the research, supervision, troubleshooting and revision of the
manuscript. R.B.: Supervision the research and revision of the manuscript. All authors read and approved the
nal manuscript.
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Funding
Open access funding provided by The Science, Technology & Innovation Funding Authority (STDF) in
cooperation with e Egyptian Knowledge Bank (EKB).
Competing interests
e authors declare no competing interests.
Additional information
Supplementary Information e online version contains supplementary material available at https:// doi. org/
10. 1038/ s41598- 024- 53441-1.
Correspondence and requests for materials should be addressed to M.I.S.orR.B.
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